Final Report Summary - FIBRO-TARGETS (Targeting cardiac fibrosis for heart failure treatment)
Executive Summary:
Cardiomyocytes, fibroblasts and vascular cells in the heart are connected by a complex matrix, principally composed of fibrillar collagen, which is instrumental in preserving structural integrity and plasticity. In the diseased heart, the matrix undergoes structural and subcellular changes that progressively influence heart function. Beyond the cardiomyocyte-centric view of heart injury, it is now accepted that alterations of the cardiac extracellular matrix (ECM) and cardiac remodelling play a major role in the development and evolution of cardiac diseases leading to heart failure (HF). These ECM alterations result in cardiac fibrosis. At the site of myocardial infarction, acute focal fibrotic scarring provides myocardial healing and prevents rupture. In contrast, chronic diffuse or focal reactive myocardial fibrosis is a consequence of either pressure or volume-overload due to persisting hypertension, valvular heart diseases, ischemic injury (in areas remote of the infarction), or diffuse myocardial diseases, such as cardiomyopathies or other diseases. Myocardial fibrosis is characterized by dysregulated collagen turnover (increased synthesis predominates over unchanged or decreased degradation) and excessive diffuse collagen accumulation in the interstitial and perivascular spaces. This dysregulation of collagen turnover takes place mainly in phenotypically transformed fibroblasts, termed myofibroblasts. Fibroblasts and particularly myofibroblasts secrete extracellular procollagen chains that assemble into collagen types I and III fibrils and become cross-linked to form the final fibres. Collagen cross-linking is an important post-translational modification because it increases myocardial tensile strength and the resistance of collagen fibers against degradation by matrix metalloproteinases. Myocardial fibrosis disrupts the myocardial architecture, contributes to myocardial disarray, and determines mechanical, electrical, and vasomotor dysfunction, thus promoting the progression of cardiac diseases to HF. Of note, although it may be decreased by mineralocortioid recetor antagonsits, fibrosis persists significantly in the myocardium of HF patients under the current treatment regimen recommended by the official guidelines. Furthermore, the severity of histologically-proven myocardial fibrosis has been reported to be associated with higher long-term mortality in patients with cardiac diseases, particularly those with HF. In this regard, the detection, prevention and repair of myocardial fibrosis have emerged as important targets for improving HF therapy. In order to achieve diagnostic and therapeutic advances, it is critical to identify new pro-fibrotic mechanisms not yet targeted by currently available therapies, and to translate these mechanisms into individualized diagnostic tools and specific therapeutic targets. Besides meaningful functional and therapeutic outcomes, valid molecular targets must not induce serious adverse effects, such as influencing inflammation processes or wound healing. Therefore, there is a need for a systematic collaboration between clinical investigators and basic scientists, together with the industry to allow integration of data from computer biomedical (in silico) and basic (in vitro) studies with preclinical (in vivo) research findings to be distilled into clinically actionable information for the fight against myocardial fibrosis. The FIBROTARGETS consortium is a multinational consortium with industrial and academic partners, funded by the European Commission and primarily aimed to characterize novel emerging mechanisms of myocardial fibrosis. Targets and biomarkers investigated include proteins, proteoglycans, and microRNAs. FIBROTARGETS has achieved its remarkable goal in identifying a number of discrete mechanistic pathways and molecular targets involved, as well as potentially anti-fibrotic agents ready to be transferred into diagnostic biomarkers and therapeutic agents amenable to improve patient care.
Project Context and Objectives:
Background and context: In Europe, 5% of hospital admissions in adults are due to heart failure (HF) – a larger proportion caused by myocardial infarction. In addition, ageing of the EU population will make incidence and prevalence of HF rise inexorably, unless effective preventive therapy is applied. So far antihypertensive therapy has had some success in preventing HF. Nevertheless, no therapy targeting pathophysiological mechanisms in HF such as fibrosis, inflammation or myocyte growth, is available for clinical use. At last, beyond the impact on myocardial interstitial fibrosis (MIF), nearly 45% of all deaths in the developed world can be attributed to some type of chronic fibro-proliferative disease. With this scenario, finding new targets, biomarkers and/or promising molecules with anti-fibrotic capabilities, would have a great impact in the society in a mid-term.
Hypothesis and objectives: FIBRO-TARGETS is based on the hypothesis that the intervention on novel fibrosis-related targets involved in the processes of fibroblast differentiation to myofibroblasts, on the collagen synthesis over degradation balance and/or on collagen maturation may allow for interstitial repair, thus providing a new strategy for the prevention and treatment of MIF.
Experimental approaches: The setting of several experiment models (for in vitro read-outs and in vivo in small and large animal) relevant to one or several studied signaling pathways has allowed to further or newly characterize the capacity of candidate biotargets to modulate collagen production and cross-linking. According to the anti- or pro- fibrotic properties of the studied biotargets, molecules (hits) were identified as potentially able to interfere with biotarget biological activity. The proposed agonist and antagonists have been selected within large libraries of molecules for most of the biotargets. When conventional structure activity relationship (SAR) analysis could not be used because of the unknown crystalline structure of OGN a different approach was taken to find homologues of OGN and use amino-acid sequence alignment and 3D protein structure overlay to identify conserved peptidic motifs that may be important for the OGN activities. Additional suitable cell-based screening assays have been developed to determine the efficacy parameters for the potential hits for several biotargets from each WP. The therapeutic potential of some of the most promising hits were tested in vitro, and challenged in vivo in small and big animal. Meanwhile, to validate therapeutic relevance of the hits identified for the studied biotargets, a common database of controls and patients susceptible of developing fibrosis or having fibrosis has been set. The merged clinical data of 1,200 individuals are associated with biosamples allowing for biomarker profiling of selected individuals for the biotargets resulting from the analysis and discoveries made experimentally. An optimization of the biosamples use has required the development and/or validation of multiplexing bioassays for selected/identified targets. A set of samples from patients and matched controls has been assayed. The resulting stratification of the patient according to signal-specific BM profile and diagnostic algorithms represents a powerful theranostic tool.
Main results:
WP 1 was dedicated to search for novel, safe and effective anti-fibrotic strategies based on the blockade of TGF-β-related fibrogenic mechanisms. We have validated the involvement of CT-1, apelin and several microRNAs in the development of MIF associated with HF, identifying them as potential novel targets for the treatment of this lesion. On the other hand, findings suggest that collagen cross-linking and LOX are key players in the development of MIF and that they are involved in the pro-fibrotic processes. Therefore, LOX emerges as potential candidate target for the treatment of MIF in HF patients.
In WP2 we investigated the biological role of non-structural extracellular matrix proteins in heart failure using multiple cell line, animal and human validation experiments. Our findings reveal that OGN reduces the proliferation of the scar tissue forming cells, fibroblasts, thereby preventing the production of the main component of scar tissue, collagen. SPARC had a protective role in murine viral myocarditis by improving heart function, reducing inflammation, cell death and mortality. We could detect small and large OGN variants in areas of fibrosis and on inflammatory cells in viral myocarditis, an inflammatory disease of the heart muscle. In ischemic patients, the expression of OGN in the heart was increased compared to healthy controls and plays a crucial role in the induction of inflammation.
In WP3 using combination of miRNA mimic library screens with a small RNA deep sequencing approach in human cardiac fibroblasts (HCFs), annotated and not annotated miRNAs were identified as potential targets for anti-fibrotic therapies. In vitro investigations uncovered repressive effects of the miRNAs on autophagy and the antioxidant system. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p, miR-132-3p as well as a novel miRNA. Consistently, we found that lead miRNA candidates were activated in murine models of fibrotic cardiac diseases, implying their possible functional importance in fibrosis-development of the diseased heart. Downstream cellular pathways were investigated such as repression of autophagy and detoxification of reactive oxygen species.
WP4 was focused on different mineralocorticoid receptors (MR) related pro-fibrotic molecules. Using bioinformatics tools, reticulocalbin RCN-3 was found to exert anti-fibrotic effects and to block the profibrotic effects of aldosterone and Gal-3. We demonstrated the role of Gal-3 as a profibrotic and also as a proinflammatory mediator and the beneficial effects of Gal-3 blockade using modified citrus pectin and the role of NGAL in high blood pressure, after MR activation as well as in myocardial infarction. We found it to be produced by immune cells in aldosterone-induced cardiac injuries in NGAL depleted mice depleted.
In WP5, using repurposing of known drugs or in silico screening, we identified hit molecules with promising anti-fibrotic potential, which, after additional developments would be used as MIF treatment. Libraries were screened, focused on the four mechanistic pathways studied within FIBRO-TARGETS. In vitro high-throughput robotic assisted screening of natural compounds was followed by in vivo validations. Anti-fibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts (HCFs) and subsequent validation and mechanistic in vitro and in vivo studies.
Hits were analyzed for dose-dependent inhibition of proliferation of HCFs, for modulation of apoptosis and extracellular matrix expression. In vitro findings were confirmed in vivo using an angiotensin 2-mediated murine model of cardiac fibrosis both in preventive and therapeutic settings, highlighting the potential translational relevance of our findings
All results have been compiled and curated and 8 compounds have been selected considering their good profile. These compounds represent an excellent starting point for further developments. In this sense, several partners within FIBRO-TARGETS have started to look for collaborative opportunities in order to push these compounds to the next stages.
After in vitro validation of the first generation of hits, we used structure-activity relationship studies for further refinement of likelihood of activity, toxicity, and solubility. All molecules were tested for efficacy under multiple newly developed cell based models (4xSBE (SMAD2/3), APJR, Col1A1 and Collagen Polymerization) and the best performing ones have been incorporated to an ADME and toxicity panel and further tested for Aqueous solubility determination, Chemical stability (acidic pH), Microsomal stability, Plasma protein binding, CYP3A4 isoform inhibition and Bi-Caco2 permeability assay.
By the end of the project, we have identified molecules with a very promising profile that will be further developed in upcoming projects. Eleven compounds have shown efficacy in at least 2 of the tests, and one has activity in all of them.
1. Potential anti-fibrotic compounds shown to inhibit the pro-fibrotic response to CT-1 and apelin and cross-linking in human cardiac fibroblasts.
2. A compound that interferes with the biological activity of OGN, resulting in a decreased inflammatory response thereby postulating it as a potential therapeutic for viral myocarditis.
3. Two compounds that interfere with the biological activity of SPARC. These compounds could be beneficial in the context of chronic fibrosis by inhibiting the binding of SPARC to collagen and thereby the big scar tissue mesh formation.
4. A series of 5 lead natural compounds interfering with key miRNAs, with confirmed cardiac anti-fibrotic activity and improved diastolic function, in hypertensive mice and the clinically highly relevant rat model of hypertensive cardiac fibrosis.
5. Small molecules interfering with NGAL in cardiac cells which are able to block its effects and could be promising therapeutic tools in the treatment of cardiac disease in which NGAL is involved.
In WP6 we have successfully established a novel pig model, which closely resembles human pathophysiology of cardiac fibrosis, induced by constant high blood pressure and heart hypertrophy. The model was comprehensively characterized through multimodal imaging, repeated transthoracic echocardiography and invasive pressure measurements. We have established a new imaging modality using 18F-FDG-PET-MRI and revealed that FDG-PET is more sensitive than cardiac MRI with late enhancement to assess dysfunctioning myocardium. We used these models for the preclinical evaluation of potential drug candidates with high translational value in the future. We have tested two potential drug candidates, which were identified in the other work packages, in rats and pigs for their effect on limiting or reversing cardiac fibrosis. Serial quantification of plasma biomarkers including miRNAs, and histological analyses revealed the values of a number of potential biomarkers and transcriptomic profiles to assess early cardiac remodelling and fibrosis.
In WP7 we have developed and validated biomarker assays for selected candidates in blood-derived matrices within a quality-controlled environment. The initial list of candidate biomarkers relevant to the various mechanistic pathways explored in the various WPs was significantly reduced because of the insufficient development of bioassays suitable for the small blood sample volumes available to the consortium. We finally assessed Thrombospondin-2, SOD2, MMP-1, SDF1-α, GDF-15, CD40L, AGRP, PIIINP, Gal-3 and NGAL.
We investigated the association of biomarkers (potentially indicative of our validated biotargets) with clinical phenotypes with potentially different fibrosis pathophysiology. Cases were age/gender matched with 2 controls, all selected from 12 patient cohorts in a common database available to individual consortium partners. Our results suggest that history of HF mainly drives the associations with the assayed biomarkers. However, in patients free from prevalent HF, distinct bioprofiles were associated with distinct phenotypes such as respectively, Galectin 3 with post MI, GDF-15 with diabetes, PIIINP with obesity and NGAL and CD40L with hypertension, suggesting that discrete biotargets may be more specifically involved in respective clinical phenotypes.
