Control of left ventricular mass by moxonidine involves reduced DNA synthesis and enhanced DNA fragmentation.

BACKGROUND AND PURPOSE: Left ventricular hypertrophy (LVH) is a maladaptive process associated with increased cardiovascular risk. Regression of LVH is associated with reduced complications of hypertension. Moxonidine is an antihypertensive imidazoline compound that reduces blood pressure primarily by central inhibition of sympathetic outflow and by direct actions on the heart to release atrial natriuretic peptide, a vasodilator and an antihypertrophic cardiac hormone. This study investigated the effect of moxonidine on LVH and the mechanisms involved in this effect. EXPERIMENTAL APPROACH: Spontaneously hypertensive rats were treated with several doses of moxonidine (s.c.) over 4 weeks. Blood pressure and heart rate were continuously monitored by telemetry. Body weight and water and food intake were measured weekly. Measurements also included left ventricular mass, DNA content, synthesis, fragmentation, and apoptotic/anti-apoptotic pathway proteins. KEY RESULTS: The decrease in mean arterial pressure stabilized at approximately -10 mm Hg after 1 week of treatment and thereafter. Compared to vehicle-treated rats (100%), left ventricular mass was dose- and time-dependently reduced by treatment. This reduction remained significantly lower after normalizing to body weight. Moxonidine reduced left ventricular DNA content and inhibited DNA synthesis. DNA fragmentation transiently, but significantly increased at 1 week of moxonidine treatment and was paralleled by elevated active caspase-3 protein. The highest dose significantly decreased the apoptotic protein Bax and all doses stimulated anti-apoptotic Bcl-2 after 4 weeks of treatment. CONCLUSIONS AND IMPLICATIONS: These studies implicate the modulation of cardiac DNA dynamics in the control of left ventricular mass by moxonidine in a rat model of hypertension.

Differential regulation of Akt, caspases and MAP kinases underlies smooth muscle cell apoptosis during aortic remodelling in SHR treated with amlodipine.

BACKGROUND AND PURPOSE: The regression of aortic hypertrophy is initiated by a transient wave of smooth muscle cell (SMC) apoptosis in spontaneously hypertensive rats (SHR) treated with antihypertensive drugs, although the molecular pathways remain unclear. EXPERIMENTAL APPROACH: Enzymes involved in apoptosis regulation were examined daily during onset aortic remodelling in SHR treated with amlodipine (20 mg kg(-1) day(-1)). KEY RESULTS: Significant reduction of aortic SMC number occurred by day 3 of amlodipine, reaching -13% at 28 days, followed by a significant regression of medial hypertrophy by day 5, reaching -13% at 28 days. ISOL-positive (apoptotic) SMC nuclei increased by 4.6-fold between days 2 and 4, in temporal correlation with the activation of caspase-8 (2.7-fold) at day 2 only, caspase-3 at days 3 and 4 (1.7-fold) and caspase-9 at day 3 only (3.1-fold). Akt phosphorylation, a pro-survival pathway, was reduced prior to apoptosis at day 1 (-52%) and until day 3. During the first 6 days of amlodipine treatment, significant reduction in phosphorylation of mitogen-activated protein (MAP) kinases was transient for p38 (-46% at day 3 only) but continuous for ERK1/2 after 3 days (-40%), and for JNK after 4 days (>-50%). CONCLUSIONS AND IMPLICATIONS: Amlodipine inhibition of Akt occurred prior to and during SMC apoptosis induction, a process mediated by the early activation of caspase-8 followed by caspase-9 and -3 and associated with MAP kinase inhibition. These findings provide insights about the molecular pathways underlying SMC apoptosis leading to vascular remodelling during amlodipine treatment of hypertension.

[3H]-thymidine labelling of DNA triggers apoptosis potentiated by E1A-adenoviral protein.

