Signaling In Endothelial Cells Research Articles

Betulinic acid accelerates diabetic wound healing by modulating hyperglycemia-induced oxidative stress, inflammation and glucose intolerance.

BackgroundDiabetes significantly delays wound healing through oxidative stress, inflammation and impaired re-epithelialization that lead to defective regulation of the healing process, although the related mechanism remains unclear. Here, we aim to investigate the potential role and mechanism for the beneficial effect of betulinic acid (BA) on diabetic wound healing.MethodsThe molecular effect of BA on hyperglycemia-mediated gene expression, oxidative stress, inflammation and glucose uptake was evaluated in endothelial, fibroblast and muscle cells. Burn injury was introduced to streptozotocin-induced diabetic rats and BA administration through either an intraperitoneal (IP) or topical (TOP) technique was used for wound treatment. Glucose tolerance was evaluated in both muscle tissue and fibroblasts, while oxidative stress and inflammation were determined in both the circulatory system and in wound tissues. The effect of BA on the wound healing process was also evaluated.ResultsBA treatment reversed hyperglycemia-induced glucose transporter type 4 (GLUT4) suppression in both muscle and fibroblast cells. This treatment also partly reversed hyperglycemia-mediated suppression of endothelial nitric oxide synthase (eNOS), nuclear factor erythroid 2-related factor 2 (Nrf2) signaling and nuclear factor NFκB p65 subunit (NFκB p65) activation in endothelial cells. An in vivo rat study showed that BA administration ameliorated diabetes-mediated glucose intolerance and partly attenuated diabetes-mediated oxidative stress and inflammation in both the circulatory system and wound tissues. BA administration by both IP and TOP techniques significantly accelerated diabetic wound healing, while BA administration by either IP or TOP methods alone had a significantly lower effect.ConclusionsBA treatment ameliorates hyperglycemia-mediated glucose intolerance, endothelial dysfunction, oxidative stress and inflammation. Administration of BA by both IP and TOP techniques was found to significantly accelerate diabetic wound healing, indicating that BA could be a potential therapeutic candidate for diabetic wound healing.

Effect of growth factors present in serum on insulin resistance and endothelial dysfunction in endothelial cells

Insulin Resistance is a pathophysiological function of Type II Diabetes Mellitus which can be comprehended by quantifying the parameters critical in the insulin signaling pathway. Serum has a profound role in evaluating cellular growth and metabolism in vitro. The growth factors present in serum such as IGF, EGF, FGF affects the regulatory components of insulin signaling pathway that leads to insulin resistance. This study explores the effect of growth factors present in Fetal Bovine Serum (FBS) in insulin signaling and endothelium regulation in endothelial cells (Ea. hyp926). The dose-dependent and time-dependent treatment of FBS on the cells displayed changes that were detected by MTT and 2-NBDG assays for assessing cell viability and glucose uptake. Spectrophotometric analysis of nitric oxide (NO) and lactate dehydrogenase (LDH) determined vascular homeostasis and no cytotoxic effects of serum, respectively, in endothelial cells. These findings indicate that FBS at higher levels could possibly lead to loss of NO activity which in turn could impair endothelium-mediated dilation. The inhibition of enzymatic activity of eNOS may activate the release of LDH in endothelial cells. In conclusion, our findings indicate a specific concentration of serum enhances insulin signaling and endothelium cell regulation by modulating glucose uptake and NO production.

Suppression of BMP signaling by PHD2 deficiency in Pulmonary Arterial hypertension.

BMP signaling deficiency is evident in the lungs of patients with pulmonary arterial hypertension. We demonstrated that PHD2 deficiency suppresses BMP signaling in the lung endothelial cells, suggesting the novel mechanisms of dysregulated BMP signaling in the development of pulmonary arterial hypertension.

AICAR Protects Vascular Endothelial Cells from Oxidative Injury Induced by the Long-Term Palmitate Excess.

