Excessive hypercholesterolemia in pregnancy impairs rat uterine artery function via activation of Toll-like receptor 4.

Hypercholesterolemia in pregnancy is a physiological process required for normal fetal development. In contrast, excessive pregnancy-specific hypercholesterolemia increases the risk of complications, such as preeclampsia. However, the underlying mechanisms are unclear. Toll-like receptor 4 (TLR4) is a membrane receptor modulated by high cholesterol levels, leading to endothelial dysfunction; but whether excessive hypercholesterolemia in pregnancy activates TLR4 is not known. We hypothesized that a high cholesterol diet (HCD) during pregnancy increases TLR4 activity in uterine arteries, leading to uterine artery dysfunction. Sprague Dawley rats were fed a control diet (n=12) or HCD (n=12) during pregnancy (gestational day 6-20). Vascular function was assessed in main uterine arteries using wire myography (vasodilation to methacholine and vasoconstriction to phenylephrine; with and without inhibitors for mechanistic pathways) and pressure myography (biomechanical properties). Exposure to a HCD during pregnancy increased maternal blood pressure, induced proteinuria, and reduced the fetal-to-placental weight ratio for both sexes. Excessive hypercholesterolemia in pregnancy also impaired vasodilation to methacholine in uterine arteries, whereby at higher doses, methacholine caused vasoconstriction instead of vasodilation in only the HCD group, which was prevented by inhibition of TLR4 or prostaglandin H synthase 1. Endothelial nitric oxide synthase expression and nitric oxide levels were reduced in HCD compared with control dams. Vasoconstriction to phenylephrine and biomechanical properties were similar between groups. In summary, excessive hypercholesterolemia in pregnancy impairs uterine artery function, with TLR4 activation as a key mechanism. Thus, TLR4 may be a target for therapy development to prevent adverse perinatal outcomes in complicated pregnancies.

A High Cholesterol Diet During Late Pregnancy Impairs Long-Term Maternal Vascular Function in Mice.

BACKGROUND: Gestational dyslipidemia is associated with pregnancy complications including preeclampsia. However, whether gestational dyslipidemia leads postpartum vascular dysfunction, which could increase the risk for cardiovascular complications later in life, is not known. Here, we aimed to determine whether a gestational dyslipidemia affects postpartum vascular health and induces early signs of atherosclerosis. METHODS: Pregnant C57BL/6 mice received a high cholesterol diet or control diet from gestational day 13.5 until term. After delivery, all mice received the control diet for ≈3 months postpartum (PP). Age-matched nulliparous females were on the same diets for equal periods. After 3 months, all mice were euthanized, serum was collected, and aortas were isolated to assess vascular function (wire myography) and markers of oxidative stress and early atherosclerosis. RESULTS: PP-high cholesterol diet females had increased circulating cholesterol levels compared with PP-control diet mice, without effect of the diet in nulliparous mice. Methacholine-induced vasodilation was impaired, and nitric oxide contribution reduced, by the high cholesterol diet in aortas of PP mice, but not in nulliparous mice. Exposure to oxidized low-density-protein cholesterol further impaired methylcholine-induced vasodilation in PP-high cholesterol diet aortas only. Compared with PP-control diet mice, aortic inducible nitric oxide synthase expression, reactive oxygen species and nitrotyrosine levels were increased in aortas from PP-high cholesterol diet mice. No differences in aortic lipid deposition and macrophage infiltration were found. CONCLUSIONS: Exposure to a high cholesterol diet in pregnancy impairs vascular function postpartum. Our results support the hypothesis that gestational dyslipidemia impacts maternal vascular function after pregnancy, which could potentially predispose these women to future cardiovascular complications.

Placenta-targeted treatment with nMitoQ prevents an endothelin receptor-A pathway cardiac phenotype observed in adult male offspring exposed to hypoxia in utero.

Prenatal hypoxia is associated with enhanced susceptibility to cardiac ischemia-reperfusion (I/R) injury in adult offspring, however, the mechanisms remain to be fully investigated. Endothelin-1 (ET-1) is a vasoconstrictor that acts via endothelin A (ETA) and endothelin B (ETB) receptors and is essential in maintaining cardiovascular (CV) function. Prenatal hypoxia alters the ET-1 system in adult offspring possibly contributing to I/R susceptibility. We previously showed that ex vivo application of ETA antagonist ABT-627 during I/R prevented the recovery of cardiac function in prenatal hypoxia-exposed males but not in normoxic males nor normoxic or prenatal hypoxia-exposed females. In this follow-up study, we examined whether placenta-targeted treatment with a nanoparticle-encapsulated mitochondrial antioxidant (nMitoQ) during hypoxic pregnancies could alleviate this hypoxic phenotype observed in adult male offspring. We used a rat model of prenatal hypoxia where pregnant Sprague-Dawley rats were exposed to hypoxia (11% O2) from gestational days (GD) 15-21 after injection with 100 µL saline or nMitoQ (125 µM) on GD15. Male offspring were aged to 4 mo and ex vivo cardiac recovery from I/R was assessed. Offspring born from hypoxic pregnancies and treated with nMitoQ had increased cardiac recovery from I/R in the presence of ABT-627 compared with their untreated counterparts where ABT-627 prevented recovery. Cardiac ETA levels were increased in males born from hypoxic pregnancies with nMitoQ treatment compared with saline controls (Western blotting). Our data indicate a profound impact of placenta-targeted treatment to prevent an ETA receptor cardiac phenotype observed in adult male offspring exposed to hypoxia in utero.NEW & NOTEWORTHY In this follow-up study, we showed a complete lack of recovery from I/R injury after the application of an ETA receptor antagonist (ABT-627) in adult male offspring exposed to hypoxia in utero while maternal treatment with nMitoQ during prenatal hypoxia exposure prevented this effect. Our data suggest that nMitoQ treatment during hypoxic pregnancies may prevent a hypoxic cardiac phenotype in adult male offspring.

