[Oxidative stress in hypertension: from the SNPs to the clinical phenotype].
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Animals↗
Biomedical subjects
G Zalba
Publications and source records attributed to G Zalba.
[Superoxide anion in the physiopathology of vascular diseases].
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Animals↗
Oxidative stress in arterial hypertension: role of NAD(P)H oxidase.
Increased vascular reactive oxygen species production, especially superoxide anion, contributes significantly in the functional and structural alterations present in hypertension. An enhanced superoxide production causes a diminished NO bioavailability by an oxidative reaction that inactivates NO. Exaggerated superoxide levels and a low NO bioavailability lead to endothelial dysfunction and hypertrophy of vascular cells. It has been shown that the enzyme NAD(P)H oxidase plays a major role as the most important source of superoxide anion in vascular cells. Several experimental observations have shown an enhanced superoxide generation as a result of the activation of vascular NAD(P)H oxidase in hypertension. Although this enzyme responds to stimuli such as vasoactive factors, growth factors, and cytokines, some recent data suggest the existence of a genetic background modulating the expression of its different components. New polymorphisms have been identified in the promoter of the p22(phox) gene, an essential subunit of NAD(P)H oxidase, influencing the activity of this enzyme. Genetic investigations of these polymorphisms will provide novel markers for determination of genetic susceptibility to oxidative stress in hypertension.
Angiotensin II↗
Cardiomyocyte apoptotic cell death in arterial hypertension: mechanisms and potential management.
Hypertensive heart disease is a progressive condition in which the compensatory left ventricular hypertrophy that maintains cardiac output leads to myocardial remodeling, characterized by fibrosis, insufficient vascularization, and alterations in cardiomyocytes, including contractile disturbances, changes in gene expression, and decrease in the number of cells. Structural abnormalities in the myocardial wall accelerate the development of diastolic and systolic dysfunction, resulting in heart failure. Many observations point to the apoptotic cell death of cardiomyocytes as a relevant factor in the transition from compensatory hypertrophy to pump failure in experimental and human hypertension. Potential inducers of cardiomyocyte apoptosis in overloaded hearts include extrinsic factors, such as mechanical forces, neurohormonal activation, oxidative stress, hypoxia, and cytokines. Some lines of evidence indicate that angiotensin II and the overstretching of cardiomyocytes are originally involved in the triggering of apoptosis in hypertension, whereas other factors are being investigated. Furthermore, intracellular changes, such as downregulation of survival proteins or activation of death proteins, seem to play an important role. The assumption that the apoptosis of cardiomyocytes worsens hypertensive heart disease prognosis brings forth new approaches to avoid or slow the transition to pump failure. In this respect, experimental data indicate that currently used antihypertensive drugs interfere with cardiomyocyte apoptosis. Moreover, the knowledge of intracellular apoptotic processes in cardiomyocytes provides novel therapeutic strategies to be added to the multimodal approach in the prevention of heart failure.
Angiotensin II↗
Polymorphisms and promoter overactivity of the p22(phox) gene in vascular smooth muscle cells from spontaneously hypertensive rats.
In a previous study, we found that the p22(phox) subunit of the NADH/NADPH oxidase is overexpressed in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) with enhanced vascular production of superoxide anion ((.)O(2)(-)). Thus, we have investigated whether changes in the sequence or activity of the promoter region of p22(phox) gene are present in SHRs. To carry out this analysis, first of all, we characterized the rat gene structure and promoter region for the p22(phox) subunit. The p22(phox) gene spans approximately 10 kb and contains 6 exons and 5 introns. Primer extension analysis indicated the transcriptional start site 100 bp upstream from the translational start site. The immediate promoter region of the p22(phox) gene does not contain a TATA box, but there are a CCAC box and putative recognition sites for nuclear factors, such as SP1, gamma-interferon, and nuclear factor-kappaB. Using reporter-gene transfection analysis, we found that this promoter was functional in VSMCs. Furthermore, we observed that p22(phox) promoter activity was significantly higher in VSMCs from SHRs than from normotensive Wistar-Kyoto rats. In addition, we found that there were 5 polymorphisms in the sequence of p22(phox) promoter between Wistar-Kyoto rats and SHRs and that they were functional. The results obtained in this study provide a tool to explore the mechanisms that regulate the expression of p22(phox) gene in rat VSMCs. Furthermore, our findings show that changes in the sequence of p22(phox) gene promoter and in the degree of activation of VSMCs are responsible for upregulated expression of p22(phox) in SHRs.
