Tumor necrosis factor-induced E-selectin expression on vascular endothelial cells

OBJECTIVE: To determine the tumor necrosis factor (TNF) receptor type involved in induction of E-selectin expression on vascular endothelial cells. DESIGN: Prospective, in vitro repeated-measures analysis of cellular responses. SETTING: Research laboratory in an academic medical center. SUBJECTS: Cultured human umbilical vein endothelial cells. INTERVENTIONS: Human umbilical vein endothelial cells were incubated with recombinant human TNF (rhTNF) to induce the expression of E-selectin on their surfaces. To block rhTNF from binding to receptors, the cells were incubated with monoclonal antibodies against TNF receptors (anti-CD120a and anti-CD120b). TNF-induced E-selectin expression of the endothelial cells, with and without blocking antibodies, was then determined using indirect immunofluorescence and flow cytometry. MEASUREMENTS AND MAIN RESULTS: Blocking of either CD120a or CD120b receptors individually resulted in inhibition of TNF-induced E-selectin expression on human umbilical vein endothelial cells. When both antibodies were added, the inhibition of TNF-induced E-selectin expression was synergistic. Inhibition of E-selectin expression was dependent on both TNF concentrations and antibody concentrations. CONCLUSIONS: Both CD120a and CD120b receptors are involved in TNF-induced E-selectin expression on human umbilical vein endothelial cells. Blocking of both or one receptor type can reduce or totally inhibit expression of E-selectin on human umbilical vein endothelial cells, but the response is dependent on both TNF and antibody concentrations.     

Effect of serum from patients with mesangial proliferative glomerulonephritis on cultured rat mesangial cells

Adenosine, an important neuromodulatory compound in the brain and retina, is a potent vasodilator in most vascular beds throughout the body. Its actions are potentiated by inhibitors of nucleoside transport into cells. Knowledge of the existence of specific adenosine uptake systems in mammalian retina and the inhibition of the uptake by nitrobenzylthioinosine (NBMPR), a potent inhibitor of nucleoside transport, raises the possibility that the associated nucleoside transport system may be of pharmacological importance in retinal function. We have characterized the binding of the nucleoside transporter probe, [3H]NBMPR, to a cultured human retinal cell line established by transfection of SV-40 T antigen plasmid-DNA. The binding was specific, saturable and reversible. Scatchard analysis of the saturation data revealed that NBMPR binds to a homogeneous population of high affinity binding sites (KD = 0.65 +/- 0.22 nM; Bmax = 466 +/- 157 fmol/mg protein) characteristically similar to the binding sites in human retinal tissue (KD = 0.32 +/- 0.01 nM; Bmax = 292 +/- 41 fmol/mg protein). Selected compounds inhibited the binding in the cell line and retinal tissue with the same rank order of potency, suggesting that the transporters in the cell line and retinal tissue are similar. The data showed that the cell line is a useful model for the study of nucleoside transporter function in human retina.     

Anti-mitogenic effects of sarpogrelate in cultured rat mesangial cells

We investigated the effects of the new 5-HT2A receptor antagonist, sarpogrelate, on DNA synthesis in renal mesangial cells stimulated with 5-HT in the presence and absence of platelet-derived growth factor (PDGF)-BB. Both 5-HT and PDGF-BB demonstrated a mitogenic effect on these cells. When mesangial cells were incubated in the absence of PDGF-BB, sarpogrelate inhibited DNA synthesis in these cells in a dose-dependent manner. In the presence of PDGF-BB, sarpogrelate had a weaker anti-mitogenic effect in mesangial cells stimulated with 5-HT. Sarpogrelate was cytotoxic at concentrations over 10(-5) M according to the results of LDH release assays, and it reduced the S1 phase in mesangial cells stimulated with 5-HT by a flow cytometry. These findings suggest that sarpogrelate may be effective in the treatment of some glomerulonephritis associated with mesangial cell proliferation.     

