Endothelial-specific Knockout Research Articles

Role of Erythropoietin Receptor Signaling in Macrophages or Choroidal Endothelial Cells in Choroidal Neovascularization.

Erythropoietin (EPO) has been proposed to reduce the progression of atrophic age-related macular degeneration (AMD) due to its potential role in neuroprotection. However, overactive EPO receptor (EPOR) signaling increased laser-induced choroidal neovascularization (CNV) and choroidal macrophage number in non-lasered mice, which raised the question of whether EPOR signaling increased CNV through the recruitment of macrophages to the choroid that released pro-angiogenic factors or through direct angiogenic effects on endothelial cells. In this study, we addressed the hypothesis that EPOR signaling increased CNV by direct effects on macrophages or endothelial cells. We used tamoxifen-inducible macrophage-specific or endothelial cell-specific EPOR knockout mice in the laser-induced CNV model, and cultured choroidal endothelial cells isolated from adult human donors. We found that macrophage-specific knockout of EPOR influenced laser-induced CNV in females only, whereas endothelial-specific knockout of EPOR reduced laser-induced CNV in male mice only. In cultured human choroidal endothelial cells, knockdown of EPOR reduced EPO-induced signal transducer and activator of transcription 3 (STAT3) activation. Taken together, our findings suggest that EPOR signaling in macrophages or choroidal endothelial cells regulates the development of CNV in a sex-dependent manner. Further studies regarding the role of EPO-induced signaling are required to assess EPO safety and to select or develop appropriate therapeutic approaches.

High fructose induces dysfunctional vasodilatation via PP2A-mediated eNOS Ser1177 dephosphorylation

BackgroundProcessed foods are popular and contain large amounts of industrial fructose, which changes people’s diet and exacerbates the negative health effects of high fructose. Several studies have shown that excessive intake of fructose has a major impact on vascular disease. However, the mechanism of the effect of high fructose on blood vessels is currently unclear.MethodsThe effect of fructose on the vasodilatation of isolated thoracic aortic rings was observed by using wire myography in wild-type (WT) mice. Cell viability and nitric oxide (NO) production were assessed by the corresponding kits in mouse vascular endothelial cells. The effect of fructose on endothelial nitric oxide synthase (eNOS) and protein phosphatase 2A (PP2A) and their changes in phosphorylation were detected by using Western blots. Moreover, a PP2A inhibitor (okadaic acid, OA) was used to evaluate the relationship between fructose and PP2A. Furthermore, PP2ACα endothelial-specific knockout (PP2A cKO) mice were used to detect the vasodilatation of in vitro fructose-incubated thoracic aortic rings by using wire myography.ResultsHigh fructose induced endothelium-dependent dysfunctional vasodilatation. High fructose reduced acetylcholine (Ach)-induced vasodilation but did not affect sodium nitroprusside (SNP)-induced vasodilation. Accordingly, NO production and the phosphorylation level of eNOS at serine (Ser) 1177 (P-eNOS) in vascular endothelial cells were remarkably reduced without changes in cell viability. The expression of protein phosphatase 2A catalytic subunit (PP2AC) was increased and the expression of phosphorylated PP2AC (P-PP2A, tyrosine [Tyr] 307) was significantly decreased. Nevertheless, these effects were reversed by OA. Moreover, knockout of the PP2A gene could recover the response of vessels to Ach under high fructose stimulation.ConclusionsOur observations demonstrate an underlying mechanism of fructose-induced dysfunctional vasodilatation. Fructose could activate PP2A, which leads to decrease in the phosphorylation of eNOS at Ser1177 and the reduction of NO release, thus leading to the occurrence of endothelium-dependent dysfunctional vasodilatation.

Abstract WMP76: Nogo-B Receptor Is Essential For Preserving HBO1-mediated Histone Acetylation In Brain Endothelial Cells And Preventing Cerebrovascular Hemorrhage

Cerebral cavernous malformations (CCMs) are enlarged leaking vascular lesions in the brain caused by loss-of-function mutations in CCM1/2/3 genes or loss of expression. Although we previously showed that Nogo-B receptor (NgBR) knockout in endothelial cells (ECs) results in CCMs-like cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NgBR regulates CCM1/2 expression has not been elucidated. Here, we show that temporal genetic depletion of NgBR in ECs at both the postnatal and adult stages results in CCM1/2 expression deficiency and consequently CCMs-like lesions such as enlarged vessels, blood-brain barrier (BBB) hyperpermeability, and intracerebral hemorrhage. These cerebrovascular defects in the brain of NgBR endothelial-specific knockout (ecKO) mice can be rescued by adeno-associated virus (AAV)-mediated overexpression of CCM1 and CCM2 genes. To reveal the molecular mechanism, we used RNA-seq analysis to examine changes in the transcriptome. Surprisingly, we found that acetyltransferase HBO1 was downregulated in NgBR deficient ECs. The mechanistic study elucidated that NgBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NgBR impairs the binding of both the HBO1 and acetylated H4K5/K12 at the promoter of CCM1 and CCM2 genes. Similarly, AAV-mediated overexpression of HBO1 restores the acetylation of H4K5/K12 and rescues the CCMs-like cerebrovascular defects in the brain of NgBR ecKO mice. Our findings on epigenetic regulation of CCM1 and CCM2 provide a perspective that NgBR and HBO1-mediated histone H4 acetylation may be targeted for preventing the onset of CCMs-like cerebrovascular disease.

