Ana Paula Dantas

Intrauterine undernutrition: expression and activity of the endothelial nitric oxide synthase in male and female adult offspring

OBJECTIVE Epidemiological studies suggest that intrauterine undernutrition plays an important role in the development of arterial hypertension in adulthood. In an attempt to define the mechanisms whereby blood pressure may be raised, we have hypothesized that arteries from offspring of nutritionally restricted dams exhibit abnormalities in the endothelial function and in nitric oxide synthesis. In order to investigate the existence of potential gender differences on the effects of intrauterine undernutrition, both male and female offspring of pregnant Wistar rats on normal and restricted diets were studied in adulthood. METHODS Female pregnant Wistar rats were fed either normal or 50% of the normal intake diets, during the whole gestational period. At 14 weeks of age, the rats were used for the study of vascular reactivity, eNOS and iNOS gene expression, eNOS activity and, in the case of females, estrogen levels. RESULTS Intrauterine undernutrition induced hypertension in both male and female offspring, but hypertension was more severe in male rats. Endothelium-intact aortic rings from male and female rats in the restricted diet group exhibited increased responses to norepinephrine, decreased vasodilation to acetylcholine and unaltered responses to sodium nitroprusside in comparison to aortic rings from control rats. No gender-related differences were observed in the vascular reactivity studies. Intrauterine undernutrition promoted decreased gene expression for eNOS in aorta isolated from male, but not female, offspring, reduction in eNOS activity in both male and female offspring and impairment in synthesis of estrogen in female offspring. CONCLUSION Our data show that intrauterine undernutrition: (1) induces hypertension both in the male and female offspring, hypertension being more severe in male than in female rats; (2) alters endothelium-dependent responses in aortas from the resulting offspring. The endothelial dysfunction is associated with a decrease in activity/expression of eNOS in aortas from male offspring. The mechanism involved in altered response to ACh in female offspring might be a consequence of reduction in estrogen levels leading to reduced eNOS activity.

Enhanced oxidative stress as a potential mechanism underlying the programming of hypertension in utero.

Maternal undernutrition during critical periods of organ development is known to impair fetal growth and predispose to the development of adulthood diseases, such as hypertension, coronary heart disease and type II diabetes that are linked to low birth weight and are characterized by endothelial dysfunction. Increased oxidative stress, in rats submitted to intrauterine undernutrition, provides a potential explanation for the endothelial dysfunction development. The aim of this study was to determine the oxidative stress and its consequence on mesenteric arteriolar responses to vasoactive agents in offspring from diet-restricted dams. For this, female pregnant Wistar rats were fed either normal or 50% of normal intake diets, during the whole gestational period. In male offspring, arterial blood pressure was determined by the tail cuff method in anesthetized rats, mesenteric arteriolar reactivity and superoxide anion generation were studied using intravital microscopy and superoxide dismutase activity was determined in mesentery by spectrophotometric assay. Intrauterine undernutrition induced hypertension, decreased vasodilation to acetylcholine and bradykinin but did not alter the responses to sodium nitroprusside. Topical application of superoxide dismutase and superoxide dismutase mimetic manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin significantly improved the altered arteriolar responses to acetylcholine and bradykinin. A decreased superoxide dismutase activity and an increased superoxide anion concentration were observed in the offspring of diet-restricted dams. This study shows for the first time that intrauterine undernutrition enhances oxidative stress in vivo and relates this to the impaired endothelium-dependent vasodilation.

Influence of Female Sex Hormones on Endothelium-Derived Vasoconstrictor Prostanoid Generation in Microvessels of Spontaneously Hypertensive Rats

