Impaired Pulmonary Arterial Vasoconstriction and Nitric Oxide-Mediated Relaxation Underlie Severe Pulmonary Hypertension in the Sugen-Hypoxia Rat Model.

Pulmonary vasoreactivity could determine the responsiveness to vasodilators and, in turn, the prognosis of pulmonary hypertension (PH). We hypothesized that pulmonary vasoreactivity is impaired, and we examined the underlying mechanisms in the Sugen-hypoxia rat model of severe PH. Male Sprague-Dawley rats were injected with Sugen (20 mg/kg s.c.) and exposed to hypoxia (9% O2) for 3 weeks, followed by 4 weeks in normoxia (Su/Hx), or treated with Sugen alone (Su) or hypoxia alone (Hx) or neither (Nx). After hemodynamic measurements, the heart was assessed for right ventricular hypertrophy (Fulton's index); the pulmonary artery, aorta, and mesenteric arteries were isolated for vascular function studies; and contractile markers were measured in pulmonary arteries using quantitative polymerase chain reaction (PCR). Other rats were used for morphometric analysis of pulmonary vascular remodeling. Right ventricular systolic pressure and Fulton's index were higher in Su/Hx versus Su, Hx, and Nx rats. Pulmonary vascular remodeling was more prominent in Su/Hx versus Nx rats. In pulmonary artery rings, contraction to high KCl (96 mM) was less in Su/Hx versus Nx and Su, and phenylephrine-induced contraction was reduced in Su/Hx versus Nx, Hx, and Su. Acetylcholine (ACh)-induced relaxation was less in Su/Hx versus Nx and Hx, suggesting reduced endothelium-dependent vasodilation. ACh relaxation was inhibited by nitric oxide synthase (NOS) and guanylate cyclase blockade in all groups, suggesting a role of the NO-cGMP pathway. Nitrate/nitrite production in response to ACh was less in Su/Hx versus Nx, supporting reduced endothelial NO production. Sodium nitroprusside (10-8 M) caused less relaxation in Su/Hx versus Nx, Hx, and Su, suggesting a decreased responsiveness of vascular smooth muscle (VSM) to vasodilators. Neither contraction nor relaxation differed in the aorta or mesenteric arteries of all groups. PCR analysis showed decreased expression of contractile markers in pulmonary artery of Su/Hx versus Nx. The reduced responsiveness to vasoconstrictors and NO-mediated vasodilation in the pulmonary, but not systemic, vessels may be an underlying mechanism of severe PH in Su/Hx rats and appears to involve attenuation of the NO relaxation pathway and a switch of pulmonary VSM cells to a synthetic less reactive phenotype.

Increases in placental nitric oxide, but not nitric oxide-mediated relaxation, underlie the improvement in placental efficiency and antihypertensive effects of hydrogen sulphide donor in hypertensive pregnancy.

Dysregulation of hydrogen sulphide (H2 S) producing enzymes has been related to hypertensive pregnancy, and H2 S donor, sodium hydrosulphide (NaHS) exerts antihypertensive effects, modulates angiogenic factors production and acts as an antioxidant. Moreover, reduction in nitric oxide (NO) bioavailability is related to hypertensive pregnancy and H2 S may interact with NO, modulating its production. We aimed to investigate the NaHS effects in hypertension-in-pregnancy and also in feto-placental parameters. Female Wistar rats (200-250 g) were mated and desoxycorticosterone acetate injections followed by replacement of water by 0.9% saline solution were used to induce hypertensive pregnancy. Rats were divided into four groups: normal pregnant (Norm-Preg), pregnant + NaHS (Preg+NaHS), hypertensive pregnant (HTN-Preg) and HTN-Preg+NaHS. Systolic blood pressure was increased in HTN-Preg and this increase was blunted in HTN-Preg+NaHS. Fetal and placental weights were decreased in HTN-Preg animals, while fetal growth restriction was improved in HTN-Preg+NaHS. Placental weight was lower in HTN-Preg+NaHS than in HTN-Preg; however, placental efficiency was re-established in HTN-Preg+NaHS rats. We observed that a partial contribution of placental NO, but not changes in anti-angiogenic factors may mediate the increases in placental efficiency in HTN-Preg+NaHS. HTN-Preg presented thoracic aorta hyperreactivity to phenylephrine while NaHS treatment blunted this hyperreactivity, which seems not to be related to NO-mediated relaxation induced by acetylcholine. Therefore, changes in vascular responsiveness promoted by NaHS treatment may underlie the beneficial effects in systolic blood pressure and feto-placental parameters in our study.