5' Untranslated Regions↗
Effects of loop diuretics on angiotensin II-stimulated vascular smooth muscle cell growth.
BACKGROUND: Torasemide and furosemide are diuretics that inhibit the Na(+), K(+), 2Cl(-) co-transporter localized in cells from the ascending limb of the loop of Henle. The effects of torasemide and furosemide on cell growth induced by angiotensin II (Ang II) were investigated in cultured vascular smooth muscle cells (VSMCs) obtained from the aorta of adult spontaneously hypertensive rats (SHR). METHODS: Cell growth was determined by DNA and protein synthesis as measured by [3H]thymidine and [3H]leucine incorporation, respectively. Proliferation of VSMCs was measured using a non-radioactive colorimetric cell proliferation assay. RESULTS: Ang II (10(-7) M) signficantly increased DNA and protein synthesis and cell proliferation in VSMCS: These effects were completely abolished by the Ang II type 1 receptor antagonist irbesartan (10(-6) M). Ang II-induced [3H]leucine incorporation was reduced in a dose-dependent way by torasemide (IC(50) value: 7.7+/-0.8x10(-7) M) but not by furosemide. Neither torasemide nor furosemide modified Ang II-stimulated [3H]thymidine incorporation or proliferation in VSMCs. CONCLUSIONS: These results indicate that torasemide, but not furosemide, inhibits Ang II-induced protein synthesis in VSMCs from SHR. Thus, it is suggested that the capacity of torasemide to block this trophic action of Ang II in rat VSMCs is not mediated by inhibition of the Na(+), K(+), 2Cl(-) co-transport mechanism.
Angiotensin II↗
The loop diuretic torasemide interferes with endothelin-1 actions in the aorta of hypertensive rats.
BACKGROUND: The direct effects of torasemide and furosemide on vasoconstriction and increases in intracellular free calcium concentration ([Ca(2+)]i) induced by endothelin-1 (ET-1) were investigated in the aorta of spontaneously hypertensive rats (SHR). METHODS: Vascular responses were assessed in endothelium-denuded aortic rings using an organ bath system. Changes of [Ca(2+)]i in cultured vascular smooth muscle cells (VSMCs) were assessed using fura-2 methodology. RESULTS: ET-1-induced vasoconstriction was reduced in a dose-dependent way by torasemide and furosemide (IC(50) values: 4.3+/-1.4x10(-5) and 9.8+/-5.6 x10(-5) M, respectively). The ET-1-induced biphasic [Ca(2+)]i increase was blocked by torasemide (IC(50)=2.0+/-0.2x10(-8) and 2.7+/-0.6x10(-6) M, respectively). Furosemide inhibited the initial rise in [Ca(2+)]i induced by ET-1, with no effect on the second rise. The specific chloride (Cl(-)) channel blocker diphenylamine-2-carboxylate inhibits both ET-1-induced responses in VSMCs (IC(50)=8.0+/-0.3x10(-9) and 2.5+/-0.7x10(-7) M, respectively). CONCLUSIONS: These results suggest that the ability of loop diuretics to interfere with the vascular actions of ET-1 may involve different molecular mechanisms. The ability of torasemide to block the vasoconstrictive action of ET-1 could include an inhibitory action on Cl(-) channels.
Animals↗
Is the balance between nitric oxide and superoxide altered in spontaneously hypertensive rats with endothelial dysfunction?
