Regulation of smooth muscle alpha-actin expression and hypertrophy in cultured mesangial cells

BACKGROUND: Mesangial cells during embryonic development and glomerular disease express smooth muscle alpha-actin (alpha-SMA). We were therefore surprised when cultured mesangial cells deprived of serum markedly increased expression of alpha-SMA. Serum-deprived mesangial cells appeared larger than serum-fed mesangial cells. We hypothesized that alpha-SMA expression may be more reflective of mesangial cell hypertrophy than hyperplasia. METHODS: Human mesangial cells were cultured in medium alone or with fetal bovine serum, thrombin, platelet-derived growth factor-BB (PDGF-BB) and/or transforming growth factor-beta1 (TGF-beta1). Alpha-SMA expression was examined by immunofluorescence, Western blot, and Northern blot analysis. Cell size was analyzed by forward light scatter flow cytometry. RESULTS: Alpha-SMA mRNA was at least tenfold more abundant after three to five days in human mesangial cells plated without serum, but beta-actin mRNA was unchanged. Serum-deprived cells contained 5.3-fold more alpha-SMA after three days and 56-fold more after five days by Western blot. Serum deprivation also increased alpha-SMA in rat and mouse mesangial cells. The effects of serum deprivation on alpha-SMA expression were reversible. Mesangial cell mitogens, thrombin or PDGF-BB, decreased alpha-SMA, but TGF-beta1 increased alpha-SMA expression and slowed mesangial cell proliferation in serum-plus medium. Flow cytometry showed that serum deprivation or TGF-beta1 treatment caused mesangial cell hypertrophy. PDGF-BB, thrombin, or thrombin receptor-activating peptide blocked hypertrophy in response to serum deprivation. CONCLUSIONS: We conclude that increased alpha-SMA expression in mesangial cells reflects cellular hypertrophy rather than hyperplasia.     

Developmental regulation of perlecan gene expression in aortic smooth muscle cells

The effect of environmental temperatures on immune competence was investigated in carp which were subjected to changes in water temperature. The activity of non-specific cytotoxic cells (NCC) against P815 target cells, and the anti-DNP antibody response were evaluated until day 56 after transfer. Low environmental temperature (12 +/- 0.5 degrees C) enhanced NCC activity and decreased antibody production. In contrast a high environmental temperature (28 +/- 0.5 degrees C) was without effect on these parameters when compared to the standard temperature (20 +/- 0.5 degrees C). The results showed a maximum effect of low environmental temperature on day 28 and an adaptation in these immune responses 56 days following transfer. Collectively, the results indicated that non-specific immunity tends to offset specific immune suppression at low environmental temperatures. To determine the mechanism(s) by which environmental temperature affects cellular immune function, membrane fluidity measurements and sialic acid titration, as well as stress assessment by plasma cortisol measurement, were determined on day 28. Taken together, the results revealed a direct effect of temperature on cellular immune function which is modulated by membrane fluidity and sugar concentration and not by stress induction.     

Regulation of 12-lipoxygenase by cytokines in vascular smooth muscle cells

Increasing evidence suggests that cytokines such as interleukin-1beta (IL-1), IL-4, and IL-8 may play an important role in the chronic inflammation and cellular growth observed in cardiovascular diseases. The lipoxygenase (LO) pathway of arachidonate metabolism has also been related to the pathology of hypertension and atherosclerosis. LO products have chemotactic, hypertrophic, and mitogenic effects in vascular cells, and the LO enzyme has been implicated in the oxidation of LDL. Furthermore, earlier studies have shown that vascular smooth muscle cell (VSMC) growth factors such as angiotensin II and platelet-derived growth factor can increase LO activity and expression in VSMCs. In the present study, we have examined whether vasoactive and inflammatory cytokines such as IL-1, IL-4, and IL-8 can modulate 12-LO activity and expression in porcine VSMCs and also whether they have growth-promoting effects in these cells. Treatment of porcine VSMCs with these cytokines led to significant increases in the levels of a cell-associated 12-LO product, 12-hydroxyeicosatetraenoic acid, as well as intracellular 12-LO enzyme activity. Furthermore, each of these cytokines led to a dose-dependent increase in 12-LO mRNA expression (333-base pair PCR product) as well as 12-LO protein expression (72 kD). In addition, all three interleukins could induce significant increases in VSMC DNA synthesis as well as proliferation. These results suggest that these cytokines have mitogenic effects in VSMCs and are also potent positive regulators of the 12-LO pathway. Thus, enhanced 12-LO activity and expression may be a key mechanism for cytokine-induced VSMC migration and proliferation.     

