High glucose-induced hyperosmolarity impacts proliferation,cytoskeleton remodeling and migration of human induced pluripotent stem cells via aquaporin-1

Background and objective: Hyperglycemia leads to adaptive cell responses in part due to hyperosmolarity. In endothelial and epithelial cells, hyperosmolarity induces aquaporin-1 (AQP1) which plays a role in cytoskeletal remodeling, cell proliferation and migration. Whether such impairments also occur in human induced pluripotent stem cells (iPS) is not known. We therefore investigated whether high glucose-induced hyperosmolarity impacts proliferation, migration, expression of pluripotency markers and actin skeleton remodeling in iPS cells in an AQP1-dependent manner. Methods and results: Human iPS cells were generated from skin fibroblasts by lentiviral transduction of four reprogramming factors (Oct4, Sox2, Klf4, c-Myc). After reprogramming, iPS cells were characterized by their adaptive responses to high glucose-induced hyperosmolarity by incubation with 5.5 mmol/L glucose, high glucose (HG) at 30.5 mM, or with the hyperosmolar control mannitol (HM). Exposure to either HG or HM increased the expression of AQP1. AQP1 co-immunoprecipitated with β-catenin. HG and HM induced the expression of β-catenin. Under these conditions, iPS cells showed increased ratios of F-actin to G-actin and formed increased tubing networks. Inhibition of AQP1 with small interfering RNA (siRNA) reverted the inducing effects of HG and HM. Conclusions: High glucose enhances human iPS cell proliferation and cytoskeletal remodeling due to hyperosmolarity-induced upregulation of AQP1.     

Enhanced secretion of endothelin-1 by elevated glucose levels from cultured bovine aortic endothelial cells

We have investigated the effect of glucose on the release of endothelin-1-like immunoreactivity (ET-1-LI) from cultured bovine aortic endothelial cells. Elevation of glucose concentrations in cultured media from 5.5 to 11.1 or 22.2 mM significantly stimulated ET-1-LI release from cultured endothelial cells. An aldose reductase inhibitor did not affect the high glucose-induced ET-1-LI release. These findings suggest the possibility that hyperglycemia in diabetic patients enhances ET-1-LI release at the local site of vascular endothelium, which might be involved in the developments of vascular complications and atherosclerosis.     

Aminosalicylic acid reduces the antiproliferative effect of hyperglycaemia,advanced glycation endproducts and glycated basic fibroblast growth factor in cultured bovine aortic endothelial cells: Comparison with aminoguanidine

Hyperglycaemia reduces proliferation of bovine aortic endothelial cells in vitro. A similar effect in vivo may contribute to long-term complications of diabetes such as impaired wound-healing and retinopathy.We report the effect of increased glucose concentrations, glycated basic fibroblast growth factor (FGF-2) and bovine serum albumin-derived advanced glycation endproducts (BSA-AGE) on the proliferation of bovine aortic endothelial cells.Glucose (30 and 50 mmol/l) had an antiproliferative effect on endothelial cells. This effect may be mediated through reduced mitogenic activity of FGF-2. The glycation of FGF-2 with 250 mmol/l glucose-6-phosphate led to reduced mitogenic activity compared to native FGF-2. BSA-AGE at concentrations of 10, 50 and 250 g/ml had an antiproliferative effect on cultured endothelial cells.Aminosalicylic acid at a concentration of 200 mol/l proved to be more effective than equimolar concentrations of aminoguanidine in protecting endothelial cells against the antiproliferative effects of both high (30 mmol/l) glucose and 50 g/ml BSA-AGE. FGF-2 glycated in the presence of 4 mmol/l aminosalicylic acid or aminoguanidine retained mitogenic activity compared to that glycated in their absence.Compounds like aminoguanidine and, in particular, aminosalicylic acid protect endothelial cells against glucose-mediated toxicity and may therefore have therapeutic potential.     