Overall, FIBRO-TARGETS results give credit to the conceptual approach of stratification of patients according to signal-specific biomarker profile identifying subsets of patients more likely to be responders to potential therapeutic molecules that we have validated as biotargets for anti-fibrotic therapy. Further experimental and clinical assessment will provide evidence to set the ground for new targeted anti-fibrotic personalized therapeutic strategies.
Project Results:
WP1
Diffuse myocardial interstitial fibrosis (MIF), caused by an excessive deposition of collagen fibers, is a common feature in heart failure (HF) patients. MIF has a detrimental impact on cardiac function and on the prognosis of these patients. Importantly, MIF is present even in those patients adequately treated according to clinical practice guidelines, indicating that current pharmacological treatments are not effectively targeting this lesion. Transforming growth factor β (TGF-β) is one of the main pro-fibrotic factors involved in MIF. However, given the broad range of TGF-β physiological actions, direct inhibition of TGF-β may have detrimental effects, explaining why the available anti-TGF-β agents aimed at treating MIF have failed so far. Therefore, the necessity exists to search for novel, safe and effective anti-fibrotic strategies based on the blockade of TGF-β-related fibrogenic mechanisms. This WP was focused on several TGF-β-related pro-fibrotic molecules: cardiotrophin-1 (CT-1), apelin, NADPH oxidases (NOX) and microRNAs.
1. Cardiotrophin-1
In patients with HF of hypertensive origin we have found an increase in the cardiac expression of the pro-hypertrophic factor CT-1 both at mRNA and protein level. Interestingly, CT-1 was directly associated with myocardial fibrosis and with collagen type I and III expression in these patients. Moreover, there was a significant association between the circulating levels of CT-1 and the serum biomarkers of collagen type I and collagen type III synthesis (PICP and PIIINP, respectively). Therefore, besides being involved in cardiomyocyte hypertrophy, CT-1 emerges as a mediator of myocardial fibrosis (Hypertension 2014;63:483-489). Reinforcing this notion, in human cardiac fibroblasts CT-1 induced collagen type I and III expression and the differentiation of fibroblasts to myofibroblasts (Hypertension 2014;63:483-489). CT-1 also increased fibronectin expression as well as metalloproteinases (-1,-2,-9) activities and the secretion of the pro-inflammatory molecule IL-6. Interestingly, CT-1 also upregulated the expression of lysyl oxidase (LOX), the main enzyme involved in collagen cross-linking. In this context, it has to be considered that increased collagen cross-linking makes collagen fibers more insoluble, stiffer and more resistant to degradation.
To investigate this association more in depth we analyzed an experimental model of HF with preserved ejection fraction (HFpEF), the Dahl salt sensitive rat, where we found MIF characterized by an increase in collagen fibers deposition both interstitial and perivascular, as well as in cardiac collagen cross-linking and LOX. Myocardial CT-1 expression, both at mRNA and protein levels, was increased in animals with HFpEF. Interestingly, CT-1 was inversely associated with the E/A ratio in all animals, a distinctive parameter of diastolic dysfunction. Of note, CT-1 was associated with MIF, and particularly with perivascular fibrosis. Importantly, the increase in CT-1 was strongly associated with the increase in LOX expression and consequently with collagen cross-linking in all animals.
Altogether, these results suggest that CT-1 may contribute to myocardial fibrosis through collagen cross-linking stimulation.
Several potential antagonists for the CT-1 receptor were developed in collaboration with WP5 and their anti-fibrotic potential was tested in human cardiac fibroblasts. Several of these compounds were able to prevent CT-1-induced procollagen type I synthesis at a concentration of 10 µM. However, the same inhibitory effect was found upon stimulation with TGF-β. Therefore, currently we cannot be certain of the specificity of the compounds.
2. Apelin
The expression of the anti-fibrotic factor apelin has been previously shown to be decreased in end-stage HF patients. In the experimental model of HFpEF (Dahl salt-sensitive rat) we found a decrease in myocardial apelin expression which was inversely associated with LOX expression and with insoluble collagen. These results support the hypothesis that the apelin system is decreased in chronic HF and that this down-regulation may contribute to MIF through the regulation of collagen cross-linking. In particular, it highlights the relevance of LOX in this process.
In different human fibroblasts (dermal, freshly isolated atrial and ventricular) apelin expression was down-regulated by TGF-β in a dose-dependent manner. On the other hand, synthetic apelin was able to partially inhibit TGF-β-induced collagen production. Several potential agonists for the apelin receptor (APJ) were developed in collaboration with WP5 and their anti-fibrotic potential was tested in human cardiac fibroblasts. Six of these compounds were able to inhibit TGF-β-induced procollagen expression at a concentration of 1 µM. Interestingly, some of them (n=6) also decreased LOX expression. Basic ADME-Tox in vitro and in vivo studies were performed for the most promising compounds and the one showing no toxicity and the best pharmacokinetic properties was selected for an in vivo proof of concept study in rodents.
3. NADPH oxidases
We evaluated the role of the pro-oxidant NADPH oxidases NOX2 and NOX4 in a murine model of essential hypertension, the spontaneously hypertensive rat (SHR), characterized by MIF and increased myocardial collagen cross-linking and LOX. We found an increased expression of both NOX2 and NOX4 expression. The 2 enzymes were associated with the extent of MIF and with procollagen type I expression. On the other hand, both isoforms were associated with LOX expression. However, in the HFpEF model of the Dahl salt sensitive rat we found a decrease in NOX4 which was associated with increased perivascular fibrosis. These apparently contradictory results suggest that the role of NOX4 may be dependent on the pathology and the main cellular source. This notion is in agreement with previously published data in experimental models and in clinical studies.
In human cardiac fibroblasts we found that NOX4 was strongly up-regulated by TGF-β in a dose-dependent manner. However, TGF-β presented no significant effects on NOX2 expression. Therefore, we focused on NOX4 for further studies. Hydrogen peroxide, which is the product of NOX4 activity, induced procollagen type I expression, as well as an increase in some of the key molecules involved in collagen processing including LOX, suggesting that NOX4-mediated oxidative stress may play a relevant role in the development of MIF. However, when we analysed the impact of NOX4 silencing on TGF-β-induced procollagen production we found that NOX4 was not an essential mediator for TGF-β effects. Taking all these data into account we decided to focus on the other molecules under study.
4. MicroRNAs
To analyze miRNAs regulated by TGF-ß, a miRNA deep sequencing approach was performed in human cardiac fibroblasts. miR-23a-5p, miR-210-5p, let-7b-3p, miR-195-5p, miR-130b-5p, miR- 423-5p levels were found to be increased in HCFs after treatment with TGF-ß. However, overexpression of these miRNAs did not induce any significant changes in the expression of fibrosis-related genes (Col1a1, Col3a1, CTGF, α-SMA, MMPs or TGF-ß) in these cells.
On the other hand, in aortic stenosis (AS) patients, who showed MIF and increased collagen cross-linking, miR-122 was found to be down-regulated in the myocardium and associated with TGF-β and with parameters related to MIF. In vitro, miR-122 was shown to directly regulate TGF-β expression (Clin Sci 2014;126:497-506). Subsequently, we analysed if any of the miRNAs reported to be related to MIF was associated with collagen cross-linking. We found that miR-19b was decreased in the myocardium of these patients and that it was inversely associated with LOX expression and consequently with collagen cross-linking. Moreover, it was associated with increased left ventricular stiffness and with the presence of HF. In human fibroblasts miR-19b inhibition increased the expression of LOX protein, suggesting that miR-19b might regulate LOX and subsequently collagen cross-linking (Sci Rep 2017;7:40696). Importantly, serum levels of miR-19b were also associated with LOX and collagen cross-linking, as well as with the myocardial expression of miR-19b, suggesting that this microRNA could be useful as a non-invasive biomarker of collagen cross-linking.
5. General Conclusion
In this WP we have validated the involvement of CT-1, apelin and several microRNAs in the development of MIF associated with HF, identifying them as potential novel targets for the treatment of this lesion. Furthermore, in collaboration with WP5 we have developed potential anti-fibrotic compounds targeting these molecules which have been shown to inhibit the pro-fibrotic response in human cardiac fibroblasts. Further in vivo studies in experimental models are needed to definitively prove the therapeutic value of these compounds.
On the other hand, findings from the different studies performed suggest that collagen cross-linking and LOX are key players in the development of MIF and that they are involved in the pro-fibrotic effects of the different molecules evaluated. Therefore, LOX emerges as potential candidate target for the treatment of MIF in HF patients.
WP2
Heart failure: a serious health problem
Within Europe, every year 3.6 million patients are diagnosed with heart failure, a condition in which the heart cannot pump sufficient blood to meet the body’s needs. As the consequence of our aging population as the burden of cardiovascular risk factors (diabetes, obesity and high blood pressure), the prevalence of heart failure is expected to increase by 50% in 2030. However, effective therapies targeting the key mechanisms, such as scar formation in the heart, underlying the development of heart failure are lacking. Therefore, there is an urgent need to develop early interventions that target key mechanisms, including cardiac scar formation, for slowing down the progression to heart failure.
The biological role of non-structural extracellular matrix proteins in heart failure
All cells in the human body are surrounded a complex meshwork of proteins and carbohydrates, called the extracellular matrix. Several non-structural proteins within this extracellular matrix, such as Osteoglycin (OGN) and SPARC, are associated with a variety of cardiovascular diseases. Interestingly, an increased expression of these non-structural extracellular matrix proteins mostly results in an improved heart pump function, survival of heart cells, function of the cells involved in scar tissue formation and inhibition of adverse inflammation, all mechanisms which are involved in the development of heart failure. However, the exact role of OGN and SPARC in heart failure, especially scar tissue formation, is unknown.
The biological role of OGN in scar tissue formation in the heart
During this consortium, we have performed multiple cell line, animal and human validation experiments to elucidate the role of OGN during fibrosis in the heart. Our findings reveal that in OGN reduces the proliferation of the scar tissue forming cells, fibroblasts, thereby preventing the production of the main component of scar tissue, collagen. High blood pressure (angiotensin II treatment) increased the cardiac scar tissue formation of young mice in both presence and absence of OGN. In old mice, absence of OGN increases scar tissue formation and decreased ability of the heart muscle to relax, one of the characteristics of heart failure, indicating that OGN protects against heart failure by reducing scar tissue formation. Absence of OGN resulted in increased rupture of infarctions, bleedings within the heart and heart dysfunction in an animal model for heart attack, while overexpression blunted heart dysfunction in an animal model for heart attack. OGN levels were significantly higher in patients with a history of heart attack. In patients with a narrowed valve opening of the major artery of the body, the aorta, OGN gene expression in the heart was significantly higher than in healthy controls, with lower OGN gene expression in patients with more severe scar tissue formation in the heart. This suggests that OGN is a negative regulator of the induction of scar tissue formation in patients with a narrowed valve opening of the aorta. In patients with a restriction in blood supply to the tissue (ischemia) the small OGN variant (34 kDa) was detected in the heart, while this variant was undetectable in control patients without scaring. Moreover, blood samples of ischemic patients showed the presence of the small OGN variant.
The biological role of OGN in the inflammatory response in the heart
In addition to the small OGN variant, we detected a large OGN variant (72 kDa) in viral myocarditis, an inflammatory disease of the heart muscle. The absence of the large OGN variant decreases inflammation in the heart of a mouse model of viral myocarditis. In viral myocarditis patients, OGN (small variant) was present in areas of fibrosis and on inflammatory cells (leukocytes; OGN large variant). In ischemic patients, the expression of the large and small OGN variant (on neutrophils) in the heart was increased compared to healthy controls. Cell line studies showed that the absence of large OGN variant decreased the activation of toll-like receptors, receptors that play a crucial role in the induction of inflammation. In addition, we identified a compound that interferes with the biological activity of OGN, resulting in a decreased inflammatory response thereby postulating it as a potential therapeutic for viral myocarditis.
The biological role of SPARC during viral myocarditis
During this consortium, we have performed multiple cell line and animal experiments to elucidate the role of SPARC during fibrosis in the heart. We demonstrated a protective role of SPARC in murine viral myocarditis by improving heart function, reducing inflammation, cell death and mortality. This protective role can be the result of the ability of SPARC to improve the endothelial barrier and thereby preventing the infiltration of immune cells in the heart. In addition, we found that SPARC improves the contraction of heart cells and heart function in healthy and viral myocarditis animal models. In addition, we identified 2 compounds that interfere with the biological activity of SPARC. These compounds could be beneficial in the context of chronic fibrosis by inhibiting the binding of SPARC to collagen and thereby the big scar tissue mesh formation.
WP3
Project #1:
In the present study, we developed natural compounds-based preventive and therapeutic approaches for cardiac fibrosis in hypertensive mice, leading to ameliorated diastolic function. Importantly, morphology of kidneys and livers remained unchanged in these animals, demonstrating toxicologically safe profiles in early evaluations. Moreover, we could confirm efficacy of the compounds in a clinically highly relevant rat model of hypertensive cardiac fibrosis; highlighting the potential translational relevance of our findings. Mechanistically, a pro-fibrotic miRNA was discovered as important key-player of the anti-fibrotic activities of the compounds.
