Bethany J. Holycross

Endothelial dysfunction and peroxynitrite formation are early events in angiotensin-induced cardiovascular disorders

Angiotensin II (ANG II) is a well‐established participant in many cardiovascular disorders, but the mechanisms involved are not clear. Vascular cell experiments suggest that ANG II is a potent stimulator of free radicals such as superoxide anion, an agent known to inactivate nitric oxide and promote the formation of peroxynitrite. Here we hypothesized that ANG II reduces the efficacy of NO‐mediated vascular relaxation and promotes vascular peroxynitrite formation in vivo. ANG II was infused in rats at sub‐pressor doses for 3 days. Systolic blood pressure and heart rate were unchanged on day 3 despite significant reductions in plasma renin activity. Thoracic aorta was isolated for functional and immunohistochemical evaluations. No difference in isolated vascular contractile responses to KCI (125 mM), phenylephrine, or ANG II was observed between groups. In contrast, relaxant response to acetylcholine (ACh) was decreased sixfold without a change in relaxant response to sodium nitroprusside. Extensive prevalence of 3‐nitrotyrosine (3‐NT, a stable biomarker of tissue peroxynitrite formation) immunoreactivity was observed in ANG II‐treated vascular tissues and was specifically confined to the endothelium. Digital image analysis demonstrated a significant inverse correlation between ACh relaxant response and 3‐NT immunoreactivity. These data demonstrate that ANG II selectively modifies vascular NO control at sub‐pressor exposures in vivo. Thus, endothelial dysfunction apparently precedes other established ANG II‐induced vascular pathologies, and this may be mediated by peroxynitrite formation in vivo. Wattanapitayakul, S., Weinstein, D. M., Holycross, B. J., Bauer, J. A. Endothelial dysfunction and peroxynitrite formation are early events in angiotensin‐induced cardiovascular disorders. FASEB J. 14, 271–278 (2000)

Adipokines: a review of biological and analytical principles and an update in dogs, cats, and horses

In addition to its role as an energy storage depot, adipose tissue is now recognized as a complex endocrine organ. Adipose tissue releases a variety of factors, termed adipokines, that regulate energy metabolism, cardiovascular function, reproductive status, and immune function. Some of the better-studied adipokines include leptin, adiponectin, and components of the renin-angiotensin system such as angiotensinogen. The function of more recently discovered adipokines such as resistin are under intense scrutiny. Abnormal production or regulation of adipokines occurs in obese individuals and is implicated in the development of a variety of associated co-morbidities including metabolic syndrome, type 2 diabetes, atherosclerosis, heart disease, and cancer in people, although evaluation in domestic species is just beginning. Adipokines are now being examined as potential biomarkers for risk assessment for development of complications related to obesity. This article summarizes the function and regulation of some better-characterized adipokines. It also reviews the current information on the characterization of adipokines in some domestic species in which rates of obesity and obesity-related disorders are increasing, such as the dog, cat, and horse.

Myocardial tumor necrosis factor-α secretion in hypertensive and heart failure-prone rats

Acute increases in blood pressure (BP) increase myocardial tumor necrosis factor (TNF)-α production, but it is not known whether chronic hypertensive stress elevates myocardial TNF-α production, possibly contributing to cardiac remodeling, decreased cardiac function, and faster progression to heart failure. BP, cardiac function, and size were evaluated in normotensive [Sprague-Dawley (SD)], spontaneously hypertensive (SHR), and spontaneously hypertensive heart failure-prone (SHHF) rats at 6, 12, 15, and 18 mo of age and in failing SHHF. Left ventricular tissues were evaluated for secretion of bioactive TNF-α and inhibition of TNF-α secretion by phosphodiesterase inhibitors. All ventricles secreted bioactive and immunoreactive TNF-α, but secretion decreased with age. SHR and SHHF rats secreted more TNF-α than SD rats at 6 mo of age, but only failing SHHF rats secreted significantly more TNF-α at 18 mo. Amrinone inhibited TNF-α secretion in all rats and was less potent but more efficacious than RO-201724 in all strains. TNF-α secretion correlated with BP and left ventricular mass in 6-mo-old rats, but this relationship disappeared with age. Results suggest that hypertension and/or cardiac remodeling is associated with elevated myocardial TNF-α, and, although hypertension, per se, did not maintain elevated cardiac TNF-α levels, SHHF rats increase TNF-α production during the end stages of failure.

Combined effects of low-dose oral spironolactone and captopril therapy in a rat model of spontaneous hypertension and heart failure.

The effects of low-dose oral spironolactone (SPIRO) in a rat model of hypertensive heart failure (spontaneously hypertensive heart failure rat) were compared with its effects when combined with captopril (CAP). Twenty-six spontaneously rats with hypertensive heart failure were treated with either placebo (CON), SPIRO (20 mg/kg/d by mouth), CAP (100 mg/kg/d by mouth), or both SPIRO and CAP for 12 weeks. This dose of oral SPIRO did not affect blood pressure, left ventricular end-diastolic diameter, left ventricular ejection fraction, plasma atrial natriuretic peptide concentration, or cardiac fibrosis; however, in combination with CAP, it exerted a significant depressor effect after 12 weeks of treatment that was accompanied by increased urine output and decreased urinary protein excretion. These effects were significantly greater than those with CAP treatment alone. A significant increase in plasma aldosterone level was observed only in CON (174 ± 21%). These data suggest that the addition of low-dose SPIRO to angiotensin I–converting enzyme inhibitor treatment may prevent progression into end-stage congestive heart failure through synergistic effects on diuresis and renoprotection.