Genetic Deletion of Vascular Endothelial Growth Factor Receptor 2 in Endothelial Cells Leads to Immediate Disruption of Tumor Vessels and Aggravation of Hypoxia

Vascular endothelial growth factor (VEGF) blockers are used widely in clinics to target various types of human cancer. Although VEGF blockers exert marked tumor suppressive effects, the therapeutic effects can be limited. Moreover, accumulating evidence shows that VEGF acts not just on endothelial cells but also on various nonendothelial cells, including tumor and immune cells, suggesting a need to revisit the bona fide action of VEGF on endothelial cells using specific genetic mouse models. Herein, tamoxifen-inducible endothelial-specific knockout mice lacking VEGF receptor 2 (Vegfr2), the major signal transducer for VEGF, were used. The initial event resulting from cessation of endothelial Vegfr2 signaling was vascular truncation and fragmentation, rather than maturation of abnormalized vessels. Although deletion of endothelial Vegfr2 suppressed intratumor hemorrhage, it enhanced hypoxia in tumor cells and reduced the number of infiltrating cytotoxic T cells, suggesting a profound reduction in intratumor blood flow. In various tissues, deletion of endothelial Vegfr2 induced regression of healthy capillaries in intestinal villi, substantiating intestinal perforation, which is one of the most common adverse effects of VEGF blockade in humans. Overall, the data suggest that some of the known effects of VEGF blockers on tumor vessels are caused by partial cessation of VEGF signaling, or by actions on nonendothelial cells. The results increasethe understanding of the mechanisms underlying anti-angiogenic therapy.

Abstract 13476: ROR2 Deficiency is Associated With Endothelial Dysfunction and Reduced Angiogenesis in Pulmonary Arterial Hypertension

Introduction: Pulmonary arterial hypertension (PAH) is characterized by endothelial dysfunction and progressive loss of lung microvessels. Our group has shown that Wnt pathways are critical for pulmonary angiogenesis but the role of the Wnt/planar cell polarity specific receptor ROR2 in lung endothelial angiogenic responses is unknown. Hypothesis: We hypothesized that ROR2 is required for proper angiogenic responses in pulmonary microvascular endothelial cells (PMVECs) and its loss contributes to endothelial dysfunction in PAH. Methods and Results: Analysis of PAH lung tissues demonstrated reduced ROR2 expression within vascular lesions and in explanted PMVECs. ROR2 deficiency was associated with reduced proliferation and tube formation in response to VEGF-A, a major inducer of angiogenesis. We found that ROR2 promotes VEGF receptor 2 activation via direct phosphorylation of cytoplasmic residues (Y1175/Y951) and that restoring ROR2 expression in PAH PMVECs led to improved angiogenic responses. Besides angiogenic sprouting, we found that ROR2 is required for lung endothelial barrier formation by promoting the assembly of mature focal adhesions and adherens junctions through activation of Src and focal adhesion kinase (FAK). Compared to wild-type controls, endothelial-specific ROR2 knockout (ROR2 ECKO) mice demonstrated more severe hemodynamics, small vessel loss, and muscularization in chronic hypoxia. Moreover, ROR2 ECKO demonstrated severe RV dysfunction associated with reduced capillary density. We validated these findings in samples of decompensated RV tissue from PAH patients, monocrotaline, and Sugen-hypoxia rats which also demonstrated a significant reduction of ROR2 expression that correlated with RV capillary loss. Conclusion: ROR2 plays a protective role in the pulmonary and RV circulations and its loss prevents vessel repair in PAH. Targeting ROR2 could promote recovery of small vessel loss and RV adaptation in PAH.

