Signaling In Endothelial Cells Research Articles

Notch Signaling in the Vasculature: Angiogenesis and Angiocrine Functions.

Formation of a functional blood vessel network is a complex process tightly controlled by pro- and antiangiogenic signals released within the local microenvironment or delivered through the bloodstream. Endothelial cells precisely integrate such temporal and spatial changes in extracellular signals and generate an orchestrated response by modulating signaling transduction, gene expression, and metabolism. A key regulator in vessel formation is Notch signaling, which controls endothelial cell specification, proliferation, migration, adhesion, and arteriovenous differentiation. This review summarizes the molecular biology of endothelial Notch signaling and how it controls angiogenesis and maintenance of the established, quiescent vasculature. In addition, recent progress in the understanding of Notch signaling in endothelial cells for controlling organ homeostasis by transcriptional regulation of angiocrine factors and its relevance to disease will be discussed.

Differentiated glioma cell-derived fibromodulin activates integrin-dependent Notch signaling in endothelial cells to promote tumor angiogenesis and growth.

Cancer stem cells (CSCs) alone can initiate and maintain tumors, but the function of non-cancer stem cells (non-CSCs) that form the tumor bulk remains poorly understood. Proteomic analysis showed a higher abundance of the extracellular matrix small leucine-rich proteoglycan fibromodulin (FMOD) in the conditioned medium of differentiated glioma cells (DGCs), the equivalent of glioma non-CSCs, compared to that of glioma stem-like cells (GSCs). DGCs silenced for FMOD fail to cooperate with co-implanted GSCs to promote tumor growth. FMOD downregulation neither affects GSC growth and differentiation nor DGC growth and reprogramming in vitro. DGC-secreted FMOD promotes angiogenesis by activating integrin-dependent Notch signaling in endothelial cells. Furthermore, conditional silencing of FMOD in newly generated DGCs in vivo inhibits the growth of GSC-initiated tumors due to poorly developed vasculature and increases mouse survival. Collectively, these findings demonstrate that DGC-secreted FMOD promotes glioma tumor angiogenesis and growth through paracrine signaling in endothelial cells and identifies a DGC-produced protein as a potential therapeutic target in glioma.

Decreased expression of ErbB2 on left ventricular epicardial cells in patients with diabetes mellitus

We investigated the cell surface expression of ErbB receptors on left ventricular (LV) epicardial endothelial cells and CD105+ cells obtained from cardiac biopsies of patients undergoing coronary artery bypass grafting surgery (CABG). Endothelial cells and CD105+ non-endothelial cells were freshly isolated from LV epicardial biopsies obtained from 15 subjects with diabetes mellitus (DM) and 8 controls. The expression of ErbB receptors was examined using flow cytometry. We found that diabetes mellitus (DM) and high levels of hemoglobin A1C are associated with reduced expression of ErbB2. To determine if the expression of ErbB2 receptors is regulated by glucose levels, we examined the effect of high Glucose in human microvascular endothelial cells (HMEC-1) and CD105+ non-endothelial cells, using a novel flow cytometric approach to simultaneously determine the total level, cell surface expression, and phosphorylation of ErbB2. Incubation of cells in the presence of 25 mM d-glucose resulted in decreased cell surface but not total levels of ErbB2. The level of ErbB2 at the cell surface is controlled by disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) that is expressed on LV epicardial cells. Inhibition of ADAM10 prevented the high glucose-dependent decrease in the cell surface expression of ErbB2. We suggest that high Glucose depresses ErbB receptor signaling in endothelial cells and cardiac progenitor cells via the promotion of ADAM10-dependent cleavage of ErbB2 at the cell surface, thus contributing to vascular dysfunction and adverse remodeling seen in diabetic patients.

Notch Signaling in Vascular Endothelial and Mural Cell Communications.

The Notch signaling pathway is a highly versatile and evolutionarily conserved mechanism with an important role in cell fate determination. Notch signaling plays a vital role in vascular development, regulating several fundamental processes such as angiogenesis, arterial/venous differentiation, and mural cell investment. Aberrant Notch signaling can result in severe vascular phenotypes as observed in cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and Alagille syndrome. It is known that vascular endothelial cells and mural cells interact to regulate vessel formation, cell maturation, and stability of the vascular network. Defective endothelial-mural cell interactions are a common phenotype in diseases characterized by impaired vascular integrity. Further refinement of the role of Notch signaling in the vascular junctions will be critical to attempts to modulate Notch in the context of human vascular disease. In this review, we aim to consolidate and summarize our current understanding of Notch signaling in the vascular endothelial and mural cells during development and in the adult vasculature.

