Decline of shear stress-induced activation of extracellular signal-regulated kinases, but not stress-activated protein kinases, in in vitro propagated endothelial cells

We investigated the involvement of mitogen-activated protein kinase (MAPK) signal transduction pathways in human endothelial cells in response to shear stress and alterations of these kinases in in vitro-propagated endothelial cells (ECs). Potent activation (10-fold) of extracellular signal-regulated kinase (ERK2), a member of the MAPK family, occurred within 10 min of shear stress (5 dynes/cm2), whereupon rapid inactivation ensued. Shear stress also induced activation of stress-activated protein kinase (SAPK) or c-Jun NH2-terminal protein kinase (JNK) in ECs. Suramin pretreatment completely inhibited shear stress stimulation of ERK2, but not SAPK/JNK, highlighting a role for growth factor receptors in ERK activation. Translocation of ERK2 from the cytoplasm to the nucleus was observed in shear-stressed endothelial cells. In addition, we compared activities of MAPKs in shear-stressed cells derived from passages 4 and 10 (older). The magnitude of ERK2 activation was significantly lower in aged ECs compared to those of passage 4, while SAPK/JNK was not altered in the in vitro aged ECs. A similar level of ERK2 activation was found in both young and older cells stimulated with phorbol-12-myristate-13-acetate (PMA), indicating an age-related alteration of the plasma membrane. Taken together, these findings suggest that MAP kinase activation may be crucial for the expression of many genes in ECs stimulated by shear stress, and that an alteration in MAPK activities could contribute to the age-related decline in proliferative capacity.     

Human immunodeficiency virus Tat protein induces interleukin 6 mRNA expression in human brain endothelial cells via protein kinase C- and cAMP-dependent protein kinase pathways

The intracellular signal transduction pathways utilized by the HIV-1-derived protein, Tat, in the activation of human central nervous system-derived endothelial cells (CNS-ECs) were examined using specific enzymatic assays. Tat induced an increase in interleukin 6 (IL-6) mRNA within 1 hr of treatment. This biological effect of Tat involved activation of both protein kinase C (PK-C) and cAMP-dependent protein kinase (PK-A) in CNS-ECs. Tat at 10 ng/ml induced a sharp, transient increase in membrane PK-C activity within 30 sec of incubation, and reached maximum levels at 2 min, declining to control values within 10 min. Tat also induced a sharp increase in intracellular cAMP levels and PK-A activity in these cells, with the PK-A activity reaching a maximum at 10 min and slowly declining to control values in 4 hr of incubation. Activation of PK-A was dependent on a Tat-induced increase in membrane PK-C activity as demonstrated by calphostin C (a PK-C inhibitor) abolishing this effect. Incubation of cells with the cyclooxygenase inhibitor indomethacin did not affect Tat-induced activation of PK-A, indicating that prostacyclins are not involved in this process. Tat-induced increase in IL-6 mRNA was abolished in the presence on PK-A inhibitor H-89, demonstrating that activation of PK-A is necessary and sufficient for the increase in IL-6 production by these cells. Both the Tat-induced increase in intracellular cAMP and IL-6 mRNA levels in CNS-ECs may play a role in altering the blood-brain barrier and thereby inducing pathology often observed in AIDS dementia.     

Ectopic expression of the calcium-binding protein parvalbumin in mouse liver endothelial cells

To elucidate the physiological role of the Ca2+ binding protein parvalbumin, we have generated transgenic mice carrying the full-length complementary DNA (cDNA) of rat parvalbumin under the control of the heavy-metal inducible metallothionein IIA promoter. Immunohistochemical and biochemical methods have been used to detect the presence of ectopic parvalbumin expression in different tissues. Here we show the expression of parvalbumin in endothelial cells lining the liver sinusoids in situ and after isolation in vitro. The hemodynamic effects of endothelin 1, a peptide hormone mediating potent vasoconstriction via calcium signalling, were investigated in the mouse liver perfused in situ. Vasoconstriction, thought to be mediated by the Ito cell, was not affected in the transgenic animals, whereas microvascular exchange, probed with the multiple indicator dilution technique, was markedly decreased in normal mice but virtually not affected in the transgenic animals. This suggests that ectopically expressed parvalbumin is involved in the regulation of Ca2+ signals in the sinusoidal endothelial cells. This animal model could be of interest to those working on the physiology of liver circulation.     