Cariporide inhibits high glucose-mediated adhesion of monocyte-endothelial cell and expression of intercellular adhesion molecule-1

The aim of this study was to examine whether cariporide, a new inhibitor of Na(+)/H(+) exchanger 1 (NHE-1), may inhibit high glucose-induced monocyte-endothelial cell adhesion and the expression of intercellular adhesion molecule-1 (ICAM-1). Cultured endothelial cells were incubated with normal glucose control (5.5 mM), cariporide control (5.5 mM glucose plus 10 microM cariporide), hyperosmolarity (5.5 mM glucose plus 16.5 mM mannitol), high glucose (HG, 22 mM), low-concentration cariporide (22 mM glucose plus 0.1 microM cariporide), medium-concentration cariporide (22 mM glucose plus 1 muM cariporide), and high-concentration cariporide (22 mM glucose plus 10 microM cariporide) for 24 h. Monocytes were isolated from peripheral human blood. Adhered monocytes were quantified by measuring their protein content. ICAM-1 expression and NHE-1 activity was determined with enzyme-linked immunosorbent assay (ELISA) and pH-sensitive fluorescent spectrophotometry. Exposure of endothelial cells to HG for 24 h caused an increase of adhesion of monocytes to endothelial cells and an increased expression of ICAM-1. However, these effects were reversed by treatment with cariporide (0.1, 1, 10 microM) in a concentration-dependent manner. Furthermore, cariporide (1 microM) was able to inhibit the activation of NHE-1 induced by HG in endothelial cells. These findings suggest that cariporide might inhibit HG-mediated monocyte-endothelial cell adhesion and expression of ICAM-1 by inhibiting the activation of NHE-1.     

Regulation of 3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

We showed earlier that insulin stimulated sugar transport in adrenal chromaffin cells (Bigornia, L. and Bihler, I. Biochim. Biophys. Acta 885, 335-344). Transport regulation and its Ca2+ -dependence was further investigated in isolated bovine adrenal chromaffin cells, serving as a model of a homogeneous neuronal cell population. Uptake of the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose was stimulated by hyperosmolar medium, and this effect was abolished in the absence of external Ca2+, or depressed in the presence of La3+ or the slow Ca2+ channel blocker methoxyverapamil. Basal transport was also stimulated by factors (acetylcholine, carbamylcholine, low-Na+ medium), which cause Ca2+ -dependent catecholamine release, and these effects were abolished in Ca2+ -free medium. In addition insulin, acetylcholine, hyperosmolar and low-Na+ medium significantly increased 45Ca uptake. Thus, glucose transport in adrenal chromaffin cells was stimulated by insulin and hyperosmolarity in a Ca2+ -dependent manner, as in muscle. Sensitivity to secretory stimuli, a regulatory feature perhaps characteristic of this cell type, was also demonstrated. In contrast to muscle, sugar transport was not affected by Na+ -pump inhibition, metabolic inhibitors or the Na+ ionophore monensin, suggesting that Ca2+ influx by Na+/Ca2+ exchange does not play a significant role in the activation of sugar transport in chromaffin cells.     

High glucose increases platelet-derived growth factor production in cultured human vascular endothelial cells and preventive effects of eicosapentaenoic acids

The regulation of the production of platelet-derived growth factor (PDGF) and the influence of high glucose concentration, eicosapentaenoic acid (EPA) were studied in cultured human umbilical vein endothelial cells (HUE). The PDGF production of HUE increased markedly depending on glucose concentration. However, EPA (3×10⁻⁴M) markedly inhibited PDGF production [27.5 mM glucose group: 123 ± 3% of control (5.5 mM glucose group), 27.5 mM glucose + EPA group: 104 ±5% of control]. These results suggested that a high glucose concentration and a high osmotic pressure-induced increase in PDGF production is involved in the development and progression of diabetic macroangiopathy. As eicosapentaenoic acid inhibits the PDGF production induced by high glucose concentration in HUE, use of this agent may exhibit anti-arteriosclerotic effects.     