BACKGROUND: Increases in oxidant stress, i.e. excessive production of superoxide anion (O2(.-)), have been reported in different models of hypertension. This study was designed to test the hypothesis that increased O2(.-) production, more than diminished nitric oxide (NO) generation, plays a critical role in endothelial dysfunction present in spontaneously hypertensive rats (SHR). METHODS: The study was performed in 30-week-old normotensive Wistar-Kyoto rats (WKY) and SHR. In addition, 16-week-old SHR were treated with oral irbesartan (average dose 20 mg/kg per day) for 14 weeks (SHR-I). Aortic nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase activity was determined by use of chemiluminescence with lucigenin. Aortic constitutive nitric oxide synthase (cNOS) activity was determined by measuring the conversion of L-arginine to L-citrulline. Vascular responses to acetylcholine were determined by isometric tension studies. RESULTS: Whereas systolic blood pressure (SBP) was significantly increased in SHR compared with WKY, no differences were observed in SBP between SHR-I and WKY. In SHR compared with WKY, we found significantly greater NADH/NADPH-driven O2(.-) production, similar cNOS-mediated NO production and an impaired vasodilation in response to acetylcholine. Treated SHR had similar NADH/NADPH oxidase activity and significantly lower cNOS activity than the WKY group. Vasodilation in response to acetylcholine was improved in SHR-I. CONCLUSIONS: These findings suggest that a diminished availability of NO secondary to an enhanced NADH/NADPH oxidase-dependent O2(.-) production may play a critical role in endothelial dysfunction of adult SHR.
Acetylcholine↗
[Vascular oxidative stress and endothelial dysfunction].
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Endothelium, Vascular↗
Vascular oxidant stress: molecular mechanisms and pathophysiological implications.
The term oxidative stress refers to a situation in which cells are exposed to excessive levels of either molecular oxygen or chemical derivatives of oxygen (ie, reactive oxygen species). Three enzyme systems produce reactive oxygen species in the vascular wall: NADH/NADPH oxidase, xanthine oxidoreductase, and endothelial nitric oxide synthase. Among vascular reactive oxygen species superoxide anion plays a critical role in vascular biology because it is the source for many other reactive oxygen species and various vascular cell functions. It is currently thought that increases in oxidant stress, namely excessive production of superoxide anion, are involved in the pathophysiology of endothelial dysfunction that accompanies a number of cardiovascular risk factors including hypercholesterolemia, hypertension and cigarette smoking. On the other hand, vascular oxidant stress plays a pivotal role in the evolution of clinical conditions such as atherosclerosis, diabetes and heart failure.
Animals↗
Vascular NADH/NADPH oxidase is involved in enhanced superoxide production in spontaneously hypertensive rats.
This study was designed to test the hypothesis that stimulation of nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase is involved in increased vascular superoxide anion (*O(2)(-)) production in spontaneously hypertensive rats (SHR). The study was performed in 16-week-old and 30-week-old normotensive Wistar-Kyoto rats (WKY(16) and WKY(30), respectively) and in 16-week-old and 30-week-old SHR (SHR(16) and SHR(30), respectively). In addition, 16-week-old SHR were treated with oral irbesartan (average dose 20 mg/kg per day) for 14 weeks (SHR(30)-I). Aortic NADH/NADPH oxidase activity was determined by use of chemiluminescence with lucigenin. The expression of p22phox messenger RNA was assessed by competitive reverse transcription-polymerase chain reaction. Vascular responses to acetylcholine were determined by isometric tension studies. Aortic wall structure was studied, determining the media thickness and the cross-sectional area by morphometric analysis. Whereas systolic blood pressure was significantly increased in the 2 groups of hypertensive animals compared with their normotensive controls, no differences were observed in systolic blood pressure between SHR(30) and SHR(16). No other differences in the parameters measured were found between WKY(16) and SHR(16). In SHR(30) compared with WKY(30), we found significantly greater p22phox mRNA level, NADH/NADPH-driven *O(2)(-) production, media thickness, and cross-sectional area and an impaired vasodilation in response to acetylcholine. Treated SHR had similar NADH/NADPH oxidase activity and p22phox expression as the WKY(30) group. The vascular functional and morphological parameters were improved in SHR(30)-I. These findings suggest that an association exists between p22phox gene overexpression and NADH/NADPH overactivity in the aortas of adult SHR. Enhanced NADH/NADPH oxidase-dependent *O(2)(-) production may contribute to endothelial dysfunction and vascular hypertrophy in this genetic model of hypertension.