In vitro high-throughput robotic assisted screening of natural compounds was followed by in vivo validations. Anti-fibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts (HCFs) subsequent validation and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of HCFs, for modulation of apoptosis and extracellular matrix expression. In vitro findings were confirmed in vivo using an angiotensin 2-mediated murine model of cardiac fibrosis both in preventive and therapeutic settings. To investigate the mechanism underlying the anti-fibrotic potential of the lead compounds, treatment-depended changes in the noncoding RNAome in primary HCFs were analyzed by RNA-deep sequencing.
To elucidate a potential influence of the identified natural substances on endogenous miRNAs, key post-transcriptional regulators of gene expression, miRNA deep-sequencing in primary HCFs was performed. Interestingly, levels of the highly conserved miR-671-5p were consistently and significantly reduced after treatment of primary HCFs with all 5 anti-fibrotic lead compounds from the initial screen in vitro.
Project #2:
In this study, we identified annotated miRNAs as potential targets for anti-fibrotic therapies. The discovery was based on a combination of miRNA mimic library screens with a small RNA deep sequencing approach in HCFs. In vitro investigations uncovered repressive effects of the miRNAs on autophagy and the antioxidant system, hereby contributing to the fibrotic actions in HCFs. The small RNA deep sequencing approach additionally allowed the identification of a not annotated miRNA that promoted HCF-migration, critical in fibrotic remodelling.
The herein described pro-fibrotic miRs therefore perturb cellular pathways of homeostasis, essential for the health of a variety of cell types and tissues, not only of the heart. Thus, inhibition of the uncovered miRNAs might be safely applied to combat cardiac fibrosis.
Taken together, both studies led to the successful discovery of potential innovative therapeutic strategies to target myocardial fibrosis, a major limitation of cardiac performance.
In detail, functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated, however unstudied pro-fibrotic miRNAs. In parallel, miRNA deep sequencing was performed after subjecting HCFs to pro- and anti-fibrotic stimuli, additionally enabling discovery of not jet annotated miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of several selected, highly conserved miRNAs as well as a novel miRNA.
Consistently, the lead miRNA candidates were found to be activated in murine models of fibrotic cardiac diseases, implying their possible functional importance in fibrosis-development of the diseased heart. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by selected miRNAs explain their profibrotic nature on a mechanistic level
WP4
Cardiac remodeling plays a major role in the development and evolution of cardiac diseases leading to heart failure (HF). During cardiac remodeling, there is an initial phase of systemic inflammation which could induce interstitial and perivascular fibrosis. Myocardial fibrosis is defined by an increase in extracellular matrix (ECM) deposition and excessive collagen accumulation in the interstitial and perivascular spaces. Aldosterone, via the activation of the mineralocorticoid receptor (MR), plays an important role promoting cardiac inflammation, fibrosis and hypertrophy. Several clinical and experimental studies have demonstrated the beneficial effects of mineralocorticoid receptor antagonists (MRAs) in mild to severe HF, suggesting that an excessive activation of the MR occurs over the course of the disease and plays an important role in the pathophysiology of HF. However, the treatment with MRAs is underuse due to the side effects of MRAs such as hyperkalemia or gynecomastia.
1. Aldo/MR.
A better understanding of the molecular mechanisms of mineralocorticoid activation pathway in cardiovascular pathophysiology remains incomplete. Indeed, a better knowledge of the underlying mechanisms may highlight novel mediators of the MR signaling cascade. These newly identified intermediates could be good candidates as biotargets for novel pharmacological approaches, especially in diseases where the aldosterone/MR pathway is involved. This WP was focused on different MR related pro-fibrotic molecules such as Galectin-3 (Gal-3), Cardiotrophin-1 (CT-1) and neutrophil gelatinase–associated lipocalin (NGAL).
We identified that Aldo up-regulated collagen type I, III and VI in human cardiac fibroblasts (Martinez-Martinez E, J Proteomics 2017). Moreover, reticulocalbin (RCN) family members are common proteins down-regulated by Aldo biotargets. The function of RCN proteins remains unknown. However, its localization in the lumen of the endoplasmic reticulum suggests a role in protein synthesis, modification, and intracellular transport. Using bioinformatics tools, we focused our study on RCN-3, demonstrating that RCN-3 exerts anti-fibrotic effects and blocks the profibrotic effects of Aldo and Gal-3 (Martinez-Martinez E, Sci Rep 2017). A patent has been filled covering the interest of RCN-3 in cardiac fibrosis.
2. Galectin-3
Gal-3 is a member of a β-galactoside–binding lectin family expressed in several tissues including the heart. It has been observed in clinical studies increased Gal-3 levels in patients with HF, which were correlated with extracellular matrix markers. In the present project, we demonstrated the role of Gal-3 as a profibrotic and also as a proinflammatory mediator. In addition, we have demonstrated the beneficial effects of Gal-3 blockade using modified citrus pectin (MCP) in experimental models of different pathologies such as hypertension (Martinez-Martinez E, Hypertension 2015a), aortic stenosis (Arrieta V, Clin Sci 2017), hyperaldosteronism (Calvier L, JACC Heart Fail 2015) and obesity (Martinez-Martinez E, Hypertension 2015b).
3.- Neutrophil Gelatinase–Associated Lipocalin
Neutrophil Gelatinase–Associated Lipocalin (NGAL) is a potent biomarker of renal injury which has been proposed to be pathogenic in cardiovascular disease since high NGAL plasma levels have been associated with mortality in HF patients. In this project, we have evaluated the role of NGAL in high blood pressure after MR activation as well as its role in myocardial infarction. In addition, we have demonstrated the importance of NGAL production by immune cells in aldosterone-induced cardiac injuries using a model of mice depleted for NGAL in their immune cells. The data have been published in Hypertension, 2017 or are currently submitted for publication.
Related with NGAL and in collaboration with WP5, we have identified small molecules interfering with NGAL in cardiac cells which are able to block its effects and could be promising therapeutic tools in the treatment of cardiac disease in which NGAL is involved.
WP5
The main objective of this WP5 is to identify hit molecules with promising anti-fibrotic potential. This list of molecules would be the starting point for additional developments that would be used as MIF treatment. To do so, three types of libraries have been screened; Prestwick Chemical Compound Library, Greenpharma natural compound Library and a dedicated set of molecules, focused on four mechanistic pathways studied within FIBRO-TARGETS. The first two libraries were used to check repurposing of known drugs whereas the latter consists in a set of molecules identified in an in silico screening, as targeting novel targets involved in MIF. The in silico approaches included mimicking known active compounds and modelling of the protein 3D structure in order to calculate the affinity of candidate molecules with the protein. The best ones in terms of affinity were selected for in vitro evaluation. This allows to reduce time and cost as we will only focus on molecules with potential to be active. After in vitro validation on the first generation of hits, we use the structure-activity relationship to design derivatives that may have better activity or less detrimental effects (eg toxicity, solubility problem...).
All these molecules have been tested for efficacy under newly developed cell based models and the best performing ones have been incorporated to a ADME and toxicity panel.
By the end of the project, we have identified up to six molecules with a very promising profile that will be further developed in upcoming projects.
In vitro screening model development.
In order to test the available molecules, we have developed several cell-based models to assess the efficacy of potential candidates by imaging. Three models were successfully generated; Col1A1 expression model, 4xSBE model (SPARC2/3 activation model) and Apelin receptor activation (APJ Receptor internalization model). The two latter, passed the requested criteria to be used in High Throughput Screening (HTS) applications (Z’ values of 0.79 and 0.784 respectively) and were used to identify sets of promising molecules.
The Col1A1 model (immortalized Human Cardiac Fibroblasts) is able to measure differences in the Col1A1 expression as well (upon TGF-beta induction, cells increased the reporter gene expression. Different time points were determined ranging from 24h through 96h). However, it did not pass HTS Z’ requirements (the response of the population was heterogeneous) and therefore it was only used to check the efficacy of the most promising compounds on this pathway.
Besides these three cell-based efficacy models, a biochemical in vitro assay has been also developed for efficacy screening. It is based on collagen self-polymerization turbidity measurements. In presence of SPARC, collagen polymerization is delayed. The original rate of polymerization restoration in presence of testing compounds is an indicator of SPARC activity inhibition.
We have screened 64 compounds from the different WPs using the Collagen polymerization assay to determine whether they are able of inhibiting SPARC action over Collagen or not. 15 appear to inhibit the SPARC activity over Collagen to some extent. The WP with more compounds positives is WP2 (7 compounds), then WP1 (5 compounds) and finally WP4 (3 compounds). This makes sense given that the compounds coming from WP2 are chemically designed to inhibit SPARC. Unfortunately, none of the compounds from WP3 showed SPARC inhibiting behaviour.
Efficacy data.
Three sets of compounds have been tested; 69 targeted compounds curated in the in-silico screening, Prestwick Chemical Compound Library (PCL 1280 compounds) and the Greenpharma Natural Compound Library (GPNCL 480 compounds). The two libraries, cover a broad chemical space and contain drugs already approved by FDA and EMEA.
From the targeted compounds, 11 have shown efficacy in at least 2 of the tests, and one has activity in all of them. On the other hand, just the 4xSBE and APJR models have been used for compounds libraries. Regarding PCL, we have identified 31 and 29 positive compounds respectively. In the case of GPNCL, just 4 and 2 compounds respectively showed efficacy in the cell-based models.
ADME-Tox data.
The toxicity of the compounds (for up to three cell types; cardiac fibroblasts, hepatocytes and cardiomyocytes) has been determined. Overall, they seem to be quite safe. Just some of them have shown to be toxic for cardiac fibroblasts (9 molecules turned out to be toxic out of 69 compounds), hepatocytes (29 compounds out of 47 were safe up to 50 μM) or cardiomyocytes (None of the tested molecules inhibited the hERG channel; crucial for cardiomyocyte function).
Compounds showing acceptable efficacy and toxicity data have entered to a panel of ADME assays designed to determine several parameters. Performed ADME assays comprise;
• Aqueous solubility determination.
70% of tested compounds showed a solubility over 60% (500 μM in PBS).
• Chemical stability (acidic pH).
92% of molecules were highly stable to acidic degradation (over 80% still present after 2h in gastric content mimicking solution).
• Microsomal stability.
Overall the compounds were extensively processed by microsomes and only the 22% of them resisted to degradation (over 65% presence after 2h). This feature by itself is not necessarily bad news. In fact, it could be desirable to avoid over dosage or mid-long term treatments.
• Plasma protein binding.
78% of the compounds bound to plasma proteins at values lower than 98%. It is broadly known that several currently marketed treatments show values around this value or even slightly higher.
• CYP3A4 isoform inhibition.
55% of the compounds seemed to be processed by another cytochrome isoform (less than 20% CYP3A4 inhibition) and 36% were mildly processed (20%
2 compounds were tested in this absorption assay and both presented permeability data in the range of the used reference molecule (Propanolol).
WP6
In this work package, we developed and characterized small and large animal models for cardiac fibrosis with high translational value and used these models for evaluation of potential new drugs. We have successfully established a novel pig model which closely resembles human pathophysiology of cardiac fibrosis induced by constant high blood pressure and heart hypertrophy. In growing, juvenile animals, a bare metal stent was implanted in the descending aorta, which consequently results in stenosis, triggering pressure overload and myocardial hypertrophy. By inducing a gradual increase of aortic pressure, this model resembles the development of hypertrophy and heart failure in human patients more closely than previously described large animal models. The model was comprehensively characterized through multimodal imaging, repeated transthoracic echocardiography and invasive pressure measurements, serial quantification of plasma biomarkers including miRNAs, and histological analyses. This work will facilitate the preclinical evaluation of potential drug candidates with high translational value in the future.
We have tested two potential drug candidates, which were identified in the other work packages, in rats and pigs for their effect on limiting or reversing cardiac fibrosis. While one of the compounds showed good efficacy and tolerability in rats, unforeseen and partially severe side effects were encountered in pigs for both potential drugs, and consequently, dosing had to be adjusted. With these lower doses, a meaningful beneficial effect of the two tested compounds failed to be proven in large animals. Although the encountered toxic effects hamper the use of the investigated compounds for therapeutic interventions, the gathered results are useful for further compound optimization.
We have established a new imaging modality to visualize cardiac remodelling based on fibrosis. We have demonstrated that 18F-FDG-PET-MRI with late enhancement investigation is useful to predict adverse cardiac remodelling, if this investigation was performed 3-day after acute myocardial infarction. We have revealed that FDG-PET is more sensitive than cardiac MRI with late enhancement to assess dysfunctioning myocardium.
