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Results for “Endothelialization”

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PubMed1999

Hepatocyte growth factor increases expression of vascular endothelial growth factor and plasminogen activator inhibitor-1 in human keratinocytes and the vascular endothelial growth factor receptor flk-1 in human endothelial cells.

The pleiotropic growth factor hepatocyte growth factor/scatter factor (HGF/SF) has been implicated by clinical and experimental studies in repair mechanisms in different organs and tissues. However, no data on the impact of HGF/SF in wound healing in the skin are yet available. Proliferating and migrating keratinocytes play a major role in repair processes in the skin by closing the wound. Recent evidence gathered from studies that used gene-deficient mice has implicated the plasminogen activator (PA)/plasmin system in wound healing, which depends on controlled matrix degradation and deposition during cell migration and proliferation. Furthermore, keratinocytes are an important source of vascular endothelial growth factor (VEGF), which is a potent inducer of angiogenesis. In this study, we show that in human keratinocytes HGF/SF but not the related cytokine macrophage stimulating protein (MSP) significantly increases expression of VEGF and plasminogen activator inhibitor-1 (PAI-1) on the level of protein and mRNA. Furthermore, we demonstrate that HGF/SF increases the expression of the VEGF receptor flk-1 in human endothelial cells and that, in an angiogenesis co-culture assay of endothelial cells and keratinocytes, HGF/SF increases endothelial cell tube formation significantly. Therefore, we propose a role for HGF/SF in wound repair in the skin: HGF/SF--produced by activated fibroblasts--increases in keratinocytes the expression of PAI-1, which leads to increased matrix stability during the repair process and which could also limit activation of HGF/SF by proteases such as urokinase-type PA (u-PA) or tissue-type PA (t-PA). Furthermore HGF/SF also increases the expression of VEGF in these cells, thereby initiating angiogenesis in a paracrine manner. This effect would be enhanced by an increased responsiveness of endothelial cells toward VEGF, resulting from the HGF/SF-induced up-regulation of flk-1 on these cells.

Animals↗

PubMed2001

Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema.

Emphysema due to cigarette smoking is characterized by a loss of alveolar structures. We hypothesize that the disappearance of alveoli involves apoptosis of septal endothelial cells and a decreased expression of lung vascular endothelial growth factor (VEGF) and its receptor 2 (VEGF R2). By terminal transferase dUTP nick end labeling (TUNEL) in combination with immunohistochemistry, we found that the number of TUNEL+ septal epithelial and endothelial cells/lung tissue nucleic acid (microg) was increased in the alveolar septa of emphysema lungs (14.2 +/- 2.0/microg, n = 6) when compared with normal lungs (6.8 +/- 1.3/microg, n = 7) (p < 0.01) and with primary pulmonary hypertensive lungs (2.3 +/- 0.8/microg, n = 5) (p < 0.001). The cell death events were not significantly different between healthy nonsmoker (7.4 +/- 1.9/microg) and smoker (5.7 +/- 0.7/microg) control subjects. The TUNEL results were confirmed by single-stranded DNA and active caspase-3 immunohistochemistry, and by DNA ligation assay. Emphysema lungs (n = 12) had increased levels of oligonucleosomal-length DNA fragmentation when compared with normal lungs (n = 11). VEGF, VEGF R2 protein, and mRNA expression were significantly reduced in emphysema. We propose that epithelial and endothelial alveolar septal death due to a decrease of endothelial cell maintenance factors may be part of the pathogenesis of emphysema.

Adolescent↗

PubMed2005

Stoichiometric relationships between endothelial tetrahydrobiopterin, endothelial NO synthase (eNOS) activity, and eNOS coupling in vivo: insights from transgenic mice with endothelial-targeted GTP cyclohydrolase 1 and eNOS overexpression.

