Isoform-specific modulation of Na+, K+-ATPase alpha-subunit gene expression in hypertension.

Sodium, potassium-adenosine triphosphatase (Na+, K+-ATPase) is hypothesized to be involved in systemic vascular hypertension through its effects on smooth muscle reactivity and myocardial contractility. By means of RNA blot analyses of cardiac, aortic, and skeletal muscle RNAs in two rat hypertensive models, Na+,K+-ATPase alpha-subunit messenger RNA isoforms (alpha 2 and alpha 3) were shown to be deinduced in response to increased intravascular pressure. The changes were observed after 48 hours or more of experimental hypertension. Under these conditions, there is coordinate induction of another alpha isoform (alpha 1) and of beta-subunit messenger RNAs, probably in response to alterations in sodium flux rather than to elevated blood pressure.

Growth factor expression in aorta of normotensive and hypertensive rats.

Hypertension causes biochemical and morphological changes in the vessel wall by unknown mechanisms. Locally produced substances may have a role in mediating these vascular changes. We have studied the expression of platelet-derived growth factor (PDGF) B chain and PDGF A chain, insulin-like growth factor (IGF)-I and IGF-II, endothelial cell growth factor (ECGF), basic fibroblast growth factor (bFGF), and transforming growth factor-beta (TGF-beta) in aortic tissue from normotensive rats and rats made hypertensive by deoxycorticosterone (DOC)/salt treatment. Using Northern blotting, we found that genes for each of these growth factors were transcriptionally active in the aorta of both normotensive and hypertensive rats. TGF-beta aortic mRNA levels increased up to threefold as a result of DOC/salt hypertension. In contrast, no major changes in the expression of either PDGF chain, IGF-I or II, ECGF, or bFGF were detectable. The results indicate that at least seven genes coding for growth factors that were shown previously to influence growth and function of vascular cells in vitro, are expressed in rat aorta in vivo. These findings support the hypothesis that synthesis and release of growth factors in the arterial wall are involved in autocrine and/or paracrine regulatory mechanisms. In addition, the increased expression of TGF-beta in vivo may have a role in mediating the aortic changes induced by hypertension.

Fibronectin biosynthesis in the rat aorta in vitro. Changes due to experimental hypertension.

This study was undertaken to determine if changes in fibronectin biosynthesis accompany the phenotypic changes that occur in aortic tissue following experimental hypertension. An in vitro procedure was developed to measure fibronectin synthesis in aortic rings obtained from normotensive or hypertensive rats. There was a three to sixfold increase in fibronectin biosynthesis by aortic rings taken from rats treated with deoxycorticosterone/salt for 7 and 21 d, the change being more pronounced at 21 d. In contrast, there was no major change at either time point in net incorporation into total protein. Studies comparing fibronectin biosynthesis in aortic rings from Wistar rats and spontaneously hypertensive rats at ages between 10 and 40 wk showed increased fibronectin biosynthesis in older animals of both strains, but only slight differences between strains. Studies using rats infused with angiotensin II showed a correlation between blood pressure elevation and increased aortic fibronectin biosynthesis. Western blot analysis of aortic extracts showed that the fibronectin content was increased in the hypertensive models. The in vitro procedure for measuring fibronectin biosynthesis appears to provide a reliable reflection of in vivo changes in fibronectin expression, and the methodology could prove useful for studying the factors influencing protein expression in vascular tissue.

Angiotensin II-induced cardiac fibrosis in the rat is increased by chronic inhibition of nitric oxide synthase.

These studies were performed to determine if the effects of angiotensin II infusion on the development of cardiac fibrosis could be modified by the chronic inhibition of nitric oxide synthase activity. NG-nitro-L-arginine-methyl ester (L-NAME) was administered to adult Wistar rats in drinking water (40 mg/kg per d). Although blood pressure was maintained at hypertensive levels after 2 wk, cardiac hypertrophy or fibrosis did not occur. Angiotensin II, given for 3 d at a dose which induced little or no blood pressure elevation and minimal if any fibrosis, caused significant fibrosis when given to a rat pretreated for 2 wk with L-NAME. This marked fibrosis did not occur if angiotensin II was given shortly after L-NAME treatment was begun or briefly after discontinuation of L-NAME. The fibrosis that occurred with combined treatment was characterized by increased immunodetectable fibronectin, the presence of inflammatory cells within interstitial and perivascular regions, and increased steady state mRNA levels for matrix genes and atrial natriuretic protein. The data indicated a regulatory role for nitric oxide in modulating the angiotensin II-induced cardiac fibrosis and suggest a potentially important autocrine or paracrine role for nitric oxide in fibroblast proliferation.

