Expression of ADAMTS1 in endothelial cells is induced by shear stress and suppressed in sprouting capillaries

ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.

Cancer therapy modulates VEGF signaling and viability in adult rat cardiac microvascular endothelial cells and cardiomyocytes

This work was motivated by the incomplete characterization of the role of vascular endothelial growth factor-A (VEGF-A) in the stressed heart in consideration of upcoming cancer treatment options challenging the natural VEGF balance in the myocardium. We tested, if the cytotoxic cancer therapy doxorubicin (Doxo) or the anti-angiogenic therapy sunitinib alters viability and VEGF signaling in primary cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). ARVM were isolated and cultured in serum-free medium. CMEC were isolated from the left ventricle and used in the second passage. Viability was measured by LDH-release and by MTT-assay, cellular respiration by high-resolution oxymetry. VEGF-A release was measured using a rat specific VEGF-A ELISA-kit. CMEC were characterized by marker proteins including CD31, von Willebrand factor, smooth muscle actin and desmin. Both Doxo and sunitinib led to a dose-dependent reduction of cell viability. Sunitinib treatment caused a significant reduction of complex I and II-dependent respiration in cardiomyocytes and the loss of mitochondrial membrane potential in CMEC. Endothelial cells up-regulated VEGF-A release after peroxide or Doxo treatment. Doxo induced HIF-1α stabilization and upregulation at clinically relevant concentrations of the cancer therapy. VEGF-A release was abrogated by the inhibition of the Erk1/2 or the MAPKp38 pathway. ARVM did not answer to Doxo-induced stress conditions by the release of VEGF-A as observed in CMEC. VEGF receptor 2 amounts were reduced by Doxo and by sunitinib in a dose-dependent manner in both CMEC and ARVM. In conclusion, these data suggest that cancer therapy with anthracyclines modulates VEGF-A release and its cellular receptors in CMEC and ARVM, and therefore alters paracrine signaling in the myocardium.

Dimethyloxalylglycine stabilizes HIF-1α in cultured human endothelial cells and increases random-pattern skin flap survival in vivo

The goal of this study was to evaluate in vitro and in vivo the effects of up-regulation of the proangiogenic hypoxia inducible factor (HIF)-1α induced by dimethyloxalylglycine on endothelial cell cultures and on skin flap survival.

Susceptibility of primary human endothelial cells to C. perfringens beta-toxin suggesting similar pathogenesis in human and porcine necrotizing enteritis

Clostridium perfringens type C causes fatal necrotizing enteritis in different mammalian hosts, most commonly in newborn piglets. Human cases are rare, but the disease, also called pigbel, was endemic in the Highlands of Papua New Guinea. Lesions in piglets and humans are very similar and characterized by segmental necro-hemorrhagic enteritis in acute cases and fibrino-necrotizing enteritis in subacute cases. Histologically, deep mucosal necrosis accompanied by vascular thrombosis and necrosis was consistently reported in naturally affected pigs and humans. This suggests common pathogenetic mechanisms. Previous in vitro studies using primary porcine aortic endothelial cells suggested that beta-toxin (CPB) induced endothelial damage contributes to the pathogenesis of C. perfringens type C enteritis in pigs. In the present study we investigated toxic effects of CPB on cultured primary human macro- and microvascular endothelial cells. In vitro, these cells were highly sensitive to CPB and reacted with similar cytopathic and cytotoxic effects as porcine endothelial cells. Our results indicate that porcine and human cell culture based in vitro models represent valuable tools to investigate the pathogenesis of this bacterial disease in animals and humans.

Rapid cytopathic effects of Clostridium perfringens beta-toxin on porcine endothelial cells

Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C beta-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis.

Shear stress modulates the expression of thrombospondin-1 and CD36 in endothelial cells in vitro and during shear stress-induced angiogenesis in vivo

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/l drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress downregulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

Rapid endocytosis of copper-zinc superoxide dismutase into human endothelial cells: role for its vascular activity

Cytosolic CuZn-SOD (SOD1) is a dimeric, carbohydrate-free enzyme with a molecular weight of about 32 kDa and also circulates in human blood plasma. Due to its molecular mass it has been believed that the enzyme cannot penetrate the cell membrane. Here we report that rapid endocytosis of FITC-CuZn-SOD into human endothelial cells occurs within 5 min. Moreover, relaxation of rat aortic rings in response to CuZn-SOD is associated with a lag time of 45-60 s and only observed in the presence of intact endothelial cells. The results indicate acute and rapid endothelial cell endocytosis of CuZn-SOD, possibly via activation of a receptor-mediated pathway. Intracellular uptake via endocytosis may contribute to the vascular effects of CuZn-SOD, including vasodilation, and is likely to play a role in regulation of vascular tone and diseases such as atherosclerosis.

Semaphorins guide PerPlexeD endothelial cells

Abrogation of the endothelial cell-specific semaphorin receptor PlexinD1 in zebrafish and mouse reveals semaphorin functions selectively affecting the cardiovascular system. Neuropilins team up with PlexinD1 to form a novel endothelial receptor complex for class 3 semaphorins.

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.

