Hemodynamic Control of Endothelial Cell Fates in Development.

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. From the onset of blood flow, the embryonic vasculature is continuously exposed to a variety of hemodynamic forces. These biomechanical stimuli are key determinants of vascular cell specification and remodeling and the establishment of vascular homeostasis. In recent years, major advances have been made in our understanding of mechano-activated signaling networks that control both spatiotemporal and structural aspects of vascular development. It has become apparent that a major site for mechanotransduction is situated at the interface of blood and the vessel wall and that this process is controlled by the vascular endothelium. In this review, we discuss the hemodynamic control of endothelial cell fates, focusing on arterial-venous specification, lymphatic development, and the endothelial-to-hematopoietic transition, and present some recent insights into the mechano-activated pathways driving these cell fate decisions in the developing embryo.

A computational model of cardiomyocyte metabolism predicts unique reperfusion protocols capable of reducing cell damage during ischemia/reperfusion.

If a coronary blood vessel is occluded and the neighboring cardiomyocytes deprived of oxygen, subsequent reperfusion of the ischemic tissue can lead to oxidative damage due to excessive generation of reactive oxygen species. Cardiomyocytes and their mitochondria are the main energy producers and consumers of the heart, and their metabolic changes during ischemia seem to be a key driver of reperfusion injury. Here, we hypothesized that tracking changes in cardiomyocyte metabolism, such as oxygen and ATP concentrations, would help in identifying points of metabolic failure during ischemia and reperfusion. To track some of these changes continuously from the onset of ischemia through reperfusion, we developed a system of differential equations representing the chemical reactions involved in the production and consumption of 67 molecular species. This model was validated and used to identify conditions present during periods of critical transition in ischemia and reperfusion that could lead to oxidative damage. These simulations identified a range of oxygen concentrations that lead to reverse mitochondrial electron transport at complex I of the respiratory chain and a spike in mitochondrial membrane potential, which are key suspects in the generation of reactive oxygen species at the onset of reperfusion. Our model predicts that a short initial reperfusion treatment with reduced oxygen content (5% of physiological levels) could reduce the cellular damage from both of these mechanisms. This model should serve as an open-source platform to test ideas for treatment of the ischemia reperfusion process by following the temporal evolution of molecular concentrations in the cardiomyocyte.

Endothelial TNF receptor 2 induces IRF1 transcription factor-dependent interferon-ß autocrine signaling to promote monocyte recruitment.

Endothelial-dependent mechanisms of mononuclear cell influx are not well understood. We showed that acute stimulation of murine microvascular endothelial cells expressing the tumor necrosis factor receptors TNFR1 and TNFR2 with the soluble cytokine TNF led to CXCR3 chemokine generation. The TNF receptors signaled through interferon regulatory factor-1 (IRF1) to induce interferon-ß (IFN-ß) and subsequent autocrine signaling via the type I IFN receptor and the transcription factor STAT1. Both TNFR2 and TNFR1 were required for IRF1-IFNß signaling and, in human endothelial cells TNFR2 expression alone induced IFN-ß signaling and monocyte recruitment. In vivo, TNFR1 was required for acute renal neutrophil and monocyte influx after systemic TNF treatment, whereas the TNFR2-IRF1-IFN-ß autocrine loop was essential only for macrophage accumulation. In a chronic model of proliferative nephritis, IRF1 and renal-expressed TNFR2 were essential for sustained macrophage accumulation. Thus, our data identify a pathway in endothelial cells that selectively recruits monocytes during a TNF-induced inflammatory response.

Recommendation on Design, Execution, and Reporting of Animal Atherosclerosis Studies: A Scientific Statement From the American Heart Association.

Animal studies are a foundation for defining mechanisms of atherosclerosis and potential targets of drugs to prevent lesion development or reverse the disease. In the current literature, it is common to see contradictions of outcomes in animal studies from different research groups, leading to the paucity of extrapolations of experimental findings into understanding the human disease. The purpose of this statement is to provide guidelines for development and execution of experimental design and interpretation in animal studies. Recommendations include the following: (1) animal model selection, with commentary on the fidelity of mimicking facets of the human disease; (2) experimental design and its impact on the interpretation of data; and (3) standard methods to enhance accuracy of measurements and characterization of atherosclerotic lesions.