Sex-specific effects of prenatal hypoxia on the cardiac endothelin system in adult offspring.

Fetal hypoxia, a major consequence of complicated pregnancies, impairs offspring cardiac tolerance to ischemia-reperfusion (I/R) insult; however, the mechanisms remain unknown. Endothelin-1 (ET-1) signaling through the endothelin A receptors (ETA) is associated with cardiac dysfunction. We hypothesized that prenatal hypoxia exacerbates cardiac susceptibility to I/R via increased ET-1 and ETA levels, whereas ETA inhibition ameliorates this. Pregnant Sprague-Dawley rats were exposed to normoxia (21% O2) or hypoxia (11% O2) on gestational days 15-21. Offspring were aged to 4 mo, and hearts were aerobically perfused or subjected to ex vivo I/R, with or without preinfusion with an ETA antagonist (ABT-627). ET-1 levels were assessed with ELISA in aerobically perfused and post-I/R left ventricles (LV). ETA and ETB levels were assessed by Western blotting in nonperfused LV. As hypothesized, ABT-627 infusion tended to improve post-I/R recovery in hypoxic females (P = 0.0528); however, surprisingly, ABT-627 prevented post-I/R recovery only in the hypoxic males (P < 0.001). ET-1 levels were increased in post-I/R LV in both sexes regardless of the prenatal exposure (P < 0.01). ETA expression was similar among all groups, whereas ETB (isoform C) levels were decreased in prenatally hypoxic females (P < 0.05). In prenatally hypoxic males, ETA signaling may be essential for tolerance to I/R, whereas in prenatally hypoxic females, ETA may contribute to cardiac dysfunction. Our data illustrate that understanding the prenatal history has critical implications for treatment strategies in adult chronic diseases.NEW & NOTEWORTHY We demonstrated that prenatal hypoxia (a common condition of pregnancy) can have profound differential effects on treatment strategies in adult cardiovascular disease. Our data using a rat model of prenatal hypoxia demonstrated that, as adults, although inhibition of endothelin (ETA) receptors before an ex vivo cardiac ischemic insult improved recovery in females, it strikingly prevented recovery in males. Our data indicate a sex-specific effect of prenatal hypoxia on the cardiac ET-1 system in adult offspring.

Advanced Maternal Age Impairs Uterine Artery Adaptations to Pregnancy in Rats.

Advanced maternal age (≥35 years) is associated with pregnancy complications. Aging impairs vascular reactivity and increases vascular stiffness. We hypothesized that uterine artery adaptations to pregnancy are impaired with advanced age. Uterine arteries of nonpregnant and pregnant (gestational day 20) young (4 months) and aged (9 months; ~35 years in humans) Sprague-Dawley rats were isolated. Functional (myogenic tone, n = 6−10/group) and mechanical (circumferential stress-strain, n = 10−24/group) properties were assessed using pressure myography and further assessment of elastin and collagen (histology, n = 4−6/group), and matrix metalloproteinase-2 (MMP-2, zymography, n = 6/group). Aged dams had worse pregnancy outcomes, including smaller litters and fetal weights (both p < 0.0001). Only in arteries of pregnant young dams did higher pressures (>100 mmHg) cause forced vasodilation. Across the whole pressure range (4−160 mmHg), myogenic behavior was enhanced in aged vs. young pregnant dams (p = 0.0010). Circumferential stress and strain increased with pregnancy in young and aged dams (p < 0.0001), but strain remained lower in aged vs. young dams (p < 0.05). Arteries from young nonpregnant rats had greater collagen:elastin ratios than the other groups (p < 0.05). In aged rats only, pregnancy increased MMP-2 active capacity. Altered functional and structural vascular adaptations to pregnancy may impair fetal growth and development with advanced maternal age.

Placental treatment improves cardiac tolerance to ischemia/reperfusion insult in adult male and female offspring exposed to prenatal hypoxia.

