Developmental origins of pregnancy-induced cardiac changes: establishment of a novel model using the atrial natriuretic peptide gene-disrupted mice.

Pregnancy evokes many challenges on the maternal cardiovascular system that may unmask predispositions for future disease. This is particularly evident for women who develop pregnancy-related disorders, for example, pre-eclampsia and gestational diabetes or hypertension. Such pregnancy-related syndromes increase the risk for cardiovascular disease (CVD) postpartum. As a result, pregnancy has been termed as a cardiovascular stress test and an indicator or marker to predict the development of CVD later in life. In addition, pregnancy-related disorders impact the development of offspring also placing them at a higher risk for disease. Utilizing pregnancy as a physiological stressor, the current investigation sought to determine whether the cardiovascular system of offspring exposed to gestational hypertension in utero would respond adversely to the stress of pregnancy. Heterozygous atrial natriuretic peptide gene-disrupted (ANP+/-) offspring were generated by either crossing male wildtype ANP+/+ with female knockout ANP-/- to produce ANP+/-KO mice or crossing female wildtype ANP+/+ with male knockout ANP-/- to produce ANP+/-WT mice. To study the cardiovascular stress induced by pregnancy, female ANP+/-WT and ANP+/-KO mice were mated with male wildtype ANP+/+ mice to initiate pregnancy. Cardiac size and molecular expression of the renin-angiotensin (RAS) and natriuretic peptide systems (NPS) were compared between offspring groups. Our data demonstrate that gestational hypertension and lack of maternal ANP did not significantly impact the progression and regression of pregnancy-induced cardiac hypertrophy over gestation and postpartum in ANP+/- offspring. Additionally, the molecular cardiac expression of the RAS and NPS did not differ between offspring groups. Future investigation should assess potential differences in cardiac function and the impact of fetal-programming on offspring cardiovascular adaptations during pregnancy in more severe models of pregnancy-related hypertensive syndrome such as angiotensin II or isoproterenol infusion.

Persistence of risk factors associated with maternal cardiovascular disease following aberrant inflammation in rat pregnancy.

INTRODUCTION: Pre-eclampsia is associated with increased risk of subsequent cardiovascular and metabolic disease in the affected mothers. While aberrant inflammation contributes to the pathophysiology of pre-eclampsia, it is unclear whether maternal inflammation contributes to the increased risk of disease. Here, we determined the effect of aberrant inflammation in pregnancy on cardiovascular and metabolic disease risk factors. METHODS: Wistar rats were administered low doses of lipopolysaccharide (LPS) on gestational days (GD) 13.5-16.5 to induce inflammation. Controls included pregnant rats treated with saline and nonpregnant rats treated with LPS or saline. We previously showed that LPS-treated pregnant rats exhibit key features of pre-eclampsia. Echocardiographic parameters, heart weight, blood pressure, blood lipids, pulse-wave velocity, and glucose tolerance, were assessed at 16 weeks postpartum. Messenger RNA levels of transcription factors associated with cardiac growth were measured in left ventricular tissue; histone modifications and global DNA methylation were determined in hearts and livers at GD 17.5 and at 16 weeks postpartum. RESULTS: Compared with saline-treated pregnant rats and nonpregnant rats treated with LPS or saline, LPS-treated pregnant rats exhibited left ventricular hypertrophy and increased blood cholesterol and low-density lipoprotein levels at 16 weeks postdelivery. LPS-treated rats had increased left ventricular mRNA levels of hypertrophy-associated transcription factors at GD 17.5 and increased levels of modified histones in hearts and livers at GD 17.5 and 16 weeks postpartum. Other parameters remained unchanged. CONCLUSION: Aberrant inflammation during pregnancy results in persistent alterations in maternal physiological parameters and epigenetic modifications that could contribute to the pathophysiology of cardiovascular disease.

Onset and Regression of Pregnancy-Induced Cardiac Alterations in Gestationally Hypertensive Mice: The Role of the Natriuretic Peptide System.