Endothelial Cell Dysfunction and the Pathobiology of Atherosclerosis.

Dysfunction of the endothelial lining of lesion-prone areas of the arterial vasculature is an important contributor to the pathobiology of atherosclerotic cardiovascular disease. Endothelial cell dysfunction, in its broadest sense, encompasses a constellation of various nonadaptive alterations in functional phenotype, which have important implications for the regulation of hemostasis and thrombosis, local vascular tone and redox balance, and the orchestration of acute and chronic inflammatory reactions within the arterial wall. In this review, we trace the evolution of the concept of endothelial cell dysfunction, focusing on recent insights into the cellular and molecular mechanisms that underlie its pivotal roles in atherosclerotic lesion initiation and progression; explore its relationship to classic, as well as more recently defined, clinical risk factors for atherosclerotic cardiovascular disease; consider current approaches to the clinical assessment of endothelial cell dysfunction; and outline some promising new directions for its early detection and treatment.

Recommendation on Design, Execution, and Reporting of Animal Atherosclerosis Studies: A Scientific Statement From the American Heart Association.

Animal studies are a foundation for defining mechanisms of atherosclerosis and potential targets of drugs to prevent lesion development or reverse the disease. In the current literature, it is common to see contradictions of outcomes in animal studies from different research groups, leading to the paucity of extrapolations of experimental findings into understanding the human disease. The purpose of this statement is to provide guidelines for development and execution of experimental design and interpretation in animal studies. Recommendations include the following: (1) animal model selection, with commentary on the fidelity of mimicking facets of the human disease; (2) experimental design and its impact on the interpretation of data; and (3) standard methods to enhance accuracy of measurements and characterization of atherosclerotic lesions.

KLF2 exerts antifibrotic and vasoprotective effects in cirrhotic rat livers: behind the molecular mechanisms of statins.

OBJECTIVE: In the liver, the transcription factor, Kruppel-like factor 2 (KLF2), is induced early during progression of cirrhosis to lessen the development of vascular dysfunction; nevertheless, its endogenous expression results insufficient to attenuate establishment of portal hypertension and aggravation of cirrhosis. Herein, we aimed to explore the effects and the underlying mechanisms of hepatic KLF2 overexpression in in vitro and in vivo models of liver cirrhosis. DESIGN: Activation phenotype was evaluated in human and rat cirrhotic hepatic stellate cells (HSC) treated with the pharmacological inductor of KLF2 simvastatin, with adenovirus codifying for this transcription factor (Ad-KLF2), or vehicle, in presence/absence of inhibitors of KLF2. Possible paracrine interactions between parenchymal and non-parenchymal cells overexpressing KLF2 were studied. Effects of in vivo hepatic KLF2 overexpression on liver fibrosis and systemic and hepatic haemodynamics were assessed in cirrhotic rats. RESULTS: KLF2 upregulation profoundly ameliorated HSC phenotype (reduced α-smooth muscle actin, procollagen I and oxidative stress) partly via the activation of the nuclear factor (NF)-E2-related factor 2 (Nrf2). Coculture experiments showed that improvement in HSC phenotype paracrinally ameliorated liver sinusoidal endothelial cells probably through a vascular endothelial growth factor-mediated mechanism. No paracrine interactions between hepatocytes and HSC were observed. Cirrhotic rats treated with simvastatin or Ad-KLF2 showed hepatic upregulation in the KLF2-Nrf2 pathway, deactivation of HSC and prominent reduction in liver fibrosis. Hepatic KLF2 overexpression was associated with lower portal pressure (-15%) due to both attenuations in the increased portal blood flow and hepatic vascular resistance, together with a significant improvement in hepatic endothelial dysfunction. CONCLUSIONS: Exogenous hepatic KLF2 upregulation improves liver fibrosis, endothelial dysfunction and portal hypertension in cirrhosis.

