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.

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.

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.

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.

Matrix metalloproteinase enhances big-endothelin-1 constriction in mesenteric vessels of pregnant rats with reduced uterine blood flow.

Preeclampsia is a leading cause of maternal and fetal morbidity/mortality; however, the pathophysiological mechanisms are unclear. Vascular endothelial dysfunction in preeclampsia has been partially attributed to changes in endothelin-1 (ET-1). Several enzymes, including matrix metalloproteinases (MMPs; particularly MMP-2), cleave the inactive precursor big ET-1 (bET-1) to active ET-1. Notably, expression levels of MMP-2 have been shown to be on the increase in women who subsequently develop preeclampsia. We hypothesized that the increased MMP-2 expression leads to increased bET-1 conversion, thereby increasing vasoconstriction in preeclampsia. A reduced uteroplacental perfusion pressure (RUPP) model of preeclampsia in the rat was used to assess mesenteric artery vascular function. Responses to bET-1 (3-310 nmol/L) and ET-1 (1-200 nmol/L) were studied in the presence or absence of inhibitors of enzymes known to cleave bET-1. Vascular contractility in response to bET-1 was greater in RUPP than Sham (P<0.001), whereas neither responses to ET-1 nor maximal contractility to high potassium salt solution (123.70 mmol/L) were different. MMP inhibition with GM6001 (30 µmol/L) significantly decreased responses to bET-1 in RUPP (P<0.001) but not Sham-operated rats. Interestingly, combined treatment with GM6001 and L-NG-nitroarginine methyl ester (100 µmol/L) revealed a NO modulation of MMPs that was reduced in RUPP. In summary, we found increased vascular contractility to bET-1 in the RUPP model of preeclampsia that was likely attributable to upstream enzymatic pathways. These data are consistent with a greater contribution of MMP to cleavage of bET-1 to ET-1 ex vivo in RUPP, suggesting that this enzyme may be partially responsible for increased bET-1-induced contractility.

Inhibition of lectin-like oxidized low-density lipoprotein-1 receptor protects against plasma-mediated vascular dysfunction associated with pre-eclampsia.

BACKGROUND: Pre-eclampsia (PE) is associated with vascular endothelial dysfunction and oxidative stress initiated by impaired trophoblast invasion. Oxidative stress modifies circulating low-density lipoprotein (LDL) to oxidized LDL (oxLDL). Lectin-like oxLDL receptor-1 (LOX-1) is a scavenger receptor for oxLDL. We hypothesized that plasma from patients with PE alters LOX-1 in normal human vessels during pregnancy, causing oxLDL-induced impairment of vascular function. METHODS: Control-matched plasma was obtained from women with PE (n = 6). Oxidized LDL and soluble LOX-1 levels were determined by enzyme-linked immunoassay (ELISA). Remaining plasma was pooled and stored at -80ºC. Human omental arteries were incubated in 3% plasma from normal pregnant (NP) women or plasma from women with PE. Expression of LOX-1 in these vessels was determined by immunohistochemistry with antibodies against LOX-1. The omental vessels were exposed to oxLDL and the LOX-1 inhibitor TS20. Vascular function was assessed in response to the vasoconstrictor U46619 and the vasodilators bradykinin (BK) and sodium nitroprusside (SNP). RESULTS: No significant differences in the concentrations of oxLDL or soluble LOX-1 (sLOX-1) were found in plasma from women with PE as compared with NP women. The expression of LOX-1 was not significantly different in either the NP or PE incubated omental vessels. Incubation of vessels from NP women in plasma from women with PE impaired their relaxation in response to BK as compared with that of NP vessels incubated in plasma from NP women. Exposure to oxLDL further impaired relaxation in NP vessels incubated with plasma from women with PE. Inhibition of LOX-1 protected against the impairment of vascular relaxation induced by plasma from women with PE. CONCLUSION: Inhibition of LOX-1 prevents endothelial dysfunction in an in vitro model of PE and may prove useful as a therapeutic target in the treatment of PE.

Prenatal hypoxia causes long-term alterations in vascular endothelin-1 function in aged male, but not female, offspring.

Prenatal hypoxia can alter the growth trajectory of the fetus and cause lasting health complications including vascular dysfunction. We hypothesized that offspring that were intrauterine growth restricted (IUGR) because of prenatal hypoxia would exhibit altered vascular endothelin-1 (ET-1) signaling in later life. Isolated mesenteric artery responses to big ET-1 (bET-1) and ET-1 were assessed by using wire myography. Male IUGR offspring had 3-fold greater bET-1-induced vasoconstriction compared with controls (n=7 per group; P<0.001); NO synthase inhibition with L-N(G)-nitro-arginine-methyl ester potentiated bET-1-induced vasoconstriction, albeit this effect was 2-fold greater (P<0.05) in male control compared with IUGR offspring. Vascular responses to bET-1 were similar between female IUGR and control offspring (n=9-11 per group). In the presence of L-N(G)-nitro-arginine-methyl ester, pretreatment with the chymase inhibitor chymostatin, the gelatinase inhibitor GM6001, or the neutral endopeptidase inhibitor thiorphan did not alter responses to bET-1; however, the ET-converting enzyme inhibitor CGS35066 almost completely abolished vascular responses to bET-1 in control and IUGR groups. Systolic blood pressure in IUGR male offspring was more responsive to ET-1 antagonism in vivo compared with controls (-9 versus -4 mm Hg; n=5 per group; P=0.02); no such differences were observed in female offspring (n=5-6 per group). These results demonstrate that vascular ET-1 function is programmed by prenatal hypoxia and provide further insights into the sex differences in the long-term vascular effects of developmental stressors.

