p160 Bcr mediates platelet-derived growth factor activation of extracellular signal-regulated kinase in vascular smooth muscle cells.

BACKGROUND: The human Bcr gene was originally identified by its presence in the chimeric Bcr/Abl oncogene, which is causative for chronic myeloblastic leukemia. Because Bcr encodes a serine/threonine protein kinase, we studied its kinase activity and determined the role of Bcr in the PDGF signaling pathway to ERK1/2 activation and DNA synthesis in rat aortic smooth muscle cells (RASMCs). METHODS AND RESULTS: In RASMCs, platelet-derived growth factor-BB (PDGF) stimulated Bcr kinase activity, with a maximum at 1 minute. Because phosphatidylinositol 3'-kinase (PI3-K) is essential for Bcr/Abl leukemogenesis, we evaluated the role of mouse PDGF-beta-receptor binding sites for PI3-K (Y708, Y719) and for phospholipase C-gamma (Y977, Y989) in PDGF-mediated Bcr kinase activation. The mutant PDGF receptor Y708F/Y719F but not Y977F/Y989F showed significantly reduced Bcr kinase activity. To determine the role of Bcr in PDGF-mediated signal transduction events leading to ERK1/2 and its downstream Elk1 transcription activation, wild-type (WT) and kinase-negative (KN) Bcr were transiently expressed in RASMCs. Bcr WT enhanced, whereas Bcr KN inhibited, PDGF-stimulated ERK1/2 and Elk1 transcriptional activity. Overexpression of Bcr also enhanced PDGF-induced Ras/Raf-1 activity and DNA synthesis, but this regulation is independent of the kinase activity of Bcr. Finally, we found that Bcr expression was increased in the neointimal layer after balloon injury of rat carotid artery. CONCLUSIONS: These results demonstrated the importance of Bcr in PDGF-mediated events, such as activation of Ras, Raf-1, ERK1/2, and Elk1, and stimulation of DNA synthesis.

Cyclophilin A is a secreted growth factor induced by oxidative stress.

Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis, hypertension, and restenosis, in part by promoting vascular smooth muscle cell (VSMC) growth. Many VSMC growth factors are secreted by VSMC and act in an autocrine manner. Here we demonstrate that cyclophilin A (CyPA), a member of the immunophilin family, is secreted by VSMCs in response to oxidative stress and mediates extracellular signal-regulated kinase (ERK1/2) activation and VSMC growth by reactive oxygen species. Human recombinant CyPA can mimic the effects of secreted CyPA to stimulate ERK1/2 and cell growth. The peptidyl-prolyl isomerase activity is required for ERK1/2 activation by CyPA. In vivo, CyPA expression and secretion are increased by oxidative stress and vascular injury. These findings are the first to identify CyPA as a secreted redox-sensitive mediator, establish CyPA as a VSMC growth factor, and suggest an important role for CyPA and enzymes with peptidyl-prolyl isomerase activity in the pathogenesis of vascular diseases.

The Gas6/Axl system: a novel regulator of vascular cell function.

Signal transduction downstream of tyrosine kinases has become an increasingly important area of study in cardiovascular biology. In this review, we consider the experimental evidence pointing to significant roles for the Axl receptor tyrosine kinase and its ligand, Gas6, in the vasculature. An introduction to the Gas6/Axl system and a discussion of its discovery is followed by a summary of the data regarding expression of Gas6/Axl in injured arteries. We conclude by discussing mechanisms by which Gas6/Axl signaling may impact on the response of blood vessels to injury, thereby contributing to the development of atherosclerosis and/or restenosis.

Increased expression of Axl tyrosine kinase after vascular injury and regulation by G protein-coupled receptor agonists in rats.

Axl is a receptor tyrosine kinase originally identified as a transforming gene product in human myeloid leukemia cells. Cultured rat vascular smooth muscle cells also express Axl, where it has been proposed that Axl may play a role in cell proliferation. In the current study, we tested the hypotheses that Axl expression would parallel neointima formation in balloon-injured rat carotid, and that Axl expression would be regulated by growth factors present at sites of vascular injury. Ribonuclease protection assay showed dynamic increases in Axl mRNA in vessels, with peak expression 7 and 14 days after injury. Immunohistochemical analysis confirmed these results and demonstrated that Axl protein expression was localized primarily to cells of the neointima after injury. Northern blot analysis indicated increased mRNA expression for the secreted Axl ligand, Gas6, in injured carotids, with a time course paralleling that of Axl upregulation. Axl and Gas6 expression were temporally correlated with neointima formation, suggesting a role for Axl signaling in this process. Other studies, performed in cultured rat vascular smooth muscle cells, revealed positive regulation of Axl mRNA expression by thrombin or angiotensin II but not by basic fibroblast growth factor, platelet-derived growth factor-BB, or transforming growth factor-ss1. Western blot analysis confirmed these results, showing that Axl protein expression was specifically increased by thrombin or angiotensin II. Our results implicate Axl as a potential mediator of vascular smooth muscle migration and proliferation caused by vascular injury and G protein-coupled receptor agonists.