Endothelial cell-specific expression of roundabout 4 is regulated by differential DNA methylation of the proximal promoter.

OBJECTIVE: The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression. METHODS AND RESULTS: Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the -42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type-specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell-derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation. CONCLUSIONS: The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.

Biomechanical forces promote embryonic haematopoiesis.

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3-5), a master regulator of haematopoiesis, and give rise to haematopoietic cells. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41(+)c-Kit(+) haematopoietic progenitor cells, concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the para-aortic splanchnopleura/aorta-gonads-mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development.

Plxnd1-mediated mechanosensing of blood flow controls the caliber of the Dorsal Aorta via the transcription factor Klf2.

The cardiovascular system generates and responds to mechanical forces. The heartbeat pumps blood through a network of vascular tubes, which adjust their caliber in response to the hemodynamic environment. However, how endothelial cells in the developing vascular system integrate inputs from circulatory forces into signaling pathways to define vessel caliber is poorly understood. Using vertebrate embryos and in vitro-assembled microvascular networks of human endothelial cells as models, flow and genetic manipulations, and custom software, we reveal that Plexin-D1, an endothelial Semaphorin receptor critical for angiogenic guidance, employs its mechanosensing activity to serve as a crucial positive regulator of the Dorsal Aorta's (DA) caliber. We also uncover that the flow-responsive transcription factor KLF2 acts as a paramount mechanosensitive effector of Plexin-D1 that enlarges endothelial cells to widen the vessel. These findings illuminate the molecular and cellular mechanisms orchestrating the interplay between cardiovascular development and hemodynamic forces.

Transcriptomic and spatial dissection of human ex vivo right atrial tissue reveals proinflammatory microvascular changes in ischemic heart disease.

Cardiovascular disease plays a central role in the electrical and structural remodeling of the right atrium, predisposing to arrhythmias, heart failure, and sudden death. Here, we dissect with single-nuclei RNA sequencing (snRNA-seq) and spatial transcriptomics the gene expression changes in the human ex vivo right atrial tissue and pericardial fluid in ischemic heart disease, myocardial infarction, and ischemic and non-ischemic heart failure using asymptomatic patients with valvular disease who undergo preventive surgery as the control group. We reveal substantial differences in disease-associated gene expression in all cell types, collectively suggesting inflammatory microvascular dysfunction and changes in the right atrial tissue composition as the valvular and vascular diseases progress into heart failure. The data collectively suggest that investigation of human cardiovascular disease should expand to all functionally important parts of the heart, which may help us to identify mechanisms promoting more severe types of the disease.

In silico modeling of shear-stress-induced nitric oxide production in endothelial cells through systems biology.

Nitric oxide (NO) produced by vascular endothelial cells is a potent vasodilator and an antiinflammatory mediator. Regulating production of endothelial-derived NO is a complex undertaking, involving multiple signaling and genetic pathways that are activated by diverse humoral and biomechanical stimuli. To gain a thorough understanding of the rich diversity of responses observed experimentally, it is necessary to account for an ensemble of these pathways acting simultaneously. In this article, we have assembled four quantitative molecular pathways previously proposed for shear-stress-induced NO production. In these pathways, endothelial NO synthase is activated 1), via calcium release, 2), via phosphorylation reactions, and 3), via enhanced protein expression. To these activation pathways, we have added a fourth, a pathway describing actual NO production from endothelial NO synthase and its various protein partners. These pathways were combined and simulated using CytoSolve, a computational environment for combining independent pathway calculations. The integrated model is able to describe the experimentally observed change in NO production with time after the application of fluid shear stress. This model can also be used to predict the specific effects on the system after interventional pharmacological or genetic changes. Importantly, this model reflects the up-to-date understanding of the NO system, providing a platform upon which information can be aggregated in an additive way.

Hemodynamic shear stress characteristic of atherosclerosis-resistant regions promotes glycocalyx formation in cultured endothelial cells.

