Periodic Reporting for period 2 - CRUCIAL (MiCrovasculaR rarefaction in vascUlar Cognitive Impairement and heArt faiLure)
Période du rapport: 2021-07-01 au 2022-12-31
CRUCIAL aims to validate methods to measure microvascular rarefaction and microvascular dysfunction in heart failure with preserved ejection fraction (HFpEF) and in vascular cognitive impairment (VCI). Both development of HFpEF and VCI is associated with the presence of comorbidities such as hypertension, hyperlipidaemia and diabetes. The second aim of CRUCIAL is to understand the mechanism by which comorbidities induce dysfunction and rarefaction in the microcirculation.
In WP1, the study protocol, information sheets and informed consent sheets have been developed and the ethics approval was obtained per 16-07-2021 (Deliverable 1.1). The first VCI patient was recruited in December 2020, up until now, in total, 10 VCI patients have been recruited. Data of brain MRI scans of the first recruited patients have been analysed, leading to further optimization of the brain MRI sequences. Images of heart MRI scans and echocardiography of the recruited patients were sent to UCL for quality control, which also leaded to further optimization of the cardiac MRI sequences. Blood samples of the first 5 recruited patients were sent to FIMA for a quality check as well.
In WP2; FIMA, UNAV and UCL have elaborated the patient-oriented protocol, prepared the informed consent forms and obtained approval from the Ethics Institutional Committees to start patient recruitment at the different sites (UNAV, UCL, FIMA). The protocol for cardiac MRI has been optimized between UCL and UNAV and tests have been run, exchanging data between partners. UNAV has implemented the technology for quantitative assessment of myocardial blood flow with the support of UCL. Scannexus helped optimize the brain MRI protocol that will be implemented on the Siemens MRI scanners at UCL and UNAV. A common database dictionary has been established with UM. UCL and UNAV, with the support of UM and SCNX, have worked together to set up an adapted (shorter and contrast-free) protocol for brain MRI in HFpEF patients. Similarly, regarding additional tools for microvascular evaluation, GlycoCheck technology has been also implemented at both UNAV and UCL, and the OCT has been acquired and set up by UCL. Protocol have been optimized for isolation of genetic material from endothelial derived vesicles.FIMA has extensive new results regarding retrospective studies.
In WP3, UCL designed a patient-oriented protocol, prepared the informed consent forms and obtained approval from the Ethics Institutional Committees to start patient recruitment (UCL), including dedicated MRI brain and heart, as well as OCT angiography in the wider INSIGHT46 protocol. The protocol for cardiac MRI has been optimized between WP3 and WP1/2 and tests have been run, exchanging data between partners. A common database dictionary has been established. UCL with the support of UM and SCNX, have worked together to set up an adapted (shorter and contrast-free) protocol for brain MRI in the NSHD 1946 Birth cohort. Similarly, regarding additional tools for microvascular evaluation, OCT has been acquired and set up by UCL.
In WP4, KU Leuven has translated all the relevant MRI protocols for the brain imaging to the small animal scanners and optimized the histology of the brain imaging using test rats (rats that were being sacrificed for other experiments).
In WP5, UM has obtained the animal ethical approval for the project. They have implemented brain laser speckle contrast imaging in mice and rats and developed a protocol for the study of neurovascular coupling in mice and rats. They have tested and optimized different vessel labelling strategies and tissue clearing protocols to gain insights into the 3D vascular architecture of brains and hearts. The imaging and image analysis pipeline are also being validated. In the meantime, UM have also performed IHC investigations on ZSF1 archived tissues from KU Leuven to assess the pericyte loss possibly associated to microvascular rarefaction. They have also started their first ZSF1 animal study aiming at characterizing change in cerebral blood flow (CBF) and the progression of cognitive impairment in Obese ZSF1 rats. Animals are studied from 15 to 34-35 weeks of age. The study will be terminated in September 2021 and 3D vascular architecture will be obtained on the collected hearts and brains thereafter.
In WP6, the ethical approval to perform experiments on rats were obtained; training on the use of R studio and BiomexTM software (Taverna et al, NAR 48: W385, 2020) for the analysis of bulk RNASeq data was done; the protocol for the isolation of ECs from rat brain via FACS enrichment wassuccessfully established. The protocols for the isolation of ECs from rat heart via MACS and FACS enrichment were also established. The collection of ECs from rat brain and heart for bulk RNASeq analysis has been initiated.
WP7 KU Leuven’s preliminary results with the ZSF1 obese rats have shown that vessel regression (assessed by staining for empty collagen sleeves) and PITX2 down-regulation were present at 14 weeks of age.
Concerning the management and dissemination activities of the project, a communication and dissemination strategy has been put in place and the Vascular Helix has been submitted. Articles highlighting the importance of the topic have been published.