Suppression of 3-deoxyglucosone and heparin-binding epidermal growth factor-like growth factor mRNA expression by an aldose reductase inhibitor in rat vascular smooth muscle cells

Reactive carbonyl compounds and oxidative stress have been recently shown to up-regulate the expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF), a potent mitogen for vascular smooth muscle cells (SMCs) produced by SMC themselves. Because the polyol pathway has been reported to influence the formation of carbonyl compounds and the oxidative stress in various cells, we conducted this study to investigate whether the polyol pathway affects HB-EGF expression along with the generation of carbonyl compounds and the oxidative stress in SMCs. We found that, compared with those cultured with 5.5mM glucose, SMCs cultured with 40 mM glucose showed the accelerated thymidine incorporation, elevated levels of intracellular sorbitol, 3-deoxyglucosone (3-DG), advanced glycation end products (AGEs), and thiobarbituric acid-reactive substances (TBARS) along with the enhanced expression of HB-EGF mRNA. An aldose reductase inhibitor (ARI), SNK-860, significantly inhibited all of these abnormalities, while aminoguanidine suppressed 3-DG levels and HB-EGF mRNA expression independent of sorbitol levels. The results suggest that the polyol pathway may play a substantial role in SMC hyperplasia under hyperglycemic condition in part by affecting HB-EGF mRNA expression via the production of carbonyl compounds and oxidative stress.     

Dissociation of 5' AMP-activated protein kinase activation and glucose uptake stimulation by mitochondrial uncoupling and hyperosmolar stress: differential sensitivities to intracellular Ca2+ and protein kinase C inhibition

2,4-dinitrophenol (DNP) compromises ATP production within the cell by disrupting the mitochondrial electron transport chain. The resulting loss of ATP leads to an increase in glucose uptake for anaerobic generation of ATP. In L6 skeletal muscle cells, DNP increases the rate of glucose uptake by twofold. We previously showed that DNP increases cell surface levels of glucose transporter 4 (GLUT4) and hexose uptake via a Ca2+-sensitive and conventional protein kinase C (cPKC)-dependent mechanism. Recently, 5' AMP-activated protein kinase (AMPK) has been proposed to mediate the stimulation of glucose uptake by energy stressors such as exercise and hypoxia. Changes in Ca2+ and cPKC have also been invoked in the stimulation of glucose uptake by exercise and hypoxia. Here we examine whether changes in cytosolic Ca2+ or cPKC lead to activation of AMPK. We show that treatment of L6 cells with DNP (0.5 mM) or hyperosmolar stress (mannitol, 0.6 M) increased AMPK activity by 3.5-fold. AMPK activation peaked by 10-15 min prior to maximal stimulation of glucose uptake. Intracellular Ca2+ chelation and cPKC inhibition prior to treatment with DNP and hyperosmolarity significantly reduced cell surface GLUT4 levels and hexose uptake but had no effect on AMPK activation. These results illustrate a break in the relationship between AMPK activation and glucose uptake in skeletal muscle cells. Activation of AMPK does not suffice to stimulate glucose uptake in response to DNP and hyperosmolarity.     

Effects of 3-hydroxybutyrate and hyperosmolarity on glucagon release from isolated perfused canine pancreas

The effects of 3-hydroxybutyrate (3-OHB) and hyperosmolarity on glucagon secretion were examined in the isolated perfused canine pancreas. When 3-OHB was infused for 15 min into the pancreas perfused with 2.8 mM glucose, 5 and 20 mM sodium 3-OHB inhibited it after a transient stimulation, whereas a similar transient stimulation was observed also by the infusion of 20 mM NaCl in a control experiment. The above inhibition was not observed under the perfusate condition of 5.5 mM glucose plus 10 mM arginine. When the isolated canine pancreas was perfused under the perfusate condition of acidosis (pH 7.1), ketoacidosis (pH 7.1 and 20 mM 3-OHB) or hyperosmolarity (+60 mOsm/kg with sucrose) throughout the experiment, the glucagon concentrations produced by 2.8 mM glucose under the ketoacidotic and hyperosmolar conditions, were less than half of those obtained under the standard condition. The insulin level was not influenced by the above perfusate conditions. These results suggest that 3-OHB inhibits glucagon secretion stimulated by glucopenia, but does not inhibit it stimulated by amino acids, and that hyperosmolarity inhibits glucagon secretion but does not inhibit insulin secretion. The pathophysiological significance of these results must be slight, considering the presence of hyperglucagonemia during prolonged starvation or diabetic ketoacidosis.     

