Mechanisms of Brain Edema Formation in Mouse Models of Intracerebral Hemorrhage

Perihematomal edema causes major neurologic deterioration following intracerebral hemorrhage (ICH), mainly resulting from the disruption of the blood-brain barrier (BBB) by multiple mediators, including inflammatory mediators and thrombin. The objective of our study was to investigate the mechanisms by which inflammation and thrombin respectively lead to the formation of brain edema following ICH. Our long-term goal is to develop new therapeutic strategies against ICH-induced brain edema by targeting: (1) VAP-1 mediated inflammatory response and (2) PDGFR-α orchestrated BBB impairment. Vascular adhesion protein-1 (VAP-1) was previously shown to promote leukocyte adhesion and transmigration. Additionally, PDGFR-α was also found to play a role in orchestrating BBB impairment. ICH injury was induced by collagenase-injection (cICH) or autologous arterial blood-injection (bICH) in mice. Two VAP-1 inhibitors, LJP1586 and semicarbazide (SCZ) were administered one hour after cICH. For mechanistic studies, VAP-1 siRNA and human recombinant VAP-1 protein were administered intracerebroventricularly. The data showed that VAP-1 inhibition reduced brain edema and neurobehavioral deficits at 24 and 72 hours after ICH induction. These two compounds were also found to decrease other adhesion molecules and cytokines expression, neutrophils infiltration and microglia/macrophage activation. The effect of VAP-1 siRNA was consistent with that of pharmacological inhibitions, whereas human recombinant VAP-1 protein abolished the protective effect of VAP-1 inhibition. The anti-inflammatory effects of VAP-1 were also corroborated using blood-induced ICH. We then proceeded to elucidate the role of PDGFR-α inhibitor-induced neuroprotection in ICH. In our ICH model, we found that PDGFR-α and its endogenous agonist PDGFAA, were upregulated in response to bICH-induced brain injury. The results showed that suppression of PDGFR-α preserved BBB integrity following bICH while activation of PDGFR-α led to BBB impairment. A p38 inhibitor reversed the effect PDGFR-α activation in naïve animals. PDGFR-α activation was suppressed by thrombin inhibition and exogenous PDGF-AA administration increased PDGFR-α activation, regardless of thrombin inhibition. In our thrombin injection model, animals receiving the treatment of a PDGF-AA neutralizing antibody or Gleevec, a PDGFR-α antagonist, showed minimized thrombin-induced BBB impairment. We concluded that anti-inflammation by targeting VAP-1 or BBB preservation by targeting PDGFR-α may serve as new treatments against brain edema following ICH

MicroRNA-210 Suppresses Junction Proteins and Disrupts Blood-Brain Barrier Integrity in Neonatal Rat Hypoxic-Ischemic Brain Injury.

Cerebral edema, primarily caused by disruption of the blood-brain barrier (BBB), is one of the serious complications associated with brain injury in neonatal hypoxic-ischemic encephalopathy (HIE). Our recent study demonstrated that the hypoxic-ischemic (HI) treatment significantly increased microRNA-210 (miR-210) in the neonatal rat brain and inhibition of miR-210 provided neuroprotection in neonatal HI brain injury. The present study aims to determine the role of miR-210 in the regulation of BBB integrity in the developing brain. miR-210 mimic was administered via intracerebroventricular injection (i.c.v.) into the brain of rat pups. Forty-eight hours after the injection, a modified Rice-Vannucci model was conducted to produce HI brain injury. Post-assays included cerebral edema analysis, western blotting, and immunofluorescence staining for serum immunoglobulin G (IgG) leakage. The results showed that miR-210 mimic exacerbated cerebral edema and IgG leakage into the brain parenchyma. In contrast, inhibition of miR-210 with its complementary locked nucleic acid oligonucleotides (miR-210-LNA) significantly reduced cerebral edema and IgG leakage. These findings suggest that miR-210 negatively regulates BBB integrity i n the neonatal brain. Mechanistically, the seed sequences of miR-210 were identified complementary to the 3' untranslated region (3' UTR) of the mRNA transcripts of tight junction protein occludin and adherens junction protein β-catenin, indicating downstream targets of miR-210. This was further validated by in vivo data showing that miR-210 mimic significantly reduced the expression of these junction proteins in rat pup brains. Of importance, miR-210-LNA preserved the expression of junction proteins occludin and β-catenin from neonatal HI insult. Altogether, the present study reveals a novel mechanism of miR-210 in impairing BBB integrity that contributes to cerebral edema formation after neonatal HI insult, and provides new insights in miR-210-LNA mediated neuroprotection in neonatal HI brain injury