Restoring placental growth factor-soluble fms-like tyrosine kinase-1 balance reverses vascular hyper-reactivity and hypertension in pregnancy.

Preeclampsia (PE) is a pregnancy-related hypertensive disorder (HTN-Preg) with unclear mechanism. An imbalance between antiangiogenic soluble fms-like tyrosine kinase-1 (sFlt-1) and angiogenic placental growth factor (PlGF) has been observed in PE, but the vascular targets and signaling pathways involved are unclear. We assessed the extent of sFlt-1/PlGF imbalance and vascular dysfunction in a rat model of HTN-Preg produced by reduction of uteroplacental perfusion pressure (RUPP), and tested whether inducing a comparable sFlt-1/PlGF imbalance by infusing sFlt-1 (10 µg·kg(-1)·day(-1)) in day 14 pregnant (Preg) rats cause similar increases in blood pressure (BP) and vascular reactivity. Using these guiding measurements, we then tested whether restoring sFlt-1/PlGF balance by infusing PIGF (20 µg·kg(-1)·day(-1)) in RUPP rats would improve BP and vascular function. On gestational day 19, BP was in Preg+sFlt-1 and RUPP > Preg, and in RUPP+PlGF < RUPP rats. Plasma sFlt-1/PlGF ratio was increased in Preg+sFlt-1, and RUPP and was reduced in RUPP+PlGF rats. In isolated endothelium-intact aorta, carotid, mesenteric, and renal artery, phenylephrine (Phe)- and high KCl-induced contraction was in Preg+sFlt-1 and RUPP > Preg, and in RUPP+PlGF < RUPP. The differences in vascular reactivity to Phe and KCl between groups were less apparent in vessels treated with the nitric oxide synthase (NOS) inhibitor l-NAME or guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or endothelium-denuded, suggesting changes in endothelial NO-cGMP pathway. In Phe precontracted vessels, ACh-induced relaxation was in Preg+sFlt-1 and RUPP < Preg, and in RUPP+PlGF > RUPP, and was blocked by N(ω)-nitro-l-arginine methyl ester (l-NAME) or ODQ treatment or endothelium removal. Western blots revealed that aortic total endothelial NOS (eNOS) and activated phosphorylated-eNOS were in Preg+sFlt-1 and RUPP < Preg and in RUPP+PlGF > RUPP. ACh-induced vascular nitrate/nitrite production was in Preg+sFlt-1 and RUPP < Preg, and in RUPP+PlGF > RUPP. Vascular relaxation to the exogenous NO donor sodium nitroprusside was not different among groups. Thus, a tilt in the angiogenic balance toward anti-angiogenic sFlt-1 is associated with decreased vascular relaxation and increased vasoconstriction and BP. Restoring the angiogenic/antiangiogenic balance using PlGF enhances endothelial NO-cGMP vascular relaxation and decreases vasoconstriction and BP in HTN-Preg rats and could offer a new approach in the management of PE.

Reductions of Circulating Nitric Oxide are Followed by Hypertension during Pregnancy and Increased Activity of Matrix Metalloproteinases-2 and -9 in Rats.

Hypertensive pregnancy has been associated with reduced nitric oxide (NO), bioavailability, and increased activity of matrix metalloproteinases (MMPs). However, it is unclear if MMPs activation is regulated by NO during pregnancy. To this end, we examined activity of MMP-2 and MMP-9 in plasma, placenta, uterus and aorta, NO bioavailability, oxidative stress, systolic blood pressure (SBP), and fetal-placental development at the early, middle, and late pregnancy stages in normotensive and Nω-Nitro-L-arginine methyl-ester (L-NAME)-induced hypertensive pregnancy in rats. Reduced MMP-2 activity in uterus, placenta, and aorta and reduced MMP-9 activity in plasma and placenta with concomitant increased NO levels were found in normotensive pregnant rats. By contrast, increased MMP-2 activity in uterus, placenta, and aorta, and increased MMP-9 activity in plasma and placenta with concomitant reduced NO levels were observed in hypertensive pregnant rats. Also, elevated oxidative stress was displayed by hypertensive pregnant rats at the middle and late stages. These findings in the L-NAME-treated pregnant rats were also followed by increases in SBP and associated with fetal growth restrictions at the middle and late pregnancy stages. We concluded that NO bioavailability may regulate MMPs activation during normal and hypertensive pregnancy.