Regulation of inducible nitric oxide synthase (iNOS) and GTP cyclohydrolase I (GTP-CH I) gene expression by ox-LDL in rat vascular smooth muscle cells

The generation of nitric oxide is regulated by several factors, including the substrates and cofactors supplementation. Decreased expression and activity of nitric oxide synthase as well as diminished amount of L-arginine or enzyme cofactors results in the inhibition of nitric oxide generation in vascular wall cells. GTP cyclohydrolase 1 is a key enzyme involved in the synthesis of tetrahydrobiopterin, one of the most important cofactors of NO synthases. We have demonstrated that oxidized LDL inhibit not only inducible nitric oxide synthase gene expression but also GTP cyclohydrolase I gene expression in interleukin-1 beta activated rat vascular smooth muscle cells in vitro. It is postulated that diminished availability of tetrahydrobiopterin may additionally impair the generation of nitric oxide in atherosclerosis.     

Regulation of Na+,K(+)-ATPase activity by dopamine in cultured rat aortic smooth muscle cells

We investigated the effect of dopamine on Na+,K(+)-ATPase activity in cultured aortic smooth muscle cells. Na+,K(+)- ATPase activity was measured by a coupled enzyme assay. Our results demonstrate that dopamine and dopamine receptor agonists, SKF-38393 (a D1 receptor agonist) and quinpirole (a D2 receptor agonist) produced 62%, 50% and 49% inhibition of Na+,K(+)-ATPase activity in aortic smooth muscle cells, respectively. The combination of the two agonists produced inhibition similar to that of dopamine. Dopamine- and the agonist-induced Na+,K(+)-ATPase inhibition was blocked by selective receptor antagonists. The Na+,K(+)-ATPase inhibition by SKF-38393 but not by quinpirole was abolished by pertussis toxin. Na+,K(+)-ATPase inhibition was also achieved by guanosine triphosphate analog GTP-gamma-S. SKF-38393 but not quinpirole stimulated phosphoinositide hydrolysis rate in rat aortic slices. SKF-38393-induced phosphoinositide hydrolysis stimulation was reversed by SCH-23390, a dopamine D1 receptor antagonist, and attenuated by pertussis toxin. In conclusion, our observations indicate that dopamine and dopamine receptor agonists inhibit Na+,K(+)-ATPase activity through specific vascular receptors. Dopamine D1 receptors are linked to pertussis toxin sensitive-mechanism(s) and a GTP-binding protein appears to be coupled to the enzyme inhibition. Finally, the inhibition of Na+,K(+)-ATPase activity in response to dopamine D1 receptor activation may be mediated by the phospholipase C signaling pathway.     

Transforming growth factor beta1 induces IL-1 receptor antagonist production and gene expression in rat vascular smooth muscle cells

BACKGROUND: We compared long-term results of coronary artery bypass grafting between 1976 and 1988 in 176 patients 40 years old or younger with a matched control group of 176 patients 25 to 30 years older. METHODS: Mean age was 37.4 +/- 2.7 years (+/- standard deviation) in the study group and 64.2 +/- 2.9 years in the control group. Matching criteria were age, sex, left ventricular ejection fraction, number of bypass grafts, and year of operation. RESULTS: The study group had more smokers (p = 0.000) and more patients with hypercholesterolemia (p = 0.026), unstable angina (p = 0.003), and preoperative myocardial infarction (p = 0.009); fewer patients had hypertension (p = 0.000) and diabetes (p = 0.005) in this group than in the control group. The internal mammary artery was used in 31% of the study patients and in 30% of the controls. The actuarial survival rates after 5, 10, and 15 years were 92%, 86%, and 72% in the study group and 92%, 86%, and 66% in the control group (p = 0.202). Young age was a predictor of cardiac reoperation. CONCLUSIONS: Late survival is similar for young and older patients, but the reintervention rate is higher in the younger group. The absence of unstable angina, a left ventricular ejection fraction greater than 0.45, and the use of internal mammary artery grafts increase survival in all patients.     

