Guidelines for in vivo models of developmental programming of cardiovascular risk.

Research utilizing animals depends on the generation of offspring for use in experiments or for the maintenance of animal colonies. While not considered by all, several different factors preceding and during pregnancy, as well as during lactation, can program various characteristics in the offspring. Here, we present the most common models of developmental programming of cardiovascular outcomes, important considerations for study design, and provide guidelines for producing and reporting rigorous and reproducible cardiovascular studies in offspring exposed to normal conditions or developmental insult. These guidelines provide considerations for the selection of the appropriate animal model and factors that should be reported to increase rigor and reproducibility, while ensuring transparent reporting of methods and results.

Impact of prenatal opioids on cardiac and autonomic development: systematic review and meta-analysis.

Prenatal opioid exposure has recently risen four-fold with limited data on the developmental effects on neonatal physiology. The objective of this systematic review is to develop an association between prenatal opioid exposure and fetal and neonatal cardiac and autonomic development and function. The review was conducted in accordance with PRISMA Guidelines, and searches were conducted using PubMed, Embase, CINAHL, and Web of Science between May 25 and October 27, 2020. Twenty studies fit inclusion criteria, in four categories: (1) fetal cardiac outcomes, (2) neonatal cardiac outcomes, (3) noninvasive autonomic outcomes, and (4) clinical and behavioral measures. For the meta-analysis, three studies (total of 210 subjects) were included. Effect sizes were measured as the mean difference in fetal heart rate between opioid-exposed and non-exposed groups. Mothers with prenatal opioid use had a significantly lower fetal heart rate as compared to mothers without prenatal opioid use, requiring further studies to determine clinical significance.

Cardiovascular Dysfunction in Intrauterine Growth Restriction.

PURPOSE OF REVIEW: We highlight important new findings on cardiovascular dysfunction in intrauterine growth restriction. RECENT FINDINGS: Intrauterine growth restriction (IUGR) is a multifactorial condition which negatively impacts neonatal growth during pregnancy and is associated with health problems during the lifespan. It affects 5-15% of all pregnancies in the USA and Europe with varying percentages in developing countries. Epidemiological studies have reported that IUGR is associated with the pathogenesis of hypertension, activation of the renin-angiotensin system (RAS), disruption in placental-mTORC and TGFß signaling cascades, and endothelial dysfunction in IUGR fetuses, children, adolescents, and adults resulting in the development of cardiovascular diseases (CVD). Experimental studies are needed to investigate therapeutic measures to treat increased blood pressure (BP) and long-term CVD problems in people affected by IUGR. We outline the mechanisms mediating fetal programming of hypertension in developing CVD. We have reviewed findings from different experimental models focusing on recent studies that demonstrate CVD in IUGR.

Insights into the Mechanisms of Fetal Growth Restriction-Induced Programming of Hypertension.

In recent decades, both clinical and animal studies have shown that fetal growth restriction (FGR), caused by exposure to adverse uterine environments, is a risk factor for hypertension as well as for a variety of adult diseases. This observation has shaped and informed the now widely accepted theory of developmental origins of health and disease (DOHaD). There is a plethora of evidence supporting the association of FGR with increased risk of adult hypertension; however, the underlying mechanisms responsible for this correlation remain unclear. This review aims to explain the current advances in the field of fetal programming of hypertension and a brief narration of the underlying mechanisms that may link FGR to increased risk of adult hypertension. We explain the theory of DOHaD and then provide evidence from both clinical and basic science research which support the theory of fetal programming of adult hypertension. In addition, we have explored the underlying mechanisms that may link FGR to an increased risk of adult hypertension. These mechanisms include epigenetic changes, metabolic disorders, vascular dysfunction, neurohormonal impairment, and alterations in renal physiology and function. We further describe sex differences seen in the developmental origins of hypertension and provide insights into the opportunities and challenges present in this field.

Macula Densa NOS1ß Modulates Renal Hemodynamics and Blood Pressure during Pregnancy: Role in Gestational Hypertension.