Mrp1 multidrug resistance-associated protein and P-glycoprotein expression in rat brain microvessel endothelial cells

Two membrane glycoproteins acting as energy-dependent efflux pumps, mdr-encoded P-glycoprotein (P-gp) and the more recently described multidrug resistance-associated protein (MRP), are known to confer cellular resistance to many cytotoxic hydrophobic drugs. In the brain, P-gp has been shown to be expressed specifically in the capillary endothelial cells forming the blood-brain barrier, but localization of MRP has not been well characterized yet. Using RT-PCR and immunoblot analysis, we have compared the expression of P-gp and Mrp1 in homogenates, isolated capillaries, primary cultured endothelial cells, and RBE4 immortalized endothelial cells from rat brain. Whereas the mdr1a P-gp-encoding mRNA was specifically detected in brain microvessels and mdr1b mRNA in brain parenchyma, mrp1 mRNA was present both in microvessels and in parenchyma. However, Mrp1 was weakly expressed in microvessels. Mrp1 expression was higher in brain parenchyma, as well as in primary cultured brain endothelial cells and in immortalized RBE4 cells. This Mrp1 overexpression in cultured brain endothelial cells was less pronounced when the cells were cocultured with astrocytes. A low Mrp activity could be demonstrated in the endothelial cell primary monocultures, because the intracellular [3H]vincristine accumulation was increased by several MRP modulators. No Mrp activity was found in the cocultures or in the RBE4 cells. We suggest that in rat brain, Mrp1, unlike P-gp, is not predominantly expressed in the blood-brain barrier endothelial cells and that Mrp1 and the mdr1b P-gp isoform may be present in other cerebral cells.     

The effect of adrenalectomy on stress-induced c-fos mRNA expression in the rat brain

Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of postranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phase high-performance liquid chromatography (RP-HPLC) we have successfully separated various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea-Triton gel electrophoresis. Our results demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to traditional separation techniques and will be useful in studying the biological function of histone acetylation.     

Oxidative stress-induced actin reorganization mediated by the p38 mitogen-activated protein kinase/heat shock protein 27 pathway in vascular endothelial cells

Vascular endothelial cells are constantly in contact with oxyradicals and must be especially well equipped to resist their toxic effects and generate appropriate physiological responses. Despite the importance of oxyradicals in the physiopathology of the vascular endothelium, the mechanisms regulating the oxidative response of endothelial cells are poorly understood. In the present study, we observed that H2O2 in concentrations that induced severe fragmentation of F-actin in fibroblasts rather induced a reorganization of F-actin in primary cultures of human umbilical vein endothelial cells (HUVECs) that was characterized by the accumulation of stress fibers, the recruitment of vinculin to focal adhesions, and the loss of membrane ruffles, H2O2 also induced in these cells a strong (10- to 14-fold) activation of the p38 mitogen-activated protein (MAP) kinase, which resulted in activation of MAP kinase-activated protein kinase-2/3 and phosphorylation of the F-actin polymerization modulator, heat shock protein 27 (HSP27). The MAP kinases extracellular-regulated kinase, and c-Jun N-terminal kinase/stress-activated protein kinase were only slightly increased by these treatments. Inhibiting p38 activity with the highly specific inhibitor SB203580 blocked the H2O2-induced endothelial microfilament responses. Moreover, fibroblasts acquired an endothelium-like SB203580-sensitive actin response when HSP27 concentration was increased by gene transfection to the same high level as found in HUVECs. The results indicate that activation of p38 MAP kinase in cells such as endothelial cells, which naturally express high level of HSP27, plays a central role in modulating microfilament responses to oxidative stress. Consequently, the p38 MAP kinase pathway may participate in the several oxyradical-activated functions of the endothelium that are associated with reorganization of microfilament network.     

