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.

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.

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.

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 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.

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.

The Long-Term Effects of Prenatal Hypoxia on Coronary Artery Function of the Male and Female Offspring.

Prenatal hypoxia predisposes the offspring to the development of cardiovascular (CV) dysfunction in adult life. Using a rat model, we assessed the effect of prenatal hypoxia on vasoconstrictive and vasodilative mechanisms in left anterior descending coronary arteries of 4- and 9.5-month-old offspring. Endothelium-dependent relaxation to methylcholine and vasoconstriction responses to endothelin-1 (ET-1) were assessed by wire myography. Prenatal hypoxia impaired endothelium-dependent vasodilation in 4- and 9.5-month-old offspring. Inhibition of nitric oxide (NO) synthase prevented coronary artery relaxation in all groups. Inhibition of prostaglandin H synthase (PGHS) improved relaxation in prenatally hypoxic males and tended to improve vasorelaxation in females, suggesting that impaired vasodilation was mediated via increased PGHS-dependent vasoconstriction. An enhanced contribution of endothelium-dependent hyperpolarization to coronary artery vasodilation was observed in prenatally hypoxic males and females. No changes in endothelial NO synthase (eNOS) and PGHS-1 expressions were observed, while PGHS-2 expression was decreased in only prenatally hypoxic males. At 4 months, ET-1 responses were similar between groups, while ETB inhibition (with BQ788) tended to decrease ET-1-mediated responses in only prenatally hypoxic females. At 9.5 months, ET-1-mediated responses were decreased in only prenatally hypoxic females. Our data suggest that prenatal hypoxia has long-term similar effects on the mechanisms of impaired endothelium-dependent vasodilation in coronary arteries from adult male and female offspring; however, coronary artery contractile capacity is impaired only in prenatally hypoxic females. Understanding the mechanistic pathways involved in the programming of CV disease may allow for the development of therapeutic interventions.

Placenta-derived extracellular vesicles from preeclamptic and healthy pregnancies impair ex vivo vascular endothelial function.

Preeclampsia (PE) is a pregnancy syndrome characterized by new-onset hypertension and end-organ dysfunction. The pathophysiology of PE remains undetermined, but it is thought that maternal vascular dysfunction plays a central role, potentially due, in part, to the release of syncytiotrophoblast-derived extracellular vesicles (STBEVs) into the maternal circulation by a dysfunctional placenta. STBEVs from normal pregnancies (NP) impair vascular function, but the effect of PE STBEVs (known to differ in composition with elevated circulating levels) on vascular function are not known. We hypothesized that PE STBEVs have more detrimental effects on vascular function compared with NP STBEVs. STBEVs were collected by perfusion of placentas from women with NP or PE. Mesenteric arteries from pregnant rats were incubated overnight with NP or PE STBEVs, and vascular function was assessed by wire myography. NP and PE STBEVs impaired endothelial function, partially by reducing nitric oxide (NO) bioavailability. Incubation of human umbilical vein endothelial cells with NP and PE STBEVs increased nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB) activation, reactive oxygen species, nitrotyrosine levels, and reduced NO levels. However, PE STBEVs increased NF-κB activation and nitrotyrosine levels to a lesser extent than NP STBEVs. Taken together, no greater impact of PE STBEVs compared with NP STBEVs on endothelial function was found. However, the impaired vascular function by PE STBEVs and increased levels of STBEVs in PE suggest PE STBEVs may contribute to maternal vascular dysfunction in PE. Our study further expands on the potential mechanisms that lead to adverse outcomes in PE and provides potential targets for future interventions.

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.

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.

Increased oxidative stress and endothelial activation in umbilical veins from pregnancies diagnosed with preeclampsia.