Increased salt sensitivity secondary to leptin resistance in SHHF rats is mediated by endothelin

A link between leptin resistance, obesity, and salt sensitivity has been suggested. SHHF/Mcc-facp rats (SHHF) were used to study the effect of gene dosage of a null mutation of the leptin receptor (cp) on salt sensitivity and response to a combined endothelin A and B receptor antagonist (bosentan). Obese (cp/cp), heterozygous (+/cp), and homozygous lean (+/+) male SHHF were fed a low salt diet (0.3% NaCl) for 7 days, followed by a high salt diet (8.0% NaCl) for 7 days. There were no significant differences in systolic blood pressure between genotypes on low salt. In response to high salt, cp/cp had significantly greater systolic pressure than +/cp and +/+. On high salt diet, cp/cp showed a significant increase in 24 h urinary endothelin excretion and increased renal expression of preproendothelin mRNA. There was no effect of high salt diet on renal excretion of nitric oxide (NOx) or on gene expression of endothelial, neuronal, or cytokine-induced nitric oxide synthase isoforms (eNOS, nNOS, iNOS, respectively). Treatment with bosentan prevented the high salt-induced increment in systolic blood pressure in cp/cp. This was associated with a doubling of renal NOx excretion, but without changes in eNOS, nNOS, or iNOS expression. Endothelin receptor antagonism did not normalize systolic pressure in any of the genotypes. Our studies indicate that obesity secondary to leptin resistance (cp/cp) results in increased salt sensitivity that is mediated by endothelin in the SHHF rat.

Comparison of Irbesartan with Captopril Effects on Cardiac Hypertrophy and Gene Expression in Heart Failure-Prone Male SHHF/Mcc-facp Rats

Angiotensin-converting enzyme (ACE) inhibitors have proven an effective means to control hypertension and manage cardiac hypertrophy. It is presently unknown if newer specific angiotensin II subtype 1 receptor (AT1R) antagonists are as effective or more effective in treating these conditions compared with ACE inhibitors. There is evidence that these classes of drugs may affect cardiac hypertrophy by different mechanisms. This study compared the effect of irbesartan, an AT1R antagonist, with that of captopril, an ACE inhibitor, on expression of early genetic markers of cardiac hypertrophy in lean male SHHF/Mcc-fa(cp) rats. SHHF/Mcc-fa(cp) rats (n = 10/group) were given captopril (100 mg/kg/day), irbesartan (50 mg/kg/day), or placebo for 16 weeks. Irbesartan and captopril significantly reduced systolic pressure and produced similar rightward shifts in the angiotensin I dose-response curve. Renal renin gene expression was increased 8.6-fold by irbesartan and 17.7-fold by captopril. The only effect on echocardiographic findings was a similar decrease in aortic peak velocity, an index of systolic function, by both treatments. Early markers of cardiac hypertrophy were significantly attenuated by both drugs. Both drugs produced marked and equivalent reductions in left ventricular atrial natriuretic peptide (ANP) messenger RNA (mRNA) levels compared with controls. This decrease in ANP gene expression was accompanied by a decrease in plasma ANP concentration in the treatment groups. The shift from V1 to V3 myosin isozymes was similarly decreased in both treatment groups, compared with controls. These data suggest that captopril and irbesartan are similarly effective in controlling expression of genes associated with ventricular hypertrophy in heart failure-prone SHHF/Mcc-fa(cp) rat.

Effect of ovariectomy in heart failure-prone SHHF/Mcc-facp rats

The importance of the loss of ovarian function to the progression of hypertension and heart disease in women is controversial. We investigated whether ovariectomy would accelerate development of hypertension, congestive heart failure, and neurohumoral activation in adult spontaneous hypertension heart failure (SHHF) rats, a genetic model of heart failure. Six months after ovariectomy, no significant differences between control and ovariectomized rats were seen in systolic or diastolic blood pressure, left ventricular fractional shortening by echocardiography, or heart weight. Percent V1 myosin isozyme was significantly lower in ovariectomized rats. Northern blot analysis failed to show significant differences between groups in expression of hepatic angiotensinogen, renal renin, or left ventricular atrial or brain natriuretic peptide mRNA. In a second experiment, serial measures of systolic pressure and left ventricular shortening fractions failed to document a significant difference between control and ovariectomized rats as they developed heart failure, although there was a significant decline in shortening fraction in both groups at the age when regular estrous cycling naturally ceases. Survival time was similar between groups. In summary, ovariectomy of adult SHHF rats does not appear to affect the progression of genetically programmed hypertension and heart failure in this model.The importance of the loss of ovarian function to the progression of hypertension and heart disease in women is controversial. We investigated whether ovariectomy would accelerate development of hypertension, congestive heart failure, and neurohumoral activation in adult spontaneous hypertension heart failure (SHHF) rats, a genetic model of heart failure. Six months after ovariectomy, no significant differences between control and ovariectomized rats were seen in systolic or diastolic blood pressure, left ventricular fractional shortening by echocardiography, or heart weight. Percent V1 myosin isozyme was significantly lower in ovariectomized rats. Northern blot analysis failed to show significant differences between groups in expression of hepatic angiotensinogen, renal renin, or left ventricular atrial or brain natriuretic peptide mRNA. In a second experiment, serial measures of systolic pressure and left ventricular shortening fractions failed to document a significant difference between control and ovariectomized rats as they developed heart failure, although there was a significant decline in shortening fraction in both groups at the age when regular estrous cycling naturally ceases. Survival time was similar between groups. In summary, ovariectomy of adult SHHF rats does not appear to affect the progression of genetically programmed hypertension and heart failure in this model.

