Final Report Summary - SMARTER (Small Artery Remodelling) Objectives and organization of SmArteRSmArteR (small artery remodelling) addressed the small arteries in our body that form the major resistance to organ perfusion. These vessels are critical for regulation of blood pressure and local supply of oxygen to tissue, and it is now clear that they play key roles in the development of cardiovascular pathologies. Despite this, these vessels remained understudied. There is a consequent lack of innovation in diagnostic and therapeutic tools based on small arteries and their structural and functional changes (remodelling) in disease states. SmArteR aimed at helping filling this gap. The scientific and technological objectives of SmArteR were:1. To train ESRs and ERs to become independent researchers and entrepreneurs in small artery remodelling.2. To increase our understanding of the molecular mechanisms of small artery remodelling and how these processes can be diagnosed at an early stage, efficiently treated and ultimately reversed.3. To generate new technology to study small artery remodelling, contributing to development ofcardiovascular therapy that specifically targets small arteries.4. To integrate the SmArteR academic partners from different disciplines and complementary expertise, with the private sector to provide both a scientifically interdisciplinary approach as well as a strong network. Our research and training program was centered around the triad Cells-Extracellular Matrix-Forces and the connections between these elements, as depicted below (see figure attachment).The work was organized in four scientific/research training workpackages, matching the various relevant levels of biological organization (WP1: molecular, WP2: vascular cell and matrix, WP3: vascular physiology, WP4: technology). While all hired fellows were active in multiple workpackages, work of the 12 ESR focused on one of the WP 1-3, while the 3 ER were employed at SME and focused on WP4. Accomplishments of the work packagesWork package 1: Molecular studiesWP 1 focussed on key molecular pathways in small artery remodelling. This encompasses mechanosensitive gene expression, integrins and biomechanics (loading on cells and ECM proteins). The identification of micro RNAs (miRNAs) as central regulators of gene expression provided new mechanistic insight into gene regulation. Their role as mechanosensitive regulators was addressed in order to provide new possible handles for future therapeutics. ESR3 studied the endothelial basement membrane laminins 411 and 511 as shear/blood flow detector. The work addressed their involvement in VE-cadherin cycling and integration with specific integrins. Affymetrix gene screens identified a critical role for the laminins on Sphingosine-1-phosphate receptor and a voltage-gated calcium channel. ESR4 studied these laminins in smooth muscle cells, focusing on contraction. While laminin α5 did not affect major master switches for SMC differentiation, a large range of proteins related to contraction and gap junctions was downregulated in SMC laminin α5 KO mice. CRISPR/Cas9 technology is being used for extension of this dataset. ESR7 demonstrated the central role of NOTCH signaling in the molecular cascades involved in vascular smooth muscle transcriptional regulation, including the inhibition of NOTCH by miRNA and soluble guanylyl cyclase as a downstream target with clear function consequences. ESR8 demonstrated the involvement of MAGI1 as a key player in assembly of endothelial focal adhesions and stress fibers, related to shear stress-dependent endothelial function. ESR10 has addressed zyxin as a mechanotransducer protein in the context of hypertension-induced vascular wall stress. She characterized the LIM-domain proteins zyxin and LPP as redundant protective molecules that help to maintain the contractile VSMC phenotype and thus contribute to impeding the development of hypertension-induced arterial remodeling. Major results: We identified several key molecular players in small artery remodeling and obtained information on their mechanisms of action. The work has been communicated mainly by peer-reviewed papers and should provide the scientific community and pharma industry with several new leads for future research and product development. Work package 2: Vascular cells and matrixThis WP addressed the two major cell types, endothelial cells and smooth muscle cells, but also included interaction with blood cells and the role of (trans-) differentiation of circulating and resident adventitial cells with pericyte-like characteristics. This choice was driven by the rapid developments in the progenitor field at large as well as by the clear identification of pluripotent cells in the vascular wall. After an initial strategic decision of the board to move away from the planned connexin studies, ESR5 addressed the central role of AMPK in vascular remodelling. While formally mainly active in WP2, he has covered the whole spectrum from molecular studies to intact vessel physiology, with a focus on calcium dynamics, cytoskeletal element dynamics and other processes of small artery remodeling. ESR11 addressed the effect of platelets and leukocytes on the small vessel wall and in particular has studied the dynamics of vWF-platelet strings and leukocyte extravasation in ADAMTS13 KO mice. ESR 12 studied the effect of Dickkopf-3 (Dkk3) in vascular stem cell migration and the underlying molecular mechanisms, with a particular focus on identifying the receptor of Dkk3, whose activation may trigger VPCs migration. The work revealed that CXCR7 serves as Dkk3 receptor, which mediates Dkk3-induced vascular progenitor migration in vitro and in tissue-engineered vessels. Several of the WP1 ESRs also contributed to WP2. Major results: Small artery research is traditionally based on endothelial cells and smooth muscle cells. Yet, much of the work in WP2 revealed that interaction of these cells with blood on the