The endothelial glycocalyx, a glycosaminoglycan layer located on the apical surface of vascular endothelial cells, has been shown to be important for several endothelial functions. Previous studies have documented that the glycocalyx is highly abundant in the mouse common carotid region, where the endothelium is exposed to laminar shear stress, and it is resistant to atherosclerosis. In contrast, the glycocalyx is scarce or absent in the mouse internal carotid sinus region, an area exposed to nonlaminar shear stress and highly susceptible to atherosclerosis. On the basis of these observations, we hypothesized that the expression of components of the endothelial glycocalyx is differentially regulated by distinct hemodynamic environments. To test this hypothesis, human endothelial cells were exposed to shear stress waveforms characteristic of atherosclerosis-resistant or atherosclerosis-susceptible regions of the human carotid, and the expression of several components of the glycocalyx was assessed. These experiments revealed that expression of several components of the endothelial glycocalyx is differentially regulated by distinct shear stress waveforms. Interestingly, we found that heparan sulfate expression is increased and evenly distributed on the apical surface of endothelial cells exposed to the atheroprotective waveform and is irregularly present in cells exposed to the atheroprone waveform. Furthermore, expression of a heparan sulfate proteoglycan, syndecan-1, is also differentially regulated by the two waveforms, and its suppression mutes the atheroprotective flow-induced cell surface expression of heparan sulfate. Collectively, these data link distinct hemodynamic environments to the differential expression of critical components of the endothelial glycocalyx.

The vascular origin of hematopoietic cells.

More than a century ago, several embryologists described sites of hematopoietic activity in the vascular wall of mid-gestation vertebrate embryos, and postulated the transient existence of a blood generating endothelium during ontogeny. This hypothesis gained significant attention in the 1970s when orthotopic transplantation experiments between quail and chick embryos revealed specific vascular areas as the site of the origin of definitive hematopoiesis. However, the vascular origin of hematopoietic precursors remained elusive and controversial for decades. Only recently, multiple experimental approaches have clearly documented that during vertebrate development definitive hematopoietic precursors arise from a subset of vascular endothelial cells. Interestingly, this differentiation is promoted by the intravascular fluid mechanical forces generated by the establishment of blood flow upon the initiation of heartbeat, and it is therefore connected with cardiovascular development in several critical aspects. In this review we present our current understanding of the relationship between vascular and definitive hematopoietic development through an historical analysis of the scientific evidence produced in this area of investigation.

The transcription factor KLF2 mediates hepatic endothelial protection and paracrine endothelial-stellate cell deactivation induced by statins.

BACKGROUND & AIMS: Statins improve hepatic endothelial function and liver fibrosis in experimental models of cirrhosis, thus they have been proposed as therapeutic options to ameliorate portal hypertension syndrome. The transcription factor Kruppel-like factor 2 (KLF2) may be induced by statins in liver sinusoidal endothelial cells (SEC), orchestrating an efficient vasoprotective response. The present study aimed at characterizing whether KLF2 mediates statins-derived hepatic protection. METHODS: Expression of KLF2 and its vasoprotective target genes was determined in SEC freshly isolated from control or CCl(4)-cirrhotic rats treated with four different statins (atorvastatin, mevastatin, simvastatin, and lovastatin), in the presence of mevalonate (or vehicle), under static or controlled shear stress conditions. KLF2-derived vasoprotective transcriptional programs were analyzed in SEC transfected with siRNA for KLF2 or siRNA-control, and incubated with simvastatin. Paracrine effects of SEC highly-expressing KLF2 on the activation status of rat and human hepatic stellate cells (HSC) were evaluated. RESULTS: Statins administration to SEC induced significant upregulation of KLF2 expression. KLF2 upregulation was observed after 6h of treatment and was accompanied by induction of its vasoprotective programs. Simvastatin vasoprotection was inhibited in the presence of mevalonate, and was magnified in cells cultured under physiological shear stress conditions. Statin-dependent induction of vasoprotective genes was not observed when KLF2 expression was muted with siRNA. SEC overexpressing KLF2 induced quiescence of HSC through a KLF2-nitric oxide-guanylate cyclase-mediated paracrine mechanism. CONCLUSIONS: Upregulation of hepatic endothelial KLF2-derived transcriptional programs by statins confers vasoprotection and stellate cells deactivation, reinforcing the therapeutic potential of these drugs for liver diseases that course with endothelial dysfunction.

