Human microvascular endothelial cells differ from macrovascular endothelial cells in their expression of matrix metalloproteinases

Matrix metalloproteinase (MMP) secretion by microvascular endothelial cells is an essential first step in the formation of new blood vessels (angiogenesis). Since angiogenesis does not occur in large blood vessels, we investigated whether the secretion of MMPs and tissue inhibitor of MMP (TIMP1) differs between micro- and macro-vascular endothelial cells. We compared the secretion of MMPs and TIMP1 by human endothelial cells derived from neonatal foreskin (FSE) and umbilical vein (HUVE) sources. The cells were incubated for 24 hr in the presence or absence of the angiogenic agents, phorbol myristate acetate (PMA, 100 ng/ml) or tumour necrosis factor-alpha (TNF, 100 ng/ml). The cell supernatants were removed and assayed for MMPs and TIMP1 using a spectrophotometric assay for MMP1, zymography, Western blotting and Northern analysis. When endothelial cells were incubated in basal medium for 24 hr they secreted MMP1, MMP2 and TIMP1 but not MMP9. HUVE secreted substantially higher levels of these proteins compared to FSE. In addition, HUVE secreted two low molecular mass bands representing activated forms of MMP2. These activated forms were not present in supernatants derived from FSE. In response to PMA, both FSE and HUVE increased secretion of MMP1 and TIMP1. However, there was a dramatic difference in level of response by the two cell types with FSE secreting substantially more TIMP1 and MMP9 compared to HUVE. These data clearly show that cultured endothelial cells derived from microvascular vs macrovascular tissues exhibit different MMP and TIMP secretory profiles.     

Hypoxia and vascular endothelial growth factor stimulate angiogenic integrin expression in bovine retinal microvascular endothelial cells

PURPOSE: Integrins alphavbeta3 and alphavbeta5 are cell-to-matrix adhesion molecules that have been reported to mediate vascular cell proliferation and migration. The authors investigated the regulation of expression of these angiogenic integrins by hypoxia and vascular endothelial growth factor (VEGF) in retinal microvascular endothelial cells in culture. METHODS: Cultured bovine retinal capillary endothelial cells were exposed to human recombinant VEGF under normoxic (95% air, 5% CO2) conditions to assess the effects of VEGF. Hypoxia studies were performed under lower oxygen concentration (0.5%-1.5% O2) induced by nitrogen replacement in constant 5% CO2 conditions. Integrin family mRNA and protein expression were assessed by northern blot analysis and immunoprecipitation. RESULTS: VEGF (25 ng/ml) increased integrin alphav, beta3, and 35 mRNA after 24 hours 6.1+/-0.8-fold (P < 0.001), 5.9+/-1.1-fold (P < 0.001), and 1.9+/-0.2-fold (P < 0.01), respectively. Similarly, hypoxia stimulated gene expression of integrin alphav and beta3 after 24 hours by 5.1+/-1.7-fold (P < 0.01) and 3.0+/-0.5-fold (P < 0.01), respectively, and integrin beta5 after 9 hours 1.4+/-0.2-fold (P < 0.05). This hypoxia-induced, integrin alphav mRNA elevation was inhibited significantly by anti-VEGF neutralizing antibody. Also, a conditioned medium from confluent endothelial cells maintained under hypoxic conditions for 24 hours produced a 7.1+/-1.1-fold increase (P < 0.001) in integrin alphav mRNA expression after 24 hours, which was reversed by anti-VEGF neutralizing antibody. Induction of integrin alphav by VEGF and hypoxia was confirmed in the protein level. CONCLUSIONS: These data suggest that hypoxia stimulates expression of vascular integrins alphavbeta3 and alphavbeta5 in retinal microvascular endothelial cells partially through autocrine-paracrine action of VEGF induced by the hypoxic state.     