BACKGROUND: Regulation of renal hemodynamics and BP via tubuloglomerular feedback (TGF) may be an important adaptive mechanism during pregnancy. Because the ß-splice variant of nitric oxide synthase 1 (NOS1ß) in the macula densa is a primary modulator of TGF, we evaluated its role in normal pregnancy and gestational hypertension in a mouse model. We hypothesized that pregnancy upregulates NOS1ß in the macula densa, thus blunting TGF, allowing the GFR to increase and BP to decrease. METHODS: We used sophisticated techniques, including microperfusion of juxtaglomerular apparatus in vitro, micropuncture of renal tubules in vivo, clearance kinetics of plasma FITC-sinistrin, and radiotelemetry BP monitoring, to determine the effects of normal pregnancy or reduced uterine perfusion pressure (RUPP) on macula densa NOS1ß/NO levels, TGF responsiveness, GFR, and BP in wild-type and macula densa-specific NOS1 knockout (MD-NOS1KO) mice. RESULTS: Macula densa NOS1ß was upregulated during pregnancy, resulting in blunted TGF, increased GFR, and decreased BP. These pregnancy-induced changes in TGF and GFR were largely diminished, with a significant rise in BP, in MD-NOS1KO mice. In addition, RUPP resulted in a downregulation in macula densa NOS1ß, enhanced TGF, decreased GFR, and hypertension. The superimposition of RUPP into MD-NOS1KO mice only caused a modest further alteration in TGF and its associated changes in GFR and BP. Finally, in African green monkeys, renal cortical NOS1ß expression increased in normotensive pregnancies, but decreased in spontaneous gestational hypertensive pregnancies. CONCLUSIONS: Macula densa NOS1ß plays a critical role in the control of renal hemodynamics and BP during pregnancy.

Sphingolipids and Kidney Disease: Possible Role of Preeclampsia and Intrauterine Growth Restriction (IUGR).

Sphingolipids are now considered not only as constitutional components of the cellular membrane but also as essential bioactive factors regulating development and physiologic functions. Ceramide is a vital intermediate of sphingolipid metabolism, synthesized by de novo and salvage pathways, producing multiple types of sphingolipids and their metabolites. Although mutations in gene-encoding enzymes regulating sphingolipid synthesis and metabolism cause distinct diseases, an abnormal sphingolipid metabolism contributes to various pathologic conditions, including kidney diseases. Excessive accumulation of glycosphingolipids and promotion of the ceramide salvage and sphingosine-1-phosphate (S1P) pathways are found in the damaged kidney. Acceleration of the sphingosine kinase/S1P/S1P receptor (SphK/S1P/S1PR) axis plays a central role in deteriorating kidney functions. The SphK/S1P/S1PR signaling impairment is also found during pregnancy complications, such as preeclampsia and intrauterine growth restriction (IUGR). This mini-review discusses the current state of knowledge regarding the role of sphingolipid metabolism on kidney diseases, and the possible involvement of preeclampsia and IUGR conditions.

Sphingosine-1-phosphate signaling in blood pressure regulation.

Sphingolipids were originally believed to play a role only as a backbone of mammalian cell membranes. However, sphingolipid metabolites, especially sphingosine-1-phosphate (S1P), are now recognized as new bioactive signaling molecules that are critically involved in numerous cellular functions of multiple systems including the immune system, central nervous system, and cardiovascular system. S1P research has accelerated in the last decade as new therapeutic drugs have emerged that target the S1P signaling axis to treat diseases of the immune and central nervous systems. There is limited knowledge of the specific effects on cardiovascular disease. This review discusses the current state of knowledge regarding the role of S1P on the regulation of blood pressure, vascular tone, and renal functions.

Androgen Receptor Blockade Differentially Regulates Blood Pressure in Growth-Restricted Versus Ovarian Deficient Rats.

Low birth weight is associated with a greater prevalence of hypertension in women by age 60; yet, the mechanisms involved are unknown. We previously reported that hypertension in female growth-restricted offspring that is associated with early reproductive senescence and a shift in the testosterone-to-estradiol ratio at 12 months of age is abolished by AR (androgen receptor) blockade in conjunction with downregulation of renal AT1aR (angiotensin type 1a receptor) mRNA expression. These data suggest androgen-mediated activation of the renin-angiotensin system contributes to the pathogenesis of hypertension that develops in female growth-restricted offspring with aging. Thus, this study tested the hypothesis that androgen-mediated increased blood pressure is specific to female growth-restricted offspring. Control and growth-restricted rats underwent sham or ovariectomy at 10 months of age. Vehicle or flutamide (8 mg/kg/day; subcutaneous), an AR antagonist, was administered at 11.5 months of age for 2 weeks followed by measurement of blood pressure. Loss of ovarian hormones was associated with a 10 mm Hg increase in blood pressure in control compared with intact counterparts accompanied by a 1.8-fold increase in renal AT1aR mRNA expression. Treatment with flutamide had no effect on blood pressure or renal AT1aR mRNA expression in ovariectomized controls. Although blood pressure was significantly decreased in flutamide-treated ovariectomized growth-restricted, flutamide had no effect on the increase in renal AT1aR mRNA expression. Therefore, these findings suggest the effect of AR blockade on blood pressure is specific to intact growth-restricted offspring and that mechanisms of postmenopausal hypertension may differ between normal and low birth weight women.