Patch Similarity Aware Data-Free Quantization for Vision Transformers

Vision transformers have recently gained great success on various computer vision tasks; nevertheless, their high model complexity makes it challenging to deploy on resource-constrained devices. Quantization is an effective approach to reduce model complexity, and data-free quantization, which can address data privacy and security concerns during model deployment, has received widespread interest. Unfortunately, all existing methods, such as BN regularization, were designed for convolutional neural networks and cannot be applied to vision transformers with significantly different model architectures. In this paper, we propose PSAQ-ViT, a Patch Similarity Aware data-free Quantization framework for Vision Transformers, to enable the generation of "realistic" samples based on the vision transformer's unique properties for calibrating the quantization parameters. Specifically, we analyze the self-attention module's properties and reveal a general difference (patch similarity) in its processing of Gaussian noise and real images. The above insights guide us to design a relative value metric to optimize the Gaussian noise to approximate the real images, which are then utilized to calibrate the quantization parameters. Extensive experiments and ablation studies are conducted on various benchmarks to validate the effectiveness of PSAQ-ViT, which can even outperform the real-data-driven methods.Comment: Accepted to ECCV 202

Repression of the Glucocorticoid Receptor Aggravates Acute Ischemic Brain Injuries in Adult Mice.

Strokes are one of the leading causes of mortality and chronic morbidity in the world, yet with only limited successful interventions available at present. Our previous studies revealed the potential role of the glucocorticoid receptor (GR) in the pathogenesis of neonatal hypoxic-ischemic encephalopathy (HIE). In the present study, we investigate the effect of GR knockdown on acute ischemic brain injuries in a model of focal cerebral ischemia induced by middle cerebral artery occlusion (MCAO) in adult male CD1 mice. GR siRNAs and the negative control were administered via intracerebroventricular (i.c.v.) injection 48 h prior to MCAO. The cerebral infarction volume and neurobehavioral deficits were determined 48 h after MCAO. RT-qPCR was employed to assess the inflammation-related gene expression profiles in the brain before and after MCAO. Western Blotting was used to evaluate the expression levels of GR, the mineralocorticoid receptor (MR) and the brain-derived neurotrophic factor/tropomyosin receptor kinase B (BDNF/TrkB) signaling. The siRNAs treatment decreased GR, but not MR, protein expression, and significantly enhanced expression levels of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) in the brain. Of interest, GR knockdown suppressed BDNF/TrkB signaling in adult mice brains. Importantly, GR siRNA pretreatment significantly increased the infarction size and exacerbated the neurobehavioral deficits induced by MCAO in comparison to the control group. Thus, the present study demonstrates the important role of GR in the regulation of the inflammatory responses and neurotrophic BDNF/TrkB signaling pathway in acute ischemic brain injuries in adult mice, revealing a new insight into the pathogenesis and therapeutic potential in acute ischemic strokes

Association between novel PLCE1variants identified in published esophageal cancer genome-wide association studies and risk of squamous cell carcinoma of the head and neck