MO469: GPR124—A Driver of Fibrotic Kidney Diseases

Abstract BACKGROUND AND AIMS Renal fibrosis is a central feature of chronic kidney disease (CKD), and the severity of CKD correlates with the magnitude of renal fibrosis. Despite the causative role attributed to fibrosis in CKD progression, there is still no treatment available that directly targets renal fibrosis. Upon kidney injury, embryonic signalling pathways are activated, promoting the repair and regeneration of injured tissue. Maladaptive repair mechanisms have been associated with the development of renal fibrosis and sustained activation of Wnt/β-catenin signalling contributes to CKD progression. GPR124 (ADGRA2 gene), has been identified as a potential fibrotic mediator through its upregulation in pericytes during pericyte to myofibroblast transition. GPR124 has been identified to function as a co-receptor for Wnt7 mediating canonical Wnt signaling in endothelial cells. We observed elevated GPR124 expression in mouse kidneys after chronic ischaemia reperfusion injury (cIRI). To further explore the role of GPR124 in CKD progression, we have studied its correlation with disease severity in human NURTuRE kidney biopsies [1]. We have also looked at GPR124 therapeutic potential in mouse cIRI. Given its potential disease relevance, we set out to develop inhibitory GPR124 antibodies as novel therapeutics to treat renal fibrosis. METHOD Diagnostic FFPE kidney cortex biopsy samples from 497 NURTuRE patients were subjected to bulk RNA-Seq. Library preparation with Illumina Enrichment Tagmentation technology was followed by sequencing and alignment of paired-end reads to the reference genome GRCh38/ENSEMBL 97 and quantification. Monoclonal Anti-GPR124 antibodies were generated against the extracellular domain of human GPR124. Top candidates were selected by FACS binding and further characterized in a TCF/LEF luciferase assay for the inhibition of Wnt7 signaling. One hu/ms cross-reactive candidate was studied in a 14-day IRI mouse model to assess its anti-fibrotic effect in vivo. Mice were exposed to 30-min unilateral renal ischaemia, followed by 14 days recovery. Kidneys were studied for gene expression by RT-PCR. Animals were divided into sham (n = 5), control IRI (no antibody, n = 12) and treated groups (each n = 12). The antibody was administered before ischemia and during the recovery phase in two doses [50 mg/kg and 150 mg/kg]. RESULTS CONCLUSION NURTuRE provides us with a unique opportunity to assess relevance of a potential target candidate gene in kidney disease patients. The potential of GPR124 as a therapeutic target was highlighted by the upregulation of expression levels along disease severity and biological pathway analysis, where our data supports a potential role in extracellular matrix reorganization. The administration of GPR124 antibodies and inhibition of the GPR124-mediated Wnt signaling in vivo inhibited the development of ischaemia-induced fibrosis in the mouse kidney. The results strongly support a role for GPR124 in human kidney disease progression, through inhibition of fibrosis, and its potential as a future clinical target.

Identification of Deubiquitinases (DUBs) that modulate PAR1‐mediated p38 MAPK inflammatory signaling in endothelial cells

Protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, induces endothelial dysfunction via multiple signal transduction pathways including p38 mitogen‐activated protein kinase (MAPK). We showed that thrombin‐stimulates K63‐linked ubiquitination of PAR1 that drives recruitment of transforming growth factor‐β‐activated kinase‐1 binding protein‐2 (TAB2), an adaptor protein that binds TAB1, which triggers p38 activation on endosomes and promotes endothelial barrier disruption. However, the regulatory processes that control thrombin‐stimulated PAR1 ubiquitin‐dependent p38 inflammatory signaling are not known. To address this gap in knowledge, we sought to identify specific deubiquitinases (DUBs) that counter thrombin‐induced ubiquitin‐mediated p38 signaling by conducting an unbiased genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells. We hypothesize that a PAR1‐specific DUB is essential for controlling proper dynamics of thrombin‐mediated p38 signaling and might be altered in endothelial dysfunction. To systematically identify DUBs, endothelial cells were transfected with pools of 4 siRNAs targeting each of the 96 DUBs and thrombin‐stimulated p38 phosphorylation was assessed by immunoblotting. In parallel, a genome‐wide DUB siRNA library screen was conducted in HeLa cells. We identified 8 different DUBs that preferentially cleave K63‐linked ubiquitin and regulate thrombin‐p38 signaling in both endothelial and HeLa cells. A time‐course analysis of thrombin‐stimulated p38 signaling confirmed that depletion of each of the 8 DUBs alone caused an increase in the magnitude and/or duration of p38 signaling. Among the candidate DUBs, USP34, USP47 and CYLD exhibited the highest expression in both endothelial and HeLa cells detected by RT‐qPCR and were further examined. Using individual siRNAs targeting each of the 3 candidate DUBs, we validated that knockdown of either USP34, USP47 or CYLD alone was sufficient to markedly enhance thrombin‐induced p38 signaling in endothelial cells. Currently, I am examining the impact of USP34‐mediated modulation of thrombin‐p38 signaling on endothelial inflammatory responses. I will present new data examining the role of USP34 on thrombin‐induced endothelial barrier disruption and induction of cytokine expression. While ubiquitin‐dependent signaling is widespread, our knowledge of the processes that control ubiquitin‐mediated GPCR signaling is limited, especially related to the function of GPCR‐specific DUBs. Here, we report the first genome‐wide DUB siRNA library screen on GPCR signaling and identified a subset of DUBs that specifically regulate PAR1‐p38 inflammatory signaling that may serve as important therapeutic targets for vascular inflammation.