Pregnancy induces cardiovascular adaptations in response to increased volume overload. Aside from the hemodynamic changes that occur during pregnancy, the maternal heart also undergoes structural changes. However, cardiac modulation in pregnancies complicated by gestational hypertension is incompletely understood. The objectives of the current investigation were to determine the role of the natriuretic peptide (NP) system in pregnancy and to assess alterations in pregnancy-induced cardiac hypertrophy between gestationally hypertensive and normotensive dams. Previously we have shown that mice lacking the expression of atrial NP (ANP; ANP(-/-)) exhibit a gestational hypertensive phenotype. In the current study, female ANP(+/+) and ANP(-/-) mice were mated with ANP(+/+) males. Changes in cardiac size and weight were evaluated across pregnancy at Gestational Days 15.5 and 17.5 and Postnatal Days 7, 14, and 28. Nonpregnant mice were used as controls. Physical measurement recordings and histological analyses demonstrated peak cardiac hypertrophy occurring at 14 days postpartum in both ANP(+/+) and ANP(-/-) dams with little to no change during pregnancy. Additionally, left ventricular expression of the renin-angiotensin system (RAS) and NP system was quantified by real-time quantitative polymerase chain reaction. Up-regulation of Agt and AT(1a) genes was observed late in pregnancy, while Nppa and Nppb genes were significantly up-regulated postpartum. Our data suggest that pregnancy-induced cardiac hypertrophy may be influenced by the RAS throughout gestation and by the NP system postpartum. Further investigations are required to gain a complete understanding of the mechanistic aspects of pregnancy-induced cardiac hypertrophy.

Placental growth factor influences maternal cardiovascular adaptation to pregnancy in mice.

In healthy human pregnancies, placental growth factor (PGF) concentrations rise in maternal plasma during early gestation, peak over Weeks 26-30, then decline. Because PGF in nongravid subjects participates in protection against and recovery from cardiac pathologies, we asked if PGF contributes to pregnancy-induced maternal cardiovascular adaptations. Cardiovascular function and structure were evaluated in virgin, pregnant, and postpartum C56BL/6-Pgf(-) (/) (-) (Pgf(-) (/) (-)) and C57BL/6-Pgf(+/+) (B6) mice using plethysmography, ultrasound, quantitative PCR, and cardiac and renal histology. Pgf(-/-) females had higher systolic blood pressure in early and late pregnancy but an extended, abnormal midpregnancy interval of depressed systolic pressure. Pgf(-/-) cardiac output was lower than gestation day (gd)-matched B6 after midpregnancy. While Pgf(-) (/) (-) left ventricular mass was greater than B6, only B6 showed the expected gestational gain in left ventricular mass. Expression of vasoactive genes in the left ventricle differed at gd8 with elevated Nos expression in Pgf(-) (/) (-) but not at gd14. By gd16, Pgf(-) (/) (-) kidneys were hypertrophic and had glomerular pathology. This study documents for the first time that PGF is associated with the systemic maternal cardiovascular adaptations to pregnancy.

Molecular adaptations in vasoactive systems during acute stroke in salt-induced hypertension.

Investigations regarding hypertension and dietary sodium, both factors that influence stroke risk, have previously been limited to using genetically disparate treatment and control groups, namely the stroke-prone, spontaneously hypertensive rat and Wistar-Kyoto rat. In this investigation, we have characterized and compared cerebral vasoactive system adaptations following stroke in genetically identical, salt-induced hypertensive, and normotensive control mice. Briefly, ANP(+/-) (C57BJ/6 × SV129 background) mice were fed chow containing either 0.8% NaCl (NS) or 8.0% NaCl (HS) for 7 weeks. Transient cerebral ischemia was induced by middle cerebral artery occlusion (MCAO). Infarct volumes were measured 24-h post-reperfusion and the mRNA expression of five major vasoactive systems was characterized using qPCR. Along with previous publications, our data validate a salt-induced hypertensive state in ANP(+/-) mice fed HS chow as they displayed left ventricular hypertrophy, increased systolic blood pressure, and increased urinary sodium excretion. Following MCAO, mice fed HS exhibited larger infarct volumes than their dietary counterparts. In addition, significant up-regulation in Et-1 and Nos3 mRNA expression in response to salt and stroke suggests implications with increased cerebral damage in this group. In conclusion, our data demonstrate increased cerebral susceptibility to stroke in salt-induced hypertensive mice. More importantly, however, we have characterized a novel method of investigating hypertension and stroke with the use of genetically identical treatment and control groups. This is the first investigation in which genetic confounding variables have been eliminated.

Maternal hypertension programs increased cerebral tissue damage following stroke in adult offspring.