Differential functions of genes regulated by VEGF-NFATc1 signaling pathway in the migration of pulmonary valve endothelial cells

We have reported that vascular endothelial growth factor (VEGF)-A induces the proliferation of human pulmonary valve endothelial cells (HPVECs) through nuclear factor in activated T cells (NFAT)c1 activation [1]. Here we show that VEGF-A increases the migration of HPVECs through NFATc1 activation, suggesting that VEGF-A/NFATc1 regulates the migration of HPVECs. To learn how this pathway may be involved in post-natal valvular repair, HPVECs were treated with VEGF-A, with or without cyclosporine A to selectively block VEGF-NFATc1 signaling. Down Syndrome critical region 1 (DSCR1) and heparin-binding EGF-like growth factor (HB-EGF) are two genes identified by DNA microarray as being up-regulated by VEGF-A in a cyclosporine-A-sensitive manner. DSCR1 silencing increased the migration of ovine valve endothelial cells, whereas HB-EGF silencing inhibited migration. This differential effect suggests that VEGF-A/NFATc1 signaling might be a crucial coordinator of endothelial cell migration in post-natal valves.     

Elevated fibroblast growth factor-2 increases tumor necrosis factor-alpha induced endothelial cell death in high glucose

Glucose and tumor necrosis factor-alpha (TNFalpha) concentrations are elevated in diabetes. Both of these factors correlate with diabetic vasculopathy and endothelial cell apoptosis, yet their combined effects have not been measured. We have previously shown that the angiogenic growth factor fibroblast growth factor-2 (FGF-2), which is generally protective against endothelial cell death, is similarly elevated in high glucose conditions. We therefore investigated the effect of TNFalpha on endothelial cell death under normal and elevated glucose conditions, with a particular focus on FGF-2. Porcine aortic endothelial cells were cultured in 5 and 30 mM glucose and stimulated with TNFalpha, together with FGF-2 or a neutralizing FGF-2 antibody. Cell death was measured via cell counts or an annexin apoptotic assay, and cell cycle phase was determined by propidium iodide labeling. TNFalpha-induced endothelial cell death increased for cells in high glucose, and cell death was enhanced with increasing FGF-2 exposure and negated by a neutralizing FGF-2 antibody. Endothelial cells were most susceptible to TNFalpha-induced cell death when stimulated with FGF-2 18 h prior to TNFalpha, corresponding to cell entry into S phase of the proliferative cycle. The FGF-2 associated increase in TNFalpha-induced cell death was negated by blocking cell entry into S phase. Endothelial cell release of FGF-2 in high glucose leads to cell cycle progression, which makes cells more susceptible to TNFalpha-induced cell death. These data suggest that growth factor outcomes in high glucose depend on secondary mediators such as cytokines and stimulation cell cycle timing.     

A case of diabetic non-ketotic hyperosmolar coma with an increase with plasma 3-hydroxybutyrate.