Hypertension, augmented activity of matrix metalloproteinases-2 and -9 and angiogenic imbalance in hypertensive pregnancy are attenuated by doxycycline.

Preeclampsia is manifested as maternal hypertension and fetal growth restriction. Matrix metalloproteinases (MMPs) are involved in hypertension and doxycycline reduces blood pressure by inhibition of MMPs. Moreover, excessive levels of MMPs and reduced nitric oxide (NO) bioavailability have been related to preeclampsia. We investigated the involvement of MMPs in hypertension in pregnancy induced by Nω-Nitro-L-arginine methyl ester (L-NAME) in rats. To this end, zimography was performed to evaluate the activity of MMPs -2 and -9 in placenta, uterus and thoracic aorta, and systolic blood pressure, feto-placental development and metabolites of NO were evaluated. Also, plasma antioxidant capacity, plasma levels of soluble fms-like tyrosine kinase-1 (sFlt-1) and placental growth factor (PLGF) were examined. Doxycycline prevented hypertensive pregnancy and significant reductions in number of pups induced by L-NAME. Low NO bioavailability was found in hypertensive pregnant rats treated (or not) with doxycycline. Increased activity of placental MMP-2 and MMP-9 and uterine MMP-2 were attenuated by doxycycline. MMP-2 activity of thoracic aorta showed no change after hypertension. Increases in PLGF with concomitant decreases in sFlt-1 levels were found with doxycycline treatment. Also, plasma antioxidant capacity was improved with doxycycline. Also, elevations of plasma antioxidant capacity were observed in hypertensive rats treated with doxycycline. Therefore, we suggest that L-NAME reduced NO and this triggered the increases in MMP-2 and -9 activities during hypertensive pregnancy. Importantly, increases in MMPs activation and angiogenic imbalance were attenuated by doxycycline and these effects were associated with decreases in systolic blood pressure.

Angiogenic imbalance and diminished matrix metalloproteinase-2 and -9 underlie regional decreases in uteroplacental vascularization and feto-placental growth in hypertensive pregnancy.

Preeclampsia is a form of hypertension-in-pregnancy (HTN-Preg) with unclear mechanism. Generalized reduction of uterine perfusion pressure (RUPP) could be an initiating event leading to uteroplacental ischemia, angiogenic imbalance, and HTN-Preg. Additional regional differences in uteroplacental blood flow could further affect the pregnancy outcome and increase the risk of preeclampsia in twin or multiple pregnancy, but the mechanisms involved are unclear. To test the hypothesis that regional differences in angiogenic balance and matrix metalloproteinases (MMPs) underlie regional uteroplacental vascularization and feto-placental development, we compared fetal and placental growth, and placental and myoendometrial vascularization in the proximal, middle and distal regions of the uterus (in relation to the iliac bifurcation) in normal pregnant (Preg) and RUPP rats. Maternal blood pressure and plasma anti-angiogenic soluble fms-like tyrosine kinase-1 (sFlt-1)/placenta growth factor (PIGF) ratio were higher, and average placentae number, placenta weight, litter size, and pup weight were less in RUPP than Preg rats. The placenta and pup number and weight were reduced, while the number and diameter of placental and adjacent myoendometrial arteries, and MMP-2 and MMP-9 levels/activity were increased, and sFlt-1/PlGF ratio was decreased in distal vs proximal uterus of Preg rats. In RUPP rats, the placenta and pup number and weight, the number and diameter of placental and myoendometrial arteries, and MMP-2 and -9 levels/activity were decreased, and sFlt-1/PlGF ratio was increased in distal vs proximal uterus. Treatment with sFlt-1 or RUPP placenta extract decreased MMP-2 and MMP-9 in distal segments of Preg uterus, and treatment with PIGF or Preg placenta extract restored MMP levels in distal segments of RUPP uterus. Thus, in addition to the general reduction in placental and fetal growth during uteroplacental ischemia, localized angiogenic imbalance and diminished MMP-2 and MMP-9 could cause further decrease in placental and myoendometrial vascularization and placental and fetal growth in distal vs proximal uterus of HTN-Preg rats. Regional differences in uteroplacental perfusion, angiogenic balance and MMPs could be a factor in the incidence of preeclampsia in multiple pregnancy.

Cardiac myeloperoxidase activity is elevated in hypertensive pregnant rats.