Urokinase expression in transduced endothelial cells

Increased thromboresistance through the release of lytic agents by endothelial cells may improve the patency of endothelial lined prosthetic grafts. We have evaluated the expression of urokinase from cells transduced with a retrovirus containing the gene for a human preprourokinase. Endothelial cells were enzymatically harvested from canine external jugular vein in nine animals and grown to confluence in culture. One-third of these cells served as controls, and the remaining two-thirds were transduced via incubation with an LXSN-type retroviral vector carrying the urokinase gene and a neomycin resistance gene. Successfully transduced cells were selected by incubation with 400 micrograms/mL G418 and pure cultures grown to confluence. Supernatants from confluent control and experimental cell cultures after 48 hours in defined, serum-free medium were assayed for human urokinase concentration and overall enzyme activity. ELISA quantitation of concentration using mouse antihuman urokinase antibody showed 0.15 +/- 0.11 ng/mL/hr/10(6) cells in the transduced cell supernatant; no measurable concentration was found in the control cells. (P < 0.01) Overall (human plus canine) enzyme activity of urokinase was determined using an indirect spectrophotometric assay based on plasminogen activation (ploug U/mL). Transduced cells showed activities of 0.12 at 10 days and 0.45 at confluence; control cell activity was 0.0 and 0.15, respectively. (P < 0.05) These data show that endothelial cells can be transduced with a urokinase expressing gene that increases the release of this thrombolytic agent. Lining small diameter prosthetic grafts with these cells may improve their thromboresistance and long-term patency.     

Expression of IL-8 mRNA in glomerular endothelial cells

OBJECTIVE: To study the expression of IL-8 mRNA in cultured human glomerular endothelial cells. METHOD: The effects of IL-1 beta and TNF alpha on the production of IL-8 by glomerular endothelial cells were also observed. RT-PCR was used. RESULTS: There was only weak expression of IL-8 mRNA in cultured human glomerular endothelial cells without the presence of any stimulating factors, whereas the expression of IL-8 mRNA in cultured human glomerular endothelial cells was significantly increased after the glomerular endothelial cells have been treated with IL-1 beta (25 u/ml) or TNF alpha (10 ng/ml) for 24 hours. CONCLUSION: Glomerular endothelial cell injury may enhance the expression of IL-8, which mediates the inflammatory reactions in glomeruli.     

Hypoxia induces vascular endothelial growth factor gene and protein expression in cultured rat islet cells

The formation of new microvasculature by capillary sprouting at the site of islet transplantation is crucial for the long-term survival and function of the graft. Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen with potent angiogenic and vascular permeability-inducing properties, may be a key factor in modulating the revascularization of islets after transplantation. In this study, we examined the gene expression of VEGF mRNA in three tumor cell lines and in isolated whole and dispersed rat islets in vitro by Northern blot hybridization in normoxic (5% CO2, 95% humidified air) and hypoxic (1% O2, 5% CO2, 94% N2) culture conditions. Increased expression of VEGF mRNA was observed in beta-TC3, RAW 264.7, and IC-21 tumor cell lines when subjected to hypoxia. With isolated whole islets in normoxic culture, a threefold increase in VEGF mRNA (P < 0.001) was seen at 48 h as compared with freshly isolated islets. This response was similar to the 3.8-fold increase observed with islets subjected to hypoxia. Dispersed rat islet cell clusters cultured on Matrigel for 24 h under hypoxic conditions showed a 3.4-fold increase (P < 0.01) in VEGF mRNA compared with those cultured in normoxia. This correlated with increased VEGF secretion as determined by enzyme-linked immunosorbent assay. Immunohistochemical studies revealed the presence of increased expression of VEGF protein near the center of islets after 24 h of normoxic culture. Islet cell clusters on Matrigel showed intense cellular localization of VEGF in both beta-cells and non-beta-cells. These findings suggest that rat islet cells, when subjected to hypoxia during the first few days after transplantation, may act as a major source of VEGF, thereby initiating revascularization and maintaining the vascular permeability of the grafted islets.     

Increase of vascular endothelial growth factor mRNA expression by 1,25-dihydroxyvitamin D3 in human osteoblast-like cells