Abstract 13881: Nogo-B Receptor is Essential for Preserving Histone Acetylation-Mediated CCM1/2 Expression in Endothelial Cells and Preventing Cerebrovascular Hemorrhage

Cerebral cavernous malformations (CCMs) are enlarged leaking vascular lesions in the brain caused by loss-of-function mutations in CCM1/2/3 genes or loss of expression. Although we previously showed that Nogo-B receptor (NgBR) knockout in endothelial cells (ECs) results in CCMs-like cerebrovascular lesions in the mouse embryo, the molecular mechanism by which NgBR regulates CCM1/2 expression has not been elucidated. Here, we show that temporal genetic depletion of NgBR in ECs at both the postnatal and adult stages results in CCM1/2 expression deficiency and consequently CCMs-like lesions such as enlarged vessels, blood-brain barrier (BBB) hyperpermeability, and cerebral hemorrhage. These cerebrovascular defects in the brain of NgBR endothelial-specific knockout (ecKO) mice can be rescued by adeno-associated virus (AAV)-mediated overexpression of CCM1 and CCM2 genes. To reveal the molecular mechanism, we used RNA-seq analysis to examine changes in the transcriptome. Surprisingly, we found that acetyltransferase HBO1 was downregulated in NgBR deficient ECs. The mechanistic study elucidated that NgBR is required for maintaining the expression of CCM1/2 in ECs via HBO1-mediated histone acetylation. ChIP-qPCR data further demonstrated that loss of NgBR impairs the binding of both the HBO1 and acetylated H4K5/K12 at the promoter of CCM1 and CCM2 genes. Similarly, AAV-mediated overexpression of HBO1 restores the acetylation of H4K5/K12 and rescues the CCMs-like cerebrovascular defects in the brain of NgBR ecKO mice. Our findings on epigenetic regulation of CCM1 and CCM2 provide a perspective that NgBR and HBO1-mediated histone H4 acetylation may be targeted for preventing the onset of CCMs-like cerebrovascular disease.

Endothelial cell regulation of systemic haemodynamics and metabolism acts through the HIF transcription factors

BackgroundThe vascular endothelium has important endocrine and paracrine roles, particularly in the regulation of vascular tone and immune function, and it has been implicated in the pathophysiology of a range of cardiovascular and inflammatory conditions. This study uses a series of transgenic murine models to explore for the first time the role of the hypoxia-inducible factors, HIF-1α and HIF-2α in the pulmonary and systemic circulations as potential regulators of systemic vascular function in normoxic or hypoxic conditions and in response to inflammatory stress. We developed a series of transgenic mouse models, the HIF-1α Tie2Cre, deficient in HIF1-α in the systemic and pulmonary vascular endothelium and the L1Cre, a pulmonary endothelium specific knockout of HIF-1α or HIF-2α. In vivo, arterial blood pressure and metabolic activity were monitored continuously in normal atmospheric conditions and following an acute stimulus with hypoxia (10%) or lipopolysaccharide (LPS). Ex vivo, femoral artery reactivity was assessed using wire myography.ResultsUnder normoxia, the HIF-1α Tie2Cre mouse had increased systolic and diastolic arterial pressure compared to litter mate controls over the day–night cycle under normal environmental conditions. VO2 and VCO2 were also increased. Femoral arteries displayed impaired endothelial relaxation in response to acetylcholine mediated by a reduction in the nitric oxide dependent portion of the response. HIF-1α L1Cre mice displayed a similar pattern of increased systemic blood pressure, metabolic rate and impaired vascular relaxation without features of pulmonary hypertension, polycythaemia or renal dysfunction under normal conditions. In response to acute hypoxia, deficiency of HIF-1α was associated with faster resolution of hypoxia-induced haemodynamic and metabolic compromise. In addition, systemic haemodynamics were less compromised by LPS treatment.ConclusionsThese data show that deficiency of HIF-1α in the systemic or pulmonary endothelium is associated with increased systemic blood pressure and metabolic rate, a pattern that persists in both normoxic conditions and in response to acute stress with potential implications for our understanding of the pathophysiology of vascular dysfunction in acute and chronic disease.

Butyrate protects endothelial function through PPARδ/miR-181b signaling

Reports of the beneficial roles of butyrate in cardiovascular diseases, such as atherosclerosis and ischemic stroke, are becoming increasingly abundant. However, the mechanisms of its bioactivities remain largely unknown. In this study, we explored the effects of butyrate on endothelial dysfunction and its potential underlying mechanism. In our study, ApoE-/- mice were fed with high-fat diet (HFD) for ten weeks to produce atherosclerosis models and concurrently treated with or without sodium butyrate daily. Thoracic aortas were subsequently isolated from C57BL/6 wild-type (WT), PPARδ-/-, endothelial-specific PPARδ wild-type (EC-specific PPARδ WT) and endothelial-specific PPARδ knockout (EC-specific PPARδ KO) mice were stimulated with interleukin (IL)-1β with or without butyrate ex vivo. Our results demonstrated that butyrate treatment rescued the impaired endothelium-dependent relaxations (EDRs) in thoracic aortas of HFD-fed ApoE-/- mice. Butyrate also rescued impaired EDRs in IL-1β-treated thoracic aorta ring ex vivo. Global and endothelial-specific knockout of PPARδ eliminated the protective effects of butyrate against IL-1β-induced impairment to EDRs. Butyrate abolished IL-1β-induced reactive oxygen species (ROS) production in endothelial cells while the inhibitory effect was incapacitated by genetic deletion of PPARδ or pharmacological inhibition of PPARδ. IL-1β increased NADPH oxidase 2 (NOX2) mRNA and protein expressions in endothelial cells, which were prevented by butyrate treatment, and the effects of butyrate were blunted following pharmacological inhibition of PPARδ. Importantly, butyrate treatment upregulated the miR-181b expression in atherosclerotic aortas and IL-1β-treated endothelial cells. Moreover, transfection of endothelial cells with miR-181b inhibitor abolished the suppressive effects of butyrate on NOX2 expressions and ROS generation in endothelial cells. To conclude, butyrate prevents endothelial dysfunction in atherosclerosis by reducing endothelial NOX2 expression and ROS production via the PPARδ/miR-181b pathway.