Although female sex hormones may attenuate endothelial dysfunction in spontaneously hypertensive rats (SHR) by increasing endothelium-derived relaxing factors (EDRFs), the influence of ovarian hormones on the generation of endothelium-derived contracting factors (EDCFs) remains unknown. The aim of this study was to evaluate the effect of estrogen and progesterone on the generation of vasoconstrictor prostanoids and superoxide anion (O2(-)) by microvessels from SHR. Vascular reactivity to norepinephrine (NE), acetylcholine (ACh), and sodium nitroprusside (SNP) were evaluated in the mesenteric arteriolar bed from estrous (OE) and ovariectomized (OVX) SHR. OVX-SHR were treated for 24 hours or 15 days with estradiol and for 15 days with estradiol+progesterone. The vascular reactivity was evaluated in the absence or presence of indomethacin (INDO, 10 micromol/L) and sodium diclofenac (DIC, 10 micromol/L), ridogrel (RID, 50 micromol/L), dazoxiben (DAZ, 10 micromol/L), or superoxide dismutase (SOD, 100 U/mL). Prostanoid levels in the arteriolar perfusate of mesenteries with or without endothelium were measured by enzyme immunoassay. An increased reactivity to NE and reduced sensitivity to ACh were observed in microvessels from OVX-SHR compared with OE-SHR. There were no differences in the responses to SNP. Treatments with estradiol and estradiol+progesterone similarly restored these altered responses. INDO, DIC, RID, and SOD also restored the NE and ACh responses in OVX-SHR. DAZ had no effect on the vascular reactivities. The release of PGF(2alpha), but not of TXB(2) and 6-keto-PGF(1alpha), was greater in OVX-SHR than in OE-SHR microvessels with endothelium when stimulated by NE. This response was normalized by hormonal treatments. Neither NE nor ACh stimulated prostanoid production by microvessels without endothelium. These results suggest that estrogen may protect female SHR against severe hypertension partly by decreasing the synthesis of EDCFs such as PGH(2)/PGF(2alpha) and O2(-).

Influence of Hypoxia on Nitric Oxide Synthase Activity and Gene Expression in Children With Congenital Heart Disease A Novel Pathophysiological Adaptive Mechanism

BackgroundChronic hypoxia has been shown to modulate nitric oxide (NO) responses in different cell models, but the relationship between hypoxia and NO synthase (NOS) regulation in humans was not studied. We studied the relationship between endothelial and inducible NOS (eNOS and iNOS) activities and expression and chronic hypoxia in children with cyanotic and acyanotic congenital heart defects. Methods and ResultsRight atrial tissue was excised from 18 patients during cardiac surgery. eNOS and iNOS activities were measured by conversion of l-[H3]arginine to l-[H3]citrulline. Gene expression of eNOS and iNOS was quantified by competitive reverse transcription–polymerase chain reaction. The eNOS activity and expression were significantly reduced in cyanotic hearts compared with acyanotic hearts: 0.38±0.14 versus 1.06±0.11 pmol · mg−1 · min−1 (P <0.0001) and 0.54±0.08 versus 0.80±0.10 relative optical density (ROD) of cDNA (P <0.0001), respectively. In contrast, iNOS activity and expression were significantly higher in cyanotic than in acyanotic children: 7.04±1.20 versus 4.17±1.10 pmol · mg−1 · min−1 (P <0.0001) and 2.55±0.11 versus 1.91±0.18 ROD of cDNA (P <0.0001), respectively. ConclusionsHypoxia downregulates eNOS activity and gene expression in cardiac tissue from patients with cyanotic congenital heart defects. By contrast, iNOS activity and expression are increased in cyanotic children and may represent an alternative mechanism to counteract the effects of hypoxia in the cardiovascular system. Therefore, a novel adaptive mechanism during hypoxia is suggested.

Sustained Decrease in Superoxide Dismutase Activity Underlies Constrictive Remodeling After Balloon Injury in Rabbits