Radiation damage to endothelial cells in vitro, as judged by the micronucleus assay

CD23 has been reported to be a macrophage/monocyte activation antigen. We focused on the expression of CD23 by peripheral blood macrophages/monocytes in 5 Kawasaki disease (KD) patients with coronary artery lesions (CAL) and compared these values with those of 35 patients without CAL. The expression of CD23 on peripheral blood macrophages/monocytes was assayed by a fluorescence-activated cell sorter using monoclonal antibodies CD23 and CD14. Absolute counts of CD23+CD14+ macrophages/monocytes in KD patients with CAL did not increase during the acute stage, while these values in KD patients without CAL increased. In addition, this decreased expression of CD23 on peripheral blood macrophages/monocytes in patients with CAL did not change during the acute stage, regardless of IVGG therapy. Our results suggest that the decreased expression of CD23 on peripheral blood macrophages/monocytes in patients with CAL is part of the regulatory system of CD23 antigen during acute KD.     

Human herpesvirus 6 infection as a risk factor for the development of severe drug-induced hypersensitivity syndrome

OBJECTIVES: To test suitability of radiographic evaluation of lung lesions as a substitute for lung lesion scores derived by examination at necropsy in challenge-exposure models of bovine pneumonia. ANIMALS: 10 calves selected by body weight from 20 multiple-source male Holstein calves approximately 1 to 2 months old enrolled in a Pasteurella multocida challenge-exposure study. PROCEDURE: Calves were paired on the basis of weight and randomly assigned within pairs to vaccine or control (saline solution) group. By use of deep tracheal cannulation, calves were challenge exposed with a culture of virulent P multocida, observed for 10 days, euthanatized, and necropsied, and the lungs were scored for pneumonic lesions. Radiographic views of the lung fields of the calves were taken before challenge exposure and before necropsy and were evaluated for alveolar disease by a veterinary radiologist. Lung lesion scores were compared with radiographic evaluations. RESULTS: There was a strong and significant correlation (R2 = 0.91, P < 0.001) between results of the evaluation of postchallenge-exposure radiographs and necropsy results. There also was also strong and significant correlation (R2 = 0.90, P < 0.001) between evaluation of the prechallenge-exposure radiographs and necropsy results. CONCLUSIONS: Radiographic evaluation of lung lesions correlates well with lung lesions found at necropsy. The findings emphasize the need for caution in interpreting the results of challenge-exposure studies of bovine respiratory tract disease in which small numbers of calves are studied.     

Role of adenosine in the hypoxic induction of vascular endothelial growth factor in porcine brain derived microvascular endothelial cells

The distribution of neuropeptide Y (NPY)-immunoreactive (IR) nerves, as well as the functional effects of NPY and the Y1- and Y2-receptor agonists, [Leu31,Pro34]NPY and NPY(13-36), respectively, have been investigated in vitro in both visceral and arterial smooth muscle of the horse intravesical ureter. NPY-IR nerve fibres were widely distributed along the entire length of the ureter, although the intravesical part was the most richly innervated region, and the only one where NPY-IR ganglion cells were found. NPY (10(-7) M) did not affect either basal tone or spontaneous rhythmic contractions of the isolated intravesical ureter, but significantly enhanced the increases in both tone and frequency of phasic activity elicited by noradrenaline (10(-6) and 10(-5) M). The Y1-receptor agonist, [Leu31,Pro34]NPY (10(-7) and 10(-6) M) did not significantly alter either ureteral basal tone or the contractile activity induced by noradrenaline, whereas the Y2-receptor agonist, NPY(13-36) (10(-7) M), mimicked the potentiating effect of NPY on noradrenaline responses. In ureteral resistance arteries (effective lumen diameters of 130-300 microm), NPY (10(-10) to 10(-7) M) elicited concentration-dependent contractions, which were inversely correlated with the arterial lumen diameter. Submaximal concentrations of NPY (10(-8) M) significantly increased the sensitivity of ureteral arteries to noradrenaline. [Leu31,Pro34]NPY (10(-10) to 10(-7) M), but not NPY(13-36), induced a contractile effect of similar magnitude and potency as those of NPY, and also potentiated noradrenaline responses. The present results demonstrate a rich NPY-innervation in the intravesical ureter and reveal functional effects of the peptide enhancing motor activity in both ureteral and arterial smooth muscles, although the receptors mediating such effects seem to be different. Thus, NPY potentiates the phasic contractions and tone elicited by noradrenaline through Y2-receptors, whereas it both contracts and potentiates noradrenaline vasoconstriction in ureteral arteries via Y1-receptors.     