Vascular endothelial growth factor (VEGF), a secreted endothelial cell-specific mitogen, is produced in endocrine organs and regulated by trophic hormones. Because angiogenesis and osteogenesis are closely regulated, we studied whether human osteoblast-like cells produce VEGF, and if so, what factors regulate VEGF mRNA expression. Human osteoblast-like cells (HObLC) derived from trabecular bone explants were cultured in alpha-MEM supplemented with 10% fetal calf serum. Northern blot analysis revealed that HObLC expressed VEGF mRNA, as did several human osteosarcoma cells. 1,25-(OH)2D3 increased the steady-state levels of VEGF mRNA in a time- and concentration-dependent manner in HObLC and one of the osteosarcoma cell lines, SaOS-2, accompanied by an increase in the concentration of immunoreactive VEGF in the conditioned medium. PTH and IGF-I also increased the level of VEGF mRNA in HObLC and SaOS-2 cells. Furthermore, 12-O-tetradecanoylphorbol ester stimulated VEGF mRNA in a time-and concentration-dependent manner. The VEGF mRNA expression induced by 1,25-(OH)2D3 was completely inhibited by H-7, but only partially by staurosporine. We have demonstrated that PTH, IGF-I, and most potently 1,25-(OH)2D3 stimulate the mRNA expression and secretion of VEGF in human osteoblast-like cells, suggesting that one of the anabolic effects of 1,25-(OH)2D3 on skeletal tissue may be mediated by VEGF produced by osteoblasts.     

Changes in multidrug transporter protein expression in endothelial cells cultured from isolated human brain microvessels

Retinol and proguanil are metabolised by the same family of hepatic cytochrome P450, i.e. CYP2C. We used proguanil as a probe to study CYP2C activity, and by implication retinol metabolism, in psoriasis. In vitro studies showed that retinol competitively inhibited the hepatic metabolism of proguanil to cycloguanil. Proguanil metabolism was assessed in 82 patients with chronic plaque psoriasis. Following proguanil orally (200 mg), urine was analysed for proguanil and cycloguanil. A proguanil to cycloguanil ratio < 1 signified extensive metabolism and a ratio > 10 poor metabolism. A wider range of ratios was observed in psoriasis than previously reported for normal subjects. The proguanil to cycloguanil ratio was assessed in 10 cases each of know severe and mild psoriasis. Low CYP2C activity was associated with severe psoriasis, poor metaboliser status occurring in 50% of the severe group, but in none of the mild cases, p < 0.01. These findings may indicate differences in retinoid metabolism in psoriasis.     

Hypoglycemia enhances the expression of mRNA encoding beta-amyloid precursor protein in rat primary cortical astroglial cells

Deposition of beta-amyloid (A beta) is a characteristic feature of the pathology of Alzheimer's disease (AD). Since glucose metabolism and the consequential ATP production are depressed in the temporal and parietal regions of the cortex in patients with AD, we designed the present study to investigate the possible role of hypometabolism in the pathogenesis of AD. We incubated rat primary cortical astroglial cells for 2 h to 4 days in a media deprived of 95% of its glucose and assessed the expression and alternative splicing of the mRNA that encoding beta-amyloid precursor protein (APP) using RT-PCR. Hypoglycemia caused a time-dependent increase in APP mRNA expression, which reaches a peak level of 173.2% of control expression (P < 0.05) at 24 h of hypoglycemia. Noteworthy, hypoglycemia favors the alternative splicing that includes the exon 7 segment, which encodes a Kunitz-type serine protease inhibitor domain. This study demonstrates that hypoglycemia increases APP mRNA expression in astroglial cells and processing of APP mRNA to a form that may encourage A beta deposits in AD. These data suggest that the observed hypometabolism in AD may contribute to its deposition of A beta in affected brain regions.     

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.     

Prolonged hypoxia increases vascular endothelial growth factor mRNA and protein in adult mouse brain

Brain hypoxia induces an increase in brain vascularity, presumably mediated by vascular endothelial growth factor (VEGF), but it is unclear whether VEGF is required to maintain the increase. In these studies, brain VEGF mRNA and protein levels were measured in adult mice kept in hypobaric chambers at 0.5 atm for 0, 0.5, 1, 2, 4, 7, and 21 days. Hypoxia was accompanied by a transient increase of VEGF mRNA expression: twofold by 0.5 day and a maximum of fivefold by 2 days; these were followed by a decrease at 4 days and a return to basal levels by 7-21 days. VEGF protein expression induced by hypoxia was bimodal, initially paralleling VEGF mRNA. There was an initial small increase at 12 h that reached a maximum by day 2, and, after a transient decrease on day 4, the protein expression increased again on day 7 before it returned to normoxic levels after 21 days. Thus, despite continued hypoxia, both VEGF mRNA and protein levels returned to basal after 7 days. These data suggest a metabolic negative-feedback system for VEGF expression during prolonged hypoxia in the brain.