Growth hormone in vascular pathology: neovascularization and expression of receptors is associated with cellular proliferation.

Vascular tumours are common lesions of the skin and subcutaneous tissue, but also occur in many other tissues and internal organs. The well-differentiated tumours consist of irregular anastomosing, blood-filled vascular channels that are lined by variably atypical endothelial cells. The less differentiated tumours may show solid strands and sheets, resembling carcinoma or lymphoma. Several growth factors, including basic fibroblast growth factor, transforming growth factors and vascular endothelial growth factor, play a role in tumour angiogenesis. Growth hormone (GH) is mitogenic for a variety of vascular tissue cells, including smooth muscle cells, fibroblasts and endothelial cells and exerts its regulatory functions in controlling metabolism, balanced growth and differentiated cell expression by acting on specific membrane-bound receptors, which trigger a phosphorylation cascade resulting in the modulation of numerous signalling pathways and of gene expression. Essential to the initiation of a cellular response to GH, the presence of receptors for this hormone may predict the adaptation of tumour cells resulting from GH exposure. To address the site/mode of action through which GH exerts its effects, a well characterized monoclonal antibody, obtained by hybridoma technology from Balb/c mice immunized with purified rabbit and rat liver GH-receptor (GHR) and directed against the hormone binding site of the receptor, was applied, using the ABC technique to determine GHR expression in a panel of vascular tumours. The GHR was cloned from a rabbit liver cDNA library with the aid of an oligonucleotide probe based on a 19 residue tryptic peptide sequence derived from 5900 fold purified rabbit liver receptor. A total of 64 benign and malignant vascular tumours were obtained from different human organ sites, including the chest wall, skin, axillary contents, duodenum, female breast, abdomen, stomach, colon, lymph node, bladder, body flank and neck regions. The tumours were of the following pathological entities: Haemangioma (n = 12); haemangioendothelioma (n = 10); Castleman's disease (n = 3), haemangiopericytoma (n = 4); angiosarcoma, (n = 11), Kaposi's sarcoma with focal infiltration by lymphoma, HIV +ve (n = 7), Kaposi's sarcoma (n = 17). The endothelial cell marker CD-31 was used to establish endothelial cell characteristics and microvascular density. To delineate tumour cell growth, immunohistochemical analysis of cycling nuclear protein and of proliferating cell nuclear antigen, using Ki-67 and PCNA polyclonal antibodies respectively, was used to demonstrate proliferative indexes. Results show that, compared to their normal tissue counterparts, nuclear and cytoplasmic expression of GHR consistently result in strong receptor immunoreactivity in the highly malignant angiosarcomas and Kaposi's sarcomas and was localized in the cell membranes and cytoplasm, but strong nuclear immunoreactivity was also identified. The presence of intracellular GHR is the result of endoplasmic reticulum and Golgi localization. Nuclear localization is due to identical nuclear GHR-binding protein. Furthermore, there was a positive correlation of GHR immunoreactivity with neoplastic cellular proliferation and cycling, as measured by Ki-67 and PCNA. In conclusion, this study shows that GHR expression in vascular tumours is a function of malignancy and cancer progression. Malignant cells, which are highly expressive of the receptor, have a greater proliferation rate and thereby also higher survival rate compared to tumours expressing lower or minimal receptor level. The presence of GHR in endothelial cells of vascular neoplasm indicates that they are target cells and GH is of importance in the proliferation of vascular tumour angiogenesis. GH is necessary not only for differentiation of progenitor cells, but also for their subsequent clonal expansion and maintenance. The results support the hypothesis that GH is involved in the paracrine-autocrine mechanism, acting locally in regulating vascular tumour growth and will be useful for site-specific studies of the evolution of vascular cancers. The use of anti-GHR antibodies to block tumour progression is an intriguing possibility.

Adriamycin-induced early changes in myocardial antioxidant enzymes and their modulation by probucol.

