Endothelial PTP1B Deletion Promotes VWF Exocytosis and Venous Thromboinflammation.

BACKGROUND: Endothelial activation promotes the release of procoagulant extracellular vesicles and inflammatory mediators from specialized storage granules. Endothelial membrane exocytosis is controlled by phosphorylation. We hypothesized that the absence of PTP1B (protein tyrosine phosphatase 1B) in endothelial cells promotes venous thromboinflammation by triggering endothelial membrane fusion and exocytosis. METHODS: Mice with inducible endothelial deletion of PTP1B (End.PTP1B-KO) underwent inferior vena cava ligation to induce stenosis and venous thrombosis. Primary endothelial cells from transgenic mice and human umbilical vein endothelial cells were used for mechanistic studies. RESULTS: Vascular ultrasound and histology showed significantly larger venous thrombi containing higher numbers of Ly6G (lymphocyte antigen 6 family member G)-positive neutrophils in mice with endothelial PTP1B deletion, and intravital microscopy confirmed the more pronounced neutrophil recruitment following inferior vena cava ligation. RT2 PCR profiler array and immunocytochemistry analysis revealed increased endothelial activation and adhesion molecule expression in primary End.PTP1B-KO endothelial cells, including CD62P (P-selectin) and VWF (von Willebrand factor). Pretreatment with the NF-κB (nuclear factor kappa B) kinase inhibitor BAY11-7082, antibodies neutralizing CD162 (P-selectin glycoprotein ligand-1) or VWF, or arginylglycylaspartic acid integrin-blocking peptides abolished the neutrophil adhesion to End.PTP1B-KO endothelial cells in vitro. Circulating levels of annexin V+ procoagulant endothelial CD62E+ (E-selectin) and neutrophil (Ly6G+) extracellular vesicles were also elevated in End.PTP1B-KO mice after inferior vena cava ligation. Higher plasma MPO (myeloperoxidase) and Cit-H3 (citrullinated histone-3) levels and neutrophil elastase activity indicated neutrophil activation and extracellular trap formation. Infusion of End.PTP1B-KO extracellular vesicles into C57BL/6J wild-type mice most prominently enhanced the recruitment of endogenous neutrophils, and this response was blunted in VWF-deficient mice or by VWF-blocking antibodies. Reduced PTP1B binding and tyrosine dephosphorylation of SNAP23 (synaptosome-associated protein 23) resulting in increased VWF exocytosis and neutrophil adhesion were identified as mechanisms, all of which could be restored by NF-κB kinase inhibition using BAY11-7082. CONCLUSIONS: Our findings show that endothelial PTP1B deletion promotes venous thromboinflammation by enhancing SNAP23 phosphorylation, endothelial VWF exocytosis, and neutrophil recruitment.

Endothelial Overexpression of TGF-ß-Induced Protein Impairs Venous Thrombus Resolution: Possible Role in CTEPH.

Endothelial cells play a critical role during venous thrombus remodeling, and unresolved, fibrotic thrombi with irregular vessels obstruct the pulmonary artery in patients with chronic thromboembolic pulmonary hypertension (CTEPH). This study sought to identify endothelial mediators of impaired venous thrombus resolution and to determine their role in the pathogenesis of the vascular obstructions in patients with CTEPH. Endothelial cells outgrown from pulmonary endarterectomy specimens (PEA) were processed for mRNA profiling, and nCounter gene expression and immunohistochemistry analysis of PEA tissue microarrays and immunoassays of plasma were used to validate the expression in CTEPH. Lentiviral overexpression in human pulmonary artery endothelial cells (HPAECs) and exogenous administration of the recombinant protein into C57BL/6J mice after inferior Vena cava ligation were employed to assess their role for venous thrombus resolution. RT2 PCR profiler analysis demonstrated the significant overexpression of factors downstream of transforming growth factor beta (TGFß), that is TGFß-Induced Protein (TGFBI or BIGH3) and transgelin (TAGLN), or involved in TGFß signaling, that is follistatin-like 3 (FSTL3) and stanniocalcin-2 (STC2). Gene expression and immunohistochemistry analysis of tissue microarrays localized potential disease candidates to vessel-rich regions. Lentiviral overexpression of TGFBI in HPAECs increased fibrotic remodeling of human blood clots in vitro, and exogenous administration of recombinant TGFBI in mice delayed venous thrombus resolution. Significantly elevated plasma TGFBI levels were observed in patients with CTEPH and decreased after PEA. Our findings suggest that overexpression of TGFBI in endothelial promotes venous thrombus non-resolution and fibrosis and is causally involved in the pathophysiology of CTEPH.

Circulating Soluble EPCR Levels Are Reduced in Patients with Ischemic Peripheral Artery Disease and Associated with Markers of Endothelial and Vascular Function.