Relationship of an abnormal plasma lipoprotein to protection from atherosclerosis in the cholesterol-fed diabetic rabbit.

Alloxan-diabetic rabbits develop a pronounced hypercholesterolemia and hypertriglyceridemia in response to cholesterol feeding. Despite higher levels of plasma cholesterol, these animals have much less atherosclerosis than cholesterol-fed nondiabetics. To determine whether this effect is due to properties of the lipoproteins, we compared chemical, physical, and metabolic characteristics of a very low density lipoprotein (VLDL) fraction (d less than 1.019 g/ml) from the diabetic and nondiabetic cholesterol-fed rabbits. The molar ratio of triglyceride to cholesteryl ester in the particles from diabetic animals ranged from 2:1 to 6:1, and this ratio remained constant in subfractions from individual rabbits. Triglyceride from nondiabetic control animals was a minor component. Differential scanning calorimetry showed a distinct order-disorder phase transition for cholesteryl ester at approximately 42 degrees C in the fractions from control animals, whereas in fractions from most of the diabetics no such transition was observed, indicating that both triglyceride and cholesteryl ester are present in the core of the same particle. The relative amount of apoprotein E in particles from diabetic animals was much less than that of cholesterol-fed controls. The ability of the lipoproteins from both groups to stimulate cholesteryl ester formation in mouse peritoneal macrophages also was tested. Lipoproteins from cholesterol-fed controls stimulated cholesteryl ester formation in a dose-dependent manner, but particles from the diabetic group had little or no effect. The results suggest that the presence of unusual VLDL particles in diabetic cholesterol-fed rabbits is responsible, at least in part, for the reduced incidence of atherosclerosis in this animal model.

Cholesteryl ester and triglyceride hydrolysis by an acid lipase from rabbit aorta.

The properties of the triglyceride- and cholesteryl ester-hydrolyzing activity by an acid lipase from rabbit aortic tissue were compared under different experimental conditions. Radiolabeled cholesteryl oleate or triolein was incorporated into phospholipid vesicles by sonication and the resulting preparations were used for in vitro studies. No distinction was observed between triglyceride lipase and cholesterol esterase activity in the aortic cytosol fraction following either thermal inactivation, inhibition by a mercurial, fractionation by ammonium sulfate or acid precipitation, or DEAE-cellulose chromatography. Addition of rabbit lipoproteins to the assay system resulted in inhibition of both cholesterol esterase and triglyceride lipase activity. Parallel changes in the hydrolysis of both substrates also were observed when exogenously added lipids were added to the incubation system in various physical states. Specificities of the enzyme system towards different cholesteryl esters were examined. No differences in the rate of hydrolysis were observed between cholesteryl oleate, palmitate and linoleate. The data suggest that a single acid lipase, presumably of lysosomal origin, has broad specificity towards triglycerides and cholesteryl esters, and may play a role in the hydrolysis of these lipids during intralysosomal degradation of lipoproteins.

Peptidyl dipeptidase in rabbit brain microvessels.

The activity of peptidyl dipeptidase (peptidyldipeptide hydrolase, EC 3.4.15.1), also known as angiotensin-converting enzyme, was studied in small blood vessel preparations isolated from rabbit brain. The vascular preparation contained arterioles and capillaries and was essentially free of extravascular material. Enzymatic activity was demonstrated in microvessel homogenates using both hippuryl-histidyl-leucine and tritium-labeled angiotensin I as substrates. Activity in the microvessels was dependent on the presence of chloride ion and was sensitive to inhibition by converting enzyme inhibitors previously shown to be effective in both vivo and in vitro. Specific activity in the micro-vessels was approximately 20 times that in homogenates of brain, and was almost 60% of that found in rat lung homogenates. The data were consistent with an endothelial localication for peptidyl dipeptidase in the cerebral vasculature and supports the proposal that this enzyme has a physiological role in extrapulmonary vascular beds.