In an additional task of this work package, we have analysed a number of potential biomarkers, including proteins and miRNA oligonucleotides, and we were able to demonstrate the predictive values of new biomarkers, that may be useful to assess early cardiac remodelling and fibrosis.
We have compared the different transcriptomic profiles of cardiac fibrosis of different diseases, which, however leads to the same clinical phenotype of heart failure and cardiac fibrosis. Overlaps and differences in gene expression signatures highlighted common and varying mechanisms leading to cardiac fibrosis. Based on these results, identification and specific targeting of the key regulator genes of the different fibrotic mechanisms and pathways can be instrumental for the development of more effective and diversified antifibrotic therapies in the future.
All results have been compiled and curated and compounds have been selected considering their promising profile. These compounds represent an excellent starting point for further developments. In this sense, several partners within FIBRO-TARGETS have started to look for collaborative opportunities in order to push these compounds to the next stages.
WP7
Immunoassay development
This primary task aimed to develop and validate biomarker assays for selected candidates in blood-derived matrices within a quality-controlled environment (ISO13485). The secondary goal was to evaluate the predictive value of these biomarkers on selected cohort of patients at risk to develop fibrosis-related heart failure.
Final selection of biomarker candidates to be assessed was agreed in April 2016 and updated in Sept 2016. These targets were subdivided in lists A (SOD2, Thrombospondin-2, AGRP, CD40L, SDF-1/CXCL12, GDF15, MMP-1, Galectin-3 and MMP9), B (SPARC, NGAL and NGAL/MMP9 complex) and C (Fibulin2, Mimecan, Biglycan, Lumican, PICP, PIIINP, CITP and CT-1) by order of priority for the consortium, market availability and foreseen biomarker assay availability from FIRALIS. Monoclonal antibodies development for those biotargets started by a literature work to find the best sequences for the protein and/or peptide of interest with the respect of the protein properties (solubility, pH, environment, the ideal bacteria strain, etc.) in order to have a good product for immunization. For some targets, the immunizations were performed using both whole protein and a peptide to increase the probability to have good clones for sandwich ELISA development. After several assays of protein production and mouse immunizations, FIRALIS succeeded to generate 220 hybridomas. Hybridomas for positive clones were grown at FIRALIS and several quantities (up to 10 mg) of each clone were purified as well as their corresponding protein for calibration range.
ELISA sandwich assay development phases combine several parameters starting with the selection of the best antibodies pair for capture and detection of target in biological fluid (plasma or serum). Then, coating conditions, incubation times and concentrations of antibodies were optimized. Upon completion of the optimization phase, lyophilization tests were performed on the standard. As soon as the final formulation of the standard was validated, the standard curve was established as well as the minimum required dilution (MRD), that is, the minimum dilution of the biological fluid (matrix) required to determine the concentration of the target independently of the matrix effect.
At this stage, when the design of the final kit is finalized, the analytical characteristics of the assay were determined. These included the concentration of the target in Quality Controls, the Linearity of dilution, the limit of detection (LOD), the lower and upper limits of quantification (LLOQ and ULOQ), Repeatability (intra-assay precision), and Reproducibility (inter-assay precision). The analytical characteristics of the assay were then confirmed by an independent team in FIRALIS, in a different laboratory. Stability studies and biological ranges in normal and 2 independent cohorts of diseased population complete this validation process. Upon completion of all these stages, the assay is ready for CE marking. Currently, the described process is completed for 3 biomarker assays.
Samples biobanking
FIRALIS has developed a biobanking facility encompassed in a fully documented tracking system linking samples to analytical results obtain from them. This biobank for research purposes is certified NF S 96-900. FIRALIS has organized the signatures of the MTA with each contributive partner. Specific MTA have been signed between the provider of cohorts and the recipient (FIRALIS) with University of Maastricht, FIMA, UCD and CHRU de Nancy (INSERM). FIRALIS has formalized the biobanking protocol.
FIRALIS received end of 2016 a first set of 60 samples (250µL plasma and 250µL serum samples from 30 heart failure patients and 30 healthy subjects) to evaluate the performance of the selected commercial bioassays to measure a maximum of BMs from the FIBRO-TARGETS lists. By end of June 2017, FIRALIS received 600 plasma and 563 serum samples for assessment using the selected platform and assays by consortium members.
Assays performance evaluation
At this stage, FIRALIS has completed full assay development for 3 targets (CNN-1, Caldesmon and clusterin). The assay development of those biomarkers had started before a final lists of 17 priority targets (Thrombospondin 2, sCD40L, GDF-15, MMP-1, Galectin-3, SPARC, Apelin, Cardiotrophin 1, MMP-2 variant 1, MMP-2 PEX, MMP-3, PINP, PIIINP, Mimecan/osteoglycin, MMP-9, NGAL, NGAL/MMP-9 complex) was issued and agreed by the consortium. Except for GDF-15 and MMP-1, FIRALIS succeeded to select at least one antibodies pair for each target showing promising data in human serum and/or plasma. Generally, pairs were first identified with using recombinant proteins spiked in fetal bovine serum and then further evaluated in human serum or plasma. More interesting, FIRALIS finalized the analytical validation for the NGAL sandwich ELISA assay.
Samples analysis
Targets from list A and PIIINP from list C were evaluated on a cohort of 60 age and sex-matched samples (30 healthy subjects and 30 HF subjects). The use of multiplexing ELLA and O-link platforms provided the consortium with data on potential 88 other predictive targets from the Cardiovascular II panel (http://www.olink.com/products/cvd-ii-panel/). Of note, due to stability issues it was agreed not to measure MMP9 in this evaluation experiment.
With the exception of Thrombospondin-2 and SOD2, expression levels of most of the biomarkers were satisfactorily detectable in both cases and controls.
Those biomarkers were analyzed for the whole cohort composed of 600 plasma samples assessed for Thrombospondin-2, SOD2, MMP-1, SDF1-α, GDF-15, CD40L, AGRP, PIIINP and Gal-3 and 563 serum samples assessed for NGAL.
The biomarkers measurements and quality check were completed by the end of Q3 2017 (mid-September 2017) and communicated to the partner in charge of statistical analysis.
Potential Impact:
FIBRO-TARGETS has achieved its remarkable goal in identifying a number of discrete mechanistic pathways and molecular targets involved, as well as potentially anti-fibrotic agents ready to be transferred into diagnostic biomarkers and therapeutic agents amenable to improve patient care. As a result of this project we have advanced in the knowledge of the mechanisms involved in the development and progression of myocardial fibrosis in HF. Furthermore, we have identified compounds with potential anti-fibrotic effects susceptible to be developed into therapeutic agents.
HF is a deadly condition that affects more than 37 million patients worldwide, with limited therapeutic possibilities. Due to the enhanced life expectancy to date, an increasing number of patients presents with diastolic HF (HF with preserved ejection fraction, HFpEF). Despite extensive research efforts during the last centuries, HFpEF still represents an unmet medical need, imposing an enormous medical and socioeconomic burden, particularly on ageing societies. Early interventions targeting key mechanisms, including myocardial interstitial fibrosis (MIF), could slow down progression to HF. No treatment has been shown to target cardiac fibrosis to counteract passive stiffness of the heart, a major contributor to HFpEF. As the current treatment approaches for HF prolong survival but do not prevent or cure HF, the FIBRO-TARGETS consortium fulfils the urgent need to develop specific effective treatments for HF. Moreover, it has to be considered that pathological fibrosis is not restricted to the heart, being present in other organs such as kidney, liver and lungs. If the mechanisms identified by the FIBRO-TARGETS consortium for cardiac fibrosis are proven to be involved in other organs fibrosis, the beneficial impact of the proposed novel anti-fibrotic strategies could be extended to other pathologies.
Therapeutic developments conducted in the last decades have led to a substantial decrease in mortality in HF with reduced ejection fraction. Therefore, demonstrating a therapeutic benefit on top of existing therapies in the logic of ‘one-size-fits-all’ trials has become increasingly difficult and cost demanding. As a consequence, in the last three decades, landmark HF trial sizes have progressed from a few hundred to many thousand patients. Operational obstacles and unbearable costs are major challenges for the successful implementation and completion of such large trials. Even with pre-specified subgroup analysis, regulators do not accept testing for responder profiles due to the risk of bias. Precision medicine has emerged as one possible solution, aiming at identifying the different strata within a disease based on a deeper understanding of the mechanisms underpinning these strata. Having this in mind, we in FIBRO-TARGETS aimed at identifying mechanistic bioprofiles using a candidate biomarker approach, that may ultimately pave the way for a precision preventive medicine approach. Specific mechanistically tailored anti-fibrotic therapies may then be administered to patients expressing corresponding biotargets, identified using biomarker profiling.
First of all, the FIBRO-TARGETS consortium offered new insights in the mechanisms involved the formation of scar tissue formation and the development of HF by executing translational research (bench-to-bedside).
We have validated the involvement of mechanistically targetable pathways in the development of myocardial fibrosis associated with HF, identifying them as potential novel targets for the treatment of fibrosis. The potential preventive, therapeutic and/or diagnostic potential of CT-1, apelin, LOX, the non-structural matrix proteins and mineralocorticoid receptor associated targets such as Gal-3, and NGAL and miRNA controlled fibrosis mechanisms will have far stretching benefits in the future for HF patients and patients with inflammation and fibrosis mediated cardiac remodelling and scar tissue formation-associated diseases. We uncovered lead drug candidates and demonstrated their potential suitability for the prevention and treatment of hypertension-induced cardiac fibrosis and diastolic dysfunction. Therefore, our results are encouraging for prospective clinical investigations to eventually reduce symptoms, hospitalizations, morbidity, mortality and medical costs.
The screening for druggable molecules paves the way for increasing the therapeutic arsenal against HF. We could identify several interesting compounds that target important proteins involved in MIF. Some are old drugs – used for other indications – that were found to be interesting in MIF. In total, 8 of them are being evaluated in animal models. They may become future drug candidates for HF. Besides, 2 natural products were patented. We plan to license these to biotech or pharmaceutical companies. A synthetic compound is under evaluation in a “big pharma” for potential co-development.
During the project, large animals have been instrumental for a better understanding of mechanisms of fibrosis. The availability of useful, practical animal models with high translational value improves the drug development process by enabling comprehensive preclinical evaluations also on the molecular level, and by discarding unsuccessful compounds before they enter clinical testing.
Precision medicine has emerged as one possible solution, aiming at identifying the different strata within a disease based on a deeper understanding of the mechanisms underpinning these strata. Accordingly, biomarker-based stratification will likely enable the development of disease-specific diagnostic tools and treatments. We have identified circulating markers, descriptive of mechanisms involving the proposed novel targets, that might be used to screen large-scale population of patients and to stratify the latter based on specific ‘fibrotic’ profiles.
During the project partners participated to congresses, external workshops and other scientific events, actively disseminating and discussing the results generated by the project with peer colleagues. A workshop was organized during the last year of the project to brainstorm on the opportunities of targeting fibrosis in cardiovascular diseases, by strengthening the ties between academic and industrial partners.
Several articles in high impact factor scientific journals have been published with the results generated by the consortium. Additionally, the results have been presented in National and International scientific and clinical meetings all along the duration of the project.
Achievements were published on FIBRO-TARGETS and other websites website and professional social network such as LinkedIn.
Partners at the FIBRO-TARGETS consortium, individually and/or collectively filed a number of patents.
Discussions started with a big pharma on a potential co-development of an apelin receptor agonist. Further in vivo efficacy tests are ongoing. We expect to have a go/no go at the end of the year from the industrial partner.
Cardior Pharmaceuticals GmbH is an academic spin-off from Medical School Hannover (MHH). Founded in 2016 by the cardiologist Prof. Thomas Thum, a Series A financing round of 15 Million EUR was successfully closed in May 2017. The company is focused on the development and clinical validation of noncoding (ncRNA) RNA therapeutics for patients with myocardial infarction and HF. In addition development of targeted delivery strategies and companion diagnostics is a major focus.
A team of six scientific co-founders from the United States and Europe, including partners form the FIBRO-TARGETS consortium, formed G3 Pharmaceuticals a newly formed biopharmaceutical company located in Lexington, MA, announcing the start of its research and development program to pursue novel galectin-3 inhibitors. The degree with which experimental galectin-3 inhibitors blocked fibrosis and disease progression across species and disease models is very promising. If it lives up to expectations, this can have a fundamental impact on HF and other fibrosis related most important conditions of our time.
FIBRO-TARGETS partners will also continue to communicate current and future results from FIBRO-TARGETS. Discussions will continue with the pharmacy industry to translate druggable molecules into clinical products.