Hyperlipidemia manifested by high blood levels of free fatty acids (FFA) and lipoprotein triglycerides is critical for the progression of type 2 diabetes (T2D) and its cardiovascular complications via vascular endothelial dysfunction. However, attempts to assess high FFA effects in endothelial culture often result in early cell apoptosis that poorly recapitulates a much slower pace of vascular deterioration in vivo and does not provide for the longer-term studies of endothelial lipotoxicity in vitro. Here, we report that palmitate (PA), a typical FFA, does not impair, by itself, endothelial barrier and insulin signaling in human umbilical vein endothelial cells (HUVEC), but increases NO release, reactive oxygen species (ROS) generation, and protein labeling by malondialdehyde (MDA) hallmarking oxidative stress and increased lipid peroxidation. This PA-induced stress eventually resulted in the loss of cell viability coincident with loss of insulin signaling. Supplementation with 5-aminoimidazole-4-carboxamide-riboside (AICAR) increased endothelial AMP-activated protein kinase (AMPK) activity, supported insulin signaling, and prevented the PA-induced increases in NO, ROS, and MDA, thus allowing to maintain HUVEC viability and barrier, and providing the means to study the long-term effects of high FFA levels in endothelial cultures. An upgraded cell-based model reproduces FFA-induced insulin resistance by demonstrating decreased NO production by vascular endothelium.

The Methyltransferase Smyd1 Mediates LPS-Triggered Up-Regulation of IL-6 in Endothelial Cells.

The lysine methyltransferase Smyd1 with its characteristic catalytic SET-domain is highly enriched in the embryonic heart and skeletal muscles, participating in cardiomyogenesis, sarcomere assembly and chromatin remodeling. Recently, significant Smyd1 levels were discovered in endothelial cells (ECs) that responded to inflammatory cytokines. Based on these biochemical properties, we hypothesized that Smyd1 is involved in inflammation-triggered signaling in ECs and therefore, investigated its role within the LPS-induced signaling cascade. Human endothelial cells (HUVECs and EA.hy926 cells) responded to LPS stimulation with higher intrinsic Smyd1 expression. By transfection with expression vectors containing gene inserts encoding either intact Smyd1, a catalytically inactive Smyd1-mutant or Smyd1-specific siRNAs, we show that Smyd1 contributes to LPS-triggered expression and secretion of IL-6 in EA.hy926 cells. Further molecular analysis revealed this process to be based on two signaling pathways: Smyd1 increased the activity of NF-κB and promoted the trimethylation of lysine-4 of histone-3 (H3K4me3) within the IL-6 promoter, as shown by ChIP-RT-qPCR combined with IL-6-promoter-driven luciferase reporter gene assays. In summary, our experimental analysis revealed that LPS-binding to ECs leads to the up-regulation of Smyd1 expression to transduce the signal for IL-6 up-regulation via activation of the established NF-κB pathway as well as via epigenetic trimethylation of H3K4.

FGD5 regulates endothelial cell PI3 kinase-β to promote neo-angiogenesis.

Angiogenesis is required in embryonic development and tissue repair in the adult. Vascular endothelial growth factor (VEGF) initiates angiogenesis, and VEGF or its receptor is targeted therapeutically to block pathological angiogenesis. Additional pro-angiogenic cues, such as CXCL12 acting via the CXCR4 receptor, co-operate with VEGF/VEGFR2 to cue vascular patterning. We studied the role of FGD5, an endothelial Rho GTP/GDP exchange factor (RhoGEF), to regulate CXCR4-dependent signals in the endothelial cell (EC). Patient-derived renal cell carcinomas produce a complex milieu of growth factors that stimulated sprouting angiogenesis and endothelial tip cell differentiation ex vivo that was blocked by EC FGD5loss. In a simplified model, CXCL12 augmented sprouting and tip gene expression under conditions where VEGF was limiting. CXCL12-stimulated tip cell differentiation was dependent on PI3kinase (PI3K)-β activity. Knockdown of EC FGD5 abolished CXCR4signaling to PI3K-β and Akt. Further, inhibition of Rac1, a Rho GTPase required for PI3K-β activity, recapitulated the signaling defects of FGD5 deficiency, suggesting that FGD5may regulate PI3K-β activity through Rac1. Overexpression of a RhoGEF deficient, Dbl domain-deleted FGD5mutant reduced CXCL12-stimulated Akt phosphorylation and failed to rescue PI3K signaling in native FGD5-deficient EC, indicating that FGD5 RhoGEF activity is required for FDG5 function. Endothelial expression of mutant PI3K-β with an inactivated Rho binding domain confirmed that CXCL12-stimulated PI3K activity in EC requires Rac1-GTP co-regulation. Together, this data identify the role of FGD5 to generate Rac1-GTP to regulate pro-angiogenic CXCR4-dependent PI3K-β signaling in EC. Inhibition of FGD5 activity may complement current angiogenesis inhibitor drugs.