Myeloperoxidase (MPO) is released from activated neutrophils. The inflammation in preeclampsia was found to be associated with endothelial dysfunction. We hypothesized that cardiac and circulating MPO levels are elevated in hypertensive pregnancy. Systolic and diastolic blood pressure and heart rate were measured on pregnancy days 14, 16, 18 and 20 in normal pregnant and hypertensive pregnant rats. Left and right ventricle weights, the number of viable fetuses, litter size, fetal and placenta weights were recorded on gestational day 21. Circulating and cardiac MPO activities, soluble fms-like tyrosine kinase-1 (sFlt-1) and vascular endothelial growth factor (VEGF) and nitric oxide (NO) were detected. The results showed increases in cardiac (left, but not right ventricle) and circulating MPO activities, and concomitantly lower number of viable fetuses, litter size, and fetal and placenta weights, and decreases in NO in hypertensive pregnant rats. Also, the increases in circulating sFlt-1 and VEGF were found in hypertensive pregnant group. In conclusion, maternal and fetal detrimental changes along with increases in circulating sFlt-1 and VEGF in hypertensive pregnancy may be associated with increases in cardiac and circulating MPO activities, confirming the causative role of inflammatory response in preeclampsia.

Metalloproteinase Inhibition Protects against Reductions in Circulating Adrenomedullin during Lead-induced Acute Hypertension.

Intoxication with lead (Pb) results in increased blood pressure by mechanisms involving matrix metalloproteinases (MMPs). Recent findings have revealed that MMP type two (MMP-2) seems to cleave vasoactive peptides. This study examined whether MMP-2 and MMP-9 levels/activities increase after acute intoxication with low lead concentrations and whether these changes were associated with increases in blood pressure and circulating endothelin-1 or with reductions in circulating adrenomedullin and calcitonin gene-related peptide (CGRP). Here, we expand previous findings and examine whether doxycycline (a MMPs inhibitor) affects these alterations. Wistar rats received intraperitoneally (i.p.) 1st dose 8 µg/100 g of lead (or sodium) acetate, a subsequent dose of 0.1 µg/100 g to cover daily loss and treatment with doxycycline (30 mg/kg/day) or water by gavage for 7 days. Similar whole-blood lead levels (9 µg/dL) were found in lead-exposed rats treated with either doxycycline or water. Lead-induced increases in systolic blood pressure (from 143 ± 2 to 167 ± 3 mmHg) and gelatin zymography of plasma samples showed that lead increased MMP-9 (but not MMP-2) levels. Both lead-induced increased MMP-9 activity and hypertension were blunted by doxycycline. Doxycycline also prevented lead-induced reductions in circulating adrenomedullin. No significant changes in plasma levels of endothelin-1 or CGRP were found. Lead-induced decreases in nitric oxide markers and antioxidant status were not prevented by doxycycline. In conclusion, acute lead exposure increases blood pressure and MMP-9 activity, which were blunted by doxycycline. These findings suggest that MMP-9 may contribute with lead-induced hypertension by cleaving the vasodilatory peptide adrenomedullin, thereby inhibiting adrenomedullin-dependent lowering of blood pressure.

Sevoflurane Induces DNA Damage Whereas Isoflurane Leads to Higher Antioxidative Status in Anesthetized Rats.

Taking into account that there are controversial antioxidative effects of inhalational anesthetics isoflurane and sevoflurane and absence of comparison of genotoxicity of both anesthetics in animal model, the aim of this study was to compare DNA damage and antioxidant status in Wistar rats exposed to a single time to isoflurane or sevoflurane. The alkaline single-cell gel electrophoresis assay (comet assay) was performed in order to evaluate DNA damage in whole blood cells of control animals (unexposed; n = 6) and those exposed to 2% isoflurane (n = 6) or 4% sevoflurane (n = 6) for 120 min. Plasma antioxidant status was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. There was no statistically significant difference between isoflurane and sevoflurane groups regarding hemodynamic and temperature variables (P > 0.05). Sevoflurane significantly increased DNA damage compared to unexposed animals (P = 0.02). In addition, Wistar rats anesthetized with isoflurane showed higher antioxidative status (MTT) than control group (P = 0.019). There were no significant differences in DNA damage or antioxidant status between isoflurane and sevoflurane groups (P > 0.05). In conclusion, our findings suggest that, in contrast to sevoflurane exposure, isoflurane increases systemic antioxidative status, protecting cells from DNA damage in rats.