Abstract 333: Tnik: A Novel Regulator Of Endothelial Interferon Signaling And Activation

Background: Endothelial cells (ECs) are prominent cell types governing vascular tone and homeostasis. Activation of ECs is the central pathophysiological process in the initiation of atherosclerosis (AS). Interferon signaling (IFN Type I and II) is activated in ECs under inflammatory conditions such as systemic lupus erythematosus and is implicated in EC activation. But the underlying mechanisms are still unknown. Through RNA sequencing and pathway analysis, we have identified TNIK, a Ste20 family of MAP kinases (MAP4K) to regulate IFN signaling in ECs. Our study is the first to report the role and function of TNIK in IFN signaling and EC activation. Hypothesis: TNIK regulates IFNs through STAT mediated signaling and downstream interferon stimulated gene (ISG) sets Methods: Cultured Human aortic endothelial cells (HAECs) were treated with control or TNIK-targeted siRNA. Later cells were subjected to static, laminar or disturbed flow conditions. Total RNA was isolated and sequenced by mRNA-seq. The differential expression data were analyzed using CLC genomics and Ingenuity Pathway Analysis (IPA). Western blotting and qRT-PCR were performed to validate STAT activation and interferon stimulated genes (ISGs) Results: We found >450 genes differentially expressed in siTNIK vs siCTRL treated ECs (fold change>2, FDR<0.05). IPA revealed that under static (ST) and disturbed flow (DF) conditions TNIK depletion lead to inhibition IFN and upregulation of eukaryotic transcription elongation factor signaling (eIF2). In addition, under DF, TNIK depletion lead to downregulation of hyperctokinemia/hyperchemokinemia. Validation through western blotting and qRT-PCR confirmed that TNIK regulates STAT expression and IFN stimulated target genes. Conclusions: Our study elucidates the unique role of TNIK in regulating STAT mediated IFN signaling and subsequent ISGs in ECs.

An siRNA library screen for endothelial PAR1‐specific deubiquitinases regulating p38 MAPK inflammatory signaling

Endothelial dysfunction is induced by inflammatory mediators that signal through GPCRs and a hallmark of inflammation. Protease‐activated receptor‐1 (PAR1), a GPCR for thrombin, induces endothelial dysfunction via multiple signal transduction pathways including p38 mitogen‐activated protein kinase (MAPK). We showed that thrombin‐stimulates K63‐linked ubiquitination of PAR1 that drives recruitment of transforming growth factor‐β‐activated kinase‐1 binding protein‐2 (TAB2), an adaptor protein that binds TAB1, which triggers p38 activation on endosomes and promotes endothelial barrier disruption. However, the regulatory processes that control thrombin‐stimulated PAR1 ubiquitin‐dependent p38 inflammatory signaling are not known. The objective of this study was to identify specific deubiquitinases (DUBs) that counter thrombin‐induced ubiquitin‐mediated p38 signaling by conducting an unbiased genome‐wide siRNA library screen targeting all 96 human DUB genes in human cultured endothelial cells. We hypothesize that a PAR1‐specific DUB is essential for controlling proper dynamics of thrombin‐mediated p38 signaling and might be altered in endothelial dysfunction. To systematically identify DUBs, endothelial cells were transfected with pools of 4 siRNAs targeting each of the 96 DUBs and thrombin‐stimulated p38 phosphorylation was assessed by immunoblotting. In parallel, a genome‐wide DUB siRNA library screen was conducted in HeLa cells. We identified 8 different DUBs that preferentially cleave K63‐linked ubiquitin and regulate thrombin‐p38 signaling in both endothelial and HeLa cells. A time‐course analysis of thrombin‐stimulated p38 signaling confirmed that depletion of each of the 8 DUBs alone caused an increase in the magnitude and/or duration of p38 signaling. Among the candidate DUBs, USP34, USP47 and CYLD exhibited the highest expression in both endothelial and HeLa cells detected by RT‐qPCR and were further examined. Using individual siRNAs targeting each of the 3 candidate DUBs, we validated that knockdown of either USP34, USP47 or CYLD alone was sufficient to markedly enhance thrombin‐induced p38 signaling in endothelial cells. New data examining the function of CYLD on thrombin‐induced inflammatory responses will be presented. While ubiquitin‐dependent signaling is widespread, our knowledge of the processes that control ubiquitin‐mediated GPCR signaling is limited, especially related to the function of GPCR‐specific DUBs. In this study, we report the first genome‐wide DUB siRNA library screen on GPCR signaling and identified a subset of DUBs that specifically regulate PAR1‐p38 inflammatory signaling that may serve as important therapeutic targets for vascular inflammation.