Offspring born from complicated pregnancies are at greater risk of cardiovascular disease in adulthood. Prenatal hypoxia is a common pregnancy complication that results in placental oxidative stress and impairs fetal development. Adult offspring exposed to hypoxia during fetal life are more susceptible to develop cardiac dysfunction, and show decreased cardiac tolerance to an ischemia/reperfusion (I/R) insult. To improve offspring cardiac outcomes, we have assessed the use of a placenta-targeted intervention during hypoxic pregnancies, by encapsulating the mitochondrial antioxidant MitoQ into nanoparticles (nMitoQ). We hypothesized that maternal nMitoQ treatment during hypoxic pregnancies improves cardiac tolerance to I/R insult in adult male and female offspring. Pregnant Sprague-Dawley rats were exposed to normoxia (21 % O2) or hypoxia (11 % O2) from gestational day 15-20, after injection with 100 µL saline or nMitoQ (125 µM) on GD15 (n=6-8/group). Male and female offspring were aged to 4 months. Both male and female offspring from hypoxic pregnancies showed reduced cardiac tolerance to I/R (assessed ex vivo using the isolated working heart technique) which was ameliorated by nMitoQ treatment. To identify potential molecular mechanisms for the changes in cardiac tolerance to I/R, cardiac levels/phosphorylation of proteins important for intracellular Ca2+ cycling were assessed with Western blotting. In prenatally hypoxic male offspring, improved cardiac recovery from I/R by nMitoQ was accompanied by increased cardiac phospholamban and phosphatase 2Ce levels, and a trend to decreased Ca2+/calmodulin-dependent protein kinase IIδ phosphorylation. In contrast, in female offspring, nMitoQ treatment in hypoxic pregnancies increased phospholamban and protein kinase Cε phosphorylation. Maternal nMitoQ treatment improves cardiac tolerance to I/R insult in adult offspring and thus has the potential to improve the later-life trajectory of cardiovascular health of adult offspring born from pregnancies complicated by prenatal hypoxia.

Increased susceptibility to cardiovascular disease in offspring born from dams of advanced maternal age.

KEY POINTS: Advanced maternal age increases the risk of pregnancy complications such as fetal growth restriction, hypertension and premature birth. Offspring born from compromised pregnancies are at increased risk of cardiovascular disease as adults. However, the effect of advanced maternal age on later-onset disease in offspring has not been investigated. In adulthood, male but not female offspring born to dams of advanced maternal age showed impaired recovery from cardiac ischaemia/reperfusion injury. Endothelium-dependent relaxation was also impaired in male but not female offspring born from aged dams. Oxidative stress may play a role in the developmental programming of cardiovascular disease in this model. Given the increasing trend toward delayed parenthood, these findings have significant population and health care implications and warrant further investigation. ABSTRACT: Exposure to prenatal stressors, including hypoxia, micro- and macronutrient deficiency, and maternal stress, increases the risk of cardiovascular disease in adulthood. It is unclear whether being born from a mother of advanced maternal age (≥35 years old) may also constitute a prenatal stress with cardiovascular consequences in adulthood. We previously demonstrated growth restriction in fetuses from a rat model of advanced maternal age, suggesting exposure to a compromised in utero environment. Thus, we hypothesized that male and female offspring from aged dams would exhibit impaired cardiovascular function as adults. In 4-month-old offspring, we observed impaired endothelium-dependent relaxation in male (P < 0.05) but not female offspring born from aged dams. The anti-oxidant polyethylene glycol superoxide dismutase improved relaxation only in arteries from male offspring of aged dams (ΔEmax : young dam -1.63 ± 0.80 vs. aged dam 11.75 ± 4.23, P < 0.05). Furthermore, endothelium-derived hyperpolarization-dependent relaxation was reduced in male but not female offspring of aged dams (P < 0.05). Interestingly, there was a significant increase in nitric oxide contribution to relaxation in females born from aged dams (ΔEmax : young dam -24.8 ± 12.1 vs. aged dam -68.7 ± 7.7, P < 0.05), which was not observed in males. Recovery of cardiac function following an ischaemia-reperfusion insult in male offspring born from aged dams was reduced by ∼57% (P < 0.001), an effect that was not evident in female offspring. These data indicate that offspring born from aged dams have an altered cardiovascular risk profile that is sex-specific. Given the increasing trend toward delaying pregnancy, these findings may have significant population and health care implications and warrant further investigation.

Sex-specific effects of advanced maternal age on cardiovascular function in aged adult rat offspring.