We have seen a case of "diabetic non-ketotic hyperosmolar coma" with ketosis. An 84-year-old man was brought into the hospital in a deeply comatous and dehydrated state. The initial blood glucose level was 1252 mg/dl with plasma osmolarity of 435 mOsm/l, but no ketonuria was detected by the nitroprusside method (Ketostix). However, the plasma 3-hydroxybutyrate (3-OHBA) level was 5 mM in a newly developed bedside film test. The serum ketone bodies were later found to be 5.56 and 0.82 mmol/l for 3-OHBA and acetoacetate (AcAc), respectively. A marked increase in glucagon, cortisol and ADH with renal dysfunction (creatinine 5.0 mg/dl) were noted. An abnormal electrocardiogram, occular convergence and chorea like movement disappeared after correction of metabolic disturbances. The moderate level of IRI (14 microU/ml) on admission and a good response to glucagon 2 months after admission also indicate that the present case is a typical hyperosmolar non-ketotic coma. Because of a preferential increase in 3-OHBA, ketonuria seemed to be absent in the regular nitroprusside test. Marked dehydration is thought to cause renal dysfunction, and the increase in ADH may have helped to prevent further aggravation of ketoacidosis. We propose to change the term hyperosmolar non-ketotic coma (HNC) to diabetic hyperosmolar coma (DHC), because sometimes patients with hyperosmolar non-ketotic diabetic coma are ketotic, as seen in the present case. Determination of 3-OHBA or individual ketone bodies in blood is important and essential for the differential diagnosis of diabetic coma. The diagnosis of either ketoacidotic or hyperosmolar coma should be made depending on the major expression of ketoacidosis or hyperglycemic hyperosmolarity.     

Stimulating effect of growth hormone on type IV collagen production by endothelial cells cultured in normal and high glucose

Collagen IV accumulation is characteristic of diabetic angiopathy. To test the possible contribution of GH, we studied its effects on collagen IV production by human umbilical vein endothelial cells at 5.5 and 16.7 mmol/l glucose. GH (100 ng/ml) markedly increased collagen IV level in the culture supernatant and in the insoluble extracellular matrix and cell fraction at both glucose concentrations. This stimulating effect of GH was additional to that of high glucose. It was more pronounced on collagen IV than on total protein synthesis. GH increased free latent gelatinase activity slightly at normal and markedly at high glucose. Using GF109203X, a PKC inhibitor, we observed that high glucose, but not GH, activated PKC. These two factors stimulating collagen IV production appear to work through different pathways, favoring an additivity of their effects. This supports the contribution of high plasma GH in diabetic vascular basement membrane thickening.     

Aquaporin-1 expression in proximal tubule epithelial cells of human kidney is regulated by hyperosmolarity and contrast agents

Primary cells of renal proximal tubule epithelium (S1 segment) of human kidney (HRPTE cells) up-regulate aquaporin-1 (AQP-1) expression in response to hyperosmolarity. NaCl and D(+)-raffinose increased (2-2.5 fold) AQP-1 expression when medium osmolarity was 400 and 500 mOsm/kg.H2O. Urea did not have this effect. Unlike our previous findings with mIMCD-3 cells, vasopressin (10(-8)M) did not affect AQP-1 expression in HRPTE cells in isosmolar or NaCl-enriched hyperosmolar conditions. Furthermore, HRPTE cells increased (3-4 fold) AQP-1 expression when exposed to hyperosmolar Reno-60 and Hypaque-76 (diatrizoates, ionic) contrast agents at 400 and 500 mOsm/kg.H2O. Isosmolar (290 mOsm/kg H2O) Visipaque (iodixanol, non-ionic) at 10% (v/v) concentrations also increased AQP-1 expression, and 25% v/v of Visipaque rendered morphological alterations of HRPTE cells and a 3-fold increase in AQP-1 expression after 24h exposure. Finally, semi-quantitative RT-PCR of HRPTE cells subjected to various isosmolar or hyperosmolar conditions demonstrated up-regulation of AQP-1 mRNA and protein levels. Our results suggest AQP-1 up-regulation in HRPTE cells exposed to environmental stresses such as hyperosmolarity and high doses of isosmolar contrast agents.     