Spectral photoacoustic imaging to estimate in vivo placental oxygenation during preeclampsia.

Preeclampsia is a pregnancy-related hypertensive disorder accounting for 14% of global maternal deaths annually. Preeclampsia - maternal hypertension and proteinuria - is promoted by placental ischemia resulting from reduced uteroplacental perfusion. Here, we assess longitudinal changes in placental oxygenation during preeclampsia using spectral photoacoustic imaging. Spectral photoacoustic images were acquired of the placenta of normal pregnant (NP) and preeclamptic reduced uterine perfusion pressure (RUPP) Sprague Dawley rats on gestational days (GD) 14, 16, and 18, corresponding to mid- to late gestation (n = 10 per cohort). Two days after implementation of the RUPP surgical model, placental oxygen saturation decreased 12% in comparison with NP. Proteinuria was determined from a 24-hour urine collection prior to imaging on GD18. Blood pressure measurements were obtained on GD18 after imaging. Placental hypoxia in the RUPP was confirmed with histological staining for hypoxia-inducible factor (HIF)-1α, a cellular transcription regulator which responds to local oxygen levels. Using in vivo, longitudinal imaging methods we determined that the placenta in the reduced uterine perfusion pressure rat model of preeclampsia is hypoxic, and that this hypoxia is maintained through late gestation. Future work will utilize these methods to assess the impact of novel therapeutics on placental ischemia and the progression of preeclampsia.

Male and Female Intrauterine Growth-Restricted Offspring Differ in Blood Pressure, Renal Function, and Glucose Homeostasis Responses to a Postnatal Diet High in Fat and Sugar.

It is well established that inadequate nutrition during fetal life followed by postnatal overabundance programs adiposity and glucose intolerance. Studies addressing sexual dimorphism in developmental responses to a dietary mismatch are limited; the effect on blood pressure and renal function is understudied. Therefore, this study tested the hypothesis that a mismatch of prenatal and postnatal nutrition heightens cardiorenal and metabolic risk, outcomes that may vary by sex. Male and female offspring from sham-operated (control) or reduced uterine perfusion dams (growth restricted) were fed regular chow or a diet high in fat and sugar (enriched diet) from weaning until 6 months of age. Male and female offspring were assessed separately; 2-way ANOVA was used to investigate interactions between intrauterine growth-restricted and enriched-diet. Blood pressure was increased in all enriched-diet groups but did not differ in enriched-diet male or female growth-restricted versus same-sex control counterparts. Glomerular filtration rate was reduced in male growth-restricted regardless of diet; a decrease exacerbated by the enriched-diet suggesting the pathogenesis of increased blood pressure induced via an enriched-diet differs between male growth-restricted versus male control. An enriched diet was associated with glucose intolerance in male and female control but not male growth-restricted; the enriched diet exacerbated glucose intolerance in female growth-restricted. Thus, these findings indicate male growth-restricted are resistant to impaired glucose homeostasis, whereas female growth-restricted are susceptible to metabolic dysfunction regardless of postnatal diet. Hence, moderation of fat and sugar intake may be warranted in those born low birth weight to ensure minimal risk for chronic disease.

Testosterone is protective against impaired glucose metabolism in male intrauterine growth-restricted offspring.