Pregnancy at an advanced maternal age has an increased risk of complications for both the mothers and their offspring. We have previously shown that advanced maternal age in a rat model leads to poor fetal outcomes, maternal vascular dysfunction, and hypertension, concordant with findings in humans. Moreover, offspring from aged dams had sex-specific cardiovascular dysfunction in young adulthood. However, the detrimental impact of aging on the cardiovascular system of the offspring in this model is unknown. We hypothesized that offspring born to aged dams (9.5-10 mo old) would have impaired cardiovascular function at 12 mo of age. Echocardiographic data revealed signs of mild left ventricular diastolic dysfunction in only male offspring from aged dams [isovolumetric relaxation time: 34.27 ± 2.04 in the young dam group vs. 27.61 ± 0.99 ms in the aged dam group, P < 0.01; mitral annular velocity ratio ( E'/ A'): 1.08 ± 0.04 in the young dam group vs. 0.96 ± 0.02 in the aged dam group, P < 0.05]. We have previously shown that in young adulthood (4 mo of age), male, but not female, offspring born to aged dams had impaired recovery from ischemia-reperfusion injury. Aging did not alter the susceptibility of female offspring to ischemia-reperfusion injury. Interestingly, wire myography data revealed that male offspring from aged dams had enhanced vascular sensitivity to methacholine (negative log of EC50: 7.4 ± 0.08 in young dams vs. 7.9 ± 0.11 in aged dams, P = 0.007) due, in part, to increased prostaglandin-mediated vasodilation. Despite intact endothelium-dependent relaxation, female offspring from aged dams had elevated systolic blood pressure (125.3 ± 4.2 mmHg in young dams vs. 144.0 ± 6.9 mmHg in aged dams, P = 0.03). These data highlight sex-specific mechanisms underlying cardiovascular programming in offspring born to dams of advanced age. NEW & NOTEWORTHY Our study demonstrated that adult male and female offspring (12 mo old) born to aged dams had impaired cardiac diastolic function and increased blood pressure, respectively, signifying sex-specific differential cardiovascular effects of advanced maternal age.

Alterations in vascular function by syncytiotrophoblast extracellular vesicles via lectin-like oxidized low-density lipoprotein receptor-1 in mouse uterine arteries.

Syncytiotrophoblast extracellular vesicles (STBEVs), released into the maternal circulation during pregnancy, have been shown to affect vascular function; however, the mechanism remains unknown. In rats, STBEVs were shown to reduce endothelium-mediated vasodilation via lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a multi-ligand scavenger receptor that has been associated with vascular dysfunction. Recently, LOX-1 was shown to interact with the angiotensin II type 1 receptor (AT-1). We hypothesized that, in pregnant mice, STBEVs would impair vascular function via LOX-1 and would specifically affect angiotensin II responses. Uterine arteries from pregnant control (C57BL/6) and LOX-1 knockout (LOX-1KO) mice were isolated on gestational day (GD) 18.5. Endothelium-dependent (methylcholine (MCh); ± N(G)-Nitro-L-arginine methyl ester to assess nitric oxide (NO) contribution), and -independent (sodium nitroprusside) vasodilation, and vasoconstriction (angiotensin II; ± AT-1 [candesartan] or angiotensin II type 2 receptor (AT-2) [PD123.319] receptor antagonists; high potassium salt solution) responses were assessed using wire myography. AT-1 and AT-2 expression was analyzed using fluorescence microscopy. Human umbilical vein endothelial cells (HUVECs) were stimulated with STBEVs ± LOX-1 blocking antibody, and superoxide and peroxynitrite production were analyzed. Although MCh-induced vasodilation was decreased (P=0.0012), NO contribution to vasodilation was greater in LOX-1KO mice (P=0.0055). STBEVs delayed angiotensin II tachyphylaxis in arteries from control but not LOX-1KO mice (P<0.0001), while AT-1 and AT-2 expression was unchanged. STBEVs increased peroxynitrite production in HUVECs via LOX-1 (P=0.0091). In summary, LOX-1 deletion altered endothelium-mediated vasodilation, suggesting that LOX-1 contributes to vascular adaptations in pregnancy. STBEVs increased angiotensin II responsiveness and oxidative stress levels via LOX-1, suggesting that increased LOX-1 expression/activation or STBEVs could adversely affect vascular function and contribute to vascular complications of pregnancy.

Cardiovascular susceptibility to in vivo ischemic myocardial injury in male and female rat offspring exposed to prenatal hypoxia.

Intrauterine growth restriction (IUGR) following prenatal hypoxia exposure leads to a higher risk of developing cardiovascular disease (CVD) in later life. Our aim was to evaluate cardiac susceptibility and its pathophysiological mechanisms following acute myocardial infarction (MI) in adult rat offspring exposed to prenatal hypoxia. Male and female rat offspring, which experienced normoxia (21% O2) or hypoxia (11% O2) in utero underwent sham or MI surgery at 12 weeks of age. Echocardiographic data revealed that both sexes had systolic dysfunction following MI surgery, independent of prenatal hypoxia. Male offspring exposed to prenatal hypoxia, however, had left ventricular dilatation, global dysfunction, and signs of diastolic dysfunction following MI surgery as evident by increased left ventricular internal diameter (LVID) during diastole (MI effect, P<0.01), Tei index (MI effect, P<0.001), and E/E' ratio (prenatal hypoxia or MI effect, P<0.01). In contrast, diastolic dysfunction in female offspring was not as evident. Cardiac superoxide levels increased only in prenatal hypoxia exposed male offspring. Cardiac sarcoendoplasmic reticulum Ca2+-ATPase2a (SERCA2a) levels, a marker of cardiac injury and dysfunction, decreased in both male and female MI groups independent of prenatal hypoxia. Prenatal hypoxia increased cardiac ryanodine receptor 2 (RYR2) protein levels, while MI reduced RYR2 in only male offspring. In conclusion, male offspring exposed to prenatal hypoxia had an increased susceptibility to ischemic myocardial injury involving cardiac phenotypes similar to heart failure involving diastolic dysfunction in adult life compared with both offspring from healthy pregnancies and their female counterparts.