Objective—The redox pathophysiology of vascular repair is incompletely understood. We assessed the role of vascular superoxide dismutase (SOD) activity in oxidative/nitrative stress and caliber loss postinjury (PI). Methods and Results—Rabbits submitted to iliac artery balloon overdistension were followed for 14 days PI. Significant decrease in vascular SOD activity occurred at 7 and 14 days PI (by 45% and 34%, respectively, versus control, 96±1 U/mg, P <0.05). Separation in concanavalin-A column showed that both extracellular SOD (ecSOD) and CuZn SOD activities were reduced, whereas Western analysis showed normal or augmented protein expression. Immunoreactivity to nitrotyrosine, neuronal NO synthase (NOS), and inducible NOS (iNOS) increased in media and neointima PI; iNOS mRNA also augmented. Administration of ecSOD from days 7 to 14 PI corrected the SOD activity decrease and minimized caliber loss by 59% (P =0.007) despite unaltered neointima. Nitrate levels markedly increased with ecSOD in injured artery homogenates (26±5 versus 4±0.3 &mgr;mol/L per mg, P =0.001). Such increase was 70% inhibited by specific iNOS antagonist 1400w. Nitrotyrosine and neuronal NOS expression decreased after ecSOD. Conclusions—Sustained low vascular SOD activity has a key role in constrictive remodeling after injury, promoting oxidative/nitrative stress and impairment of iNOS-derived NO bioavailability. SOD function may critically determine whether iNOS induction is beneficial or deleterious in vivo. (Arterioscler Thromb Vasc Biol. 2003;23:2197-2202.)

Vascular aging in women: is estrogen the fountain of youth?

Aging is associated with structural and functional changes in the vasculature, including endothelial dysfunction, arterial stiffening and remodeling, impaired angiogenesis, and defective vascular repair, and with increased prevalence of atherosclerosis. Cardiovascular risk is similar for older men and women, but lower in women during their fertile years. This age- and sex-related difference points to estrogen as a protective factor because menopause is marked by the loss of endogenous estrogen production. Experimental and some clinical studies have attributed most of the protective effects of estrogen to its modulatory action on vascular endothelium. Estrogen promotes endothelial-derived NO production through increased expression and activity of endothelial nitric oxide synthase, and modulates prostacyclin and thromboxane A2 release. The thromboxane A2 pathway is key to regulating vascular tone in females. Despite all the experimental evidence, some clinical trials have reported no cardiovascular benefit from estrogen replacement therapy in older postmenopausal women. The “Timing Hypothesis,” which states that estrogen-mediated vascular benefits occur only before the detrimental effects of aging are established in the vasculature, offers a possible explanation for these discrepancies. Nevertheless, a gap remains in current knowledge of cardiovascular aging mechanisms in women. This review comprises clinical and experimental data on the effects of aging, estrogens, and hormone replacement therapy on vascular function of females. We aim to clarify how menopause and aging contribute jointly to vascular aging and how estrogen modulates vascular response at different ages.