Cardiovascular complications.

This report summarizes the current state of knowledge concerning the cardiovascular system in various animal models of diabetes and presents their major strengths and weaknesses for studying the important research questions in the field. Nonhuman primates have many desirable features for studies on the macrovascular and cardiac complications of the disease as well as risk factor alterations, but their availability, cost, and maintenance present practical disadvantages. The spontaneous rodent models of diabetes currently are not considered very useful for cardiovascular research, but they have not been well characterized with respect to most aspects of their cardiovascular system. Alloxan-diabetic rabbits offer some promise for examining the effects of diabetes on atherogenesis, lipoprotein metabolism, and cardiomyopathy, but additional research is required to validate their usefulness. Insufficient data are available on canine and swine models of diabetes to judge their merits for cardiovascular research. The Task Force recommends: (1) additional longterm investigations to determine the extent and severity of cardiovascular complications in the well-characterized rodent models and in diabetic rodents with multiple risk factor abnormalities; (2) further studies on the macrovascular disease and lipoprotein abnormalities of the alloxan-diabetic rabbit and the development of rabbit colonies with spontaneous diabetes; (3) increased emphasis on such potentially important but neglected areas of research in diabetic animals as the intramyocardial circulation, adventitial blood vessels, blood pressure, platelet function, blood coagulation, blood rheology, and autonomic nervous function; (4) long-term studies on the influence of control of hyperglycemia and of insulin therapy on cardiovascular complications in diabetic animals; and (5) encouragement of use of diabetic nonhuman primates for cardiovascular research and institution of measures to increase their supply and availability by expanding current colonies, screening newly imported animals for diabetes, and establishing a visiting scientist's program allowing investigators to study diabetic primates at resource centers.

Exacerbation of atherosclerosis by hypertension. Potential mechanisms and clinical implications.

Recent experimental data suggest marked similarities between the effects of hypertension and hypercholesterolemia on the arterial intima. Both conditions also seem to exert proinflammatory effects on the artery, resulting in the recruitment of monocytes into the intima. These effects may be due to production of oxygen-free radicals, which in turn may stimulate genes involved in the recruitment of inflammatory cells into the arterial wall. Plaque rupture and acute myocardial infarction are related to local accumulation of inflammatory cells in vulnerable areas of the plaque. Recent clinical trials using cholesterol-lowering or antihypertensive therapies have shown a decrease in cardiovascular events that may have resulted from withdrawal of inflammatory effects on the arterial wall. Angiotensin-converting enzyme inhibitors decrease the rate of myocardial infarction in patients with overt congestive heart failure or left ventricular dysfunction. These drugs probably affect several mechanisms related to the inhibition of angiotensin production and the potentiation of bradykinin and resultant enhancement of nitric oxide and prostacyclin. The mechanisms could include reversing the proinflammatory effects of angiotensin and hypercholesterolemia on the arterial wall. Future therapeutic strategies of vascular protection in hypertension may include direct attacks on proinflammatory or pro-oxidant vascular mechanisms.

1989 Corcoran lecture: adaptive and maladaptive responses of the arterial wall to hypertension.

This study reviews recent experimental data from our own and other laboratories on the effects of hypertension on the arterial wall and the potential mechanisms by which hypertension can induce vascular injury and accelerate atherosclerosis. The findings suggest that the responses of the arterial media to hypertension reflect appropriate adaptations to increased intramural tension with resultant medial thickening secondary to an increase in both cellular mass and extracellular matrix. The role of growth factors in this process and their effects on arterial contractility are discussed as well as the potential importance of the changes in extracellular matrix constituents. The intimal changes induced by hypertension have many similarities to those caused by aging or hypercholesterolemia and can in part reflect general arterial responses to injury. They make the arterial wall more vulnerable to the effects of hypercholesterolemia, however, and as noted in our studies with the Watanabe heritable hyperlipidemic rabbit, pronounced acceleration of atherosclerosis is induced when hypertension is combined with hypercholesterolemia. Antihypertensive drugs can affect the arterial response to hypercholesterolemia. In the present study, new data are provided indicating that captopril inhibits aortic atherosclerosis in the Watanabe heritable hyperlipidemic rabbit in association with a pronounced reduction in cellularity of lesions.