[(3)H]-thymidine is commonly used to analyze the accumulation of [(3)H]-labeled chromatin fragments in cells undergoing apoptosis. This study shows that [(3)H]-thymidine incorporation within DNA is sufficient per se to inhibit growth and to induce apoptosis in canine kidney epithelial cells and porcine aorta endothelial cells. Despite high-level [(3)H]-thymidine-DNA labeling, rat vascular smooth muscle cells (VSMC) showed only modest inhibition of growth and induction of apoptosis compared to other cell types. Similarly to serum deprivation, apoptosis triggered by [(3)H]-thymidine labeling was sharply potentiated by VSMC transfection with a functional analogue of c-myc, E1A-adenoviral protein (VSMC-E1A), and was suppressed by stimulation of cAMP signaling with forskolin as well as by and Na/K pump inhibition with ouabain. Both apoptosis induction and growth suppression seen in [(3)H]-thymidine-treated VSMC-E1A were reduced by the pan-caspase inhibitor z-VAD.fmk. Thus, our results show that the differential efficiency of the apoptotic machinery determines cell type-specific attenuation of growth in cells with [(3)H]-thymidine-labeled DNA. They also demonstrate that [(3)H]-thymidine-treated and serum-deprived VSMC employ common intermediates of the apoptotic machinery, including steps that are potentiated by E1A-adenoviral protein and inhibited by activation of cAMP signaling as well as by inversion of the intracellular [Na(+)](i)/[K(+)](i) ratio.

Apoptosis in Barrett's oesophagus following antireflux surgery.

BACKGROUND: Intestinal metaplasia persists in Barrett's mucosa despite control of reflux. Tissue homeostasis is maintained by the balance between apoptosis and proliferation. There is an unexplained temporary increase in proliferation in patients with Barrett's mucosa after antireflux surgery, and the long-term effect of any therapy in altering this balance remains unclear. The aim of this study was to assess apoptosis in Barrett's oesophagus following antireflux surgery. METHODS: Apoptosis was evaluated in endoscopic biopsy specimens from 19 patients with Barrett's oesophagus 4 years after Collis-Nissen gastroplasty using an in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) method. RESULTS: Intestinal metaplasia had a lower apoptosis index than gastric metaplasia (0.27 versus 2.14 per cent; P < 0.001). After operation there was a steady increase of apoptosis in intestinal metaplasia over time (from 0.23 per cent before operation to 0.42 per cent within 2 years and to 0.59 per cent 4 years after operation; P = 0.015). Patients with persistent acid exposure did not show any increase in apoptosis in comparison with patients without acid exposure (0.41 versus 0.59 per cent; P = 0.91). CONCLUSION: Apoptosis is less in intestinal metaplasia than in gastric metaplasia, although there is an increase after antireflux surgery. Persistent acid reflux may predispose to malignancy.

Norepinephrine-induced aortic hyperplasia and extracellular matrix deposition are endothelin-dependent.

BACKGROUND: Sympathetic hyperactivity is observed in several disease states and may contribute to cardiovascular hypertrophic remodeling. Endothelin has been suggested to be a mediator of hypertrophy. OBJECTIVE: To examine the involvement of endothelin in maintaining the growth response induced by exogenous norepinephrine. DESIGN AND METHODS: Rats were treated with norepinephrine (2.5 microg/Kg per min subcutaneously) for 2 and 4 weeks, alone or in association with the selective endothelin-A (ETA) receptor antagonist, darusentan (LU135252, 30 mg/Kg per day orally) for weeks 3 and 4. RESULTS: Increases in medial cell number and accumulation of collagen and elastin characterized norepinephrine-induced aortic remodeling. These effects occurred without marked changes of mean arterial pressure, but may be related to enhanced pressure variability in addition to direct effects of norepinephrine. Inhibition of ETA receptors by darusentan reversed aortic alterations produced by infusion of norepinephrine. Evaluation of medial apoptosis did not reveal any significant change in any group at 4 weeks. CONCLUSIONS: Antagonism of ETA receptors effectively and rapidly reversed norepinephrine-induced aortic structural and compositional changes, suggesting a central role of endothelin in mediating this response. Thus, ETA receptor antagonists may help to regress large artery remodeling in conditions of increased circulating catecholamine concentrations.