Mechanism of vascular dysfunction due to circulating factors in women with pre-eclampsia.

Circulating factors have been proposed to play a major role in the pathophysiology of endothelial dysfunction in pre-eclampsia (PE), which is defined as new-onset hypertension with proteinuria after 20 weeks of gestation. However, the mechanisms leading to altered vascular reactivity remain unclear. We hypothesized that circulating factors lead to endothelial dysfunction by increasing oxidative stress and reducing nitric oxide (NO) and prostaglandin (PG) bioavailability. Pregnant rat uterine and mesenteric arteries were incubated overnight with 3% normotensive (NP) or PE plasma collected from women upon admission to hospital. Responses to methacholine (MCh) were obtained using wire myography to assess endothelial function pathways. Vascular superoxide level was measured via dihydroethidium staining and nitric oxide synthase (NOS) expression via Western blots. PE plasma significantly increased superoxide levels and impaired endothelial dysfunction in uterine arteries (Emax 79.9±5.6% compared with 44.9±6.3%, P=0.0004), which was restored in the presence of oxidant scavengers or PG synthesis inhibition. Uterine artery vasodilation was abolished in the presence of pan-NOS inhibitor (P<0.0001) in both NP- and PE-treated vessels, but inducible nitric oxide synthase (iNOS)-dependent vasodilation was present only in NP-treated arteries. Uterine arteries exposed to PE plasma exhibit an increased endothelial NOS expression and a decreased iNOS expression. PE plasma did not alter endothelial function in mesenteric arteries, suggesting that the effect of circulating factors was vascular-bed-specific. We have shown that circulating factors lead to endothelial dysfunction via altered oxidative stress and vasodilator pathways. The present study contributes to our understanding of the pathophysiology and finding a potential target for intervention in PE.

Effect of sodium tanshinone IIA sulfonate treatment in a rat model of preeclampsia.

Preeclampsia is a disorder of pregnancy with a significant impact on maternal and fetal health. The complexity of this multifactorial condition has precluded development of effective therapies and, although many potential pathways have been investigated, the etiology still requires clarification. Our group has investigated the scavenger lectin-like oxidized LDL (LOX-1) receptor, which may respond to factors released from the distressed placenta that contribute to the vascular pathologies observed in preeclampsia. Given the known beneficial effects of sodium tanshinone IIA sulfonate (STS; a component of Salvia miltiorrhiza) on vasodilation, reduction of oxidative stress, and lipid profiles, we have investigated its role as a potential treatment strategy. We hypothesized that STS would improve vascular endothelial function and, combined with a reduction in oxidative stress, would improve pregnancy outcomes in a rat model of preeclampsia (reduced uteroplacental perfusion pressure, RUPP). We further hypothesized this may occur via the action of STS on the LOX-1 and/or platelet-activating factor (PAF) receptor axes. The RUPP model increased maternal blood pressure, vascular oxidative stress, and involvement of the vascular PAF receptor. Treatment with STS during pregnancy decreased both oxidative stress and involvement of the PAF receptor; however, it also increased involvement of the LOX-1 receptor, which is in line with the concept that scavenger receptors, such as LOX-1 and PAF, are upregulated in response to ligand binding and/or under pathological conditions. In this model of preeclampsia, however, the vascular actions of STS did not lead to improvements in pregnancy outcome such as fetal biometrics or maternal blood pressure.

Aerobic exercise training reduces cardiac function in adult male offspring exposed to prenatal hypoxia.

Intrauterine growth restriction (IUGR) has been associated with increased susceptibility to myocardial ischemia-reperfusion (I/R) injury. Exercise is an effective preventive intervention for cardiovascular diseases; however, it may be detrimental in conditions of compromised health. The aim of this study was to determine whether exercise training can improve cardiac performance after I/R injury in IUGR offspring. We used a hypoxia-induced IUGR model by exposing pregnant Sprague-Dawley rats to 21% oxygen (control) or hypoxic (11% oxygen; IUGR) conditions from gestational day 15 to 21. At 10 wk of age, offspring were randomized to a sedentary group or to a 6-wk exercise protocol. Transthoracic echocardiography assessments were performed after 6 wk. Twenty-four hours after the last bout of exercise, ex vivo cardiac function was determined using a working heart preparation. With exercise training, there was improved baseline cardiac performance in male control offspring but a reduced baseline cardiac performance in male IUGR exercised offspring (P < 0.05). In male offspring, exercise decreased superoxide generation in control offspring, while in IUGR offspring, it had the polar opposite effect (interaction P ≤ 0.05). There was no effect of IUGR or exercise on cardiac function in female offspring. In conclusion, in male IUGR offspring, exercise may be a secondary stressor on cardiac function. A reduction in cardiac performance along with an increase in superoxide production in response to exercise was observed in this susceptible group.