Change in pressor responsiveness to angiotensin II as a determinant of blood pressure after unclipping in two-kidney, one clip hypertensive rats.

The hypotensive effect of correction of renal artery stenosis in humans or experimental animals with renovascular hypertension is commonly attributed to decreasing renin secretion from the formerly stenotic kidney. However, plasma renin activity is normal in 50% of individuals with renovascular hypertension. We studied conscious, chronically instrumented two-kidney, one clip (2K1C) rats. The renal artery clip was removed and mean arterial pressure measured for 3 days. The majority of the fall in blood pressure occurred between 2 and 48 hours after unclipping. Measurement of water balance and urinary sodium excretion revealed no effect of unclipping. In another experiment, 2K1C hypertensive rats were chronically treated with enalapril and concomitant infusion of angiotensin II (3.8 pmol/min [4 ng/min] IV) to maintain blood pressure at hypertensive levels and prevent a fall in angiotensin II levels on unclipping. After 5 days, the clip was removed or sham removed, and treatment was continued. Blood pressure was recorded for 7 days. Blood pressure remained elevated in the sham unclipped rats. Unclipped rats exhibited a dichotomous response; blood pressure fell significantly within 48 hours in the majority of rats (responders) but remained elevated in a minority (nonresponders). All treatment was then withdrawn for 2 days. Sham unclipped rats remained hypertensive, and responders exhibited a further small decline in blood pressure. Blood pressure fell to normal in the nonresponders. Blood pressure falls after correction of renal artery stenosis in renovascular hypertension in part because of a decrease in pressor responsiveness to angiotensin II.

Role of ANG II in hypertension produced by chronic inhibition of nitric oxide synthase in conscious rats.

These experiments tested the hypothesis that hypertension caused by chronic inhibition of nitiric oxide synthase (NOS) is associated with augmented pressor responsiveness to angiotensin II (ANG II). Antagonism of ANG II AT1 receptors with losartan caused a greater fall in blood pressure (BP) in rats treated for 2 wk with the NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) than in normotensive rats. The delayed time course of the decline in BP implicated the slow pressor effect (SPE) of ANG II in L-NAME hypertension. Further experiments showed that direct elicitation of the SPE by continuous low-dose (4 ng/min) intravenous infusion of ANG II in enalapril-treated rats resulted in a larger chronic increase in BP if NOS was inhibited. However, L-NAME alone also caused a significant increase in BP in enalapril-treated rats. The combined effect on BP of ANG II and L-NAME was merely additive. These results confirm that ANG II is involved in L-NAME hypertension. However, chronic pressor responsiveness to the peptide is not augmented by L-NAME.

Inhibition of the slow pressor effect of angiotensin II contributes to the antihypertensive effect of angiotensin-converting enzyme inhibitors in renovascular hypertension.

The authors recently reported that renovascular hypertension (RVH) in rats is associated with enhanced responsiveness to the slow pressor effect of angiotensin II (AngII). It was concluded that the contribution of AngII to the development of hypertension could not be inferred solely from measurements of plasma peptide concentrations. This experiment was performed to test the corollary hypothesis that the antihypertensive efficacy of angiotensin-converting enzyme inhibitors in established RVH could be caused in part by inhibition of the slow pressor effect. After five control days of measurement of mean arterial pressure (MAP) and other variables, RVH rats were given the angiotensin-converting enzyme inhibitor enalapril in their drinking water. Half of these rats also received a continuous infusion of AngII, 4 ng/min i.v., an infusion rate previously shown to restore normal MAP in normal rats rendered hypotensive by chronic treatment with enalapril. Other RVH rats were given the potent vasodilator minoxidil in their drinking water, and half of these received AngII. Treatments lasted 7 days and were followed by 5 days without intervention. Enalapril significantly reduced MAP, but concurrent AngII infusion overcame this effect; MAP remained at control hypertensive levels. AngII did not affect the antihypertensive response to minoxidil. These results confirm that addition of relatively modest amounts of AngII to the circulation is able to maintain elevated blood pressure in RVH. Thus, inhibition of the slow pressor effect of AngII may partially explain the antihypertensive effect of angiotensin-converting enzyme inhibitors in RVH.

