Sonja Zafirovic

Regulation of Endothelial Nitric Oxide Synthase and High-Density Lipoprotein Quality by Estradiol in Cardiovascular Pathology

Estrogens have been recognized, in the last 3 decades, as important hormones in direct and indirect modulation of vascular health. In addition to their direct benefit on cardiovascular health, the presence of esterified estrogen in the lipid core of high-density lipoprotein (HDL) particles indirectly contributes to atheroprotection by significantly improving HDL quality and functionality. Estrogens modulate their physiological activity via genomic and nongenomic mechanisms. Genomic mechanisms are thought to be mediated directly by interaction of the hormone receptor complex with the hormone response elements that regulate gene expression. Nongenomic mechanisms are thought to occur via interaction of the estrogen with membrane-bound receptors, which rapidly activate intracellular signaling without binding of the hormone receptor complex to its hormone response elements. Estradiol in particular mediates early and late endothelial nitric oxide synthase (eNOS) activation via interaction with estrogen receptors through both nongenomic and genomic mechanisms. In the vascular system, the primary endogenous source of nitric oxide (NO) generation is eNOS. Nitric oxide primarily influences blood vessel relaxation, the heart rate, and myocyte contractility. The abnormalities in expression and/or functions of eNOS lead to the development of cardiovascular diseases, both in animals and in humans. Although considerable research efforts have been dedicated to understanding the mechanisms of action of estradiol in regulating cardiac eNOS, more research is needed to fully understand the details of such mechanisms. This review focuses on recent findings from animal and human studies on the regulation of eNOS and HDL quality by estradiol in cardiovascular pathology.

In vivo effects of 17β-estradiol on cardiac Na(+)/K(+)-ATPase expression and activity in rat heart.

In this study the in vivo effects of estradiol in regulating Na(+)/K(+)-ATPase function in rat heart was studied. Adult male Wistar rats were treated with estradiol (40μg/kg, i.p.) and after 24h the animals were sacrificed and the heart excised. Following estradiol administration, cardiac Na(+)/K(+)-ATPase activity, expression of the α1 subunit, and phosphorylation of the α1 subunit were significantly increased. These animals also had significantly decreased levels of digoxin-like immunoreactive factor(s). Na(+) levels were also significantly reduced but to a level that was still within the normal physiological range, highlighting the ability of the Na(+)/K(+)-ATPase to balance the ionic composition following treatment with estradiol. Estradiol treated rats also showed increased phosphorylation of protein kinase B (Akt), and extracellular-signal-regulated kinase 1/2 (ERK1/2). We therefore suggest a role for Akt and/or ERK1/2 in estradiol-mediated regulation of cardiac Na(+)/K(+)-ATPase expression and activity in rat heart.

Estradiol In Vivo Induces Changes in Cardiomyocytes Size in Obese Rats

We studied the in vivo effects of estradiol on size and biochemical parameters of cardiomyocytes in pathophysiological conditions such as obesity and insulin resistance. Male Wistar rats were normally fed (controls, n = 7) or fed with high-fat diet (obese, n = 14). Half of the obese rats (obese + estradiol, n = 7) were treated with a single dose of estradiol (40 μg/kg, intraperitoneally) and 24 hours after treatment all the rats were killed. Estradiol in vivo in obese rats resulted in a significant increase in protein kinase B (Akt) activation (P < .05) and decrease in heart mass (P < .05), ratio of the heart mass/body mass (P < .05), transverse diameters of cardiomyocytes (P < .001), concentration of serum high-sensitivity C-reactive protein (P < .001), and total cholesterol (P < .01) compared with obese nontreated rats. Our results suggest that estradiol in obese/IR rats affects the size of cardiomyocytes and its actions lead in vivo to a reduction in obesity-induced cardiac hypertrophy, via Akt.

A high fat diet induces sex-specific differences in hepatic lipid metabolism and nitrite/nitrate in rats