Novel Mediators of Reelin Signaling in Human Vascular Endothelial Cells

IntroductionThe formation of coronary vessels is fundamental in heart development. Our lab has demonstrated that Reelin, an extracellular matrix glycoprotein, is involved in the formation of coronary vessels. Reelin expression is localized to the vascular endothelial cells of coronary vessels in embryonic mice. Human dermal microvascular endothelial cells (HDMECs) were used to examine the potential role of Reelin in vascular development as they appropriately model events involved in vessel formation and endogenously express RELN mRNA. Previous studies from our lab indicated that small interfering RNA (siRNA)‐mediated gene silencing of RELNin HDMECs reduced cell migration and basal cell membrane permeability, and increased capillary‐like tube formation. Based on these results, we believe Reelin is involved in vasculogenesis and angiogenesis. This prompted us to analyze expression of angiogenic transcripts by qPCR array in negative control and RELN knockdown (KD) HDMECs. Our results indicated significant upregulation of ADAMTS1 (a disintegrin and metalloproteinase with thrombospondin motifs), and significant changes in expression of other angiogenesis‐related transcripts.Study ObjectiveThis study aimed to validate the angiogenesis array data by individual TaqMan qPCR analysis of mRNA expression of the significantly altered transcripts. Also, we aimed to analyze the encoded protein expression of these transcripts in negative control and RELN KD cells. We hypothesized that significant alterations in expression of ADAMTS1 and other angiogenic transcripts leads to changes in their protein expression in our RELN KD cells and mediates Reelin signaling in human vascular endothelial cells.MethodsTo validate our angiogenesis qPCR array data, we utilized HDMECs as an in vitro system to examine mRNA and protein expression. Gene‐specific siRNAs were used to target and knockdown endogenous expression of RELN in HDMECs. Total RNA was isolated from negative control and RELNKD HDMECs and it was analyzed by qPCR with TaqMan probes for RELN, ADAMTS1, GAPDH, 18SRNA and other angiogenic transcripts. Also, protein expression of the encoded transcripts was analyzed by immunofluorescence and western blot in negative control and RELN KD HDMECs.ResultsRELN KD HDMECs exhibited a >90% reduction in Reelin protein expression compared to negative control HDMECs. ADAMTS1 mRNA was significantly upregulated in RELN KD cells while expression of other angiogenic transcripts were also altered in these cells. We demonstrated by immunofluorescence that ADAMTS1 is localized to the nucleus, and it is detected in HDMEC lysates by western blot analysis. In addition, ADAMTS1 protein expression differed between negative control and RELN KD HDMECs.ConclusionsWe conclude that ADAMTS1 mRNA is upregulated in RELN KD HDMECs versus negative control cells. Knockdown of RELN in HDMECS altered cell migration, membrane permeability, capillary‐like tube formation and expression of angiogenic transcripts. These findings provide key evidence that ADAMTS1 and other angiogenic transcripts may be novel mediators of Reelin signaling in human vascular endothelial cells.