Plasminogen activator expression in rat arterial smooth muscle cells depends on their phenotype and is modulated by cytokines

Cultured rat aortic smooth muscle cells (SMCs) exhibit at least 2 phenotypic variants: (1) a spindle-shaped phenotype, obtained from normal adult media, and (2) an epithelioid phenotype, obtained from intimal thickening 15 days after endothelial injury. Both phenotypes can be cloned from each location, with normal media yielding a majority of spindle-shaped clones and intimal thickening yielding a majority of epithelioid clones. These findings suggest that intimal thickening develops essentially from a subpopulation of medial SMCs exhibiting epithelioid features in vitro. Using zymographic and Northern blot analyses, we have studied plasminogen activator (PA) expression by these SMCs. Our results show that epithelioid SMCs, cultured as whole SMC populations or as clones, display higher PA activity than do spindle-shaped SMCs, irrespective of their origin. This is mainly due to differences in the expression of tissue PA and, to a lesser extent, urokinase PA and is accompanied by a decrease in PA inhibitor 1. Tissue PA activity is increased by basic fibroblast growth factor and platelet-derived growth factor-BB, particularly in epithelioid SMCs. Taken together, these results indicate that SMCs are heterogeneous with respect to their proteolytic profile, at least as far as the PA system is concerned. Proteolytic activity of the different SMC populations is modulated by cytokines that play a role in intimal thickening. Our results are in agreement with the suggestion that epithelioid SMCs are mainly responsible for intimal thickening.     

Elevated expression of membrane-type 1 and 3 matrix metalloproteinases in rat vascular smooth muscle cells activated by arterial injury

Matrix metalloproteinases (MMPs) play critical roles in tissue remodeling under various physiologic and pathologic conditions. We recently reported the expression of three membrane-type MMPs (MT-MMPs) by cultured vascular smooth muscle cells (SMCs) of rats (Shofuda et al, 1997). To investigate the roles of the MT-MMPs in the matrix remodeling of blood vessels, expression of MT1-MMP and MT3-MMP was examined in normal and balloon-injured rat carotid arteries by in situ hybridization and immunohistochemistry. Both MT-MMP mRNAs were detected in the intimal-dedifferentiated SMCs, but were negligible in the medial SMCs or in any of normal vascular cells. To elucidate the regulatory mechanism for the MT-MMPs expression, effects of various factors on cultured rat SMCs were also examined. MT1-MMP mRNA was constantly expressed at a high level, and its expression was weakly increased by treatment with interleukin-1beta or tumor necrosis factor-alpha. When the cells were incubated with type IV collagen, the MT1 -MMP expression was markedly decreased. On the other hand, expression of MT3-MMP mRNA was strongly increased by platelet-derived growth factor and fibronectin. These results suggest that type IV collagen may act as a negative regulator for the expression of MT1-MMP in the medial SMCs, whereas platelet-derived growth factor and fibronectin may up-regulate MT3-MMP expression under pathologic conditions. Furthermore, the elevated expression of MT1-MMP and MT3-MMP in SMCs was well associated with their dedifferentiated phenotype.     

Regulation of mitogen-activated protein kinase phosphatase-1 in vascular smooth muscle cells