CaMKIIδ is upregulated by pro-inflammatory cytokine IL-6 in a JAK/STAT3-dependent manner to promote angiogenesis.

Ca2+ /calmodulin-dependent protein kinase II (CaMKII) is a ubiquitous serine threonine kinase with established roles in physiological and pathophysiological vascular remodeling. Based on our previous study demonstrating that CaMKIIδ promotes thrombin-induced endothelial permeability and recent reports that CaMKII may contribute to inflammatory remodeling in the heart, we investigated CaMKIIδ-dependent regulation of endothelial function downstream of an interleukin-6 (IL-6)/JAK/STAT3 signaling axis. Upon treatment with IL-6 and its soluble receptor (sIL-6r), CaMKIIδ expression is significantly induced in HUVEC. Using pharmacological inhibitors of JAK and siRNA targeting STAT3, we demonstrated that activation of STAT3 is sufficient to induce CaMKIIδ expression. Under these conditions, rather than promoting IL-6-induced permeability, we found that CaMKIIδ promotes endothelial cell migration as measured by live cell imaging of scratch wound closure and single-cell motility analysis. In a similar manner, endothelial cell proliferation was attenuated upon knockdown of CaMKIIδ as determined by growth curves, cell cycle analysis, and capacitance of cell-covered electrodes as measured by ECIS. Using inducible endothelial-specific STAT3 knockout mice, we demonstrate that STAT3 signaling promotes developmental angiogenesis in the neonatal mouse retina assessed at postnatal day 6. CaMKIIδ expression in retinal endothelium was attenuated in these animals as measured by qPCR. STAT3's effects on angiogenesis were phenocopied by the endothelial-specific knockout of CaMKIIδ, with significantly reduced vascular outgrowth and number of junctions in the developing P6 retina. For the first time, we demonstrate that transcriptional regulation of CaMKIIδ by STAT3 promotes endothelial motility, proliferation, and in vivo angiogenesis.

Abstract 14787: Loss of Wnt7a is Associated With Reduced Vegf-a/vegfr2 Mediated Endothelial Tip Cell Formation and Angiogenesis in Pulmonary Arterial Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is characterized by loss of microvessels. The Wnt pathways control pulmonary angiogenesis but their role in PAH is incompletely understood. Objective: We hypothesized that Wnt activation in pulmonary microvascular endothelial cells (PMVECs) is required for pulmonary angiogenesis and its loss contributes to PAH. Methods: Lung tissue and PMVECs from healthy donors and PAH patients were screened for Wnt ligand production. Tip cell dynamics were analyzed using 3D spheroid assays and in vivo studies were carried out with tamoxifen-inducible Wnt knockout mice exposed to chronic hypoxia and Sugen-hypoxia (SuHx). Results: Healthy PMVECs demonstrated ~6-fold Wnt7a expression that was absent in PAH PMVECs and vascular lesions. Wnt7a expression correlated with formation of tip cells, an endothelial phenotype critical for angiogenesis. To analyze PMVEC tip cell formation, we developed a 3D PMVEC spheroid model combined with live imaging to monitor the behavior of healthy and PAH PMVECs in response to VEGF-A and Wnt7a. We found that PAH PMVECs demonstrated reduced tip cell formation as evidenced by reduced filopodia formation and random motility, which could be partially rescued by recombinant Wnt7a. The mechanism by which Wnt7a promotes VEGF signaling activity is dependent on its capacity to activate the small GTPase cdc42 and facilitate phosphorylation of the Y951 and Y1175 tyrosine residues in VEGFR2 following VEGF-A stimulation. While there was no phenotypic difference between wild type and mice with endothelial-specific Wnt7a knockout (Wnt7a EC/EC ) under chronic hypoxia and SuHx, we found that global Wnt7a +/- mice demonstrated higher pressures and vascular remodeling compared to wild type mice. Interestingly, we found that global Wnt7a knockout (Wnt7a KO/KO ) mice succumbed 10 days after tamoxifen injection demonstrating interstitial thickening associated with expansion of the pulmonary capillaries, an unexpected phenotype resembling alveolar capillary dysplasia. Conclusions: We have identified Wnt7a as a novel angiogenic factor that plays a pivotal role in pulmonary angiogenesis by promoting VEGF-A mediated tip cell formation. Targeting Wnt7a could promote recovery of small vessel loss in PAH.

