Effects of Drugs, Phytoestrogens, Nutrients and Probiotics on Endothelial Dysfunction in the Estrogen-Deficient State.

BACKGROUND: Endothelial dysfunction is commonly present in estrogen-deficient states, e.g., after menopause. In the search for alternatives to hormone replacement therapy (HRT), treatments based on phytoestrogens or in non-hormonal mechanisms have been under evaluation. OBJECTIVE: Here we aim to present an overview of innovative potential treatments for endothelial dysfunction in estrogen-deficient states, introducing our own preliminary data about the probiotic kefir. METHODS: We conducted a review based on a PubMed database search for keywords of interest (Menopause, Ovariectomy, Vascular dysfunction, Hot flashes, Metformin, Statins, Phytoestrogens, Omega-3, Vitamin D, Probiotics). RESULTS: Vascular parameters were found to be improved by both metformin and statins through pleiotropic effects, being related to a decrease in oxidative stress and restoration of the nitric oxide pathway. Phytoestrogens such as genistein and resveratrol have also been shown to improve vascular dysfunction, which seems to involve their estrogenic-like actions. Omega-3, vitamin D and its analogues, as well as probiotics, have shown similar vascular beneficial effects in both postmenopausal women and an animal model of ovariectomy (OVX), which could be related to antioxidant and/or anti-inflammatory effects. Moreover, our preliminary data on the probiotic kefir treatment in OVX rats suggested a vascular antioxidant effect. In particular, some evidence points to statins and vitamin D having anti-atherogenic effects. CONCLUSION: Pleiotropic effects of common medications and natural compounds could have therapeutic potential for endothelial dysfunction in estrogen-deficient states. They could, therefore, work as future complementary or alternative treatments to HRT.

Long-term treatment with Nandrolone Decanoate impairs mesenteric vascular relaxation in both sedentary and exercised female rats.

Nandrolone Decanoate (ND) is an Anabolic Androgenic Steroid (AAS) that under abusive regimen can lead to multiple physiological adverse effects. Studies of AAS-mediated cardiovascular (CV) alterations were mostly taken from male subjects, even though women are also susceptible to the effects of AAS and gender-specific differences in susceptibility to vascular diseases exist. Here we investigate ND-induced vascular reactivity alterations in both sedentary and exercised female rats and whether these alterations depend on endothelium-derived factors. We show that chronic exposure of female Wistar rats to ND (20mg/Kg/week for 4weeks) impaired the vascular mesenteric bed (MVB) reactivity to vasodilator (acetylcholine) agonist. The endothelium-dependent Nitric Oxide (NO) component was reduced in ND-treated rats, whereas neither the endothelium-derived hyperpolarizing factor (EDHF) component nor prostanoids were altered in the MVBs. Endothelial dysfunction observed in ND-treated rats was associated with decreased eNOS (Ser1177) and Akt (Ser473) phosphorylation sites and upregulation of iNOS and NADPH oxidase expression. Exercise training by weight lifting in water did not improve the vascular alterations induced by ND treatment. ND treatment also significantly reduced the serum levels of estradiol in females, overriding its CV protective effect. These results help uncover the role of ND modulating endothelial function in the setting of CV disease caused by the abuse of AAS in females. If this translates to humans, young women abusing AAS can potentially lose the cardio protective effect rendered by estrogen and be more susceptible to CV alterations.

Low dose of methyltestosterone in ovariectomised rats improves baroreflex sensitivity without geno- and cytotoxicity.