(1) Background: Endothelial dysfunction initiates cardiovascular pathologies, including peripheral artery disease (PAD). The pathophysiology of impaired new vessel formation in the presence of angiogenic stimuli, such as ischemia and inflammation, is unknown. We have recently shown in mice that reduced endothelial protein C receptor (EPCR) expression results in defective angiogenesis following experimental hindlimb ischemia. (2) Purpose: To determine soluble (s)EPCR levels in the plasma of patients with PAD and to compare them with the protein C activity and biomarkers of endothelial function, inflammation, and angiogenesis. (3) Methods and Results: Clinical tests of vascular function and immunoassays of plasma from patients with PAD stage II were compared to age- and sex-matched individuals with and without cardiovascular risk factors or PAD stage III/IV patients. sEPCR levels were significantly lower in PAD stage II patients compared to subjects with risk factors, but no PAD, and further decreased in PAD stage III/IV patients. Plasma protein C activity or levels of ADAM17, a mediator of EPCR shedding, did not differ. Significant associations between sEPCR and the ankle-brachial index (p = 0.0359), age (p = 0.0488), body mass index (p = 0.0110), and plasma sE-selectin levels (p = 0.0327) were observed. High-sensitive CRP levels and white blood cell counts were significantly elevated in PAD patients and associated with serum glucose levels, but not sEPCR. In contrast, plasma TNFα or IL1ß levels did not differ. Circulating levels of VEGF were significantly elevated in PAD stage II patients (p = 0.0198), but not associated with molecular (sE-selectin) or functional (ankle-brachial index) markers of vascular health. (4) Conclusions: Our findings suggest that circulating sEPCR levels may be useful as biomarkers of endothelial dysfunction, including angiogenesis, in persons older than 35 years and that progressive loss of endothelial protein C receptors might be involved in the development and progression of PAD.

Deletion of endothelial leptin receptors in mice promotes diet-induced obesity.

Obesity promotes endothelial dysfunction. Endothelial cells not only respond, but possibly actively promote the development of obesity and metabolic dysfunction. Our aim was to characterize the role of endothelial leptin receptors (LepR) for endothelial and whole body metabolism and diet-induced obesity. Mice with tamoxifen-inducible, Tie2.Cre-ERT2-mediated deletion of LepR in endothelial cells (End.LepR knockout, KO) were fed high-fat diet (HFD) for 16 weeks. Body weight gain, serum leptin levels, visceral adiposity and adipose tissue inflammation were more pronounced in obese End.LepR-KO mice, whereas fasting serum glucose and insulin levels or the extent of hepatic steatosis did not differ. Reduced brain endothelial transcytosis of exogenous leptin, increased food intake and total energy balance were observed in End.LepR-KO mice and accompanied by brain perivascular macrophage accumulation, whereas physical activity, energy expenditure and respiratory exchange rates did not differ. Metabolic flux analysis revealed no changes in the bioenergetic profile of endothelial cells from brain or visceral adipose tissue, but higher glycolysis and mitochondrial respiration rates in those isolated from lungs. Our findings support a role for endothelial LepRs in the transport of leptin into the brain and neuronal control of food intake, and also suggest organ-specific changes in endothelial cell, but not whole-body metabolism.

Arginase-1 Deletion in Erythrocytes Promotes Vascular Calcification via Enhanced GSNOR (S-Nitrosoglutathione Reductase) Expression and NO Signaling in Smooth Muscle Cells.

BACKGROUND: Erythrocytes (red blood cells) participate in the control of vascular NO bioavailability. The purpose of this study was to determine whether and how genetic deletion of ARG1 (arginase-1) affects vascular smooth muscle cell NO signaling, osteoblastic differentiation, and atherosclerotic lesion calcification. METHODS: Atherosclerosis-prone mice with conditional, erythrocyte-restricted deletion of ARG1 (apoE-/- red blood cell.ARG1 knockout) were generated and vascular calcification studied using molecular imaging of the osteogenic activity agent OsteoSense, Alizarin staining or immunohistochemistry, qPCR of osteogenic markers and ex vivo assays. RESULTS: Atherosclerotic lesion size at the aortic root did not differ, but calcification was significantly more pronounced in apoE-/- mice lacking erythrocyte ARG1. Incubation of murine and human VSMCs with lysed erythrocyte membranes from apoE-/- red blood cell. ARG1-knockout mice accelerated their osteogenic differentiation, and mRNA transcripts of osteogenic markers decreased following NO scavenging. In addition to NO signaling via sGC (soluble guanylyl cyclase), overexpression of GSNOR (S-nitrosoglutathione reductase) enhanced degradation of S-nitrosoglutathione to glutathione and reduced protein S-nitrosation of HSP (heat shock protein)-70 were identified as potential mechanisms of vascular smooth muscle cell calcification in mice lacking ARG1 in erythrocytes, and calcium phosphate deposition was enhanced by heat shock and prevented by GSNOR inhibition. Messenger RNA levels of enzymes metabolizing the arginase products L-ornithine and L-proline also were elevated in VSMCs, paralleled by increased proliferation, myofibroblast marker and collagen type 1 expression. CONCLUSIONS: Our findings support an important role of erythrocyte ARG1 for NO bioavailability and L-arginine metabolism in VSMCs, which controls atherosclerotic lesion composition and calcification.