Vascular Endothelial Growth Factor Signaling in Models of Oxygen-Induced Retinopathy: Insights Into Mechanisms of Pathology in Retinopathy of Prematurity.

Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide. Blindness can occur from retinal detachment caused by pathologic retinal angiogenesis into the vitreous, termed intravitreal neovascularization (IVNV). Although agents that interfere with the bioactivity of vascular endothelial growth factor (VEGF) are now used to treat IVNV, concerns exist regarding the identification of optimal doses of anti-VEGF for individual infants and the effect of broad VEGF inhibition on physiologic angiogenesis in external organs or in the retina of a preterm infant. Therefore, it is important to understand VEGF signaling in both physiologic and pathologic angiogenesis in the retina. In this manuscript, we review the role of receptors that interact with VEGF in oxygen-induced retinopathy (OIR) models that represent features of ROP pathology. Specifically, we discuss our work regarding the regulation of VEGFR2 signaling in retinal endothelial cells to not only reduce severe ROP but also facilitate physiologic retinal vascular and neuronal development.

STAT3-YAP/TAZ signaling in endothelial cells promotes tumor angiogenesis.

The nuclear translocation and activity of the cotranscriptional activators YAP and TAZ (YAP/TAZ) in endothelial cells (ECs) are crucial during developmental angiogenesis. Here, we studied the role of YAP/TAZ signaling in ECs in tumor angiogenesis and found that the expression of YAP/TAZ and downstream target genes in ECs correlated with tumor vascularization in human colorectal carcinomas and skin melanoma. Treatment with the YAP/TAZ inhibitor verteporfin reduced vessel density and tumor progression in a mouse colorectal cancer (CRC) model. Conditional deletion of YAP/TAZ in ECs reduced tumor angiogenesis and growth in a mouse B16-F10 melanoma model. Using cultured ECs and mice with EC-specific ablation, we showed that signal transducer and activator of transcription 3 (STAT3) was required for the activation of YAP/TAZ in tumor-associated ECs. Moreover, we showed that STAT3-mediated signaling promoted YAP/TAZ activity and that the nuclear shuttling machinery for STAT3 was also required for YAP/TAZ nuclear translocation. Together, our data highlight the role of YAP/TAZ as critical players in ECs during tumor angiogenesis and provide insight into the signaling pathways leading to their activation.

Potentiation of polyunsaturated fatty acids anti-inflammatory action through redox signaling in fructose-treated endothelial cells

Potentiation of polyunsaturated fatty acids anti-inflammatory action through redox signaling in fructose-treated endothelial cells

Neuropilin 1 regulates bone marrow vascular regeneration and hematopoietic reconstitution