Endothelial dysfunction in vascular disease states is associated with reduced NO bioactivity and increased superoxide (O2*-) production. Some data suggest that an important mechanism underlying endothelial dysfunction is endothelial NO synthase (eNOS) uncoupling, whereby eNOS generates O2*- rather than NO, possibly because of a mismatch between eNOS protein and its cofactor tetrahydrobiopterin (BH4). However, the mechanistic relationship between BH4 availability and eNOS coupling in vivo remains undefined because no studies have investigated the regulation of eNOS by BH4 in the absence of vascular disease states that cause pathological oxidative stress through multiple mechanisms. We investigated the stoichiometry of BH4-eNOS interactions in vivo by crossing endothelial-targeted eNOS transgenic (eNOS-Tg) mice with mice overexpressing endothelial GTP cyclohydrolase 1 (GCH-Tg), the rate-limiting enzyme in BH4 synthesis. eNOS protein was increased 8-fold in eNOS-Tg and eNOS/GCH-Tg mice compared with wild type. The ratio of eNOS dimer:monomer was significantly reduced in aortas from eNOS-Tg mice compared with wild-type mice but restored to normal in eNOS/GCH-Tg mice. NO synthesis was elevated by 2-fold in GCH-Tg and eNOS-Tg mice but by 4-fold in eNOS/GCH-Tg mice compared with wild type. Aortic BH4 levels were elevated in GCH-Tg and maintained in eNOS/GCH-Tg mice but depleted in eNOS-Tg mice compared with wild type. Aortic and cardiac O2*- production was significantly increased in eNOS-Tg mice compared with wild type but was normalized after NOS inhibition with Nomega-nitro-L-arginine methyl ester hydrochloride (L-NAME), suggesting O2*- production by uncoupled eNOS. In contrast, in eNOS/GCH-Tg mice, O2*- production was similar to wild type, and L-NAME had no effect, indicating preserved eNOS coupling. These data indicate that eNOS coupling is directly related to eNOS-BH4 stoichiometry even in the absence of a vascular disease state. Endothelial BH4 availability is a pivotal regulator of eNOS activity and enzymatic coupling in vivo.

Animals↗

PubMed2003

Quantitative evaluation of endothelial progenitors and cardiac valve endothelial cells: proliferation and differentiation on poly-glycolic acid/poly-4-hydroxybutyrate scaffold in response to vascular endothelial growth factor and transforming growth factor beta1.

Three-dimensional scaffolds made of bioabsorbable polymeric constituents are currently being tested for use in tissue engineering of various tissues. A composite scaffold of poly-glycolic acid (PGA) non-woven mesh dip-coated in a 1% solution of poly-4-hydroxybutyrate (P4HB) was shown to be suitable as a scaffold for creation of tissue-engineered trileaflet pulmonic valve replacements in sheep [Hoerstrup, S.P., et al., Circulation 102(Suppl. 3), III44, 2000]. However, little is known about how cells seeded on PGA/P4HB respond in vitro to soluble factors supplied in the culture medium. To optimize tissue development in vitro, before implantation, we set out to develop quantitative biochemical assays to measure how cells seeded on PGA/P4HB respond to growth and differentiation factors. Herein we show that ovine aortic valvular endothelial cells and circulating endothelial progenitor cells (EPCs) seeded onto PGA/P4HB proliferate in response to vascular endothelial growth factor and transdifferentiate to a mesenchymal phenotype in response to transforming growth factor beta(1). Transdifferentiation from an endothelial to mesenchymal phenotype is a critical step during embryonic development of cardiac valves. Our results demonstrate that valvular endothelial cells and EPCs isolated from peripheral blood can recapitulate critical developmental steps on PGA/P4HB. These results demonstrate that PGA/P4HB provides a conducive environment for cellular proliferation, differentiation, and tissue development.

Cell Differentiation↗

PubMed2002

Analysis of coagulation cascade and endothelial cell activation during inhibition of vascular endothelial growth factor/vascular endothelial growth factor receptor pathway in cancer patients.