List of Websites:
http://www.fibrotargets.eu/
Cardiomyocytes, fibroblasts and vascular cells in the heart are connected by a complex matrix, principally composed of fibrillar collagen, which is instrumental in preserving structural integrity and plasticity. In the diseased heart, the matrix undergoes structural and subcellular changes that progressively influence heart function. Beyond the cardiomyocyte-centric view of heart injury, it is now accepted that alterations of the cardiac extracellular matrix (ECM) and cardiac remodelling play a major role in the development and evolution of cardiac diseases leading to heart failure (HF). These ECM alterations result in cardiac fibrosis. At the site of myocardial infarction, acute focal fibrotic scarring provides myocardial healing and prevents rupture. In contrast, chronic diffuse or focal reactive myocardial fibrosis is a consequence of either pressure or volume-overload due to persisting hypertension, valvular heart diseases, ischemic injury (in areas remote of the infarction), or diffuse myocardial diseases, such as cardiomyopathies or other diseases. Myocardial fibrosis is characterized by dysregulated collagen turnover (increased synthesis predominates over unchanged or decreased degradation) and excessive diffuse collagen accumulation in the interstitial and perivascular spaces. This dysregulation of collagen turnover takes place mainly in phenotypically transformed fibroblasts, termed myofibroblasts. Fibroblasts and particularly myofibroblasts secrete extracellular procollagen chains that assemble into collagen types I and III fibrils and become cross-linked to form the final fibres. Collagen cross-linking is an important post-translational modification because it increases myocardial tensile strength and the resistance of collagen fibers against degradation by matrix metalloproteinases. Myocardial fibrosis disrupts the myocardial architecture, contributes to myocardial disarray, and determines mechanical, electrical, and vasomotor dysfunction, thus promoting the progression of cardiac diseases to HF. Of note, although it may be decreased by mineralocortioid recetor antagonsits, fibrosis persists significantly in the myocardium of HF patients under the current treatment regimen recommended by the official guidelines. Furthermore, the severity of histologically-proven myocardial fibrosis has been reported to be associated with higher long-term mortality in patients with cardiac diseases, particularly those with HF. In this regard, the detection, prevention and repair of myocardial fibrosis have emerged as important targets for improving HF therapy. In order to achieve diagnostic and therapeutic advances, it is critical to identify new pro-fibrotic mechanisms not yet targeted by currently available therapies, and to translate these mechanisms into individualized diagnostic tools and specific therapeutic targets. Besides meaningful functional and therapeutic outcomes, valid molecular targets must not induce serious adverse effects, such as influencing inflammation processes or wound healing. Therefore, there is a need for a systematic collaboration between clinical investigators and basic scientists, together with the industry to allow integration of data from computer biomedical (in silico) and basic (in vitro) studies with preclinical (in vivo) research findings to be distilled into clinically actionable information for the fight against myocardial fibrosis. The FIBROTARGETS consortium is a multinational consortium with industrial and academic partners, funded by the European Commission and primarily aimed to characterize novel emerging mechanisms of myocardial fibrosis. Targets and biomarkers investigated include proteins, proteoglycans, and microRNAs. FIBROTARGETS has achieved its remarkable goal in identifying a number of discrete mechanistic pathways and molecular targets involved, as well as potentially anti-fibrotic agents ready to be transferred into diagnostic biomarkers and therapeutic agents amenable to improve patient care.
Project Context and Objectives:
Background and context: In Europe, 5% of hospital admissions in adults are due to heart failure (HF) – a larger proportion caused by myocardial infarction. In addition, ageing of the EU population will make incidence and prevalence of HF rise inexorably, unless effective preventive therapy is applied. So far antihypertensive therapy has had some success in preventing HF. Nevertheless, no therapy targeting pathophysiological mechanisms in HF such as fibrosis, inflammation or myocyte growth, is available for clinical use. At last, beyond the impact on myocardial interstitial fibrosis (MIF), nearly 45% of all deaths in the developed world can be attributed to some type of chronic fibro-proliferative disease. With this scenario, finding new targets, biomarkers and/or promising molecules with anti-fibrotic capabilities, would have a great impact in the society in a mid-term.
Hypothesis and objectives: FIBRO-TARGETS is based on the hypothesis that the intervention on novel fibrosis-related targets involved in the processes of fibroblast differentiation to myofibroblasts, on the collagen synthesis over degradation balance and/or on collagen maturation may allow for interstitial repair, thus providing a new strategy for the prevention and treatment of MIF.
Experimental approaches: The setting of several experiment models (for in vitro read-outs and in vivo in small and large animal) relevant to one or several studied signaling pathways has allowed to further or newly characterize the capacity of candidate biotargets to modulate collagen production and cross-linking. According to the anti- or pro- fibrotic properties of the studied biotargets, molecules (hits) were identified as potentially able to interfere with biotarget biological activity. The proposed agonist and antagonists have been selected within large libraries of molecules for most of the biotargets. When conventional structure activity relationship (SAR) analysis could not be used because of the unknown crystalline structure of OGN a different approach was taken to find homologues of OGN and use amino-acid sequence alignment and 3D protein structure overlay to identify conserved peptidic motifs that may be important for the OGN activities. Additional suitable cell-based screening assays have been developed to determine the efficacy parameters for the potential hits for several biotargets from each WP. The therapeutic potential of some of the most promising hits were tested in vitro, and challenged in vivo in small and big animal. Meanwhile, to validate therapeutic relevance of the hits identified for the studied biotargets, a common database of controls and patients susceptible of developing fibrosis or having fibrosis has been set. The merged clinical data of 1,200 individuals are associated with biosamples allowing for biomarker profiling of selected individuals for the biotargets resulting from the analysis and discoveries made experimentally. An optimization of the biosamples use has required the development and/or validation of multiplexing bioassays for selected/identified targets. A set of samples from patients and matched controls has been assayed. The resulting stratification of the patient according to signal-specific BM profile and diagnostic algorithms represents a powerful theranostic tool.
Main results:
WP 1 was dedicated to search for novel, safe and effective anti-fibrotic strategies based on the blockade of TGF-β-related fibrogenic mechanisms. We have validated the involvement of CT-1, apelin and several microRNAs in the development of MIF associated with HF, identifying them as potential novel targets for the treatment of this lesion. On the other hand, findings suggest that collagen cross-linking and LOX are key players in the development of MIF and that they are involved in the pro-fibrotic processes. Therefore, LOX emerges as potential candidate target for the treatment of MIF in HF patients.
In WP2 we investigated the biological role of non-structural extracellular matrix proteins in heart failure using multiple cell line, animal and human validation experiments. Our findings reveal that OGN reduces the proliferation of the scar tissue forming cells, fibroblasts, thereby preventing the production of the main component of scar tissue, collagen. SPARC had a protective role in murine viral myocarditis by improving heart function, reducing inflammation, cell death and mortality. We could detect small and large OGN variants in areas of fibrosis and on inflammatory cells in viral myocarditis, an inflammatory disease of the heart muscle. In ischemic patients, the expression of OGN in the heart was increased compared to healthy controls and plays a crucial role in the induction of inflammation.
In WP3 using combination of miRNA mimic library screens with a small RNA deep sequencing approach in human cardiac fibroblasts (HCFs), annotated and not annotated miRNAs were identified as potential targets for anti-fibrotic therapies. In vitro investigations uncovered repressive effects of the miRNAs on autophagy and the antioxidant system. In-depth in vitro analysis confirmed the pro-fibrotic nature of selected, highly conserved miRNAs miR-20a-5p, miR-132-3p as well as a novel miRNA. Consistently, we found that lead miRNA candidates were activated in murine models of fibrotic cardiac diseases, implying their possible functional importance in fibrosis-development of the diseased heart. Downstream cellular pathways were investigated such as repression of autophagy and detoxification of reactive oxygen species.
WP4 was focused on different mineralocorticoid receptors (MR) related pro-fibrotic molecules. Using bioinformatics tools, reticulocalbin RCN-3 was found to exert anti-fibrotic effects and to block the profibrotic effects of aldosterone and Gal-3. We demonstrated the role of Gal-3 as a profibrotic and also as a proinflammatory mediator and the beneficial effects of Gal-3 blockade using modified citrus pectin and the role of NGAL in high blood pressure, after MR activation as well as in myocardial infarction. We found it to be produced by immune cells in aldosterone-induced cardiac injuries in NGAL depleted mice depleted.
In WP5, using repurposing of known drugs or in silico screening, we identified hit molecules with promising anti-fibrotic potential, which, after additional developments would be used as MIF treatment. Libraries were screened, focused on the four mechanistic pathways studied within FIBRO-TARGETS. In vitro high-throughput robotic assisted screening of natural compounds was followed by in vivo validations. Anti-fibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts (HCFs) and subsequent validation and mechanistic in vitro and in vivo studies.
Hits were analyzed for dose-dependent inhibition of proliferation of HCFs, for modulation of apoptosis and extracellular matrix expression. In vitro findings were confirmed in vivo using an angiotensin 2-mediated murine model of cardiac fibrosis both in preventive and therapeutic settings, highlighting the potential translational relevance of our findings
All results have been compiled and curated and 8 compounds have been selected considering their good profile. These compounds represent an excellent starting point for further developments. In this sense, several partners within FIBRO-TARGETS have started to look for collaborative opportunities in order to push these compounds to the next stages.
After in vitro validation of the first generation of hits, we used structure-activity relationship studies for further refinement of likelihood of activity, toxicity, and solubility. All molecules were tested for efficacy under multiple newly developed cell based models (4xSBE (SMAD2/3), APJR, Col1A1 and Collagen Polymerization) and the best performing ones have been incorporated to an ADME and toxicity panel and further tested for Aqueous solubility determination, Chemical stability (acidic pH), Microsomal stability, Plasma protein binding, CYP3A4 isoform inhibition and Bi-Caco2 permeability assay.
By the end of the project, we have identified molecules with a very promising profile that will be further developed in upcoming projects. Eleven compounds have shown efficacy in at least 2 of the tests, and one has activity in all of them.
1. Potential anti-fibrotic compounds shown to inhibit the pro-fibrotic response to CT-1 and apelin and cross-linking in human cardiac fibroblasts.
2. A compound that interferes with the biological activity of OGN, resulting in a decreased inflammatory response thereby postulating it as a potential therapeutic for viral myocarditis.
3. Two compounds that interfere with the biological activity of SPARC. These compounds could be beneficial in the context of chronic fibrosis by inhibiting the binding of SPARC to collagen and thereby the big scar tissue mesh formation.
4. A series of 5 lead natural compounds interfering with key miRNAs, with confirmed cardiac anti-fibrotic activity and improved diastolic function, in hypertensive mice and the clinically highly relevant rat model of hypertensive cardiac fibrosis.
5. Small molecules interfering with NGAL in cardiac cells which are able to block its effects and could be promising therapeutic tools in the treatment of cardiac disease in which NGAL is involved.
In WP6 we have successfully established a novel pig model, which closely resembles human pathophysiology of cardiac fibrosis, induced by constant high blood pressure and heart hypertrophy. The model was comprehensively characterized through multimodal imaging, repeated transthoracic echocardiography and invasive pressure measurements. We have established a new imaging modality using 18F-FDG-PET-MRI and revealed that FDG-PET is more sensitive than cardiac MRI with late enhancement to assess dysfunctioning myocardium. We used these models for the preclinical evaluation of potential drug candidates with high translational value in the future. We have tested two potential drug candidates, which were identified in the other work packages, in rats and pigs for their effect on limiting or reversing cardiac fibrosis. Serial quantification of plasma biomarkers including miRNAs, and histological analyses revealed the values of a number of potential biomarkers and transcriptomic profiles to assess early cardiac remodelling and fibrosis.
In WP7 we have developed and validated biomarker assays for selected candidates in blood-derived matrices within a quality-controlled environment. The initial list of candidate biomarkers relevant to the various mechanistic pathways explored in the various WPs was significantly reduced because of the insufficient development of bioassays suitable for the small blood sample volumes available to the consortium. We finally assessed Thrombospondin-2, SOD2, MMP-1, SDF1-α, GDF-15, CD40L, AGRP, PIIINP, Gal-3 and NGAL.
We investigated the association of biomarkers (potentially indicative of our validated biotargets) with clinical phenotypes with potentially different fibrosis pathophysiology. Cases were age/gender matched with 2 controls, all selected from 12 patient cohorts in a common database available to individual consortium partners. Our results suggest that history of HF mainly drives the associations with the assayed biomarkers. However, in patients free from prevalent HF, distinct bioprofiles were associated with distinct phenotypes such as respectively, Galectin 3 with post MI, GDF-15 with diabetes, PIIINP with obesity and NGAL and CD40L with hypertension, suggesting that discrete biotargets may be more specifically involved in respective clinical phenotypes.
Overall, FIBRO-TARGETS results give credit to the conceptual approach of stratification of patients according to signal-specific biomarker profile identifying subsets of patients more likely to be responders to potential therapeutic molecules that we have validated as biotargets for anti-fibrotic therapy. Further experimental and clinical assessment will provide evidence to set the ground for new targeted anti-fibrotic personalized therapeutic strategies.