Direct interactions between platelets and cultured rat mesangial cells

Platelets seem to be involved in the pathogenesis of some kidney diseases, but the exact relationships between platelets and the changes in renal function are incompletely known. Mesangial cells (MC) were incubated with platelet-supernatants (PS) and cellular surface area (CSA) and myosin light-chain phosphorylation (MLCP) were measured. CSA of PS-incubated MC (PS-MC) significantly diminished, as compared to control MC (70 +/- 6% vs. 100 +/- 5%). PS induced a significant increase in MLCP with respect to control cells (150 +/- 23% vs. 100 +/- 18%). When platelets were pretreated with indomethacin, the PS-dependent contraction was abolished. Pretreatment with sulotroban (SU) or BN-52021 (BN), a thromboxane A2 (TXA2) and a platelet-activating factor (PAF) receptor blocker respectively, also completely blocked the PS effects. In other experiments, platelets were activated with thrombin (T), adding the so obtained PS to MC. Moreover, cells were also preincubated with T and then added PS. No changes in CSA were observed in either case. It may be concluded that PS contracted cultured MC, and these changes could be related to the decreased glomerular filtration rate (GFR) observed in some diseases in which platelets seem to be involved. TXA2 and PAF may be responsible for this effect. In contrast, T incubation inhibited the effect of PS, perhaps through a direct relaxing effect of T in MC.     

Glomerular mesangial cells: electrophysiology and regulation of contraction

Mesangial cells are smooth muscle-like pericytes that abut and surround the filtration capillaries within the glomerulus. Studies of the fine ultrastructure of the glomerulus show that the mesangial cell and the capillary basement membrane form a biomechanical unit capable of regulating filtration surface area as well as intraglomerular blood volume. Structural and functional studies suggest that mesangial cells regulate filtration rate in both a static and dynamic fashion. Mesangial excitability enables a homeostatic intraglomerular stretch reflex that integrates an increase in filtration pressure with a reduction in capillary surface area. In addition, mesangial tone is regulated by diverse vasoactive hormones. Agonists, such as angiotensin II, contract mesangial cells through a signal transduction pathway that releases intracellular stores of Ca2+, which subsequently activate nonselective cation channels and Cl- channels to depolarize the plasma membrane. The change in membrane potential activates voltage-gated Ca2+ channels, allowing Ca2+ cell entry and further activation of depolarizing conductances. Contraction and entry of cell Ca2+ are inhibited only when Ca2+-activated K+ channels (BK(Ca)) are activated and the membrane is hyperpolarized toward the K+ equilibrium potential. The mesangial BK(Ca) is a weak regulator of contraction in unstimulated cells; however, the gain of the feedback is increased by atrial natriuretic peptide, nitric oxide, and the second messenger cGMP, which activates protein kinase G and decreases both the voltage and Ca2+ activation thresholds of BK(Ca) independent of sensitivity. This enables BK(Ca) to more effectively counter membrane depolarization and voltage-gated Ca2+ influx. After hyperpolarizing the membrane, BK(Ca) rapidly inactivates because of dephosphorylation by protein phosphatase 2A. Regulation of ion channels has been linked casually to hyperfiltration during early stages of diabetes mellitus. Determining the signaling pathways controlling the electrophysiology of glomerular mesangial cells is important for understanding how glomerular filtration rate is regulated in health and disease.     

High glucose inhibits cytosolic calcium signaling in cultured rat mesangial cells

Glomerular vasodilatation in the early stages of type I diabetes mellitus apparently results from arteriolar insensitivity to vasoconstrictors. Since cytosolic free calcium ([Ca2+]i) is a major signaling mechanism for smooth muscle contraction, we studied whether growth of smooth muscle-like rat glomerular mesangial cells in media with high glucose concentration affects [Ca2+]i responses to vasoconstrictors. In cells grown for five days in 22 mM glucose, we observed blunted responsiveness to three structurally unrelated vasoconstrictors that elevate [Ca2+]i via a phospholipase C-dependent mechanism, angiotensin II, prostaglandin F2 alpha, and arginine vasopressin. Inhibition of [Ca2+]i responses was not due to an osmotic effect of high glucose, since it was not mimicked by hypertonic mannitol. While the size of intracellular Ca2+ pools was unaffected by elevated glucose, Na+/Ca2+ exchange was markedly inhibited, thus ruling out both impaired filling of Ca2+ stores and enhanced counter-regulatory mechanisms. Impaired myoinositol transport or intracellular sorbitol accumulation were not responsible for the effects of high glucose, since supplementation of media with myo-inositol or with the aldose reductase inhibitor. Alcon 1576, failed to reverse insensitivity to vasoconstrictors. On the other hand, down-regulation or pharmacological inhibition of protein kinase C completely reversed the effects of high glucose, thus indicating involvement of this signal transduction pathway. These data suggest a possible intracellular mechanism for the impaired vascular sensitivity underlying early renal hemodynamic changes in diabetes mellitus.     

