We previously reported that apoptosis is increased m smooth muscle cells cultured from the aorta of spontaneously hypertensive rats versus normotenslve controls. As an mtttal m viva exploration, we now exammed smooth muscle cell apoptosis regulation during the regression of vascular hypertrophy m the thoracic aorta media of spontaneously hypertensive rats receiving the antihypertensive drug enalapnl (30 mg kg-' d-'), losartan (30 mg kg-' d-'), mfedipme (35 mg kg-' d-l), hydralazme (40 mg kg-' d-'), propranolol(50 mg kg-' d-'), or hydrochlorothiazide (75 mg kg-' d-') for 1 to 4 weeks starting at 10 to 11 weeks of age Three criteria were used to evaluate smooth muscle cell apoptosis (1) ohgonucleosomal fragmentation of the extracted aortm DNA, (2) reduction in aortic DNA content, and (3) depletion of smooth muscle cells m the arterial media Arterial DNA synthesis was evaluated by [3H]thymidme mcorporation m viva After 4 weeks of treatment, systohc blood pressure was reduced stgmficantly by >42% wtth losartan, enalapnl, and hydralazme, and by 23% with mfedipme, versus control values of 22025 mm Hg. However, these agents affected vascular growth and apoptosis differently Losartan, enalapnl, and mfedipme stimulated smooth muscle cell apoptosrs threefold to fivefold before there was a significant reduction m DNA synthesis (>25%), vascular mass (>19%), or vascular DNA content (>38%), and these treatments markedly reduced (by 38% to 50%) medial cell number as measured at 4 weeks by the three-dimensional disector method Losartan and mfedipme stimulated smooth muscle cell apoptosis before reducing blood pressure In contrast, hydralazme did not affect vascular mass, apoptosis, or DNA synthesis, although blood pressure was lowered Propranolol or hydrochlorothiazlde failed to affect hypertension or vascular growth Thus, smooth muscle cell apoptosls represents a novel therapeutic target for the control of hypertensive vessel remodeling m response to therapeutic agents (Hypertension. 1997;29[part 2]:340-349.) Key Words l apoptosts l smooth muscle cell . angtotensm II l calcmm channel antagonist S tudies m humans and in experimental models of hypertension have clearly demonstrated the importance of vascular structure m the regulation of blood pressure i-6 Increased vascular mass is an important feature of hypertensive vessels. At the level of small muscular arteries, vascular hypertrophy accompanied by smooth muscle cell (SMC) hypertrophy or hyperplasia acts as an amplifier for elevated vascular resistance and blood pressure.7%8 At the level of the aorta and its main branches, increased mass and ngidtty of the arterial wall contribute to systolic hypertension and represent an mdependent risk factor for left ventricular hypertrophy.9 The remodeling of vascular structure, mcludmg the regression of vascular hypertrophy, is now considered a key therapeutic target in the effort to reduce mortahty and morbidity associated with high blood pressure lo-13Increased vascular mass associated with the replication and accumulatio...

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1The mechanisms by which agents modulate the induction ofkinin B,-receptors were investigated by studying the effects of kinins in vitro, by use of the rabbit isolated aorta, and in vivo by measuring the blood pressure of anaesthetized rabbits. 2 The contractile response ofthe rabbit isolated aorta to kinins increased in a time-dependent manner in vitro. This effect was abolished by continuous exposure to the protein synthesis inhibitor cycloheximide (71 gM).3 Several substances were found to increase specifically the rate of sensitization to des-Arg9-bradykinin (des-Arg9-Bk), when applied continuously in vitro to tissues isolated from normal animals: bacterial lipopolysaccharide (LPS; I ;Lg ml'), muramyl-dipeptide (MDP; 2 pg ml-'), phorbol myristate acetate (PMA; 320 nM), epidermal growth factor (EGF; 100 ng ml-') and endothelial cell growth factor (150 l~g ml-'). 5 It has been demonstrated that LPS induces B,-receptors in rabbits enabling des-Arg9-Bk to act as a hypotensive agent. In these experiments neutropenia induced by nitrogen mustard, did not prevent the in vivo effect of LPS. MDP (300 pg) and PMA (1I00 jg) were also found to induce a state of responsiveness to des-Arg9-Bk in vivo. FMLP (I mg i.v.) induced a temporary decrease in blood neutrophil counts but had no effect on the induction of responses to des-Arg9-Bk. 6 The development of responses mediated by the B,-receptor in the two experimental systems seems to be unrelated to the activation of neutrophil leukocytes, but may be related to the activation of tissue macrophages. Approximately 3% of cultured adherent cells derived from rabbit aorta strips following protease digestion were stained for non-specific esterase, supporting such a possibility.

