The evolutionary conserved miR-137/325 tandem mediates obesity-induced hypogonadism and metabolic comorbidities by repressing hypothalamic kisspeptin.

BACKGROUND: Obesity-induced hypogonadism (OIH) is a prevalent, but often neglected condition in men, which aggravates the metabolic complications of overweight. While hypothalamic suppression of Kiss1-encoded kisspeptin has been suggested to contribute to OIH, the molecular mechanisms for such repression in obesity, and the therapeutic implications thereof, remain unknown. METHODS: A combination of bioinformatic, expression and functional analyses was implemented, assessing the role of the evolutionary-conserved miRNAs, miR-137 and miR-325, in mediating obesity-induced suppression of hypothalamic kisspeptin, as putative mechanism of central hypogonadism and metabolic comorbidities. The implications of such miR-137/325-kisspeptin interplay for therapeutic intervention in obesity were also explored using preclinical OIH models. RESULTS: MiR-137/325 repressed human KISS1 3'-UTR in-vitro and inhibited hypothalamic kisspeptin content in male rats, while miR-137/325 expression was up-regulated, and Kiss1/kisspeptin decreased, in the medio-basal hypothalamus of obese rats. Selective over-expression of miR-137 in Kiss1 neurons reduced Kiss1/ kisspeptin and partially replicated reproductive and metabolic alterations of OIH in lean mice. Conversely, interference of the repressive actions of miR-137/325 selectively on Kiss1 3'-UTR in vivo, using target-site blockers (TSB), enhanced kisspeptin content and reversed central hypogonadism in obese rats, together with improvement of glucose intolerance, insulin resistance and cardiovascular and inflammatory markers, despite persistent exposure to obesogenic diet. Reversal of OIH by TSB miR-137/325 was more effective than chronic kisspeptin or testosterone treatments in obese rats. CONCLUSIONS: Our data disclose that the miR-137/325-Kisspeptin repressive interaction is a major player in the pathogenesis of obesity-induced hypogonadism and a putative druggable target for improved management of this condition and its metabolic comorbidities in men suffering obesity. SIGNIFICANCE STATEMENT: Up to half of the men suffering obesity display also central hypogonadism, an often neglected complication of overweight that can aggravate the clinical course of obesity and its complications. The mechanisms for such obesity-induced hypogonadism remain poorly defined. We show here that the evolutionary conserved miR137/miR325 tandem centrally mediates obesity-induced hypogonadism via repression of the reproductive-stimulatory signal, kisspeptin; this may represent an amenable druggable target for improved management of hypogonadism and other metabolic complications of obesity.

Connecting nutritional deprivation and pubertal inhibition via GRK2-mediated repression of kisspeptin actions in GnRH neurons.

BACKGROUND: Perturbations in the timing of puberty, with potential adverse consequences in later health, are increasingly common. The underlying neurohormonal mechanisms are unfolded, but nutritional alterations are key contributors. Efforts to unveil the basis of normal puberty and its metabolic control have focused on mechanisms controlling expression of Kiss1, the gene encoding the puberty-activating neuropeptide, kisspeptin. However, other regulatory phenomena remain ill-defined. Here, we address the putative role of the G protein-coupled-receptor kinase-2, GRK2, in GnRH neurons, as modulator of pubertal timing via repression of the actions of kisspeptin, in normal maturation and conditions of nutritional deficiency. METHODS: Hypothalamic RNA and protein expression analyses were conducted in maturing female rats. Pharmacological studies involved central administration of GRK2 inhibitor, ßARK1-I, and assessment of gonadotropin responses to kisspeptin or phenotypic and hormonal markers of puberty, under normal nutrition or early subnutrition in female rats. In addition, a mouse line with selective ablation of GRK2 in GnRH neurons, aka G-GRKO, was generated, in which hormonal responses to kisspeptin and puberty onset were monitored, in normal conditions and after nutritional deprivation. RESULTS: Hypothalamic GRK2 expression increased along postnatal maturation in female rats, especially in the preoptic area, where most GnRH neurons reside, but decreased during the juvenile-to-pubertal transition. Blockade of GRK2 activity enhanced Ca+2 responses to kisspeptin in vitro, while central inhibition of GRK2 in vivo augmented gonadotropin responses to kisspeptin and advanced puberty onset. Postnatal undernutrition increased hypothalamic GRK2 expression and delayed puberty onset, the latter being partially reversed by central GRK2 inhibition. Conditional ablation of GRK2 in GnRH neurons enhanced gonadotropin responses to kisspeptin, accelerated puberty onset, and increased LH pulse frequency, while partially prevented the negative impact of subnutrition on pubertal timing and LH pulsatility in mice. CONCLUSIONS: Our data disclose a novel pathway whereby GRK2 negatively regulates kisspeptin actions in GnRH neurons, as major regulatory mechanism for tuning pubertal timing in nutritionally-compromised conditions.

