Angiotensin II-induced growth effects in vascular smooth muscle in cell culture and in the aortic tunica media in organ culture.

Several different studies have investigated the growth effects of angiotensin II on vascular smooth muscle cells in culture. However, smooth muscle cells change their phenotype when placed in culture. The objective of the present study was to investigate the effects of angiotensin II on (3)H-thymidine and (3)H-proline incorporation in vascular smooth muscle cells in culture and in the tunica media of blood vessels perfused at normal physiological pressures in organ culture, thus avoiding the phenotypic changes observed in cell culture. The perfusion system consisted of a peristaltic pump and a closed circuit of plastic tubing connected to a culture media bottle where thoracic rat aortae were placed. Angiotensin II induced an increase in (3)H-thymidine and (3)H-proline incorporation in both culture systems. The results suggest that angiotensin II may play a role in mediating cell growth in vascular smooth muscle cells in their 'contractile' as well as in their 'synthetic' phenotype.

Platelet-derived growth factor mediates angiotensin II-induced DNA synthesis in vascular smooth muscle cells.

In order to elucidate the mechanism of angiotensin II (ANG II)-induced proliferation in vascular smooth muscle cells in culture, growth rates and 3H-thymidine incorporation into DNA in response to ANG II treatment were examined in cultured rat aortic smooth muscle cells. ANG II-treated and control cells were exposed to the ANG II receptor antagonists [Sar1, Val5, Ala8]-ANG II (Sar) and DUP753 and to antibody against platelet-derived growth factor (PDGF). In growing cells, ANG II acted as a moderate mitogen, inducing an increase in growth rate during the first two days of treatment. ANG II induced a marked increase in 3H-thymidine incorporation in cultured vascular smooth muscle cells. The effect was blocked by the ANG II inhibitors Sar and DUP753 and by the PDGF antibody. ANG II was able to stimulate vascular smooth muscle growth in cell culture. The effect seemed to be mediated, at least in part, by PDGF. These results are in agreement with a possible role of ANG II in promoting vascular growth in physiological and/or pathological situations.

Increased intraluminal pressure induces DNA synthesis and c-fos expression in perfused rat aorta.

The molecular mechanisms responsible for the vascular hypertrophy observed in the arteries of hypertensive subjects are poorly understood. In this study, we tested the hypothesis that an increase in intraluminal pressure could by itself induce some of the vascular changes associated with hypertension, such as increased DNA synthesis and c-fos expression. We perfused rat thoracic aortae at different pressures for up to 4 h. The perfusion system consisted of a peristaltic pump and a closed circuit of plastic tubing connected to a culture media bottle where rat thoracic aortae were placed. After a 30 min equilibration period at 20 mm Hg, the perfusion pressure was adjusted to "normotensive levels" (132 +/- 3/59 +/- 4 mm Hg) or "hypertensive levels" (204 +/- 5/74 +/- 8 mm Hg). 3H-Thymidine was added at this time. After 4 h, the arteries were removed from the apparatus. Tunica media and adventitia were separated and processed for scintillation counting. 3H-Thymidine incorporation was 39% higher in the "hypertensive" than in the "normotensive" arteries. In separate experiments, after a 20 min equilibration period, the arteries were perfused for an additional 30 min at 50/10, 100/35, or 150/50 mm Hg. After being removed from the perfusion apparatus, the arteries were homogenized and total RNA was isolated. c-fos Expression was analyzed by Northern blot. c-fos Expression corresponded directly with the perfusion pressure. The highest levels of c-fos expression were detected in the arteries exposed to the highest pressures. These findings support the hypothesis that hemodynamic and/or mechanical factors can influence cell growth and function.

Interactions between angiotensin II and adenosine 3':5'-cyclic monophosphate in the regulation of amino acid transport by vascular smooth muscle cells.

The regulation of amino acid transport by angiotensin II (AII) and cyclic AMP (cAMP) was assessed in cultured vascular smooth muscle cells, using a nonmetabolizable amino acid, alpha-[3H]aminoisobutyric acid (AIB). An exposure time in excess of 2 h was required for AII to elicit a stimulatory response, the magnitude of which increased in a time-dependent manner for 12 h. AII-induced transport was blocked by [1-sarcosine, 8-isoleucine]AII, a competitive inhibitor of AII binding. The effect of AII was not abolished by downregulation protein kinase C with phorbol 12,13-dibutyrate or by use of a protein kinase C inhibitor, suggesting that transport in response to AII can be mediated by a protein kinase C independent pathway. In contrast, the elimination of calcium from the incubation medium reduced AII-stimulated AIB uptake. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide partially inhibited AIB uptake in response to AII, suggesting that calmodulin may be involved in the modulation of AII-stimulated amino acid transport. AIB transport was also increased by elevating intracellular cAMP levels via beta-adrenergic receptor stimulation, the use of a cAMP analog (N6-monobutyryl cAMP), or a phosphodiesterase inhibitor (3-isobutyl-1-methylxanthine) or by direct stimulation of adenylate cyclase with forskolin. cAMP-induced AIB transport was evident within 10 min and peaked within 1 h. At 1 h AII enhanced cAMP-stimulated AIB transport. A possible mechanism for this effect is suggested by the observation that AII potentiated cAMP production in response to isoproterenol and 3-isobutyl-1-methylxanthine.

