A7r5 Vascular Smooth Muscle Research Articles

Smooth muscle--specific expression of CYP4A1 induces endothelial sprouting in renal arterial microvessels.

Cytochrome P450 (CYP) 4A1 has been characterized as the most efficient arachidonic acid omega-hydroxylase catalyzing the formation of 20-hydroxyeicosatetraenoic acid (20-HETE), a potent constrictor of the renal and cerebral microcirculation and a mitogen for smooth muscle cells. We constructed adenoviruses expressing the CYP4A1 cDNA or LacZ under the control of the smooth muscle cell-specific promoter SM22alpha (Ad-SM22-4A1 and Ad-SM22-nLacZ, respectively). Beta-galactosidase expression was detected in Ad-SM22-nLacZ-transduced vascular smooth muscle A7r5 and PAC1 cells, but not in Ad-SM22-nLacZ-transduced 3T3 fibroblasts or vascular endothelial cells. Likewise, CYP4A1 mRNA and protein were detected in Ad-SM22-4A1-transduced A7r5 and PAC1 cells. Ad-SM22-4A1-transduced A7r5 cells metabolized lauric acid to 12-hydroxy-lauric acid at a rate 5 times greater than that of cells transduced with Ad-SM22-nLacZ (4.79+/-1.77 versus 0.97+/-0.57 nmol 12-hydroxy lauric acid/10(6) cells per h). Smooth muscle-specific LacZ expression was also detected in microdissected renal interlobar arteries transduced with Ad-SM22-nLacZ. Arteries transduced with Ad-SM22-4A1 produced higher levels of 20-HETE (4.04+/-0.29 and 13.43+/-2.84 ng/mg protein in Ad-SM22-nLacZ-transduced and Ad-SM22-4A1-transduced arteries, respectively) and demonstrated a marked angiogenic activity measured as the total length of sprouting neovessels (12.63+/-3.66 mm in Ad-SM22-4A1-transduced vessels versus 1.79+/-0.89 mm in Ad-SM22-nLacZ-transduced vessels). This angiogenic activity represented endothelial cell sprouting and was fully blocked by treatment with HET0016, a selective inhibitor of CYP4A-catalyzed reactions. The inhibitory effect of HET0016 was reversed by addition of a 20-HETE agonist. We conclude that Ad-SM22-4A1 drives a smooth muscle-specific functional expression of CYP4A1 and demonstrates increased angiogenesis, presumably via increased production of 20-HETE.

Regulation of Corticotropin-Releasing Factor Receptor Type 2β Messenger Ribonucleic Acid by Interleukin-1β in Rat Vascular Smooth Muscle Cells

Objective: Corticotropin-releasing factor receptor type 2 (CRF R2) messenger RNA (mRNA) expression in the rodent heart or vessels is modulated by exposure to urocortin and glucocorticoids. In addition, we previously found that incubation with a variety of cytokines, such as interleukin (IL)-1 and IL-6, and tumor necrosis factor-α also reduced CRF R2β mRNA expression, with IL-1β being the most effective. In this study, we further explored the regulation of CRF R2β mRNA levels by IL-1β in the rat vascular smooth muscle A7r5 cells. Methods: A7r5 cells were incubated with IL-1β, urocortin, or both for 6 h, after pre-incubating with or without anti-IL-1β antibody (Ab) for 30 min, and then CRF R2β mRNA levels were measured by RNase protection assay. Cells were incubated with lipopolysaccharides, IL-1β, IL-6 , dexamethasone, forskolin, or urocortin for 20 min, and then intracellular cAMP was measured by cAMP RIA. Results: IL-1β produced a significant time-dependent decrease in CRF R2β mRNA levels. Combined urocortin and IL-1β administration did not have synergistic effects on the decrease in CRF R2β mRNA levels. IL-1β Ab failed to block the ability of urocortin to regulate CRF R2β mRNA levels, suggesting that urocortin regulated CRF R2β mRNA levels via another pathway than IL-1β production. Urocortin induced the intracellular cAMP production in A7r5 cells, while IL-1β failed to induce it. Conclusion: The multifactorial regulation of CRF R2β mRNA expression in the A7r5 cells serves to limit the inotropic and chronotropic effects of CRF R2 agonists such as urocortin during prolonged physical or immune challenge.

Transcriptional Regulation of the Human iNOS Gene in Vascular-Smooth-Muscle Cells and Macrophages: Evidence for Tissue Specificity

We have cloned the 5′ upstream −1034 to +88 fragment of the human inducible nitric oxide synthase (hiNOS) gene and demonstrate its competence to promote luciferase gene transcription in vascular-smooth-muscle (VSM) cells and macrophages. Sequential 5′ end-deletions localized positive regulatory elements of hiNOS transcription in VSM A7r5 cells downstream of nucleotide −205 and demonstrated the functional importance of the resident NF-κB site (nucleotides −115 to −106). The hiNOS promoter/enhancer was induced strongly by LPS and IFN-γ and modestly by IL-1β in RAW 264.7 cells, but not in VSM cells. Truncation of the NF-κB site markedly diminished, but did not eliminate, LPS-inducibility. Sodium salicylate and ibuprofen down-regulated the basal transcriptional activity of the hiNOS promoter/enhancer in VSM but not in RAW 264.7 cells. These results indicate that the transcriptional regulation of the hiNOS gene features considerable complexity and tissue specificity.

Manidipine inhibits endothelin-1-induced [Ca 2+] i signaling but potentiates endothelin's effect on c-fos and c-jun induction in vascular smooth muscle and glomerular mesangial cells

We studied the effects of manidipine, a newly synthesized dihydropyridine Ca 2+ channel antagonist, on endothelin-1 (ET-1)-induced [Ca 2+] i signaling and immediate-early gene induction in vascular smooth muscle A7r5 cells and glomerular mesangial cells (MCs). ET-1-induced a dose-dependent, biphasic increase in [Ca 2+] i in both A7r5 cells and MCs. Manidipine inhibited the ET-1-induced [Ca 2+] i increase by reducing both the transient and sustained Ca 2+ increments in A7r5 cells and MCs. At concentrations of 10 −10 to 10 −6 mol/L, inhibition was dose dependent with a median effective dose (ED 50) of 10 −9 mol/L. A 20-minute preincubation period was required to observe the maximal inhibitory effect of manidipine. In contrast, manidipine (10 −5 mol/L) potentiated ET-1-induced c-fos and c-jun expression in A7r5 cells. This potentiating effect appeared 30 minutes after manidipine was added to the media and lasted for 15 hours. Manidipine alone had no detectable effect on c-fos steady-state messenger ribonucleic acid level. Manidipine induced c-jun expression starting at 60 minutes but with much lower potency. These data demonstrate that manidipine is a potent inhibitor for ET-1-induced [Ca 2+] i signaling and that manidipine has multiple effects on ET-1-induced signaling, including potentiating the immediate-early gene response.

The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na+/H+ and Cl-/HCO3- antiporters. The Na+/H+ exchanger is a major route of H+ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H+ into the extracellular space in exchange for Na+. The Cl-/HCO3- exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na+/H+ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl-/HCO3- exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in 'sensing' changes in intracellular pH, to which these antiporters respond.