Abstract 525: Adult Inducible Deletion of Endothelial Hypoxia-inducible Factor-2α Exaggerates Myocardial Infarction Induced Heart Failure and Cardiac Microvascular Barrier Dysfunction

Background: Myocardial ischemia occurs during myocardial infarction results in the stabilization of Hypoxia-inducible factors (HIFs). Hif2α expresses profoundly in vascular endothelial cells (EC), and its embryonic deletion increases vessel permeability. It has been shown that HIF2a is protective from renal and pulmonary injury. However, the direct role of ecHIF2α in ischemia heart disease is unknown. We hypothesized that ecHIF2a expression in response to myocardial infarction (MI) protects cardiac barrier dysfunction and against heart failure. Methods and Results: We generated the Inducible endothelial-specific knockout mice (ecHIF2a -/- ) by crossing Hif2a flox/flox mice with Cre ERT2 mice under the VE-cadherin promoter. Followed with MI, ecHIF2a -/- mice displayed worsened cardiac function determined by echocardiography, and they had increased mortality as compared to the controls. In vitro, we used primary mouse cardiac microvascular endothelial cells (mCMVEC) from ecHIF2a -/- mouse hearts. We found that under hypoxia condition or 1mM Dimethyloxalylglycine treatment, the deficiency of HIF2a in the mCMVEC increased endothelial permeability determined by trans-endothelial electrical resistance. The knocking down of HIF2a in HUVECs induced by a HIF2a siRNA led to impaired tube formation accessed by the significant reduction in total node counts, junctions, meshes, and full tube length compared to control-siRNA treated cells. HIF2α deletion and hypoxia both reduced endothelial cell migration, and interestingly, the retarded HIF2α-/- ECs migration seems to be independent of hypoxia. Moreover, apoptosis assay showed that ecHIF2a -/- ECs increased cell early apoptotic stage compared to WT in hypoxic conditions, but not in normoxia indicating the critical role of HIF2α in ECs survival during cardiac ischemia. Finally, several increased markers of inflammation, such as ICAM-1 and VCAM-1, are associated with HIF2a deletion. Conclusion: These data revealed an essential role of HIF2α in protecting cardiac remodeling in response to MI, which might through promoting endothelial cell migration, barrier function, as well as vascularization. Thus, HIF2α is a potential therapeutic target in the treatment of ischemic heart disease.

P717Glucagon-like peptide 1 (GLP-1) improves endothelial dysfunction and vascular inflammation in polymicrobial sepsis induced by cecal ligation and puncture (CLP)

Abstract Objective Sepsis causes severe hypotension, accompanied by high mortality in the setting of septic shock. LEADER, SUSTAIN-6 and other clinical trials revealed cardioprotective and anti-inflammatory properties of GLP-1 analogs like Liraglutide (Lira). We already demonstrated improved survival by amelioration of disseminated intravasal coagulation (DIC) in lipopolysaccharide (LPS)-induced endotoxemia by inhibition of the GLP-1 degrading enzyme dipeptidylpeptidase-4 (DPP-4). With the present study we aim to investigate the mechanism of protective effects of the GLP-1 analog Lira and the DPP4 inhibitor Linagliptin (Lina) in the clinically relevant sepsis model cecal ligation and puncture (CLP). Methods C57/BL6j and endothelial cell-specific GLP-1 receptor knockout mice (Cdh5crexGLP-1rfl/flmice) were used and sepsis was induced by cecal ligation and puncture (CLP). DPP4 inhibitor (Lina, 5mg/kg/d; 3 days) and GLP-1 analog (Lira, 200μg/kg/d; 3 days) were applied subcutaneously. Aortic vascular function was tested by isometric tension recording. Aorta and heart tissue was used for Western blotting, dot blot and qRT-PCR. Endogenous GLP-1 (7–36 and 9–36) and insulin was determined by ELISA. Blood samples were collected for examination of cell count, oxidative stress and glucose levels. Results Body temperature was increased by CLP and normalized by Lina and Lira. Sham- and Lira- but not Lina-treated septic mice showed low blood glucose levels compared to healthy controls. Acetylcholine-induced (endothelium-dependent) vascular relaxation in aorta was impaired by CLP. This was accompanied by vascular inflammation and elevation of IL-6, iNOS, ICAM-1, and TNF-alpha mRNA levels in aortic tissue. Vascular, cardiac and whole blood oxidative stress were increased by CLP. Furthermore, we detected higher levels of IL-6, 3-nitrotyrosine (3-NT) and 4-hydroxynonenal (4-NHE) in plasma of CLP animals. Lina and Lira reduced oxidative stress and vascular inflammation, which was accompanied by improved endothelial function. In addition, CLP treatment in endothelial specific knockout mice of the GLP-1r strongly induced mortality compared to WT mice, with the effect being strongest in the Lira-treated group. Conclusion The present study demonstrates that Lina (DPP4 inhibitor) and the GLP-1 analog Lira ameliorate sepsis-induced endothelial dysfunction by reduction of vascular inflammation and oxidative stress. Clinical trials like LEADER and SUSTAIN-6 proved that GLP-1 analogs like Lira have cardioprotective effects in T2DM patients. The present study, performed in a clinically relevant model of polymicrobial sepsis, reveals that the known cardioprotective effects of GLP-1 might be translated to other diseases which affect the cardiovascular system like sepsis, underlining the potent anti-inflammatory effects of GLP-1 analogs.