Morphometric study of cerebral arteries from spontaneously hypertensive and stroke-prone spontaneously hypertensive rats.

OBJECTIVE: The importance of sympathetic innervation for the development of structural changes in the cerebral arteries of hypertensive animals was studied. DESIGN: Sympathetic denervation was induced with combined treatment from birth of antibody against nerve growth factor and guanethidine. Previous studies from our laboratory showed that this procedure not only caused a permanent denervation of the mesenteric arteries, but also prevented the development of hypertension in spontaneously hypertensive rats (SHR). METHODS: Morphometric measurement of the structural changes was carried out in the basilar, superior cerebellar, posterior cerebral and middle cerebral arteries from 28-week-old SHR, stroke-prone SHR, and normotensive Wistar-Kyoto rats. The results were compared with those obtained from cerebral arteries of sympathectomized rats. RESULTS: Total vascular wall cross-sectional area was significantly larger in the basilar and superior cerebellar arteries from hypertensive rats compared with normotensives. The change was characterized by an increase in the number of smooth muscle cell layers. There were also differences between the two hypertensive groups in some arteries. Sympathetic denervation attenuated the development of hypertension and vascular changes in some arteries. There was a positive linear correlation between blood pressure and medial cross-sectional area, and between blood pressure and the number of smooth muscle cell layers for the four arteries analysed. CONCLUSION: Sympathetic nerves have a trophic influence upon the remodelling of some cerebral arteries during the development of genetic hypertension.

Morphological and contractile characteristics of rat aortae perfused for 3 or 6 days in vitro.

We studied the morphological and contractile characteristics of rat thoracic aortic segments perfused for 3 or 6 days under pulsatile conditions. Light microscopic examination of the segments revealed the presence of an unchanged tunica media. However, the intimal surface was mostly devoid of endothelial cells. The perfused aortic segments showed a dramatic increase in spontaneous tone when compared to fresh and sham-treated aortic segments. Maximum responses to potassium and norepinephrine were reduced after 3 days of perfusion (20-40% reduction), while maximum responses to 5-hydroxytryptamine and angiotensin II were not significantly different. After 6 days of perfusion, maximum responses to all agonist were reduced (50-60%). Sensitivity to norepinephrine was not affected by the treatment, while sensitivity to 5-hydroxytryptamine was reduced. The perfused aortic segments relaxed well in response to isoproterenol. Our system provides a useful experimental model for short-term studies of hypertension- and atherosclerosis-related vascular changes. Further refinement and characterization could improve the performance of the system for longer-term studies.

Hypertensive factor in different models of experimental hypertension.

A hypertensive factor (HF), isolated from rat erythrocytes, has been shown to stimulate in vitro calcium uptake in aortic rings and to elevate blood pressure when injected into normotensive rats. In the present study, we investigated tissue responsiveness to HF in spontaneously hypertensive rats (SHR), Wistar Kyoto rats (WKY), 2-kidney, 1-clip renovascular hypertensive rats and uninephrectomized rats that were given water or saline to drink or that were treated with DOCA and given water or saline to drink (DOCA-salt). Tissue responsiveness was determined by incubating aortic rings from the rats in the different groups with a constant amount of HF and measuring "lanthanum-resistant" calcium uptake. Tissue sensitivity to HF was greater in SHR than in WKY. In contrast, tissue sensitivity to HF was not enhanced in 2-kidney, 1-clip renovascular and DOCA-salt hypertensive rats relative to their appropriate controls. These results suggest that the increased tissue responsiveness to HF found in SHR is not universally associated with elevated blood pressure; increased tissue sensitivity seems to be a specific characteristic of genetic hypertension.

Effects of cyclic adenosine 3',5'-monophosphate on amino acid transport and incorporation into protein in the rat aorta.

To assess the effects of cyclic AMP on amino acid transport and incorporation into aortic tissue protein, rat aortic rings were incubated with the cyclic AMP analog, N6-monobutyryl cyclic AMP (MBcAMP), the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (MIX), and radiolabeled amino acids. Subsequently, the aortic rings were homogenized in 5% trichloroacetic acid (TCA) and processed for liquid scintillation counting. Radioactivity present in the TCA supernatant following centrifugation was used to estimate amino acid transport. TCA-precipitable radioactivity was used as a measure of amino acid incorporation into protein. MBcAMP induced an increase in the uptake of [3H]alpha-aminoisobutyric acid into aortic rings and an increase in the incorporation of radiolabeled proline and leucine into TCA-precipitable protein. Similar effects were observed with low concentrations of MIX (0.025-0.25 mM); however, at higher concentrations of MIX, there was an attenuation of the effect or frank inhibition. Maximum stimulation of transport was observed within 90-120 min of the addition of MIX or MBcAMP to the incubation medium, whereas the effect on amino acid incorporation was not detectable until after 12 h of exposure to MIX or MBcAMP. The effects of cyclic AMP on transport were observed in both the tunica media and the tunica adventitia, whereas the effects on amino acid incorporation into protein were observed only in the tunica media. These data are consistent with a possible role for cyclic AMP in promoting changes in the tunica media that could lead to the development of vascular hypertrophy.