Project Results:
WP1
Diffuse myocardial interstitial fibrosis (MIF), caused by an excessive deposition of collagen fibers, is a common feature in heart failure (HF) patients. MIF has a detrimental impact on cardiac function and on the prognosis of these patients. Importantly, MIF is present even in those patients adequately treated according to clinical practice guidelines, indicating that current pharmacological treatments are not effectively targeting this lesion. Transforming growth factor β (TGF-β) is one of the main pro-fibrotic factors involved in MIF. However, given the broad range of TGF-β physiological actions, direct inhibition of TGF-β may have detrimental effects, explaining why the available anti-TGF-β agents aimed at treating MIF have failed so far. Therefore, the necessity exists to search for novel, safe and effective anti-fibrotic strategies based on the blockade of TGF-β-related fibrogenic mechanisms. This WP was focused on several TGF-β-related pro-fibrotic molecules: cardiotrophin-1 (CT-1), apelin, NADPH oxidases (NOX) and microRNAs.
1. Cardiotrophin-1
In patients with HF of hypertensive origin we have found an increase in the cardiac expression of the pro-hypertrophic factor CT-1 both at mRNA and protein level. Interestingly, CT-1 was directly associated with myocardial fibrosis and with collagen type I and III expression in these patients. Moreover, there was a significant association between the circulating levels of CT-1 and the serum biomarkers of collagen type I and collagen type III synthesis (PICP and PIIINP, respectively). Therefore, besides being involved in cardiomyocyte hypertrophy, CT-1 emerges as a mediator of myocardial fibrosis (Hypertension 2014;63:483-489). Reinforcing this notion, in human cardiac fibroblasts CT-1 induced collagen type I and III expression and the differentiation of fibroblasts to myofibroblasts (Hypertension 2014;63:483-489). CT-1 also increased fibronectin expression as well as metalloproteinases (-1,-2,-9) activities and the secretion of the pro-inflammatory molecule IL-6. Interestingly, CT-1 also upregulated the expression of lysyl oxidase (LOX), the main enzyme involved in collagen cross-linking. In this context, it has to be considered that increased collagen cross-linking makes collagen fibers more insoluble, stiffer and more resistant to degradation.
To investigate this association more in depth we analyzed an experimental model of HF with preserved ejection fraction (HFpEF), the Dahl salt sensitive rat, where we found MIF characterized by an increase in collagen fibers deposition both interstitial and perivascular, as well as in cardiac collagen cross-linking and LOX. Myocardial CT-1 expression, both at mRNA and protein levels, was increased in animals with HFpEF. Interestingly, CT-1 was inversely associated with the E/A ratio in all animals, a distinctive parameter of diastolic dysfunction. Of note, CT-1 was associated with MIF, and particularly with perivascular fibrosis. Importantly, the increase in CT-1 was strongly associated with the increase in LOX expression and consequently with collagen cross-linking in all animals.
Altogether, these results suggest that CT-1 may contribute to myocardial fibrosis through collagen cross-linking stimulation.
Several potential antagonists for the CT-1 receptor were developed in collaboration with WP5 and their anti-fibrotic potential was tested in human cardiac fibroblasts. Several of these compounds were able to prevent CT-1-induced procollagen type I synthesis at a concentration of 10 µM. However, the same inhibitory effect was found upon stimulation with TGF-β. Therefore, currently we cannot be certain of the specificity of the compounds.
2. Apelin
The expression of the anti-fibrotic factor apelin has been previously shown to be decreased in end-stage HF patients. In the experimental model of HFpEF (Dahl salt-sensitive rat) we found a decrease in myocardial apelin expression which was inversely associated with LOX expression and with insoluble collagen. These results support the hypothesis that the apelin system is decreased in chronic HF and that this down-regulation may contribute to MIF through the regulation of collagen cross-linking. In particular, it highlights the relevance of LOX in this process.
In different human fibroblasts (dermal, freshly isolated atrial and ventricular) apelin expression was down-regulated by TGF-β in a dose-dependent manner. On the other hand, synthetic apelin was able to partially inhibit TGF-β-induced collagen production. Several potential agonists for the apelin receptor (APJ) were developed in collaboration with WP5 and their anti-fibrotic potential was tested in human cardiac fibroblasts. Six of these compounds were able to inhibit TGF-β-induced procollagen expression at a concentration of 1 µM. Interestingly, some of them (n=6) also decreased LOX expression. Basic ADME-Tox in vitro and in vivo studies were performed for the most promising compounds and the one showing no toxicity and the best pharmacokinetic properties was selected for an in vivo proof of concept study in rodents.
3. NADPH oxidases
We evaluated the role of the pro-oxidant NADPH oxidases NOX2 and NOX4 in a murine model of essential hypertension, the spontaneously hypertensive rat (SHR), characterized by MIF and increased myocardial collagen cross-linking and LOX. We found an increased expression of both NOX2 and NOX4 expression. The 2 enzymes were associated with the extent of MIF and with procollagen type I expression. On the other hand, both isoforms were associated with LOX expression. However, in the HFpEF model of the Dahl salt sensitive rat we found a decrease in NOX4 which was associated with increased perivascular fibrosis. These apparently contradictory results suggest that the role of NOX4 may be dependent on the pathology and the main cellular source. This notion is in agreement with previously published data in experimental models and in clinical studies.
In human cardiac fibroblasts we found that NOX4 was strongly up-regulated by TGF-β in a dose-dependent manner. However, TGF-β presented no significant effects on NOX2 expression. Therefore, we focused on NOX4 for further studies. Hydrogen peroxide, which is the product of NOX4 activity, induced procollagen type I expression, as well as an increase in some of the key molecules involved in collagen processing including LOX, suggesting that NOX4-mediated oxidative stress may play a relevant role in the development of MIF. However, when we analysed the impact of NOX4 silencing on TGF-β-induced procollagen production we found that NOX4 was not an essential mediator for TGF-β effects. Taking all these data into account we decided to focus on the other molecules under study.
4. MicroRNAs
To analyze miRNAs regulated by TGF-ß, a miRNA deep sequencing approach was performed in human cardiac fibroblasts. miR-23a-5p, miR-210-5p, let-7b-3p, miR-195-5p, miR-130b-5p, miR- 423-5p levels were found to be increased in HCFs after treatment with TGF-ß. However, overexpression of these miRNAs did not induce any significant changes in the expression of fibrosis-related genes (Col1a1, Col3a1, CTGF, α-SMA, MMPs or TGF-ß) in these cells.
On the other hand, in aortic stenosis (AS) patients, who showed MIF and increased collagen cross-linking, miR-122 was found to be down-regulated in the myocardium and associated with TGF-β and with parameters related to MIF. In vitro, miR-122 was shown to directly regulate TGF-β expression (Clin Sci 2014;126:497-506). Subsequently, we analysed if any of the miRNAs reported to be related to MIF was associated with collagen cross-linking. We found that miR-19b was decreased in the myocardium of these patients and that it was inversely associated with LOX expression and consequently with collagen cross-linking. Moreover, it was associated with increased left ventricular stiffness and with the presence of HF. In human fibroblasts miR-19b inhibition increased the expression of LOX protein, suggesting that miR-19b might regulate LOX and subsequently collagen cross-linking (Sci Rep 2017;7:40696). Importantly, serum levels of miR-19b were also associated with LOX and collagen cross-linking, as well as with the myocardial expression of miR-19b, suggesting that this microRNA could be useful as a non-invasive biomarker of collagen cross-linking.
5. General Conclusion
In this WP we have validated the involvement of CT-1, apelin and several microRNAs in the development of MIF associated with HF, identifying them as potential novel targets for the treatment of this lesion. Furthermore, in collaboration with WP5 we have developed potential anti-fibrotic compounds targeting these molecules which have been shown to inhibit the pro-fibrotic response in human cardiac fibroblasts. Further in vivo studies in experimental models are needed to definitively prove the therapeutic value of these compounds.
On the other hand, findings from the different studies performed suggest that collagen cross-linking and LOX are key players in the development of MIF and that they are involved in the pro-fibrotic effects of the different molecules evaluated. Therefore, LOX emerges as potential candidate target for the treatment of MIF in HF patients.
WP2
Heart failure: a serious health problem
Within Europe, every year 3.6 million patients are diagnosed with heart failure, a condition in which the heart cannot pump sufficient blood to meet the body’s needs. As the consequence of our aging population as the burden of cardiovascular risk factors (diabetes, obesity and high blood pressure), the prevalence of heart failure is expected to increase by 50% in 2030. However, effective therapies targeting the key mechanisms, such as scar formation in the heart, underlying the development of heart failure are lacking. Therefore, there is an urgent need to develop early interventions that target key mechanisms, including cardiac scar formation, for slowing down the progression to heart failure.
The biological role of non-structural extracellular matrix proteins in heart failure
All cells in the human body are surrounded a complex meshwork of proteins and carbohydrates, called the extracellular matrix. Several non-structural proteins within this extracellular matrix, such as Osteoglycin (OGN) and SPARC, are associated with a variety of cardiovascular diseases. Interestingly, an increased expression of these non-structural extracellular matrix proteins mostly results in an improved heart pump function, survival of heart cells, function of the cells involved in scar tissue formation and inhibition of adverse inflammation, all mechanisms which are involved in the development of heart failure. However, the exact role of OGN and SPARC in heart failure, especially scar tissue formation, is unknown.
The biological role of OGN in scar tissue formation in the heart
During this consortium, we have performed multiple cell line, animal and human validation experiments to elucidate the role of OGN during fibrosis in the heart. Our findings reveal that in OGN reduces the proliferation of the scar tissue forming cells, fibroblasts, thereby preventing the production of the main component of scar tissue, collagen. High blood pressure (angiotensin II treatment) increased the cardiac scar tissue formation of young mice in both presence and absence of OGN. In old mice, absence of OGN increases scar tissue formation and decreased ability of the heart muscle to relax, one of the characteristics of heart failure, indicating that OGN protects against heart failure by reducing scar tissue formation. Absence of OGN resulted in increased rupture of infarctions, bleedings within the heart and heart dysfunction in an animal model for heart attack, while overexpression blunted heart dysfunction in an animal model for heart attack. OGN levels were significantly higher in patients with a history of heart attack. In patients with a narrowed valve opening of the major artery of the body, the aorta, OGN gene expression in the heart was significantly higher than in healthy controls, with lower OGN gene expression in patients with more severe scar tissue formation in the heart. This suggests that OGN is a negative regulator of the induction of scar tissue formation in patients with a narrowed valve opening of the aorta. In patients with a restriction in blood supply to the tissue (ischemia) the small OGN variant (34 kDa) was detected in the heart, while this variant was undetectable in control patients without scaring. Moreover, blood samples of ischemic patients showed the presence of the small OGN variant.
The biological role of OGN in the inflammatory response in the heart
In addition to the small OGN variant, we detected a large OGN variant (72 kDa) in viral myocarditis, an inflammatory disease of the heart muscle. The absence of the large OGN variant decreases inflammation in the heart of a mouse model of viral myocarditis. In viral myocarditis patients, OGN (small variant) was present in areas of fibrosis and on inflammatory cells (leukocytes; OGN large variant). In ischemic patients, the expression of the large and small OGN variant (on neutrophils) in the heart was increased compared to healthy controls. Cell line studies showed that the absence of large OGN variant decreased the activation of toll-like receptors, receptors that play a crucial role in the induction of inflammation. In addition, we identified a compound that interferes with the biological activity of OGN, resulting in a decreased inflammatory response thereby postulating it as a potential therapeutic for viral myocarditis.
The biological role of SPARC during viral myocarditis
During this consortium, we have performed multiple cell line and animal experiments to elucidate the role of SPARC during fibrosis in the heart. We demonstrated a protective role of SPARC in murine viral myocarditis by improving heart function, reducing inflammation, cell death and mortality. This protective role can be the result of the ability of SPARC to improve the endothelial barrier and thereby preventing the infiltration of immune cells in the heart. In addition, we found that SPARC improves the contraction of heart cells and heart function in healthy and viral myocarditis animal models. In addition, we identified 2 compounds that interfere with the biological activity of SPARC. These compounds could be beneficial in the context of chronic fibrosis by inhibiting the binding of SPARC to collagen and thereby the big scar tissue mesh formation.
WP3
Project #1:
In the present study, we developed natural compounds-based preventive and therapeutic approaches for cardiac fibrosis in hypertensive mice, leading to ameliorated diastolic function. Importantly, morphology of kidneys and livers remained unchanged in these animals, demonstrating toxicologically safe profiles in early evaluations. Moreover, we could confirm efficacy of the compounds in a clinically highly relevant rat model of hypertensive cardiac fibrosis; highlighting the potential translational relevance of our findings. Mechanistically, a pro-fibrotic miRNA was discovered as important key-player of the anti-fibrotic activities of the compounds.