Ionizing radiation and chemotherapy deplete hematopoietic stem cells and damage the vascular niche wherein hematopoietic stem cells reside. Hematopoietic stem cell regeneration requires signaling from an intact bone marrow (BM) vascular niche, but the mechanisms that control BM vascular niche regeneration are poorly understood. We report that BM vascular endothelial cells secrete semaphorin 3 A (SEMA3A) in response to myeloablation and SEMA3A induces p53 – mediated apoptosis in BM endothelial cells via signaling through its receptor, Neuropilin 1 (NRP1), and activation of cyclin dependent kinase 5. Endothelial cell – specific deletion of Nrp1 or Sema3a or administration of anti-NRP1 antibody suppresses BM endothelial cell apoptosis, accelerates BM vascular regeneration and concordantly drives hematopoietic reconstitution in irradiated mice. In response to NRP1 inhibition, BM endothelial cells increase expression and secretion of the Wnt signal amplifying protein, R spondin 2. Systemic administration of anti - R spondin 2 blocks HSC regeneration and hematopoietic reconstitution which otherwise occurrs in response to NRP1 inhibition. SEMA3A – NRP1 signaling promotes BM vascular regression following myelosuppression and therapeutic blockade of SEMA3A – NRP1 signaling in BM endothelial cells accelerates vascular and hematopoietic regeneration in vivo.

Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage

Background and purposeMitochondria play a central role in the host response to viral infection and immunity, being key to antiviral signaling and exacerbating inflammatory processes. Mitochondria and Toll-like receptor (TLR) have been suggested as potential targets in SARS-CoV-2 infection. However, the involvement of TLR9 in SARS-Cov-2-induced endothelial dysfunction and potential contribution to cardiovascular complications in COVID-19 have not been demonstrated. This study determined whether infection of endothelial cells by SARS-CoV-2 affects mitochondrial function and induces mitochondrial DNA (mtDNA) release. We also questioned whether TLR9 signaling mediates the inflammatory responses induced by SARS-CoV-2 in endothelial cells. Experimental approachHuman umbilical vein endothelial cells (HUVECs) were infected by SARS-CoV-2 and immunofluorescence was used to confirm the infection. Mitochondrial function was analyzed by specific probes and mtDNA levels by real-time polymerase chain reaction (RT-PCR). Inflammatory markers were measured by ELISA, protein expression by western blot, intracellular calcium (Ca2+) by FLUOR-4, and vascular reactivity with a myography. Key resultsSARS-CoV-2 infected HUVECs, which express ACE2 and TMPRSS2 proteins, and promoted mitochondrial dysfunction, i.e. it increased mitochondria-derived superoxide anion, mitochondrial membrane potential, and mtDNA release, leading to activation of TLR9 and NF-kB, and release of cytokines. SARS-CoV-2 also decreased nitric oxide synthase (eNOS) expression and inhibited Ca2+ responses in endothelial cells. TLR9 blockade reduced SARS-CoV-2-induced IL-6 release and prevented decreased eNOS expression. mtDNA increased vascular reactivity to endothelin-1 (ET-1) in arteries from wild type, but not TLR9 knockout mice. These events were recapitulated in serum samples from COVID-19 patients, that exhibited increased levels of mtDNA compared to sex- and age-matched healthy subjects and patients with comorbidities. Conclusion and applicationsSARS-CoV-2 infection impairs mitochondrial function and activates TLR9 signaling in endothelial cells. TLR9 triggers inflammatory responses that lead to endothelial cell dysfunction, potentially contributing to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathways may help to define novel therapeutic strategies for COVID-19.

Abstract 11462: A Surprising Role of TNIK in Regulating Endothelial Interferon Signaling and Activation

Background: Endothelial cells (ECs) predominantly govern 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. Yet, 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 and EC activation. Hypothesis: TNIK regulates IFNs through focal adhesion kinase (FAK) mediated STAT signaling in EC activation (Fig 1A). 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 then RNA-sequencing was performed. 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 led to inhibition IFN (-log P-value >11) and upregulation of eukaryotic transcription elongation factor signaling (eIF2, -log P-value >7) (Fig 1B, C). In addition, under DF, TNIK depletion led to downregulation of hypercytokinemia/hyperchemokinemia (-log P-value >8, Fig 1B). Validation through western blotting and qRT-PCR (Fig 1D, E) confirmed that TNIK regulates FAK and STAT phosphorylation and IFN stimulated gene targets. Conclusions: Our study elucidates the unique role of TNIK in regulating FAK-STAT mediated IFN signaling and subsequently EC activation.