Mechanical Tension‐induced Vascular and Alveolar Morphogenesis during Lung Regeneration

Mechanical forces altered after unilateral pneumonectomy (PNX) control post‐PNX vascular and alveolar morphogenesis. This study aims to understand the mechanosensitive mechanism of lung vascular and alveolar regeneration in the post‐PNX mouse lungs. Vascular formation is stimulated at the subpleural region in the remaining mouse lung after unilateral PNX, where mechanical stretch is enhanced, while inhibition of mechanical tension by insertion of prosthesis attenuates these effects. The focal adhesion protein, paxillin senses mechanical stretch after PNX and the levels of paxillin are upregulated in SPB‐positive alveolar epithelial type II (ATII) cells and endothelial cells (ECs), which stimulates the expression of angiogenic factor angiopoietin‐1 (Ang1) in ATII cells and Ang2 in ECs to induce EC directional motility to the subpleural region. Cellular stretching increases the expression of paxillin, which induces expression of Ang2 in ECs and Ang1 in ATII cells in vitro. Post‐PNX lung growth and vascular formation toward the subpleural region are suppressed in Pxnfl/fl‐Sftpc‐CreERT2 mouse lungs, in which paxillin expression is attenuated in ATII cells. Structure of lamellar bodies that control surfactant homeostasis is also disrupted in post‐PNX Pxnfl/fl‐Sftpc‐CreERT2 mouse lung ATII cells. These results suggest that cooperative action of mechanosensitive paxillin signaling in ECs and ATII cells mediates vascular and alveolar epithelial formation during post‐PNX lung growth.

Abstract 496: The Cardiometabolic Disease Risk Protein SVEP1 Activates AKT/mTOR By Signaling Through The Orphan Receptor PEAR1

Background: Sushi, von Willebrand factor type A, EGF and pentraxin domain containing 1 (SVEP1) is an extracellular matrix protein that circulates in plasma and is causally related to cardiovascular disease, hypertension, and type 2 diabetes. A recent genome wide association study (GWAS) also implicates SVEP1 in platelet reactivity. The gene most strongly associated with platelet reactivity in the GWAS is Platelet and Endothelial Cell Receptor 1 ( PEAR1 ), a gene that encodes an orphan receptor tyrosine kinase-like protein that also associates with cardiovascular disease. Little is known about the molecular interactions and disease mechanisms of these proteins, despite their associations with cardiovascular disease and platelet reactivity. Methods: We used Mendelian Randomization (MR) and phenome-wide association studies to identify candidate SVEP1 protein interactions. We tested candidate interactions using recombinant proteins with molecular, cellular, and ex vivo assays. Results: A coding variant within the ectodomain of PEAR1 alters plasma levels of SVEP1 in humans (p=1.7x10^-17), suggesting these proteins may interact. MR demonstrates that genetically determined, increased plasma PEAR1 levels causally associate with decreased plasma SVEP1 (p=1.3x10^-11), further supporting an interaction. PEAR1 co-immunoprecipitates with SVEP1. Exposure to SVEP1 induces PEAR1 phosphorylation and activation of AKT/mTOR signaling in endothelial cells, vascular smooth muscle cells, and platelets. siRNA knockdown of PEAR1 abrogates this signaling. Endothelial cells adhere and proliferate in response to immobilized SVEP1. Platelets isolated from mice lacking SVEP1 have lower activation relative to controls and phenocopy platelets from mice lacking PEAR1. Conclusions: Despite robust evidence for the role of SVEP1 and PEAR1 in cardiovascular disease, critical gaps remain in our understanding of their disease mechanisms. Here, we identify SVEP1 as the first biological ligand of PEAR1 and a potent activator of AKT/mTOR signaling. The interaction between SVEP1 and PEAR1 is particularly intriguing, given the prominent role of AKT/mTOR in cardiovascular disease, and disrupting the interaction may be therapeutically beneficial.

Multi‐scale Computational Model of Endothelial Cell‐Pericyte Coupling in Idiopathic Pulmonary Fibrosis