OBJECTIVE: The angiogenesis inhibitor SU5416 is a potent inhibitor of vascular endothelial growth factor (VEGF) receptor-1 and -2. VEGF may be involved in hemostasis by altering the hemostatic properties of endothelial cells. We analyzed the effects of SU5416 on the coagulation cascade and the vessel wall in patients with advanced cancer. METHODS AND RESULTS: Markers for thrombin generation, activation of the protein C pathway, fibrinolysis, and endothelial cell activation were measured in patients with renal cell carcinoma, soft tissue sarcoma, or melanoma on days 0, 14, and 28 of treatment with SU5416. Three of 17 sampled patients developed a thromboembolic event in the fifth week of treatment. Markers for thrombin generation and fibrinolysis did not show significant changes. We observed a significant increase in endogenous thrombin potential and of parameters reflecting endothelial cell activation (von Willebrand antigen, soluble tissue factor, and soluble E-selectin) in all patients (P< or =0.001). In patients experiencing a thromboembolic event, endogenous thrombin potential, soluble tissue factor, and soluble E-selectin increased to a significantly greater extent (P=0.029, P=0.021, and P=0.007, respectively). CONCLUSIONS: VEGF is not only a permeability, proliferation, and migration factor, but it is also a maintenance and protection factor for endothelial cells.

Angiogenesis Inhibitors↗

PubMed2006

Circulating endothelial cells, endothelial progenitor cells, and endothelial microparticles in cancer.

Cancer, a proliferative disease hallmarked by abnormal cell growth and spread, is largely dependent on tumor neoangiogenesis, with evidence of vascular endothelial dysfunction. Novel ways to assess vascular function in cancer include measuring levels of circulating endothelial cells (CEC). Rare in healthy individuals, increased CEC in peripheral blood reflects significant vascular damage and dysfunction. They have been documented in many human diseases, including different types of cancers. An additional circulating cell population are endothelial progenitor cells (EPC), which have the ability to form endothelial colonies in vitro and may contribute toward vasculogenesis. At present, there is great interest in evaluating the role of EPC as novel markers for tumor angiogenesis and drug therapy monitoring. Recently, exocytic procoagulant endothelial microparticles (EMP) have also been identified. CEC, EPC, and EMP research works may have important clinical implications but are often impeded by methodological issues and a lack of consensus on phenotypic identification of these cells and particles. This review aims to collate existing literature and provide an overview on the current position of CEC, EPC, and EMP in cell biology terms and to identify their significance to clinical medicine, with particular emphasis on relationship with cancer.

Apoptosis↗

PubMed2002

Anti-vascular endothelial growth factor receptor 2 antibody reduces tumorigenicity and metastasis in orthotopic prostate cancer xenografts via induction of endothelial cell apoptosis and reduction of endothelial cell matrix metalloproteinase type 9 production.

PURPOSE: Vascular endothelial growth factor (VEGF), which is produced by tumor cells, is a potent endothelial cell mitogen. The aim of the present study was to evaluate the response of orthotopic prostate cancer xenografts and prostate cancer bone metastasis to anti-VEGF receptor (flk-1) antibody (DC101) treatment. EXPERIMENTAL DESIGN: Orthotopic prostate cancer models (PC-3M-MM2 and LNCaP-LN3 prostate carcinoma cells) and a prostate cancer bone metastasis model (PC-3M-MM2) were used for these experiments. Early and established tumors were treated with saline, paclitaxel, DC101, or a DC101-plus-paclitaxel combination for 5 weeks (PC-3M-MM2) and 12 weeks (LNCaP-LN3). At the end of therapy, tumors were removed and weighed. Apoptosis, tumor cell proliferation, and angiogenesis- and metastasis-related gene expression were evaluated using immunohistochemistry, in situ hybridization, and terminal deoxynucleotidyl transferase-ediated nick end labeling (TUNEL). RESULTS: After treatment of early tumors (PC-3M-MM2), median prostate tumor weights (+/-SE) were 1230 +/- 210 mg in untreated controls, 482 +/- 121 mg in mice treated with paclitaxel (P = 0.009), 148 +/- 27 mg in mice treated with DC101 (P < 0.001), and 48 +/- 10 mg in mice treated with the combination of DC101 and paclitaxel (P < 0.001). Lymph node metastasis occurred in 7 of the 9 control mice, 5 of the 9 paclitaxel-treated animals, 5 of the 12 DC101-treated animals, and 2 of the 11 animals in the combination therapy group. Treatment with DC101 alone or in combination with paclitaxel reduced tumor-induced neovascularity measured by microvessel density and tumor cell proliferation (by proliferating cell nuclear antigen) and enhanced apoptosis (measured by TUNEL) in tumor cells and endothelial cells compared with controls. In the tibial prostate cancer metastasis model, significant inhibition of tumor growth was observed. In the LNCaP-LN3 orthotopic prostate cancer model, tumors occurred in 7 of the 10 control mice, 4 of the 10 paclitaxel-treated animals, 5 of the 10 DC101-treated animals, and 2 of the 11 animals in the combination therapy group (P < 0.05). The efficacy of DC101 was much greater in the treatment of early tumors, which suggests that tumor burden may be a critical factor in determining the response to DC101. In vitro and in vivo analysis of endothelial cell function showed reduced matrix metalloproteinase type 9 production in endothelial cells treated with DC101. CONCLUSIONS: This study confirms the principle of tumor growth inhibition by targeting angiogenesis within tumors and supports the use of anti-VEGF receptor agents.