Abstract 13033: Cavin/Caveolin Interaction Modulates BMP/Smad Signaling in Pulmonary Artery Endothelial Cells

Background: Pulmonary hypertension (PH) is a progressive disease associated with poor prognosis. Caveolin-1 (Cav1) and Cavin-1 are components of caveolae, and both interact with each other and influence the composition and stabilization of caveolae. Cav1 is identified as a related gene of pulmonary arterial hypertension (PAH). Gene mutation of bone morphogenetic protein type II receptor (BMPRII) is the most common cause of PAH. BMPRII is localized in caveolae and associates with Cav1. However, the role of the Caveolin-Cavin system on the BMP/Smad signaling and the PAH progression has not been well-known. Methods and Results: As in previous reports, Cav1 knockout mice exhibited PH with pulmonary vascular remodeling and right ventricular hypertrophy. Caveolae disappeared in pulmonary microvascular endothelial cells of Cav1 knockout mice. We then examined the role of Caveolin-Cavin and the association with BMP/Smad signaling in human pulmonary artery endothelial cells (hPAECs). Cav1 knockdown in hPAECs reduced BMPRII at the plasma membrane and Smad 1/5/9 phosphorylation. Cav1 knockdown also significantly increased hypoxia-induced apoptosis in hPAECs. Cav1 was associated with BMPRII at the membrane of hPAECs. In addition, Cavin-1 inhibited the interaction of BMPRII with Cav1 and reduced BMPRII localization on the membrane of hPAECs. Cavin-1 knockdown reversed BMPRII localization at the plasma membrane and Smad 1/5/9 phosphorylation induced by Cav1 knockdown. Both Cavin-1 and BMPRII were associated with the Cav1 scaffolding domain. These results suggest that Cavin-1 interacts with Cav1 and inhibits the association of Cav1 with BMPRII, resulting in modulating Smad signaling pathway. Conclusions: Cavin-1 and BMPRII are competitively associated with Cav1 at the plasma membrane and modulating BMP/Smad signal transduction in PAECs. Our findings reveal a previously undescribed mechanism via Cavin-Caveolin interaction in BMP/Smad signaling-modulated PH development.

Abstract 9196: Innocuous Levels of Pulmonary Thrombosis Protect Against Inflammatory Lung Injury via Endothelial Arachidonate 15-Lipoxygenase and Hypoxia-Inducible Factor 1α

Introduction: Sepsis patients commonly suffer from lung thrombosis and sepsis-induced acute lung injury (ALI), but clinical trials of anti-coagulants in sepsis and ALI have failed, and the impact of varying severities of lung thrombosis on ALI is unknown. Hypothesis: Pulmonary thrombosis regulates the severity of sepsis-induced ALI through modulation of pulmonary endothelial cell (EC) viability. In this study, we aimed to determine the impact of varying levels of lung thrombosis on ALI and to delineate the underlying mechanisms. Methods: We assessed inflammatory lung injury in mouse models of pulmonary thrombosis combined with sepsis-induced lung injury and thrombocytopenia. We also used RNA sequencing and nanoparticle-mediated EC-targeted gene editing to delineate and target thrombosis-induced EC signaling pathways. Results: In wild type mice, excessive increases in lung thrombosis worsened ALI, but the induction of a mild and innocuous level of lung thrombosis instead reduced ALI and mortality. We then showed that this mild level of lung thrombosis increases the expression of multiple factors that regulate cell survival, including arachidonate 15-lipoxygenase (Alox15) and hypoxia-inducible factor (HIF) 1α in ECs. Next, we used nanoparticle-mediated EC gene editing to show that the protective impact of mild lung thrombosis on ALI is dependent upon EC-specific Alox15 and HIF1α. From a therapeutic perspective, the mild level of lung thrombosis could be induced up to 8 hours after sepsis and still maintain its protective impact on ALI. Furthermore, the induction of mild lung thrombosis completely prevented thrombocytopenia-induced increases and ALI and mortality. Conclusions: Mild levels of lung thrombosis protect against inflammatory lung injury via EC Alox15 and HIF1α. Promoting lung EC survival through Alox15 or HIF1α signaling represents a promising therapeutic strategy against ALI.