Addition of simvastatin to cold storage solution prevents endothelial dysfunction in explanted rat livers.

UNLABELLED: Pathophysiological alterations in the endothelial phenotype result in endothelial dysfunction. Flow cessation, occurring during organ procurement for transplantation, triggers the endothelial dysfunction characteristic of ischemia/reperfusion injury, partly due to a reduction in the expression of the vasoprotective transcription factor Kruppel-like Factor 2 (KLF2). We aimed at (1) characterizing the effects of flow cessation and cold storage on hepatic endothelial phenotype, and (2) ascertaining if the consequences of cold stasis on the hepatic endothelium can be pharmacologically modulated, improving liver graft function. Expression of KLF2 and its vasoprotective programs was determined in (i) hepatic endothelial cells (HEC) incubated under cold storage conditions with or without the KLF2-inducer simvastatin, and (ii) rat livers not cold stored or preserved in cold University of Wisconsin solution (UWS) supplemented with simvastatin or its vehicle. In addition, upon warm reperfusion hepatic vascular resistance, endothelial function, nitric oxide vasodilator pathway, apoptosis, inflammation, and liver injury were evaluated in not cold stored livers or livers preserved in cold UWS supplemented with simvastatin or vehicle. Expression of KLF2 and its vasoprotective programs decrease in HEC incubated under cold storage conditions. Cold-stored rat livers exhibit a time-dependent decrease in KLF2 and its target genes, liver injury, increased hepatic vascular resistance, and endothelial dysfunction. The addition of simvastatin to the storage solution, maintained KLF2-dependent vasoprotective programs, prevented liver damage, inflammation, and oxidative stress and improved endothelial dysfunction. CONCLUSION: Our results provide a rationale to evaluate the beneficial effects of a vasoprotective preservation solution on human liver procurement for transplantation.

Endothelial cell dysfunction in cardiac disease: driver or consequence?

The vascular endothelium is a multifunctional cellular system which directly influences blood components and cells within the vessel wall in a given tissue. Importantly, this cellular interface undergoes critical phenotypic changes in response to various biochemical and hemodynamic stimuli, driving several developmental and pathophysiological processes. Multiple studies have indicated a central role of the endothelium in the initiation, progression, and clinical outcomes of cardiac disease. In this review we synthesize the current understanding of endothelial function and dysfunction as mediators of the cardiomyocyte phenotype in the setting of distinct cardiac pathologies; outline existing in vivo and in vitro models where key features of endothelial cell dysfunction can be recapitulated; and discuss future directions for development of endothelium-targeted therapeutics for cardiac diseases with limited existing treatment options.

Engineering microvascular networks using a KLF2 reporter to probe flow-dependent endothelial cell function.

Shear stress generated by the flow of blood in the vasculature is a potent regulator of endothelial cell phenotype and vascular structure. While vascular responses to flow are complex and context-dependent, endothelial cell signaling in response to shear stress induced by laminar flows is coordinated by the transcription factor KLF2. The expression of KLF2 in endothelial cells is associated with a quiescent, anti-inflammatory phenotype and has been well characterized in two-dimensional systems, but has not been studied in three-dimensional in vitro systems. Here we develop engineered microvascular networks (MVNs) with a KLF2-based endothelial cell sensor within a microfluidic chip, apply continuous flow using an attached microfluidic pump, and study the effects of this flow on vascular structure and function. We found that culture of MVNs exposed to flow for 48 hours that resulted in increased expression of the KLF2-GFP-reporter display larger vessel diameters and decreased vascular branching and resistance. Additionally, vessel diameters after the application of flow were independent of initial MVN morphologies. Finally, we found that MVNs exposed to flow have improved vascular barrier function and decreased platelet adhesion. The MVNs with KLF2-based flow sensors represent a powerful tool for evaluating the structural and functional effects of flow on engineered three-dimensional vascular systems.