Resistance to cardiomyocyte hypertrophy in ae3-/- mice, deficient in the AE3 Cl-/HCO3- exchanger.

BACKGROUND: Cardiac hypertrophy is central to the etiology of heart failure. Understanding the molecular pathways promoting cardiac hypertrophy may identify new targets for therapeutic intervention. Sodium-proton exchanger (NHE1) activity and expression levels in the heart are elevated in many models of hypertrophy through protein kinase C (PKC)/MAPK/ERK/p90RSK pathway stimulation. Sustained NHE1 activity, however, requires an acid-loading pathway. Evidence suggests that the Cl-/HCO3- exchanger, AE3, provides this acid load. Here we explored the role of AE3 in the hypertrophic growth cascade of cardiomyocytes. METHODS: AE3-deficient (ae3-/-) mice were compared to wildtype (WT) littermates to examine the role of AE3 protein in the development of cardiomyocyte hypertrophy. Mouse hearts were assessed by echocardiography. As well, responses of cultured cardiomyocytes to hypertrophic stimuli were measured. pH regulation capacity of ae3-/- and WT cardiomyocytes was assessed in cultured cells loaded with the pH-sensitive dye, BCECF-AM. RESULTS: ae3-/- mice were indistinguishable from wild type (WT) mice in terms of cardiovascular performance. Stimulation of ae3-/- cardiomyocytes with hypertrophic agonists did not increase cardiac growth or reactivate the fetal gene program. ae3-/- mice are thus protected from pro-hypertrophic stimulation. Steady state intracellular pH (pHi) in ae3-/- cardiomyocytes was not significantly different from WT, but the rate of recovery of pHi from imposed alkalosis was significantly slower in ae3-/- cardiomyocytes. CONCLUSIONS: These data reveal the importance of AE3-mediated Cl-/HCO3- exchange in cardiovascular pH regulation and the development of cardiomyocyte hypertrophy. Pharmacological antagonism of AE3 is an attractive approach in the treatment of cardiac hypertrophy.

Sex-specific differences in the mechanisms for enhanced thromboxane A2-mediated vasoconstriction in adult offspring exposed to prenatal hypoxia.

BACKGROUND: Prenatal hypoxia, a common pregnancy complication, leads to impaired cardiovascular outcomes in the adult offspring. It results in impaired vasodilation in coronary and mesenteric arteries of the adult offspring, due to reduced nitric oxide (NO). Thromboxane A2 (TxA2) is a potent vasoconstrictor increased in cardiovascular diseases, but its role in the impact of prenatal hypoxia is unknown. To prevent the risk of cardiovascular disease by prenatal hypoxia, we have tested a maternal treatment using a nanoparticle-encapsulated mitochondrial antioxidant (nMitoQ). We hypothesized that prenatal hypoxia enhances vascular TxA2 responses in the adult offspring, due to decreased NO modulation, and that this might be prevented by maternal nMitoQ treatment. METHODS: Pregnant Sprague-Dawley rats received a single intravenous injection (100 µL) of vehicle (saline) or nMitoQ (125 µmol/L) on gestational day (GD)15 and were exposed to normoxia (21% O2) or hypoxia (11% O2) from GD15 to GD21 (term = 22 days). Coronary and mesenteric arteries were isolated from the 4-month-old female and male offspring, and vasoconstriction responses to U46619 (TxA2 analog) were evaluated using wire myography. In mesenteric arteries, L-NAME (pan-NO synthase (NOS) inhibitor) was used to assess NO modulation. Mesenteric artery endothelial (e)NOS, and TxA2 receptor expression, superoxide, and 3-nitrotyrosine levels were assessed by immunofluorescence. RESULTS: Prenatal hypoxia resulted in increased U46619 responsiveness in coronary and mesenteric arteries of the female offspring, and to a lesser extent in the male offspring, which was prevented by nMitoQ. In females, there was a reduced impact of L-NAME in mesenteric arteries of the prenatal hypoxia saline-treated females, and reduced 3-nitrotyrosine levels. In males, L-NAME increased U46619 responses in mesenteric artery to a similar extent, but TxA2 receptor expression was increased by prenatal hypoxia. There were no changes in eNOS or superoxide levels. CONCLUSIONS: Prenatal hypoxia increased TxA2 vasoconstrictor capacity in the adult offspring in a sex-specific manner, via reduced NO modulation in females and increased TP expression in males. Maternal placental antioxidant treatment prevented the impact of prenatal hypoxia. These findings increase our understanding of how complicated pregnancies can lead to a sex difference in the programming of cardiovascular disease in the adult offspring.