Endothelial cell and platelet bioenergetics: effect of glucose and nutrient composition

It has been suggested that cells that are independent of insulin for glucose uptake, when exposed to high glucose or other nutrient concentrations, manifest enhanced mitochondrial substrate oxidation with consequent enhanced potential and generation of reactive oxygen species (ROS); a paradigm that could predispose to vascular complications of diabetes. Here we exposed bovine aortic endothelial (BAE) cells and human platelets to variable glucose and fatty acid concentrations. We then examined oxygen consumption and acidification rates using recently available technology in the form of an extracellular oxygen and proton flux analyzer. Acute or overnight exposure of confluent BAE cells to glucose concentrations from 5.5 to 25 mM did not enhance or change the rate of oxygen consumption (OCR) under basal conditions, during ATP synthesis, or under uncoupled conditions. Glucose also did not alter OCR in sub-confluent cells, in cells exposed to low serum, or in cells treated with added pyruvate. Likewise, overnight exposure to fatty acids of varying saturation had no such effects. Overnight exposure of BAE cells to low glucose concentration decreased maximal uncoupled respiration, but not basal or ATP related oxygen consumption. Labeled glucose oxidation to CO(2) increased, but only marginally after high glucose exposure while oleate oxidation to CO(2) decreased. Overnight exposure to linolenic acid, but not oleic or linoleic acid increased extracellular acidification consistent with enhanced glycolytic metabolism. We were unable to detect an increase in production of reactive oxygen species (ROS) from BAE cells exposed to high medium glucose. Like BAE cells, exposure of human platelets to glucose did not increase oxygen consumption. As opposed to BAE cells, platelet mitochondria demonstrate less respiratory reserve capacity (beyond that needed for basal metabolism). Our data do not support the concept that exposure to high glucose or fatty acids accelerates mitochondrial oxidative metabolism in endothelial cells or platelets.     

High glucose blocks the effects of estradiol on human vascular cell growth: differential interaction with estradiol and raloxifene

Because diabetic women appear not to be protected by estrogen in terms of propensity to cardiovascular disease, we tested the possibility that chronic hyperglycemia modulates the effects of E(2) on vascular cell growth in vitro. Human endothelial cells (E304) and vascular smooth muscle cells (VSMC) were grown in normal glucose (5.5 mmol/l), high glucose (22 mmol/l) or high manitol (22 nmol/l; an osmotic control) for 7 days. In endothelial cells glucose per se stimulated DNA synthesis. However E(2)- (but not RAL-) stimulated [3H] thymidine incorporation was attenuated in the presence of high glucose. In parallel, E(2)-dependent MAP-kinase-kinase activity was blocked in the presence of high glucose. High glucose increased basal creatine kinase (CK) specific activity, but E(2)-stimulated CK was not significantly impaired in the presence of high glucose. In VSMC, high glucose prevented the inhibitory effect of high E(2) (but not of high RAL) concentrations on DNA synthesis. High glucose also prevented E(2)-induced MAP-kinase-kinase activity. In contrast, while high glucose augmented basal CK, the relative E(2)-induced changes were roughly equal in normal and high high glucose media. Hence, high glucose blocks several effects of E(2) on vascular cell growth, which are mediated, in part, via the MAP-kinase system and are likely contributors to E(2)'s anti-atherosclerotic properties. Since RAL's estrogen-mimetic effects on human vascular cell growth were independent of MAP-kinase activation and were not affected by hyperglycemia, the potential use of RAL to circumvent the loss of estrogen function induced by hyperglycemia and diabetes in the human vasculature should be further explored.     

Comparison of protective effects of aspirin, D-penicillamine and vitamin E against high glucose-mediated toxicity in cultured endothelial cells

