Excessive hypercholesterolemia in pregnancy is a risk factor for the development of preeclampsia, but its ensuing impact on offspring cardiovascular health remains not fully understood. In the present study, Sprague Dawley rats were fed a control or high cholesterol diet (HCD) from gestational days 6-20 (term=22 days). Female and male offspring were aged to adulthood (4 months old), and in vivo and ex vivo cardiovascular function were assessed. In female offspring, body weight gain was greater in the HCD group compared with controls after 2 months of age. Blood pressure, echocardiographic parameters, cardiac capacity to recover from an ischemic insult, and endothelium-dependent vasodilation were similar between the female groups. However, vasoconstriction to a thromboxane analog (U46619) was reduced in thoracic aortas, but not in mesenteric, coronary, or carotid arteries, from the HCD females compared with controls, and was associated with lower phosphorylated myosin phosphatase target subunit 1 (MYPT1Thr855) levels. In male offspring, body weight gain, blood pressure, and echocardiographic parameters were similar between the groups, and there was no impact of HCD on vasoconstriction to U46619 or vasodilation in mesenteric arteries, carotid arteries, or thoracic aortas. However, endothelium-dependent vasodilation was reduced in coronary arteries of HCD males compared with controls and was associated with increased reactive oxygen species levels. There was also reduced cardiac capacity of the HCD males to recover from an ischemic insult. In summary, excessive hypercholesterolemia in pregnancy impaired the cardiovascular health of adult female and male offspring, but the mechanisms and vascular beds affected were specific to each sex.

Excessive hypercholesterolemia (eHC) in pregnancy is associated with complications such as preeclampsia, yet its impact on maternal cardiac adaptation and long-term cardiovascular health remains unclear. We hypothesized that eHC impairs maternal cardiac structure and function during pregnancy and that these effects persist postpartum. Sprague Dawley rats were fed either a control diet (CTL) or high cholesterol diet (eHC) from gestational day (GD) 6-20 (term=22 days). Echocardiography was performed before pregnancy, at GD20, and 3 months postpartum. Plasma and left ventricular tissue were collected at GD20 and postpartum for molecular analyses. CTL dams demonstrated normal pregnancy-associated cardiac adaptations, including increased diastolic left ventricular internal diameter, stroke volume, cardiac output, and diastolic left ventricular volume in late pregnancy, with normalization postpartum. These adaptations were attenuated in eHC dams. In late pregnancy, eHC dams exhibited reduced stroke volume and cardiac output compared with CTL, along with alterations in diastolic function, including reduced mitral valve A' velocity. Postpartum, eHC dams showed changes in diastolic indices, including reduced mitral valve A-wave velocity and E'/A' ratio. Measures of systolic performance (ejection fraction and fractional shortening) were unchanged between groups. Molecular analyses revealed increased cardiomyocyte size, altered collagen remodeling characterized by an increased COL1-to-COL3 ratio, and elevated CD31 expression in eHC dams during pregnancy. Postpartum, collagen remodeling was evident, but now with reduced COL1 and increased COL3 expression. Together, these findings demonstrate that eHC disrupts normal maternal cardiac adaptation during pregnancy and promotes adverse remodeling and cardiac dysfunction postpartum, with potential implications for long-term cardiovascular disease risk.