Animals↗
Mechanisms of increased susceptibility to angiotensin II-induced apoptosis in ventricular cardiomyocytes of spontaneously hypertensive rats.
Previous findings have shown that hypotensive doses of losartan prevent the excess of apoptosis present in the hypertrophied left ventricle of adult spontaneously hypertensive rats (SHR). This study was designed to determine whether angiotensin II facilitates apoptosis in cardiomyocytes of adult SHR. Primary cultures of ventricular cardiomyocytes from 30-week-old normotensive Wistar-Kyoto rats (WKY) and SHR with left ventricular hypertrophy were exposed to 10(-)(9) mol/L angiotensin II for 24 hours. Apoptotic cells were assessed by terminal deoxynucleotidyl transferase assay and confirmed by Annexin V detection. The expression of Bax-alpha, Bcl-2, p53, and caspase-3 proteins was assessed by Western blot assays. The expression of BAX gene was assessed by Northern blot. Angiotensin II increased (P<0.01) cardiomyocyte apoptosis, and this effect was higher (P<0.001) in SHR cells than in WKY cells. Whereas losartan (10(-7) mol/L) blocked the apoptotic effect of the octapeptide in cells from the two strains of rats, PD123319 (10(-7) mol/L) inhibited angiotensin II-mediated apoptosis only in SHR cells. Angiotensin II stimulated (P<0.01) Bax-alpha protein, and this effect was higher (P<0.01) in SHR cells than in WKY cells. Angiotensin II did not modify Bcl-2, p53, and BAX mRNA in cells from the two strains of rats. Angiotensin II induced a similar increase (P<0.05) in the ratio caspase-3/procaspase-3 (an index of caspase-3 activation) in cardiomyocytes from the two strains of rats. The present in vitro results indicate that SHR cardiomyocytes exhibit enhanced susceptibility to angiotensin II-induced apoptosis. Ligand binding to angiotensin II type 1 and type 2 receptors leading to changes in posttranscriptional processing of Bax-alpha and accumulation of this proapoptotic protein may be involved in the abnormal response of SHR cardiomyocytes. These data support a role for angiotensin II in apoptosis observed in the left ventricle of these rats.
Angiotensin II↗
Losartan inhibits the post-transcriptional synthesis of collagen type I and reverses left ventricular fibrosis in spontaneously hypertensive rats.
OBJECTIVE: Previous studies have shown that as well as left ventricular hypertrophy, myocardial fibrosis develops early in rats with spontaneous hypertension (SHR). The present study was designed to investigate whether chronic treatment with the angiotensin II type 1 (AT1) receptor antagonist losartan modifies collagen type I metabolism and reverses left ventricular fibrosis in young SHR with left ventricular hypertrophy. DESIGN: The study was performed in 30-week-old normotensive Wistar-Kyoto (WKY) rats, untreated SHR and SHR treated with losartan (20 mg/mg per day, orally) for 14 weeks before they were killed. METHODS: Ventricular pro-alpha 1 (I) collagen messenger RNA was analyzed by Northern blot. Serum levels of the carboxy-terminal propeptide of procollagen type I (PIP) and the pyridoline cross-linked telopeptide domain of collagen type I (CITP) were determined by specific radioimmunoassays as markers of collagen type I synthesis and degradation, respectively. Collagen volume fraction was determined in the left ventricle by quantitative morphometry. RESULTS: Compared with WKY rats, SHR exhibited increased (P < 0.05) mean arterial pressure, pro-alpha 1 (I) collagen messenger RNA, PIP and left ventricular collagen volume fraction, and similar CITP values. After the treatment period, mean arterial pressure was higher (P < 0.05) in losartan-treated SHR than in WKY rats. Compared with untreated SHR, treated SHR showed no left ventricular hypertrophy and diminished (P < 0.05) values of mean arterial pressure, PIP and left ventricular collagen volume fraction. No changes in pro-alpha 1 (I) collagen messenger RNA and CITP values were observed with treatment in SHR. No significant differences in the left ventricular collagen volume fraction were observed between treated SHR with normal blood pressure and treated SHR with abnormally high blood pressure at the end of the treatment period. CONCLUSIONS: These results suggest that chronic AT1 blockade with losartan decreases the post-transcriptional synthesis of fibril-forming collagen type I molecules in young SHR. This effect may be involved in the ability of this drug to reverse left ventricular fibrosis in young rats with genetic hypertension. Apart from its antihypertensive action, other mechanisms may mediate the antifibrotic effect of losartan in this animal model.