BACKGROUND: The clinical usefulness of adriamycin is restricted by the development of congestive heart failure. It has been suggested that probucol, a strong antioxidant, completely prevents adriamycin-induced cardiomyopathy without interfering with its antitumor properties. The present study investigated the effects of adriamycin and probucol on myocardial antioxidant enzyme activities and immunoreactive protein levels in rats. METHODS AND RESULTS: Activities and protein levels of glutathione peroxidase (GSHPx) were significantly decreased from 2 to 24 hours, and those of manganese superoxide dismutase were decreased at 1 and 2 hours after adriamycin treatment. These changes were prevented by probucol. Catalase activity was increased from 2 to 24 hours after adriamycin treatment, but its protein levels were not significantly changed. Copper zinc superoxide dismutase activity and protein level were not changed at any time. Myocardial lipid peroxidation was found to be significantly higher at all time points, and this change was also prevented by probucol. Treatment with probucol alone increased GSHPx activity at 2 weeks, and in these hearts, lipid peroxidation was lower than the control value. Within 24 hours, there was no mortality in any of the groups. CONCLUSIONS: It is suggested that an early and persistent decrease in GSHPx activity and protein may play an important role in the pathogenesis of adriamycin-induced cardiomyopathy, worsening heart failure and mortality.

Beneficial effects of verapamil in diabetic cardiomyopathy.

It has been suggested that the occurrence of an intracellular Ca2+ overload may result in the development of diabetic cardiomyopathy, which is associated with depletion of high-energy phosphate stores and a derangement of ultrastructure and cardiac dysfunction. Accordingly, the effects of verapamil, a Ca2+ antagonist, on cardiac function, ultrastructure, and high-energy phosphate stores in the myocardium were evaluated in rats made diabetic by an intravenous injection of streptozocin (65 mg/kg). Four weeks after the induction of diabetes, the animals were treated with three doses (2, 4, or 8 mg.kg-1.day-1) of verapamil for 4 wk until they were used for the measurement of different parameters. Untreated diabetic animals had slower heart rates, depressed rate of contraction and rate of relaxation, lower peak left ventricular systolic pressure, and elevated left ventricular diastolic pressure. All of these changes were significantly improved in diabetic rats receiving verapamil treatment. The beneficial effects of verapamil were more evident with higher doses (8 mg.kg-1.day-1) than with the lower doses (2 mg.kg-1.day-1). The diabetic animals also showed alterations in myocardial high-energy phosphate stores and exhibited evidence of ultrastructural damage; these abnormalities were improved by verapamil treatment without affecting their hyperglycemic status. Our results demonstrate that verapamil is capable of preventing diabetes-induced myocardial changes and support the involvement of Ca2+ in the cardiac pathology during diabetes.

Pressure overload-induced cardiac hypertrophy with and without dilation.

OBJECTIVES: The present study was designed to produce a small animal model showing compensated hypertrophy followed by congestive heart failure within a reasonable time period. BACKGROUND: Although there are various large animal experimental models of hypertrophy and heart failure, the occurrence of these two stages within a reasonable time period has not been shown very successfully in small animals. METHODS: A mildly constricting band was placed around the ascending aorta of very young guinea pigs (mean age 25 +/- 3 days) to impose a gradually increasing pressure overload. The animals were examined at different postoperative intervals up to 20 weeks. RESULTS: At 10 weeks, there was a 56% increase in ventricular weight/body weight ratio, a 33% increase in left ventricular wall thickness and a significant increase in left ventricular systolic pressure. The animals with 20 weeks of banding had developed various clinical symptoms of congestive heart failure including dyspnea, cyanotic appearance of the extremities, hydrothorax and ascites. Although at this stage there was 86% hypertrophy, the increase in wall thickness was only 20%, indicating cardiac dilation. Depressed left ventricular systolic pressure and increased left ventricular end-diastolic pressure and the increase in wet weight/dry weight ratio in the lungs and liver at 20 weeks also indicated the occurrence of heart failure. The collagen content in the heart of animals with banding for 10 and 20 weeks was 160% and 240%, respectively, of that in corresponding sham control animals. CONCLUSIONS: The data suggest that the heart was in a stage of compensated hypertrophy for up to 10 weeks, whereas heart failure was seen at 20 weeks. The two functional stages, compensatory hypertrophy followed by prolonged failure, make this model appropriate for studies on the transition of heart hypertrophy to congestive heart failure.

Effects of afterload-reducing drugs on pathogenesis of antioxidant changes and congestive heart failure in rats.