Effect of steroid hormones and retinoids on the formation of capillary-like tubular structures of human microvascular endothelial cells in fibrin matrices is related to urokinase expression

Angiogenesis, the formation of new capillary blood vessels, is a feature of a variety of pathological processes. To study the effects of a specific group of hormones (all ligands of the steroid/retinoid/thyroid hormone receptor superfamily) on the angiogenic process in humans, we have used a model system in which human microvascular endothelial cells from foreskin (hMVEC) are cultured on top of a human fibrin matrix in the presence of basic fibroblast growth factor and tumor necrosis factor-alpha. This model mimics the in vivo situation where fibrin appears to be a common component of the matrix present at sites of chronic inflammation and tumor stroma. Our results show that testosterone and dexamethasone are strong inhibitors and all-trans retinoic acid (at-RA) and 9-cis retinoic acid (9-cis RA) are potent stimulators of the formation of capillary-like tubular structures. These effects are mediated by their respective nuclear hormone receptors as demonstrated by the use of specific synthetic receptor agonists and antagonists. 17beta-estradiol, progesterone, and 1,25-dihydroxyvitamin D3 did not affect or only weakly affected in vitro angiogenesis, which may be related to the lack of significant nuclear receptor expression. Although hMVEC express both thyroid hormone receptors alpha and beta, no effect of thyroid hormone on tube formation was found. The effects of testosterone, dexamethasone, at-RA, and 9-cis RA on tube formation were accompanied by parallel changes in urokinase-type plasminogen activator (u-PA) expression, at both mRNA and antigen levels. Exogenous suppletion of the medium with single chain u-PA enhances tube formation in our in vitro model, whereas quenching of u-PA activity (but not of tissue-type plasminogen activator activity) or of u-PA binding to u-PA receptor by specific antibodies suppressed basal and retinoid-stimulated tube formation. Moreover, addition of scu-PA to testosterone- or dexamethasone-treated hMVEC restored the suppressed angiogenic activity for a substantial part. Aprotinin, an inhibitor of plasmin activity, completely inhibited tube formation, indicating that the proteolytic properties of the u-PA/u-PA receptor complex are crucial in this process. Our results show that steroid hormones (testosterone and dexamethasone) and retinoids have strong, but opposite effects on tube formation in a human in vitro model reflecting pathological angiogenesis in the presence of fibrin and inflammatory mediators. These effects can be explained by hormone-receptor-mediated changes in u-PA expression, resulting in enhanced local proteolytic capacity of the u-PA/u-PA receptor complex.     

Hepatic veno-occlusive disease in a case of polymyositis associated with thrombotic thrombocytopenic purpura/hemolytic uremic syndrome

A 50-year-old woman was treated with prednisolone for polymyositis. During the therapy, thrombotic thrombocytopenic purpura/hemolytic uremic syndrome (TTP/HUS) occurred. Neither plasma infusion nor plasma exchange could relieve the clinical manifestations of TTP/HUS. Moreover, massive ascites appeared and worsened her condition. She died approximately one year after the diagnosis of polymyositis. The autopsy revealed centri-lobular hepatic necrosis and nonthrombotic obliteration of hepatic small veins. The diagnosis of hepatic veno-occlusive disease (VOD) was made. It was suspected that common factors other than cytoreductive therapy had damaged the endothelium and caused TTP/HUS and VOD in a case of polymyositis.     

Fibrin induction of tissue factor expression in human vascular endothelial cells

BACKGROUND: For the present study, we hypothesized that fibrin is an inducer of tissue factor (TF) expression in vascular endothelial cells in vitro and in vivo. METHODS AND RESULTS: To test the in vitro aspect of this hypothesis, human umbilical vein endothelial cells (HUVECs) were cocultured with physiologically relevant concentrations of fibrin (0.03 to 1.0 mg fibrin/mL) for various times (0.5 to 24 hours), and TF expression was compared with that in unstimulated HUVECs (media control). Results demonstrated that fibrin induced a time- and dose-dependent increase in TF antigen expression, functional TF procoagulant activity, and TF mRNA in HUVECs. CONCLUSIONS: These studies demonstrate that fibrin can directly regulate TF expression in HUVECs in vitro.     