Mitogen-activated protein (MAP) kinase cascades are major signaling systems by which cells transduce extracellular cues into intracellular responses. In general, MAP kinases are activated by phosphorylation on tyrosine and threonine residues and inactivated by dephosphorylation. Therefore, MAP kinase phosphatase-1 (MKP-1), a dual-specificity protein tyrosine phosphatase that exhibits catalytic activity toward both regulatory sites on MAP kinases, is suggested to be responsible for the downregulation of extracellular signal-regulated kinase (ERK), stress-activated protein kinase (SAPK), and p38 MAP kinase. In the present study, we examined the role of these MAP kinases in the induction of MKP-1 in vascular smooth muscle cells (VSMCs). Extracellular stimuli such as platelet-derived growth factor (PDGF), 12-O-tetradecanoylphorbol 13-acetate (TPA), and angiotensin II, which activated ERK but not SAPK/p38 MAP kinase, induced a transient induction of MKP-1 mRNA and its intracellular protein. In addition, PD 098059, an antagonist of MEK (MAP kinase/ERK kinase), the upstream kinase of ERK, significantly reduced the PDGF-induced activation of ERK and potently inhibited the expression of MKP-1 after stimulation with PDGF, thereby demonstrating the induction of MKP-1 in response to activation of the ERK signaling cascade. Furthermore, anisomycin, a potent stimulus of SAPK and p38 MAP kinase, also induced MKP-1 mRNA expression. This effect of anisomycin was significantly inhibited in the presence of the p38 MAP kinase antagonist SB 203580. These data suggest the induction of MKP-1, not only after stimulation of the cell growth promoting ERK pathway but also in response to activation of stress-responsive MAP kinase signaling cascades. We suggest that this pattern of MKP-1 induction may be a negative feedback mechanism in the control of MAP kinase activity in VSMCs.     

G protein-coupled receptor kinase 5 in cultured vascular smooth muscle cells and rat aorta. Regulation by angiotensin II and hypertension

GRK5, a recently cloned member of the G protein-coupled receptor kinase family, has been shown to phosphorylate and participate in the desensitization of angiotensin II (Ang II) type 1A (AT1A) receptors. In this study, the effect of angiotensin II on GRK5 expression was examined in cultured vascular smooth muscle cells and aortas of Ang II-infused hypertensive rats. In vascular smooth muscle cells, Ang II (100 nM) up-regulated GRK5 mRNA as early as 1 h, with a peak at 16 h. This up-regulation was dose- and calcium-dependent. The increase in GRK5 mRNA was reflected in a smaller increase in protein expression, which nonetheless had functional significance since AT1 receptor phosphorylation was increased and phospholipase C activation was decreased following prolonged incubation with Ang II. In aortas of Ang II-infused hypertensive rats, both GRK5 mRNA and protein levels increased approximately 3-fold compared with sham-operated rats at 5 and 7 days, respectively. This up-regulation was blocked either by losartan or by the nonspecific vasodilator hydralazine. Since a subpressor dose of Ang II did not increase GRK5 mRNA levels and norepinephrine infusion also increased GRK5 mRNA expression, we conclude that Ang II-induced GRK5 up-regulation in rat aortas may be due to hypertension per se. Hormone- and hemodynamic stress-induced GRK5 regulation may provide a novel molecular basis for long-term regulation of agonist sensitivity of vascular cells.     

Peroxisome proliferator-activated receptor gamma activators inhibit gene expression and migration in human vascular smooth muscle cells

Mesenchymal hamartomas of the liver are the second most common benign liver tumor of childhood. The experience with this tumor at Egleston's Children Hospital at Emory University from 1989 to 1994 is reviewed. Eight patients presented with abdominal distention or an upper abdominal mass. Six patients presented at a mean age of 8 months, and two patients presented at 17 and 23 years of age, respectively. Four patients displayed normal alpha-fetoprotein levels, whereas one patient had an elevated level. Liver function studies were normal in all patients. Abdominal ultrasonography and CT scans revealed a cystic, septated mass within the liver or on a pedicle in all patients. Five patients had simple excision of the tumor, and two had major hepatic resections. The cysts were multiloculated and lined with cuboidal bile duct epithelium surrounded by stroma containing proliferating bile ducts, blood vessels, and compressed liver tissue with no calcifications. In one patient, some pathologists favored the diagnosis of malignant myxoid fibrous histiocytoma because of similar-appearing stroma. Follow-up (mean, 35 months) revealed one symptomatic recurrence after initial resection was incomplete. There were no other recurrences and no malignant transformations. A septated, noncalcified, cystic hepatic mass in an infant with normal liver function studies and characteristic ultrasound or CT is likely a benign mesenchymal hamartoma that can be cured by total local excision.