Blood pressure lowering diminishes age-related changes in the rat aortic intima.

These studies were undertaken to determine whether age-related changes in the aortic intima can be inhibited by prolonged blood pressure lowering. Normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) were treated with a combination of chlorothiazide, reserpine, and hydralazine beginning at 20 weeks of age and continuing until 62 weeks. Aortic morphology was assessed at the termination of the study by light and electron microscopy. Average systolic blood pressures during the final 6 months of therapy were: for untreated WKY, 147 +/- 5 mm Hg (mean +/- SE); for treated WKY, 110 +/- 2; for untreated SHR, 206 +/- 3; and for treated SHR, 104 +/- 4. Intimal and medial abnormalities were maximal in untreated SHR, while untreated WKY showed moderate changes consistent with their age. In contrast, both treatment groups exhibited relatively minimal alterations despite their advanced age. The occurrence of intimal lesions in individual animals correlated with the average level of blood pressure during therapy. These data suggest that blood pressure reduction, even in normotensive animals, may reduce the effects of aging on the arterial wall.

Are some antihypertensive therapies more efficacious than others in preventing complications and prolonging life?

Antihypertensive therapy has been used for almost 35 years to reduce blood pressure and prevent morbidity and mortality related to the hypertensive state. Malignant, severe, and moderate hypertension have all been shown to be worthy of drug treatment, but controversy remains as to the degree of benefit that is achievable by treating milder hypertension. A variety of clinical trials have demonstrated that antihypertensive therapy reduces the incidence of stroke, congestive heart failure, and left ventricular hypertrophy and the progression in severity of hypertension. The benefits with respect to prevention of coronary heart disease (CHD) have been much less impressive. Thiazide diuretics have been the base therapy for the bulk of the hypertensive subjects studied to date who have not demonstrated reduced incidence of CHD. Therapy with beta-blockers has the potential for reducing CHD, but an analysis of four studies finds only two with positive results. On the other hand, since that study found reduced total mortality as well as CHD compared with thiazide diuretic, its findings cannot be ignored. Other questions deserving further investigation include how other antihypertensive therapies compare with respect to the risk reduction found with thiazide diuretics and beta-blockers, the optimal posttreatment blood pressure, whether persons with mild hypertension benefit from therapy, whether women should be treated differently, and whether atherosclerosis may be affected by specific antihypertensive therapies.

Inhibition of nuclear polyploidy by propranolol in aortic smooth muscle cells of hypertensive rats.

The ability of propranolol to inhibit the development of polyploidy in aortic vascular smooth muscle cells associated with hypertension was studied in deoxycorticosterone (DOC)-salt treated rats. Six-week treatment with DOC-salt resulted in significant increases in systolic blood pressure, heart weight, and aortic weight in treated animals compared to increases in uninephrectomized controls. Additionally, the percentage of tetraploid nuclei in aortic smooth muscle cells increased to 17.0 +/- 0.2% in DOC-salt treated rats versus 7.8 +/- 0.3% in normotensive controls. Administration of propranolol (500 mg/L in drinking water) did not inhibit the development of hypertension for up to 4 weeks or the associated increase in cardiac or aortic weight in DOC-salt-treated rats, but did prevent the increase in polyploidy of aortic smooth muscle cell nuclei (8.9 +/- 0.9% in propranolol-treated rats compared to 7.8 +/- 0.3% in normotensive controls). These results indicate that propranolol inhibits the development of hypertension-induced polyploidy in aortic smooth muscle cells of DOC-salt-treated rats and that factors other than blood pressure may be important in this change.

Effects of hypertension on migration and proliferation of smooth muscle in culture.

Migratory and proliferative characteristics of explanted rat aortic smooth muscle cells were studied in response to hypertension induced by 4 weeks of deoxycorticosterone-salt administration. Under low serum conditions (0.1% fetal bovine serum), over 80% of aortic medial explants from hypertensive rats yielded smooth muscle cell colonies after 8 days of culture while fewer than 10% of the control explants were positive. Time lapse video analysis of subsequent growth in the presence of 10% serum revealed that interdivision times of smooth muscle cells from hypertensive animals were significantly shorter than those in controls (p less than 0.01). Significant differences in proliferative capacity of smooth muscle cells were evident, even after one subculture (p less than 0.01). Comparison of these results with data from mechanical injury suggests that 4 weeks of deoxycorticosterone-salt hypertension can potentiate subsequent smooth muscle cell migration and growth in vitro to an extent similar to that observed with the combined effects of total endothelial denudation and wall distention by a balloon catheter.