Deregulation of miR-324/KISS1/kisspeptin in early ectopic pregnancy: mechanistic findings with clinical and diagnostic implications.

BACKGROUND: Ectopic pregnancy is a life-threatening condition for which novel screening tools that would enable early accurate diagnosis would improve clinical outcomes. Kisspeptins, encoded by KISS1, play an essential role in human reproduction, at least partially by regulating placental function and possibly embryo implantation. Kisspeptin levels are elevated massively in normal pregnancy and reportedly altered in various gestational pathologic diseases. Yet, the pathophysiologic role of KISS1/kisspeptin in ectopic pregnancy has not been investigated previously. OBJECTIVE: The purpose of this study was to evaluate changes of KISS1/kisspeptin levels in ectopic pregnancy and their underlaying molecular mechanisms and to ascertain the diagnostic implications of these changes. STUDY DESIGN: A total of 122 women with normal pregnancy who underwent voluntary termination of pregnancy and 84 patients who experienced tubal ectopic pregnancy were recruited. Measurements of plasma kisspeptins and KISS1 expression analyses in human embryonic/placental tissue were conducted in ectopic pregnancy and voluntary termination of pregnancy control subjects during the early gestational window (<12 weeks). Putative microRNA regulators of KISS1 were predicted in silico, followed by expression analyses of selected microRNAs and validation of repressive interactions in vitro. Circulating levels of these microRNAs were also assayed in ectopic pregnancy vs voluntary termination of pregnancy. RESULTS: Circulating kisspeptins gradually increased during the first trimester of normal pregnancy but were reduced markedly in ectopic pregnancy. This profile correlated with the expression levels of KISS1 in human embryonic/placental tissue, which increased in voluntary termination of pregnancy but remained suppressed in ectopic pregnancy. Bioinformatic predictions and expression analyses identified miR-27b-3p and miR-324-3p as putative repressors of KISS1 in human embryonic/placental tissue at <12 weeks gestation, when expression of microRNAs was low in voluntary termination of pregnancy control subjects but significantly increased in ectopic pregnancy. Yet, a significant repressive interaction was documented only for miR-324-3p, occurring at the predicted 3'-UTR of KISS1. Interestingly, circulating levels of miR-324-3p, but not of miR-27b-3p, were suppressed distinctly in ectopic pregnancy, despite elevated tissue expression of the pre-microRNA. A decision-tree model that used kisspeptin and miR-324-3p levels was successful in discriminating ectopic pregnancy vs voluntary termination of pregnancy, with a receiver-operating characteristic area under the curve of 0.95±0.02 (95% confidence interval). CONCLUSION: Our results document a significant down-regulation of KISS1/kisspeptins in early stages of ectopic pregnancy via, at least partially, a repressive interaction with miR-324-3p. Our data identify circulating kisspeptins and miR-324-3p as putative biomarkers for accurate screening of ectopic pregnancy at early gestational ages.

mPGES-1 (Microsomal Prostaglandin E Synthase-1) Mediates Vascular Dysfunction in Hypertension Through Oxidative Stress.

mPGES-1 (microsomal prostaglandin E synthase-1), the downstream enzyme responsible for PGE2 (prostaglandin E2) synthesis in inflammatory conditions and oxidative stress are increased in vessels from hypertensive animals. We evaluated the role of mPGES-1-derived PGE2 in the vascular dysfunction and remodeling in hypertension and the possible contribution of oxidative stress. We used human peripheral blood mononuclear cells from asymptomatic patients, arteries from untreated and Ang II (angiotensin II)-infused mPGES-1-/- and mPGES-1+/+ mice, and vascular smooth muscle cells exposed to PGE2 In human cells, we found a positive correlation between mPGES-1 mRNA and carotid intima-media thickness (r=0.637; P<0.001) and with NADPH oxidase-dependent superoxide production (r=0.417; P<0.001). In Ang II-infused mice, mPGES-1 deletion prevented all of the following: (1) the augmented wall:lumen ratio, vascular stiffness, and altered elastin structure; (2) the increased gene expression of profibrotic and proinflammatory markers; (3) the increased vasoconstrictor responses and endothelial dysfunction; (4) the increased NADPH oxidase activity and the diminished mitochondrial membrane potential; and (5) the increased reactive oxygen species generation and reduced NO bioavailability. In vascular smooth muscle cells or aortic segments, PGE2 increased NADPH oxidase expression and activity and reduced mitochondrial membrane potential, effects that were abolished by antagonists of the PGE2 receptors (EP), EP1 and EP3, and by JNK (c-Jun N-terminal kinase) and ERK1/2 (extracellular-signal-regulated kinases 1/2) inhibition. Deletion of mPGES-1 augmented vascular production of PGI2 suggesting rediversion of the accumulated PGH2 substrate. In conclusion, mPGES-1-derived PGE2 is involved in vascular remodeling, stiffness, and endothelial dysfunction in hypertension likely through an increase of oxidative stress produced by NADPH oxidase and mitochondria.