Animals↗

PubMed2001

Hsp90 ensures the transition from the early Ca2+-dependent to the late phosphorylation-dependent activation of the endothelial nitric-oxide synthase in vascular endothelial growth factor-exposed endothelial cells.

Vascular endothelial growth factor (VEGF) exerts its angiogenic effects partly through the activation of endothelial nitric-oxide synthase (eNOS). Association with heat shock protein 90 (hsp90) and phosphorylation by Akt were recently shown to separately activate eNOS upon VEGF stimulation in endothelial cells. Here, we examined the interplay between these different mechanisms in VEGF-exposed endothelial cells. We documented that hsp90 binding to eNOS is, in fact, the crucial event triggering the transition from the Ca(2+)-dependent activation of eNOS to the phosphorylation-mediated potentiation of its activity by VEGF. Accordingly, we showed that early VEGF stimulation first leads to the Ca(2+)/calmodulin disruption of the caveolin-eNOS complex and promotes the association between eNOS and hsp90. eNOS-bound hsp90 can then recruit VEGF-activated (phosphorylated) Akt to the complex, which in turn can phosphorylate eNOS. Further experiments in transfected COS cells expressing either wild-type or S1177A mutant eNOS led us to identify the serine 1177 as the critical residue for the hsp90-dependent Akt-mediated activation of eNOS. Finally, we documented that although the VEGF-induced phosphorylation of eNOS leads to a sustained production of NO independently of a maintained increase in [Ca(2+)](i), this late stage of eNOS activation is strictly conditional on the initial VEGF-induced Ca(2+)-dependent stimulation of the enzyme. These data establish the critical temporal sequence of events leading to the sustained activation of eNOS by VEGF and suggest new ways of regulating the production of NO in response to this cytokine through the ubiquitous chaperone protein, hsp90.

Animals↗

PubMed1997

Endothelial-dependent mechanisms regulate leukocyte transmigration: a process involving the proteasome and disruption of the vascular endothelial-cadherin complex at endothelial cell-to-cell junctions.

Although several adhesion molecules expressed on leukocytes (beta1 and beta2 integrins, platelet endothelial cell adhesion molecule 1 [PECAM-1], and CD47) and on endothelium (intercellular adhesion molecule 1, PECAM-1) have been implicated in leukocyte transendothelial migration, less is known about the role of endothelial lateral junctions during this process. We have shown previously (Read, M.A., A.S. Neish, F.W. Luscinskas, V.J. Palambella, T. Maniatis, and T. Collins. 1995. Immunity. 2:493-506) that inhibitors of the proteasome reduce lymphocyte and neutrophil adhesion and transmigration across TNF-alpha-activated human umbilical vein endothelial cell (EC) monolayers in an in vitro flow model. The current study examined EC lateral junction proteins, principally the vascular endothelial (VE)-cadherin complex and the effects of proteasome inhibitors (MG132 and lactacystin) on lateral junctions during leukocyte adhesion, to gain a better understanding of the role of EC junctions in leukocyte transmigration. Both biochemical and indirect immunofluorescence analyses of the adherens junction zone of EC monolayers revealed that neutrophil adhesion, not transmigration, induced disruption of the VE-cadherin complex and loss of its lateral junction localization. In contrast, PECAM-1, which is located at lateral junctions and is implicated in neutrophil transmigration, was not altered. These findings identify new and interrelated endothelial-dependent mechanisms for leukocyte transmigration that involve alterations in lateral junction structure and a proteasome-dependent event(s).