The maternal system is challenged with many physiological changes throughout pregnancy to prepare the body to meet the metabolic needs of the fetus and for delivery. Many pregnancies, however, are faced with pathological stressors or complications that significantly impact maternal health. A shift in this paradigm is now beginning to investigate the implication of pregnancy complications on the fetus and their continued influence on offspring disease risk into adulthood. In this investigation, we sought to determine whether maternal hypertension during pregnancy alters the cerebral response of adult offspring to acute ischemic stroke. Atrial natriuretic peptide gene-disrupted (ANP(-/-)) mothers exhibit chronic hypertension that escalates during pregnancy. Through comparison of heterozygote offspring born from either normotensive (ANP(+/-WT)) or hypertensive (ANP(+/-KO)) mothers, we have demonstrated that offspring exposed to maternal hypertension exhibit larger cerebral infarct volumes following middle cerebral artery occlusion. Observation of equal baseline cardiovascular measures, cerebrovascular structure, and cerebral blood volumes between heterozygote offspring suggests no added influences on offspring that would contribute to adverse cerebral response post-stroke. Cerebral mRNA expression of endothelin and nitric oxide synthase vasoactive systems demonstrated up-regulation of Et-1 and Nos3 in ANP(+/-KO) mice and thus an enhanced acute vascular response compared to ANP(+/-WT) counterparts. Gene expression of Na(+)/K(+) ATPase channel isoforms, Atp1a1, Atp1a3, and Atp1b1, displayed no significant differences. These investigations are the first to demonstrate a fetal programming effect between maternal hypertension and adult offspring stroke outcome. Further mechanistic studies are required to complement epidemiological evidence of this phenomenon in the literature.

A cross-sectional study of global DNA methylation and risk of colorectal adenoma.

BACKGROUND: The methylation of DNA is recognized as a key epigenetic mechanism and evidence for its role in the development of several malignancies is accumulating. We evaluated the relationship between global methylation in DNA derived from normal appearing colon mucosal tissue and blood leukocytes, and colorectal adenoma risk. METHODS: Patients, aged 40 to 65, scheduled for a screening colonoscopy were recruited. During the colonoscopy, two pinch biopsies of healthy, normal appearing mucosa were obtained from the descending colon. A fasting blood sample was also collected. The methylation status of LINE-1 (long interspersed nuclear element-1) repetitive sequences, as a surrogate measure of global methylation, was quantified in DNA extracted from normal colon mucosa and blood leukocytes. Statistical analysis of the relationship between global DNA methylation and adenoma risk was conducted on 317 participants, 108 subjects with at least one pathologically confirmed adenoma and 209 subjects with a normal colonoscopy. RESULTS: A statistically significant inverse relationship was observed between LINE-1 methylation in colon tissue DNA and adenoma risk for males and for both sexes combined for the lowest methylation quartile compared to the highest (adjusted ORs = 2.94 and 2.26 respectively). For blood, although the overall pattern of odds ratio estimates was towards an increase in risk for lower methylation quartiles compared to the highest methylation quartile, there were no statistically significant relationships observed. A moderate correlation was found between LINE-1 methylation levels measured in tissue and blood (Pearson correlation 0.36). CONCLUSIONS: We observed that lower levels of LINE-1 DNA methylation in normal appearing background colon mucosa were associated with increased adenoma risk for males, and for both sexes combined. Though these findings provide some support for a relationship between LINE-1 DNA methylation in colon mucosal tissue and adenoma risk, large prospective cohort studies are needed to confirm results. Until such investigations are done, the clinical usefulness of LINE-1 methylation as a biomarker of increased adenoma risk is uncertain. Regardless, this study contributes to a better understanding of the role of global DNA methylation as an early event in CR carcinogenesis with implications for future etiologic research.

Gestational hypertension and the developmental origins of cardiac hypertrophy and diastolic dysfunction.