IntroductionMicrovascular stability is highly dependent on endothelial cell‐pericyte coupling. In fibrotic diseases, such as idiopathic pulmonary fibrosis (IPF), extracellular matrix stiffening and elevated concentrations of profibrotic factors, such as TGF‐β, can disrupt this cell‐cell communication. Treatment of IPF with the antifibrotic drug Nintedanib, which targets PDGF‐βR, FGF‐R, and VEGF‐R signaling, may counteract these effects to strengthen endothelial cell‐pericyte coupling and promote microvessel stability1,2. We have developed a multi‐scale computational model that simulates intracellular signaling in endothelial cells and pericytes, heterotypic cell‐cell communication, and the dynamic lung microenvironment to study how Nintedanib2 and other drugs affect microvascular network remodeling in IPF. We hypothesize that blocking PDGF‐βR and FGF‐R signaling with Nintedanib is sufficient to rescue TGF‐β‐induced endothelial cell‐pericyte decoupling.Materials and MethodsThe multi‐scale model is comprised of logic‐based ordinary differential equations representing intracellular signaling networks in both endothelial cells and pericytes integrated into an agent‐based model representing the lung environment wherein the simulated cells interact with one another, sensing and dynamically altering their microenvironment (Figure 1). The logic‐based network signaling models were developed using the Netflux3 toolkit in MATLAB. The agent‐based model of the spatiotemporal 2D lung environment was constructed in NetLogo4. The multi‐scale model was created by linking the logic‐based network models with the agent‐based model using the NL4Py5 package in Python.Results and DiscussionEndothelial cell‐pericyte decoupling was signified by decreased N‐cadherin expression in pericytes, increased αSMA and Col1mRNA (indicating possible pericyte‐to‐myofibroblast transition), and increased physical separation and distances between endothelial cells and pericytes. The multi‐scale model predicted that increasing TGF‐β concentrations significantly elevated αSMA and Col1mRNA expression in simulated pericytes. N‐cadherin levels did not change in response to TGF‐β or Nintedanib treatment in the network model of the pericyte alone but were affected by the presence of endothelial cells in the agent‐based model, highlighting the importance of endothelial cell‐pericyte interactions in determining pericyte behaviors.ConclusionsOur multi‐scale model demonstrates the importance of considering endothelial cell‐pericyte communication in predicting cellular responses to pro‐fibrotic environments. By representing intracellular biochemical signaling networks, heterotypic cell interactions, and the dynamic multi‐cell microenvironment, we were able to demonstrate a potential mechanism through which Nintedanib treatment affects endothelial cell‐pericyte coupling in IPF.References1) Hanumegowda, C., et al.; Chest. 2012 2) Wollin, L., et al.; Eur Respir J. 2015 3) Kraeutler, MJ., et al.; BMC Syst Biol. 2010 4) Sklar, E., Artif. Life 2007 5) Gunaratne, C.; 2018. NL4Py.

PAR2, not PAR1, regulates endothelium‐dependent vascular tone in resistance arteries

Protease activated receptors (PARs), a subfamily of G‐protein‐coupled receptors, are activated by cleavage of its extracellular domains. Four mammalian PAR members have been identified (PAR1‐4). PAR1 is the thrombin receptor, while PAR2 is activated physiologically by peptides or N‐terminal proteolytic cleavage. The resulting cleaved N‐termini acts as a tethered ligand that initiates receptor signaling. PARs may regulate vascular tone, cell permeability and cell proliferation. However, the precise roles of PAR1 and PAR2 in regulating vascular tone via the endothelium, and how PAR‐evoked signals transmit extracellularly and intracellularly, are incompletely understood.To explore the physiological functions, and the underlying signalling pathways in native endothelium, PAR1 (TFLLR‐NH2) and PAR2 (2‐Furoyl‐LIGRLO‐amide; 2fLI) activating peptides were used, and changes in intracellular Ca2+ and contractility measured in en face rat mesenteric arteries. Surprisingly, PAR1 activation (TFLLR‐NH2or trypsin) neither triggered Ca2+ signals in endothelial cells or alter vascular tone. On the other hand, PAR2 activation (2fLI) evoked large global propagating Ca2+ waves in endothelial cells and endothelium‐dependent dilation of mesenteric arteries. The Ca2+ signals evoked by PAR2 activation were kinetically distinctive when compared to those of muscarinic receptor activation. PAR2 activation (2fLI) also evoked responses in different clusters of endothelial cells from those of muscarinic receptor (acetylcholine) activation. PAR2 activation with 2fLI or trypsin failed to evoke Ca2+ signals after depletion of the internal store with the Ca2+‐ATPase (SERCA) inhibitor, CPA. This result suggests that PAR2 evokes Ca2+ release from internal stores. Pharmacological block of inositol 1,4,5‐trisphosphate (IP3) with either 2‐APB or caffeine, also blocked PAR2‐evoked Ca2+ signals. Phospholipase C inhibition (U73122) also blocked the Ca2+ signals evoked by PAR2 activation. Therefore, PAR2 is a PLC linked G protein coupled receptor which evokes Ca2+ release from the internal store via IP3 receptors in endothelial cells. The TRPC3 blocker, PYR3 and store‐operated Ca2+ entry (SOCE) blocker, YM58483 each significantly supressed PAR2 mediated Ca2+ signals, suggesting internal store was replenished via TRPC3 channel and SOCE.In conclusion, PAR2 rather than PAR1, regulates vascular tone in mesenteric arteries by evoking Ca2+ release from the internal store via IP3 receptors to generate propagating Ca2+ waves in distinct sub‐populations of endothelial cells.

