Donna Lombardi

Angiotensin II induces smooth muscle cell proliferation in the normal and injured rat arterial wall.

The present study was undertaken to explore the possibility that neointimal smooth muscle cells, the characteristic cells of restenosis and atherosclerosis, are selectively stimulated to replicate by a hypertensive stimulus. Angiotensin II (AII) was infused by osmotic minipumps for 2 weeks in 4.5-month-old rats. Group A received AII (200 ng/min) 2 weeks after a balloon catheter-induced injury of the thoracic aorta and left common carotid artery. Group B received only AII, group C only balloon denudation, and group D neither balloon injury nor AII. During the AII or Ringers solution infusion, all animals received [3H]thymidine via a second minipump to measure DNA synthesis. AII increased the systolic pressure by more than 40 mm Hg. AII significantly increased DNA synthesis in the media of the carotid artery from 0.2 +/- 0.2% in group C to 2.5 +/- 1.5% in group A (mean +/- SD, n = 5 or 6). DNA synthesis in the neointima of the carotid artery significantly increased with AII from 4.8 +/- 4.2% in group C to 19.8 +/- 13.9% in group A. Cross-sectional area of the neointima almost doubled during AII infusion, and it increased approximately 25% in the media. Comparable results were obtained in the aorta. In a second experiment, AII was infused (125 ng/min) for 2 weeks in 11-week-old rats. Concomitantly, [3H]thymidine was given. Control rats received Ringers solution and [3H]thymidine in their pumps. Blood pressures were elevated to the same extent as in the older animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Renal injury from angiotensin II-mediated hypertension.

Angiotensin II (Ang II)-mediated hypertension induces vascular smooth muscle cell hypertrophy and hyperplasia in systemic blood vessels, but the effects of Ang II on the intrinsic cell populations within the kidney have been less well characterized. We infused Ang II for 14 days into rats by minipump at doses (200 ng/min) that resulted in moderate hypertension (mean systolic blood pressure 156-172 mm Hg). Small renal arterial vessels of Ang II-infused rats demonstrated focal injury with fibrinoid necrosis and medial hyperplasia, whereas the glomerular capillaries demonstrated only rare segmental hyalinosis. Proliferation of vascular smooth muscle cells was pronounced (fourfold to 20-fold increase in [3H]thymidine incorporation) as opposed to a minimal proliferation of glomerular cells in Ang II-infused rats. In contrast, the principal effect of Ang II in glomeruli was to increase the expression of alpha-smooth muscle actin by mesangial cells and desmin by visceral glomerular epithelial cells. Ang II-infused rats also developed focal tubulointerstitial injury, with tubular atrophy and dilation, cast formation, an interstitial monocytic infiltrate, and mild interstitial fibrosis with increased type IV collagen deposition. The injury was associated with a proliferation of distal tubule, collecting duct, and interstitial cells as determined by immunostaining for proliferating cell nuclear antigen, and was accompanied by an increase in platelet-derived growth factor B-chain messenger RNA in the area of interstitial injury as localized by in situ hybridization. Renal interstitial cells also underwent phenotypic modulation in which they expressed alpha-smooth muscle actin. Vehicle-infused control rats displayed no tubular injury, proliferation, or phenotypic modulation. Thus, Ang II in doses that cause moderate hypertension induces marked vascular, glomerular, and tubulointerstitial injury with cell proliferation, leukocyte recruitment, phenotypic modulation with the upregulation of proteins normally associated with smooth muscle cells, and interstitial fibrosis.

Mesangial cell apoptosis: the major mechanism for resolution of glomerular hypercellularity in experimental mesangial proliferative nephritis.

Increases in mesangial cell number may herald glomerular scarring, but they are not irreversible. This study sought mechanisms by which surplus glomerular mesangial cells can be cleared. A small proportion of cultured mesangial cells exhibited typical morphological features of apoptosis (programmed cell death), which was increased by growth factor deprivation or exposure to cycloheximide, stimuli known to increase apoptosis in other cell types. Apoptosis was confirmed by typical internucleosomal chromatin cleavage. In vivo, clear morphological evidence of mesangial apoptosis leading to phagocytosis by neighboring mesangial cells was obtained in self-limited mesangial proliferation induced in rats by Thy1.1 antibody, apoptosis occurring approximately 10-fold more frequently than in the healthy rat glomerulus. Indeed, changes in glomerular cell number in Thy1.1 nephritis strongly suggested that apoptosis is the major cell clearance mechanism counterbalancing cell division, thereby mediating resolution of glomerular hypercellularity in experimental mesangial proliferation.

Molecular cloning and characterization of 2B7, a rat mRNA which distinguishes smooth muscle cell phenotypes in vitro and is identical to osteopontin (secreted phosphoprotein I, 2aR).