BACKGROUND: Phospholipase C epsilon 1 (PLCE1) (an effector of Ras) belonging to the phospholipase family plays crucial roles in carcinogenesis and progression of several cancers, including squamous cell carcinoma of the head and neck (SCCHN). A single nucleotide polymorphism (SNP, rs2274223) in PLCE1 has been identified as a novel susceptibility locus in genome-wide association studies (GWAS) of esophageal squamous cell carcinoma (ESCC) and gastric cardia adenocarcinoma (GCA) that share similar risk factors with SCCHN. Therefore, we investigated the association between potentially functional SNPs in PLCE1 and susceptibility to SCCHN. METHODS: We genotyped three potentially functional SNPs (rs2274223A/G, rs3203713A/G and rs11599672T/G) of PLCE1 in 1,098 SCCHN patients and 1,090 controls matched by age and sex in a non-Hispanic white population. RESULTS: Although none of three SNPs was alone significantly associated with overall risk of SCCHN, their combined effects of risk alleles (rs2274223G, rs3203713G and rs11599672G) were found to be associated with risk of SCCHN in a locus-dose effect manner (P(trend )= 0.046), particularly for non-oropharyngeal tumors (P(trend )= 0.017); specifically, rs2274223 was associated with a significantly increased risk (AG vs. AA: adjusted OR = 1.29, 95% CI = 1.01-1.64; AG/GG vs. AA: adjusted OR = 1.30, 95% CI = 1.03-1.64), while rs11599672 was associated with a significantly decreased risk (GG vs. TT: adjusted OR = 0.54, 95% CI = 0.34-0.86; TG/GG vs. TT: adjusted OR = 0.76, 95% CI = 0.61-0.95). CONCLUSIONS: Our findings suggest that PLCE1 variants may have an effect on risk of SCCHN associated with tobacco and alcohol exposure, particularly for those tumors arising at non-oropharyngeal sites. These findings, although need to be validated by larger studies, are consistent with those in esophageal and gastric cancers

Blood-brain barrier-associated pericytes internalize and clear aggregated amyloid-β42 by LRP1-dependent apolipoprotein E isoform-specific mechanism

Table S1. Demographic and clinical features of human subjects used in this study. Figure S1. Aβ deposition in microvessels in AD patients and APPSw/0 mice. Figure S2. Biochemical analysis of Aβ42 aggregates. Figure S3. Cy3-Aβ42 cellular uptake in wild type mouse brain slices within 30 min. Figure S4. Pericyte coverages in Lrp1lox/lox and Lrp1lox/lox; Cspg4-Cre mice. Figure S5.. LRP1 and apoE suppression with siRNA. (DOCX 1454 kb

PO-305 An 8-week, low carbohydrate, high fat, ketogenic diet enhanced exercise capacity through improved ketolysis and lipolysis in mice