Endothelial‐specific nanodelivery of Rho kinase inhibitors: Targeting tumor angiogenesis and metastasis

ObjectiveThe limitations of current vascular endothelial growth factor (VEGF)‐centered anti‐angiogenic therapy include the inhibition of physiological angiogenesis, development of evasive resistance, and increased vascular leakage, which drives the need for a novel therapy for tumor angiogenesis and metastasis. RhoA pathway inhibition blocks VEGF‐ and sphingosine‐1 phosphate (S1P)‐induced angiogenesis and vascular leakage without hindering endogenous angiogenesis. Fasudil, a clinically relevant Rho‐associated kinase (ROCK) inhibitor, blocks the RhoA downstream signaling, but adverse events (hypotension, skin reactions) exist. Here, we propose endothelial ROCK targeting with Fasudil‐containing immune‐related nanodelivery.HypothesisFasudil‐encapsulated CD31‐targeting liposomal formulation is a potential tumor antiangiogenic therapy by blocking RhoA signaling via ROCK inhibition in the endothelial cells.MethodsFasudil‐liposomal nanoparticles were prepared by DPPC, cholesterol, 16:0 PEG2000PE, DSPE‐PEG2000‐mal, DSPE‐PEG‐FITC, and CHCl3: MeOH (4:1) combination, thin‐film formation, downsizing after Fasudil incorporation, filtering, and conjugation with IgG and CD31‐targeting antibodies. We used dynamic light scattering (DLS) for the liposomal characterization, dose‐response experiments on primary Human Umbilical Vein Endothelial Cells (HUVEC) and Bend3 cells for in vitro toxicity evaluation, antibody (Ab)‐to‐liposome binding evaluation and pathway inhibition evaluation by western blot, and UPLC analysis (EE)% for the evaluation of encapsulation efficiency. The binding affinity of the CD31‐conjugated liposomes and their time‐dependent cell incorporation were evaluated by competitive binding assay via immunohistochemistry (IHC) and flow cytometry. Morphometric analysis was assessed by Livecyte imaging analysis.ResultsSize and PDI measurement by DLS confirmed the stability of our formulation, which presented limited toxicity (concentration: 0.625‐5120 µM; IC50 at 43.05 µM [HUVECs over 24 hours of treatment]) with a significantly higher overall survival rate than Fasudil. Efficient CD31 and mouse IgG Ab conjugation to Fasudil liposomes and blockade of the RhoA downstream signaling in endothelial cells were confirmed. The specific binding of CD31‐targeting liposomes on endothelial cells was verified in the presence and absence of excess CD31 Ab. The time‐dependent study confirmed the kinetics of liposome incorporation in endothelial cells and the enhanced uptake of CD31 liposomes compared to IgG. Cell morphometric and physiological analysis revealed comparable alterations caused by Fasudil‐ and endothelial‐specific Fasudil‐containing liposomes.ConclusionWe have successfully developed Fasudil‐encapsulated, CD31‐targeting liposomes. Future studies will be directed to study the formulation’s effect on endothelial physiology in vitro and in vivo, tumor growth, angiogenesis, and metastasis.

SARS-CoV-2 and type I interferon signaling in brain endothelial cells: Blurring the lines between friend or foe

A recent paper published in Stem Cell Reports (Krasemann et al., 2022) described gene-, pathway-, and tissue-resolution evidence on the mechanisms involved in SARS-CoV-2 CNS entry via the blood-brain-barrier (BBB). The work provided further evidence on how the virus may interact with the neurovascular unit, resulting in enhanced inflammatory signaling in affected cells.

Small-world connectivity dictates collective endothelial cell signaling

Every blood vessel is lined by a single layer of highly specialized, yet adaptable and multifunctional endothelial cells. These cells, the endothelium, control vascular contractility, hemostasis, and inflammation and regulate the exchange of oxygen, nutrients, and waste products between circulating blood and tissue. To control each function, the endothelium processes endlessly arriving requests from multiple sources using separate clusters of cells specialized to detect specific stimuli. A well-developed but poorly understood communication system operates between cells to integrate multiple lines of information and coordinate endothelial responses. Here, the nature of the communication network has been addressed using single-cell Ca2+ imaging across thousands of endothelial cells in intact blood vessels. Cell activities were cross-correlated and compared to a stochastic model to determine network connections. Highly correlated Ca2+ activities occurred in scattered cell clusters, and network communication links between them exhibited unexpectedly short path lengths. The number of connections between cells (degree distribution) followed a power-law relationship revealing a scale-free network topology. The path length and degree distribution revealed an endothelial network with a “small-world” configuration. The small-world configuration confers particularly dynamic endothelial properties including high signal-propagation speed, stability, and a high degree of synchronizability. Local activation of small clusters of cells revealed that the short path lengths and rapid signal transmission were achieved by shortcuts via connecting extensions to nonlocal cells. These findings reveal that the endothelial network design is effective for local and global efficiency in the interaction of the cells and rapid and robust communication between endothelial cells in order to efficiently control cardiovascular activity.