OBJECTIVES: The present study sought to evaluate the effects of the afterload-reducing drugs captopril and prazosin on changes in antioxidants as well as oxidative stress in relation to hemodynamic function in congestive heart failure (CHF) subsequent to myocardial infarction (MI). BACKGROUND: Afterload reduction therapy has been shown to reduce morbidity and mortality in patients with MI. CHF subsequent to MI in rats is associated with a decrease in myocardial endogenous antioxidants and an increase in oxidative stress. METHODS: The left anterior descending coronary artery in male Sprague-Dawley rats was ligated. Sham and experimental (post-MI [PMI]) animals were assessed for hemodynamic function as well as lung and liver weights at 1, 4 and 16 weeks after operation. At 4 weeks, some rats were also treated with captopril (2 g/liter in drinking water daily) or prazosin (0.2 mg/kg body weight subcutaneously daily) and assessed at 16 weeks. Hearts were isolated to study the activity of superoxide dismutase (SOD), glutathione peroxidase (GSHPx) and catalase as well as for thiobarbituric acid reactive substances (TBARS). RESULTS: CHF at 4 and 16 weeks in the infarcted rats was indicated by an increase in left ventricular end-diastolic pressure and wet/dry weight lung and liver ratios and depressed left ventricular systolic pressure and dyspnea. All these changes were attenuated in both the captopril- and prazosin-treated groups. SOD, GSHPx and catalase activity in the untreated PMI groups was decreased at 4 and 16 weeks. However, treatment with captopril resulted in a significant improvement in SOD, GSHPx and catalase activity in the 16-week PMI group. With prazosin, only SOD activity was improved in the treated 16-week PMI group. Lipid peroxidation as indicated by TBARS was significantly increased in the 16-week PMI group, and both captopril and prazosin modulated this increase. CONCLUSIONS: Occurrence of an antioxidant deficit and an increase in oxidative stress in the myocardium may play a role in the pathogenesis of CHF subsequent to MI. Attenuation of these changes in antioxidant activity with vasodilator (or antioxidant?) therapy mitigates the process of heart failure.

Modulation of oxidative stress by a selective inhibition of angiotensin II type 1 receptors in MI rats.

OBJECTIVES: To examine whether blocking of the renin-angiotensin system (RAS) at the angiotensin II type 1 (AT1) receptor site is accompanied by changes in the oxidative stress parameters. BACKGROUND: Congestive heart failure in rats after myocardial infarction (MI) has been shown to correlate with a decrease in antioxidant enzyme activities and an increase in oxidative stress. Inhibition of the RAS with captopril improves cardiac function and survival in MI rats with a reduction in oxidative stress. METHODS: Myocardial infarction in rats was produced by ligation of the left coronary artery. At four weeks after surgery, animals from the sham as well as MI groups were treated with losartan (2 mg/ml in drinking water daily). At 16 weeks after surgery, the animals were examined for hemodynamic function and the hearts were analyzed for antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase and catalase) and oxidative stress (lipid hydroperoxides, reduced and oxidized glutathione and redox ratio). RESULTS: Congestive heart failure was characterized by dyspnea, depressed hemodynamic function and presence of lung and liver congestion. This was also associated with a decrease in the myocardial catalase (-25%), glutathione peroxidase (-38%) and superoxide dismutase (-42%) activities. An increase in oxidative stress in these hearts was indicated by an increase in lipid hydroperoxides (+67%) and reduction in the redox ratio (-75%). Hemodynamic function was better maintained and there were no indications of dyspnea or lung or liver congestion in the losartan-treated MI rats. In these animals, myocardial oxidative stress was markedly reduced and glutathione peroxidase and catalase activities were significantly improved compared with the untreated MI group. CONCLUSIONS: Blocking of RAS at the AT1 receptor site without the inhibition of angiotensin-converting enzymes modulates heart failure after MI, and this beneficial effect is associated with a decrease in oxidative stress. This study suggests a newer role for losartan in the treatment of heart failure.

Role of oxidative stress in transition of hypertrophy to heart failure.