Loss of endothelial sulfatase-1 after experimental sepsis attenuates subsequent pulmonary inflammatory responses.

Sepsis patients are at increased risk for hospital-acquired pulmonary infections, potentially due to postseptic immunosuppression known as the compensatory anti-inflammatory response syndrome (CARS). CARS has been attributed to leukocyte dysfunction, with an unclear role for endothelial cells. The pulmonary circulation is lined by an endothelial glycocalyx, a heparan sulfate-rich layer essential to pulmonary homeostasis. Heparan sulfate degradation occurs early in sepsis, leading to lung injury. Endothelial synthesis of new heparan sulfates subsequently allows for glycocalyx reconstitution and endothelial recovery. We hypothesized that remodeling of the reconstituted endothelial glycocalyx, mediated by alterations in the endothelial machinery responsible for heparan sulfate synthesis, contributes to CARS. Seventy-two hours after experimental sepsis, coincident with glycocalyx reconstitution, mice demonstrated impaired neutrophil and protein influx in response to intratracheal lipopolysaccharide (LPS). The postseptic reconstituted glycocalyx was structurally remodeled, with enrichment of heparan sulfate disaccharides sulfated at the 6-O position of glucosamine. Increased 6-O-sulfation coincided with loss of endothelial sulfatase-1 (Sulf-1), an enzyme that specifically removes 6-O-sulfates from heparan sulfate. Intravenous administration of Sulf-1 to postseptic mice restored the pulmonary response to LPS, suggesting that loss of Sulf-1 was necessary for postseptic suppression of pulmonary inflammation. Endothelial-specific knockout mice demonstrated that loss of Sulf-1 was not sufficient to induce immunosuppression in non-septic mice. Knockdown of Sulf-1 in human pulmonary microvascular endothelial cells resulted in downregulation of the adhesion molecule ICAM-1. Taken together, our study indicates that loss of endothelial Sulf-1 is necessary for postseptic suppression of pulmonary inflammation, representing a novel endothelial contributor to CARS.

2005 - GATA3 PROMOTES THE ENDOTHELIAL-TO-HEMATOPOIETIC TRANSITION VIA REGULATION OF THE CELL CYCLE

Hematopoietic stem cells (HSCs) are known to emerge in the dorsal aorta at E10.5 of mouse embryo development. They derive from hemogenic endothelial cells via a process known as endothelial-to-hematopoietic transition (EHT), which comprises a number of intermediate steps and the molecular details of which have not been fully characterized. Our lab has previously demonstrated that embryos deficient for the transcription factor Gata3 have a defect in HSC generation in the dorsal aorta. This was linked to a lack of catecholamine synthesis in these embryos, which demonstrated that neurotransmitters released from the co-developing sympathetic nervous system promote embryonic HSC production. We now have evidence that Gata3 plays an additional role in EHT. Gata3 expression was found to enrich for hemogenic endothelial cell activity, and an endothelial-specific knockout of Gata3 severely impairs HSC generation in the dorsal aorta. Furthermore, with the use of co-aggregation assays developed in the Medvinsky lab, we were able to determine that Gata3 is expressed in the pro-HSC and pre-HSC type I stage, but is downregulated thereafter. RNA-Seq analysis of Gata3-positive and negative cell populations highlighted a cell cycle signature, with cell cycle inhibitors, such as p57Kip2, specifically enriched in Gata3-expressing cells. This suggests that a cell cycle arrest may be required for endothelial cells to complete the transition to a hematopoietic fate, which is supported by our preliminary results with embryos deficient for p57Kip2. Our results thus provide further insights into the molecular mechanisms of HSC generation from endothelial cells.