Increased sympathetic innervation in the cerebral and mesenteric arteries of hypertensive rats.

The density of catecholamine-containing nerve fibers was studied in the cerebral and mesenteric arteries from normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and stroke-prone SHR (SHRSP) in the growing (SHR, WKY) and adult (SHR, SHRSP, WKY) animals. Cerebral arteries from SHR showed an increased adrenergic innervation from day 1. The nerve plexuses reached an adult pattern earlier in SHR than in WKY. The arteries from adult SHR and SHRSP (22 weeks old) showed a markedly higher nerve density than WKY. There was a positive linear correlation between blood pressure and nerve density for four cerebral arteries. The mesenteric arteries were not innervated at birth. However, hyperinnervation of these arteries in the SHR was already present at 10 days of age as compared with WKY. Sympathectomy with anti-nerve growth factor and guanethidine caused a complete disappearance of fluorescent fibers in the mesenteric arteries from SHR and WKY, and in the cerebral arteries of WKY. The same procedure caused only partial denervation of the cerebral arteries from hypertensive animals. We postulate that the increase in nerve density in the cerebral arteries from the hypertensive rats may contribute to the development of arterial hypertrophy in chronic hypertension through the trophic effect of the sympathetic innervation on vascular structure.

Effect of dietary calcium on in vitro aortic tissue responsiveness to a hypertensive factor.

It has been proposed that calcium supplementation in the diet is associated with a reduction in blood pressure. In the present study, we investigated vascular tissue sensitivity to a hypertensive factor (HF) in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY) fed a high calcium diet, a low calcium diet and a food restricted diet. HF, which has been isolated from erythrocytes, increases blood pressure when injected into normotensive rats and stimulates calcium uptake by aortic rings in vitro. Five-week-old rats were divided into the following groups: SHR and WKY fed a regular diet (1% calcium), SHR and WKY fed a high calcium diet (4% calcium), SHR and WKY fed a low calcium diet (0.02% calcium) and SHR and WKY fed a regular diet (1% calcium) in which food intake was restricted to 65% of ad libitum intake. Food intake, body weight, urine phosphate excretion and blood pressure development were followed for 8 weeks. At sacrifice, plasma levels of calcium and phosphate were determined. Tissue responsiveness to HF was calculated by incubating aortic rings from the rats in the different groups with HF and measuring lanthanum-resistant calcium uptake. A 4-fold increase in dietary calcium reduced blood pressure and tissue responsiveness to HF in SHR. Neither parameter was affected by the high calcium diet in WKY. The low calcium diet had no effect on either blood pressure or tissue responsiveness to HF in SHR or WKY. Restriction of food intake induced a reduction in blood pressure and in tissue responsiveness to HF in SHR. It did not affect the same parameters in WKY. The results suggest that the increased tissue responsiveness to HF in the SHR may be associated with high blood pressure.

Aortic cyclic AMP levels in hypertensive rats.

The levels of adenosine 3':5' cyclic monophosphate (cyclic AMP) were measured in aortae from 2-kidney, 1-clip renovascular hypertensive and DOCA-salt hypertensive rats. Cyclic AMP concentration and content were increased in the arteries from the hypertensive animals compared to their normotensive controls. There was a significant positive linear correlation between aortic cyclic AMP concentration and blood pressure and between aortic cyclic AMP content and blood pressure. Since cyclic AMP has been shown to be involved in cellular proliferation and protein synthesis, a perturbation in the normal functioning of this second messenger system could be related to some of the vascular changes associated with the hypertensive process.

Denervation increases myogenic tone in a resistance artery in the growing rabbit ear.

The effect of chronic sympathetic and sensory denervation of the growing rabbit ear vasculature on myogenic tone in a resistance artery was studied. Unilateral superior cervical ganglionectomy and section of greater and anterior auricular nerves were performed at 4 wk of age. Compared with the contralateral control, 2 and 6 wk later, the denervated artery developed greater stretch-dependent myogenic tone. This phenomenon may partially account for the return of tone described in the denervated ear vasculature.

Altered endothelium-mediated relaxation after denervation of growing rabbit ear artery.

The relaxation response to methacholine and sodium nitroprusside was examined in ring segments from the posterior auricular artery and its continuation the central ear artery in growing rabbits 2, 4, 6 and 8 weeks following unilateral adrenergic and sensory denervation. The maximal relaxation achieved by methacholine (endothelium-dependent) was significantly depressed in the denervated arteries compared with the contralateral controls. Sodium nitroprusside (endothelium-independent relaxant agent) elicited the maximal relaxation in all tissues. These results demonstrated impaired arterial endothelium-dependent relaxation to methacholine after chronic denervation.