OBJECTIVES: In an attempt to define the role of increased oxidative stress in the transition from compensatory hypertrophy to heart failure, this study examined the effects of long-term vitamin E therapy on the occurrence of heart failure subsequent to chronic pressure overload in guinea pigs. BACKGROUND: Hyperfunctional heart hypertrophy has been shown to be accompanied by an increase in the endogenous antioxidant reserve, whereas congestive heart failure is accompanied by a decrease in this reserve. The effects of vitamin E, a naturally occurring antioxidant, on the development of heart failure from a hypertrophic stage were examined. METHODS: The ascending aorta in guinea pigs was coarcted. For vitamin treatment, slow-release pellets were implanted at the time of the operation. The animals were assessed at 10 and 20 weeks for hemodynamic function, myocardial structure, antioxidant agents and oxidative stress. RESULTS: Banding of the ascending aorta in guinea pigs resulted in hyperfunctional hypertrophy at 10 weeks, which was followed by congestive heart failure at 20 weeks. Hypertrophied hearts showed decreased oxidative stress, as evidenced by a higher oxidation-reduction (redox) state and less lipid peroxidation, whereas the failure stage was characterized by increased oxidative stress. Supplementation of animals with timed-release vitamin E tablets resulted in an increased myocardial content of the vitamin, and the banded animals did not develop any signs of heart failure at 20 weeks. Hemodynamic function at 20 weeks in these vitamin E-treated animals was also better maintained. The myocardial reduced glutathione/oxidized glutathione ratio of vitamin E-treated animals at 20 weeks was higher and lipid peroxidation was less compared with the untreated animals. Ultrastructural abnormalities were significantly less in the vitamin E-treated hearts compared with the untreated failing hearts at 20 weeks. CONCLUSIONS: An improved myocardial redox state with vitamin E therapy, coupled with the modulation of the development of heart failure, may indicate a pathophysiologic role for increased oxidative stress in the pathogenesis of heart failure. This study suggests the potential therapeutic value of long-term antioxidant treatment in modulating or preventing the pathogenesis of heart failure.

Effects of probucol on changes of antioxidant enzymes in adriamycin-induced cardiomyopathy in rats.

OBJECTIVE: The clinical usefulness of doxorubicin (adriamycin, ADR) is restricted by the risk of developing congestive heart failure. Probucol has been reported to completely prevent ADR cardiomyopathy without interfering with its antitumor effects. The current study investigated the effects of ADR and probucol on antioxidant enzyme gene expression during adriamycin-induced cardiomyopathy in a rat model. METHODS: The mRNA abundance by Northern and immunoreactive protein levels by Western blotting of myocardial antioxidant enzymes, glutathione peroxidase (GSHPx), manganese superoxide dismutase (MnSOD) and catalase (CAT) were examined in relation to the enzyme activities in hemodynamically assessed control and treated animals. RESULTS: At 3 weeks post-treatment duration, ADR caused heart failure which was prevented by probucol. MnSOD mRNA abundance as well as protein levels were depressed by ADR treatment by 45% and 20%, respectively, and this change was prevented by probucol. However, the mRNA and protein levels of GSHPx and CAT were not significantly changed by ADR or probucol. ADR had no effect on SOD activity but this enzyme activity was increased by probucol and probucol plus ADR. GSHPx enzyme activity was decreased and oxidative stress as indicated by TBARS was increased by ADR and these changes were also modulated by probucol. CONCLUSION: An increase in oxidative stress, GSHPx inactivation and MnSOD downregulation during ADR cardiomyopathy were prevented by probucol treatment.

The role of oxidative stress in the genesis of heart disease.

Although researchers in radiation and cancer biology have known about the existence of free radicals and their potential role in pathobiology for several decades, cardiac biologists only began to take notice of these noxious species in the 1970s. Exponential growth of free radical research occurred after the discovery of superoxide dismutase in 1969. This antioxidant enzyme is responsible for the dismutation of superoxide radical--a free radical chain initiator. A fine balance between free radicals and a variety of endogenous antioxidants is believed to exist. Any disturbance in this equilibrium in favour of free radicals causes an increase in oxidative stress and initiates subcellular changes leading to cardiomyopathy and heart failure. Our knowledge about the role of free radicals in the pathogenesis of cardiac dysfunction is fast approaching the point where newer therapies employing antioxidants are in sight.

Antioxidant potentials of vitamin A and carotenoids and their relevance to heart disease.