Silencing of STE20-type kinase STK25 in human aortic endothelial and smooth muscle cells is atheroprotective

Recent studies highlight the importance of lipotoxic damage in aortic cells as the major pathogenetic contributor to atherosclerotic disease. Since the STE20-type kinase STK25 has been shown to exacerbate ectopic lipid storage and associated cell injury in several metabolic organs, we here investigate its role in the main cell types of vasculature. We depleted STK25 by small interfering RNA in human aortic endothelial and smooth muscle cells exposed to oleic acid and oxidized LDL. In both cell types, the silencing of STK25 reduces lipid accumulation and suppresses activation of inflammatory and fibrotic pathways as well as lowering oxidative and endoplasmic reticulum stress. Notably, in smooth muscle cells, STK25 inactivation hinders the shift from a contractile to a synthetic phenotype. Together, we provide several lines of evidence that antagonizing STK25 signaling in human aortic endothelial and smooth muscle cells is atheroprotective, highlighting this kinase as a new potential therapeutic target for atherosclerotic disease.

Piezo1 Is a Mechanosensor Channel in Central Nervous System Capillaries.

Capillaries are equipped to sense neurovascular coupling agents released onto the outer wall of a capillary, translating these external signals into electrical/Ca2+ changes that play a crucial role in blood flow regulation and ensuring that neuronal demands are met. However, control mechanisms attributable to forces imposed onto the lumen are less clear. Here, we show that Piezo1 channels act as mechanosensors in central nervous system capillaries. Electrophysiological analyses confirmed expression and function of Piezo1 channels in brain cortical and retinal capillaries. Activation of Piezo1 channels evoked currents that were sensitive to endothelial cell-specific Piezo1 deletion. Using genetically encoded Ca2+ indicator mice and an ex vivo pressurized retina preparation, we found that activation of Piezo1 channels by mechanical forces triggered Ca2+ signals in capillary endothelial cells. Collectively, these findings indicate that Piezo1 channels are capillary mechanosensors that initiate crucial Ca2+ signals and could, therefore, have a profound impact on central nervous system blood flow control.

Structural and Functional Changes in Aged Skin Lymphatic Vessels.

Lymphatic structure and function play a critical role in fluid transport, antigen delivery, and immune homeostasis. A dysfunctional lymphatic system is associated with chronic low-grade inflammation of peripheral tissues, poor immune responses, and recurrent infections, which are also hallmarks of aging pathology. Previous studies have shown that aging impairs lymphatic structure and function in a variety of organ systems, including the intestines and central nervous system. However, previous studies are mostly limited to qualitative analysis of lymphatic structural changes and quantification of intestinal collecting vessel contractile function. It is not clear whether decreased lymphatic function contributes to pathological conditions related to aging, nor how it affects the skin immune microenvironment. Further, the effects of aging on skin initial and collecting lymphatic vessels, dendritic cell (DC) migration, cutaneous lymphatic pumping, and VEGFR-3 signaling in lymphatic endothelial cells (LECs) have not been quantitatively analyzed. Here, using fluorescent immunohistochemistry and flow cytometry, we confirm that aging decreases skin initial and collecting lymphatic vessel density. Indocyanine green (ICG) lymphangiography and DC migration assays confirm that aging decreases both fluid pumping and cell migration via lymphatic vessels. At the cellular level, aging causes decreased VEGFR-3 signaling, leading to increased LEC apoptosis and senescence. Finally, we determined that aging causes decreased lymphatic production of chemokines and alters LEC expression of junctional and adhesion molecules. This in turn leads to increased peri-lymphatic inflammation and nitrosative stress that might contribute to aging pathology in a feed-forward manner. Taken together, our study, in addition to quantitatively corroborating previous findings, suggests diverse mechanisms that contribute to lymphatic dysfunction in aging that in turn exacerbate the pathology of aging in a feed-forward manner.

The Beauty and Complexity of Blood Vessel Patterning.

This review highlights new concepts in vascular patterning in the last 10 years, with emphasis on its beauty and complexity. Endothelial cell signaling pathways that respond to molecular or mechanical signals are described, and examples of vascular patterning that use these pathways in brain, skin, heart, and kidney are highlighted. The pathological consequences of patterning loss are discussed in the context of arteriovenous malformations (AVMs), and prospects for the next 10 years presented.