In vitro high-throughput robotic assisted screening of natural compounds was followed by in vivo validations. Anti-fibrotic drug candidates were identified by functional screening of 480 chemically diverse natural compounds in primary human cardiac fibroblasts (HCFs) subsequent validation and mechanistic in vitro and in vivo studies. Hits were analyzed for dose-dependent inhibition of proliferation of HCFs, for modulation of apoptosis and extracellular matrix expression. In vitro findings were confirmed in vivo using an angiotensin 2-mediated murine model of cardiac fibrosis both in preventive and therapeutic settings. To investigate the mechanism underlying the anti-fibrotic potential of the lead compounds, treatment-depended changes in the noncoding RNAome in primary HCFs were analyzed by RNA-deep sequencing.
To elucidate a potential influence of the identified natural substances on endogenous miRNAs, key post-transcriptional regulators of gene expression, miRNA deep-sequencing in primary HCFs was performed. Interestingly, levels of the highly conserved miR-671-5p were consistently and significantly reduced after treatment of primary HCFs with all 5 anti-fibrotic lead compounds from the initial screen in vitro.
Project #2:
In this study, we identified annotated miRNAs as potential targets for anti-fibrotic therapies. The discovery was based on a combination of miRNA mimic library screens with a small RNA deep sequencing approach in HCFs. In vitro investigations uncovered repressive effects of the miRNAs on autophagy and the antioxidant system, hereby contributing to the fibrotic actions in HCFs. The small RNA deep sequencing approach additionally allowed the identification of a not annotated miRNA that promoted HCF-migration, critical in fibrotic remodelling.
The herein described pro-fibrotic miRs therefore perturb cellular pathways of homeostasis, essential for the health of a variety of cell types and tissues, not only of the heart. Thus, inhibition of the uncovered miRNAs might be safely applied to combat cardiac fibrosis.
Taken together, both studies led to the successful discovery of potential innovative therapeutic strategies to target myocardial fibrosis, a major limitation of cardiac performance.
In detail, functional miRNA mimic library screens were applied in human cardiac fibroblasts (HCFs) to identify annotated, however unstudied pro-fibrotic miRNAs. In parallel, miRNA deep sequencing was performed after subjecting HCFs to pro- and anti-fibrotic stimuli, additionally enabling discovery of not jet annotated miRNAs. In-depth in vitro analysis confirmed the pro-fibrotic nature of several selected, highly conserved miRNAs as well as a novel miRNA.
Consistently, the lead miRNA candidates were found to be activated in murine models of fibrotic cardiac diseases, implying their possible functional importance in fibrosis-development of the diseased heart. To determine downstream cellular pathways and their role in the fibrotic response, targets of the annotated miRNA candidates were modulated. We here provide evidence that repression of autophagy and detoxification of reactive oxygen species by selected miRNAs explain their profibrotic nature on a mechanistic level
WP4
Cardiac remodeling plays a major role in the development and evolution of cardiac diseases leading to heart failure (HF). During cardiac remodeling, there is an initial phase of systemic inflammation which could induce interstitial and perivascular fibrosis. Myocardial fibrosis is defined by an increase in extracellular matrix (ECM) deposition and excessive collagen accumulation in the interstitial and perivascular spaces. Aldosterone, via the activation of the mineralocorticoid receptor (MR), plays an important role promoting cardiac inflammation, fibrosis and hypertrophy. Several clinical and experimental studies have demonstrated the beneficial effects of mineralocorticoid receptor antagonists (MRAs) in mild to severe HF, suggesting that an excessive activation of the MR occurs over the course of the disease and plays an important role in the pathophysiology of HF. However, the treatment with MRAs is underuse due to the side effects of MRAs such as hyperkalemia or gynecomastia.
1. Aldo/MR.
A better understanding of the molecular mechanisms of mineralocorticoid activation pathway in cardiovascular pathophysiology remains incomplete. Indeed, a better knowledge of the underlying mechanisms may highlight novel mediators of the MR signaling cascade. These newly identified intermediates could be good candidates as biotargets for novel pharmacological approaches, especially in diseases where the aldosterone/MR pathway is involved. This WP was focused on different MR related pro-fibrotic molecules such as Galectin-3 (Gal-3), Cardiotrophin-1 (CT-1) and neutrophil gelatinase–associated lipocalin (NGAL).
We identified that Aldo up-regulated collagen type I, III and VI in human cardiac fibroblasts (Martinez-Martinez E, J Proteomics 2017). Moreover, reticulocalbin (RCN) family members are common proteins down-regulated by Aldo biotargets. The function of RCN proteins remains unknown. However, its localization in the lumen of the endoplasmic reticulum suggests a role in protein synthesis, modification, and intracellular transport. Using bioinformatics tools, we focused our study on RCN-3, demonstrating that RCN-3 exerts anti-fibrotic effects and blocks the profibrotic effects of Aldo and Gal-3 (Martinez-Martinez E, Sci Rep 2017). A patent has been filled covering the interest of RCN-3 in cardiac fibrosis.
2. Galectin-3
Gal-3 is a member of a β-galactoside–binding lectin family expressed in several tissues including the heart. It has been observed in clinical studies increased Gal-3 levels in patients with HF, which were correlated with extracellular matrix markers. In the present project, we demonstrated the role of Gal-3 as a profibrotic and also as a proinflammatory mediator. In addition, we have demonstrated the beneficial effects of Gal-3 blockade using modified citrus pectin (MCP) in experimental models of different pathologies such as hypertension (Martinez-Martinez E, Hypertension 2015a), aortic stenosis (Arrieta V, Clin Sci 2017), hyperaldosteronism (Calvier L, JACC Heart Fail 2015) and obesity (Martinez-Martinez E, Hypertension 2015b).
3.- Neutrophil Gelatinase–Associated Lipocalin
Neutrophil Gelatinase–Associated Lipocalin (NGAL) is a potent biomarker of renal injury which has been proposed to be pathogenic in cardiovascular disease since high NGAL plasma levels have been associated with mortality in HF patients. In this project, we have evaluated the role of NGAL in high blood pressure after MR activation as well as its role in myocardial infarction. In addition, we have demonstrated the importance of NGAL production by immune cells in aldosterone-induced cardiac injuries using a model of mice depleted for NGAL in their immune cells. The data have been published in Hypertension, 2017 or are currently submitted for publication.
Related with NGAL and in collaboration with WP5, we have identified small molecules interfering with NGAL in cardiac cells which are able to block its effects and could be promising therapeutic tools in the treatment of cardiac disease in which NGAL is involved.
WP5
The main objective of this WP5 is to identify hit molecules with promising anti-fibrotic potential. This list of molecules would be the starting point for additional developments that would be used as MIF treatment. To do so, three types of libraries have been screened; Prestwick Chemical Compound Library, Greenpharma natural compound Library and a dedicated set of molecules, focused on four mechanistic pathways studied within FIBRO-TARGETS. The first two libraries were used to check repurposing of known drugs whereas the latter consists in a set of molecules identified in an in silico screening, as targeting novel targets involved in MIF. The in silico approaches included mimicking known active compounds and modelling of the protein 3D structure in order to calculate the affinity of candidate molecules with the protein. The best ones in terms of affinity were selected for in vitro evaluation. This allows to reduce time and cost as we will only focus on molecules with potential to be active. After in vitro validation on the first generation of hits, we use the structure-activity relationship to design derivatives that may have better activity or less detrimental effects (eg toxicity, solubility problem...).
All these molecules have been tested for efficacy under newly developed cell based models and the best performing ones have been incorporated to a ADME and toxicity panel.
By the end of the project, we have identified up to six molecules with a very promising profile that will be further developed in upcoming projects.
In vitro screening model development.
In order to test the available molecules, we have developed several cell-based models to assess the efficacy of potential candidates by imaging. Three models were successfully generated; Col1A1 expression model, 4xSBE model (SPARC2/3 activation model) and Apelin receptor activation (APJ Receptor internalization model). The two latter, passed the requested criteria to be used in High Throughput Screening (HTS) applications (Z’ values of 0.79 and 0.784 respectively) and were used to identify sets of promising molecules.
The Col1A1 model (immortalized Human Cardiac Fibroblasts) is able to measure differences in the Col1A1 expression as well (upon TGF-beta induction, cells increased the reporter gene expression. Different time points were determined ranging from 24h through 96h). However, it did not pass HTS Z’ requirements (the response of the population was heterogeneous) and therefore it was only used to check the efficacy of the most promising compounds on this pathway.
Besides these three cell-based efficacy models, a biochemical in vitro assay has been also developed for efficacy screening. It is based on collagen self-polymerization turbidity measurements. In presence of SPARC, collagen polymerization is delayed. The original rate of polymerization restoration in presence of testing compounds is an indicator of SPARC activity inhibition.
We have screened 64 compounds from the different WPs using the Collagen polymerization assay to determine whether they are able of inhibiting SPARC action over Collagen or not. 15 appear to inhibit the SPARC activity over Collagen to some extent. The WP with more compounds positives is WP2 (7 compounds), then WP1 (5 compounds) and finally WP4 (3 compounds). This makes sense given that the compounds coming from WP2 are chemically designed to inhibit SPARC. Unfortunately, none of the compounds from WP3 showed SPARC inhibiting behaviour.
Efficacy data.
Three sets of compounds have been tested; 69 targeted compounds curated in the in-silico screening, Prestwick Chemical Compound Library (PCL 1280 compounds) and the Greenpharma Natural Compound Library (GPNCL 480 compounds). The two libraries, cover a broad chemical space and contain drugs already approved by FDA and EMEA.
From the targeted compounds, 11 have shown efficacy in at least 2 of the tests, and one has activity in all of them. On the other hand, just the 4xSBE and APJR models have been used for compounds libraries. Regarding PCL, we have identified 31 and 29 positive compounds respectively. In the case of GPNCL, just 4 and 2 compounds respectively showed efficacy in the cell-based models.
ADME-Tox data.
The toxicity of the compounds (for up to three cell types; cardiac fibroblasts, hepatocytes and cardiomyocytes) has been determined. Overall, they seem to be quite safe. Just some of them have shown to be toxic for cardiac fibroblasts (9 molecules turned out to be toxic out of 69 compounds), hepatocytes (29 compounds out of 47 were safe up to 50 μM) or cardiomyocytes (None of the tested molecules inhibited the hERG channel; crucial for cardiomyocyte function).
Compounds showing acceptable efficacy and toxicity data have entered to a panel of ADME assays designed to determine several parameters. Performed ADME assays comprise;
• Aqueous solubility determination.
70% of tested compounds showed a solubility over 60% (500 μM in PBS).
• Chemical stability (acidic pH).
92% of molecules were highly stable to acidic degradation (over 80% still present after 2h in gastric content mimicking solution).
• Microsomal stability.
Overall the compounds were extensively processed by microsomes and only the 22% of them resisted to degradation (over 65% presence after 2h). This feature by itself is not necessarily bad news. In fact, it could be desirable to avoid over dosage or mid-long term treatments.
• Plasma protein binding.
78% of the compounds bound to plasma proteins at values lower than 98%. It is broadly known that several currently marketed treatments show values around this value or even slightly higher.
• CYP3A4 isoform inhibition.
55% of the compounds seemed to be processed by another cytochrome isoform (less than 20% CYP3A4 inhibition) and 36% were mildly processed (20%
2 compounds were tested in this absorption assay and both presented permeability data in the range of the used reference molecule (Propanolol).
WP6
In this work package, we developed and characterized small and large animal models for cardiac fibrosis with high translational value and used these models for evaluation of potential new drugs. We have successfully established a novel pig model which closely resembles human pathophysiology of cardiac fibrosis induced by constant high blood pressure and heart hypertrophy. In growing, juvenile animals, a bare metal stent was implanted in the descending aorta, which consequently results in stenosis, triggering pressure overload and myocardial hypertrophy. By inducing a gradual increase of aortic pressure, this model resembles the development of hypertrophy and heart failure in human patients more closely than previously described large animal models. The model was comprehensively characterized through multimodal imaging, repeated transthoracic echocardiography and invasive pressure measurements, serial quantification of plasma biomarkers including miRNAs, and histological analyses. This work will facilitate the preclinical evaluation of potential drug candidates with high translational value in the future.
We have tested two potential drug candidates, which were identified in the other work packages, in rats and pigs for their effect on limiting or reversing cardiac fibrosis. While one of the compounds showed good efficacy and tolerability in rats, unforeseen and partially severe side effects were encountered in pigs for both potential drugs, and consequently, dosing had to be adjusted. With these lower doses, a meaningful beneficial effect of the two tested compounds failed to be proven in large animals. Although the encountered toxic effects hamper the use of the investigated compounds for therapeutic interventions, the gathered results are useful for further compound optimization.