Despite being one of the first vitamins to be discovered, the full range of biological activities for vitamin A remains to be defined. Structurally similar to vitamin A, carotenoids are a group of nearly 600 compounds. Only about 50 of these have provitamin A activity. Recent evidence has shown vitamin A, carotenoids and provitamin A carotenoids can be effective antioxidants for inhibiting the development of heart disease. Vitamin A must be obtained from the diet: green and yellow vegetables, dairy products, fruits and organ meats are some of the richest sources. Within the body, vitamin A can be found as retinol, retinal and retinoic acid. Because all of these forms are toxic at high concentrations, they are bound to proteins in the extracellular fluids and inside cells. Vitamin A is stored primarily as long chain fatty esters and as provitamin carotenoids in the liver, kidney and adipose tissue. The antioxidant activity of vitamin A and carotenoids is conferred by the hydrophobic chain of polyene units that can quench singlet oxygen , neutralize thiyl radicals and combine with and stabilize peroxyl radicals. In general, the longer the polyene chain, the greater the peroxyl radical stabilizing ability. Because of their structures, vitamin A and carotenoids can autoxidize when O2 tension increases, and thus are most effective antioxidants at low oxygen tensions that are typical of physiological levels found in tissues. Overall, the epidemiological evidence suggests that vitamin A and carotenoids are important dietary factors for reducing the incidence of heart disease. Although there is considerable discrepancy in the results from studies in humans regarding this relationship, carefully controlled experimental studies continue to indicate that these compounds are effective for mitigating and defending against many forms of cardiovascular disease. More work, especially concerning the relevance of how tissue concentrations, rather than plasma levels, relate to the progression of tissue damage in heart disease is required. This review assembles information regarding the basic structure and metabolism of vitamin A and carotenoids as related to their antioxidant activities. Epidemiological, intervention trials and experimental evidence about the effectiveness of vitamin A and carotenoids for reducing cardiovascular disease is also reviewed.

Metabolism of vitamin A in the heart increases after a myocardial infarction.

The supply of vitamin A to the myocardium by storage organs during increased oxidative stress subsequent to myocardial infarction (MI) was examined in hemodynamically assessed rats using compartment analysis of a radio-labeled vitamin A. 3H-Vitamin A was injected into two groups of rats: an MI group and a control group. There were no differences in the plasma or myocardial content of total vitamin A (unlabeled + labeled) between the two groups. However, the proportion of 3H-vitamin A was greater in the myocardium as well as plasma of MI rats. Rats with MI also had significantly lower 3H-vitamin A levels in liver and kidney than sham controls. The greatest difference in vitamin A content was in the concentrations of 3H-labeled storage forms of vitamin A in the liver of MI animals. Activity of bile salt-dependent retinyl ester hydrolase, an enzyme responsible for hydrolyzing vitamin A storage forms, was significantly increased in the liver of MI animals. These data indicate that analysis of plasma concentrations of vitamin A to ascertain links to cardiac conditions may be inappropriate. Specifically, during MI, increased amounts of vitamin A are mobilized from the liver to the heart without changing plasma concentrations. This is facilitated by an increase in the activity of an enzyme that hydrolyzes vitamin A storage forms.

Apoptosis in adriamycin cardiomyopathy and its modulation by probucol.

The dose-dependent cardiomyopathy and heart failure due to adriamycin have been shown to be due to increased oxidative stress and loss of myocytes. We examined the incidence of myocardial apoptosis as well as changes in the expression of apoptotic regulatory gene products in an established animal model of adriamycin cardiomyopathy. Rats were treated with adriamycin (cumulative dose, 15 mg/kg), and the hearts were examined for apoptosis as well as expression of Bax, caspase 3, and Bcl-2 at 0, 4, 10, 16, and 21 days after the treatment. A significant increase in the incidence of apoptosis was seen at 4 days, followed by a decline at 10 and 16 days of posttreatment. At 21 days, the number of apoptotic cells increased again and included cells of the conducting system. Expression of Bax corresponded to these biphasic changes, whereas the converse was true for the expression of Bcl-2. The latter peaked at 10 days followed by a decline at 16 and 21 days. The Bax/Bcl-2 ratio also correlated with the incidence of apoptosis. Expression of caspase 3 correlated with increased apoptosis, but only at early time points. Probucol (cumulative dose, 120 mg/kg), a known antioxidant as well as promoter of endogenous antioxidants, significantly reduced the incidence of apoptosis as well as expression of Bax. Adriamycin-induced hemodynamic changes were also prevented by probucol. These data suggest that adriamycin-induced apoptosis is mediated by oxidative stress and may play a role in the development of heart failure.

Susceptibility of LDL to oxidative stress in obstructive sleep apnea.

