Incongruence between transcriptional and vascular pathophysiological cell states

Research in R.B.’s laboratory was supported by the European Research Council Starting Grant AngioGenesHD (638028) and Consolidator Grant AngioUnrestUHD (101001814), the CNIC Intramural Grant Program Severo Ochoa (11-2016-IGP-SEV-2015-0505), the Ministerio de Ciencia e Innovación (MCIN) (SAF2013-44329-P, RYC-2013- 13209, and SAF2017-89299-P) and ‘La Caixa’ Banking Foundation (HR19-00120). J.V.’s laboratory was supported by MCIN (PGC2018- 097019-B-I00 and PID2021-122348NB-I00) and La Caixa (HR17-00247 and HR22-00253). K.G.’s laboratory was supported by Knut and Alice Wallenberg Foundation (2020.0057) and Vetenskapsrådet (2021-04896). The CNIC is supported by Instituto de Salud Carlos III, MCIN, and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MCIN/ AEI/10.13039/501100011033). Microscopy experiments were performed at the Microscopy and Dynamic Imaging Unit, CNIC, ICTS-ReDib, co-funded by MCIN/AEI/10.13039/501100011033 and FEDER ‘Una manera de hacer Europa’ (ICTS-2018-04-CNIC-16). M.F.-C. was supported by PhD fellowships from La Caixa (CX_E-2015-01) and Boehringer Ingelheim travel grants. S.M. was supported by the Austrian Science Fund (J4358). A.R. was supported by the Youth Employment Initiative (PEJD-2019-PRE/BMD-16990). L.G.-O. was supported by the Spanish Ministry of Economy and Competitiveness (PRE2018-085283). We thank S. Bartlett (CNIC) for English editing, as well as the members of the Transgenesis, Microscopy, Genomics, Citometry and Bioinformatic units at CNIC. We also thank F. Radtke (Swiss Institute for Experimental Cancer Research), R. H. Adams (Max Planck Institute for Molecular Biomedicine), F. Alt (Boston Children’s Hospital, Harvard Medical School), T. Honjo (Kyoto University Institute for Advanced Studies), I. Flores (CNIC), J. Lewis (Cancer Research UK London Research Institute), S. Habu (Tokai University School of Medicine), T. Gridley (Maine Health Institute for Research) and C. Brakebusch (Biotech Research and Innovation Centre) for sharing the Dll4floxed, Notch1floxed, Notch2floxed, Cdh5(PAC)-creERT2, Myc floxed, Rbpj floxed, p21−/−, Jag1floxed, Dll1floxed, Jag2floxed and Rac1floxed mice.S

Mural Serum Response Factor (SRF) Deficiency Provides Insights into Retinal Vascular Functionality and Development

Serum response factor (SRF) controls the expression of muscle contraction and motility genes in mural cells (MCs) of the vasculature. In the retina, MC-SRF is important for correct angiogenesis during development and the continuing maintenance of the vascular tone. The purpose of this study was to provide further insights into the effects of MC SRF deficiency on the vasculature and function of the mature retina in SrfiMCKO mice that carry a MC-specific deletion of Srf. Retinal morphology and vascular integrity were analyzed in vivo via scanning laser ophthalmoscopy (SLO), angiography, and optical coherence tomography (OCT). Retinal function was evaluated with full-field electroretinography (ERG). We found that retinal blood vessels of these mutants exhibited different degrees of morphological and functional alterations. With increasing severity, we found vascular bulging, the formation of arteriovenous (AV) anastomoses, and ultimately, a retinal detachment (RD). The associated irregular retinal blood pressure and flow distribution eventually induced hypoxia, indicated by a negative ERG waveform shape. Further, the high frequency of interocular differences in the phenotype of individual SrfiMCKO mice points to a secondary nature of these developments far downstream of the genetic defect and rather dependent on the local retinal context

Age‐Associated Changes in Endothelial Transcriptome and Epigenetic Landscapes Correlate With Elevated Risk of Cerebral Microbleeds

Background Stroke is a leading global cause of human death and disability, with advanced aging associated with elevated incidences of stroke. Despite high mortality and morbidity of stroke, the mechanisms leading to blood‐brain barrier dysfunction and development of stroke with age are poorly understood. In the vasculature of brain, endothelial cells (ECs) constitute the core component of the blood‐brain barrier and provide a physical barrier composed of tight junctions, adherens junctions, and basement membrane. Methods and Results We show, in mice, the incidents of intracerebral bleeding increases with age. After isolating an enriched population of cerebral ECs from murine brains at 2, 6, 12, 18, and 24 months, we studied age‐associated changes in gene expression. The study reveals age‐dependent dysregulation of 1388 genes, including many involved in the maintenance of the blood‐brain barrier and vascular integrity. We also investigated age‐dependent changes on the levels of CpG methylation and accessible chromatin in cerebral ECs. Our study reveals correlations between age‐dependent changes in chromatin structure and gene expression, whereas the dynamics of DNA methylation changes are different. Conclusions We find significant age‐dependent downregulation of the Aplnr gene along with age‐dependent reduction in chromatin accessibility of promoter region of the Aplnr gene in cerebral ECs. Aplnr is associated with positive regulation of vasodilation and is implicated in vascular health. Altogether, our data suggest a potential role of the apelinergic axis involving the ligand apelin and its receptor to be critical in maintenance of the blood‐brain barrier and vascular integrity