Slow pressor effect of angiotensin II in normotensive rats with renal artery stenosis.

1. We have shown previously that renal artery stenosis in rats causes enhanced responsiveness to the slow pressor effect of angiotensin II (AngII) and suggested that two-kidney, one clip (2K1C) hypertension may depend, in part, on changes in responsiveness to the peptide. 2. The present experiment was performed in order to investigate whether a degree of renal artery stenosis that was insufficient to raise blood pressure was able to enhance responsiveness to the slow pressor effect of AngII. 3. Two to four weeks after placement of a 0.2 mm clip over the left renal artery (2K1C) or a sham operation, some 2K1C rats were normotensive. These rats and the sham rats then received an intravenous infusion of AngII (4 ng/min) for 10 days. 4. AngII caused the 2K1C rats to attain significantly higher mean arterial pressure than the sham rats (152 +/- 7 vs 133 +/- 7 mmHg) and did not result in water or electrolyte retention in the 2K1C rats. 5. These results indicate that normotensive 2K1C rats exhibit enhanced responsiveness to the slow pressor effect of AngII and that the arterial pressure response to renal ischaemia may depend on both AngII formation and responsiveness to the chronic actions of the peptide.

Enhanced slow pressor effect of angiotensin II in two-kidney, one clip rats.

Phase II of two-kidney, one clip (2K1C) Goldblatt hypertension in the rat is characterized by elevated blood pressure and near-normal plasma concentrations of angiotensin II (Ang II) but is reversed by inhibition of the renin-angiotensin system. We hypothesized that this angiotensin dependence is due to enhanced responsiveness to the slow pressor effect of Ang II caused by renal artery stenosis. To test this idea, we submitted rats to either renal artery clipping or sham operation. These groups were immediately subdivided; some animals received enalapril in their drinking water (508 mumol/L), and the rest drank distilled water only. After 10 to 14 days, catheters were inserted into the aorta and vena cava, and the rats were housed in metabolism cages. After 3 control days of measurement of mean arterial pressure and other variables, the enalapril-treated groups received an intravenous infusion of Ang II at a dose of 3.8 pmol/min (4 ng/min) for 14 days. Rats not drinking enalapril received only saline vehicle (2 mmol Na+ per day). After 3 days of Ang II infusion, the enalapril-treated 2K1C rats had attained a significantly higher level of mean arterial pressure than the enalapril-treated sham rats. At the end of the Ang II infusion, mean arterial pressure in enalapril-treated 2K1C rats was 151 +/- 6 mm Hg versus 107 +/- 7 mm Hg in enalapril-treated sham rats. Mean arterial pressure in the enalapril-treated sham rats after Ang II infusion was not significantly different from that of untreated sham rats (109 +/- 2 mm Hg).(ABSTRACT TRUNCATED AT 250 WORDS)

Time course of losartan blockade of angiotensin II hypertension versus blockade of angiotensin II fast pressor effects.

This study investigated the ability of the selective angiotensin II (AII)-type (AT)1 receptor antagonist losartan to reverse the fast (< 30 sec) pressor effect of AII, and the hypertension produced by chronic (2 weeks) i.v. infusion of AII (AII hypertension). We hypothesized the following: if AII hypertension is caused solely by stimulation of AT1 receptors mediating the fast pressor effect, then the time course of the antihypertensive effect of losartan in AII hypertension should parallel the time course of losartan inhibition of pressor responses to acute, bolus injection of AII. Thus, in one group of rats, pressor responses to bolus injections of AII (10 ng, n = 10) were measured before and subsequently at numerous time points after losartan administration (3 mg.kg-1 i.v.). Other groups of rats received continuous infusions of AII i.v. for 15 days at 2 ng.min-1 (n = 8), 4 ng.min-1 (n = 8) or 10 ng.min-1 (n = 6). On days 2, 7 and 12 of the AII infusion, rats received a bolus injection of losartan (3 mg.kg-1, i.a.). Mean arterial pressure (MAP) was then measured at numerous time points after losartan administration. Within 5 min of administration, losartan caused almost complete inhibition of fast pressor responses to acute injections of AII, and the magnitude of this inhibition did not change for over 24 hr. On the other hand, in AII hypertension, losartan lowered MAP significantly within 5 min only in rats receiving 10 ng.min-1 AII, but in all three groups caused a slower decline in MAP that peaked around 2 hr after losartan injection.(ABSTRACT TRUNCATED AT 250 WORDS)