We have identified a rat smooth muscle cell mRNA, 2B7, which distinguishes smooth muscle cell phenotypes in vitro. Sequence and tissue distribution data strongly suggest this mRNA to be identical to osteopontin (secreted phosphoprotein I, 2aR). In vivo, 2B7 mRNA is expressed in normal rat aorta and carotid arteries at levels 50-60-fold greater than heart, and about 3-4 times the levels found in adult rat kidney on a per micrograms of total RNA basis. Of the other smooth muscle sources surveyed, significant levels of 2B7 mRNA were detectable in total RNA prepared from rat uterus and stomach, but not intestine. 2B7 mRNA levels in both carotid and aortic artery increase with age, and are elevated approximately 5-fold in the carotid artery 48 h after balloon angioplasty. The presence of osteopontin (secreted phosphoprotein I, 2aR) in the normal artery wall and its increased expression after injury suggests a previously unappreciated role for this molecule in the vascular system.

Neutralizing Antibodies Directed Against Osteopontin Inhibit Rat Carotid Neointimal Thickening After Endothelial Denudation

Osteopontin is an arginine-glycine-aspartate-containing acidic glycoprotein with adhesive and migratory activities in vitro. We previously showed that osteopontin was highly expressed in injured rat arteries as well as in human atherosclerotic plaques. In contrast, uninjured blood vessels make very little osteopontin. In this report, we have investigated the role of osteopontin in rat neointima formation using neutralizing antibodies. Rats were treated with either nonimmune or antiosteopontin antibody and subjected to endothelial denudation of the carotid artery by using a balloon catheter. Two weeks after injury, intimal areas and cell numbers were significantly decreased (33% and 31%, respectively) in the antiosteopontin group compared with the nonimmune IgG group. No differences in carotid medial areas or cell numbers were observed. Intimal and medial replication rates, as measured by continuous bromodeoxyuridine infusion during the final week of the experimental protocol, were not significantly different between the two groups. No gross histological changes were noted in the intimas formed in the presence of either neutralizing or nonimmune antibody. In addition, no difference in early carotid medial cell replication rate was observed when antibodies were infused for 4 days after angioplasty. These data demonstrate for the first time a functional role for osteopontin in the process of carotid neointimal thickening in vivo and suggest that osteopontin plays an active role in the remodeling processes important for human atherosclerotic and restenotic lesion development.

Peripheral Vascular Stenosis in Apolipoprotein E-Deficient Mice: Potential Roles of Lipid Deposition, Medial Atrophy, and Adventitial Inflammation

A systematic analysis of the distribution of advanced atherosclerotic lesions was undertaken in chow-fed, 9-month-old apolipoprotein (apo) E-deficient mice to identify sites amenable for study of mechanisms of formation of stenotic lesions. The arterial tree was dissected intact and included medium-sized arteries in the extremities as well as arteries of the head and neck. The most reproducible lesions were seen in the ascending aorta and in the carotid, femoral, and popliteal arteries. Casting of the vascular tree provided additional verification of the presence of lumen narrowing in the external branches of the carotid artery. Consistent with what has been observed in human atherosclerotic arteries, there was dilation in response to lesion growth and no correlation between lesion mass and lumen loss in the mouse arteries. This adaptation was especially true in the ascending aorta, where normal lumen size was maintained at atherosclerotic sites. In contrast, the external carotid arteries were stenotic in 9 of 12 animals. Here too, however, loss of lumen did not correlate with lesion mass but did correlate with adventitial inflammation and medial atrophy. Lumen narrowing also occurred most frequently at sites where extracellular cholesterol clefts were a prominent part of the lesion. These data suggest that the stenotic process in advanced atherosclerotic vessels may depend on death of medial smooth muscle cells, possibly in response to inflammatory changes in the plaque or adventitia.

Angiotensin II Induction of Osteopontin Expression and DNA Replication in Rat Arteries

We recently identified the adhesive protein osteopontin as a novel smooth muscle cell product overexpressed in rat developing neointima and human atheroma. Although osteopontin is a candidate stimulant for intimal lesion progression because of its chemotactic and calcium binding functions, factors controlling osteopontin expression in arteries remain poorly defined. In vitro, smooth muscle cell expression of osteopontin is associated with cell cycle transit or alterations in cell phenotype, and it is increased by angiotensin II (Ang II) stimulation. In the present studies, we investigated both osteopontin expression and DNA replication in the arterial wall in response to chronic Ang II infusion in vivo. Rat carotid arteries with or without intimal thickening (induced by balloon catheterization) were examined. Ang II (250 ng/kg per minute) or vehicle was coinfused with bromodeoxyuridine (to label replicating DNA in vivo) for 2 weeks beginning 4 weeks after injury. With Ang II, smooth muscle cells overexpressed osteopontin as shown by protein immunohistochemistry, in situ hybridization, and Northern blot analyses. Osteopontin mRNA levels were increased markedly (approximately fivefold) in the normal artery media and injured artery neointima, but levels remained low in the injured artery media, in positive correlation (R2 = 0.88, P < .001) with DNA replication in the smooth muscle layers, further suggesting that osteopontin may be a growth-associated, phenotype-dependent gene for smooth muscle cells. However, osteopontin expression in neointima was not restricted to areas showing DNA replication, suggesting a nonobligatory association. Ang II induced severe hypertension. Arterial osteopontin expression was increased also by chronic catecholamine infusion, a model of vascular growth stimulation showing labile pressure elevations. Osteopontin induction in smooth muscle cells may contribute to Ang II-dependent intimal lesion progression and vascular remodeling events associated with renovascular diseases or hyperadrenergic disorders.