Transcriptional profile of the rat cardiovascular system at single cell resolution.

Background: Despite the critical role of the cardiovascular system, our understanding of its cellular and transcriptional diversity remains limited. We therefore sought to characterize the cellular composition, phenotypes, molecular pathways, and communication networks between cell types at the tissue and sub-tissue level across the cardiovascular system of the healthy Wistar rat, an important model in preclinical cardiovascular research. We obtained high quality tissue samples under controlled conditions that reveal a level of cellular detail so far inaccessible in human studies. Methods and Results: We performed single nucleus RNA-sequencing in 78 samples in 10 distinct regions including the four chambers of the heart, ventricular septum, sinoatrial node, atrioventricular node, aorta, pulmonary artery, and pulmonary veins (PV), which produced an aggregate map of 505,835 nuclei. We identified 26 distinct cell types and additional subtypes, including a number of rare cell types such as PV cardiomyocytes and non-myelinating Schwann cells (NMSCs), and unique groups of vascular smooth muscle cells (VSMCs), endothelial cells (ECs) and fibroblasts (FBs), which gave rise to a detailed cell type distribution across tissues. We demonstrated differences in the cellular composition across different cardiac regions and tissue-specific differences in transcription for each cell type, highlighting the molecular diversity and complex tissue architecture of the cardiovascular system. Specifically, we observed great transcriptional heterogeneities among ECs and FBs. Importantly, several cell subtypes had a unique regional localization such as a subtype of VSMCs enriched in the large vasculature. We found the cellular makeup of PV tissue is closer to heart tissue than to the large arteries. We further explored the ligand-receptor repertoire across cell clusters and tissues, and observed tissue-enriched cellular communication networks, including heightened Nppa - Npr1/2/3 signaling in the sinoatrial node. Conclusions: Through a large single nucleus sequencing effort encompassing over 500,000 nuclei, we broadened our understanding of cellular transcription in the healthy cardiovascular system. The existence of tissue-restricted cellular phenotypes suggests regional regulation of cardiovascular physiology. The overall conservation in gene expression and molecular pathways across rat and human cell types, together with our detailed transcriptional characterization of each cell type, offers the potential to identify novel therapeutic targets and improve preclinical models of cardiovascular disease.

Fibroblast activation in response to TGFß1 is modulated by co-culture with endothelial cells in a vascular organ-on-chip platform.

Background: Tissue fibrosis is a major healthcare burden that affects various organs in the body for which no effective treatments exist. An underlying, emerging theme across organs and tissue types at early stages of fibrosis is the activation of pericytes and/or fibroblasts in the perivascular space. In hepatic tissue, it is well known that liver sinusoidal endothelial cells (EC) help maintain the quiescence of stellate cells, but whether this phenomenon holds true for other endothelial and perivascular cell types is not well studied. Methods: The goal of this work was to develop an organ-on-chip microvascular model to study the effect of EC co-culture on the activation of perivascular cells perturbed by the pro-fibrotic factor TGFß1. A high-throughput microfluidic platform, PREDICT96, that was capable of imparting physiologically relevant fluid shear stress on the cultured endothelium was utilized. Results: We first studied the activation response of several perivascular cell types and selected a cell source, human dermal fibroblasts, that exhibited medium-level activation in response to TGFß1. We also demonstrated that the PREDICT96 high flow pump triggered changes in select shear-responsive factors in human EC. We then found that the activation response of fibroblasts was significantly blunted in co-culture with EC compared to fibroblast mono-cultures. Subsequent studies with conditioned media demonstrated that EC-secreted factors play at least a partial role in suppressing the activation response. A Luminex panel and single cell RNA-sequencing study provided additional insight into potential EC-derived factors that could influence fibroblast activation. Conclusion: Overall, our findings showed that EC can reduce myofibroblast activation of perivascular cells in response to TGFß1. Further exploration of EC-derived factors as potential therapeutic targets in fibrosis is warranted.