Evidence of an altered in vivo vascular cell turnover in spontaneously hypertensive rats and its modulation by long-term antihypertensive treatment.

The aims of this study were to measure in vivo cell turnover in the thoracic aorta from spontaneously hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) and to investigate how it could be modulated by chronic antihypertensive treatment. Cell turnover was estimated in rats in which DNA had been prelabeled in utero with [ 3 H]-thymidine, by the rate of disappearance of total [ 3 H]-DNA from birth to 20 weeks of age. In SHR compared with WKY, neonatal relative aortic mass was transiently elevated and was reversed to hypotrophy at 8 weeks. At 20 weeks of age, aortic hypertrophy reappeared. Aortic DNA content reflected the morphologic changes observed with age. In both SHR and WKY, the decline with time in [ 3 H]-prelabeled aortic DNA coupled with the increase in total organ DNA demonstrated that cells prelabeled in utero died and were replaced. Decline in [ 3 H]-DNA from birth to 8 weeks of age was approximately threefold faster in the aorta from SHR than in WKY. In older SHR, the decrease in [ 3 H]-DNA was then slower and similar to that of WKY. Chronic treatment of SHR for 15 weeks from the age of 5 weeks, with hydralazine, enalapril, or nifedipine prevented the rise in systolic blood pressure, aortic mass, and DNA content. This was associated with an unchanged residual radioactivity of [ 3 H]-prelabeled aortic DNA over time, suggesting that the treatment did not stimulate cumulative cell death. We propose that the altered cell turnover is a component of aortic remodeling observed in hypertension. Our data also suggest that it is possible to modulate in vivo cell turnover and affect vascular remodeling by pharmacologic therapy.

Regression of neointimal lesions in the carotid artery of nifedipine-treated SHR and WKY rats: possible role of apoptosis.

We previously observed that nifedipine reduces aortic hypertrophy in spontaneously hypertensive rats (SHR) partly by inducing smooth muscle cell (SMC) apoptosis. The present study examined nifedipine regulation of SMC apoptosis in carotids with or without a neointima. A neointima was produced by endothelial denudation of the left carotid of SHR and WKY rats. The contralateral carotid remained uninjured. Beginning at week 6 after injury, rats received nifedipine or placebo for 5 and 7 additional weeks. In situ terminal deoxynucleotidyl transferase (TdT)-mediated DNA labeling was used to mark apoptotic nuclei. Nifedipine reduced blood pressure in SHR but not WKY rats. Nifedipine had antihypertrophic effects in both SHR and WKY rats. In each strain, the greater reduction in cross-sectional area was seen in the neointima. In this tissue, nifedipine significantly increased TdT-positive SMC (4-fold at week 5 in SHR and 5-fold at week 7 in WKY rats) and reduced SMC number (70% in SHR and 29% in WKY rats) at week 7 compared to week 0. The effects were less pronounced in the injured and uninjured media. Thus, the antihypertrophic action of nifedipine is amplified in the neointima of SHR and WKY rat carotids, where histological evidence suggests SMC deletion via apoptosis.

Antiproliferative effect of brief exposure to cholera toxin in vascular smooth muscle cells: role of cAMP and protein kinase A.

The effect of cholera toxin (CTX), an activator of the adenylate cyclase-coupled G protein alpha(s) subunit, was studied on cultured vascular smooth muscle cell (VSMC) proliferation. Continuous exposure (48 h) to CTX as well as 2-min pretreatment of VSMC with CTX led to the same level of cAMP production, inhibition of DNA synthesis, and arrest in the G1 phase without induction of necrosis or apoptosis in VSMC. Protein kinase A (PKA) activity in CTX-pretreated cells was transiently elevated by 3-fold after 3 h of incubation, whereas after 48 h it was reduced by 2-fold compared with baseline values without modulation of the expression of its catalytic alpha subunit. The PKA inhibitors H89 and KT 5720 did not protect VSMC from the antiproliferative effect of CTX. Two-dimensional electrophoresis was used to analyze the influence of CTX on protein phosphorylation. After 3 h of incubation of CTX-pretreated cells, we observed both newly-phosphorylated and dephosphorylated proteins (77 and 50 protein species, respectively). After 24 h of incubation, the number of phosphorylated proteins in CTX-treated cells was decreased to 39, whereas the number of dephosphorylated proteins was increased to 106. In conclusion, brief exposure to CTX leads to full-scale activation of cAMP signaling and evokes VSMC arrest in the G1 phase.