Protein Tyrosine Phosphatase 1B Deficiency in Vascular Smooth Muscle Cells Promotes Perivascular Fibrosis following Arterial Injury.

BACKGROUND: Smooth muscle cell (SMC) phenotype switching plays a central role during vascular remodeling. Growth factor receptors are negatively regulated by protein tyrosine phosphatases (PTPs), including its prototype PTP1B. Here, we examine how reduction of PTP1B in SMCs affects the vascular remodeling response to injury. METHODS: Mice with inducible PTP1B deletion in SMCs (SMC.PTP1B-KO) were generated by crossing mice expressing Cre.ERT2 recombinase under the Myh11 promoter with PTP1Bflox/flox mice and subjected to FeCl3 carotid artery injury. RESULTS: Genetic deletion of PTP1B in SMCs resulted in adventitia enlargement, perivascular SMA+ and PDGFRß+ myofibroblast expansion, and collagen accumulation following vascular injury. Lineage tracing confirmed the appearance of Myh11-Cre reporter cells in the remodeling adventitia, and SCA1+ CD45- vascular progenitor cells increased. Elevated mRNA expression of transforming growth factor ß (TGFß) signaling components or enzymes involved in extracellular matrix remodeling and TGFß liberation was seen in injured SMC.PTP1B-KO mouse carotid arteries, and mRNA transcript levels of contractile SMC marker genes were reduced already at baseline. Mechanistically, Cre recombinase (mice) or siRNA (cells)-mediated downregulation of PTP1B or inhibition of ERK1/2 signaling in SMCs resulted in nuclear accumulation of KLF4, a central transcriptional repressor of SMC differentiation, whereas phosphorylation and nuclear translocation of SMAD2 and SMAD3 were reduced. SMAD2 siRNA transfection increased protein levels of PDGFRß and MYH10 while reducing ERK1/2 phosphorylation, thus phenocopying genetic PTP1B deletion. CONCLUSION: Chronic reduction of PTP1B in SMCs promotes dedifferentiation, perivascular fibrosis, and adverse remodeling following vascular injury by mechanisms involving an ERK1/2 phosphorylation-driven shift from SMAD2 to KLF4-regulated gene transcription.

EPCR-PAR1 biased signaling regulates perfusion recovery and neovascularization in peripheral ischemia.

Blood clot formation initiates ischemic events, but coagulation roles during postischemic tissue repair are poorly understood. The endothelial protein C receptor (EPCR) regulates coagulation, as well as immune and vascular signaling, by protease activated receptors (PARs). Here, we show that endothelial EPCR-PAR1 signaling supports reperfusion and neovascularization in hindlimb ischemia in mice. Whereas deletion of PAR2 or PAR4 did not impair angiogenesis, EPCR and PAR1 deficiency or PAR1 resistance to cleavage by activated protein C caused markedly reduced postischemic reperfusion in vivo and angiogenesis in vitro. These findings were corroborated by biased PAR1 agonism in isolated primary endothelial cells. Loss of EPCR-PAR1 signaling upregulated hemoglobin expression and reduced endothelial nitric oxide (NO) bioavailability. Defective angiogenic sprouting was rescued by the NO donor DETA-NO, whereas NO scavenging increased hemoglobin and mesenchymal marker expression in human and mouse endothelial cells. Vascular specimens from patients with ischemic peripheral artery disease exhibited increased hemoglobin expression, and soluble EPCR and NO levels were reduced in plasma. Our data implicate endothelial EPCR-PAR1 signaling in the hypoxic response of endothelial cells and identify suppression of hemoglobin expression as an unexpected link between coagulation signaling, preservation of endothelial cell NO bioavailability, support of neovascularization, and prevention of fibrosis.

Activated Endothelial TGFß1 Signaling Promotes Venous Thrombus Nonresolution in Mice Via Endothelin-1: Potential Role for Chronic Thromboembolic Pulmonary Hypertension.