Interaction of Listeria monocytogenes with human brain microvascular endothelial cells: InlB-dependent invasion, long-term intracellular growth, and spread from macrophages to endothelial cells

Invasion of endothelial tissues may be crucial in a Listeria monocytogenes infection leading to meningitis and/or encephalitis. Internalization of L. monocytogenes into endothelial cells has been previously demonstrated by using human umbilical vein endothelial cells as a model system. However, during the crossing of the blood-brain barrier, L. monocytogenes most likely encounters brain microvascular endothelial cells which are strikingly different from macrovascular or umbilical vein endothelial cells. In the present study human brain microvascular endothelial cells (HBMEC) were used to study the interaction of L. monocytogenes with endothelial cells, which closely resemble native microvascular endothelial cells of the brain. We show that L. monocytogenes invades HBMEC in an InlB-dependent and wortmannin-insensitive manner. Once within the HBMEC, L. monocytogenes replicates efficiently over a period of at least 18 h, moves intracellularly by inducing actin tail formation, and spreads from cell to cell. Using a green fluorescent protein-expressing L. monocytogenes strain, we present direct evidence that HBMEC are highly resistant to damage by intracellularly growing L. monocytogenes. Infection of HBMEC with L. monocytogenes results in foci of heavily infected, but largely undamaged endothelial cells. Heterologous plaque assays with L. monocytogenes-infected P388D1 macrophages as vectors demonstrate efficient spreading of L. monocytogenes into HBMEC, fibroblasts, hepatocytes, and epithelial cells, and this phenomenon is independent of the inlC gene product.     

Signal transduction in human hematopoietic cells by vascular endothelial growth factor related protein, a novel ligand for the FLT4 receptor

We have recently identified a novel ligand of the vascular endothelial growth factor (VEGF) family termed VEGF-related protein (VRP), which specifically binds to the FLT4 receptor. To characterize the signaling events after VRP engagement of its cognate receptor in hematopoietic cells, a population of human erythroleukemia (HEL) cells, termed HEL-JW, expressing high levels of FLT4 receptor was isolated. Stimulation of HEL-JW cells with VRP alone and in combination with the c-kit ligand/stem cell factor increased cell growth. VRP induced tyrosine phosphorylation of various proteins, including the FLT4 receptor. Further characterization of these tyrosine phosphorylated molecules revealed that Shc, Grb2, and SOS form a complex with the activated FLT4 receptor. HEL-JW cells also expressed RAFTK, a recently identified member of the focal adhesion kinase family. RAFTK was phosphorylated and activated upon VRP treatment, and there was an enhanced association of this kinase with the adaptor protein Grb2. Furthermore, the c-Jun NH2-terminal kinase (JNK), involved in growth activation and shown to mediate RAFTK signaling in other cell types, was activated by VRP stimulation. We also observed that VRP treatment of HEL-JW cells resulted in the phosphorylation of the cytoskeletal protein paxillin. This treatment resulted in an increased association of paxillin with RAFTK, which was mediated by the C-terminal region of RAFTK. These studies indicate that VRP stimulation induced the formation of a signaling complex at its activated receptor as well as activation of RAFTK. VRP-mediated activation of RAFTK may facilitate signal transduction to the cytoskeleton and downstream to the JNK pathway in FLT4-expressing blood cells.     