Abstract Background Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of morbidity and mortality worldwide. Despite effective lipid‐lowering treatments, substantial residual risks remain. In atherosclerosis, vascular smooth muscle cells (SMCs) undergo dedifferentiation, promoting disease progression. Membrane‐type I matrix metalloproteinase (MT1‐MMP/MMP14) promotes SMC dedifferentiation. However, the effect of inhibiting MMP14 in adults, particularly those with existing atherosclerotic plaques, is unclear. Methods We developed an inducible conditional SMC‐specific MMP14 knockout mouse model. Cardiac and vascular function were assessed using echocardiography and wire myography, respectively. Atherosclerosis progression and regression were evaluated in Ldlr−/− mice with or without MMP14 deficiency. snRNA‐seq of the aortas from Ldlr−/− mice was performed to determine the effect on SMC populations. Results MMP14 expression was elevated in SMCs within fibroatheroma compared with the pathological intima thickening in coronary aortas from patients with ASCVD. Conditional knockout of SMC MMP14 in adult mice did not change plasma cholesterol levels or basic cardiac and vascular function. However, atherosclerosis development was reduced, and the regression of existing plaques was enhanced in Ldlr−/− mice lacking SMC MMP14. snRNA‐seq revealed increased fibroblast‐like SMCs and reduced foam cell‐like SMCs in MMP14‐deficient Ldlr−/− mice compared to Ldlr−/− mice. Furthermore, SMC MMP14 deficiency decreased SMC proliferation and migration, accompanied by reduced platelet‐derived growth factor receptor (PDGFR) β levels and attenuated PDGF signalling. Conclusion SMC MMP14 promotes atherosclerosis in adult mice, likely through reducing PDGF signalling and inhibiting SMC migration and proliferation.

Low-dose aspirin is recommended to pregnant individuals at increased risk of preeclampsia to improve outcomes. We recently showed that low-dose aspirin improves uterine artery function in a rat model of excessive hypercholesterolemia (eHC) in pregnancy, a known risk factor for preeclampsia. However, its effects on placentas from male and female offspring remain unclear. Here, we examined how low-dose aspirin affects various placental inflammatory and angiogenic markers, as well as the maternal soluble fms-like tyrosine kinase receptor-1 (sFlt-1)/placental growth factor (PlGF) ratio, in eHC pregnancies. We hypothesized that low-dose aspirin reduces placental inflammation, leading to angiogenic balance in these pregnancies. Sprague-Dawley rats were fed a control diet or a high cholesterol diet (to model eHC) from gestational day (GD) 6 to 20, with placebo or low-dose aspirin administered from GD10 to 20. On GD20, placentas were collected and separated based on the fetal sex. eHC in pregnancy elevated maternal plasma sFlt-1 without altering PlGF, resulting in an increased sFlt-1/PlGF ratio; that did not occur with low-dose aspirin treatment. Moreover, placental sFlt-1 was increased in male, but not in female, fetuses, and was reduced by low-dose aspirin. Placental PlGF was reduced in males, but increased in females, of eHC pregnancies; low-dose aspirin restored placental PlGF in only the female placentas. NLRP3 (a major placental inflammatory pathway) levels were increased in only eHC male placentas and normalized by low-dose aspirin. These findings reveal that low-dose aspirin restores the maternal plasma sFlt-1/PlGF ratio and suppresses placental inflammation through sex-specific mechanisms in eHC pregnancies.NEW & NOTEWORTHY This study demonstrates that pregnancies complicated by excessive hypercholesterolemia have a higher maternal plasma sFlt-1/PlGF ratio driven by increased sFlt-1 levels, indicating an angiogenic imbalance. Importantly, this imbalance can be prevented with low-dose aspirin, an actionable therapeutic option during pregnancy. Low-dose aspirin suppressed placental inflammation (evidenced by decreased NLRP3 levels) and lowered placental-derived sFlt-1 via sex-specific mechanisms.