RATIONALE: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGFß (transforming growth factor-ß) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGFß in the pathophysiology of CTEPH is unknown. OBJECTIVE: To determine whether defective TGFß signaling in endothelial cells contributes to thrombus nonresolution and fibrosis. METHODS AND RESULTS: Venous thrombosis was induced by inferior vena cava ligation in mice with genetic deletion of TGFß1 in platelets (Plt.TGFß-KO) or TGFß type II receptors in endothelial cells (End.TGFßRII-KO). Pulmonary endarterectomy specimens from CTEPH patients were analyzed using immunohistochemistry. Primary human and mouse endothelial cells were studied using confocal microscopy, quantitative polymerase chain reaction, and Western blot. Absence of TGFß1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGFßRII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGFß1 levels, endothelial TGFßRI/ALK1 (activin receptor-like kinase), and TGFßRI/ALK5 expression were detected in End.TGFßRII-KO mice, and activated TGFß signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGFßRII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGFß1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGFßRII-KO endothelial cells, murine venous thrombi, or endarterectomy specimens and plasma of CTEPH patients, and endothelin-1 overexpression was prevented by inhibition of ALK5, and to a lesser extent of ALK1. ALK5 inhibition and endothelin receptor antagonization inhibited mesenchymal lineage conversion in TGFß1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGFßRII-KO mice. CONCLUSIONS: Endothelial TGFß1 signaling via type I receptors and endothelin-1 contribute to mesenchymal lineage transition and thrombofibrosis, which were prevented by blocking endothelin receptors. Our findings may have relevant implications for the prevention and management of CTEPH.

Increased Lymphangiogenesis and Lymphangiogenic Growth Factor Expression in Perivascular Adipose Tissue of Patients with Coronary Artery Disease.

Experimental and human autopsy studies have associated adventitial lymphangiogenesis with atherosclerosis. An analysis of perivascular lymphangiogenesis in patients with coronary artery disease is lacking. Here, we examined lymphangiogenesis and its potential regulators in perivascular adipose tissue (PVAT) surrounding the heart (C-PVAT) and compared it with PVAT of the internal mammary artery (IMA-PVAT). Forty-six patients undergoing coronary artery bypass graft surgery were included. Perioperatively collected C-PVAT and IMA-PVAT were analyzed using histology, immunohistochemistry, real time PCR, and PVAT-conditioned medium using cytokine arrays. C-PVAT exhibited increased PECAM-1 (platelet endothelial cell adhesion molecule 1)-positive vessel density. The number of lymphatic vessels expressing lymphatic vessel endothelial hyaluronan receptor-1 or podoplanin was also elevated in C-PVAT and associated with higher inflammatory cell numbers, increased intercellular adhesion molecule 1 (ICAM1) expression, and fibrosis. Significantly higher expression of regulators of lymphangiogenesis such as vascular endothelial growth factor (VEGF)-C, VEGF-D, and VEGF receptor-3 was observed in C-PVAT compared to IMA-PVAT. Cytokine arrays identified angiopoietin-2 as more highly expressed in C-PVAT vs. IMA-PVAT. Findings were confirmed histologically and at the mRNA level. Stimulation of human lymphatic endothelial cells with recombinant angiopoietin-2 in combination with VEGF-C enhanced sprout formation. Our study shows that PVAT surrounding atherosclerotic arteries exhibits more extensive lymphangiogenesis, inflammation, and fibrosis compared to PVAT surrounding a non-diseased vessel, possibly due to local angiopoietin-2, VEGF-C, and VEGF-D overexpression.

Angiogenic Endothelial Cell Signaling in Cardiac Hypertrophy and Heart Failure.

Endothelial cells are, by number, one of the most abundant cell types in the heart and active players in cardiac physiology and pathology. Coronary angiogenesis plays a vital role in maintaining cardiac vascularization and perfusion during physiological and pathological hypertrophy. On the other hand, a reduction in cardiac capillary density with subsequent tissue hypoxia, cell death and interstitial fibrosis contributes to the development of contractile dysfunction and heart failure, as suggested by clinical as well as experimental evidence. Although the molecular causes underlying the inadequate (with respect to the increased oxygen and energy demands of the hypertrophied cardiomyocyte) cardiac vascularization developing during pathological hypertrophy are incompletely understood. Research efforts over the past years have discovered interesting mediators and potential candidates involved in this process. In this review article, we will focus on the vascular changes occurring during cardiac hypertrophy and the transition toward heart failure both in human disease and preclinical models. We will summarize recent findings in transgenic mice and experimental models of cardiac hypertrophy on factors expressed and released from cardiomyocytes, pericytes and inflammatory cells involved in the paracrine (dys)regulation of cardiac angiogenesis. Moreover, we will discuss major signaling events of critical angiogenic ligands in endothelial cells and their possible disturbance by hypoxia or oxidative stress. In this regard, we will particularly highlight findings on negative regulators of angiogenesis, including protein tyrosine phosphatase-1B and tumor suppressor p53, and how they link signaling involved in cell growth and metabolic control to cardiac angiogenesis. Besides endothelial cell death, phenotypic conversion and acquisition of myofibroblast-like characteristics may also contribute to the development of cardiac fibrosis, the structural correlate of cardiac dysfunction. Factors secreted by (dysfunctional) endothelial cells and their effects on cardiomyocytes including hypertrophy, contractility and fibrosis, close the vicious circle of reciprocal cell-cell interactions within the heart during pathological hypertrophy remodeling.