Vascular dysfunction is a hallmark of cardiovascular disease (CVD). Offspring from preeclamptic pregnancies are at risk of CVD later in life. Whether fetal vasculature from preeclamptic pregnancies displays signs of vascular dysfunction (i.e., oxidative/nitrosative stress, endothelial activation) associated with increased expression of lectin-like oxidized LDL receptor-1 (LOX-1) and angiotensin-II type-1 receptor (AT1) is unknown. We demonstrated increased superoxide, nitrotyrosine and ICAM-1 levels in umbilical vein tissues of preeclamptic vs. normal pregnancies; without changes in LOX-1 and AT1 levels. Our findings suggest that the fetal vasculature may be impacted in preeclampsia, which could contribute to an increased risk of offspring CVD.

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.

Role of Lectin-like Oxidized LDL Receptor-1 and Syncytiotrophoblast Extracellular Vesicles in the Vascular Reactivity of Mouse Uterine Arteries During Pregnancy.

Vascular complications in pregnancy (e.g. preeclampsia) are a major source of maternal and foetal morbidity and mortality, and may be due to excessive release of placental syncytiotrophoblast-derived extracellular vesicles (STBEVs) into the maternal circulation. Increased activity of the multi-ligand scavenger receptor Lectin-like Oxidized LDL Receptor-1 (LOX-1) is associated with vascular dysfunction, and LOX-1 has been shown to interact with angiotensin II receptor type 1 (AT1). We hypothesized that STBEVs contribute to vascular dysfunction via LOX-1 and AT1 receptors during pregnancy. Uterine arteries from late pregnant wildtype and LOX-1 overexpressing mice were incubated overnight with or without STBEVs and vascular function was assessed using wire myography. STBEV-incubation decreased angiotensin II responsiveness only in wildtype mice, which coincided with decreased AT1 contribution and expression. Thus, STBEVs reduced angiotensin II responsiveness in normal pregnancy, but not in conditions of increased LOX-1 expression, suggesting that STBEVs (via LOX-1) play a role in normal adaptations to pregnancy. Oxidized LDL (a LOX-1 ligand) increased angiotensin II-induced vasoconstriction in STBEV-incubated arteries from both mouse strains, suggesting that the LOX-1 pathway may be involved in complicated pregnancies with elevated STBEVs and oxidized LDL levels (such as preeclampsia). These data increase our understanding of vascular complications during pregnancy.

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.

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.

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.

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.

Syncytiotrophoblast extracellular vesicles impair rat uterine vascular function via the lectin-like oxidized LDL receptor-1.

Syncytiotrophoblast extracellular vesicles (STBEVs) are placenta derived particles that are released into the maternal circulation during pregnancy. Abnormal levels of STBEVs have been proposed to affect maternal vascular function. The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a multi-ligand scavenger receptor. Increased LOX-1 expression and activation has been proposed to contribute to endothelial dysfunction. As LOX-1 has various ligands, we hypothesized that, being essentially packages of lipoproteins, STBEVs are able to activate the LOX-1 receptor thereby impairing vascular function via the production of superoxide and decreased nitric oxide bioavailability. Uterine arteries were obtained in late gestation from Sprague-Dawley rats and incubated for 24h with or without human STBEVs (derived from a normal pregnant placenta) in the absence or presence of a LOX-1 blocking antibody. Vascular function was assessed using wire myography. Endothelium-dependent maximal vasodilation to methylcholine was impaired by STBEVs (MCh Emax: 57.7±5.9% in STBEV-incubated arteries vs. 77.8±2.9% in controls, p<0.05). This was prevented by co-incubation of STBEV-incubated arteries with LOX-1 blocking antibodies (MCh Emax: 78.8±4.3%, p<0.05). Pre-incubation of the vessels with a nitric oxide synthase inhibitor (L-NAME) demonstrated that the STBEV-induced impairment in vasodilation was due to decreased nitric oxide contribution (ΔAUC 12.2±11.7 in STBEV-arteries vs. 86.5±20 in controls, p<0.05), which was abolished by LOX-1 blocking antibody (ΔAUC 98.9±17, p<0.05). In STBEV-incubated vessels, LOX-1 inhibition resulted in an increased endothelial nitric oxide synthase expression (p<0.05), to a level similar to control vessels. The oxidant scavenger, superoxide dismutase, did not improve this impairment, nor were vascular superoxide levels altered. Our data support an important role for STBEVs in impairment of vascular function via activation of LOX-1 and reduced nitric oxide mediated vasodilation. Moreover, we postulate that the LOX-1 pathway could be a potential therapeutic target in pathologies associated with vascular dysfunction during pregnancy.