Mural Cell SRF Controls Pericyte Migration, Vessel Patterning and Blood Flow

Background: Pericytes and vascular smooth muscle cells, collectively known as mural cells, are recruited through PDGFB (platelet-derived growth factor B)-PDGFRB (platelet-derived growth factor receptor beta) signaling. MCs are essential for vascular integrity, and their loss has been associated with numerous diseases. Most of this knowledge is based on studies in which MCs are insufficiently recruited or fully absent upon inducible ablation. In contrast, little is known about the physiological consequences that result from impairment of specific MC functions. Here, we characterize the role of the transcription factor SRF (serum response factor) in MCs and study its function in developmental and pathological contexts. Methods: We generated a mouse model of MC-specific inducible Srf gene deletion and studied its consequences during retinal angiogenesis using RNA-sequencing, immunohistology, in vivo live imaging, and in vitro techniques. Results: By postnatal day 6, pericytes lacking SRF were morphologically abnormal and failed to properly comigrate with angiogenic sprouts. As a consequence, pericyte-deficient vessels at the retinal sprouting front became dilated and leaky. By postnatal day 12, also the vascular smooth muscle cells had lost SRF, which coincided with the formation of pathological arteriovenous shunts. Mechanistically, we show that PDGFB-dependent SRF activation is mediated via MRTF (myocardin-related transcription factor) cofactors. We further show that MRTF-SRF signaling promotes pathological pericyte activation during ischemic retinopathy. RNA-sequencing, immunohistology, in vivo live imaging, and in vitro experiments demonstrated that SRF regulates expression of contractile SMC proteins essential to maintain the vascular tone. Conclusions: SRF is crucial for distinct functions in pericytes and vascular smooth muscle cells. SRF directs pericyte migration downstream of PDGFRB signaling and mediates pathological pericyte activation during ischemic retinopathy. In vascular smooth muscle cells, SRF is essential for expression of the contractile machinery, and its deletion triggers formation of arteriovenous shunts. These essential roles in physiological and pathological contexts provide a rationale for novel therapeutic approaches through targeting SRF activity in MCs

Time to Peak Glucose and Peak C-Peptide During the Progression to Type 1 Diabetes in the Diabetes Prevention Trial and TrialNet Cohorts

OBJECTIVE To assess the progression of type 1 diabetes using time to peak glucose or C-peptide during oral glucose tolerance tests (OGTTs) in autoantibody-positive relatives of people with type 1 diabetes. RESEARCH DESIGN AND METHODS We examined 2-h OGTTs of participants in the Diabetes Prevention Trial Type 1 (DPT-1) and TrialNet Pathway to Prevention (PTP) studies. We included 706 DPT-1 participants (mean ± SD age, 13.84 ± 9.53 years; BMI Z-score, 0.33 ± 1.07; 56.1% male) and 3,720 PTP participants (age, 16.01 ± 12.33 years; BMI Z-score, 0.66 ± 1.3; 49.7% male). Log-rank testing and Cox regression analyses with adjustments (age, sex, race, BMI Z-score, HOMA-insulin resistance, and peak glucose/C-peptide levels, respectively) were performed. RESULTS In each of DPT-1 and PTP, higher 5-year diabetes progression risk was seen in those with time to peak glucose >30 min and time to peak C-peptide >60 min (P < 0.001 for all groups), before and after adjustments. In models examining strength of association with diabetes development, associations were greater for time to peak C-peptide versus peak C-peptide value (DPT-1: χ2 = 25.76 vs. χ2 = 8.62; PTP: χ2 = 149.19 vs. χ2 = 79.98; all P < 0.001). Changes in the percentage of individuals with delayed glucose and/or C-peptide peaks were noted over time. CONCLUSIONS In two independent at-risk populations, we show that those with delayed OGTT peak times for glucose or C-peptide are at higher risk of diabetes development within 5 years, independent of peak levels. Moreover, time to peak C-peptide appears more predictive than the peak level, suggesting its potential use as a specific biomarker for diabetes progression