Prenatal hypoxia, when the fetus does not receive enough oxygen, is a common problem during pregnancy that impacts the developing fetus. It is associated with an increased risk of cardiovascular disease in the offspring in adulthood. While the mechanisms are not fully understood, the blood vessel function in the offspring may be impacted by prenatal hypoxia. We hypothesize that prenatal hypoxia increases the constriction of the blood vessels in the offspring. The placenta, an essential organ for fetal development, supplies oxygen and nutrients to the fetus. In prenatal hypoxia pregnancies, the placenta does not work properly. We have been studying a placental treatment (called nMitoQ) to improve placenta function and thereby the blood vessel function of the offspring. We used a rat model of prenatal hypoxia, where pregnant rats (dams) were placed in a low oxygen environment (hypoxia) during the last trimester of pregnancy. Control rats were kept in normal oxygen conditions. The dams were treated with nMitoQ, or with saline (control). Next, we studied the blood vessels of the offspring in adulthood. We found that prenatal hypoxia increases the constriction of the blood vessels, which was prevented by treating the dams with nMitoQ. Interestingly, this impact was more severe in females compared to males, and the mechanisms were different between the sexes. This study helps in the understanding of how complicated pregnancies can impair cardiovascular health in the offspring, and in a potential development of targeted and sex-specific therapies for those offspring at high risk for future cardiovascular disease.

Quantification of carbonic anhydrase gene expression in ventricle of hypertrophic and failing human heart.

BACKGROUND: Carbonic anhydrase enzymes (CA) catalyze the reversible hydration of carbon dioxide to bicarbonate in mammalian cells. Trans-membrane transport of CA-produced bicarbonate contributes significantly to cellular pH regulation. A body of evidence implicates pH-regulatory processes in the hypertrophic growth pathway characteristic of hearts as they fail. In particular, Na+/H+ exchange (NHE) activation is pro-hypertrophic and CA activity activates NHE. Recently Cardrase (6-ethoxyzolamide), a CA inhibitor, was found to prevent and revert agonist-stimulated cardiac hypertrophy (CH) in cultured cardiomyocytes. Our goal thus was to determine whether hypertrophied human hearts have altered expression of CA isoforms. METHODS: We measured CA expression in hypertrophied human hearts to begin to examine the role of carbonic anhydrase in progression of human heart failure. Ventricular biopsies were obtained from patients undergoing cardiac surgery (CS, n = 14), or heart transplantation (HT, n = 13). CS patients presented mild/moderate concentric left ventricular hypertrophy and normal right ventricles, with preserved ventricular function; ejection fractions were ~60%. Conversely, HT patients with failing hearts presented CH or ventricular dilation accompanied by ventricular dysfunction and EF values of 20%. Non-hypertrophic, non-dilated ventricular samples served as controls. RESULTS: Expression of atrial and brain natriuretic peptide (ANP and BNP) were markers of CH. Hypertrophic ventricles presented increased expression of CAII, CAIV, ANP, and BNP, mRNA levels, which increased in failing hearts, measured by quantitative real-time PCR. CAII, CAIV, and ANP protein expression also increased approximately two-fold in hypertrophic/dilated ventricles. CONCLUSIONS: These results, combined with in vitro data that CA inhibition prevents and reverts CH, suggest that increased carbonic anhydrase expression is a prognostic molecular marker of cardiac hypertrophy.

Increased stiffness of omental arteries from late pregnant women at advanced maternal age.

Advanced maternal age (≥35 years) is a risk factor for poor pregnancy outcomes. Pregnancy requires extensive maternal vascular adaptations, and with age, our blood vessels become stiffer and change in structure (collagen and elastin). However, the effect of advanced maternal age on the structure of human resistance arteries during pregnancy is unknown. As omental resistance arteries contribute to blood pressure regulation, assessing their structure in pregnancy may inform on the causal mechanisms underlying pregnancy complications in women of advanced maternal age. Omental fat biopsies were obtained from younger (<35 years) or advanced maternal age (≥35 years) women during caesarean delivery (n = 7-9/group). Arteries (200-300 µm) were isolated and passive mechanical properties (circumferential stress and strain) assessed with pressure myography. Collagen (Masson's Trichrome) and elastin (Verhoff) were visualized histologically and % positively-stained area was assessed. Median maternal age was 32 years (range 25-34) for younger, and 38 years (range 35-42) for women of advanced maternal age. Circumferential strain was lower in arteries from advanced maternal age versus younger women but circumferential stress was not different. Omental artery collagen levels were similar, while elastin levels were lower with advanced maternal age versus younger pregnancies. The collagen:elastin ratio was greater in arteries from advanced maternal age versus younger women. In conclusion, omental arteries from women of advanced maternal age were less compliant with less elastin compared with arteries of younger controls, which may affect how vascular stressors are tolerated during pregnancy. Understanding how vascular aging affects pregnancy adaptations may contribute to better pregnancy outcomes.