Salt-Induced Cardiac Hypertrophy Is Independent of Blood Pressure and Endothelin in Obese, Heart Failure-Prone SHHF Rats

The interaction of salt sensitivity and obesity in development of cardiac hypertrophy is incompletely understood. The SHHF/Mcc-facp (SHHF) rat model was used to examine the effect of high salt on cardiac hypertrophy and expression of endothelin (ET) and nitric oxide synthase (NOS) isoforms. Homozygous lean (+/+) and obese (facp/facp) SHHF were fed a low-salt diet (0.3% NaCl) for seven days followed by a high-salt diet (8.0% NaCl) for seven days. To assess the role of ET in mediating cardiac hypertrophy and gene expression with high salt, additional groups were treated with an ETA/ETB receptor antagonist (bosentan) while on high salt. Obese SHHF showed an increase in systolic blood pressure and cardiac hypertrophy in response to the high-salt diet. High salt resulted in decreased expression of preproET as well as all three NOS isoforms in the Obese, while cytokine induced NOS (iNOS) and neuronal NOS (nNOS) increased in Leans. Though the salt-sensitive component of the hypertension observed in the Obese was prevented by bosentan, cardiac hypertrophy still occurred and expression of all NOS isoforms remained lower in Obese compared to Lean. Endothelial NOS (eNOS) expression increased in the Lean with bosentan. These studies suggest that cardiac hypertrophy is independent of the level of hypertension and may be mediated by altered production of NOS isoforms in salt-sensitive, obese SHHF.

OBESE FEMALE SHHF/Mcc-facp RATS RESIST ANTIHYPERTENSIVE EFFECTS OF RENIN-ANGIOTENSIN SYSTEM INHIBITION

Gender and obesity may influence response to pharmacological modulation of the renin-angiotensin system. We used SHHF/Mcc-facp rats to study effect of obesity and gender on the ability of an AT1 receptor antagonist to decrease blood pressure. After 2 weeks treatment with irbesartan (50 mg/kg), only lean and obese males showed significant decreases in blood pressure, while obese females were completely resistant. Lean females showed a trend toward lowering of pressure (p = 0.06). However, irbesartan similarly shifted angiotensin II dose response curves to the right in all groups. Twelve weeks of irbesartan also failed to decrease blood pressure, but did significantly reduce heart weight in obese females. In untreated rats, obese females had lower plasma renin activity and serum angiotensin converting enzyme activity compared to lean males, while lean and obese females had increased urinary endothelin excretion. Despite an otherwise similar genetic background contributing to hypertension and heart failure, obese females have different patterns of humoral activation compared to lean males, which may contribute to their resistance to the depressor effects of irbesartan.

Endurance exercise training reduces cardiac sodium/calcium exchanger expression in animals susceptible to ventricular fibrillation.

Aim: Increased sodium/calcium exchanger activity (NCX1, an important regulator of cardiomyocyte cystolic calcium) may provoke arrhythmias. Exercise training can decrease NCX1 expression in animals with heart failure improving cytosolic calcium regulation, and could thereby reduce the risk for ventricular fibrillation (VF). Methods: To test this hypothesis, a 2-min coronary occlusion was made during the last minute of exercise in dogs with healed myocardial infarctions; 23 had VF (S, susceptible) and 13 did not (R, resistant). The animals were randomly assigned to either 10-week exercise training (progressively increasing treadmill running; S n = 9; R n = 8) or 10-week sedentary (S n = 14; R n = 5) groups. At the end of the 10-week period, the exercise + ischemia test provoked VF in sedentary but not trained susceptible dogs. On a subsequent day, cardiac tissue was harvested and NCX1 protein expression was determined by Western blot. Results: In the sedentary group, NCX1 expression was significantly (ANOVA, P < 0.05) higher in susceptible compared to resistant dogs. In contrast, NCX1 levels were similar in the exercise trained resistant and susceptible animals. Conclusion: These data suggest that exercise training can restore a more normal NCX1 level in dogs susceptible to VF, improving cystolic calcium regulation and could thereby reduce the risk for sudden death following myocardial infarction.