The developmental origins of health and disease refer to the theory that adverse maternal environments influence fetal development and the risk of cardiovascular disease in adulthood. We used the chronically hypertensive atrial natriuretic peptide knockout (ANP-/-) mouse as a model of gestational hypertension, and attempted to determine the effect of gestational hypertension on left ventricular (LV) structure and function in adult offspring. We crossed normotensive ANP+/+ females with ANP-/- males (yielding ANP+/-(WT) offspring) and hypertensive ANP-/- females with ANP+/+ males (yielding ANP+/-(KO) offspring). Cardiac gene expression was measured using real-time quantitative PCR. Cardiac function was assessed using echocardiography. Daily injections of isoproterenol (ISO) were used to induce cardiac stress. Collagen deposition was assessed using picrosirius red staining. All mice were 10 weeks of age. Gestational hypertension resulted in significant LV hypertrophy in offspring, with no change in LV function. Treatment with ISO resulted in significant LV diastolic dysfunction with a restrictive filling pattern (increased E/A ratio and E/e') and interstitial myocardial fibrosis only in ANP+/-(KO) and not ANP+/-(WT) offspring. Gestational hypertension programs adverse LV structural and functional remodeling in offspring. These data suggest that adverse maternal environments may increase the risk of heart failure in offspring later in life.

Implantation of scaffold-free engineered cartilage constructs in a rabbit model for chondral resurfacing.

Joint resurfacing techniques offer an attractive treatment for damaged or diseased cartilage, as this tissue characteristically displays a limited capacity for self-repair. While tissue-engineered cartilage constructs have shown efficacy in repairing focal cartilage defects in animal models, a substantial number of cells are required to generate sufficient quantities of tissue for the repair of larger defects. In a previous study, we developed a novel approach to generate large, scaffold-free cartilaginous constructs from a small number of donor cells (20 000 cells to generate a 3-cm(2) tissue construct). As comparable thicknesses to native cartilage could be achieved, the purpose of the present study was to assess the ability of these constructs to survive implantation as well as their potential for the repair of critical-sized chondral defects in a rabbit model. Evaluated up to 6 months post-implantation, allogenic constructs survived weight bearing without a loss of implant fixation. Implanted constructs appeared to integrate near-seamlessly with the surrounding native cartilage and also to extensively remodel with increasing time in vivo. By 6 months post-implantation, constructs appeared to adopt both a stratified (zonal) appearance and a biochemical composition similar to native articular cartilage. In addition, constructs that expressed superficial zone markers displayed higher histological scores, suggesting that transcriptional prescreening of constructs prior to implantation may serve as an approach to achieve superior and/or more consistent reparative outcomes. As the results of this initial animal study were encouraging, future studies will be directed toward the repair of chondral defects in more mechanically demanding anatomical locations.

Expression of Chondrogenic Potential Markers in Cultured Chondrocytes from the Human Knee Joint.

OBJECTIVES: While substantial progress has been made in engineering cartilaginous constructs for animal models, further research is needed to translate these methodologies for human applications. Evidence suggests that cultured autologous chondrocytes undergo changes in phenotype and gene expression, thereby affecting their proliferation and differentiation capacity. This study was designed to evaluate the expression of chondrogenic markers in cultured human articular chondrocytes from passages 3 (P3) and 7 (P7), beyond the current clinical recommendation of P3. METHODS: Cultured autologous chondrocytes were passaged from P3 up to P7, and quantitative polymerase chain reaction (qPCR) was used to assess mRNA expression of chondrogenic markers, including collagen type I (COLI), collagen type II (COLII), aggrecan (AGG), bone morphogenetic protein 4 (BMP4), transcription factor SOX-9 (SOX9), proteoglycan 4 (PGR4), and transformation-related protein 53 (p53), between P3 and P7. RESULTS: Except for AGG, no significant differences were found in the expression of markers between passages, suggesting the maintenance of chondrogenic potential in cultured chondrocytes. Differential expression identified between SOX9 and PGR4, as well as between COLI and SOX9, indicates that differences in chondrogenic markers are present between age groups and sexes, respectively. CONCLUSIONS: Overall, expression profiles of younger and male chondrocytes exhibit conversion of mature cartilage characteristics compared to their counterparts, with signs of dedifferentiation and loss of phenotype within-group passaging. These results may have implications in guiding the use of higher passaged chondrocytes for engineering constructs and provide a foundation for clinical recommendations surrounding the repair and treatment of articular cartilage pathology in both sexes.

Sex-specific differences in natriuretic peptide and nitric oxide synthase expression in ANP gene-disrupted mice.

