Primary cultures of human arterial SMCs were initiated by explant outgrowth from unused segments of internal thoracic artery retrieved at the time of coronary artery bypass surgery, as previously described.4142 The identity of vascular SMCs was confirmed morphologically and by positive immunostaining for smooth muscle α-actin. The samples were incubated for 90 minutes at 37°C and undigested proteins precipitated on ice with 10% trichloroacetic acid. The rate of collagen synthesis, relative to that of all proline-containing proteins, was calculated assuming that the number of proline residues in collagen is 5.4-fold higher than that in noncollagen proteins.44 The relative rate of collagen synthesis was thus determined as: SMCs were stimulated with various concentrations of angiotensin II, in the presence or absence of angiotensin II receptor antagonists, anti-TGF-β antibody, and tyrosine kinase inhibitors. Procollagen mRNA levels were normalized to glyceraldehyde 3-phosphate dehydrogenase mRNA abundance and expressed as a percentage of vehicle-treated controls. We next determined if angiotensin II–stimulated production of TGF-β mediated the observed increase in collagen expression in SMCs. Angiotensin II (ANG II) stimulates production of superoxide (O 2 −) by NADPH oxidase (NOX) in medullary thick ascending limbs (TALs).There are three isoforms of the catalytic subunit (NOX1, 2, and 4) known to be expressed in the kidney. To determine if stimulation of SMCs with angiotensin II resulted in elaboration of TGF-β, a mink lung epithelial cell TGF-β bioassay was used. Neutralizing antibody to TGF-β (30 μg/mL) but not a control IgG (30 μg/mL) abrogated the increase in proα1(I) collagen mRNA expression (normalized to changes in glyceraldehyde 3-phosphate dehydrogenase mRNA levels) induced by 10−8 mol/L angiotensin II. The amount of proα1(I) collagen mRNA per sample was determined from a standard curve, generated from hybridization reactions by using known concentrations of a proα1(I) collagen mRNA 300-base fragment. Role of TGF-β in angiotensin II (Ang II)-stimulated collagen expression by human SMCs. To determined if tyrosine phosphorylation was functionally linked to collagen synthesis, we studied the effect of 2 mechanistically distinct inhibitors of tyrosine kinase, genistein, and tyrphostin A25. The adenovirus expressing the bacterial β-galactosidase (AdLacZ) was used as a control. Figure 7. Published by Elsevier Inc. on behalf of American Society for Biochemistry and Molecular Biology. We studied the effect of angiotensin II on collagen production by human arterial SMCs, using uptake of [3H]proline into collagenase-digestible proteins, and by ribonuclease protection assay for mRNA encoding the proα1 chain of type I collagen, the major collagen in arteries. Labeled riboprobe (120 pg) was added to 2.0 to 3.0 μg of total RNA from each experimental sample. 1-800-AHA-USA-1 Inhibition of phospholipase C (PLC) or protein kinase C (PKC) also abolished the stimulatory effect of AngII on Cl channels. The effect is mediated by the AT1 receptor, suggesting that pharmacological therapy with AT1 receptor antagonists may modulate collagen accumulation during vascular disease. In similar manner, tyrphostin A25, but not the inactive homolog tyrphostin A1, blocked angiotensin II–stimulated procollagen expression. Anti-smooth muscle α-actin antibody (clone 1A4) was obtained from Dako. To test this, SMCs were incubated for 48 hours with angiotensin II and either genistein, tyrphostin A25, or the inactive homolog tyrphostin A1, and conditioned medium was assayed for TGF-β activity. Angiotensin II, long recognized as a regulator of vascular tone, has also garnered interest because of its capacity to act as a vascular cell growth factor. All experiments were performed by using human SMCs in the third or fourth subculture. A, Dose–response curve of effect of angiotensin II on proα1(I) collagen mRNA abundance, as determined by RNase protection assay. To explore the possibility that tyrosine kinases may be involved in upregulation of type I collagen by angiotensin II, we used 2 mechanistically distinct inhibitors of tyrosine phosphorylation. As shown in Figure 6, 10 μmol/L genistein as well as 50 μmol/L tyrphostin A25 significantly inhibited angiotensin II–stimulated TGF-β production, whereas 50 μmol/L tyrphostin A1 had no effect.
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