NCK-dependent pericyte migration promotes pathological neovascularization in ischemic retinopathy

Pericytes are mural cells that surround capillaries and control angiogenesis and capillary barrier function. During sprouting angiogenesis, endothelial cell-derived platelet-derived growth factor-B (PDGF-B) regulates pericyte proliferation and migration via the platelet-derived growth factor receptor-β (PDGFRβ). PDGF-B overexpression has been associated with proliferative retinopathy, but the underlying mechanisms remain poorly understood. Here we show that abnormal, α-SMA-expressing pericytes cover angiogenic sprouts and pathological neovascular tufts (NVTs) in a mouse model of oxygen-induced retinopathy. Genetic lineage tracing demonstrates that pericytes acquire α-SMA expression during NVT formation. Pericyte depletion through inducible endothelial-specific knockout of Pdgf-b decreases NVT formation and impairs revascularization. Inactivation of the NCK1 and NCK2 adaptor proteins inhibits pericyte migration by preventing PDGF-B-induced phosphorylation of PDGFRβ at Y1009 and PAK activation. Loss of Nck1 and Nck2 in mural cells prevents NVT formation and vascular leakage and promotes revascularization, suggesting PDGFRβ-Y1009/NCK signaling as a potential target for the treatment of retinopathies.

Abstract 043: Endothelial Adenosine Kinase Deficiency Ameliorates Diet-induced Insulin Resistance

Background: Obesity is associated with endothelial dysfunction characterized by reduced bioavailability of nitric oxide (NO). It is well known that adenosine regulates endothelial nitric oxide release and vasodilation. However, it is unclear whether intracellular adenosine is able to modulate endothelial nitric oxide production and diet-induced metabolic disorders. This study is to investigate the effect of elevated intracellular adenosine caused by adenosine kinase (ADK) deletion or knockdown on endothelial nitric oxide production and diet-induced insulin resistance as well as the pathways associated to this effect. Methods and results: Endothelial-specific ADK knockout mice fed a high-fat diet show decreased fasting blood glucose and insulin, suppressed adipose tissue inflammation and hepatic steatosis, increased skeletal muscle arteriole vasodilation in an eNOS dependent manner. Mechanistically, ADK knockdown in Human Umbilical Vein Endothelial Cells (HUVECs) with adenoviral ADK shRNA elevated both intracellular and extracellular adenosine, and consequently increased endothelial nitric oxide synthase (eNOS) expression and activation, resulting in an increase in NO production, insulin uptake and vasodilatory-stimulated phosphoprotein (VASP) phosphorylation. Treatment of HUVECs with adenosine A2b receptor antagonist MRS 1754 abolished ADK-knockdown induced eNOS expression. eNOS phosphorylation in ADK-knockdown HUVECs were not interfered with adenosine receptors antagonists. Conclusion: ADK knockdown-mediated elevation of intracellular adenosine in endothelial cells ameliorates diet-induced insulin resistance and metabolic disorders. This is attributed to an enhancement of NO production caused by increased eNOS activation and expression, the latter is regulated via endothelial adenosine A2b receptor.

P064 INTERFERON-GAMMA INDUCED VASCULAR IMPAIRMENT CONTRIBUTES TO THE PATHOGENESIS OF INFLAMMATORY BOWEL DISEASES

Inflammatory Bowel Diseases (IBD) are characterized by upregulation of Interferon-gamma (IFN-γ). Recently we showed that IFN-γ blockade by a specific antibody in Dextran Sodium Sulfate (DSS)-colitis mouse model results in increased angiogenesis and reduced vessel permeability(1). The aim of the present project was to validate the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD. For examination of vascular IFN-γ effects in vivo, we established an endothelial cell-specific Ifngr2-knock-out (Ifngr2ΔEndoC) mouse model. Vessel sprouting after IFN-γ stimulation of metatarsals was used for functional validation of the knock out. In order to analyze the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD, Ifngr2ΔEndoC and control mice were subjected to DSS-colitis. Angiogenesis and vascular barrier function of the colon were compared. Comparison of metatarsal angiogenic sprouting from Ifngr2ΔEndoC and control mice showed that endothelial-specific knock-out of Ifngr2 abrogates the angiostatic effects of IFN-γ. Induction of DSS-colitis revealed that Ifngr2ΔEndoC mice, compared to control mice, had a reduced inflammation of the colon. Investigation of specific vascular alterations showed an increased angiogenesis rate for Ifngr2ΔEndoC mice accompanied by an improved vascular barrier function, marked by higher vascular pericyte coverage and reduced vessel permeability. Our results indicate for first time an important pathofunction of the blood vessels in IBD. Specifically an IFN-γ-induced disruption of the vascular barrier function exaggerated the course of disease. This may open new therapy options. 1. Haep et al. Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice. Inflamm Bowel Dis. 2015.