Sex-specific differences in hormone-mediated gene regulation may influence susceptibility to cardiac hypertrophy, a primary risk factor for cardiovascular disease. Under hormonal influence, natriuretic peptide (NP) and nitric oxide synthase (NOS) systems modulate cardio-protective gene programs through common downstream production of cyclic guanosine 3'-5' monophosphate (cGMP). Ablation of either system can adversely affect cardiac adaptation to stresses and insults. This study elucidates sex-specific differences in cardiac NP and NOS system gene expression and assesses the impact of the estrous cycle on these systems using the atrial natriuretic peptide gene-disrupted (ANP(-/-)) mouse model. Left ventricular expression of the NP and NOS systems was analyzed using real-time quantitative polymerase chain reaction in 13- to 16-week-old male, proestrous and estrous female ANP(+/+) and ANP(-/-) mice. Left ventricular and plasma cGMP levels were measured to assess the convergent downstream effects of the NP and NOS systems. Regardless of genotype, males had higher expression of the NP system while females had higher expression of the NOS system. In females, transition from proestrus to estrus lowered NOS system expression in ANP(+/+) mice while the opposite was observed in ANP(-/-) mice. No significant changes in left ventricular cGMP levels across gender and genotype were observed. Significantly lower plasma cGMP levels were observed in ANP(-/-) mice compared to ANP(+/+) mice. Regardless of genotype, sex-specific differences in cardiac NP and NOS system expression exist, each sex enlisting a predominant system to conserve downstream cGMP. Estrous cycle-mediated alterations in NOS system expression suggests additional hormone-mediated gene regulation in females.

Aberrant inflammation in rat pregnancy leads to cardiometabolic alterations in the offspring and intrauterine growth restriction in the F2 generation.

Children of women with pre-eclampsia have increased risk of cardiovascular (CV) and metabolic disease in adult life. Furthermore, the risk of pregnancy complications is higher in daughters born to women affected by pre-eclampsia than in daughters born after uncomplicated pregnancies. While aberrant inflammation contributes to the pathophysiology of pregnancy complications, including pre-eclampsia, the contribution of maternal inflammation to subsequent risk of CV and metabolic disease as well as pregnancy complications in the offspring remains unclear. Here, we demonstrate that 24-week-old female rats (F1) born to dams (F0) exposed to lipopolysaccharide (LPS) during pregnancy (to induce inflammation) exhibited mild systolic dysfunction, increased cardiac growth-related gene expression, altered glucose tolerance, and coagulopathy; whereas male F1 offspring exhibited altered glucose tolerance and increased visceral fat accumulation compared with F1 sex-matched offspring born to saline-treated dams. Both male and female F1 offspring born to LPS-treated dams had evidence of anemia. Fetuses (F2) from F1 females born to LPS-treated dams were growth restricted, and this reduction in fetal growth was associated with increased CD68 positivity (indicative of macrophage presence) and decreased expression of glucose transporter-1 in their utero-placental units. These results indicate that abnormal maternal inflammation can contribute to increased risk of CV and metabolic disease in the offspring, and that the effects of inflammation may cross generations. Our findings provide evidence in support of early screening for CV and metabolic disease, as well as pregnancy complications in offspring affected by pre-eclampsia or other pregnancy complications associated with aberrant inflammation.

Altered expression of the natriuretic peptide system in genetically modified heme oxygenase-1 mice treated with high dietary salt.

Heme oxygenase-1 (HO-1) has been well established as a cytoprotective molecule, and has been shown to exert cardioprotective effects in both hypertension and cardiac hypertrophy. However, the precise mechanism of the cardioprotective effect of HO-1 has yet to be fully elucidated. With the natriuretic peptide system (NPS) as a key player in cardiovascular homeostasis and tissue dynamics, we sought to examine the effect of high dietary salt treatment in genetic models of HO-1 expression, and assessed the expression of the NPS in the left ventricle (LV), to determine if the effects of altered HO-1 expression may be due to modified levels of the NPS. Age-matched 12-week old male HO-1 knockout (HO-1(-/-)) and HO-1 cardiomyocyte-specific transgenic overexpressing (HO-1(Tg)) mice were treated with either normal salt (NS; 0.8%) or high salt (HS; 8.0%) chow for 5 weeks. LV mRNA expression was determined using quantitative real-time PCR. ANP peptide level was measured in the LV and plasma using radioimmunoassay, and LV cyclic 3'-5' guanosine monophosphate level was measured using an enzyme immunoassay kit. HO-1(-/-) fed HS diet had significantly higher left ventricle-to-body weight ratio (LV/BW) compared to HO-1(+/+) mice fed NS diet. HO-1(-/-) mice had significantly reduced expression of the NPS compared to controls, and these mice did not exhibit a salt-induced increase in ANP expression. HS treatment had no noticeable effect on LV/BW in HO-1(Tg) mice compared to controls. HO-1(Tg) mice had significantly higher ANP and BNP expression compared to controls. There were no differences in LV cGMP levels among all genotypes and dietary treatments. HO-1 ablation resulted in significantly lower mRNA expression of the NPS, whereas HO-1 overexpression resulted in higher mRNA expression of the NPS. Both were substantiated by peptide levels as measured by RIA. These data indicate that the detrimental effect of reduced HO-1 expression and the cardioprotective effect of increased HO-1 expression may be due, in part, to altered expression of the NPS.