Placental insufficiency alters the intrauterine environment leading to increased risk for chronic disease including impaired glucose metabolism in low birth weight infants. Using a rat model of low birth weight, we previously reported that placental insufficiency induces a significant increase in circulating testosterone in male intrauterine growth-restricted offspring (mIUGR) in early adulthood that is lost by 12 months of age. Numerous studies indicate testosterone has a positive effect on glucose metabolism in men. Female growth-restricted littermates exhibit glucose intolerance at 6 months of age. Thus, the aim of this paper was to determine whether mIUGR develop impaired glucose metabolism, and whether a decrease in elevated testosterone levels plays a role in its onset. Male growth-restricted offspring were studied at 6 and 12 months of age. No impairment in glucose tolerance was observed at 6 months of age when mIUGR exhibited a 2-fold higher testosterone level compared to age-matched control. Fasting blood glucose was significantly higher and glucose tolerance was impaired with a significant decrease in circulating testosterone in mIUGR at 12 compared with 6 months of age. Castration did not additionally impair fasting blood glucose or glucose tolerance in mIUGR at 12 months of age, but fasting blood glucose was significantly elevated in castrated controls. Restoration of elevated testosterone levels significantly reduced fasting blood glucose and improved glucose tolerance in mIUGR. Thus, our findings suggest that the endogenous increase in circulating testosterone in mIUGR is protective against impaired glucose homeostasis.

Intrauterine growth restriction programs an accelerated age-related increase in cardiovascular risk in male offspring.

Placental insufficiency programs an increase in blood pressure associated with a twofold increase in serum testosterone in male growth-restricted offspring at 4 mo of age. Population studies indicate that the inverse relationship between birth weight and blood pressure is amplified with age. Thus, we tested the hypothesis that intrauterine growth restriction programs an age-related increase in blood pressure in male offspring. Growth-restricted offspring retained a significantly higher blood pressure at 12 but not at 18 mo of age compared with age-matched controls. Blood pressure was significantly increased in control offspring at 18 mo of age relative to control counterparts at 12 mo; however, blood pressure was not increased in growth-restricted at 18 mo relative to growth-restricted counterparts at 12 mo. Serum testosterone levels were not elevated in growth-restricted offspring relative to control at 12 mo of age. Thus, male growth-restricted offspring no longer exhibited a positive association between blood pressure and testosterone at 12 mo of age. Unlike hypertension in male growth-restricted offspring at 4 mo of age, inhibition of the renin-angiotensin system with enalapril (250 mg/l for 2 wk) did not abolish the difference in blood pressure in growth-restricted offspring relative to control counterparts at 12 mo of age. Therefore, these data suggest that intrauterine growth restriction programs an accelerated age-related increase in blood pressure in growth-restricted offspring. Furthermore, this study suggests that the etiology of increased blood pressure in male growth-restricted offspring at 12 mo of age differs from that at 4 mo of age.

Chronic Blockade of the Androgen Receptor Abolishes Age-Dependent Increases in Blood Pressure in Female Growth-Restricted Rats.

Intrauterine growth restriction induced via placental insufficiency programs a significant increase in blood pressure at 12 months of age in female growth-restricted rats that is associated with early cessation of estrous cyclicity, indicative of premature reproductive senescence. In addition, female growth-restricted rats at 12 months of age exhibit a significant increase in circulating testosterone with no change in circulating estradiol. Testosterone is positively associated with blood pressure after menopause in women. Thus, we tested the hypothesis that androgen receptor blockade would abolish the significant increase in blood pressure that develops with age in female growth-restricted rats. Mean arterial pressure was measured in animals pretreated with and without the androgen receptor antagonist, flutamide (8 mg/kg/day, SC for 2 weeks). Flutamide abolished the significant increase in blood pressure in growth-restricted rats relative to control at 12 months of age. To examine the mechanism(s) by which androgens contribute to increased blood pressure in growth-restricted rats, blood pressure was assessed in rats untreated or treated with enalapril (250 mg/L for 2 weeks). Enalapril eliminated the increase in blood pressure in growth-restricted relative to vehicle- and flutamide-treated controls. Furthermore, the increase in medullary angiotensin type 1 receptor mRNA expression was abolished in flutamide-treated growth-restricted relative to untreated counterparts and controls; cortical angiotensin-converting enzyme mRNA expression was reduced in flutamide-treated growth-restricted versus untreated counterparts. Thus, these data indicate that androgens, via activation of the renin-angiotensin system, are important mediators of increased blood pressure that develops by 12 months of age in female growth-restricted rats.

Glucose intolerance develops prior to increased adiposity and accelerated cessation of estrous cyclicity in female growth-restricted rats.