Objective Carbohydrates, lipids and proteins are utilized both for energy production and structure of body. Among them, protein is the most important component of our body, carbohydrates and lipids are more flexible for energy supply system. Due to carbohydrate pitfall and lipid reserve abundance, coaches and elite athletes aspirate for an effective way to enhance fat utilization. Meanwhile, intramuscular triacylglycerol (IMTG) is a special way for skeletal muscle to store lipids. During exercise, IMTG may contribute up to 20% of total energy turnover, thus contribute significantly for ATP synthesis during exercise. However, abnormal or excessive fat deposition in skeletal muscle may induce insulin resistance as well. Intramuscular lipolysis regulation is crucial for energy supply system during exercise. It is reported by Amati and colleagues that well-trained athletes exhibit higher levels of IMTG and diacylglycerol (DAG) as well as well-preserved sensitivity to insulin, indicating lipolysis ability may be enhanced during exercise. In our previous study, we reported that an 8-week, a low carbohydrate, ketogenic diet increased running time till exhausted in male C57BL6/J mice, presuming the mechanism to be enhanced fat utilization. In the present study, we observed the alternation pattern of messenger RNAs related to lipid mobilization, fatty acid utilization and ketone body oxidation in muscle and adipose tissue immediately after exercise in both Type 1 and Type 2 muscle fibers. Materials and Methods Male C57BL/6J mice (n = 35) were purchased from Takasugi Experimental Animals Supply (Kasukabe, Japan) at 7 weeks of age. All mice were randomly divided into four groups: chow diet (control: Con), including chow diet, sedentary (n = 8) and chow diet plus exercise (Con + Ex, n = 9), ketogenic diet (KD), including KD, sedentary, n = 9, and KD plus exercise (KD + Ex, n = 9) groups. A KD diet TP-201450 (consisting of 76.1% fat, 8.9% protein and 3.5% carbohydrate, 7.342 kcal/g) and a chow diet AIN93G (consisting of 7% fat, 17.8% protein and 64.3% carbohydrate, 3.601 kcal/g) wt/wt were obtained from Trophic (TROPHIC Animal Feed High-tech Co., Ltd., Nantong, Jiangsu, China). Mice were maintained on ad libitum chow diet or KD. Total RNA was extracted from the gastrocnemius muscle, soleus muscle and epididymal adipose tissue using the RNeasy Mini Kit or RNeasy Lipid Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Total RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. PCR was performed with the Fast 7500 real-time PCR system (Applied Biosystems) using the Fast SYBR® Green PCR Master Mix (Applied Biosystems). Plasma IL-6 was measured using a R&D Mouse ELISA Duo set (R&D Systems, Minneapolis, USA) according to the manufacture’s instructions. Plasma glycerol was measured using Glycerol Colorimetric Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA). Results and Discussion 3.1 IL-6 concentration and exercise-induced myokine IL-6 mRNA alternation in both muscle fiber IL-6 plays essential roles in immune responses. However, exercise induced IL-6 is reported to be able to stimulate lipolysis both in IMTG pool intramuscularly and adipocytes. Defined as exercise factor, or so-called myokines, muscle-derived IL-6 exhibits regulating function in various experiment circumstances. Recombinant human IL-6 infusion showed an enhanced lipolysis and fat oxidation capacity in human subjects. Genetically IL-6 deficient mice presented a reduced ability on lipolysis and fatty acid oxidation. During KD administration, fat oxidation is no doubt the predominant, if not the only origin of energy, this make us to suspect IL-6 may be altered by acute exercise. As shown in Figure 1, IL-6 mRNA increased rapidly, with a nearly 100-fold change in slow-twitch muscle fiber, and KD helped to this up-regulation. Transcription IL-6 level increased significantly in KD, compared to Con group subject, in soleus muscle, under the context of exhaustive exercise. This makes us to suspect that IL-6 may contribute to enhanced lipolysis and fatty acid mobilization. However, the effect is not observed in fast-twitch muscle. The result indicated that IL-6 mRNA expression exhibited a muscle fiber specification. Slow-twitch muscle fiber contributes more to endurance exercise, as fast-twitch muscle fiber mainly contributes to explosive strength and acceleration. The difference of fiber function leads to a different secretion mode of IL-6. The error bar is high in both exercise group. We observed an interesting phenomenon: the mice who quit at around 200 minutes has the highest IL-6 mRNA expression (gastrocnemius muscle) and plasma IL-6 concentration in both groups. One reason may be that, as the exercise taking on and gradually reach final fatigue, the call for fatty acid decreases with time. As shown in Figure 2, both muscular IL-6 protein and plasma IL-6 were increased by exhaustive exercise. However, plasma IL-6 is significantly lower in the KD plus exercise group, though IL-6 rose nearly 5-fold in control feed group, it only rose to 2.5-fold in the KD group after exercise.  For this phenomenon, the best explanation is that the well-adaption of lipid-centered metabolism, including metabolic flexibility and increased IMTG reservoir weakened the need to pull the trigger; this may also be the answer why KD mice had lower weight. 