Lysed Erythrocyte Membranes Promote Vascular Calcification.

BACKGROUND: Intraplaque hemorrhage promotes atherosclerosis progression, and erythrocytes may contribute to this process. In this study we examined the effects of red blood cells on smooth muscle cell mineralization and vascular calcification and the possible mechanisms involved. METHODS: Erythrocytes were isolated from human and murine whole blood. Intact and lysed erythrocytes and their membrane fraction or specific erythrocyte components were examined in vitro using diverse calcification assays, ex vivo by using the murine aortic ring calcification model, and in vivo after murine erythrocyte membrane injection into neointimal lesions of hypercholesterolemic apolipoprotein E-deficient mice. Vascular tissues (aortic valves, atherosclerotic carotid artery specimens, abdominal aortic aneurysms) were obtained from patients undergoing surgery. RESULTS: The membrane fraction of lysed, but not intact human erythrocytes promoted mineralization of human arterial smooth muscle cells in culture, as shown by Alizarin red and van Kossa stain and increased alkaline phosphatase activity, and by increased expression of osteoblast-specific transcription factors (eg, runt-related transcription factor 2, osterix) and differentiation markers (eg, osteopontin, osteocalcin, and osterix). Erythrocyte membranes dose-dependently enhanced calcification in murine aortic rings, and extravasated CD235a-positive erythrocytes or Perl iron-positive signals colocalized with calcified areas or osteoblast-like cells in human vascular lesions. Mechanistically, the osteoinductive activity of lysed erythrocytes was localized to their membrane fraction, did not involve membrane lipids, heme, or iron, and was enhanced after removal of the nitric oxide (NO) scavenger hemoglobin. Lysed erythrocyte membranes enhanced calcification to a similar extent as the NO donor diethylenetriamine-NO, and their osteoinductive effects could be further augmented by arginase-1 inhibition (indirectly increasing NO bioavailability). However, the osteoinductive effects of erythrocyte membranes were reduced in human arterial smooth muscle cells treated with the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide or following inhibition of NO synthase or the NO receptor soluble guanylate cyclase. Erythrocytes isolated from endothelial NO synthase-deficient mice exhibited a reduced potency to promote calcification in the aortic ring assay and after injection into murine vascular lesions. CONCLUSIONS: Our findings in cells, genetically modified mice, and human vascular specimens suggest that intraplaque hemorrhage with erythrocyte extravasation and lysis promotes osteoblastic differentiation of smooth muscle cells and vascular lesion calcification, and also support a role for erythrocyte-derived NO.

Endothelial Leptin Receptor Deletion Promotes Cardiac Autophagy and Angiogenesis Following Pressure Overload by Suppressing Akt/mTOR Signaling.

BACKGROUND: Cardiac remodeling is modulated by overnutrition or starvation. The adipokine leptin mediates energy balance between adipose tissue and brain. Leptin and its receptors are expressed in the heart. METHODS AND RESULTS: To examine the importance of endothelial leptin signaling in cardiac hypertrophy, transverse aortic constriction was used in mice with inducible endothelium-specific deletion of leptin receptors (End.LepR-KO) or littermate controls (End.LepR-WT). End.LepR-KO was associated with improved left ventricular function (fractional shortening, 28.4% versus 18.8%; P=0.0114), reduced left ventricular dilation (end-systolic inner left ventricular diameter, 3.59 versus 4.08 mm; P=0.0188) and lower heart weight (133 versus 173 mg; P<0.0001) 20 weeks after transverse aortic constriction. Histology and quantitative polymerase chain reaction analysis confirmed reduced cardiomyocyte hypertrophy. STAT3 (signal transducer and activator of transcription) activation was reduced, and Akt (protein kinase B) and mTOR (mammalian target of rapamycin) phosphorylation after transverse aortic constriction were blunted in End.LepR-KO hearts. Elevated LC3 (microtubule associated protein 1 light chain 3)-I/-II conversion ( P=0.0041) and increased (LC3II-positive) endothelial cells ( P=0.0042) in banded hearts of End.LepR-KO mice suggested improved cardiac angiogenesis because of activated autophagy. Microscopy confirmed autophagosome accumulation after genetic or small interfering RNA-mediated LepR downregulation. Enhanced sprouting angiogenesis was observed in endothelial cells ( P<0.0001) and aortic rings ( P=0.0060) from End.LepR-KO mice, and murine and human endothelial sprouting angiogenesis was reduced after mTOR inhibition using rapamycin or autophagy inhibition using 3-methyladenine. Banded End.LepR-KO mouse hearts exhibited less apoptosis ( P=0.0218), inflammation ( P=0.0251), and fibrosis ( P=0.0256). Reduced endothelial autophagy was also observed in myocardial biopsies of heart failure patients with cardiac fibrosis. CONCLUSIONS: Our findings suggest that endothelial leptin signaling contributes to cardiac fibrosis and functional deterioration by suppressing endothelial autophagy and promoting endothelial dysfunction in a chronic pressure overload model.