Prenatal hypoxia, a common pregnancy complication, leads to cardiac and vascular dysfunction, thereby increasing the risk of cardiovascular disease in the adult offspring. Carotid arteries are responsible for the majority of the blood flow to the brain/head, and carotid artery dysfunction is associated with life-threatening cardiovascular events, such as stroke. However, whether prenatal hypoxia exposure impacts the function of the carotid arteries in the adult offspring is not known. We hypothesize that prenatal hypoxia impairs carotid artery function in the adult male and female offspring. Sprague Dawley rats were exposed to normoxia (21% O-2) or hypoxia (11% O-2) from gestational day 15 to 21 (term = 22 days; n = 9 or 10/group). Carotid arteries were isolated from the 4-mo-old male and female offspring. Vasoconstrictor and vasodilatory properties were assessed by wire myography, and biomechanical properties (myogenic tone, circumferential stress, and strain) were assessed by pressure myography. Collagen deposition (Masson's trichrome stain) and elastin density (Verhoeff stain) were measured in carotid artery cryosections. Prenatal hypoxia did not impact vasoconstriction or vasorelaxation responses in carotid arteries from both offspring. However, in males, prenatal hypoxia reduced carotid artery myogenic tone development and increased circumferential strain, which coincided with lower collagen deposition and higher elastin density. In females, prenatal hypoxia tended to lower carotid artery circumferential strain (i.e., increased stiffness), without differences in myogenic tone or collagen/elastin density. Altogether, these data show that exposure to prenatal hypoxia affects the carotid arteries of the adult offspring in a sex-specific manner, which may impact the blood flow regulation to the brain. NEW & NOTEWORTHY Little is known about the (long-lasting) impact of pregnancy complications on offspring carotid artery function. We showed that, in adult male offspring, prenatal hypoxia decreased carotid artery myogenic tone and stiffness and changed collagen and elastin densities, whereas in females, prenatal hypoxia increased stiffness. These findings contribute to understanding sex-specific differences of adult offspring exposed to a suboptimal in utero environment on the carotid arteries, an important/easily accessible vascular bed for patient disease evaluation.

Excessive hypercholesterolemia (eHC) in pregnancy reduces placental efficiency in both fetal sexes, but the mechanisms are not known. In this study, Sprague Dawley rats received a control or high cholesterol diet (to model eHC) during pregnancy, after which various markers of placental function were assessed. Lipid levels, but not reactive oxygen species levels, were increased in both the male and female eHC placentas vs. controls. However, compared to control placentas, eHC reduced cholesterol receptors, increased cholesterol transporters, lowered fetal cholesterol levels, and altered the unfolded protein response in a sex-specific manner. Moreover, NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) levels were increased in only the male eHC placentas, and associated with reduced interleukin 1β levels, likely due to its rapid secretion into the circulation. The levels of caspase 8, but not caspase 1, were increased in only the male eHC placentas vs. controls, suggesting that the processing of interleukin 1β may have happened via a non-canonical pathway. In conclusion, eHC in pregnancy impacts the placentas of both the male and female offspring, but the activation of the NLRP3 inflammasome in only the male placentas suggests that the male offspring may be more susceptible to excessive increases in maternal cholesterol levels.

BACKGROUND:Excessive hypercholesterolemia in pregnancy increases the risk of preeclampsia (HC-PE), though the mechanisms remain unclear. We recently showed that uterine artery function is impaired in HC-PE pregnancies via activation of the TLR4 (toll-like receptor 4)/PGHS1 (prostaglandin H synthase 1) pathway. Low-dose aspirin lowers preeclampsia risk in high-risk pregnancies by inhibiting PGHS1, but its effects in HC-PE pregnancies are not known. Moreover, oxidized low-density lipoprotein (oxLDL) levels rise in HC-PE, potentially activating TLR4 and LOX-1 (lectin-like oxLDL receptor-1; scavenger receptor linked to vascular dysfunction in preeclampsia). However, whether this occurs in HC-PE is not known. METHODS:Sprague Dawley rats received a control or high-cholesterol diet (to induce HC-PE) from gestational day 6 to 20, with placebo or low-dose aspirin (1.5 mg/kg daily) given from gestational day 10 to 20. On gestational day 20, pregnancy outcomes and uterine artery function were assessed. RESULTS:Uterine artery blood flow velocity and placental weights were higher in HC-PE placebo-treated dams versus controls, but these were reduced by low-dose aspirin. Endothelium-dependent vasodilation was impaired in the uterine arteries of the HC-PE placebo group versus controls and was corrected by low-dose aspirin. Ex vivo inhibition of TLR4, PGHS1, or LOX-1 also normalized endothelium-dependent vasodilation in the HC-PE placebo-treated dams. Exposure to oxLDL in the bath (modeling a secondary hit) further impaired endothelium-dependent vasodilation in the uterine arteries of the HC-PE placebo group, partially via TLR4 and LOX-1, which was prevented by low-dose aspirin. CONCLUSIONS:Low-dose aspirin improved uterine artery endothelial function in HC-PE pregnancies; likely by suppressing the TLR4/LOX-1/PGHS1 pathway.