3.2 Fatty acid mobilation related RNA alternation after exhaustive exercise under endogenous ketosis in epididymal adipose tissue Adipose triglyceride lipase (ATGL) is also known as desnutrin in the first place, is a kind of lipase whose substance is patatin-like phospholipase domain-containing protein. Hormone-sensitive lipase (HSL) is also known as cholesteryl ester hydrolase (CEH), is another intracellular neutral lipase. ATGL and HSL cooperated to break apart fatty acids from TG, after which IMTG-origin fatty acid will be directly used for beta-oxidation, or lipid drop-origin fatty acids will be transported though lipoprotein shipping in the form of VLDL from adipose tissue into muscle fibers during exercise. As shown in Figure 3, mRNA expression levels of lipase were significantly enhanced by KD or exercise, indicating the up-regulated lipid mobilization and utilization ability is enhanced by exercise in adipocytes. However, KD plus exercise reversed this increase. One plausible explain for this phenomenon is the lack of plasma IL-6, thus the ability to mobilize fatty acid from adipose tissue is reduced. Adrenergic blocking agents are reported to harm fatty acid mobilization during fasting, and IL-6 is reported to function as adrenergic hormone. Adipocyte-specific HSL deficiency mice present lowered submaximal exercise capacity. Our experiment design, the protocol for treadmill running is similar to a submaximal exercise. Under this circumstance, fat mobilization seems to be critical. Loss of this mobilizing ability, while exercise capacity is yet enhanced, makes us to suspect whether IMTG plays dominant role in this process. 3.3 Ketolytic RNA alternation after exhaustive exercise under conditions of endogenous ketosis in Type 1 and Type 2 muscle fiber Ketolysis is a complete oxidation of ketone bodies. Ketone bodies are utilized by mitochondria of extrahepatic tissues via a series of enzymatic reactions. Ketolysis is regulated by a rate-limiting enzyme 3-oxoacid CoA-transferase 1 (OXCT)-1 and hydroxybutyrate dehydrogenase (HBDH). Thus, we measured the transcriptional alternation of these enzymes in different muscle tissues. In our previous study, plasma ketone body (KB) increased rapidly in the sedentary KD group. However, after exhaustive exercise, blood KB of those KD mice dropped dramatically, while situation of blood KD in the Con mice showed a different figure. These results indicated that 1n 8-week KD administration has improved ketolysis, the ability for subjects to utilize KB. To investigate the mechanism of this enhancement, we assessed key enzymes in ketolysis in both fiber types. As shown in Figure 4, gene expressions of these enzymes also present a fiber-specification. Since fast-twitch muscle fiber plays a second role in endurance exercise, exercise did not alter ketolytic enzymes in the transcriptional level, in gastrocnemius tissue. However, in the slow-twitch muscle fiber, it was changed. HBDH is up-regulated significantly in the case of KD plus exercise. Results here indicated that HBDH plays the key role in the improvement of exercise capacity by an 8-week KD. 3.4 Lipolysis- and fatty acid oxidation related RNA alternation after exhaustive exercise during endogenous ketosis in Type 1 and Type 2muscle fiber After reaching working site, muscle lipoprotein lipase (LPL) hydrolyzes VLDL and harvests fatty acids at last, which will be finally utilized as primary fuel. Carnitine palmitoyl transferase (CPT)-1A, acyl-CoA oxidase (ACO), hydroxyacyl-coenzyme A dehydrogenase (HADH), medium chain acyl-CoA dehydrogenase (MCAD) and malonyl-CoA decarboxylase (MCD) are key regulating enzymes during fatty acid beta-oxidation. And for IMTG, the fatty acid is harvest by intramuscular lipase, ATGL and HSL. In an article published several years ago, the authors called adipose ATGL and HSL, “the mover and shaker of muscle lipolysis”. As shown in Figure 5, both ATGL and HSL mRNA expression are up-regulated by KD, but not by exhaustive exercise. Combined with results in the above part, enhanced mobilization of fatty acid intramuscularly is the main factor, but not the mobilization from adipocyte. LPL mRNA expression in gastrocnemius exhibited the same pattern of lipase mRNA synthesized by adipose tissue. Combined together, the reduced fatty mobilization from adipocyte, partly being the results of higher blood NEFA and TG, accompanied with enhanced fatty acid gain from IMTG pool, need for LPL was reduced.  As shown in Figure 6, in fast-twitch muscle fiber, CPT-1a, ACO and HADH mRNA expressions are enhanced by KD during exhaustive exercise. In slow-twitch muscle fiber, feed played as a main factor regulating fatty acid oxidation. CPT1a, MCAD and MCD mRNA expressions are enhanced. In summary, though tissue specific specificity were observed, overall ability of intramuscular fatty acid mobilization and fatty acid oxidation were enhanced by an 8-week KD feeding, thus contributed to exercise capacity. Compared to a glucose-centered metabolic system, a long-term KD feeding leads to establishment of a fatty acid oxidation-centered metabolic system. Metabolic flexibility is used as a term for the ability to adapt to conditional change in metabolic demand, and an 8-week KD helped established lipid-focused metabolic system through keto-adaption, thus increasing the metabolic flexibility. This is not a denial for the conception “glycogen loading” before competitions, while adequate KD meal may help our body to be more flexible during fuel choosing. Moderate training may enhance the ability to utilize ketone bodies as well as fatty acid, or to increase fatty acid mobilization from adipose tissue. Further investigation is urged to be carried out.   Conclusions In the present study, we investigated how an 8-week KD remodeled adipose and muscle metabolic adaptation towards ketolysis, lipolysis and fatty acid oxidation under the circumstance of exhaustive exercise. Along with enhanced fatty acid oxidation capacity, KD also enhanced fatty acid mobilization capacity, ketolysis and lipolysis. These results revealed that an 8-week KD administration enhance exercise performance by up-regulated ketolytic and free fatty acid oxidation ability, indicating KD being a promising diet approach in athletes