Men and women differ substantially with regard to the severity of insulin resistance (IR) but the underlying mechanism(s) of how this occurs is poorly characterized. We investigated whether a high fat (HF) diet resulted in sex-specific differences in nitrite/nitrate production and lipid metabolism and whether these variances may contribute to altered obesity-induced IR. Male and female Wistar rats were fed a standard laboratory diet or a HF diet for 10 weeks. The level of plasma nitrite/nitrate, as well as free fatty acid (FFA), in both plasma and liver lysates were assessed. The levels of inducible nitric oxide (NO) synthase (iNOS), p65 subunit of NFκB, total and phosphorylated forms of Akt, mTOR and PDK-1 in lysates, and the levels of glucose transporter 2 (Glut-2) and fatty acid translocase/cluster of differentiation 36 (FAT/CD36) in plasma membrane fractions of liver were assessed. HF-fed male rats exhibited a significant increase in plasma nitrite/nitrate, and hepatic FFA and FAT/CD36 levels compared with controls. They also displayed a relative decrease in iNOS and Glut-2 levels in the liver. Phosphorylation of Akt (at Ser(473) and Thr(308)), mTOR and PDK-1 was also reduced. HF-fed female rats exhibited increased levels of NFκB-p65 in liver compared with controls, while levels of Glut-2, FAT/CD36 and Akt phosphorylation at Thr(308) and PDK-1 were decreased. Our results reveal that altered lipid and glucose metabolism in obesity, lead to altered iNOS expression and nitrite/nitrate production. It is likely that this mechanism contributes to sex-specific differences in the development of IR.

Apoptosis and Acute Brain Ischemia in Ischemic Stroke

Apoptosis may contribute to a significant proportion of neuron death following acute brain ischemia (ABI), but the underlying mechanisms are still not fully understood. Brain ischemia may lead to stroke, which is one of the main causes of long-term morbidity and mortality in both developed and developing countries. Therefore, stroke prevention and treatment is clinically important. There are two important separate areas of the brain during ABI: the ischemic core and the ischemic penumbra. The ischemic core of the brain experiences a sudden reduction of blood flow, just minutes after ischemic attack with irreversible injury and subsequent cell death. On the other hand, apoptosis within the ischemic penumbra may occur after several hours or days, while necrosis starts in the first hours after the onset of ABI in the ischemic core. ABI is characterized by key molecular events that initiate apoptosis in many cells, such as overproduction of free radicals, Ca2+ overload and excitotoxicity. These changes in cellular homeostasis may trigger either necrosis or apoptosis, which often depends on cell type, cell age, and location in the brain. Apoptosis results in DNA fragmentation, degradation of cytoskeletal and nuclear proteins, cross-linking of proteins, formation of apoptotic bodies, expression of ligands for phagocytic cell receptors and finally uptake by phagocytic cells. This review focuses on recent findings based on animal and human studies regarding the apoptotic mechanisms of neuronal death following ABI and the development of potential neuroprotective agents that reduce morbidity. The effects of statins on stroke prevention and treatment as well as on apoptotic mediators are also considered.

Effects of 17β-estradiol on cardiac Na(+)/K(+)-ATPase in high fat diet fed rats.

The aim of this study was to investigate in vivo effects of estradiol on Na(+)/K(+)-ATPase activity/expression in high fat (HF) diet fed rats. Adult male Wistar rats were fed normally (Control, n = 7) or with a HF diet (Obese, n = 14) for 10 weeks. After 10 weeks, half of the obese rats were treated with estradiol (Obese + Estradiol, n = 7, 40 μg/kg, i.p.) as a bolus injection and 24 h after treatment all the rats were sacrificed. Estradiol in vivo in obese rats in comparison with obese non-treated rats led to a statistically significant increase in concentration of serum Na(+) (p < 0.05), Na(+)/K(+)-ATPase activity (p < 0.01), expression of α1 (p < 0.01) and α2 (p < 0.05) subunit of Na(+)/K(+)-ATPase, both PI3K subunits p85 (p < 0.01), p110 (p < 0.05), and association of IRS-1 with p85 (p < 0.05), while significantly decrease expression of AT1 (p < 0.05) and Rho A (p < 0.01) proteins. Our results suggest that estradiol in vivo in pathophysiological conditions, such as obesity accompanied with insulin resistance stimulates activity and expression of Na(+)/K(+)-ATPase by a mechanism that involves the participation of IRS-1/PI3K/Akt signaling. In addition, the decreasing level of AT1 and Rho A proteins estradiol probably attenuates the detrimental effect of obesity to decreased IRS-1/PI3K association and consequently reduce Na(+)/K(+)-ATPase activity/expression.