High glucose inhibits nitric oxide production in cultured rat mesangial cells

Hyperglycemia directly contributes to the development of diabetic nephropathy. A high-serum glucose concentration alters intraglomerular hemodynamics and promotes deposition of extracellular matrix in the kidney. Nitric oxide (NO) is a short-lived messenger molecule that participates in the regulation of renal blood flow, GFR, and mesangial matrix accumulation. Therefore, in this study it was tested whether high glucose directly modulates NO synthesis by rat mesangial cells in vitro by measuring the accumulation of nitrite, the stable metabolite of NO, in the incubation media. Raising the external glucose concentration to 33.3 mM for 24 to 72 h reduced nitrite levels in cell supernatants in a time-dependent manner to a nadir of 14 +/- 3% of the amount in normal glucose media (5.6 mM) (P < 0.01). The decline in NO synthesis in high glucose media was paralleled by decreased cyclic guanosine monophosphate generation; however, there was no alteration in rat mesangial cell expression of inducible NO synthase protein. The suppressive effect of high glucose on NO production by mesangial cells was not modified by inhibition of protein kinase C (H-7), the addition of antioxidants (vitamin E or superoxide dismutase), or a pan-specific anti-transforming growth factor-beta antibody. An elevated ambient glucose caused a time-dependent reduction in mesangial cell L-arginine content. Addition of L-arginine (10 to 20 mM) to external media partially reversed the inhibitory effect of high glucose on mesangial cell NO production in a dose-dependent manner. The highest dose of L-arginine (20 mM) increased mesangial cell L-arginine content to comparable levels in normal and high glucose media. These results indicate that high glucose causes depletion of L-arginine in mesangial cells and compromises NO synthesis. Limitation in the metabolic precursor and other, as yet unidentified, factors act to reduce NO production by mesangial cells in the presence of an elevated ambient glucose level, a change that may play a role in the development of diabetic glomerulosclerosis.     

Effect of emodin on c-myc proto-oncongen expression in cultured rat mesangial cells

We report a patient with chronic asymptomatic Chagas' disease that presented Trypanosoma cruzi reactivation after kidney transplantation and immune depression. The only clinical manifestation of the disease was ulcerative skin lesions, which is unusual in Chagas' disease.     

Receptor-mediated endocytosis of angiotensin II in rat myometrial cells

The events involved in the processing of the angiotensin II (Ang II)-receptor complex were studied in primary cultures of rat myometrial cells. Ang II bound to rat myometrial cells in a specific, time- and temperature-dependent fashion. Pretreatment with cycloheximide did not interfere with binding up to 3 hr, but inhibited increases in binding observed over longer periods. The [3H]Ang II binding to intact cells was inhibited by dithiothreitol (DTT), and the rank order of potency of Ang II and nonpeptide antagonists to inhibit the [3H]Ang II binding was Ang II > Losartan > PD 123319 or CGP 42112B, indicating the presence of the AT1 receptor type. Whereas most of the [3H]Ang II binding at 4 degrees was susceptible to acid or pronase treatment, binding at 35 degrees was resistant to both treatments, suggesting an internalization of the Ang II-receptor complex. Phenylarsine oxide (PAO) and N-ethylmaleimide (NEM) caused a concentration-dependent inhibition when the binding assay was performed at 35 degrees, but no effect was observed at 4 degrees, indicating that these agents did not alter cell-surface binding but actually prevented the internalization process. Simultaneous treatment with 1 mM DTT or beta-mercaptoethanol prevented the inhibitory effect of NEM, but only DTT could prevent the inhibition caused by PAO, indicating that two closely located sulfhydryl groups must be involved in the internalization process. Chloroquine (100 microM) inhibited the [3H]Ang II dissociation from cells, and monensin (25 microM) induced a 30% inhibition of [3H]Ang II binding (35 degrees, 3 hr), suggesting endosomal processing of the Ang II-receptor complex with receptor recycling to the cell surface. These results indicate that Ang II binding to AT1 receptors in rat myometrial cells is followed by internalization of the Ang II-receptor complex and recycling of the receptor to the cell surface.     