Endothelial and myocyte apoptosis--role of angiotensin II.

Recent studies have assessed the role of angiotensin II in apoptosis and the effects of the angiotensin II type 1 (AT1) and type 2 (AT2) receptor subtypes. A complex picture is emerging with cell-specific effects of AT1 and AT2 receptors, and differential effects dependent on pathology. In vitro studies have shown that angiotensin II mediated apoptosis in myocytes and endothelium. More recent in vivo studies have shown the prevention and regression of vascular lesions by apoptosis induction with medications (including AT1 receptor inhibitor).

Workshop: excess growth and apoptosis: is hypertension a case of accelerated aging of cardiovascular cells?

Several groups including ours have demonstrated cardiac hyperplasia in neonates from genetically hypertensive rat strains. We have shown that similar problems exist in the kidney as well. More recently, we found that excessive heart and kidney weight is neonatally related to inhibition of apoptosis. Using recombinant inbred strains derived from a reciprocal cross between Brown Norway and spontaneously hypertensive rat progenitor strains, we mapped the inhibition of neonatal apoptosis to 2 distinct loci on chromosomes 1 (Myl 2) and 18 (Abrb 2). Positional candidate genes at these loci are being explored. These studies have also demonstrated that the loci determining kidney and heart weights in neonates are distinct from those determining increased organ weight in adults. The impact of blood pressure per se is also divergent because adult kidney weight is negatively correlated whereas heart weight is positively correlated with it. Analyses by extremes of low and high percentiles from fetal life to adulthood identified a single locus determining heart weight at Acaa on chromosome 8 in newborn (P=0.0003) and adult (P=0.016) rats. The Acaa region contains a DNA mismatch repair gene (hMLH1). The kinetics of neonatal growth through adulthood by prelabeling DNA with [(3)H]thymidine in pregnant mares showed that although the growth process is complex and nonlinear in the kidney of hypertensive rats, there is an increased turnover of cells, that is, reduced half-life of DNA. This observation is supported by the presence of shorter telomere fragments in kidneys of spontaneously hypertensive rats. These studies suggest that cardiovascular cells from hypertensive animals are subject to accelerated turnover, potentially leading to their accelerated aging.

Endotoxin sensitization to kinin B(1) receptor agonist in a non-human primate model: haemodynamic and pro-inflammatory effects.

1. Although endotoxaemia induces kinin B(1) receptors in several animal models, this condition is not documented in primates. This study examined the up-regulation of haemodynamic and pro-inflammatory responses to the B(1) agonist des-Arg(10)-kallidin (dKD) in a non-human primate model. 2. Green monkeys (Cercopithecus aethiops St Kitts) received lipopolysaccharide (LPS; 90 microg kg(-1)) or saline intravenously. After 4 h, anaesthetized monkeys were cannulated via the carotid artery to monitor blood pressure changes following intra-arterial injections of dKD or the B(2) agonist bradykinin (BK). Oedema induced by subcutaneous kinin administration was evaluated as the increase in ventral skin folds in anaesthetized monkeys injected with captopril at 4 h to 56 days post-LPS. 3. LPS increased rectal temperature but did not affect blood pressure after 4 h. dKD reduced blood pressure (E(max): 27+/-4 mmHg; EC(50): 130 pmol kg(-1)) and increased heart rate (E(max): 33 b.p.m.) only after LPS. In contrast, the dose-dependent fall in blood pressure with BK was comparable in all groups. The selective B(1) antagonist [Leu(9)]dKD (75 ng kg(-1) min(-1), intravenously) abolished responses to dKD but not BK. 4. dKD injection induced oedema dose-dependently (2.4+/-0.1 mm at 150 nmol) only following LPS (at 4 h to 12 days but not 56 days). In contrast, BK-induced oedema was present and stable in all monkeys. Co-administration of [Leu(9)]dKD (150 nmol) significantly reduced oedema induced by dKD (50 nmol). 5. These results suggest LPS up-regulation of B(1) receptor effects in green monkeys. This non-human primate model may be suitable for testing new, selective B(1) antagonists with therapeutic potential as anti-inflammatory agents.