Benefits of L-Arginine on Cardiovascular System

The amino acid, L-Arginine (L-Arg) plays an important role in the cardiovascular system. Data from the literature show that L-Arg is the only substrate for the production of nitric oxide (NO), from which L-Arg develops its effects on the cardiovascular system. As a free radical, NO is synthesized in all mammalian cells by L-Arg with the activity of NO synthase (NOS). In states of hypertension, diabetes, hypercholesterolemia and vascular inflammation a disorder occurs in the metabolic pathway of the synthesis of NO from L-Arg which all together bring alterations of blood vessels. Experimental results obtained on animals, as well as clinical studies show that L-Arg has an effect on thrombocytes, on the process of coagulation and on the fibrolytic system. This mini review represents a summary of the latest scientific animal and human studies related to L-Arg and its mechanisms of actions with a focus on the role of L-Arg via NO pathway in cardiovascular disorders. Moreover, here we present data from recent animal and clinical studies suggesting that L-Arg could be one of the possible therapeutic molecules for improving the treatment of different cardiovascular disorders.

Influence of a High-Fat Diet on Cardiac iNOS in Female Rats

BACKGROUND Overexpression of inducible nitric oxide synthase (iNOS) is a key link between high-fat (HF) diet induced obesity and cardiovascular disease. Oestradiol has cardioprotective effects that may be mediated through reduction of iNOS activity/expression. METHODS In the present study, female Wistar rats were fed a standard diet or a HF diet (42% fat) for 10 weeks. iNOS gene and protein expressions were measured in heart tissue. HF-fed rats exhibited a significant increase in cardiac iNOS mRNA by 695% (p<.05), iNOS protein level by 248% (p<0.01), without changes in nitrate/nitrite levels. Expression of CD36 protein in plasma membranes was increased by 37% (p<0.05), while the concentration of free fatty acids (FFA) was reduced by 25% (p<0.01) in HF-fed rats. Expression of the p50 subunit of nuclear factor-kB (NFkB-p50) in heart was increased by 77% (p<0.01) in HF-fed rats. Expression of protein kinase B (Akt) and extracellular signalregulated kinases 1/2 (ERK1/2) were unchanged between the groups. There was a significant increase in the ratio of phospho-Akt/total Akt but not for phospho-ERK1/2/total ERK1/2 in HF-fed rats. Estrogen receptor-α levels (by 50%; p<0.05) and serum oestradiol concentrations (by 35%; p<0.05) were shown to be significantly reduced in HF-fed rats. RESULTS AND CONCLUSION Our results revealed that a HF diet led to increased iNOS expression, most likely via a mechanism involving Akt and NFκB-p50 proteins. Decreased levels of oestradiol and ERα protein in the HF-fed group, in combination with increased iNOS levels are consistent with the hypothesis that oestradiol has a cardioprotective effect through its ability to regulate iNOS expression.

Effects of Levothyroxine Replacement Therapy on Parameters of Metabolic Syndrome and Atherosclerosis in Hypothyroid Patients: A Prospective Pilot Study

The aim of this study was to investigate the effect of levothyroxine (LT4) replacement therapy during three months on some parameters of metabolic syndrome and atherosclerosis in patients with increased thyroid-stimulating hormone (TSH) level. This study included a group of 30 female patients with TSH level >4 mIU/L and 15 matched healthy controls. Intima media complex thickness (IMCT) and peak systolic flow velocity (PSFV) of superficial femoral artery were determined by Color Doppler scan. In hypothyroid subjects, BMI, SBP, DBP, and TSH were significantly increased versus controls and decreased after LT4 administration. FT4 was significantly lower in hypothyroid subjects compared with controls and significantly higher by treatment. TC, Tg, HDL-C, and LDL-C were similar to controls at baseline but TC and LDL-C were significantly decreased by LH4 treatment. IMCT was significantly increased versus controls at baseline and significantly reduced by treatment. PSFV was similar to controls at baseline and significantly decreased on treatment. In this study, we have demonstrated the effects of LT4 replacement therapy during three months of treatment on correction of risk factors of metabolic syndrome and atherosclerosis.

Effects of altered hepatic lipid metabolism on regulation of hepatic iNOS

Abstract An altered hepatic lipid metabolism involves multifactorial pathologies such as hepatic inflammation, insulin resistance and oxidative stress. Immunity has an essential role in the regulation of glucose and lipid metabolism in the liver. Inducible nitric oxide (NO) synthase (iNOS) has been proposed as an important factor that interplays between immunity and energy metabolism and also in the pathogenesis of obesity-linked insulin resistance. In the liver, locally produced NO plays a protective role during inflammation, and the balance of NO protective and cytotoxic effects is very important. This review is focused on understanding the molecular mechanisms of iNOS regulation in the state of altered hepatic lipid metabolism, which is critical for developing new strategies for treatment of hepatic disorders.