Animals↗
p53-mediated upregulation of BAX gene transcription is not involved in Bax-alpha protein overexpression in the left ventricle of spontaneously hypertensive rats.
An association of increased apoptosis with overexpression of the proapoptotic protein Bax-alpha has been reported in the left ventricle of adult spontaneously hypertensive rats (SHR). Both alterations were corrected in SHR that received long-term treatment with the AT1 antagonist losartan. To gain insight into the regulation of cardiac Bax-alpha protein in genetic hypertension, we investigated the expression of the protein p53 (a BAX gene transcription factor) and BAX mRNA in the left ventricle of 30-week-old Wistar-Kyoto rats (WKY), SHR, and SHR treated with losartan (20 mg. kg-1. d-1) during 14 weeks before death. The expression of p53 and Bax proteins was assessed by Western blot analysis. The expression of BAX mRNA was assessed by Northern blot analysis. The density of apoptotic cells was assessed by direct immunoperoxidase detection of biotin-labeled deoxyuridine nucleotides. Compared with WKY, untreated SHR exhibited increased apoptosis (P<0.05), increased Bax-alpha protein (P<0.05), and similar levels of p53 protein and BAX mRNA. Losartan given long term was associated with the normalization of apoptosis and Bax-alpha protein expression. The expression of BAX mRNA was decreased (P<0. 05) in treated SHR compared with untreated SHR. No changes in the expression of p53 protein were observed in losartan-treated SHR. These results suggest that overexpression of the Bax-alpha protein seen in the left ventricle of adult SHR with increased apoptosis is not related to a p53-mediated upregulation of BAX gene transcription. Our data also suggest that normalization of Bax-alpha protein observed in SHR after long-term blockade of angiotensin II type 1 receptors may be due to the inhibition of BAX gene transcription.
Animals↗
Torasemide inhibits angiotensin II-induced vasoconstriction and intracellular calcium increase in the aorta of spontaneously hypertensive rats.
Torasemide is a loop diuretic that is effective at low once-daily doses in the treatment of arterial hypertension. Because its antihypertensive mechanism of action may not be based entirely on the elimination of salt and water from the body, a vasodilator effect of this drug can be considered. In the present study, the ability of different concentrations of torasemide to modify angiotensin II (Ang II)-induced vascular responses was examined, with the use of an organ bath system, in endothelium-denuded aortic rings from spontaneously hypertensive rats. Ang II-induced increases of intracellular free calcium concentration ([Ca(2+)](i)) were also examined by image analysis in cultured vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats. A dose-response curve to Ang II was plotted for cumulative concentrations (from 10(-9) to 10(-6) mol/L) in endothelium-denuded aortic rings (pD(2)=7.5+/-0.3). Isometric contraction induced by a submaximal concentration of Ang II (10(-7) mol/L) was reduced in a dose-dependent way by torasemide (IC(50)=0.5+/-0.04 micromol/L). Incubation of VSMCs with different concentrations of Ang II (from 10(-10) to 10(-6) mol/L) resulted in a dose-dependent rise of [Ca(2+)](i) (pD(2)=7.5+/-0.3). The stimulatory effect of [Ca(2+)](i) induced by a submaximal concentration of Ang II (10(-7) mol/L) was blocked by torasemide (IC(50)=0.5+/-0.3 nmol/L). Our findings suggest that torasemide blocks the vasoconstrictor action of Ang II in vitro. This action can be related to the ability of torasemide to block the increase of [Ca(2+)](i) induced by Ang II in VSMCs. It is proposed that these actions might be involved in the antihypertensive effect of torasemide observed in vivo.