Vascularized Carotid Atherosclerotic Plaque Models for the Validation of Novel Methods of Quantifying Intraplaque Neovascularization.

BACKGROUND: Intraplaque neovascularization (IPN) in advanced lesions of the carotid artery has been linked to plaque progression and risk of rupture. Quantitative measurement of IPN may provide a more powerful tool for the detection of such "vulnerable" plaque than the current visual scoring method. The aim of this study was to develop a phantom platform of a neovascularized atherosclerotic plaque within a carotid artery to assess new methods of quantifying IPN. METHODS: Ninety-two synthetic plaque models with various IPN architectures representing different ranges of IPN scoring were created and assessed using contrast-enhanced ultrasound. Intraplaque neovascularization volume was calculated from contrast infiltration in B mode. The plaque models were used to develop a testing platform for IPN quantification. A neovascularized enhancement ratio (NER) was calculated using commercially available software. The plaque model NERs were then compared to human plaque NERs (n = 42) to assess score relationship. Parametric mapping of dynamic intensity over time was used to differentiate IPN from calcified plaque regions. RESULTS: A positive correlation between NER and IPN volume (rho = 0.45; P < .0001) was found in the plaque models. Enhancement of certain plaque model types showed that they resembled human plaques, with visual grade scores of 0 (NER mean difference = 1.05 ± SE 2.45; P = .67), 1 (NER mean difference = 0.22 ± SE 3.26; P = .95), and 2 (NER mean difference = -0.84 ± SE 3.33; P = .80). An optimal cutoff for NER (0.355) identified grade 2 human plaques with a sensitivity of 95% and specificity of 91%. CONCLUSIONS: We developed a carotid artery model of neovascularized plaque and established a quantitative method for IPN using commercially available technology. We also developed an analysis method to quantify IPN in calcified plaques. This novel tool has the potential to improve clinical identification of vulnerable plaques, providing objective measures of IPN for cardiovascular risk assessment.

Altered regulation of renal interstitial hydrostatic pressure and the renal renin-angiotensin system in the absence of atrial natriuretic peptide.

BACKGROUND: Although it has been well established that atrial natriuretic peptide gene-disrupted (ANP-/-) mice are a useful model of salt-sensitive hypertension, surprisingly little is known about the control of their intrarenal renin-angiotensin system (RAS) and pressure-natriuresis mechanism, key components in blood pressure, fluid and electrolyte regulation. The aim of this study was to determine whether ANP disruption results in changes in the renal and adrenal local RAS and the acute pressure-natriuresis mechanism. METHODS: Renal and adrenal renin, angiotensin type 1 (AT1)(A and B) and angiotensin type 2 (AT2) receptor messenger RNA expression levels were determined by northern blotting or real time reverse transcriptase-polymerase chain reaction. Plasma aldosterone and renal and adrenal angiotensin II peptide levels were determined by radioimmunoassay. To examine the acute pressure-natriuresis response, changes in renal interstitial hydrostatic pressure (RIHP) were assessed after manipulations of renal arterial pressure (RAP) in anaesthetized mice. RESULTS: Renal and adrenal renin mRNA and angiotensin II levels were lower in ANP-/- and +/- mice compared with +/+mice. ANP-/- mice also had greater renal AT1A and adrenal AT2 mRNA levels compared with the other genotypes. RAP and RIHP were significantly higher in -/-mice compared with +/+mice. Furthermore, there was a blunted slope of the RAP-RIHP relationship after increases in RAP in ANP-/- mice. CONCLUSION: These data indicate that ANP disruption results in a blunting of the dynamic properties of the acute pressure-natriuresis mechanism at increased levels of RAP, as well as a reduced expression of renal and adrenal local RAS.