Acetylcysteine↗

PubMed2005

Differential effects of vascular endothelial growth factor receptor-2 inhibitor ZD6474 on circulating endothelial progenitors and mature circulating endothelial cells: implications for use as a surrogate marker of antiangiogenic activity.

PURPOSE: Circulating endothelial cells (CEC) comprise at least two distinct populations: bone marrow-derived circulating endothelial progenitors (CEP) and mature CECs derived from existing vasculature. We hypothesized that antiangiogenic agents may have differential effects on CEPs and mature CECs and that these changes may serve as a marker of biological activity. EXPERIMENTAL DESIGN: The effect of angiogenesis inhibitors on CECs was evaluated by flow cytometry after vascular endothelial growth factor (VEGF)-induced mobilization and in mice bearing Lewis lung carcinoma (LLC). Tumor angiogenesis was evaluated in parallel by immunohistochemistry. RESULTS: In nontumor-bearing mice, VEGF administration increased both mature CECs and CEPs. This increase was inhibited by the VEGF receptor 2 inhibitor ZD6474 as well as the VEGF inhibitor-soluble Flt-1. ZD6474 had no significant effect on CECs in the absence of exogenous VEGF stimulation. In contrast, LLC-bearing mice had an increase in mature CECs but not CEPs after 3 days of treatment with ZD6474. The increase in mature CECs was dose-dependent, accompanied by a decrease in tumor microvessel density, and preceded reduction in tumor volume. Treatment of LLC-bearing mice with the vascular targeting agent ZD6126 also increased mature CECs. CONCLUSIONS: VEGF inhibitors can have differential effects on mature CECs and CEPs, and agents inhibiting tumor angiogenesis may cause a concomitant increase in mature CECs. This increase occurs in tumor-bearing but not in nontumor-bearing mice, suggesting that tumor endothelium is a potential source of mature CECs. Therefore, assessing both mature CECs and CEPs may provide insights into the mechanism of antiangiogenic agents and serve as an early surrogate marker of biological activity.

Animals↗

PubMed2002

Vascular endothelial growth factor induces SHC association with vascular endothelial cadherin: a potential feedback mechanism to control vascular endothelial growth factor receptor-2 signaling.

Vascular endothelial (VE)-cadherin is endothelium specific, mediates homophilic adhesion, and is clustered at intercellular junctions. VE-cadherin is required for normal development of the vasculature in the embryo and for angiogenesis in the adult. Here, we report that VE-cadherin is associated with VE growth factor (VEGF) receptor-2 (VEGFR-2) on the exposure of endothelial cells to VEGF. The binding parallels receptor phosphorylation on tyrosine residues, which is maximal at 5 minutes and then declines within 30 minutes. Tyrosine phosphorylation of VE-cadherin was maximal at 30 minutes after the addition of the growth factor. At this time point, the protein could be coimmunoprecipitated with the adaptor protein Shc. Pull-down experiments with different Shc domains and mutants of the VE-cadherin cytoplasmic tail have shown that Shc binds to the carboxy-terminal domain of the VE-cadherin tail through its Src homology 2 domain (SH2). We found that Shc phosphorylation lasts longer in endothelial cells carrying a targeted null mutation in the VE-cadherin gene than in VE-cadherin-positive cells. These data suggest that VE-cadherin expression exerts a negative effect on Shc phosphorylation by VEGFR-2. We speculate that VE-cadherin binding to Shc promotes its dephosphorylation through associated phosphatases.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Rapid transactivation of the vascular endothelial growth factor receptor KDR/Flk-1 by the bradykinin B2 receptor contributes to endothelial nitric-oxide synthase activation in cardiac capillary endothelial cells.