Cardiovascular diseases are more common in patients with obstructive sleep apnea (OSA) than in the general population. We hypothesized that severe hypoxemia during sleep in these patients may cause an imbalance between reactive oxygen species and the antioxidant reserve that is important for the detoxification of these molecules. We tested the hypothesis that low-density lipoproteins (LDL) in hypoxic OSA patients may be more susceptible to oxidative stress than LDL of nonhypoxic OSA patients and normal controls. Fifteen OSA patients were included in this study, six with severe hypoxia (hypoxic group) who spent more than 10 minutes during sleep with SaO2 < 85% (mean 96 minutes), and nine OSA patients (nonhypoxic group) who spent less than 10 minutes during sleep with SaO2 < 85% (mean 1.1 minutes). Six healthy nonsmoking males of the same age group were included as a control group. The susceptibility of each individual's LDL to oxidative stress was examined after free-radical challenge in vitro by assessing changes in levels of conjugated dienes. The LDL in OSA patients with severe hypoxia was not more susceptible to oxidative stress compared to the LDL of nonhypoxic OSA patients and normal controls. After 6 hours of exposure to an oxidative agent, the changes in the mean conjugated diene were not different among the three groups (p = 0.75). The time required to reach 50% of maximal absorbance was also not different, p = 0.199. Glutathione peroxidase and catalase activities in red blood cells in the hypoxic and nonhypoxic patient groups were not significantly different. One night of CPAP therapy in each patient group did not significantly change the level of the antioxidant enzymes. Our results did not show any difference in the susceptibility to oxidative stress between hypoxic and nonhypoxic OSA patients and normal controls.

Complete stunning in hypertrophied guinea pig heart. Defects in sarcolemmal calcium influx.

Isolated sham control as well as hypertrophied guinea pig hearts were subjected to global ischemia and reperfusion. Developed force declined to zero during 5 min of ischemia without any significant change in resting tension in both sham control and hypertrophied hearts. Upon reperfusion, control hearts showed nearly complete recovery of developed force within 20 min, whereas hypertrophied hearts during this time showed no contractile function, i.e., "a complete stunning" was observed. A continued reperfusion of the stunned hypertrophied hearts ultimately resulted in complete recovery of force within 40-60 min. Data on myocardial cation content showed a relative calcium deficiency in the stunned hearts (3.4 mumol/gm dry wt) as compared to sham control hearts (5.3 mumol/gm dry wt). Stunning could be reversed sooner by isoproterenol (100 microns), and low Na+ (35 and 60 mM) perfusion. Recovery of contractile function by low Na+ was blocked by amiloride (0.17-1.2 mM) in a dose-dependent manner. Perfusion with Bay K8644 (0.1-10 microM) as well as low (0.62 mM) and high (2.5 mM) extracellular calcium concentrations failed to reverse stunning. The pharmacological interventions that were able to reverse the stunning condition also increased the myocardial calcium content. Although the possibilities of a sarcoplasmic reticular dysfunction and/or reduced sensitivity of myofilaments are not excluded, data suggest that a defect in calcium influx across the sarcolemma is an important factor in "complete stunning." It is suggested that this "potential sarcolemmal defect" in the hypertrophied heart, which is unmasked by the ischemic stress, may also represent an early abnormality in the pathogenesis of heart failure.

Free radicals and the heart.

Because of the molecular configuration, most free radicals are highly reactive and can cause cell injury. Protective mechanisms have evolved to provide defense against free-radical injury. Any time these defense systems are overwhelmed, such as during disease states, cell dysfunction may occur. In this review we discuss cellular sources as well as the significance of free radicals, oxidative stress, and antioxidants. A probable role of oxidative stress in various cardiac pathologies has been also analyzed. Although some methods for the detection of free radicals as well as oxidative stress have been cited, better methods to study the quantity as well as subcellular distribution of free radicals are needed in order to understand fully the role of free radicals in both health and disease.

Myocardial oxidative stress and antioxidants in hypertension as a result of nitric oxide synthase inhibition.

Rats were made hypertensive by the administration of the nitric oxide synthase inhibitor nitro-L-arginine (LNA, 2.74 mmol/L) in drinking water for 7 d. Hearts from hemodynamically assessed animals were analyzed for lipid peroxidation (LPO), gamma-glutamylcysteine-synthetase (gamma-GCS), glutathione disulfide reductase (GR), glutathione peroxidase (GSHPx), catalase (CAT), superoxide dismutase (SOD), and total radical trapping potential (TRAP) activities. LNA treatment increased the mean arterial blood pressure by 46% and the heart rate by 22% without changing plasma renin activity. LNA treatment resulted in a 30% increase in LPO. gamma-GCS was reduced by 48% and GR by 36% in the cardiac tissue of hypertensive rats as compared to controls. The activity of nonselenium GSHPx was reduced by 27%, and selenium-dependent GSHPx activity in the heart was not affected by LNA treatment. In hypertensive rats, SOD activity was increased by 16%, and CAT was decreased by 46%. TRAP was lower (27%) in the myocardium of hypertensive rats than in that of controls. These data suggest that LNA-induced hypertension is associated with increased myocardial oxidative stress.