In WP2; FIMA, UNAV and UCL have elaborated the patient-oriented protocol, prepared the informed consent forms and obtained approval from the Ethics Institutional Committees to start patient recruitment at the different sites (UNAV, UCL, FIMA). The protocol for cardiac MRI has been optimized between UCL and UNAV and tests have been run, exchanging data between partners. UNAV has implemented the technology for quantitative assessment of myocardial blood flow with the support of UCL. Scannexus helped optimize the brain MRI protocol that will be implemented on the Siemens MRI scanners at UCL and UNAV. A common database dictionary has been established with UM. UCL and UNAV, with the support of UM and SCNX, have worked together to set up an adapted (shorter and contrast-free) protocol for brain MRI in HFpEF patients. Similarly, regarding additional tools for microvascular evaluation, GlycoCheck technology has been also implemented at both UNAV and UCL, and the OCT has been acquired and set up by UCL. Protocol have been optimized for isolation of genetic material from endothelial derived vesicles.FIMA has extensive new results regarding retrospective studies.
In WP3, UCL designed a patient-oriented protocol, prepared the informed consent forms and obtained approval from the Ethics Institutional Committees to start patient recruitment (UCL), including dedicated MRI brain and heart, as well as OCT angiography in the wider INSIGHT46 protocol. The protocol for cardiac MRI has been optimized between WP3 and WP1/2 and tests have been run, exchanging data between partners. A common database dictionary has been established. UCL with the support of UM and SCNX, have worked together to set up an adapted (shorter and contrast-free) protocol for brain MRI in the NSHD 1946 Birth cohort. Similarly, regarding additional tools for microvascular evaluation, OCT has been acquired and set up by UCL.
In WP4, KU Leuven has translated all the relevant MRI protocols for the brain imaging to the small animal scanners and optimized the histology of the brain imaging using test rats (rats that were being sacrificed for other experiments).
In WP5, UM has obtained the animal ethical approval for the project. They have implemented brain laser speckle contrast imaging in mice and rats and developed a protocol for the study of neurovascular coupling in mice and rats. They have tested and optimized different vessel labelling strategies and tissue clearing protocols to gain insights into the 3D vascular architecture of brains and hearts. The imaging and image analysis pipeline are also being validated. In the meantime, UM have also performed IHC investigations on ZSF1 archived tissues from KU Leuven to assess the pericyte loss possibly associated to microvascular rarefaction. They have also started their first ZSF1 animal study aiming at characterizing change in cerebral blood flow (CBF) and the progression of cognitive impairment in Obese ZSF1 rats. Animals are studied from 15 to 34-35 weeks of age. The study will be terminated in September 2021 and 3D vascular architecture will be obtained on the collected hearts and brains thereafter.
In WP6, the ethical approval to perform experiments on rats were obtained; training on the use of R studio and BiomexTM software (Taverna et al, NAR 48: W385, 2020) for the analysis of bulk RNASeq data was done; the protocol for the isolation of ECs from rat brain via FACS enrichment wassuccessfully established. The protocols for the isolation of ECs from rat heart via MACS and FACS enrichment were also established. The collection of ECs from rat brain and heart for bulk RNASeq analysis has been initiated.
WP7 KU Leuven’s preliminary results with the ZSF1 obese rats have shown that vessel regression (assessed by staining for empty collagen sleeves) and PITX2 down-regulation were present at 14 weeks of age.
Concerning the management and dissemination activities of the project, a communication and dissemination strategy has been put in place and the Vascular Helix has been submitted. Articles highlighting the importance of the topic have been published.
The impact of CRUCIAL’s work is to develop methods to assess microvascular rarefaction in people with comorbidities such that they can be identified as having early symptoms of HFpEF and/or VCI. Our second impact is to study mechanisms of rarefaction, to investigate how it will be possible to stop rarefaction from occurring. In the short term, the most important impact of CRUCIAL is to validate current MRI protocols used in clinical research but not yet accepted by the wider medical community. CRUCIAL will test other possible technologies to measure microvascular rarefaction and dysfunction. This includes the GlycoCheck sublingual videomicroscopy, optical coherence tomography angiography (OCTa) of the retina, and gene expression in circulating endothelial-derived microvesicles. The clinical aspects of CRUCIAL aim to measure the presence of microvascular rarefaction, thereby giving doctors an additional diagnostic tool to identify HFpEF and VCI earlier, whereas the experimental aspects of CRUCIAL aim to understand why rarefaction occurs in the presence of comorbidities such that we may be able to prevent or reverse it therapeutically. We have already made significant progress in understanding the changes that comorbidities produce in endothelial cells. We have performed single cell RNA-Seq from brain and heart endothelial cells and are currently performing the bioinformatic analysis of the data (WP6). Interestingly, we have already found that there is an increase in the number of endothelial cells in the brain of the hypertensive/obese rats.