Cardiovascular disease (CVD) is the leading cause of death globally in both men and women. Sex differences exist in the onset, progression, and outcomes of CVD, with sex-specific factors such as pregnancy-related conditions likely contributing to these disparities. Preeclampsia (PE) and gestational diabetes mellitus (GDM) are two of the most common medical complications of pregnancy, affecting approximately 3–5% and 5–18%, respectively. Women with a history of PE or GDM are at a higher risk of developing CVD later in life. Still, the underlying mechanisms are not fully understood. This review presents an overview of the mechanisms underlying the development of PE and GDM, and discusses pathophysiological pathways revealed through animal models that could contribute to long-term maternal cardiovascular dysfunction.

Background Preeclampsia is a risk factor for the development of later‐life cardiovascular disease. However, the underlying mechanisms are poorly understood. Excessive hypercholesteremia in pregnancy induces a preeclampsia‐like phenotype, but whether this also impacts maternal vascular function later in life has not been fully characterized. Methods and Results Sprague Dawley rats received a control diet (CD) or a high‐cholesterol (HCD) diet from gestational day 6 to 20, after which maternal vascular function was assessed 3 months postpartum. Exposure to an HCD in pregnancy reduced later‐life endothelium‐dependent vasodilation in carotid arteries (−15.24±3.27%), which was mediated via prostaglandin H synthase 2. There were no differences in vasodilation between CD and HCD postpartum rats in the mesenteric arteries, coronary arteries, or aortas. Vasoconstriction to phenylephrine increased in carotid arteries (61.02±21.48%) and reduced in aortas (−23.24±6.19%) of the HCD postpartum group versus CD dams, without differences in mesenteric and coronary arteries. The increased vasoconstriction in carotid arteries was due to lower nitric oxide modulation of constriction. Moreover, carotid artery myogenic response was reduced (−37.68±10.07%) and stiffness was increased (19.67±6.21%) in the HCD postpartum rats compared with CD along with decreased elastin density (−20.85±4.52%). The impact of the HCD on vascular function did not occur in age‐matched never‐pregnant female rats. Conclusions Excessive hypercholesterolemia in pregnancy impairs later‐life maternal vascular function in rats with varying impacts across different vascular beds. Understanding mechanisms for pregnancy‐specific excessive hypercholesterolemia provides avenues for targeted intervention strategies to reduce the burden of cardiovascular disease in women who had a complicated pregnancy.