one side and the adventitia on the other side is a crucial element in vascular remodelling. These complex interactions are only beginning to be understood, but make clear that a still more integrative approach is needed for studying cellular behavior in and around the vascular wall. Work package 3: Vascular physiologyWP3 studied small artery remodelling at the integrated level of intact arteries and tissues, focusing on the consequences for perfusion and vascular biomechanics. While most of SmArteR addressed small arteries in general, in WP3 partial focus was on cerebral vessels, considering that vascular cognitive impairment is a growing burden in the aging population. The work of ESR1 started from an extensive gene expression study delivered by the previous ITN, SMART. Mechanistic studies were performed on a range of candidate gene products that were highly differentially expressed between normotensive and hypertensive rat strains. The work included Thrombospondin 4 as a new key player in remodeling, and demonstrated the involvement in remodeling and interaction with other networks. In addition, integration of functional and structural remodeling along vessels was studied in newly developed technology. ESR2 addressed cerebral vessel by focusing on their involvement in interstitial water transport and brain drainage. Vessels of hypertensive animals were found to have a larger active water transport and the work provided an understanding of interstitial fluid flow as a key cause of local vascular and neurological events. ESR6 studied the chloride channel TMEM16A that was also differentially expressed in the above SMART study. The work revealed that in addition to being a chloride channel, TMEM16A has importance for the inflammatory status of the vascular wall, which likely has consequences for the contractility. ESR9 worked on the complexity of the regulation of cerebral circulation and particularly the role of hemodynamic factors in acute adaptation and chronic remodelling following hypertension and traumatic brain injury. She demonstrated a key role for the P450 pathway. Major results: The studies in WP3 have on the one hand demonstrated the involvement of novel remodeling pathways in hypertension, and on the other hand have made clear that more integration is needed, not only as concerns other cell types as was found in WP2, but also other mechanisms and roles of the small vessels, such as the active transvascular transport of water in the hypertensive brain. Work package 4: TechnologyIn a collaboration between the academic partners and the three technology SMEs, this WP contributed to developing new technology and new applications addressing biomechanics, ECM and cells of the vascular wall. ER1 and ER2 were consecutively hired at the same premise (Lifetec) and worked on the use of slaughter house pig blood vessels. Technology was developed to keep these vessels perfused and pressurized in organoid culture, such that they can serve as a platform for testing new drugs and devices. The work has mainly used large vessels and was performed in close collaboration with the group in Amsterdam using small arteries. Standard protocols were derived for maintaining the vessel viable and for assessing their state of remodeling at the functional and molecular level. ER3 employed a novel in vivo approach for resistance arteries, based on control of the vessel environment while allowing normal perfusion and flow measurement by laser speckle technology. She studied effects of different anesthesia as a use case and furthermore evaluated the role of endothelial cells in tone control in this setting. Several ESR have furthermore contributed to the work of the SME. Major results: These studies have helped developing novel technology that is of relevance to not only the small artery researchers but also to the cardiovascular field at large. Accomplishments of the training programmeAn extensive training program was executed as defined in our Grant Agreement. This included all levels from local hands on, local courses to summer schools and network symposia. All ESR received training amounting at least 18, as planned. The training involved all of the SmArteR PI’s but in addition also many academic and industrial seniors. We believe that the network events formed a major asset as they helped building the team and establishing many formal and informal collaborations. While many graduated by now, the team of ESRs and ERs still form a strong community maintaining very regular scientific and social contact. The expected final results and their potential impact and use We believe that SmArteR has delivered what it promised: a wealth of improved understanding of the role and mechanisms of small artery remodelling in relation to cardiovascular diseases, and 15 young investigators with a unique European experience who found or are finding their way in the continuation of their academic careers. The work has been published in peer-reviewed journals as detailed elsewhere. All Ph.D. defenses are either done or planned within the next few months. The final results are used by us and many others for continuous basic and translational studies. Clearly, an important step will be testing the relevance of the current animal work for humans. The step towards clinical trials based on current work is still too large, but we do believe that we have provided the pharma industry with potential leads for drug development. We also believe we have helped giving the small arteries the scientific and public attention that is needed for progress in cardiovascular diagnostics and therapy. Target groups for whom the research could be relevant Scientists in cardiovascular research, cardiovascular research societies and communities, cardiovascular pharma industry, patient groups and national foundations on cardiovascular diseases, aspirant Ph.D. students. More information about the SmArteR consortium is available at our website, www.smallartery.eu.