Development and validation of a method for precise dating of female puberty in laboratory rodents: The puberty ovarian maturation score (Pub-Score).

Puberty is a key developmental event whose primary regulatory mechanisms remain poorly understood. Precise dating of puberty is crucial for experimental (preclinical) studies on its complex neuroendocrine controlling networks. In female laboratory rodents, external signs of puberty, such as vaginal opening (VO) and epithelial cell cornification (i.e., first vaginal estrus, FE), are indirectly related to the maturational state of the ovary and first ovulation, which is the unequivocal marker of puberty. Whereas in rats, VO and FE are almost simultaneous with the first ovulation, these events are not so closely associated in mice. Moreover, external signs of puberty can be uncoupled with first ovulation in both species under certain experimental conditions. We propose herein the Pubertal Ovarian Maturation Score (Pub-score), as novel, reliable method to assess peripubertal ovarian maturation in rats and mice. This method is founded on histological evaluation of pre-pubertal ovarian maturation, based on antral follicle development, and the precise timing of first ovulation, by retrospective dating of maturational and regressive changes in corpora lutea. This approach allows exact timing of puberty within a time-window of at least two weeks after VO in both species, thus facilitating the identification and precise dating of advanced or delayed puberty under various experimental conditions.

Lysyl Oxidase Induces Vascular Oxidative Stress and Contributes to Arterial Stiffness and Abnormal Elastin Structure in Hypertension: Role of p38MAPK.

AIMS: Vascular stiffness, structural elastin abnormalities, and increased oxidative stress are hallmarks of hypertension. Lysyl oxidase (LOX) is an elastin crosslinking enzyme that produces H2O2 as a by-product. We addressed the interplay between LOX, oxidative stress, vessel stiffness, and elastin. RESULTS: Angiotensin II (Ang II)-infused hypertensive mice and spontaneously hypertensive rats (SHR) showed increased vascular LOX expression and stiffness and an abnormal elastin structure. Mice over-expressing LOX in vascular smooth muscle cells (TgLOX) exhibited similar mechanical and elastin alterations to those of hypertensive models. LOX inhibition with ß-aminopropionitrile (BAPN) attenuated mechanical and elastin alterations in TgLOX mice, Ang II-infused mice, and SHR. Arteries from TgLOX mice, Ang II-infused mice, and/or SHR exhibited increased vascular H2O2 and O2.- levels, NADPH oxidase activity, and/or mitochondrial dysfunction. BAPN prevented the higher oxidative stress in hypertensive models. Treatment of TgLOX and Ang II-infused mice and SHR with the mitochondrial-targeted superoxide dismutase mimetic mito-TEMPO, the antioxidant apocynin, or the H2O2 scavenger polyethylene glycol-conjugated catalase (PEG-catalase) reduced oxidative stress, vascular stiffness, and elastin alterations. Vascular p38 mitogen-activated protein kinase (p38MAPK) activation was increased in Ang II-infused and TgLOX mice and this effect was prevented by BAPN, mito-TEMPO, or PEG-catalase. SB203580, the p38MAPK inhibitor, normalized vessel stiffness and elastin structure in TgLOX mice. INNOVATION: We identify LOX as a novel source of vascular reactive oxygen species and a new pathway involved in vascular stiffness and elastin remodeling in hypertension. CONCLUSION: LOX up-regulation is associated with enhanced oxidative stress that promotes p38MAPK activation, elastin structural alterations, and vascular stiffness. This pathway contributes to vascular abnormalities in hypertension. Antioxid. Redox Signal. 27, 379-397.

Increased nitric oxide bioavailability in adult GRK2 hemizygous mice protects against angiotensin II-induced hypertension.