Sphingosine kinase-1 is a hypoxia-regulated gene that stimulates migration of human endothelial cells

Sphingosine kinases (SK) catalyze the production of sphingosine-1-phosphate which in turn regulates cell responses such as proliferation and migration. Here, we show that exposure of the human endothelial cell line EA.hy 926 to hypoxia stimulates a increased SK-1, but not SK-2, mRNA, protein expression, and activity. This effect was due to stimulated SK-1 promoter activity which contains two putative hypoxia-inducible factor-responsive-elements (HRE). By deletion of one of the two HREs, hypoxia-induced promoter activation was abrogated. Furthermore, hypoxia upregulated the expression of HIF-1alpha and HIF-2alpha, and both contributed to SK-1 gene transcription as shown by selective depletion of HIF-1alpha or HIF-2alpha by siRNA. The hypoxia-stimulated SK-1 upregulation was functionally coupled to increased migration since the selective depletion of SK-1, but not of SK-2, by siRNAs abolished the migratory response. In summary, these data show that hypoxia upregulates SK-1 activity and results in an accelerated migratory capacity of endothelial cells. SK-1 may thus serve as an attractive therapeutic target to treat diseases associated with increased endothelial migration and angiogenesis such as cancer growth and progression.

Reactivity of human natural antibodies to endothelial cells from Galalpha(1,3)Gal-deficient pigs

BACKGROUND: Xenoreactive human natural antibodies (NAb) are predominantly directed against galactose-alpha(1,3)galactose (Gal). Binding of immunoglobulin (Ig) G and IgM NAb activates porcine endothelial cells (pEC) and triggers complement lysis responsible for hyperacute xenograft rejection. In vitro, IgG NAb induce human natural killer (NK) cell-mediated lysis of pEC by antibody-dependent cell-mediated cytotoxicity (ADCC). The present study examined the levels of anti-porcine NAb in a large number of individuals and addressed the functional role of non-Gal anti-porcine NAb. METHODS: Sera from 120 healthy human blood donors were analyzed for the presence of anti-porcine NAb by flow cytometry using porcine red blood cells (pRBC), lymphoblastoid cells (pLCL), and pEC derived from control or Gal-deficient pigs. Xenogeneic complement lysis was measured by flow cytometry using human serum and rabbit complement. ADCC was analyzed by chromium-release assays using human serum and freshly isolated NK cells. RESULTS: Human IgM binding to pRBC was found in 93% and IgG binding in 86% of all samples. Non-Gal NAb comprised 13% of total IgM and 36% of total IgG binding to pEC. NAb/complement-induced lysis and ADCC of Gal-deficient compared to Gal-positive pEC were 21% and 29%, respectively. The majority of anti-Gal and non-Gal IgG NAb were of the IgG2 subclass. CONCLUSIONS: The generation of Gal-deficient pigs has overcome hyperacute anti-Gal-mediated xenograft rejection in nonhuman primates. Non-Gal anti-porcine NAb represent a potentially relevant immunological hurdle in a subgroup of individuals by inducing endothelial damage in xenografts.

Anti-HLA I antibodies induce VEGF production by endothelial cells, which increases proliferation and paracellular permeability

Anti-human leukocyte antigen class I (HLA I) antibodies were shown to activate several protein kinases in endothelial cells (ECs), which induces proliferation and cell survival. An important phenomenon in antibody-mediated rejection is the occurrence of interstitial edema. We investigated the effect of anti-HLA I antibodies on endothelial proliferation and permeability, as one possible underlying mechanism of edema formation. HLA I antibodies increased the permeability of cultured ECs isolated from umbilical veins. Anti-HLA I antibodies induced the production of vascular endothelial growth factor (VEGF) by ECs, which activated VEGF receptor 2 (VEGFR2) in an autocrine manner. Activated VEGFR2 led to a c-Src-dependent phosphorylation of vascular endothelial (VE)-cadherin and its degradation. Aberrant VE-cadherin expression resulted in impaired adherens junctions, which might lead to increased endothelial permeability. This effect was only observed after cross-linking of HLA I molecules by intact antibodies. Furthermore, our results suggest that increased endothelial proliferation following anti-HLA I treatment occurs via autocrine VEGFR2 activation. Our data indicate the ability of anti-HLA I to induce VEGF production in ECs. Transactivation of VEGFR2 leads to increased EC proliferation and paracellular permeability. The autocrine effect of VEGF on endothelial permeability might be an explanation for the formation of interstitial edema after transplantation.

Lack of galactose-alpha-1,3-galactose expression on porcine endothelial cells prevents complement-induced lysis but not direct xenogeneic NK cytotoxicity

The galactose-alpha-1,3-galactose (alphaGal) carbohydrate epitope is expressed on porcine, but not human cells, and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models, NAb binding to porcine endothelial cells will likely induce complement activation, lysis, and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts, either by direct recognition of activating molecules on target cells or by FcgammaRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of alphaGal protects porcine endothelial cells from NAb/complement-induced lysis, direct xenogeneic NK lysis, NAb-dependent ADCC, and adhesion of human NK cells under shear stress. Homologous recombination, panning, and limiting dilution cloning were used to generate an alphaGal-negative porcine endothelial cell line, PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the alphaGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However, direct xenogeneic lysis of PED2*3.51, mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92, was not reduced. Furthermore, adhesion of IL-2-activated human NK cells did not rely on alphaGal expression. In conclusion, removal of alphaGal leads to a clear reduction in complement-induced lysis and ADCC, but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity, indicating that alphaGal is not a dominant target for direct human NK cytotoxicity against porcine cells.