Inducible Knockdown of Endothelial Protein Tyrosine Phosphatase-1B Promotes Neointima Formation in Obese Mice by Enhancing Endothelial Senescence.

AIMS: Protein tyrosine phosphatase-1B (PTP1B) is a negative regulator of receptor tyrosine kinase signaling. In this study, we determined the importance of PTP1B expressed in endothelial cells for the vascular response to arterial injury in obesity. RESULTS: Morphometric analysis of vascular lesions generated by 10% ferric chloride (FeCl3) revealed that tamoxifen-inducible endothelial PTP1B deletion (Tie2.ERT2-Cre × PTP1Bfl/fl; End.PTP1B knockout, KO) significantly increased neointima formation, and reduced numbers of (endothelial lectin-positive) luminal cells in End.PTP1B-KO mice suggested impaired lesion re-endothelialization. Significantly higher numbers of proliferating cell nuclear antigen (PCNA)-positive proliferating cells as well as smooth muscle actin (SMA)-positive or vascular cell adhesion molecule-1 (VCAM1)-positive activated smooth muscle cells or vimentin-positive myofibroblasts were detected in neointimal lesions of End.PTP1B-KO mice, whereas F4/80-positive macrophage numbers did not differ. Activated receptor tyrosine kinase and transforming growth factor-beta (TGFß) signaling and oxidative stress markers were also significantly more abundant in End.PTP1B-KO mouse lesions. Genetic knockdown or pharmacological inhibition of PTP1B in endothelial cells resulted in increased expression of caveolin-1 and oxidative stress, and distinct morphological changes, elevated numbers of senescence-associated ß-galactosidase-positive cells, and increased expression of tumor suppressor protein 53 (p53) or the cell cycle inhibitor cyclin-dependent kinase inhibitor-2A (p16INK4A) suggested senescence, all of which could be attenuated by small interfering RNA (siRNA)-mediated downregulation of caveolin-1. In vitro, senescence could be prevented and impaired re-endothelialization restored by preincubation with the antioxidant Trolox. INNOVATION: Our results reveal a previously unknown role of PTP1B in endothelial cells and provide mechanistic insights how PTP1B deletion or inhibition may promote endothelial senescence. CONCLUSION: Absence of PTP1B in endothelial cells impairs re-endothelialization, and the failure to induce smooth muscle cell quiescence or to protect from circulating growth factors may result in neointimal hyperplasia.

Age-Dependent and -Independent Effects of Perivascular Adipose Tissue and Its Paracrine Activities during Neointima Formation.

Cardiovascular risk factors may act by modulating the composition and function of the adventitia. Here we examine how age affects perivascular adipose tissue (PVAT) and its paracrine activities during neointima formation. Aortic tissue and PVAT or primary aortic smooth muscle cells from male C57BL/6JRj mice aged 52 weeks ("middle-aged") were compared to tissue or cells from mice aged 16 weeks ("adult"). Vascular injury was induced at the carotid artery using 10% ferric chloride. Carotid arteries from the middle-aged mice exhibited smooth muscle de-differentiation and elevated senescence marker expression, and vascular injury further aggravated media and adventitia thickening. Perivascular transplantation of PVAT had no effect on these parameters, but age-independently reduced neointima formation and lumen stenosis. Quantitative PCR analysis revealed a blunted increase in senescence-associated proinflammatory changes in perivascular tissue compared to visceral adipose tissue and higher expression of mediators attenuating neointima formation. Elevated levels of protein inhibitor of activated STAT1 (PIAS1) and lower expression of STAT1- or NFκB-regulated genes involved in adipocyte differentiation, inflammation, and apoptosis/senescence were present in mouse PVAT, whereas PIAS1 was reduced in the PVAT of patients with atherosclerotic vessel disease. Our findings suggest that age affects adipose tissue and its paracrine vascular activities in a depot-specific manner. PIAS1 may mediate the age-independent vasculoprotective effects of perivascular fat.

The endothelial tumor suppressor p53 is essential for venous thrombus formation in aged mice.