Sustained effect of angiotensin II on tyrosine phosphorylation of annexin I in glomerular mesangial cells

By means of selective extraction in a Ca(2+)-chelating medium and immunoblotting, four annexins (I, II, V, and VI) were identified in both isolated rat renal glomeruli and rat glomerular mesangial cells. Upon 32P labeling of these cells in culture, annexin I was immunoprecipitated using a specific polyclonal antibody and was found to incorporate radioactivity in a constitutive manner. However, as with epidermal growth factor (200 ng/ml), addition of angiotensin II (10(-7) M), arginine-vasopressin (10(-7) M), or endothelin I (10(-7) M) resulted in a 2-3-fold stimulation of annexin I phosphorylation. The basal phosphorylation as well as the stimulating effect of angiotensin II were also detected by immunoblotting annexin extracts using an antiphosphotyrosine antibody. In addition, among various phosphotyrosyl proteins isolated from EGTA extracts by adsorption onto an anti-phosphotyrosine antibody, annexin I was specifically recognized by Western blotting using a monoclonal anti-annexin I antibody, and displayed the same increase upon cell stimulation with angiotensin II. Moreover, thin layer chromatographic analysis of phosphoamino acids present in immunoprecipitated [32P]annexin I showed an exclusive labeling of phosphotyrosine residue(s). Finally, the effect of angiotensin II was detectable after 10 min, maximal at 6 h, and present until 12 h of incubation. Using 12-h stimulation, tyrosine phosphorylation of annexin I displayed a maximum at 10(-7) to 10(-6) M angiotensin II. These data report for the first time the stimulation of annexin I tyrosine phosphorylation by biologically active peptides acting via receptors belonging to the superfamily of seven hydrophobic domain, G-protein-linked receptors, which lack an intrinsic protein tyrosine kinase. This suggests a possible role of annexin I in the mitogenic effect of angiotensin II, arginine-vasopressin, and endothelin I, which was previously observed on rat glomerular mesangial cells as well as on other cells.     

Up-regulation of angiotensin type 2 receptor mRNA by angiotensin II in rat cortical cells

The present experiment demonstrates that the exposure of angiotensin II (AII) produced an up-regulation of the AT2 receptor mRNA level in rat cortical cells. AII (10(-9)-10(-5) M) exerted a marked increase of AT2 receptor mRNA in a dose-dependent manner. The maximum increase was observed at 3 hr of AII stimulation and lasted 3 hr. The up-regulation of AT2 receptor mRNA was antagonized by PD123319, an AT2 receptor antagonist, but not by SC-52458, an AT1 receptor antagonist, thus suggesting that the increase in AT2 receptor mRNA is mediated via AT2 receptor. This increase is blocked by serine/threonine phosphatase inhibitor okadaic acid, but not by the phosphotyrosine phosphatase inhibitor sodium vanadate, thus suggesting the involvement of serine/threonine phosphatase in this process. Protein kinase C inhibitor, H-7 and calphostin C, did not inhibit the AII-induced up-regulation significantly. In addition, calcium ionophore, A23187 had no effect. These findings suggest that the AT2 receptor mRNA expression by AII is regulated by the activity of serine/threonine phosphatase in the cortical neurons. This observation is also the first example concerning the regulation of AT2 receptor within the brain.     

Sodium appetite elicited by intracerebroventricular infusion of angiotensin II in the rat: II. Synergistic interaction with systemic mineralocorticoids.

Angiotensin and mineralocorticoids, the hormones of sodium conservation, acted together to arouse in male Sprague-Dawley rats a sodium appetite with shorter latency and greater magnitude than is produced by larger amounts of each acting alone. This potentiation was selective for sodium ingestion and occurred in the absence of significant changes in sodium balance. Therefore, because endogenous angiotensin and mineralocorticoids are concurrently elevated during sodium deficiency, sodium appetite may be aroused by a synergy of the peptide and the steroid. (29 ref) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Nitric oxide modulates the synthesis of extracellular matrix proteins in cultured rat mesangial cells