Angiotensin II↗
Molecular cloning and characterization of the human p44 mitogen-activated protein kinase gene.
The complete genomic structure of the human p44(mapk) gene (HMGW-approved symbol PRKM3) has been determined. The gene covers 9 kb and is composed of nine exons and eight introns. This structure is identical to the previously reported mouse p44(mapk) gene, indicating a high degree of evolutionary conservation. A sequence differing by one nucleotide from the consensus TATA box is present 132 positions upstream of the main transcription initiation point. This point has been located 415 nucleotides upstream of the translation initiation codon ATG and perfectly meets the consensus criteria for an initiator element (Inr). Multiple consensus sequences for factors that regulate either basal transcription or gene expression during cell differentiation and proliferation can be found in the putative promoter region. Some of them, such as several G/C boxes located downstream from the transcription initiation point, are also present in the homologous mouse gene, where they were shown to be functional.
5' Untranslated Regions↗
Altered regulation of smooth muscle cell proliferation and apoptosis in small arteries of spontaneously hypertensive rats.
AIM: It has been proposed that alterations of the balance between programmed cell death and cell replication might be involved in abnormalities of smooth muscle cell growth in arterial hypertension. This study was designed to analyse some regulators of apoptosis and proliferation in smooth muscle cells of small intra-myocardial arteries from the left ventricle of adult normotensive Wistar-Kyoto rats (WKY) and adult spontaneously hypertensive rats (SHR). Therefore, we assessed the expression of the cytoplasmic proteins Bax and Bcl-2, respectively a promoter and an inhibitor of apoptosis, and the expression of cyclin A, a nuclear protein that induces proliferation of smooth muscle cells. METHODS AND RESULTS: We measured the percentages of smooth muscle cells expressing these proteins using monoclonal antibodies and the avidin-biotin immunoperoxidase method. Compared with WKY, cells from SHR exhibited normal Bax expression, increased (P < 0.001) Bcl-2 expression and increased (P < 0.001) cyclin A expression. The ratio of Bax to Bcl-2, an index of cell susceptibility to apoptosis, was lower (P < 0.001) in SHR than in WKY. Systolic blood pressure was directly correlated (P < 0.01) with Bcl-2 and cyclin A in SHR. CONCLUSION: These results suggest that apoptosis and proliferation of smooth muscle cells might be inhibited and stimulated, respectively, in small arteries of adult SHR. The imbalance between these two processes may account for abnormalities of smooth muscle cell growth in the arterial wall in genetic hypertension.
Animals↗
Isolation of a cDNA encoding the rat MAP-kinase homolog of human p63mapk.
Using a combination of screening, RACE, and RT-PCR, we have isolated a new rat brain cDNA, we refer to as rMNK2, that showed strong homology to known MAP-kinases. The deduced amino acid sequence of rMNK2 indicated that it is the rat homolog of human p63(mapk), showing 94.5% identity. rMNK2 showed 77% homology with rat ERK3 and its human homolog p97(mapk), and 43% homology with both rat genes rMNK1(ERK1) and ERK2, within the kinase domain. This suggest that rMNK2 and ERK3 belong to a separate subfamily within the rat MAP-kinase multigene family. The most interesting difference lies in subdomain VIII, where this new subfamily contain a SEG/SPR motif instead of the TEY/APE found in the ERK subfamily, the TPY/APE found in the JNK/SAPK subfamily or the TGY/APE found in the p38/RK subfamily. The human homologs of ERK3 and rMNK2 (p97(mapk) and p63(mapk)) also show this significant change. Expression of rMNK2 has been detected in brain and to a lesser extent in lung by reverse transcription/PCR (RT-PCR). In situ hybridization of rat brain slices demonstrated a restricted expression of rMNK2 in the choroid plexus and hippocampus. This is interesting because the human homolog p63(mapk) maps to 18q12-21, a region that might be implicated in manic-depressive illness.
Amino Acid Sequence↗