Computational model of brain endothelial cell signaling pathways predicts therapeutic targets for cerebral pathologies

Brain endothelial cells serve many critical homeostatic functions. In addition to sensing and regulating blood flow, they maintain blood-brain barrier function, including precise control of nutrient exchange and efflux of xenobiotics. Many signaling pathways in brain endothelial cells have been implicated in both health and disease; however, our understanding of how these signaling pathways functionally integrate is limited. A model capable of integrating these signaling pathways could both advance our understanding of brain endothelial cell signaling networks and potentially identify promising molecular targets for endothelial cell-based drug or gene therapies. To this end, we developed a large-scale computational model, wherein brain endothelial cell signaling pathways were reconstructed from the literature and converted into a network of logic-based differential equations. The model integrates 63 nodes (including proteins, mRNA, small molecules, and cell phenotypes) and 82 reactions connecting these nodes. Specifically, our model combines signaling pathways relating to VEGF-A, BDNF, NGF, and Wnt signaling, in addition to incorporating pathways relating to focused ultrasound as a therapeutic delivery tool. To validate the model, independently established relationships between selected inputs and outputs were simulated, with the model yielding correct predictions 73% of the time. We identified influential and sensitive nodes under different physiological or pathological contexts, including altered brain endothelial cell conditions during glioma, Alzheimer's disease, and ischemic stroke. Nodes with the greatest influence over combinations of desired model outputs were identified as potential druggable targets for these disease conditions. For example, the model predicts therapeutic benefits from inhibiting AKT, Hif-1α, or cathepsin D in the context of glioma – each of which are currently being studied in clinical or pre-clinical trials. Notably, the model also permits testing multiple combinations of node alterations for their effects on the network and the desired outputs (such as inhibiting AKT and overexpressing the P75 neurotrophin receptor simultaneously in the context of glioma), allowing for the prediction of optimal combination therapies. In all, our approach integrates results from over 100 past studies into a coherent and powerful model, capable of both revealing network interactions unapparent from studying any one pathway in isolation and predicting therapeutic targets for treating devastating brain pathologies.

Piezo1 is a mechanosensor channel in CNS capillaries

Cerebral blood flow (CBF) is exquisitely controlled to meet the ever-changing demands of active neurons in the brain. Brain capillaries are equipped with sensors of neurovascular coupling agents released from neurons/astrocytes onto the outer wall of a capillary. While capillaries can translate external signals into electrical and Ca2+ changes, control mechanisms from the lumen are less clear. The continuous flux of red blood cells and plasma through narrow-diameter capillaries imposes mechanical forces on the luminal (inner) capillary wall. Whether-and, if so, how-the ever-changing CBF could be mechanically sensed in capillaries is not known. Here, we propose and provide evidence that the mechanosensitive Piezo1 channels operate as mechanosensors in CNS capillaries to ultimately regulate CBF. Patch clamp electrophysiology confirmed the expression and function of Piezo1 channels in brain cortical and retinal capillary endothelial cells. Mechanical or pharmacological activation of Piezo1 channels evoked currents that were sensitive to Piezo1 channel blockers. Using genetically encoded Ca2+ indicator (Cdh5-GCaMP8) mice, we observed that Piezo1 channel activation triggered Ca2+ signals in endothelial cells. An ex vivo pressurized retina preparation was employed to further explore the mechanosensitivity of capillary Piezo1-mediated Ca2+ signals. Genetic and pharmacologic manipulation of Piezo1 in endothelial cells had significant impacts on CBF, reemphasizing the crucial role of mechanosensation in blood flow control. In conclusion, this study shows that Piezo1 channels act as mechanosensors in capillaries, and that these channels initiate crucial Ca2+ signals. We further show that Piezo1 modulates CBF, an observation of profound significance for the control of brain blood flow in health and in disorders where hemodynamic forces are disrupted, such as hypertension.

Growth Arrest Specific-6 and Axl Coordinate Inflammation and Hypertension.