Nitric oxide (NO) is an important effector molecule of the inflammatory response. It is synthesized by mesangial cells and has been proposed to contribute to glomerular injury in various disease states. We studied whether NO modulates extracellular matrix production in cultured rat mesangial cells. Stimulation of rat mesangial cell NO release with gamma-interferon and lipopolysaccharide resulted in reduced production of collagen (by 35%) fibronectin (by 48%) (P < 0.05). In contrast, laminin synthesis was enhanced two-fold by the same maneuver (P < 0.05). These changes were reversed by the addition of L-NAME, a selective inhibitor of inducible nitric oxide synthase. This is the first demonstration that NO regulates the synthesis of extracellular matrix by mesangial cells. The results indicate that increased renal production of NO in glomerular diseases may attenuate the production and accumulation of matrix proteins and limit the severity of glomerulosclerosis.     

Effect of lipoproteins on cultured human mesangial cells

It was recently reported that low-density lipoprotein (LDL) promotes mesangial cell proliferation, and oxidized LDL is cytotoxic for mesangial cells. However, there have been few studies about the effects of other lipoproteins on mesangial cells. Accordingly, we investigated the effect of various lipoproteins on cultured human mesangial cells using 3H-thymidine (3H-TdR) incorporation and cell counting assays. We also investigated the levels of several cytokines in mesangial cell culture supernatants after stimulation by the lipoproteins. Addition of very-low-density lipoprotein (VLDL) at concentrations up to 100 micrograms/mL, intermediate-density lipoprotein (IDL) at up to 50 micrograms/mL, and LDL at up to 50 micrograms/mL induced the proliferation of cultured human mesangial cells, whereas cell growth was inhibited at higher concentrations. Oxidized LDL caused a concentration-dependent decrease of 3H-TdR incorporation. High-density lipoprotein (HDL) had no proliferative effective effect at any concentration. Exposure to VLDL, IDL, LDL, or a high concentration of HDL enhanced the secretion of interleukin-6, platelet-derived growth factor, and transforming growth factor-beta by mesangial cells, whereas tumor necrosis factor-alpha secretion was stimulated by oxidized LDL. These finding indicate that triglyceride (TG)-rich lipoproteins (VLDL and IDL) promote mesangial cell proliferation as well as LDL, whereas oxidized LDL has the reverse effect. These effects of lipoproteins may be related to modulation of various cytokines. Accordingly, TG-rich lipoproteins, LDL, and oxidized LDL may be involved in mesangial cell proliferation and injury in patients with mesangial proliferative glomerulonephritis.     

Cyclic stretching force selectively up-regulates transforming growth factor-beta isoforms in cultured rat mesangial cells

Glomerular distention from increased intraglomerular pressure stretches mesangial cells (MCs). Stretching MCs in culture stimulates extracellular matrix accumulation, suggesting that this may be a mechanism for glomerular hypertension-associated glomerulosclerosis. We examined whether mechanical stretching serves as a stimulus for the synthesis and activation of the prosclerotic molecule transforming growth factor (TGF)-beta, thus providing a potential system for auto-induction of extracellular matrix. Rat MCs cultured on flexible-bottom plates were subjected to cyclic stretching for up to 3 days and then assayed for TGF-beta mRNA, secretion of TGF-beta, and localization of active TGF-beta by immunostaining. MCs contained mRNA for all three mammalian isoforms of TGF-beta. Cyclic stretching for 36 hours increased TGF-beta1 and TGF-beta3 mRNA levels approximately twofold, without altering the levels of TGF-beta2 mRNA. This was followed at 48 to 72 hours by the increased secretion of both latent and active TGF-beta1. Latent, but not active, TGF-beta3 secretion also increased whereas the levels of TGF-beta2 were unaffected by mechanical force. The stretching force in this system is unequally distributed over the culture membrane. Localization of active TGF-beta by immunostaining demonstrated that the quantity of cell-associated cytokine across the culture was directly proportional to the zonal amplitude of the stretching force. These results demonstrate that stretching force stimulates MCs to selectively release and activate TGF-beta1. This mechanical induction of TGF-beta1 may help explain the increased extracellular matrix associated with intraglomerular hypertension.     