Apoptosis in cardiovascular remodeling--effect of medication.

In the last decade, apoptosis has emerged as a key determinant of target organ damage in cardiovascular diseases. The suggestion that increased cardiomyocyte apoptosis participates in the etiology of heart failure probably contributed to the negative view of the prevalence of apoptosis in the field of cardiovascular diseases. However, we and others have shown that up-regulation of apoptosis in certain cardiovascular cells may contribute to the beneficial action of antihypertensive drugs on target-organ structure. As an explanation for this apparent discrepancy, the same stimulus, e.g. angiotensin II, can induce apoptosis or stimulate cell growth in different cell types (e.g., cardiomyocytes and fibroblasts, respectively). Using the angiotensin pathway as a paradigm, this review proposes an integrative view of cell growth and cell death regulation in cardiovascular cells in order to illustrate how cell-specific responses to the same stimulus may in part explain the patterns of cell population dynamics during the development and treatment of target organ damage in hypertension.

Proapoptotic and growth-inhibitory role of angiotensin II type 2 receptor in vascular smooth muscle cells of spontaneously hypertensive rats in vivo.

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 converting-enzyme inhibition.

Evidence that nitric oxide regulates AT1-receptor agonist and antagonist efficacy in rat injured carotid artery.

Vascular injury stimulates AT1-receptor expression and nitric oxide (NO) production in smooth muscle cells (SMCs). We examined the ability of AT1 agonists and antagonists to regulate vascular tone ex vivo in injured arteries and the possible modulation by SMC-derived NO. Rings of rat carotid arteries were isolated at day 7 after endothelial denudation and stimulated with angiotensin (Ang) II in the absence or presence of the AT1 antagonists losartan, L-158,809, or EXP-3174. Freshly denuded contralateral arteries were used as controls. AngII-induced contractions were similar in control and injured arteries. Losartan caused an insurmountable inhibition of AngII-induced contractions in injured but not control arteries. Enhanced inhibition of AngII in injured arteries also was observed in the presence of L-158,809 and EXP-3174. In the presence of the NO synthesis inhibitor nitromonomethyl-L-arginine (L-NMMA), maximal contractions to AngII were greater in injured than in control vessels, and AT1-receptor blockade with losartan was surmountable in all vessels. Mechanical removal of superficial neointimal SMCs attenuated NO production and normalized the efficacy of losartan in injured arteries. These results suggest a role for NO in reducing the biologic effects of AT1-receptor agonists and potentiating the efficacy of AT1 antagonists in vessels undergoing remodeling after injury.

Angiotensin I-converting enzyme activity and vascular sensitivity to angiotensin I in rat injured carotid artery.

We used a vasoreactivity assay to examine the functional significance of angiotensin I-converting enzyme overexpression in smooth muscle cells after vascular injury. Rat carotid arteries isolated at days 2 to 14 after in vivo endothelial denudation were compared with the contralateral freshly denuded (control) vessels. Arterial rings were constricted ex vivo with angiotensin I in the absence or presence of the angiotensin I-converting enzyme inhibitors captopril (300 nM and 3 microM) or perindoprilate (1 nM). Angiotensin I-converting enzyme activity was determined by cleavage of the chromogenic substrate Hip-His-Leu. Angiotensin I-converting enzyme activity in injured arteries was increased (2-fold) at day 7 only after vascular injury. Contractions to angiotensin I were unaffected after injury. Inhibition by captopril and perindoprilate of angiotensin I-induced contractions was significantly less potent in injured arteries at day 7 as compared to control vessels. Mechanical removal of neointimal smooth muscle cells normalized the inhibition by captopril in injured arteries at day 7. Captopril did not affect angiotensin II-induced contractions. Thus, upregulation of angiotensin I-converting enzyme after arterial injury confers resistance to angiotensin I-converting enzyme inhibitors.