Vascular Aging: Facts and Factors

“Man is as old as his arteries.” This old aphorism has been widely confirmed by epidemiological and observational studies establishing that cardiovascular diseases can be age-related in terms of their onset and progression. Besides, with aging come a number of physiological and morphological changes that alters cardiovascular function and lead to subsequently increased risk of cardiovascular disease, even in health asymptomatic individuals. Even though different adaptive mechanisms to protect blood vessels against mild stress have been described, the aging process induces a progressive failure of protective mechanisms, leading to vascular changes. The outcomes of the aging-related modifications are the impairment of homeostasis of the irrigated organs and resultant target organ damage. The increasing mean age of the population in industrialized countries has turned out to be an economic and public health problem, as the increase in life expectancy goes in parallel with high incidence of several pathological conditions, despite unprecedented advances in prevention, diagnostics, and treatment. Of all aging-related illness, cardiovascular diseases remain the leading cause of morbidity and mortality in the elderly, and thus it is imperative to understand the mechanism underlying cardiovascular senescence. This Research Topic presents a forum of comprehensive reviews on distinct aspects of the pathophysiology of vascular aging to provide insights into the causes and consequences of this complex process and attempts to propose new therapeutic strategies for the management of vascular senescence. Such as the idea proposed by Ming et al. (2012) and discussed by Cau and Tostes (2012), which suggests that targeting mTORC1-S6K1 signaling could be a promising therapeutic modality to retard the aging process and treat cardiovascular disease in the elderly. Vascular aging could be simply described as a consequence of natural physical stress and fatigue that could account for the major physical changes seen in elderly: dilation (after fracture of load-bearing material) and stiffening (by transfer of stress to the more rigid collagenous component of the arterial wall). Although aging-associated changes on vascular functioning are considered a set up for cardiovascular disease, modifications on cardiac, and central function could slow down or accelerate this set point. Therefore, it is crucial to understand how aging and other pathophysiological states affect the interaction between the heart and arterial network. The article by Wojciechowska et al. (2012) provides information on the changes with age in central and peripheral systolic blood pressure, based on data collected from randomly recruited European and Chinese subjects supporting a vicious circle between age-related stiffness, increasing systolic blood pressure, and cardiovascular complications. Chantler and Lakatta (2012) elegantly describe the concept of arterial ventricular coupling and provide valuable information on how aging, in the absence and presence of cardiovascular disease, affects the coupling both at rest and during exercise, and its pathophysiological consequences. In addition, vascular aging has been largely associated with senescence of the vascular endothelium. El Assar et al. (2012) present a wide overview of the mechanisms that participate on endothelial dysfunction that accompanies vascular aging, analyzing the synergisms, and interactions between them, some of the cellular mechanisms related to endothelial senescence as well as the prevention or reversion of those mechanisms that produce endothelial dysfunction. Cau et al. (2012) based on evidence from experimental models review the contribution of NO synthase (NOS) isoform alterations on aging-associated vascular dysfunctions, addressing the potential prevention by some drugs that modulate the expression/activity of NOS. Finally, Blanco and Bernabeu (2012) summarize data supporting the link between the splicing factor SRSF1 and endothelial cell senescence and suggest the existence of a common genetic program involving alternative splicing of a cluster of genes, which contributes to a senescent environment in the vessels. Nonetheless, aging-associated damage to the endothelium may not simply be a consequence of the endothelial cell malfunctioning, but also as a result of impaired maintenance repair systems by endothelial progenitor cell (EPC). As discussed by Williamson et al. (2012), a deterioration of endogenous EPC function with age may culminate in a decreased capacity for neovascularization and/or reduced re-endothelialization of vascular lesions, facilitating the development, progression, and clinical sequelae of cardiovascular disease. Growing evidence show that the progress of vascular aging in women follows a different chronology than in men. The gender-associated difference in the pathophysiology of cardiovascular disease has generated heated discussion, although a general consensus has validated a role of sex hormones in the modulation of vascular function and dysfunction. This research topic covers two fronts in this field: Novella et al. (2012) review clinical and experimental data to clarify how menopause and aging contribute jointly to vascular senescence and how estrogen modulates vascular response at different ages; Lopes et al. (2012) discuss the conflicting information on the role of testosterone to the regulation of vascular function in elder men and women. Last but not least, as suggested by Gragasin et al. (2012) the elderly is more frequently represented in common medical procedures and surgeries. Thus, understanding the circulatory changes that accompany the aging process is therefore becoming increasingly timely and relevant. These authors discuss aspects of vascular control in aging that are particularly relevant in the maintenance of intraoperative hemodynamic stability reviewing the effect of certain notable anesthetic agents with respect to the aging vasculature. Given the growing clinical relevance of the subject, we are pleased that our Research Topic has brought together basic and clinical scientists to spotlight on one of the greatest enemies of elder population: i.e., the vascular senescence. We hope that this Research Topic also place a special call on the need of studies to establish treatments and procedures to reduce the detrimental effects of vascular aging. It has been a great pleasure to be involved in this Research Topic and we would like to thank all of the authors, reviewers, and Frontiers staff for helping to make this Research Topic possible.