Mitochondrial NADP؉ -isocitrate dehydrogenase activity is crucial for cardiomyocyte energy and redox status, but much remains to be learned about its role and regulation. We obtained data in spontaneously hypertensive rat hearts that indicated a partial inactivation of this enzyme before hypertrophy development. We tested the hypothesis that cardiac mitochondrial NADP ؉ -isocitrate dehydrogenase is a target for modification by the lipid peroxidation product 4-hydroxynonenal, an aldehyde that reacts readily with protein sulfhydryl and amino groups. This hypothesis is supported by the following in vitro and in vivo evidence. In isolated rat heart mitochondria, enzyme inactivation occurred within a few minutes upon incubation with 4-hydroxynonenal and was paralleled by 4-hydroxynonenal/ NADP ؉ -isocitrate dehydrogenase adduct formation. Enzyme inactivation was prevented by the addition of its substrate isocitrate or a thiol, cysteine or glutathione, suggesting that 4-hydroxynonenal binds to a cysteine residue near the substrate's binding site. Using an immunoprecipitation approach, we demonstrated the formation of 4-hydroxynonenal/NADP ؉ -isocitrate dehydrogenase adducts in the heart and their increased level (210%) in 7-week-old spontaneously hypertensive rats compared with control Wistar Kyoto rats. To the best of our knowledge, this is the first study to demonstrate that mitochondrial NADP ؉ -isocitrate dehydrogenase is a target for inactivation by 4-hydroxynonenal binding. Furthermore, the pathophysiological significance of our finding is supported by in vivo evidence. Taken altogether, our results have implications that extend beyond mitochondrial NADP ؉ -isocitrate dehydrogenase. Indeed, they emphasize the implication of post-translational modifications of mitochondrial metabolic enzymes by 4-hydroxynonenal in the early oxidative stress-related pathophysiological events linked to cardiac hypertrophy development.Mitochondrial dysfunction is considered to play a key role in the pathogenesis of cardiac hypertrophy and failure. Chronic alterations of fuel metabolism and oxidative stress status are factors that could impair the capacity of the mitochondria to fulfil their crucial role in energy production (1) and thereby contribute to the activation of signaling pathways governing cell death by apoptosis and/or necrosis (2, 3). Although the role of mitochondrial energy fuel deficits or of oxidative stress are often investigated separately, accumulating evidence indicates that these two factors are linked. For example, several metabolic enzymes can be inactivated through post-translational modifications by oxidative stress-related molecular components (4 -11). The latter molecules include oxygen-and nitrogen-derived reactive species or aldehydes produced from lipid peroxidation. Of specific interest to this study is 4-hydroxynonenal (HNE), 1 the major ␣-␤-unsaturated aldehyde formed from peroxidation of both -3 and -6 polyunsaturated fatty acids, whose formation is enhanced in hypertrophied and ischemic/ reperfused...

