The role of Nrf2 and PPARgamma in the improvement of oxidative stress in hypertension and cardiovascular diseases.

Reactive oxygen species are an important element of redox regulation in cells and tissues. During physiological processes, molecules undergo chemical changes caused by reduction and oxidation reactions. Free radicals are involved in interactions with other molecules, leading to oxidative stress. Oxidative stress works two ways depending on the levels of oxidizing agents and products. Excessive action of oxidizing agents damages biomolecules, while a moderate physiological level of oxidative stress (oxidative eustress) is necessary to control life processes through redox signaling required for normal cellular operation. High levels of reactive oxygen species (ROS) mediate pathological changes. Oxidative stress helps to regulate cellular phenotypes in physiological and pathological conditions. Nrf2 (nuclear factor erythroid 2-related factor 2, NFE2L2) transcription factor functions as a target nuclear receptor against oxidative stress and is a key factor in redox regulation in hypertension and cardiovascular disease. Nrf2 mediates transcriptional regulation of a variety of target genes. The Keap1-Nrf2-ARE system regulates many detoxification and antioxidant enzymes in cells after the exposure to reactive oxygen species and electrophiles. Activation of Nrf2/ARE signaling is differentially regulated during acute and chronic stress. Keap1 normally maintains Nrf2 in the cytosol and stimulates its degradation through ubiquitination. During acute oxidative stress, oxidized molecules modify the interaction of Nrf2 and Keap1, when Nrf2 is released from the cytoplasm into the nucleus where it binds to the antioxidant response element (ARE). This triggers the expression of antioxidant and detoxification genes. The consequence of long-term chronic oxidative stress is activation of glycogen synthase kinase 3beta (GSK-3beta) inhibiting Nrf2 activity and function. PPARgamma (peroxisome proliferator-activated receptor gamma) is a nuclear receptor playing an important role in the management of cardiovascular diseases, hypertension and metabolic syndrome. PPARgamma targeting of genes with peroxisome proliferator response element (PPRE) has led to the identification of several genes involved in lipid metabolism or oxidative stress. PPARgamma stimulation is triggered by endogenous and exogenous ligands - agonists and it is involved in the activation of several cellular signaling pathways involved in oxidative stress response, such as the PI3K/Akt/NOS pathway. Nrf2 and PPARgamma are linked together with their several activators and Nrf2/ARE and PPARgamma/PPRE pathways can control several types of diseases.

Chronic NOS Inhibition Affects Oxidative State and Antioxidant Response Differently in the Kidneys of Young Normotensive and Hypertensive Rats.

Deficiency of nitric oxide (NO) and oxidative stress can be a cause, a consequence, or, more often, a potentiating factor for hypertension and hypertensive renal disease. Both NO and superoxide anions are radical molecules that interact with each other, leading to oxidative damage of such organs as the kidney. In the present study, we investigated the effect of chronic-specific (neuronal NOS inhibition) and nonspecific NOS inhibition on the oxidative state and antioxidant response and associated oxidative damage of the kidney of young normotensive and hypertensive rats. Young male normotensive Wistar rats (WRs, age 4 weeks) and spontaneously hypertensive rats (SHRs, age 4 weeks) were divided into three groups for each strain by the type of administered compounds. The first group was treated with 7-nitroindazole (WR+7-NI; SHR+7-NI), the second group was treated with N(G)-nitro-L-arginine-methyl ester (WR+L-NAME; SHR+L-NAME), and the control group was treated with pure drinking water (WR; SHR) continuously for up to 6 weeks. Systolic blood pressure increased in WR+L-NAME after the first week of administration and increased slightly in SHR+L-NAME in the third week of treatment. 7-NI had no effect on blood pressure. While total NOS activity was not affected by chronic NOS inhibition in any of the WR groups, it was attenuated in SHR+7-NI and SHR+L-NAME. Nitration of proteins (3-nitrotyrosine expression) was significantly reduced in WR+7NI but not in WR+L-NAME and increased in SHR+7-NI and SHR+L-NAME. Immunoblotting analysis of SOD isoforms showed decreased SOD2 and SOD3 expressions in both WR+7-NI and WR+L-NAME followed by increased SOD activity in WR+L-NAME. Conversely, increased expression of SOD2 and SOD3 was observed in SHR+L-NAME and SHR+7-NI, respectively. SOD1 expression and total activity of SOD did not change in the SHR groups. Our results show that the antioxidant defense system plays an important role in maintaining the oxidative state during NO deficiency. While the functioning antioxidant system seeks to balance the oxidation state in the renal cortex of normotensive WRs, the impaired antioxidant activity leads to the development of oxidative damage of proteins in the kidney induced by peroxynitrite in SHRs.

The role of perivascular adipose tissue and endogenous hydrogen sulfide in vasoactive responses of isolated mesenteric arteries in normotensive and spontaneously hypertensive rats.