Mitogenic Effect of Angiotensin II on Rat Carotid Arteries and Type II or III Mesenteric Microvessels but Not Type I Mesenteric Microvessels Is Mediated by Endogenous Basic Fibroblast Growth Factor

In this study, anti-basic fibroblast growth factor (anti-bFGF) antibody was used to determine whether the mitogenic effect of angiotensin II in vivo could be blocked by neutralizing bFGF in the vessel wall. Animals, divided into six experimental groups, were given (1) angiotensin II, (2) angiotensin II + anti-bFGF antibody, (3) angiotensin II + normal goat IgG (ngIgG), (4) anti-bFGF antibody, (5) ngIgG, and (6) Ringers solution. Angiotensin II at 435 ng x kg(-1) x min(-1) was infused into rats continuously for 1 week to induce smooth muscle cell replication, and anti-bFGF antibody or ngIgG was injected intravenously 4 times over the 1-week period at a dose of 60 mg/injection. Bromodeoxyuridine (30 mg/mL) was also continuously infused during the 1-week period. The left carotid artery of all animals was balloon-injured on day 4 of the treatment, and all groups were killed for study on day 7. The results showed that angiotensin II significantly stimulated smooth muscle replication in the balloon-injured carotid artery, intact carotid artery, and three branch levels of the mesenteric vascular tree. Anti-bFGF was able to block the mitogenic effect of angiotensin II in larger vessels but not the smallest (type I) microvessels of the mesenteric arterial tree. This differential response may be attributable to the nature of the lesions in type I vessels versus larger vessels: the type I vascular lesion has a large component of proliferating macrophages, whereas the larger vessels show less injury, few macrophages, and varying levels of smooth muscle replication. Our data suggest that the vessel wall remodeling in the angiotensin II-treated larger vessels involves DNA replication that is dependent on the presence of bFGF.

Chronic α1-adrenoreceptor stimulation increases DNA synthesis in rat arterial wall : Modulation of responsiveness after vascular injury

Despite indirect evidence from studies using adrenergic antagonists or sympathectomy, catecholamines have never been shown directly to stimulate vascular smooth muscle cell (SMC) DNA replication in vivo. We studied whether a chronic infusion of catecholamine stimulates SMC replication in vivo in both uninjured arteries and arteries with a neointima formed after vascular injury. Animals were killed after 2 weeks of continuous infusion of bromodeoxyuridine (to label replicating DNA) and either phenylephrine, norepinephrine, or vehicle solution, starting early (third week) or late (ninth week) after balloon injury to the left common carotid artery. In catecholamine-infused animals, the uninjured carotid artery or thoracic aorta showed a marked increase in cross-sectional area (>25%) and frequency of cells undergoing DNA synthesis among medial SMCs (4- to 10-fold) and endothelial cells (13-fold). With catecholamine infusion at 9 to 10 weeks after injury, the media or neointima of the injured carotid artery sh...

Renal and vascular injury induced by exogenous angiotensin II is AT1 receptor-dependent.

Angiotensin II (Ang II) infusion in rats augments vascular injury in balloon-injured carotid arteries and induces marked vascular and tubulointerstitial injury in kidneys. We examined how the AT1 receptor is modulated and whether blockade of the receptor with losartan could prevent the phenotypic and cellular changes. We also examined the role of the local renin-angiotensin system (RAS) by examining the expression of angiotensin-converting enzyme (ACE) and the effect of treatment with the ACE inhibitor, ramipril. Ang II infusion resulted in systemic hypertension and accelerated intimal and medial thickening in balloon-injured carotid arteries. Renal injury was manifested by proteinuria, glomerular phenotypic changes (mesangial expression of α-actin and podocyte expression of desmin), and tubulointerstitial injury with the tubular upregulation of the macrophage-adhesive protein, osteopontin, the interstitial accumulation of macrophages and myofibroblasts, and the deposition of collagen types III and IV. Ang II infusion decreased AT1 receptor number in the renal interstitium but not in glomeruli. Losartan completely blocked the Ang II-mediated hypertension, proteinuria, and injury to both carotid and kidney. Ang II infusion was also associated with an increase in ACE protein in both the proximal tubular brush border as well as at interstitial sites of injury, but despite evidence for activation of the local RAS, treatment with ramipril was without effect. These studies demonstrate that the renal and vascular injury induced by Ang II infusion is mediated by the AT1 receptor despite downregulation of the receptor in the interstitium. In addition, although there is evidence for local RAS activation, the injury appears to be mediated solely by the exogenous Ang II.