G protein-coupled receptor kinase 2 (GRK2) is a ubiquitous serine/threonine protein kinase able to phosphorylate and desensitize the active form of several G protein-coupled receptors. Given the lack of selective inhibitors for GRK2, we investigated the effects elicited by GRK2 inhibition in vascular responses using global adult hemizygous mice (GRK2(+/-)). The vasodilator responses to acetylcholine or isoproterenol were increased in aortas and mesenteric resistance arteries from GRK2(+/-) mice compared with wild-type (WT) littermates. After angiotensin II (AngII) infusion, GRK2(+/-) mice were partially protected against hypertension, vascular remodeling, and mechanical alterations, even when resting basal blood pressures were not significantly different. AngII infusion also (1) increased GRK2 levels in WT but not in GRK2(+/-) vessels; (2) increased vasoconstrictor responses to phenylephrine in WT but not in GRK2(+/-) mice; and (3) decreased vasodilator responses to acetylcholine and vascular pAkt and eNOS levels more in WT than in GRK2(+/-) animals. Vascular NO production and the modulation of vasoconstrictor responses by endothelial-derived NO remained enhanced in GRK2(+/-) mice infused with AngII. Thus, GRK2(+/-) mice are resistant to the development of vascular remodeling and mechanical alterations, endothelial dysfunction, increased vasoconstrictor responses, and hypertension induced by AngII at least partially through the preservation of NO bioavailability. In conclusion, our results describe an important role for GRK2 in systemic hypertension and further establish that an inhibition of GRK2 could be a beneficial treatment for this condition.

Aerobic exercise reduces oxidative stress and improves vascular changes of small mesenteric and coronary arteries in hypertension.

BACKGROUND AND PURPOSE: Regular physical activity is an effective non-pharmacological therapy for prevention and control of hypertension. We investigated the effects of aerobic exercise training in vascular remodelling and in the mechanical and functional alterations of coronary and small mesenteric arteries from spontaneously hypertensive rats (SHR). EXPERIMENTAL APPROACH: Normotensive Wistar Kyoto (WKY), SHR and SHR trained on a treadmill for 12 weeks were used to evaluate vascular structural, mechanical and functional properties. KEY RESULTS: Exercise did not affect lumen diameter, wall thickness and wall/lumen ratio but reduced vascular stiffness of coronary and mesenteric arteries from SHR. Exercise also reduced collagen deposition and normalized altered internal elastic lamina organization and expression of MMP-9 in mesenteric arteries from SHR. Exercise did not affect contractile responses of coronary arteries but improved the endothelium-dependent relaxation in SHR. In mesenteric arteries, training normalized the increased contractile responses induced by U46619 and by high concentrations of acetylcholine. In vessels from SHR, exercise normalized the effects of the NADPH oxidase inhibitor apocynin and the NOS inhibitor l-NAME in vasodilator or vasoconstrictor responses, normalized the increased O(2) (-) production and the reduced Cu/Zn superoxide dismutase expression and increased NO production. CONCLUSIONS AND IMPLICATIONS: Exercise training of SHR improves endothelial function and vascular stiffness in coronary and small mesenteric arteries. This might be related to the concomitant decrease of oxidative stress and increase of NO bioavailability. Such effects demonstrate the beneficial effects of exercise on the vascular system and could contribute to a reduction in blood pressure.

Reciprocal relationship between reactive oxygen species and cyclooxygenase-2 and vascular dysfunction in hypertension.

AIMS: This study evaluates a possible relationship between reactive oxygen species (ROS) and cyclooxygenase (COX)-2-derived products in conductance and resistance arteries from hypertensive animals. Angiotensin II (Ang II)-infused mice or spontaneously hypertensive rats treated with the NAD(P)H Oxidase inhibitor apocynin, the mitochondrion-targeted SOD2 mimetic Mito-TEMPO, the superoxide dismutase analog tempol, or the COX-2 inhibitor Celecoxib were used. RESULTS: Apocynin, Mito-TEMPO, and Celecoxib treatments prevented Ang II-induced hypertension, the increased vasoconstrictor responses to phenylephrine, and the reduced acetylcholine relaxation. The NOX-2 inhibitor gp91ds-tat, the NOX-1 inhibitor ML171, catalase, and the COX-2 inhibitor NS398 abolished the ex vivo effect of Ang II-enhancing phenylephrine responses. Antioxidant treatments diminished the increased vascular COX-2 expression, prostanoid production, and/or participation of COX-derived contractile prostanoids and thromboxane A(2) receptor (TP) in phenylephrine responses, observed in arteries from hypertensive models. The treatment with the COX-2 inhibitor normalized the increased ROS production (O(2)·(-) and H(2)O(2)), NAD(P)H Oxidase expression (NOX-1, NOX-4, and p22phox) and activity, MnSOD expression, and the participation of ROS in vascular responses in both hypertensive models. Apocynin and Mito-TEMPO also normalized these parameters of oxidative stress. Apocynin, Mito-TEMPO, and Celecoxib improved the diminished nitric oxide (NO) production and the modulation by NO of phenylephrine responses in the Ang II model. INNOVATION: This study provides mechanistic evidence of circuitous relationship between COX-2 products and ROS in hypertension. CONCLUSION: The excess of ROS from NAD(P)H Oxidase and/or mitochondria and the increased vascular COX-2/TP receptor axis act in concert to induce vascular dysfunction and hypertension.