Postnatal resveratrol supplementation improves cardiovascular function in male and female intrauterine growth restricted offspring.

Intrauterine growth restriction (IUGR) may predispose offspring to an increased susceptibility of developing cardiovascular disease (CVD) in adult life. The window of opportunity to treat later life CVD programmed in fetal life is critical. The aim of this study was to identify the effect of resveratrol treatment of IUGR offspring at a time of known CV dysfunction. Sprague-Dawley male and female rat offspring who experienced normoxia (21% O2; control) or hypoxia (11% O2; IUGR) in utero were fed a high-fat (HF) diet (3-21 weeks of age) or a HF diet (3-21 weeks of age) supplemented with resveratrol from 13 to 21 weeks of age. At 21 weeks of age, echocardiographic data showed that male IUGR offspring had mild in vivo diastolic dysfunction, whereas female IUGR offspring had early signs of cardiac diastolic dysfunction that was not altered by resveratrol treatment. Notably, male and female IUGR offspring demonstrated equal susceptibility to ex vivo cardiac dysfunction recovery after ischemia/reperfusion (I/R) injury and this was improved by resveratrol treatment, independent of sex. Resveratrol increased cardiac phospho-adenosine monophosphate kinase (p-AMPK) levels in only female IUGR offspring. IUGR or resveratrol did not alter cardiac superoxide levels. However, in male offspring, an overall effect of IUGR in reducing cardiac catalase levels was observed that was not altered by resveratrol. Interestingly, in only female IUGR offspring, resveratrol significantly increased cardiac superoxide dismutase (SOD) 2 levels. In conclusion, resveratrol treatment of adult IUGR offspring, at the time of known CV dysfunction, improved cardiac function recovery in both sexes and the mechanisms involved were partially sex-specific.

Postpartum Vascular Dysfunction in the Reduced Uteroplacental Perfusion Model of Preeclampsia.

Preeclampsia is a disorder affecting 2-8% of all pregnancies, characterized by gestational hypertension (≥ 140/90 mmHg) and proteinuria (≥300 mg over 24 hours) diagnosed following the 20th week of pregnancy, and for which there is currently no available treatment. While the precise cause of preeclampsia is unknown, placental ischemia/hypoxia resulting from abnormal trophoblast invasion and maternal endothelial dysfunction are central characteristics. Preeclampsia is a major cause of both maternal and fetal morbidity and mortality in the perinatal period. In addition, women who have experienced preeclampsia are more likely to suffer cardiovascular disease later in life. The cause of this elevation in cardiovascular risk postpartum, however, is unknown. We hypothesize that there may be lasting vascular dysfunction following exposure to reduced uteroplacental perfusion during pregnancy that may contribute to increased cardiovascular risk postpartum. Using the rat reduced utero-placental perfusion pressure (RUPP) model of preeclampsia, blood pressure was assessed in dams at gestational day 20, one and three months postpartum. Mesenteric artery and aortic function were assessed using wire myography. We demonstrated hypertension and increased mesenteric artery responses to phenylephrine at gestational day 20, with the latter due to a decreased contribution of nitric oxide without any change in methylcholine-induced relaxation. At one month postpartum, we demonstrated a small but significant vasoconstrictive phenotype that was due to an underlying loss of basal nitric oxide contribution. At three months postpartum, endothelium-dependent relaxation of the aorta demonstrated sensitivity to oxLDL and mesenteric arteries demonstrated decreased nitric oxide bioavailability with impaired methylcholine-induced relaxation; indicative of an early development of endothelial dysfunction. In summary, we have demonstrated impaired vascular function following exposure to a RUPP pregnancy that continued into the postpartum period; suggesting that a pregnancy complicated by preeclampsia may predispose women to later life cardiovascular disease via ongoing vascular dysfunction.