BACKGROUND: The incidence of metabolic disease increases in early menopause. Low birth weight influences the age at menopause. Thus, this study tested the hypothesis that intrauterine growth restriction programs early reproductive aging and impaired glucose homeostasis in female rats. METHODS: Estrous cyclicity, body composition, and glucose homeostasis were determined in female control and growth-restricted rats at 6 and 12 mo of age; sex steroids at 12 mo. RESULTS: Glucose intolerance was present at 6 mo of age prior to cessation of estrous cyclicity and increased adiposity in female growth-restricted rats. However, female growth-restricted rats exhibited persistent estrus and a significant increase in adiposity, fasting glucose, and testosterone at 12 mo of age (P < 0.05). Insulin release in response to a glucose challenge was blunted in conjunction with a reduction in protein expression of pancreatic glucose transporter type 2 and estrogen receptor-α at 12 mo of age in female growth-restricted rats (P < 0.05). CONCLUSION: This study demonstrated that slow fetal growth programmed glucose intolerance that developed prior to early estrous acyclicity; yet, fasting glucose levels were elevated in conjunction with increased adiposity, accelerated cessation of estrous cyclicity and a shift toward testosterone excess at 12 mo of age in female growth-restricted rats.

Sex-Specific Effect of Endothelin in the Blood Pressure Response to Acute Angiotensin II in Growth-Restricted Rats.

The renal endothelin system contributes to sex differences in blood pressure with males demonstrating greater endothelin type-A receptor-mediated responses relative to females. Intrauterine growth restriction programs hypertension and enhance renal sensitivity to acute angiotensin II in male growth-restricted rats. Endothelin is reported to work synergistically with angiotensin II. Thus, this study tested the hypothesis that endothelin augments the blood pressure response to acute angiotensin II in male growth-restricted rats. Systemic and renal hemodynamics were determined in response to acute angiotensin II (100 mg/kg per minute for 30 minutes) with and without the endothelin type-A receptor antagonist, Atrasentan (ABT-627; 10 ng/kg per minute for 30 minutes), in rats pretreated with enalapril (250 mg/L for 1 week) to normalize the endogenous renin-angiotensin system. Endothelin type-A receptor blockade reduced angiotensin II-mediated increases in blood pressure in male control and male growth-restricted rats. Endothelin type-A receptor blockade also abolished hyper-responsiveness to acute angiotensin II in male growth-restricted rats. Yet, blood pressure remained significantly elevated above baseline after endothelin type-A receptor blockade, suggesting that factors in addition to endothelin contribute to the basic angiotensin II-induced pressor response in male rats. We also determined sex-specific effects of endothelin on acute angiotensin II-mediated hemodynamic responses. Endothelin type-A receptor blockade did not reduce acute angiotensin II-mediated increases in blood pressure in female control or growth-restricted rats, intact or ovariectomized. Thus, these data suggest that endothelin type-A receptor blockade contributes to hypersensitivity to acute angiotensin II in male growth-restricted rats and further supports the sex-specific effect of endothelin on blood pressure.

Fetal programming and cardiovascular pathology.

Low birth weight serves as a crude proxy for impaired growth during fetal life and indicates a failure for the fetus to achieve its full growth potential. Low birth weight can occur in response to numerous etiologies that include complications during pregnancy, poor prenatal care, parental smoking, maternal alcohol consumption, or stress. Numerous epidemiological and experimental studies demonstrate that birth weight is inversely associated with blood pressure and coronary heart disease. Sex and age impact the developmental programming of hypertension. In addition, impaired growth during fetal life also programs enhanced vulnerability to a secondary insult. Macrosomia, which occurs in response to maternal obesity, diabetes, and excessive weight gain during gestation, is also associated with increased cardiovascular risk. Yet, the exact mechanisms that permanently change the structure, physiology, and endocrine health of an individual across their lifespan following altered growth during fetal life are not entirely clear. Transmission of increased risk from one generation to the next in the absence of an additional prenatal insult indicates an important role for epigenetic processes. Experimental studies also indicate that the sympathetic nervous system, the renin angiotensin system, increased production of oxidative stress, and increased endothelin play an important role in the developmental programming of blood pressure in later life. Thus, this review will highlight how adverse influences during fetal life and early development program an increased risk for cardiovascular disease including high blood pressure and provide an overview of the underlying mechanisms that contribute to the fetal origins of cardiovascular pathology.