Bradykinin (BK) and vascular endothelial growth factor (VEGF)-165 stimulate vasodilatation, microvascular permeability, and angiogenesis via the activation of the B2-type and KDR/Flk-1 receptors. To delineate the signal transduction pathways distal to the receptor activation in microvascular permeability, we compared their effects on two downstream targets, i.e. endothelial nitric-oxide (NO) synthase (eNOS) and F-actin, in primary cultures of cardiac capillary endothelial cells. The two mediators induced a similar cytoskeletal reorganization and both the translocation and activation of eNOS, leading to NO release within the first minutes of cell exposure. At the same time, BK produced the tyrosine phosphorylation and internalization of KDR/Flk-1 as did VEGF itself. This transactivation was blocked by the selective inhibitor of VEGF receptor tyrosine kinase activity but not by inhibitors of epidermal growth factor receptor or protein kinase C activity. The selective inhibitor of VEGF receptor tyrosine kinase activity totally prevented the effects of VEGF but only partially inhibited NO release induced by BK without affecting the concomitant cytoskeletal reorganization. Thus, BK transactivated KDR/Flk-1 through an intrinsic kinase activity of KDR/Flk-1, resulting in a further eNOS activation in endothelial cells. This represents a novel mechanism whereby a G protein-coupled receptor activates a receptor tyrosine kinase to generate biological response.

Animals↗

PubMed1997

Endothelial cells as target for antiphospholipid antibodies. Human polyclonal and monoclonal anti-beta 2-glycoprotein I antibodies react in vitro with endothelial cells through adherent beta 2-glycoprotein I and induce endothelial activation.

OBJECTIVE: To investigate the ability of human anti-beta 2-glycoprotein I (anti-beta 2 GPI) antibodies to recognize the cofactor adherent on endothelial cells (EC) and to modulate endothelial functions. METHODS: Six human affinity-purified polyclonal anti-beta 2 GPI IgG and 2 IgM monoclonal antibodies (MAb) were obtained from patients with the antiphospholipid syndrome. The antibodies were tested for their ability to 1) bind to endothelial monolayers through the adherent beta 2 GPI and 2) modulate endothelial adhesion molecule expression and interleukin-6 (IL-6) and 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) secretion. RESULTS: The affinity-purified IgG and the MAb with anti-beta 2 GPI activity, but not the respective controls, displayed EC binding, which declined on cells incubated in serum-free medium and was restored in a dose-dependent manner by exogenous human beta 2 GPI. After EC binding, both polyclonal and monoclonal antibodies up-regulated adhesion molecule expression. Anti-beta 2 GPI MAb also significantly increased IL-6 and 6-keto-PGF1 alpha secretion. CONCLUSION: These findings support the hypothesis that anti-beta 2 GPI antibodies bind and activate EC through the adherent cofactor beta 2 GPI, likely leading to a procoagulant state.

6-Ketoprostaglandin F1 alpha↗

PubMed2006

Endothelial cell superoxide anion radical generation is not dependent on endothelial nitric oxide synthase-serine 1179 phosphorylation and endothelial nitric oxide synthase dimer/monomer distribution.