Perivascular adipose tissue (PVAT) and hydrogen sulfide (H2S) play important roles in the modulation of vasoactive responses and can interfere with the ethiopathogenesis of essential hypertension. The aim of this study was to evaluate the mutual relationship between PVAT and H2S (endogenously produced, exogenous) in vasoactive responses of isolated mesenteric arteries (MA) in adult normotensive (Wistar) and spontaneously hypertensive rats (SHR). In SHR, hypertension was associated with cardiac hypertrophy and increased contractility; however, there were no differences in the amount of retroperitoneal fat between strains. PVAT revealed the anti-contractile effect on vasoconstriction induced by exogenous noradrenaline in both strains, but surprisingly, this effect was stronger in SHR. Concurrently; PVAT exhibited a pro-contractile effect on contractions to endogenous noradrenaline released from arterial sympathetic nerves in SHR, but not in Wistar rats. We confirmed the anti-contractile effect of H2S in both, the vascular wall and PVAT of Wistar rats because the pre-treatment with propargylglycine (PPG), an inhibitor of H2S producing enzyme, significantly increased the noradrenaline-induced contraction. In SHR, H2S in the vascular wall exhibited a pro-contractile effect that was eliminated by the presence of PVAT; however, the pre-treatment with PPG did not affect noradrenaline contraction farther. Nevertheless, unlike in Wistar rats, the presence of PVAT potentiated the vasorelaxant effect of exogenously applied H2S in SHR. Our results confirmed that PVAT of MA and endogenously produced H2S could manifest as pro-contractile or as anti-contractile. In SHR, unlike in Wistar rats, the pro-contractile effect of PVAT associated with the stimulation of perivascular nerves, and the pro-contractile effect of H2S in the arterial wall could represent pathologic features. On the other hand, PVAT of SHR is endowed with compensatory vasoactive mechanisms, which include stronger anti-contractile action of an unknown factor (other than H2S) and potentiation of the vasorelaxant effect of exogenous H2S.

The peroxisome proliferator-activated receptor gamma agonist pioglitazone improves nitric oxide availability, renin-angiotensin system and aberrant redox regulation in the kidney of pre-hypertensive rats.

The peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent nuclear receptor. It plays an important role in kidney physiology, where it might contribute to arterial blood pressure regulation and hypertension development by modulation of several signaling pathways. In our study we focused on the effect of PPARγ agonist pioglitazone on changes in the nitric oxide synthase (NOS) expression and activity, the renin-angiotensin system (RAS) cascade, and redox homeostasis signaling pathways in the renal cortex of young pre hypertensive rat models. Young (5-weeks old) spontaneously hypertensive (SHR) and borderline hypertensive (BHR) rats were treated by pioglitazone (PIO, 10 mg/kg/day) during 10 days. Blood pressure (BP) was determined by plethysmography method. Changes in lipid profile were detected in plasma with standard kits using biochemical analyser. Gene expression has been detected by qRT-PCR and protein level was determined using Western blot analysis. Superoxide dismutase (SOD) and catalase (CAT) activities were determined spectrophotometrically and the total enzyme activity of NOS was measured using a radioactive assay based on conversion of [3H] L-arginine to [3H] L- citrulline. Administration of pioglitazone decreased BP in BHR and slowed down the development of BP increase in young SHR animals. For NOS, activation by PPARγ correlated with increase in gene and protein expression of NOS isoforms and in total enzyme activity both in BHR and SHR. In the AT1R/Nox pathway, the treatment did not significantly influence mRNA expression of the p22phox subunit of NADPH oxidase (Nox) and AT1R, but up-regulated the 'pro-vasodilatatory' Mas and AT2R receptors in both BHR and SHR groups. Pioglitazone treatment affected redox regulation. Increase in gene expression of nuclear factor E2-related factor 2 (Nrf2) and SOD isoforms correlated with SOD and CAT enzyme activities. The group treatment-to-control ratios, BHR Pioglitazone to BHR control and SHR Pioglitazone to SHR control for gene expression increased by 10% to 230%. The largest effect of PPARγ has been observed in SOD1, SOD3 and the Mas receptor gene treatment-to-control ratios. The most prominent differences between BHR and SHR were observed in SOD1 and Mas receptor expressions, with large effects of opposite sign in BHR versus SHR. Our data indicate that an increase of NO release activates signaling in the renal cortex of pre-hypertensive rats after pioglitazone treatment. Improvement of NO availability, AT2R, Mas receptors and aberrant redox regulation is thought to be the major correlated mechanisms mediating the BP decrease affected by the PPARγ agonist treatment. We also observed that the most sensitive tissue responses to PPARγ-dependent activation of Nrf2 have been primarily found in the kidney of young hypertensive animals.

The role of PPARgamma in cardiovascular diseases.

The peroxisome proliferator-activated receptors (PPAR) belong to the nuclear superfamily of ligand-activated transcription factors. PPARgamma acts as a nutrient sensor that regulates several homeostatic functions. Its disruption can lead to vascular pathologies, disorders of fatty acid/lipid metabolism and insulin resistance. PPARgamma can modulate several signaling pathways connected with blood pressure regulation. Firstly, it affects the insulin signaling pathway and endothelial dysfunction by modulation of expression and/or phosphorylation of signaling molecules through the PI3K/Akt/eNOS or MAPK/ET-1 pathways. Secondly, it can modulate gene expression of the renin- angiotensin system - cascade proteins, which potentially slow down the progression of atherosclerosis and hypertension. Thirdly, it can modulate oxidative stress response either directly through PPAR or indirectly through Nrf2 activation. In this context, activation and functioning of PPARgamma is very important in the regulation of several disorders such as diabetes mellitus, hypertension and/or metabolic syndrome.