Sex-Specific Differences in the Placental Unfolded Protein Response in a Rodent Model of Gestational Hypoxia.

Gestational hypoxia is a major contributor to fetal growth restriction (FGR) and perinatal morbidity and mortality and has been closely linked to the activation of the unfolded protein response (UPR) in the placenta. Recent studies on adverse pregnancy conditions show differential adaptive responses in pregnancies carrying male or female fetuses. Here, we use an established rat model of hypoxic pregnancy and FGR to test the hypothesis that chronic hypoxia promotes sexually dimorphic activation of the placental UPR. Our data showed that gestational hypoxia increased glucose regulatory protein 78 (GRP78) expression in male placentae, increased activating transcription factor 6 activation (ATF6) in female placentae, and did not induce changes in other UPR markers. In addition, gestational hypoxia reduced fetal weight only in males and ATF6 activation correlated with an increase in the fetal crown-rump-length/body weight ratio only in females. These results suggest sex-specific divergence in the placental adaptive response to gestational hypoxia, which may account for the sexual dimorphism observed in placental function and pregnancy outcomes in complicated pregnancies.

Placenta-Derived Extracellular Vesicles From Preeclamptic Pregnancies Impair Vascular Endothelial Function via Lectin-Like Oxidized LDL Receptor-1.

BACKGROUND: Preeclampsia is a complex syndrome that includes maternal vascular dysfunction. Syncytiotrophoblast-derived extracellular vesicles from preeclampsia placentas (preeclampsia-STBEVs) were shown to induce endothelial dysfunction, but an endothelial transmembrane mediator is still unexplored. The LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is a transmembrane scavenger receptor that can cause endothelial dysfunction, and its expression is increased in the endothelium of preeclampsia women. In this study, we hypothesized that LOX-1 mediates the effects of preeclampsia-STBEVs on endothelial function. METHODS: Preeclampsia-STBEVs were collected by perfusion of placentas from women with preeclampsia and in vitro and ex vivo endothelial cell function were assessed. RESULTS: In human umbilical vein endothelial cells, inhibition of LOX-1 with LOX-1 blocking antibody (TS20) reduced the uptake of preeclampsia-STBEVs (61.3±8.8%). TS20 prevented the activation of ERK (extracellular signal-regulated kinase, a kinase downstream of LOX-1) and reduced the activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells; 21.1±8.0%) and nitrative stress (23.2±10.3%) that was induced by preeclampsia-STBEVs. Vascular function was assessed by wire myography in isolated mesenteric arteries from pregnant rats that were incubated overnight with preeclampsia-STBEVs±TS20. TS20 prevented endothelium-dependent vasodilation impairment induced by preeclampsia-STBEVs. Nitric oxide contribution to the relaxation was reduced by preeclampsia-STBEVs, which was prevented by TS20. Superoxide dismutase or apocynin, an inhibitor of NOX (nicotinamide adenine dinucleotide phosphate oxidase), restored the impaired endothelium-dependent vasodilation in arteries exposed to preeclampsia-STBEVs. CONCLUSIONS: Taken together, our findings demonstrate that LOX-1 mediates the endothelial dysfunction induced by preeclampsia-STBEVs. Our study further expands on the mechanisms that may lead to adverse outcomes in preeclampsia and proposes LOX-1 as a potential target for future interventions.

Oligomerization of SLC4A11 protein and the severity of FECD and CHED2 corneal dystrophies caused by SLC4A11 mutations.

Mutations in the SLC4A11 gene, which encodes a plasma membrane borate transporter, cause recessive congenital hereditary endothelial corneal dystrophy type 2 (CHED2), corneal dystrophy and perceptive deafness (Harboyan syndrome), and dominant late-onset Fuchs endothelial corneal dystrophy (FECD). We analyzed missense SLC4A11 mutations identified in FECD and CHED2 patients and expressed in transfected HEK 293 cells. Chemical cross-linking and migration in nondenaturing gels showed that SLC4A11 exists as a dimer. Furthermore, co-immunoprecipitation of epitope-tagged proteins revealed heteromeric interactions between wild-type (WT) and mutant SLC4A11 proteins. When expressed alone, FECD- and CHED2-causing mutant SLC4A11 proteins are primarily retained intracellularly. Co-expression with WT SLC4A11 partially rescued the cell surface trafficking of CHED2 mutants, but not FECD mutants. CHED2 alleles of SLC4A11 did not affect cell surface processing of WT SLC4A11. In contrast, FECD mutants reduced WT cell surface processing efficiency, consistent with dominant inheritance of FECD. The reduction in movement of WT protein to the cell surface caused by FECD SLC4A11 helps to explain the dominant inheritance of this disorder. Similarly, the failure of CHED2 mutant SLC4A11 to affect the processing of WT protein, explains the lack of symptoms found in CHED2 carriers and the recessive inheritance of the disorder.