Venous thromboembolism (VTE) is a leading cause of morbidity and mortality in elderly people. Increased expression of tumor suppressor protein 53 (p53) has been implicated in vascular senescence. Here, we examined the importance of endothelial p53 for venous thrombosis and whether endothelial senescence and p53 overexpression are involved in the exponential increase of VTE with age. Mice with conditional, endothelial-specific deletion of p53 (End.p53-KO) and their wild-type littermates (End.p53-WT) underwent subtotal inferior vena cava (IVC) ligation to induce venous thrombosis. IVC ligation in aged (12-month-old) End.p53-WT mice resulted in higher rates of thrombus formation and greater mean thrombus size vs adult (12-week-old) End.p53-WT mice, whereas aged End.p53-KO mice were protected from vein thrombosis. Analysis of primary endothelial cells from aged mice or human vein endothelial cells after induction of replicative senescence revealed significantly increased early growth response gene-1 (Egr1) and heparanase expression, and plasma factor Xa levels were elevated in aged End.p53-WT, but not in End.p53-KO mice. Increased endothelial Egr1 and heparanase expression also was observed after doxorubicin-induced p53 overexpression, whereas p53 inhibition using pifithrin-α reduced tissue factor (TF) expression. Importantly, inhibition of heparanase activity using TF pathway inhibitor-2 (TFPI2) peptides prevented the enhanced venous thrombus formation in aged mice and restored it to the thrombotic phenotype of adult mice. Our findings suggest that p53 accumulation and heparanase overexpression in senescent endothelial cells are critically involved in mediating the increased risk of venous thrombosis with age and that heparanase antagonization may be explored as strategy to ameliorate the prothrombotic endothelial phenotype with age.

Selective Deletion of Leptin Signaling in Endothelial Cells Enhances Neointima Formation and Phenocopies the Vascular Effects of Diet-Induced Obesity in Mice.

OBJECTIVE: Obesity is associated with elevated circulating leptin levels and hypothalamic leptin resistance. Leptin receptors (LepRs) are expressed on endothelial cells, and leptin promotes neointima formation in a receptor-dependent manner. Our aim was to examine the importance of endothelial LepR (End.LepR) signaling during vascular remodeling and to determine whether the cardiovascular consequences of obesity are because of hyperleptinemia or endothelial leptin resistance. APPROACH AND RESULTS: Mice with loxP-flanked LepR alleles were mated with mice expressing Cre recombinase controlled by the inducible endothelial receptor tyrosine kinase promoter. Obesity was induced with high-fat diet. Neointima formation was examined after chemical carotid artery injury. Morphometric quantification revealed significantly greater intimal hyperplasia, neointimal cellularity, and proliferation in End.LepR knockout mice, and similar findings were obtained in obese, hyperleptinemic End.LepR wild-type animals. Analysis of primary endothelial cells confirmed abrogated signal transducer and activator of transcription-3 phosphorylation in response to leptin in LepR knockout and obese LepR wild-type mice. Quantitative PCR, ELISA, and immunofluorescence analyses revealed increased expression and release of endothelin-1 in End.LepR-deficient and LepR-resistant cells, and ET receptor A/B antagonists abrogated their paracrine effects on murine aortic smooth muscle cell proliferation. Reduced expression of peroxisome proliferator-activated receptor-γ and increased nuclear activator protein-1 staining was observed in End.LepR-deficient and LepR-resistant cells, and peroxisome proliferator-activated receptor-γ antagonization increased endothelial endothelin-1 expression. CONCLUSIONS: Our findings suggest that intact endothelial leptin signaling limits neointima formation and that obesity represents a state of endothelial leptin resistance. These observations and the identification of endothelin-1 as soluble mediator of the cardiovascular risk factor obesity may have relevant therapeutic implications.

Endothelial deletion of protein tyrosine phosphatase-1B protects against pressure overload-induced heart failure in mice.

AIMS: Cardiac angiogenesis is an important determinant of heart failure. We examined the hypothesis that protein tyrosine phosphatase (PTP)-1B, a negative regulator of vascular endothelial growth factor (VEGF) receptor-2 activation, is causally involved in the cardiac microvasculature rarefaction during hypertrophy and that deletion of PTP1B in endothelial cells prevents the development of heart failure. METHODS AND RESULTS: Cardiac hypertrophy was induced by transverse aortic constriction (TAC) in mice with endothelial-specific deletion of PTP1B (End.PTP1B-KO) and controls (End.PTP1B-WT). Survival up to 20 weeks after TAC was significantly improved in mice lacking endothelial PTP1B. Serial echocardiography revealed a better systolic pump function, less pronounced cardiac hypertrophy, and left ventricular dilation compared with End.PTP1B-WT controls. Histologically, banded hearts from End.PTP1B-KO mice exhibited increased numbers of PCNA-positive, proliferating endothelial cells resulting in preserved cardiac capillary density and improved perfusion as well as reduced hypoxia, apoptotic cell death, and fibrosis. Increased relative VEGFR2 and ERK1/2 phosphorylation and greater eNOS expression were present in the hearts of End.PTP1B-KO mice. The absence of PTP1B in endothelial cells also promoted neovascularization following peripheral ischaemia, and bone marrow transplantation excluded a major contribution of Tie2-positive haematopoietic cells to the improved angiogenesis in End.PTP1B-KO mice. Increased expression of caveolin-1 as well as reduced NADPH oxidase-4 expression, ROS generation and TGFß signalling were observed and may have mediated the cardioprotective effects of endothelial PTP1B deletion. CONCLUSIONS: Endothelial PTP1B deletion improves cardiac VEGF signalling and angiogenesis and protects against chronic afterload-induced heart failure. PTP1B may represent a useful target to preserve cardiac function during hypertrophy.