Tumor necrosis factor-induced apoptosis stimulates p53 accumulation and p21WAF1 proteolysis in ME-180 cells

Tumor necrosis factor (TNF)-mediated apoptotic signaling has been characterized by activation of specific protease or protein kinase cascades that regulate the onset of apoptosis. TNF has also been shown to induce oxidative or genotoxic stress in some cell types, and apoptotic potential may be determined by the cellular response to this stress. To determine the role of genotoxic stress in TNF-mediated apoptosis, we examined cellular accumulation of p53 in TNF-treated ME-180 cells selected for apoptotic sensitivity (ME-180S) or resistance (ME-180R) to TNF. Although TNF was able to activate receptor-mediated signaling in either cell line, p53 accumulation was measurable only in apoptotically sensitive ME-180S cells. TNF-induced changes in p53 levels were detected 1 h after treatment, and peak levels were measurable 4-8 h after TNF exposure. TNF was unable to induce p21WAF1 in either cell line but affected the stability of this protein in apoptotically responsive ME-180S cells. Evidence of p21WAF1 proteolysis was detected by monitoring the appearance of a 16-kDa immunoblottable p21WAF1 fragment, which became detectable 4 h after TNF addition and increased in content before the onset of DNA fragmentation (16-24 h). The kinetics of p21WAF1 proteolysis closely paralleled those of poly(ADP-ribose) polymerase, suggesting cleavage of p21WAF1 by activation of an apoptotic protease. Pretreatment of ME-180S cells with the apoptotic protease inhibitor YVAD blocked TNF-induced apoptosis and prevented both poly(ADP-ribose) polymerase and p21WAF1 degradation but did not affect p53 induction. These results provide evidence for the early onset of genotoxic stress in cells committed to TNF-mediated apoptosis and for divergence in propagation of this signal in non-responsive cells. In addition, TNF-induced p21WAF1 proteolysis may be mediated by an apoptotic protease and may contribute to the apoptotic process by disrupting p53 signaling, altering cell cycle inhibition, and limiting cellular recovery from genotoxic stress.     

Effects of low density lipoprotein on cytosolic [Ca++] in cultured rat mesangial cells

BACKGROUND: Recently, a low density lipoprotein (LDL) receptor has been identified in mesangial cells. However, the nature of intracellular signals in mesangial cells after exposure to LDL is unclear. METHODS: We studied the effect of LDL on cultured rat mesangial cell [Ca++]i using spectrofluorometry. RESULTS: Addition of LDL (15 micrograms/mL) produced a rapid, transient, and dose-dependent rise in [Ca++]i within seconds, returning to baseline in 6 minutes. No further rise was observed at higher LDL concentrations. No significant rise in [Ca++]i was observed with LDL in cells placed in a Ca(++)-free medium. The [Ca++]i rise was greatly attenuated in magnitude and duration when lanthanum was used. In contrast, verapamil failed to block the LDL-induced rise in [Ca++]i. Addition of LDL did not alter production of inositol 1,4,5-triphosphate (IP3) by mesangial cells. CONCLUSIONS: Low density lipoprotein caused a transient rise in [Ca++]i in cultured rat mesangial cells. The observed rise in [Ca++]i was largely caused by influx of extracellular Ca++ through receptor-gated channels. Mobilization from intracellular stores and activation of IP3 were not involved. The rise in [Ca++]i may mediate the effects of LDL on mesangial cell function.     

The effect of angiotensin II on myosin heavy chain expression in cultured myocardial cells

Angiotensin II (AII), the principal mediator of the renin-angiotensin system, is an important regulator of vascular and cardiac homeostasis. AII has also been shown to be a regulator of cardiac hypertrophy and of the corresponding changes in amount and composition of certain tissue proteins. We examined the trophic effects of AII on cultured myocytes derived from neonatal rat ventricles and followed, by Northern blot analysis and polyacrylamide gel electrophoresis, the expression of alpha- and beta-myosin heavy chain iso-mRNAs and isoproteins. Our findings show that a single administration of AII is sufficient to induce a trophic response in cultured beating myocytes and to enhance the expression of beta-myosin heavy chain iso-mRNA and isoprotein, having no effect on alpha-myosin heavy chain. Induction of alpha-myosin heavy chain expression by thyroid hormone before AII was administered showed that AII could not potentiate a shift from alpha- to beta-myosin heavy chain predominance. We suggest that the potency of AII to regulate the expression of myosin heavy chain isogenes is restricted to the beta isoform and is overridden by thyroid hormone.