This study compared the protective effects of three different anti-glycation compounds, aspirin, D-penicillamine and vitamin E, against high glucose and advanced glycation endproduct (AGE) mediated toxicity in cultured bovine aortic endothelial cells using two approaches. Their proliferation was assessed in culture in different concentrations of glucose (5.5-100 mmol/l) with and without these inhibitors. A monolayer of cultured endothelial cells was wounded and recovery at the wound site was measured following exposure to different concentrations of glucose with and without inhibitors. The ability of these compounds to protect cultured endothelial cells following exposure to bovine serum albumin-derived advanced glycation endproducts (BSA-AGE) was also studied. Addition of glucose to cultured endothelial cells inhibited their proliferation in a dose dependent manner. All three compounds protected against the anti-proliferative effects of high glucose, with vitamin E being the most effective. The migration of cultured endothelial cells following wounding was inhibited by increasing concentrations of glucose but was maintained in the presence of all three anti-glycation compounds with vitamin E, again giving the greatest protection. Vitamin E was also the most effective at protecting against the anti-proliferative effects of BSA-AGE. D-penicillamine was not as effective as vitamin E whereas aspirin offered no significant protection against AGE-induced cellular toxicity. Our studies suggest that compounds, such as vitamin E, with combined antiglycation and antioxidant properties offer maximum therapeutic potential in protection against high glucose and AGE-mediated cellular toxicity.     

CD44 isoforms containing exon V3 are responsible for the presentation of heparin-binding growth factor

Glycosaminoglycan-modified isoforms of CD44 have been implicated in growth factor presentation at sites of inflammation. In the present study we show that COS cell transfectants expressing CD44 isoforms containing the alternatively spliced exon V3 are modified with heparan sulfate (HS). Binding studies with three HS-binding growth factors, basic-fibroblast growth factor (b-FGF), heparin binding-epidermal growth factor (HB-EGF), and amphiregulin, showed that the HS-modified CD44 isoforms are able to bind to b-FGF and HB-EGF, but not AR. b-FGF and HB-EGF binding to HS-modified CD44 was eliminated by pretreating the protein with heparitinase or by blocking with free heparin. HS- modified CD44 immunoprecipitated from keratinocytes, which express a CD44 isoform containing V3, also bound to b-FGF. We examined whether HS- modified CD44 isoforms were expressed by activated endothelial cells where they might present HS-binding growth factors to leukocytes during an inflammatory response. PCR and antibody-binding studies showed that activated cultured endothelial cells only express the CD44H isoform which does not contain any of the variably spliced exons including V3. Immunohistological studies with antibodies directed to CD44 extracellular domains encoded by the variably spliced exons showed that vascular endothelial cells in inflamed skin tissue sections do not express CD44 spliced variants. Keratinocytes, monocytes, and dendritic cells in the same specimens were found to express variably spliced CD44. 35SO4(-2)-labeling experiments demonstrated that activated cultured endothelial cells do not express detectable levels of chondroitin sulfate or HS-modified CD44. Our results suggest that one of the functions of CD44 isoforms expressing V3 is to bind and present a subset of HS-binding proteins. Furthermore, it is probable that HS- modified CD44 is involved in the presentation of HS-binding proteins by keratinocytes in inflamed skin. However, our data suggests that CD44 is not likely to be the proteoglycan principally involved in presenting HS- binding growth factors to leukocytes on the vascular cell wall.     

Osmotic stress induces HB-EGF gene expression via Ca(2+)/Pyk2/JNK signal cascades in rat aortic smooth muscle cells

The present study was undertaken in an attempt to clarify the pathway by which hyperosmotic stress induces HB-EGF gene expression in rat aortic smooth muscle cells (RASMC). Hyperosmotic stress induced by a high concentration of glucose or mannitol resulted in an increase in HB-EGF mRNA level in a dose- and time-dependent manner. HB-EGF induction was blocked by curcumin, a c-jun/fos antisense oligonucleotide and a dominant-negative mutant of JNK1. Electrophoretic mobility shift assay also showed the involvement of AP-1 in HB-EGF gene expression by glucose. In addition, hyperosmotic stress induced rapid phosphorylation of Pyk2 in RASMC. TPA and calcium chelating agents (BAPTA-AM and EGTA) blocked Pyk2 phosphorylation and HB-EGF gene expression. Furthermore, HB-EGF gene expression and JNK activation by hyperosmotic stress were sensitive to PP2, an Src kinase-specific inhibitor. These findings indicate that hyperosmotic stress activates JNK via calcium-Pyk2 signaling cascades, which in turn induce HB-EGF gene expression.