Tetrahydrobiopterin (BH4) and heat shock protein 90 (hsp90) have been anticipated to regulate endothelial nitric oxide synthase (eNOS)-dependent superoxide anion radical (O2*-) generation in endothelial cells. It is not known, however, whether hsp90 and BH4 increase O2*- in a synergistic manner, or whether this increase is a consequence of downstream changes in eNOS phosphorylation on serine 1179 (eNOS-S1179) and changes in dimer/monomer distribution. Here O2*- production from purified BH4 -free eNOS and eNOS:hsp90 complexes determined by spin-trapping methodology showed that hsp90 neither inhibits O2*- nor alters the requirement of BH4 to inhibit radical release from eNOS. In endothelial cells, O2*- detection with the novel high-performance liquid chromatography assay of 2-hydroxyethidium showed that inhibition of hsp90 did not increase O2*-, while a significant increase in O2*- was detected in BH4 -depleted cells. Radicicol, a hsp90 inhibitor, disrupted eNOS:hsp90 association, decreased eNOS-S1179, but increased biopterin production in a dose-dependent fashion. These changes were followed by an increase in eNOS activity, demonstrating that high biopterin levels offset inhibition of eNOS phosphorylation and diminished interaction with hsp90. In contrast, depletion of biopterin did not affect hsp90 levels or interaction with eNOS or eNOS dimer/monomer ratio in bovine aorta endothelial cells (BAECs). We conclude that low BH4 but not inhibition of hsp90 increases O2*- in BAECs by mechanism(s) that unlikely involve phosphorylation to eNOS-S1179 or eNOS monomerization.

Animals↗

PubMed2002

Aprotinin impairs coronary endothelial function and down-regulates endothelial NOS in rat coronary microvascular endothelial cells.

OBJECTIVE: The non-specific serine protease inhibitor aprotinin is currently used to reduce blood loss and the need for blood transfusion after cardiopulmonary bypass. We have recently reported that aprotinin impairs endothelium-dependent but not endothelium-independent relaxations in rat thoracic aortic rings due to its inhibitory effect on endothelial nitric oxide (NO) production. In light of these findings, the current study was designed to investigate the effects of aprotinin on coronary endothelial function in isolated rat hearts and on the expression of endothelial NO synthase (eNOS) in cultured rat coronary microvascular endothelial cells (CMEC). METHODS: Hearts obtained from Sprague-Dawley rats were perfused on a constant flow Langendorff isolated heart system and coronary perfusion pressure and cardiac parameters were recorded. The coronary relaxant responses to bolus infusions of bradykinin (BK) and sodium nitroprusside (SNP) were recorded in the absence and presence of aprotinin. Total RNA and protein samples were extracted from CMEC incubated with aprotinin or the vehicle, 0.9% NaCl. RT-PCR-Southern blotting and Western analyses were carried out to assess eNOS mRNA and protein levels, respectively. RESULTS: Aprotinin (125 and 250 kIU/ml) increased coronary perfusion pressure without changing the heart rate and cardiac contractility. Aprotinin inhibited BK-induced coronary vasodilatation at 250 kIU/ml, but not at 125 kIU/ml concentrations. The relaxant response to SNP did not change in response to either concentration of the drug. Incubation of CMEC with aprotinin down-regulated eNOS mRNA and protein at 250 kIU/ml, but not at 125 kIU/ml concentration. CONCLUSION: These data suggest that aprotinin selectively inhibits NO synthesis at higher doses (> or = 250 kIU/ml) and therefore impairs endothelium-dependent coronary vascular tone. This effect of the drug may contribute to its 'blood-sparing' action, but may also account for the increase in the incidence of postoperative graft thrombosis observed in clinical practice during aprotinin therapy.

Animals↗

PubMed1991

Growth characteristics of retinal capillary endothelial cells compared with pulmonary vein endothelial cells in culture. The effect of pericytes on differentiation of endothelial cells.

Bovine retinal capillary endothelial cells (RCECs) and pulmonary vein endothelial cells (PVECs) were isolated and investigated in plate culture, three-dimensional culture and in co-culture with pericytes. In plate culture, RCECs required growth factor in the medium for growth whereas PVECs did not. Phenotypic modulation (a tendency to become similar morphologically to smooth muscle cells, and to accumulate into thread-like structures) was observed in PVECs but not in RCECs. In three-dimensional culture, RCECs contracted, aggregated and were unable to proliferate. Proliferation was elicited when the gel matrix was adsorbed by fibronectin or upon co-culture with pericytes. In contrast, PVECs not only proliferated but also formed tubular structures. In co-culture with pericytes, PVECs in close contact with, or in near apposition to pericytes formed tubular structures earlier than those without contact in the same dish. These results provide new findings about differences in the growth characteristics of endothelial cells between microvessels and large vessels. In addition, it is considered that pericytes may promote tube formation by endothelial cells in three-dimensional culture.