Losartan-induced apoptosis as a novel mechanism for the prevention of vascular lesion formation after injury.

Smooth muscle cell (SMC) apoptosis is transiently increased at the onset of the regression of aortic hypertrophy in spontaneously hypertensive rats (SHR) treated with the angiotensin II AT(1) antagonist losartan. We postulated that losartan induction of SMC apoptosis contributes to suppression of neointimal hyperplasia after vascular injury. Losartan or placebo treatment was initiated two days before balloon injury in the SHR aorta. Compared with time-matched placebo, losartan decreased neointimal cross-sectional area at Days 5 and 10 after injury by 50% and 64% respectively, without affecting medial mass. At Day 10, losartan significantly decreased SMC number (by 56%) in the neointima, but not in the media. DNA synthesis was significantly inhibited at Day 5 but not at Day 10. Losartan significantly increased aortic DNA fragmentation by 2.6- and 4.1-fold, at Days 5 and 10, respectively. In situ labeling of SMC with terminal deoxynucleotidyltransferase revealed significant 61% and 68% increases in apoptotic SMC at Days 5 and 10 with losartan treatment, predominantly in the neointima. Thus, losartan suppressed neointima formation in part by the induction of SMC apoptosis, which may be dissociated from the inhibition of DNA synthesis. Therefore, losartan-induced SMC apoptosis may be a potential therapeutic approach to control occlusive vascular disorders.

Apoptosis during regression of cardiac hypertrophy in spontaneously hypertensive rats. Temporal regulation and spatial heterogeneity.

We previously reported that increased apoptosis participates in the regression of aortic hypertrophy in spontaneously hypertensive rats. To further document the potential role of apoptosis in cardiovascular therapy, we examined apoptosis during regression of hypertrophy in the heart of spontaneously hypertensive rats receiving the antihypertensive drug enalapril (30 mg. kg(-1). d(-1)), losartan (30 mg. kg(-1). d(-1)), nifedipine (35 mg. kg(-1). d(-1)), hydralazine (40 mg. kg(-1). d(-1)), propranolol (50 mg. kg(-1). d(-1)), or hydrochlorothiazide (75 mg. kg(-1). d(-1)) for 1 to 4 weeks, starting at 10 to 11 weeks of age. Systolic blood pressure and heart rate were measured by the tail-cuff method. Markers of apoptosis included oligonucleosomal DNA fragmentation in extracted cardiac DNA or in situ in ventricular cross sections labeled with terminal deoxynucleotidyl transferase. Cardiac DNA synthesis was evaluated by [(3)H]-thymidine incorporation in vivo. All drugs reduced cardiac workload, defined as the product of blood pressure and heart rate, by >20% at 4 weeks. However, only nifedipine, enalapril, losartan, and propranolol reduced cardiac mass (>19%) within 4 weeks. Regression of cardiac hypertrophy was accompanied by a 50% to 300% increase in DNA fragmentation and a >20% reduction in DNA synthesis, resulting in a >20% reduction in cardiac DNA content after 4 weeks. Apoptosis induction occurred early and was transient within 4 weeks of nifedipine, enalapril, or losartan administration. With all regression-inducing drugs, the increase in DNA fragmentation occurred mainly in the subepicardium. Thus, transient induction of apoptosis in the subepicardium appears to be a characteristic feature of the early response to drug-induced regression of cardiac hypertrophy in spontaneously hypertensive rats.

Heparinase III limits rat arterial smooth muscle cell proliferation in vitro and in vivo.