Endothelial dysfunction of rat coronary arteries after exposure to low concentrations of mercury is dependent on reactive oxygen species.

BACKGROUND AND PURPOSE: Exposure to mercury is known to increase cardiovascular risk but the underlying mechanisms are not well explored. We analysed whether chronic exposure to low mercury doses affects endothelial modulation of the coronary circulation. EXPERIMENTAL APPROACH: Left coronary arteries and hearts from Wistar rats treated with either HgCl(2) (first dose 4.6 µg·kg(-1) , subsequent doses 0.07 µg·kg(-1) day(-1) , 30 days) or vehicle were used. Endothelial cells from pig coronary arteries incubated with HgCl(2) were also used. KEY RESULTS: Mercury treatment increased 5-HT-induced vasoconstriction but reduced acetylcholine-induced vasodilatation. It also reduced nitric oxide (NO) production and the effects of NO synthase inhibition with L-NAME (100 µmol·L(-1) ) on 5-HT and acetylcholine responses. Superoxide anion production and mRNA levels of NOX-1 and NOX-4 were all increased. The superoxide anion scavenger tiron (1 mmol·L(-1)) reduced 5-HT responses and increased acetylcholine responses only in vessels from mercury-treated rats. In isolated hearts from mercury-treated rats, coronary perfusion and diastolic pressure were unchanged, but developed isovolumetric systolic pressure was reduced. In these hearts, L-NAME increased coronary perfusion pressure and diastolic pressure while it further reduced developed systolic pressure. CONCLUSIONS AND IMPLICATIONS: Chronic exposure to low doses of mercury promotes endothelial dysfunction of coronary arteries, as shown by decreased NO bioavailability induced by increased oxidative stress. These effects on coronary function increase resistance to flow, which under overload conditions might cause ventricular contraction and relaxation impairment. These findings provide further evidence that mercury, even at low doses, could be an environmental risk factor for cardiovascular disease.

Losartan and tempol treatments normalize the increased response to hydrogen peroxide in resistance arteries from hypertensive rats.

OBJECTIVE: To analyse the role of angiotensin II, via AT1 receptors, and oxidative stress in the mechanisms underlying the increased response to hydrogen peroxide (H2O2) of mesenteric resistance arteries from spontaneously hypertensive rats (SHRs). METHODS: Arteries from normotensive and SHRs untreated or treated with the AT1 receptor antagonist, losartan (15 mg/kg per day, 12 weeks), or with the superoxide dismutase analogue, tempol (1 mmol/l, 17 days), were used. Arteries were mounted in microvascular myographs for isometric tension recording; superoxide anion (O2(*-)) production was evaluated by dihydroethidium fluorescence, thromboxane A2 production by enzyme immunoassay and plasma nitrite levels by the Griess method. RESULTS: H2O2 (1-100 micromol/l) induced higher contractile responses in mesenteric resistance arteries from hypertensive than normotensive rats. In SHRs, losartan and tempol treatments induced the following effects: normalized the increased H2O2 contractile responses observed; modified neither the inhibitory effects of the cyclooxygenase inhibitor, indomethacin [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1-H-indole-3-acetic acid] (1 micromol/l), and the thromboxane A2/prostaglandin H2 receptor antagonist, SQ 29 548 (1 micromol/l), on H2O2 contraction, nor the increase in thromboxane A2 production in response to H2O2; abolished the increased vascular O2(*-) production; increased both the potentiatory effect of the nitric oxide inhibitor, N(G)-nitro-L-arginine methyl ester (100 micromol/l), on H2O2 responses and the acetylcholine-induced relaxation. Moreover, losartan treatment abolished the effect of the O2(*-) scavenger, tiron (1 mmol/l), on H2O2 responses and increased plasma nitrite levels. CONCLUSION: Nitric oxide removal by an excessive O2(*-) production, probably from an upregulated renin-angiotensin system, participates in the increased response to H2O2 in mesenteric resistance arteries from SHRs.