We have established a new imaging modality to visualize cardiac remodelling based on fibrosis. We have demonstrated that 18F-FDG-PET-MRI with late enhancement investigation is useful to predict adverse cardiac remodelling, if this investigation was performed 3-day after acute myocardial infarction. We have revealed that FDG-PET is more sensitive than cardiac MRI with late enhancement to assess dysfunctioning myocardium.
In an additional task of this work package, we have analysed a number of potential biomarkers, including proteins and miRNA oligonucleotides, and we were able to demonstrate the predictive values of new biomarkers, that may be useful to assess early cardiac remodelling and fibrosis.
We have compared the different transcriptomic profiles of cardiac fibrosis of different diseases, which, however leads to the same clinical phenotype of heart failure and cardiac fibrosis. Overlaps and differences in gene expression signatures highlighted common and varying mechanisms leading to cardiac fibrosis. Based on these results, identification and specific targeting of the key regulator genes of the different fibrotic mechanisms and pathways can be instrumental for the development of more effective and diversified antifibrotic therapies in the future.
All results have been compiled and curated and compounds have been selected considering their promising profile. These compounds represent an excellent starting point for further developments. In this sense, several partners within FIBRO-TARGETS have started to look for collaborative opportunities in order to push these compounds to the next stages.
WP7
Immunoassay development
This primary task aimed to develop and validate biomarker assays for selected candidates in blood-derived matrices within a quality-controlled environment (ISO13485). The secondary goal was to evaluate the predictive value of these biomarkers on selected cohort of patients at risk to develop fibrosis-related heart failure.
Final selection of biomarker candidates to be assessed was agreed in April 2016 and updated in Sept 2016. These targets were subdivided in lists A (SOD2, Thrombospondin-2, AGRP, CD40L, SDF-1/CXCL12, GDF15, MMP-1, Galectin-3 and MMP9), B (SPARC, NGAL and NGAL/MMP9 complex) and C (Fibulin2, Mimecan, Biglycan, Lumican, PICP, PIIINP, CITP and CT-1) by order of priority for the consortium, market availability and foreseen biomarker assay availability from FIRALIS. Monoclonal antibodies development for those biotargets started by a literature work to find the best sequences for the protein and/or peptide of interest with the respect of the protein properties (solubility, pH, environment, the ideal bacteria strain, etc.) in order to have a good product for immunization. For some targets, the immunizations were performed using both whole protein and a peptide to increase the probability to have good clones for sandwich ELISA development. After several assays of protein production and mouse immunizations, FIRALIS succeeded to generate 220 hybridomas. Hybridomas for positive clones were grown at FIRALIS and several quantities (up to 10 mg) of each clone were purified as well as their corresponding protein for calibration range.
ELISA sandwich assay development phases combine several parameters starting with the selection of the best antibodies pair for capture and detection of target in biological fluid (plasma or serum). Then, coating conditions, incubation times and concentrations of antibodies were optimized. Upon completion of the optimization phase, lyophilization tests were performed on the standard. As soon as the final formulation of the standard was validated, the standard curve was established as well as the minimum required dilution (MRD), that is, the minimum dilution of the biological fluid (matrix) required to determine the concentration of the target independently of the matrix effect.
At this stage, when the design of the final kit is finalized, the analytical characteristics of the assay were determined. These included the concentration of the target in Quality Controls, the Linearity of dilution, the limit of detection (LOD), the lower and upper limits of quantification (LLOQ and ULOQ), Repeatability (intra-assay precision), and Reproducibility (inter-assay precision). The analytical characteristics of the assay were then confirmed by an independent team in FIRALIS, in a different laboratory. Stability studies and biological ranges in normal and 2 independent cohorts of diseased population complete this validation process. Upon completion of all these stages, the assay is ready for CE marking. Currently, the described process is completed for 3 biomarker assays.
Samples biobanking
FIRALIS has developed a biobanking facility encompassed in a fully documented tracking system linking samples to analytical results obtain from them. This biobank for research purposes is certified NF S 96-900. FIRALIS has organized the signatures of the MTA with each contributive partner. Specific MTA have been signed between the provider of cohorts and the recipient (FIRALIS) with University of Maastricht, FIMA, UCD and CHRU de Nancy (INSERM). FIRALIS has formalized the biobanking protocol.
FIRALIS received end of 2016 a first set of 60 samples (250µL plasma and 250µL serum samples from 30 heart failure patients and 30 healthy subjects) to evaluate the performance of the selected commercial bioassays to measure a maximum of BMs from the FIBRO-TARGETS lists. By end of June 2017, FIRALIS received 600 plasma and 563 serum samples for assessment using the selected platform and assays by consortium members.
Assays performance evaluation
At this stage, FIRALIS has completed full assay development for 3 targets (CNN-1, Caldesmon and clusterin). The assay development of those biomarkers had started before a final lists of 17 priority targets (Thrombospondin 2, sCD40L, GDF-15, MMP-1, Galectin-3, SPARC, Apelin, Cardiotrophin 1, MMP-2 variant 1, MMP-2 PEX, MMP-3, PINP, PIIINP, Mimecan/osteoglycin, MMP-9, NGAL, NGAL/MMP-9 complex) was issued and agreed by the consortium. Except for GDF-15 and MMP-1, FIRALIS succeeded to select at least one antibodies pair for each target showing promising data in human serum and/or plasma. Generally, pairs were first identified with using recombinant proteins spiked in fetal bovine serum and then further evaluated in human serum or plasma. More interesting, FIRALIS finalized the analytical validation for the NGAL sandwich ELISA assay.
Samples analysis
Targets from list A and PIIINP from list C were evaluated on a cohort of 60 age and sex-matched samples (30 healthy subjects and 30 HF subjects). The use of multiplexing ELLA and O-link platforms provided the consortium with data on potential 88 other predictive targets from the Cardiovascular II panel (http://www.olink.com/products/cvd-ii-panel/). Of note, due to stability issues it was agreed not to measure MMP9 in this evaluation experiment.
With the exception of Thrombospondin-2 and SOD2, expression levels of most of the biomarkers were satisfactorily detectable in both cases and controls.
Those biomarkers were analyzed for the whole cohort composed of 600 plasma samples assessed for Thrombospondin-2, SOD2, MMP-1, SDF1-α, GDF-15, CD40L, AGRP, PIIINP and Gal-3 and 563 serum samples assessed for NGAL.
The biomarkers measurements and quality check were completed by the end of Q3 2017 (mid-September 2017) and communicated to the partner in charge of statistical analysis.
Potential Impact:
FIBRO-TARGETS has achieved its remarkable goal in identifying a number of discrete mechanistic pathways and molecular targets involved, as well as potentially anti-fibrotic agents ready to be transferred into diagnostic biomarkers and therapeutic agents amenable to improve patient care. As a result of this project we have advanced in the knowledge of the mechanisms involved in the development and progression of myocardial fibrosis in HF. Furthermore, we have identified compounds with potential anti-fibrotic effects susceptible to be developed into therapeutic agents.
HF is a deadly condition that affects more than 37 million patients worldwide, with limited therapeutic possibilities. Due to the enhanced life expectancy to date, an increasing number of patients presents with diastolic HF (HF with preserved ejection fraction, HFpEF). Despite extensive research efforts during the last centuries, HFpEF still represents an unmet medical need, imposing an enormous medical and socioeconomic burden, particularly on ageing societies. Early interventions targeting key mechanisms, including myocardial interstitial fibrosis (MIF), could slow down progression to HF. No treatment has been shown to target cardiac fibrosis to counteract passive stiffness of the heart, a major contributor to HFpEF. As the current treatment approaches for HF prolong survival but do not prevent or cure HF, the FIBRO-TARGETS consortium fulfils the urgent need to develop specific effective treatments for HF. Moreover, it has to be considered that pathological fibrosis is not restricted to the heart, being present in other organs such as kidney, liver and lungs. If the mechanisms identified by the FIBRO-TARGETS consortium for cardiac fibrosis are proven to be involved in other organs fibrosis, the beneficial impact of the proposed novel anti-fibrotic strategies could be extended to other pathologies.
Therapeutic developments conducted in the last decades have led to a substantial decrease in mortality in HF with reduced ejection fraction. Therefore, demonstrating a therapeutic benefit on top of existing therapies in the logic of ‘one-size-fits-all’ trials has become increasingly difficult and cost demanding. As a consequence, in the last three decades, landmark HF trial sizes have progressed from a few hundred to many thousand patients. Operational obstacles and unbearable costs are major challenges for the successful implementation and completion of such large trials. Even with pre-specified subgroup analysis, regulators do not accept testing for responder profiles due to the risk of bias. Precision medicine has emerged as one possible solution, aiming at identifying the different strata within a disease based on a deeper understanding of the mechanisms underpinning these strata. Having this in mind, we in FIBRO-TARGETS aimed at identifying mechanistic bioprofiles using a candidate biomarker approach, that may ultimately pave the way for a precision preventive medicine approach. Specific mechanistically tailored anti-fibrotic therapies may then be administered to patients expressing corresponding biotargets, identified using biomarker profiling.
First of all, the FIBRO-TARGETS consortium offered new insights in the mechanisms involved the formation of scar tissue formation and the development of HF by executing translational research (bench-to-bedside).
We have validated the involvement of mechanistically targetable pathways in the development of myocardial fibrosis associated with HF, identifying them as potential novel targets for the treatment of fibrosis. The potential preventive, therapeutic and/or diagnostic potential of CT-1, apelin, LOX, the non-structural matrix proteins and mineralocorticoid receptor associated targets such as Gal-3, and NGAL and miRNA controlled fibrosis mechanisms will have far stretching benefits in the future for HF patients and patients with inflammation and fibrosis mediated cardiac remodelling and scar tissue formation-associated diseases. We uncovered lead drug candidates and demonstrated their potential suitability for the prevention and treatment of hypertension-induced cardiac fibrosis and diastolic dysfunction. Therefore, our results are encouraging for prospective clinical investigations to eventually reduce symptoms, hospitalizations, morbidity, mortality and medical costs.
The screening for druggable molecules paves the way for increasing the therapeutic arsenal against HF. We could identify several interesting compounds that target important proteins involved in MIF. Some are old drugs – used for other indications – that were found to be interesting in MIF. In total, 8 of them are being evaluated in animal models. They may become future drug candidates for HF. Besides, 2 natural products were patented. We plan to license these to biotech or pharmaceutical companies. A synthetic compound is under evaluation in a “big pharma” for potential co-development.
During the project, large animals have been instrumental for a better understanding of mechanisms of fibrosis. The availability of useful, practical animal models with high translational value improves the drug development process by enabling comprehensive preclinical evaluations also on the molecular level, and by discarding unsuccessful compounds before they enter clinical testing.
Precision medicine has emerged as one possible solution, aiming at identifying the different strata within a disease based on a deeper understanding of the mechanisms underpinning these strata. Accordingly, biomarker-based stratification will likely enable the development of disease-specific diagnostic tools and treatments. We have identified circulating markers, descriptive of mechanisms involving the proposed novel targets, that might be used to screen large-scale population of patients and to stratify the latter based on specific ‘fibrotic’ profiles.
During the project partners participated to congresses, external workshops and other scientific events, actively disseminating and discussing the results generated by the project with peer colleagues. A workshop was organized during the last year of the project to brainstorm on the opportunities of targeting fibrosis in cardiovascular diseases, by strengthening the ties between academic and industrial partners.
Several articles in high impact factor scientific journals have been published with the results generated by the consortium. Additionally, the results have been presented in National and International scientific and clinical meetings all along the duration of the project.
Achievements were published on FIBRO-TARGETS and other websites website and professional social network such as LinkedIn.
Partners at the FIBRO-TARGETS consortium, individually and/or collectively filed a number of patents.
Discussions started with a big pharma on a potential co-development of an apelin receptor agonist. Further in vivo efficacy tests are ongoing. We expect to have a go/no go at the end of the year from the industrial partner.
Cardior Pharmaceuticals GmbH is an academic spin-off from Medical School Hannover (MHH). Founded in 2016 by the cardiologist Prof. Thomas Thum, a Series A financing round of 15 Million EUR was successfully closed in May 2017. The company is focused on the development and clinical validation of noncoding (ncRNA) RNA therapeutics for patients with myocardial infarction and HF. In addition development of targeted delivery strategies and companion diagnostics is a major focus.
A team of six scientific co-founders from the United States and Europe, including partners form the FIBRO-TARGETS consortium, formed G3 Pharmaceuticals a newly formed biopharmaceutical company located in Lexington, MA, announcing the start of its research and development program to pursue novel galectin-3 inhibitors. The degree with which experimental galectin-3 inhibitors blocked fibrosis and disease progression across species and disease models is very promising. If it lives up to expectations, this can have a fundamental impact on HF and other fibrosis related most important conditions of our time.
FIBRO-TARGETS partners will also continue to communicate current and future results from FIBRO-TARGETS. Discussions will continue with the pharmacy industry to translate druggable molecules into clinical products.
List of Websites:
http://www.fibrotargets.eu/