Effects of deoxycorticosterone-salt hypertension on cell ploidy in the rat aorta.

We investigated the effect of deoxycorticosterone-salt hypertension and its reversal on nuclear ploidy in rat aortic smooth muscle cells, using flow cytometry. Systolic blood pressure and the percentage of cells with tetraploid nuclei increased over a 4-week period and then remained constant. In control rats 6.0 +/- 0.6% of aortic cells had tetraploid nuclei, and this increased to 27.0 +/- 1.1 and 26.6 +/- 2.4% after 4 and 8 weeks of deoxycorticosterone-salt treatment, respectively. Computer analysis of the forward light scatter data obtained from the cell sorter indicated that the average tetraploid cell was approximately 1.6-fold larger than the average diploid cell in both normotensive and hypertensive animals. Analysis of the DNA content of intact cells and isolated nuclei indicated that the increased DNA content per cell was predominantly attributable to tetraploid nuclei rather than binucleated cells. Reversal of hypertension with either a low sodium diet for 8 to 13 weeks or chlorothiazide administration failed to reverse the abnormalities seen in arterial smooth muscle cell ploidy, despite normalization of the blood pressure and the ratio of heart to body weight.

Hypertension-induced changes of platelet-derived growth factor receptor expression in rat aorta and heart.

Hypertension-associated growth of vascular smooth muscle cells might be mediated in vivo by platelet-derived growth factor (PDGF). Our previous investigations in hypertensive rats failed to demonstrate changes in aortic steady-state mRNA levels of PDGF A or B chains. The current studies were performed to determine whether hypertension might affect the expression of PDGF receptors. We studied PDGF alpha- and beta-receptor gene expression by Northern analysis using human and rat cDNA probes. Studies of tissue distribution revealed that PDGF beta-receptor mRNA was most abundant in total aorta and aortic media, whereas the PDGF alpha-receptor mRNA was most abundant in the lung and was expressed at low levels in aortic tissue. Deoxycorticosterone acetate (DOCA)-salt hypertension induced a threefold increase in aortic steady-state PDGF beta-receptor mRNA levels. Aortic PDGF beta-receptor expression also was higher in spontaneously hypertensive rats (SHRs) when compared with age-matched normotensive Wistar-Kyoto (WKY) controls. Aortic PDGF alpha-receptor steady-state mRNA levels were unchanged in DOCA-salt hypertension and were expressed at similar levels in WKY rats and SHRs. Unlike the findings with aorta, cardiac PDGF beta- and alpha-receptor and PDGF B-chain expressions were unchanged in the DOCA-salt model and were decreased in SHRs. These findings indicate that hypertension can increase aortic steady-state mRNA levels for PDGF beta-receptor. They also indicate that tissue-specific expression of the genes of the PDGF ligand/receptor system are differentially regulated in hypertension.

Dissociation between the antiatherosclerotic effect of trandolapril and suppression of serum and aortic angiotensin-converting enzyme activity in the Watanabe heritable hyperlipidemic rabbit.

This study was undertaken to determine whether low doses of the angiotensin-converting enzyme (ACE) inhibitor trandolapril affected atherosclerosis in the Watanabe heritable hyperlipidemic (WHHL) rabbit. Trandolapril (10 micrograms/kg body weight per 48 hours) was begun at 3 months of age and continued for 9 months. The selected dose reduced serum ACE activity but did not influence blood pressure. Both serum and aortic ACE activity were reduced by more than 80% in the trandolapril-treated compared with control WHHL rabbits, similar to the suppression achieved with the 25-fold-higher dose that in our previous studies induced marked inhibition of aortic atherosclerotic lesions in the WHHL rabbit. In contrast to these prior findings, low-dose trandolapril had no effect on aortic surface involvement by atherosclerosis, aortic cholesterol content, or aortic morphology. The data suggest that the antiatherosclerotic action of ACE inhibitors in the WHHL rabbit may not be related directly to arterial enzyme inhibition.