The Protein Interaction of RNA Helicase B (RhlB) and Polynucleotide Phosphorylase (PNPase) Contributes to the Homeostatic Control of Cysteine in Escherichia coli.

PNPase, one of the major enzymes with 3' to 5' single-stranded RNA degradation and processing activities, can interact with the RNA helicase RhlB independently of RNA degradosome formation in Escherichia coli. Here, we report that loss of interaction between RhlB and PNPase impacts cysteine homeostasis in E. coli. By random mutagenesis, we identified a mutant RhlB(P238L) that loses 75% of its ability to interact with PNPase but retains normal interaction with RNase E and RNA, in addition to exhibiting normal helicase activity. Applying microarray analyses to an E. coli strain with impaired RNA degradosome formation, we investigated the biological consequences of a weakened interaction between RhlB and PNPase. We found significant increases in 11 of 14 genes involved in cysteine biosynthesis. Subsequent Northern blot analyses showed that the up-regulated transcripts were the result of stabilization of the cysB transcript encoding a transcriptional activator for the cys operons. Furthermore, Northern blots of PNPase or RhlB mutants showed that RhlB-PNPase plays both a catalytic and structural role in regulating cysB degradation. Cells expressing the RhlB(P238L) mutant exhibited an increase in intracellular cysteine and an enhanced anti-oxidative response. Collectively, this study suggests a mechanism by which bacteria use the PNPase-RhlB exosome-like complex to combat oxidative stress by modulating cysB mRNA degradation.

Circulating Endothelial Cells Expressing the Angiogenic Transcription Factor Krüppel-Like Factor 4 are Decreased in Patients with Coronary Artery Disease.

OBJECTIVE: The zinc finger transcription factor KLF4 is known to control diverse EC functions. METHODS: The functional role of KLF4 for angiogenesis and its association with CAD was examined in HUVECs and human CECs. RESULTS: In two different angiogenesis assays, siRNA-mediated KLF4 downregulation impaired HUVEC sprouting and network formation. Conversely, KLF4 overexpression increased HUVEC sprouting and network formation. Similar findings were observed after incubation of HUVECs with CdM from KLF4 cDNA-transfected cells, suggesting a role of paracrine factors for mediating angiogenic KLF4 effects. In this regard, VEGF expression was increased in KLF4-overexpressing HUVECs, whereas its expression was reduced in HUVECs transfected with KLF4 siRNA. To examine the relevance of our in vitro findings for human endothelial dysfunction, we analyzed the expression of KLF4 in CECs of patients with stable CAD. Flow cytometry analyses revealed decreased numbers of KLF4-positive CECs in peripheral blood from CAD patients compared to healthy controls. CONCLUSIONS: Our findings suggest that KLF4 may represent a potential biomarker for EC dysfunction. In the future, (therapeutic) modulation of KLF4 may be useful in regulating EC function during vascular disease processes.

Endothelial p53 deletion improves angiogenesis and prevents cardiac fibrosis and heart failure induced by pressure overload in mice.

BACKGROUND: Cardiac dysfunction developing in response to chronic pressure overload is associated with apoptotic cell death and myocardial vessel rarefaction. We examined whether deletion of tumor suppressor p53 in endothelial cells may prevent the transition from cardiac hypertrophy to heart failure. METHODS AND RESULTS: Mice with endothelial-specific deletion of p53 (End.p53-KO) were generated by crossing p53fl/fl mice with mice expressing Cre recombinase under control of an inducible Tie2 promoter. Cardiac hypertrophy was induced by transverse aortic constriction. Serial echocardiography measurements revealed improved cardiac function in End.p53-KO mice that also exhibited better survival. Cardiac hypertrophy was associated with increased p53 levels in End.p53-WT controls, whereas banded hearts of End.p53-KO mice exhibited lower numbers of apoptotic endothelial and non-endothelial cells and altered mRNA levels of genes regulating cell cycle progression (p21), apoptosis (Puma), or proliferation (Pcna). A higher cardiac capillary density and improved myocardial perfusion was observed, and pharmacological inhibition or genetic deletion of p53 also promoted endothelial sprouting in vitro and new vessel formation following hindlimb ischemia in vivo. Hearts of End.p53-KO mice exhibited markedly less fibrosis compared with End.p53-WT controls, and lower mRNA levels of p53-regulated genes involved in extracellular matrix production and turnover (eg, Bmp-7, Ctgf, or Pai-1), or of transcription factors involved in controlling mesenchymal differentiation were observed. CONCLUSIONS: Our analyses reveal that accumulation of p53 in endothelial cells contributes to blood vessel rarefaction and fibrosis during chronic cardiac pressure overload and suggest that endothelial cells may be a therapeutic target for preserving cardiac function during hypertrophy.