Actins↗

PubMed2005

Losartan metabolite EXP3179 activates Akt and endothelial nitric oxide synthase via vascular endothelial growth factor receptor-2 in endothelial cells: angiotensin II type 1 receptor-independent effects of EXP3179.

BACKGROUND: Recent studies suggest that angiotensin type 1 receptor (AT1R) blockers have vascular protective effects beyond blood pressure lowering. Because of the importance of endothelial nitric oxide synthase (eNOS) in vascular and platelet function, we hypothesized that losartan and its metabolites would stimulate eNOS and its upstream activators Akt and phosphatidylinositol 3-kinase (PI3K). METHODS AND RESULTS: Losartan is metabolized into EXP3174 (AT1R-blocking metabolite) and EXP3179 (no AT1R-blocking properties). Treatment of endothelial cells (ECs) with losartan and both metabolites stimulated phosphorylation of Akt and eNOS in the absence of angiotensin II. However, the magnitude for EXP3179 was much greater than EXP3174, and the EC50 was significantly lower (-logEC50, 8.2+/-0.1 versus 5.4+/-0.2 mol/L), suggesting an AT1R-independent effect. Inhibiting PI3K or vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine phosphorylation abrogated EXP3179-induced eNOS phosphorylation. In endothelium of intact rat aorta, EXP3179 also stimulated Akt and eNOS phosphorylation. VEGFR2 activation was shown to be calcium and Src family kinase dependent by use of specific drug inhibitors and dominant negative kinase transfection. EXP3179 significantly inhibited tumor necrosis factor alpha-induced apoptosis by approximately 60% (from 30.1+/-5.8% to 12.2+/-2.0% TUNEL-positive cells), which was abolished by pretreatment with the PI3K inhibitor LY294002. Cleaved caspase-3 was suppressed by 48% with EXP3179. CONCLUSIONS: The losartan metabolite EXP3179 stimulates eNOS phosphorylation and suppresses tumor necrosis factor alpha-induced EC apoptosis by activating the VEGFR2/PI3K/Akt pathway.

Angiotensin II Type 1 Receptor Blockers↗

PubMed1998

Vascular permeability in experimental diabetes is associated with reduced endothelial occludin content: vascular endothelial growth factor decreases occludin in retinal endothelial cells. Penn State Retina Research Group.

Blood-retinal barrier (BRB) breakdown is a hallmark of diabetic retinopathy, but the molecular changes that cause this pathology are unclear. Occludin is a transmembrane component of interendothelial tight junctions that may regulate permeability at the BRB. In this study, we examined the effects of vascular endothelial growth factor (VEGF) and diabetes on vascular occludin content and barrier function. Sprague-Dawley rats were made diabetic by intravenous streptozotocin injection, and age-matched animals served as controls. After 3 months, BRB permeability was quantified by intravenous injection of fluorescein isothiocyanate-bovine serum albumin (FITC-BSA), Mr 66 kDa, and 10-kDa rhodamine-dextran (R-D), followed by digital image analysis of retinal sections. Retinal fluorescence intensity for FITC-BSA increased 62% (P < or = 0.05), but R-D fluorescence did not change significantly. Occludin localization at interendothelial junctions was confirmed by immunofluorescence, and relative protein content was determined by immunoblotting of retinal homogenates. Retinal occludin content decreased approximately 35% (P < or = 0.03) in the diabetic versus the control animals, whereas the glucose transporter GLUT1 content was unchanged in rat retinas. Additionally, treatment of bovine retinal endothelial cells in culture with 0.12 nmol/l or 12 nmol/l VEGF for 6 h reduced occludin content 46 and 54%, respectively. These data show that diabetes selectively reduces retinal occludin protein expression and increases BRB permeability. Our findings suggest that the elevated VEGF in the vitreous of patients with diabetic retinopathy increases vascular permeability by downregulating occludin content. Decreased tight junction protein expression may be an important means by which diabetes causes increased vascular permeability and contributes to macular edema.

Animals↗