Metallothioneins, oxidative stress and the cardiovascular system.

Metallothioneins (MTs) discovered four decades ago as metal binding low molecular weight thiol proteins, in recent years, have generated immense interest due to their involvement in various physiological and pathophysiological events. In spite of the recent advances in the knowledge base in MTs, no specific study on the isolation or characterization of MTs isoforms and their precise function in the heart has been conducted. Although most stress conditions in the heart do not produce much change in myocardial MTs, TNF-alpha and IL-6 do induce MT in the heart to the extent comparable with the liver. Cardiotoxicity of anticancer drugs and vulnerability of the heart to other oxidative stress conditions may partially be due to lack of inducibility of MT in the heart by these interventions. A clear understanding of induction of MTs in the heart may help in the development of better approaches to modulate the pathogenesis of cardiomyopathies and heart failure.

Probucol improves antioxidant activity and modulates development of diabetic cardiomyopathy.

To examine the role of free radicals in diabetic cardiomyopathy, myocardial antioxidants as well as lipid peroxide content were examined in rats made diabetic with a single injection of streptozotocin (65 mg/kg i.v). At 4 wk, the left ventricular peak systolic (LVSP) as well as aortic pressures were depressed in the diabetic group. Hearts from diabetic animals showed about a 100% increase in thiobarbituric acid reactive substances (TBARS), indicating increased lipid peroxidation. This was accompanied by about a 50% decrease in superoxide dismutase (SOD) and 60% decrease in glutathione peroxidase (GSHPx) enzyme activities. Catalase activity in these hearts showed a small but significant increase. Treatment with probucol (10 mg/kg i.p., on alternate days), a known lipid-lowering drug with strong antioxidant properties, was initiated 1 d after the induction of diabetes and was continued for 4 wk. In probucol-treated diabetic animals, LVSP was not different from controls. Probucol treatment caused a small but significant improvement in serum insulin and decrease in glucose levels as well as increased myocardial SOD, GSHPx, and catalase activities with a concomitant decrease in TBARS in the diabetic animals. These data provide evidence that diabetic cardiomyopathy is associated with an antioxidant deficit, and a better cardiac function due to treatment with probucol may be related to the improved insulin levels as well as maintenance of the antioxidant status of the heart.

A relative deficit in antioxidant reserve may contribute in cardiac failure.

Antioxidant enzyme activities, including superoxide dismutase, glutathione peroxidase and catalase, are known to be altered under various physiological and pathophysiological conditions. There is a significant increase in some of these activities in the myocardium during stable hyperfunctional heart hypertrophy subsequent to pressure overload, as well as after exercise training in rats. Hearts with increased antioxidant capacity have been reported to be more resistant to in vivo and in vitro oxidative stress. On the other hand, cardiomyopathy and heart failure under a variety of conditions are accompanied by increased free radicals and lipid peroxidation. These data lead to the hypothesis that maintained or improved function during compensated heart hypertrophy may be supported by an increased antioxidant capacity, and a relative deficit in this 'antioxidant reserve' may contribute in the decompensated state.

Effects of increase in extracellular calcium on frequency-dependent inotropism in cardiac muscle.

Frequency-dependent effects on contractility in isolated rabbit papillary muscles were compared at different calcium concentrations ranging between 1 and 12.5 mM. Increase in extracellular calcium up to 3 mM resulted in an increase in the frequency-dependent positive inotropy. A further increase in extracellular calcium, up to 5 mM, brought about a quantitative decrease in the frequency-dependent positive inotropy. Still higher concentrations of extracellular calcium, 7.5 and 12.5 mM, altogether abolished the frequency-dependent increase in contractility. The latter at these concentrations of calcium was rather depressed in response to frequency increases. These characteristic changes in the frequency-dependent inotropism at different extracellular calcium concentrations may have been due to the occurrence of a moderate to severe intracellular calcium overload. It is suggested that heart function in certain cardiac diseases, associated with intracellular calcium overload, may not improve with an increase in heart rate.