Cardiac hypertrophy in anion exchanger 1-null mutant mice with severe hemolytic anemia.

Anion exchanger 1 (AE1; SLC4A1), the plasma membrane Cl(-)/HCO(3)(-) exchanger of erythrocytes, is also expressed in heart. The aim of this study was to assess the role of AE1 in heart function through study of AE1-null (AE1(-/-)) mice, which manifest severe hemolytic anemia resulting from erythrocyte fragility. Heart weight-to-body weight ratios were significantly higher in the AE1(-/-) mice than in wild-type (AE1(+/+)) littermates at both 1-3 days postnatal (3.01 +/- 0.38 vs. 1.45 +/- 0.04) and at 7 days postnatal (9.45 +/- 0.53 vs. 4.13 +/- 0.41), indicating that loss of AE1 led to cardiac hypertrophy. Heterozygous (AE1(+/-)) mice had no signs of cardiac hypertrophy. Morphology of the adult AE1(-/-) mutant heart revealed an increased left ventricular mass, accompanied by increased collagen deposition and fibrosis. M-mode echocardiography revealed dysfunction of the AE1(-/-) hearts, including dilated left ventricle end diastole and systole and expanded left ventricular mass compared with AE1(+/+) hearts. Expression of intracellular pH-regulatory mechanisms in the hypertrophic myocardium of neonate AE1(-/-) mutant mice was indistinguishable from AE1(+/-) and AE1(+/+) mice, as assessed by quantitative real-time RT-PCR. Confocal immunofluorescence revealed that, in normal mouse myocardium, AE1 is sarcolemmal, whereas AE3 and slc26a6 are found both at the sarcolemma and in internal membranes (T tubules and sarcoplasmic reticulum). These results indicate that AE1(-/-) mice, which suffer from severe hemolytic anemia and spherocytosis, display cardiac hypertrophy and impaired cardiac function, reminiscent of findings in patients with hereditary abnormalities of red blood cells. No essential role for AE1 in heart function was found.

Slc26a6: a cardiac chloride-hydroxyl exchanger and predominant chloride-bicarbonate exchanger of the mouse heart.

Bicarbonate facilitate more than 50% of pH recovery in the acidotic myocardium, and have roles in cardiac hypertrophy and steady-state pH regulation. To determine which bicarbonate transporters are responsible for this activity, we measured the expression levels of all known HCO3(-)-anion exchange proteins in mouse heart, by quantitative real time RT-PCR. Bicarbonate-anion exchangers are members of either the SLC4A or the SLC26A gene families. In neonatal and adult myocardium, AE1 (Slc4a1), AE2 (Slc4a2), AE3 (Slc4a3) (AE3fl and AE3c variants), Slc26a3 and Slc26a6 were expressed. Adult hearts expressed Slc26a3 and Slc4a1-3 mRNAs at similar levels, while Slc26a6 mRNA was about seven-fold higher than AE3, which was more abundant than any other. Immunohistochemistry revealed that Slc26a6 and AE3 are present in the plasma membrane of ventricular myocytes. Slc26a6 expression levels were higher in ventricle than atrium, whereas AE3 was detected only in ventricle. Cl(-)-HCO(3)(-) and Cl(-)-OH(-) exchange activity of SLC26A6 and AE3 were investigated in transfected HEK293 cells, using intracellular fluorescence measurements of 2',7'-bis (2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), to monitor intracellular pH (pH(i)). Rates of pH(i) change were measured under HCO3(-)-containing (Cl(-)-HCO(3)(-)) or nominally HCO3(-)-free (Cl(-)-OH(-)) conditions. HCO3(-) fluxes were similar for cells expressing AE3fl, SLC26A6 or Slc26a3, suggesting that they have similar transport activity. However, only SLC26A6 and Slc26a3 functioned as Cl(-)-OH(-) exchangers. Activation of alpha-adrenergic receptors, which stimulates protein kinase C, inhibited SLC26A6 Cl(-)-HCO(3)(-) exchange activity. We conclude that Slc26a6 is the predominant Cl(-)-HCO(3)(-) and Cl(-)-OH(-) exchanger of the myocardium and that Slc26a6 is negatively regulated upon alpha-adrenergic stimulation.