P064 INTERFERON-GAMMA INDUCED VASCULAR IMPAIRMENT CONTRIBUTES TO THE PATHOGENESIS OF INFLAMMATORY BOWEL DISEASES

Inflammatory Bowel Diseases (IBD) are characterized by upregulation of Interferon-gamma (IFN-γ). Recently we showed that IFN-γ blockade by a specific antibody in Dextran Sodium Sulfate (DSS)-colitis mouse model results in increased angiogenesis and reduced vessel permeability(1). The aim of the present project was to validate the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD. For examination of vascular IFN-γ effects in vivo, we established an endothelial cell-specific Ifngr2-knock-out (Ifngr2ΔEndoC) mouse model. Vessel sprouting after IFN-γ stimulation of metatarsals was used for functional validation of the knock out. In order to analyze the specific contribution of vascular effects of IFN-γ to the pathogenesis of IBD, Ifngr2ΔEndoC and control mice were subjected to DSS-colitis. Angiogenesis and vascular barrier function of the colon were compared. Comparison of metatarsal angiogenic sprouting from Ifngr2ΔEndoC and control mice showed that endothelial-specific knock-out of Ifngr2 abrogates the angiostatic effects of IFN-γ. Induction of DSS-colitis revealed that Ifngr2ΔEndoC mice, compared to control mice, had a reduced inflammation of the colon. Investigation of specific vascular alterations showed an increased angiogenesis rate for Ifngr2ΔEndoC mice accompanied by an improved vascular barrier function, marked by higher vascular pericyte coverage and reduced vessel permeability. Our results indicate for first time an important pathofunction of the blood vessels in IBD. Specifically an IFN-γ-induced disruption of the vascular barrier function exaggerated the course of disease. This may open new therapy options.

Prostaglandin E receptor-4 receptor mediates endothelial barrier-enhancing and anti-inflammatory effects of oxidized phospholipids.

Unlike other agonists that cause transient endothelial cell (EC) response, the products of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) oxidation that contain cyclopenthenone groups, which recapitulate prostaglandin-like structure, cause sustained enhancement of the pulmonary EC barrier. The mechanisms that drive the sustained effects by oxidized PAPC (OxPAPC) remain unexplored. On the basis of the structural similarity of isoprostanoid moieties that are present in full-length oxygenated PAPC species, we used an inhibitory approach to perform the screening of prostanoid receptors as potential candidates that mediate OxPAPC effects. Results show that only prostaglandin E receptor-4 (EP4) was involved and mediated the sustained phase of the barrier-enhancing effects of OxPAPC that are associated with the activation of Rac GTPase and its cytoskeletal targets. EC incubation with OxPAPC also induced EP4 mRNA expression in pulmonary ECs and lung tissue. EP4 knockdown using gene-specific small interfering RNA did not affect the rapid phase of OxPAPC-induced EC barrier enhancement or the protective effects against thrombin-induced EC permeability, but abolished the advanced barrier enhancement phase and suppressed the protective effects of OxPAPC against more sustained EC barrier dysfunction and cell inflammatory response caused by TNF-α. Endothelial-specific knockout of the EP4 receptor in mice attenuated the protective effect of intravenous OxPAPC administration in the model of acute lung injury caused by intratracheal injection of LPS. Taken together, these results demonstrate a novel role for prostaglandin receptor EP4 in the mediation of barrier-enhancing and anti-inflammatory effects caused by oxidized phospholipids.-Oskolkova, O., Gawlak, G., Tian, Y., Ke, Y., Sarich, N., Son, S., Andreasson, K., Bochkov, V. N., Birukova, A. A., Birukov, K. G. Prostaglandin E receptor-4 receptor mediates endothelial barrier-enhancing and anti-inflammatory effects of oxidized phospholipids.

Atg7 Regulates Brain Angiogenesis via NF-κB-Dependent IL-6 Production.

The formation of brain vasculature is an essential step during central nervous system development. The molecular mechanism underlying brain angiogenesis remains incompletely understood. The role of Atg7, an autophagy-related protein, in brain angiogenesis was investigated in this study. We found that the microvessel density in mice brains with endothelial-specific knockout of Atg7 (Atg7 EKO) was significantly decreased compared to wild-type control. Consistently, in vitro angiogenesis assays showed that Atg7 knockdown impaired angiogenesis in brain microvascular endothelial cells. Further results indicated that knockdown of Atg7 reduced interleukin-6 (IL-6) expression in brain microvascular endothelial cells, which is mediated by NF-κB-dependent transcriptional control. Interestingly, exogenous IL-6 restored the impaired angiogenesis and reduced cell motility caused by Atg7 knockdown. These results demonstrated that Atg7 has proangiogenic activity in brain angiogenesis which is mediated by IL-6 production in a NF-κB-dependent manner.