Thyroxine Increases Collagen Type II Expression and Accumulation in Scaffold-Free Tissue-Engineered Articular Cartilage.

Low collagen accumulation in the extracellular matrix is a pressing problem in cartilage tissue engineering, leading to a low collagen-to-glycosaminoglycan (GAG) ratio and poor mechanical properties in neocartilage. Soluble factors have been shown to increase collagen content, but may result in a more pronounced increase in GAG content. Thyroid hormones have been reported to stimulate collagen and GAG production, but reported outcomes, including which specific collagen types are affected, are variable throughout the literature. Here we investigated the ability of thyroxine (T4) to preferentially stimulate collagen production, as compared with GAG, in articular chondrocyte-derived scaffold-free engineered cartilage. Dose response curves for T4 in pellet cultures showed that 25 ng/mL T4 increased the total collagen content without increasing the GAG content, resulting in a statistically significant increase in the collagen-to-GAG ratio, a fold change of 2.3 ± 1.2, p < 0.05. In contrast, another growth factor, TGFß1, increased the GAG content in excess of threefold more than the increase in collagen. In large scaffold-free neocartilage, T4 also increased the total collagen/DNA at 1 month and at 2 months (fold increases of 2.1 ± 0.8, p < 0.01 and 2.1 ± 0.4, p < 0.001, respectively). Increases in GAG content were not statistically significant. The effect on collagen was largely specific to collagen type II, which showed a 2.8 ± 1.6-fold increase of COL2A1 mRNA expression (p < 0.01). Western blots confirmed a statistically significant increase in type II collagen protein at 1 month (fold increase of 2.2 ± 1.8); at 2 months, the fold increase of 3.7 ± 3.3 approached significance (p = 0.059). Collagen type X protein was less than the 0.1 µg limit of detection. T4 did not affect COL10A1 and COL1A2 gene expression in a statistically significant manner. Biglycan mRNA expression increased 2.6 ± 1.6-fold, p < 0.05. Results of this study show that an optimized dosage of T4 is able to increase collagen type II content, and do so preferential to GAG. Moreover, the upregulation of COL2A1 gene expression and type II collagen protein accumulation, without a concomitant increase in collagens type I or type X, signifies a direct enhancement of chondrogenesis of hyaline articular cartilage without the induction of terminal differentiation.

Estrogen protects against the development of salt-induced cardiac hypertrophy in heterozygous proANP gene-disrupted mice.

Cardiovascular disease is the leading cause of morbidity and mortality in both men and women, but the incidence for women rises sharply after menopause. This has been mainly attributed in the reduction of the female sex hormone estrogen during menopause, suggesting that estrogen may have cardioprotective effects, although how estrogen exerts its cardioprotective effects is not fully understood. Moreover, the beneficial effect of estrogen on end-organ damage such as cardiac hypertrophy (CH) remains unclear. The aim of the present study was to examine the interaction between estrogen and the natriuretic peptide system (NPS) and their possible roles during the development of CH by using the proANP heterozygous atrial natriuretic peptide (ANP +/-) mouse as a model of salt-sensitive CH. Male, female ANP +/- mice, and also ovariectomized (Ovx) female ANP +/- mice treated with oil or estrogen were fed either a normal or high salt (HS) diet. After a 5-week treatment period, marked CH was noted in the male and oil-injected Ovx female ANP +/- mice treated with HS. The cardiac NPS, i.e. ANP, B-type natriuretic peptide, and natriuretic peptide receptor-A, was activated in these ANP +/- mice. Interestingly, the female and estrogen-injected Ovx female ANP +/- mice did not exhibit CH, and the cardiac NPS remained unchanged. Collectively, we provide direct evidence that estrogen has the ability to resist the induction of salt-induced CH in ANP +/- mice. Furthermore, the development of hypertrophy may be activating the cardiac NPS in an attempt to blunt these structural changes.

Elevated renal norepinephrine in proANP gene-disrupted mice is associated with increased tyrosine hydroxylase expression in sympathetic ganglia.