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Abstract-Perinatal conditions (such as preterm birth) can affect adult health and disease, particularly the cardiovascular system. Transient neonatal high O 2 exposure in rat in adulthood (a model of preterm birth-related complications) leads to elevated blood pressure, vascular rigidity, and dysfunction with renin-angiotensin system activation. We postulate that neonatal hyperoxic stress also affects myocardial structure, function, and expression of renin-angiotensin system components. Sprague-Dawley pups were kept with their mother in 80% O 2 or in room air (control) from days 3 to 10 of life. Left ventricular function was assessed in 4-, 7-, 12-week-old (echocardiography) and in 16-week-old (intraventricular catheterization) male O 2 -exposed versus control rats. At 16 weeks, hearts from O 2 -exposed rats showed cardiomyocyte hypertrophy, enhanced fibrosis, and increased expression of transforming growth factor-β1, senescence-associated proteins p53 and Rb, upregulation of angiotensin II type 1 (AT1) receptor expression (protein and AT1a/b mRNA), and downregulation of AT2 receptors. At 4 weeks (before blood pressure increase), the expression of cardiomyocyte surface area, fibrosis, p53, and AT1b was significantly increased and AT2 decreased in O 2 -exposed animals. After 4 weeks of continuous angiotensin II infusion (starting at 12 weeks), O 2 -exposed rats developed severe heart failure, with impaired myocardial mechanical properties compared with saline-infused rats. Transient neonatal O 2 exposure in rats leads to left ventricular hypertrophy, fibrosis and dysfunction, and increased susceptibility to heart failure under pressure overload. These results are relevant to the growing population of individuals born preterm who may be at higher risk of cardiac dysfunction when faced with increased peripheral resistance associated with hypertension, vascular diseases, and In cardiovascular disease, RAS plays a major role in vascular dysfunction, cardiac hypertrophy, and fibrosis through Ang II acting predominantly on its type I receptor (AT1) 10,11 triggering downstream mechanisms such as the profibrotics transforming growth factor-β1 (TGF-β1) and hypoxic-inducible factor-1α (HIF-1α), 12 as well as senescence-associated pathways. 13Activation of the RAS in the kidneys, brain, and systemic vasculature plays a key role in the elevation and maintenance of BP in experimental models associated with deleterious perinatal conditions including transient neonatal high O 2 exposure. 6,14 However, the effect of neonatal pro-oxidant conditions on cardiac RAS components has not been reported. To determine whether neonatal hyperoxia exposure impaired cardiac development and predisposed to cardiac dysfunction, myocardial structure, and function, expression of cardiac RAS components, as well as TGF-β1, HIF-1α, and senescenceassociated proteins, was determined in young, 4-week-old, and adult, 16-week-old rats infused or not with Ang II. Materials and MethodsSprague-Dawley pups were kept with their mother in 80% O 2 using an oxyc...

Abstract-Angiotensin type 2 (AT 2 ) receptors for angiotensin II suppress cell growth and induce apoptosis in vitro, but their role is poorly defined in vivo. We reported that transient induction of smooth muscle cell (SMC) apoptosis precedes DNA synthesis inhibition and aortic hypertrophy regression in spontaneously hypertensive rats treated with the AT 1 antagonist losartan or the converting-enzyme inhibitor enalapril. Although both drugs are equipotent in reducing SMC number, apoptosis occurs significantly earlier with losartan than enalapril. To examine the role of AT 2 receptors in this model, spontaneously hypertensive rats were given valsartan, an AT 1 antagonist, or enalapril, in combination or not with the AT 2 antagonist PD123319 for 1 or 2 weeks. Control rats received vehicle. Systolic blood pressure was reduced similarly by valsartan and enalapril but it was not significantly affected by PD123319. Angiotensin II plasma levels were increased (6-fold) with valsartan and reduced (80%) with enalapril but unaffected by PD123319. Valsartan significantly increased internucleosomal DNA fragmentation indicative of apoptosis at 1 week only (2.7-fold) and significantly reduced aortic mass (18%), SMC number (33%), and DNA synthesis (24%, measured by 3 H-thymidine incorporation) at 2 weeks. These valsartan-induced changes were prevented by PD123319. In contrast, enalapril-induced DNA fragmentation (2-fold increase at 2 weeks) was not affected by PD123319. PD123319 given alone did not affect growth or apoptosis. AT 1 and AT 2 receptor mRNAs were detected in the aorta by reverse transcription-polymerase chain reaction. Together, these results provide the first evidence that AT 2 receptors mediate vascular mass regression by stimulating SMC apoptosis in vivo, an effect seen during AT 1 receptor blockade but not during convertingenzyme inhibition.