Heparinase III degrades heparan sulfate proteoglycans, which are co-receptors for growth factors that stimulate arterial proliferation. We assessed the ability of locally-delivered heparinase III to limit medial vascular smooth muscle cell proliferation induced by balloon catheter injury in rat carotid arteries. Whereas vehicle-treated arteries showed 12% of smooth muscle cells proliferating after 2 days, heparinase III (0.022-5.7 mg/kg) treated arteries showed 0.8-4%. Chemically-inactivated heparinase III did not limit proliferation. In isolated rat A10 vascular smooth muscle cells, heparinase III (1 IU/ml) inhibited both PDGF-BB and bFGF mediated increases in proliferation and migration. These results suggest that heparinase III can limit proliferation by affecting heparan sulfate proteoglycan binding growth factors following arterial injury.

Effect of phosphorothioated oligonucleotides on neointima formation in the rat carotid artery. Dissecting the mechanism of action.

Several studies have shown that single-dose administration of agents that inhibit medial cell replication, such as antisense oligonucleotides to cell replication genes, can inhibit neointima formation after arterial injury. However, the precise mechanism of action of these agents is unknown. We analyzed the effect of phosphorothioated oligonucleotides delivered periadventitially on the response to injury in the balloon-injured rat carotid artery. Antisense oligonucleotides to c-myc suppressed medial replication 2 days after injury, but this effect was not present at 4 or 14 days. Endothelial cell proliferation was not affected by antisense oligonucleotides. There was, however, a significant suppression of intimal area and intima/media ratio at 14 days and an increase in lumen area in the antisense-treated group. Indeed, an increase in the number of medial cells at 14 days in the antisense group indicated that most of the effect of the agent was due to the suppression that most of the effect of the agent was due to the suppression of cell migration. No effect was noted on expression of two genes, osteopontin and tropoelastin, used as markers of modulation of smooth muscle cells to a "neonatal" phenotype at 4 days after injury. Because no effect on cell proliferation could be demonstrated after 2 days, our data indicate that an early effect of the antisense agent mediates its longer-term effects. We suggest that this effect may be due to the suppression of migration of medial smooth muscle cells rather than the suppression of medial or intimal cell proliferation.

Smooth muscle apoptosis during vascular regression in spontaneously hypertensive rats.

We previously reported that apoptosis is increased in smooth muscle cells cultured from the aorta of spontaneously hypertensive rats versus normotensive controls. As an initial in vivo exploration, we now examined smooth muscle cell apoptosis regulation during the regression of vascular hypertrophy in the thoracic aorta media of spontaneously hypertensive rats receiving the antihypertensive drug enalapril (30 mg.kg-1.d-1), losartan (30 mg.kg-1.d-1), nifedipine (35 mg.kg-1.d-1), hydralazine (40 mg.kg-1.d-1), propranolol (50 mg.kg-1.d-1), or hydrochlorothiazide (75 mg.kg-1.d-1) for 1 to 4 weeks starting at 10 to 11 weeks of age. Three criteria were used to evaluate smooth muscle cell apoptosis: (1) oligonucleosomal fragmentation of the extracted aortic DNA, (2) reduction in aortic DNA content, and (3) depletion of smooth muscle cells in the arterial media. Arterial DNA synthesis was evaluated by [3H]thymidine incorporation in vivo. After 4 weeks of treatment, systolic blood pressure was reduced significantly by > 42% with losartan, enalapril, and hydralazine, and by 23% with nifedipine, versus control values of 220 +/- 5 mm Hg. However these agents affected vascular growth and apoptosis differently. Losartan, enalapril, and nifedipine stimulated smooth muscle cell apoptosis threefold to fivefold before there was a significant reduction in 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 nifedipine stimulated smooth muscle cell apoptosis before reducing blood pressure. In contrast, hydralazine did not affect vascular mass, apoptosis, or DNA synthesis, although blood pressure was lowered. Propranolol or hydrochlorothiazide failed to affect hypertension or vascular growth. Thus, smooth muscle cell apoptosis represents a novel therapeutic target for the control of hypertensive vessel remodeling in response to therapeutic agents.