The sympatholytic properties of atrial natriuretic peptide (ANP) contribute to its vasodilatory and natriuretic effects. High circulating catecholamine levels, along with renal dysfunction, present in proANP gene-disrupted (-/-) mice are thought to contribute to the hypertension characteristic of this model. To further understand the mechanism by which the absence of ANP leads to stimulation of sympathetic activity we measured tyrosine hydroxylase expression in mice with and without ANP. The adrenal and prevertebral ganglionic expression of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine production, was significantly increased in ANP -/- mice. ANP's sympatholytic properties include the depression of ganglionic and adrenal TH expression and catecholamine production. Thus, these results suggest that the absence of ANP's sympatholytic effects is not completely compensated for in ANP -/- mice. In addition, mice devoid of ANP display an increase in renal sympathetic nerve activity from birth through to adulthood which may lead to structural and/or functional changes in the early postnatal kidney that contribute to the hypertensive phenotype of ANP -/- mice. The over-activation of the sympathetic nervous system in mice lacking ANP confirms the important role of this peptide in the modulation of sympathetic nerve activity and its contribution to blood pressure homeostasis.

Protection of human vascular smooth muscle cells from H2O2-induced apoptosis through functional codependence between HO-1 and AKT.

OBJECTIVE: Oxidative stress (OS) induces smooth muscle cell apoptosis in the atherosclerotic plaque, leading to plaque instability and rupture. Heme oxygenase-1 (HO-1) exerts cytoprotective effects in the vessel wall. Recent evidence suggests that PKB/Akt may modulate HO-1 activity. This study examined the role of Akt in mediating the cytoprotective effects of HO-1 in OS-induced apoptosis of human aortic smooth muscle cells (HASMCs). METHODS AND RESULTS: HASMCs were transduced with retroviral vectors expressing HO-1, Akt, or GFP and exposed to H2O2. Cell viability was assessed by MTT assay. OS was determined by CM-H2DCFDA fluorescence, and apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL), caspase-3 activity, and Bcl-2/Bad levels. Mitochondrial membrane potential (delta psi(m)) was assessed by fluorescence-activated cell sorter (FACS) using JC-1. HO-1 reduced H2O2-induced OS and apoptosis. Akt knockdown removed the protective effect of HO-1 on delta psi(m) during exposure to H2O2. Conversely, HO-1 knockdown removed the protective effect of Akt on delta psi(m). Inhibition of PI3K-Akt reduced induction of HO-1 protein expression by H2O2 and blocked its anti-apoptotic effects. The Akt-mediated upregulation of HO-1 was dependent on activation of HO-1 promoter by Nrf2. CONCLUSIONS: HO-1 and Akt exert codependent cytoprotective effects against OS-induced apoptosis in HASMCs. These findings may have implications for the design of novel therapeutic strategies for plaque stabilization.

Sex Differences of the Natriuretic Peptide Polymorphism Associated With Angiographic Coronary Atherosclerosis.

BACKGROUND: Polymorphisms within natriuretic peptide (NP) genes have been associated with clinical outcomes for cardiovascular disease (CVD), but no previous study has compared the effect of these polymorphisms between men and women. This study aimed to investigate the association between single nucleotide polymorphisms (SNPs) in key genes of the NP system and coronary angiographic outcomes, with the focus on the sexual dimorphism in the effects of these SNPs. METHODS: Patients undergoing clinically indicated coronary angiography (n = 513, 328 men and 185 women) were consented and genotyped for NPPA rs5065, NPPB rs198389 and NPR2 rs10758325. Patients were stratified into having normal coronaries, non-obstructive coronary artery disease (CAD) or obstructive CAD, based on the highest stenosis in any epicardial artery. Average luminal narrowing across all four arteries was derived to represent the overall atherosclerotic burden. RESULTS: The frequency of NPPB rs198389 AA genotype was significantly higher in women with obstructive CAD (P = 0.014). The same association was not observed in males. With respect to atherosclerotic burden, an association was found between the AA genotype and average luminal narrowing in women (P = 0.005), but not in men. CONCLUSIONS: The current study identified an association between an SNP of the NPPB gene and coronary atherosclerotic burden through angiographic evidence in women but not in men. These results suggest that B-type natriuretic peptide (BNP) may have more important involvement in the development of CAD in women compared to men, and as such, genotyping of the NPPB gene may serve as a potential biomarker to identify women with high risk for CAD.