This study evaluated the effects of the isolated use of a low dose of methyltestosterone (MT) on cardiovascular reflexes and hormonal levels and its geno- and cytotoxic safety in ovariectomized rats. Female Wistar rats were divided into four groups (n = 6), respectively: SHAM (received vehicle methylcellulose 0.5%), SHAM + MT (received MT 0.05 mg/kg), OVX (received vehicle), and OVX + MT (received MT). Twenty-one days after ovariectomy, treatment was given orally daily for 28 days. The Bezold-Jarisch reflex (BJR) was analyzed by measuring the bradycardic and hypotensive responses elicited by phenylbiguanide (PBG) administration. The baroreflex sensitivity (BRS) was evaluated by phenylephrine and sodium nitroprussite. Myocyte hypertrophy was determined by morphometric analysis of H&E stained slides. Biochemical data were analyzed, as well as micronucleus assay. MT improved BRS and increased testosterone values, but did not change estradiol in the OVX group. MT did not promote changes in mean arterial pressure, heart rate, BJR, serum concentrations of troponin I, weight and histopathology of the heart. MT was able to restore the BRS in OVX rats. The geno- and cytotoxic safety of the MT was demonstrated by the absence of an increase in the micronucleus (PCEMN) or change in the ratio between normochromatic erythrocytes and polychromatic erythrocytes (NCE/PCE).

Replacement of specific markers for apoptosis and necrosis by nuclear morphology for affordable cytometry.

OBJECTIVE: The objective of the present study was to employ high throughput image analysis to detect necrosis and apoptosis. Specific markers were replaced by morphological parameters of cells and nuclei. METHOD: Fresh blood was taken from a healthy female and given a treatment to induce cell necrosis and apoptosis. Afterward, the samples were stained with AnnexinV-FITC, DRAQ5 and DAPI. Slides were made and analyzed using the cytometer iCys. Pictures were scanned. The analyzed sample consisted of 73 sets of images of DAPI, DRAQ5 and AnnexinV-FITC, respectively. For image analysis and subsequent statistical processing, the CellProfiler and CellProfilerAnalyst were used. Each sample was analyzed twice. The first analysis was conducted using the markers (DAPI, DRAQ5 and Annexin) for an unequivocal identification and subsequent count of necrotic, apoptotic and live cells (gold standard). Thereafter, a second analysis was performed for the nuclear morphology and texture (morphometric analysis). After the machine learning process was completed, the software calculated the quantity of cells in each of the three groups. A comparison between the result of the gold standard and the morphometric analysis was performed using linear regression and a Bland-Altman test. RESULTS: The linear regression between the two compared analyses was r(2)=0.57 for apoptosis, r(2)=0.84 for necrosis and r(2)=0.79 for living cells. CONCLUSION: It may be concluded that it is possible to replace specific markers against morphology without losing the reproducible high-throughput character of a cytometric analysis.

Metformin ameliorates ovariectomy-induced vascular dysfunction in non-diabetic Wistar rats.

Metformin is an antihyperglycaemic drug with pleiotropic effects that result in cardiovascular improvement. The aim of the present study was to evaluate the effects of metformin treatment on vascular dysfunction in ovariectomized rats. At 8 weeks of age, female Wistar rats were subjected to ovariectomy or a sham surgery. After 21 days, the animals were divided into three groups: SHAM (sham-operated rats), OVX (ovariectomized rats) and MET (ovariectomized rats treated with metformin at 300 mg/kg of body weight per day), and treated for 14 days. The vasorelaxation responses to ACh (acetylcholine) and SNP (sodium nitroprusside) were evaluated in mesenteric vascular beds, oxidative stress was evaluated and Western blot analysis of eNOS (endothelial NO synthase) and the NADPH oxidase Nox2 was performed. ACh-induced relaxation was reduced in the OVX group and partially restored in the MET group. L-NAME (NG-nitro-L-arginine methyl ester) attenuated and equalized the ACh-induced response in all groups. Attenuation of the ACh-induced responses by 4-aminopyridine (a blocker of voltage-gated potassium channels) was greater in the MET group compared with the OVX group. The SNP-induced responses were reduced in the OVX group and restored in the MET group. Inhibition of NADPH oxidase by apocynin (10 µM) restored the SNP-induced responses in the OVX group, enhanced these responses in the MET group and had no effect in the SHAM group. The OVX group exhibited reduced levels of eNOS protein and increased levels of oxidative stress and Nox2 protein; metformin treatment corrected all of these parameters. In conclusion, the pathophysiological changes observed in the mesenteric beds of ovariectomized rats were ameliorated by metformin. If this translates to humans, metformin could have additional benefits for post-menopausal women treated with this drug for glycaemic control.