L-selectin-dependent leukocyte adhesion to microvascular but not to macrovascular endothelial cells of the human coronary system

To characterize L-selectin-dependent cell adhesion to human vascular endothelium, human cardiac microvascular endothelial cells (HCMEC) and human coronary endothelial cells (HCEC) were isolated from explanted human hearts. The adhesion behavior of human (NALM-6) and mouse (300.19) pre-B cells transfected with cDNA encoding for human L-selectin was compared with that of the respective nontransfected cells in a flow chamber in vitro. More than 80% of the adhesion to tumor necrosis factor-alpha (TNF-alpha)-stimulated HCMEC at shear stresses >2 dyne/cm2 was L-selectin dependent and could be equally well blocked by an anti-L-selectin antibody or a L-selectin-IgG-chimera. No L-selectin dependent adhesion to HCEC could be shown. The L-selectin dependent adhesion to HCMEC was insensitive to neuraminidase, but greatly inhibited by addition of NaClO3, which inhibits posttranslational sulfation and remained elevated for at least 24 hours of stimulation. E-selectin dependent adhesion of HL60 cells to HCMEC was blocked by neuraminidase, but not by NaClO3 and returned to control levels within 18 hours of HCMEC stimulation. It is concluded that microvascular, but not macrovascular endothelial cells express TNF-alpha-inducible sulfated ligand(s) for L-selectin, which differ from known L-selectin ligands, because sialylation is not required. The prolonged time course of L-selectin dependent adhesion suggests a role in sustained leukocyte recruitment into inflammatory sites in vivo.     

Differential reactivity of brain microvascular endothelial cells to TNF reflects the genetic susceptibility to cerebral malaria

Upon infection with Plasmodium berghei ANKA (PbA), various inbred strains of mice exhibit different susceptibility to the development of cerebral malaria (CM). Tumor necrosis factor-alpha (TNF) and interferon-gamma (IFN-gamma) have been shown to be crucial mediators in the pathogenesis of this neurovascular complication. Brain microvascular endothelial cells (MVEC) represent an important target of both cytokines. In the present study, we show that brain MVEC purified from CM-susceptible (CM-S) CBA/J mice and CM-resistant (CM-R) BALB/c mice exhibit a different sensitivity to TNF. CBA/J brain MVEC displayed a higher capacity to produce IL-6 and to up-regulate intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in response to TNF than BALB/c brain MVEC. In contrast, no difference was found in the induction of E-selectin after TNF challenge. CM-S brain MVEC were also significantly more sensitive to TNF-induced lysis. This differential reactivity to TNF was further substantiated by comparing TNF receptor expression on CM-S and CM-R brain MVEC. Although the constitutive expression of TNF receptors was comparable on cells from the two origins, TNF induced an up-regulation of both p55 and p75 TNF receptors in CM-S, but not in CM-R brain MVEC. A similar regulation was found at the level of TNF receptor mRNA, but not for receptor shedding. Although a protein kinase C inhibitor blocked the response to TNF in both the brain MVEC, an inhibitor of protein kinase A selectively abolished the response to TNF in CM-R, but not CM-S brain MVEC, suggesting a differential protein kinase involvement in TNF-induced activation of CM-S and CM-R brain MVEC. These results indicate that brain MVEC purified from CM-S and CM-R mice exhibit distinctive sensitivity to TNF This difference may be partly due to a differential regulation of TNF receptors and via distinct protein kinase pathways.     

Thrombopoietin and its receptor, c-mpl, are constitutively expressed by mouse liver endothelial cells: evidence of thrombopoietin as a growth factor for liver endothelial cells

Present data suggest that the primary site of thrombopoietin (TPO) mRNA is the liver. Previously, we reported that specific murine liver endothelial cells (LEC-1) located in the hepatic sinusoids support in vitro megakaryocytopoiesis from murine hematopoietic stem cells suggesting that these cells may be a source of TPO. We report here that TPO and its receptor, c-mpl, are coexpressed on cloned LEC-1. Enzyme-linked immunosorbent assay (ELISA), biological assay, and flow cytometry studies confirmed the expression of both TPO and its receptor, respectively, at the protein level. TPO activity was enhanced in supernatants from LEC-1 treated with tumor necrosis factor (TNF)-alpha and gamma-interferon (INF). Our results show that TPO through its receptor stimulated the growth of LEC-1 in vitro. These observations establish LEC-1 as a novel source of TPO in the liver. To our knowledge, this is the first report that liver endothelial cells express both TPO and its receptor, c-mpl, and our findings indicate that this cytokine constitutes a growth factor for liver endothelial cells in vitro.