Incongruence between transcriptional and vascular pathophysiological cell states.

The Notch pathway is a major regulator of endothelial transcriptional specification. Targeting the Notch receptors or Delta-like ligand 4 (Dll4) dysregulates angiogenesis. Here, by analyzing single and compound genetic mutants for all Notch signaling members, we find significant differences in the way ligands and receptors regulate liver vascular homeostasis. Loss of Notch receptors caused endothelial hypermitogenic cell-cycle arrest and senescence. Conversely, Dll4 loss triggered a strong Myc-driven transcriptional switch inducing endothelial proliferation and the tip-cell state. Myc loss suppressed the induction of angiogenesis in the absence of Dll4, without preventing the vascular enlargement and organ pathology. Similarly, inhibition of other pro-angiogenic pathways, including MAPK/ERK and mTOR, had no effect on the vascular expansion induced by Dll4 loss; however, anti-VEGFA treatment prevented it without fully suppressing the transcriptional and metabolic programs. This study shows incongruence between single-cell transcriptional states, vascular phenotypes and related pathophysiology. Our findings also suggest that the vascular structure abnormalization, rather than neoplasms, causes the reported anti-Dll4 antibody toxicity.

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.