Background: Preeclampsia is a significant risk factor for the development of later-life cardiovascular disease; however, the underlying molecular mechanisms are not fully understood. Studies have shown that excessive hypercholesteremia occurring during pregnancy increases the risk of vascular pregnancy complications, such as preeclampsia. Bioavailability of nitric oxide (NO), a critical endothelium-derived vasodilator in pregnancy, is reduced in both hypercholesteremia and preeclampsia. However, the later-life vascular impact of excessive pregnancy-specific hypercholesteremia is not known. Hypothesis: Excessive hypercholesteremia in pregnancy impairs later-life maternal vascular function by reducing NO availability. Methods: Sprague-Dawley rats were fed a control diet (CD) or high cholesterol diet (HCD; 2% cholesterol + 0.5% cholic acid) from gestational day 6 to 20 (term=22 days; n=10-12). After pregnancy, all dams received a CD. Three months after pregnancy (equal to ~10 years in humans), mesenteric (systemic resistance arteries) and carotid (vascular bed that supplies blood to the cerebral vasculature) arteries were isolated, and endothelium-dependent (to methacholine, MCh) and -independent (to sodium nitroprusside, SNP) vasodilation, plus vasoconstriction responses (to phenylephrine, PE) were assessed using wire myography. NO contribution was assessed using a pan-NO synthase inhibitor (L-NAME; 100 μmol). Data were summarized as maximum responses (Emax), sensitivity (pEC50), or delta Emax (control vs. L-NAME). The statistical tests applied were a Student’s t-test or two-way ANOVA with Sidak’s post-hoc test (significance: p<0.05). Results: In mesenteric arteries, vasodilation to MCh or SNP was not different between the CD and HCD groups three months after the insult. However, L-NAME reduced maximum vasodilation to MCh in the HCD group (p=0.0433) but not in CD animals. Similarly, vasoconstriction to PE was not different between groups, but L-NAME increased the PE sensitivity in the HCD group (p=0.0423), without effects in the CD animals. In carotid arteries, a HCD during pregnancy reduced vasodilation to MCh (p=0.0345), and while L-NAME prevented vasodilation to MCh in both groups (p<0.0001), this effect was more pronounced in the HCD compared to CD group (delta Emax, p=0.0422). Vasodilation to SNP was similar between groups. Vasoconstriction responses to PE were increased in carotid arteries of HCD compared to CD animals (p=0.0357). L-NAME increased PE-mediated vasoconstriction in both groups (CD: 4.32-fold and HCD: 2.71-fold), but this NO modulation was significantly less in the HCD group compared to CD (delta Emax, p=0.0337). Conclusion: Exposure to a HCD, during pregnancy only, impaired later-life maternal vascular function in carotid, but not mesenteric arteries, suggesting vascular-bed specific effects in animals with a history of preeclampsia. Moreover, a HCD during pregnancy altered the NO pathway in both vascular beds, indicating that long-term alterations in NO were established during pregnancy. In summary, excessive pregnancy-specific hypercholesterolemia contributes to later-life maternal vascular dysfunction via modulation of the NO pathway, which may help explain the increased cardiovascular risk after preeclampsia. This work was supported by a foundation grant from the Canadian Institutes of Health Research (CIHR FS154313) and by the Women and Children’s Health Research Institute (WCHRI) through the generosity of the Stollery Children’s Hospital Foundation and the Alberta Women’s Health Foundation. A.A.O. is supported by a WCHRI postdoctoral fellowship through the generosity of the Stollery Children’s Hospital Foundation and the Alberta Women’s Health Foundation. E.E. was supported by a summer research studentship from Alberta Innovates. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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.

Abstract 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. Graphical Abstract

Prenatal hypoxia is associated with placental oxidative stress, leading to impaired fetal growth and an increased risk of cardiovascular disease in the adult offspring; however, the mechanisms are unknown. Alterations in mitochondrial function may result in impaired cardiac function in offspring. In this study, we hypothesized that cardiac mitochondrial function is impaired in adult offspring exposed to intrauterine hypoxia, which can be prevented by placental treatment with a nanoparticle-encapsulated mitochondrial antioxidant (nMitoQ). Cardiac mitochondrial respiration was assessed in 4-month-old rat offspring exposed to prenatal hypoxia (11% O2) from gestational day (GD)15–21 receiving either saline or nMitoQ on GD 15. Prenatal hypoxia did not alter cardiac mitochondrial oxidative phosphorylation capacity in the male offspring. In females, the NADH + succinate pathway capacity decreased by prenatal hypoxia and tended to be increased by nMitoQ. Prenatal hypoxia also decreased the succinate pathway capacity in females. nMitoQ treatment increased respiratory coupling efficiency in prenatal hypoxia-exposed female offspring. In conclusion, prenatal hypoxia impaired cardiac mitochondrial function in adult female offspring only, which was improved with prenatal nMitoQ treatment. Therefore, treatment strategies targeting placental oxidative stress in prenatal hypoxia may reduce the risk of cardiovascular disease in adult offspring by improving cardiac mitochondrial function in a sex-specific manner.