Effects of Estrogen on Vascular Inflammation: A Matter of Timing

Objective—Our study aims to determine the role of time of menopause on vascular inflammation biomarkers and how it affects their modulation by estrogen and raloxifene in postmenopausal women. Methods and Results—Uterine arteries from 68 postmenopausal women were divided into 3 segments and cultured for 24 hours in tissue culture media containing 17&bgr;-estradiol (100 nmol/L), raloxifene (100 nmol/L), or vehicle. Assessment of arterial concentration of 13 inflammatory biomarkers was performed by multiplex immunobead-based assay. Aging per se has a positive correlation with the generation of several proinflammatory markers. Although short-term estradiol exposure correlates with lower expression of tumor necrosis factor-&agr;, vascular endothelial growth factor, and interleukin-1&bgr; in all age groups, for most biomarkers aging was associated with a switch from a beneficial anti-inflammatory action by estrogen, at earlier stages of menopause, to a proinflammatory profile after 5 years past its onset. Raloxifene has no significant effect on the expression of all proinflammatory markers. Western blot analysis of estrogen receptor expression (estrogen receptor-&agr; and estrogen receptor-&bgr;) showed that estrogen receptor-&bgr; increases with aging, and this increase has a positive correlation with the generation of several proinflammatory markers. Conclusion—Aging alters estrogen-mediated effects on the modulation of inflammatory biomarkers in women. How aging affects estrogen responses on vascular inflammation is not clear, but our data show a positive association between increased estrogen receptor-&bgr; expression with aging and proinflammatory effects by estrogen.

Equine Estrogens Impair Nitric Oxide Production and Endothelial Nitric Oxide Synthase Transcription in Human Endothelial Cells Compared With the Natural 17β-Estradiol

Conjugated equine estrogen therapy is the most common hormone replacement strategy used to treat postmenopausal women. However, the ability of an individual conjugated equine estrogen to modulate NO production and, therefore, to induce cardiovascular protection is largely unknown. The effects of equine and naturally occurring estrogens on NO generation were evaluated in human aortic endothelial cells by measuring in vivo NO production, as well as NO synthase (eNOS) activity and expression. The transcriptional activity on the eNOS gene was determined by the ability of estrogen receptors (&agr; and &bgr;) to activate the eNOS promoter and induce transcription. Docking and molecular dynamics simulations were used to study structural features of the interaction between estrogenic compounds and estrogen receptor-&agr;. After 24 hours of incubation, we found that estrone upregulated NO production almost as effectively as estradiol by increasing eNOS activity and expression. However, the effect of equine estrogens (equilin, equilenin, and their metabolites) were marked decreased. eNOS promoter activity by equine estrogens was 30% to 50% lower than the naturally occurring estrogens. Computational analysis of estrogen molecules revealed that position 17 and the saturation of estrogenic compounds in ring B are important determinants for estrogen receptor-&agr; transcriptional activity. Equine estrogens increase NO production less effectively than naturally occurring estrogens, partially because of their lesser ability to activate the eNOS promoter and induce transcription. Differences in NO production by different estrogens may account for the differences in cardiovascular benefits achieved by the distinct estrogen replacement therapies.

Aging Negatively Affects Estrogens-Mediated Effects on Nitric Oxide Bioavailability by Shifting ERα/ERβ Balance in Female Mice

Aims Aging is among the major causes for the lack of cardiovascular protection by estrogen (E2) during postmenopause. Our study aims to determine the mechanisms whereby aging changes E2 effects on nitric oxide (NO) production in a mouse model of accelerated senescence (SAM). Methods and Results Although we found no differences on NO production in females SAM prone (SAMP, aged) compared to SAM resistant (SAMR, young), by either DAF-2 fluorescence or plasmatic nitrite/nitrate (NO2/NO3), in both cases, E2 treatment increased NO production in SAMR but had no effect in SAMP. Those results are in agreement with changes of eNOS protein and gene expression. E2 up-regulated eNOS expression in SAMR but not in SAMP. E2 is also known to increase NO by decreasing its catabolism by superoxide anion (O2 -). Interestingly, E2 treatment decreased O2 − production in young females, while increased O2 − in aged ones. Furthermore, we observed that aging changed expression ratio of estrogen receptors (ERβ/ERα) and levels of DNA methylation. Increased ratio ERβ/ERα in aged females is associated to a lack of estrogen modulation of NO production and with a reversal in its antioxidant effect to a pro-oxidant profile. Conclusions Together, our data suggest that aging has detrimental effects on E2-mediated benefits on NO bioavailability, partially by affecting the ability of E2 to induce up regulation of eNOS and decrease of O2 −. These modifications may be associated to aging-mediated modifications on global DNA methylation status, but not to a specific methylation at 5′flanking region of ERα gene.