Properties of angiotensin-converting enzyme in intact cerebral microvessels.

Angiotensin-converting enzyme (ACE) was studied in preparations of microvessels isolated from rabbit cerebral cortex. Activity was determined by measuring the degradation of hippuryl-histidyl-leucine (Hip-His-Leu) by the intact microvessels in a physiological salt solution at pH 7.4. ACE activity was dependent on both substrate and chloride ion concentration and was inhibited by captopril in a manner similar to that observed previously with tissue homogenates. Angiotensin I was rapidly degraded by the intact microvessels, even in the presence of 10(-6)M captopril. An advantage of the methodology employed was the ability to pretreat the microvessels and then assess the effect of pretreatment by transfer to a postincubation assay system. Pretreatment with a hyperosmolar urea solution did not change ACE activity or cause release of ACE from the microvessels, although lactic dehydrogenase and lysosomal enzymes were released. Pretreatment with captopril caused a lag in the subsequent degradation of Hip-His-Leu, presumably reflecting dissociation of inhibitor from the cell-associated enzyme. ACE activity was unaffected by hypoxic or anoxic incubation conditions. The ability to measure ACE activity of the microvessels in vitro provides a unique opportunity to study the properties of the enzyme in intact cerebrovascular endothelial cells.

Aortic endothelial and subendothelial cells in experimental hypertension and aging.

The endothelial and subendothelial cellular changes occurring as a result of hypertension and aging were characterized in deoxycorticosterone/salt-treated (D/S), spontaneously hypertensive (SHR), and Wistar-Kyoto (WKY) rats. An increase in the number of subendothelial cells occurred with both hypertension and aging and was most dramatic with D/S hypertension. Many of the cells found in the widened subendothelium showed morphological characteristics of mononuclear cells (both macrophages and lymphocytes), and of smooth muscle cells. Normalization of blood pressure by withdrawal of D/S and maintenance of rats on a low salt diet reversed the number of subendothelial cells to levels of control animals of comparable age. Significant alterations were seen in the aortic endothelial cells of D/S animals. Within 2 to 4 weeks of D/S administration, the endothelial cells doubled in number and often assumed bizarre shapes with nuclear folding and bulging toward the lumen. Some similar abnormalities in endothelial cell shape and appearance occurred with increasing age in the SHR and control WKY, although the number of endothelial cells increased only slightly in these groups. These results suggest that profound cellular changes in the aortic intima occur with an increase in blood pressure. These changes are pronounced in the D/S model of hypertension, while virtually absent in SHR. Comparable alterations also may be seen in aged normotensive animals, but to a lesser extent and with slower progression.

Hypertension induces alternatively spliced forms of fibronectin in rat aorta.

Fibronectin expression was shown recently to increase in the rat aorta in response to experimental hypertension. Fibronectin is known to alter the phenotype of vascular smooth muscle and endothelial cells, and relative changes in the expression of different isoforms of fibronectin, generated by alternative splicing and distinguished by the absence or presence of inserts designated as EIIIA, EIIIB, and V, may reflect a change in cell phenotype. In the present study we examined the expression of alternatively spliced forms of aortic fibronectin during deoxycorticosterone-salt hypertension. Aortic RNA was analyzed quantitatively using Northern blot analysis and ribonuclease protection assays. Using Northern blot analysis, deoxycorticosterone-salt treatment for 21 days led to a 4.9-fold increase in EIIIA fibronectin messenger RNA, while EIIIB and V forms increased by 2.6- and 2.5-fold, respectively. As determined by ribonuclease protection assays, the percentage of fibronectin transcripts containing either EIIIA, EIIIB, or V in control aorta was 7.3%, 19%, and 40%, respectively. The percentage of EIIIA transcripts increased 42% over control levels after 21 days of deoxycorticosterone-salt treatment, whereas no proportionate change in the other alternatively spliced forms was found. Thus, all forms increased, but a selective increase in the EIIIA form was induced. Analogous increases in each of the fibronectin isoforms were found in the spontaneously hypertensive rats when compared with age-matched Wistar-Kyoto or Wistar rats, and 40-week-old animals showed increases over 10-week-old animals in all strains, consistent with an age-dependent increase in aortic fibronectin expression.