[Figure: see text].

PEDF is an endogenous inhibitor of VEGF-R2 angiogenesis signaling in endothelial cells

Pigment epithelium derived factor (PEDF), an endogenous inhibitor of angiogenesis, targets the growth of aberrant blood vessels in many tissues, including the eye. In this study we show that PEDF prevented early mitogenic signals of vascular endothelial growth factor (VEGF-A) in primate retinal endothelial cells, blocking proliferation, migration and tube formation. PEDF inhibited the phosphorylation and activation of five major downstream VEGF-A signaling partners, namely phosphoinositide-3-OH Kinase (PI3K), AKT, FAK, Src (Y416), and PLC-γ. It did so by binding to the extracellular domain of VEGF-R2, blocking VEGF-A-induced tyrosine phosphorylation (Tyr 951 and Tyr 1175), and inhibiting VEGF-R2 receptor kinase activity. PEDF had no effect on the transcription or translation of VEGF-R2 in cultured HUVECs. PEDF also bound to the extracellular domain of VEGF-R1. We conclude that PEDF blocks the growth of new blood vessels, in part, by reducing VEGF-A activation of its key mitogenic receptor, VEGF-R2, and by preventing its downstream signals in endothelial cells.

Identification of Endothelial Proteins in Plasma Associated With Cardiovascular Risk Factors.

Endothelial cell (EC) dysfunction is a well-established response to cardiovascular disease risk factors, such as smoking and obesity. Risk factor exposure can modify EC signaling and behavior, leading to arterial and venous disease development. Here, we aimed to identify biomarker panels for the assessment of EC dysfunction, which could be useful for risk stratification or to monitor treatment response. Approach and Results: We used affinity proteomics to identify EC proteins circulating in plasma that were associated with cardiovascular disease risk factor exposure. Two hundred sixteen proteins, which we previously predicted to be EC-enriched across vascular beds, were measured in plasma samples (N=1005) from the population-based SCAPIS (Swedish Cardiopulmonary Bioimage Study) pilot. Thirty-eight of these proteins were associated with body mass index, total cholesterol, low-density lipoprotein, smoking, hypertension, or diabetes. Sex-specific analysis revealed that associations predominantly observed in female- or male-only samples were most frequently with the risk factors body mass index, or total cholesterol and smoking, respectively. We show a relationship between individual cardiovascular disease risk, calculated with the Framingham risk score, and the corresponding biomarker profiles. EC proteins in plasma could reflect vascular health status.

Tie2 activation protects against prothrombotic endothelial dysfunction in COVID-19.

Endothelial dysfunction accompanies the microvascular thrombosis commonly observed in severe COVID-19. Constitutively, the endothelial surface is anticoagulant, a property maintained at least in part via signaling through the Tie2 receptor. During inflammation, the Tie2 antagonist angiopoietin-2 (Angpt-2) is released from endothelial cells and inhibits Tie2, promoting a prothrombotic phenotypic shift. We sought to assess whether severe COVID-19 is associated with procoagulant endothelial dysfunction and alterations in the Tie2/angiopoietin axis. Primary HUVECs treated with plasma from patients with severe COVID-19 upregulated the expression of thromboinflammatory genes, inhibited the expression of antithrombotic genes, and promoted coagulation on the endothelial surface. Pharmacologic activation of Tie2 with the small molecule AKB-9778 reversed the prothrombotic state induced by COVID-19 plasma in primary endothelial cells. Lung autopsies from patients with COVID-19 demonstrated a prothrombotic endothelial signature. Assessment of circulating endothelial markers in a cohort of 98 patients with mild, moderate, or severe COVID-19 revealed endothelial dysfunction indicative of a prothrombotic state. Angpt-2 concentrations rose with increasing disease severity, and the highest levels were associated with worse survival. These data highlight the disruption of Tie2/angiopoietin signaling and procoagulant changes in endothelial cells in severe COVID-19. Our findings provide rationale for current trials of Tie2-activating therapy with AKB-9778 in COVID-19.