Associations between obesity, smoking behaviors, reproductive traits and spontaneous abortion: a univariable and multivariable Mendelian randomization study

BackgroundThe correlation between potential risk factors such as obesity (leg fat percentage (left), arm fat percentage (left), waist circumference, body fat percentage, trunk fat percentage), smoking behaviors (past tobacco smoking, smoking initiation, smoking/smokers in household, current tobacco smoking) and reproductive traits (age first had sexual intercourse (AFS), age at menarche (AAM), and age at first birth (AFB)) have been linked to the occurrence of spontaneous abortion (SA). However, the causal associations between these factors and SA remain unclear.MethodsWe conducted univariable and multivariable Mendelian randomization (MR) analyses to evaluate the associations of obesity, smoking behavior and reproductive traits with SA. To select appropriate genetic instruments, we considered those that had reached the genome-wide significance level (P < 5 × 10–8) in their corresponding genome-wide association studies (GWAS) involving a large number of individuals (ranging from 29,346 to 1,232,091). SA was obtained from the FinnGen consortium, which provided summary-level data for 15,073 SA cases and 135,962 non-cases.ResultsAssessed individually using MR, the odds ratios (ORs) of SA were 0.728 (P = 4.3608×10-8), 1.063 (P = 0.0321), 0.926 (P = 9.4205×10-4), 1.141 (P = 7.9882×10-3), 5.154 (P = 0.0420), 1.220 (P = 0.0350), 1.228 (P = 0.0117), 0.795 (P = 0.0056), 1.126 (P = 0.0318), for one standard deviation (SD) increase in AFS, AAM, AFB, smoking initiation, smoking/smokers in household, arm fat percentage (left), leg fat percentage (left), waist circumference and body fat percentage, 0.925 (P = 0.4158) and 1.075 (P = 0.1479) for one SD increase in past tobacco smoking, trunk fat percentage for one SD increase in SA. In multivariable MR (MVMR), only AFS (OR = 0.802; P = 0.0250), smoking initiation (OR = 1.472, P = 0.0258), waist circumference (OR = 0.813, P = 0.0220) and leg fat percentage (left) (OR = 4.446, P = 0.043) retained a robust effect.ConclusionSmoking behaviors, reproductive traits and obesity-related anthropometric indicators are potential causal factors for SA. Higher leg fat percentage; smoking initiation; and lower waist circumference and AFS may increase the risk of SA. Understanding the causal relationship for SA may provide more information for SA intervention and prevention strategies

Antenatal Hypoxia and Programming of Glucocorticoid Receptor Expression in the Adult Rat Heart

Glucocorticoid receptor (GR) signaling is critical for development and function of the heart. Our previous study demonstrated that gestational hypoxia induced epigenetic repression of the GR gene in the developing heart. The present study aims to determine that the alterations of promoter methylation level and epigenetic repression of the GR gene in the developing heart in response to maternal hypoxia is sustained in adult offspring and potential gender differences in the programming of GR gene. Pregnant rats were treated with 10.5% O2 from gestational day 15 (E15) to 21 (E21). Hearts were isolated from 5-month-old male and female offspring with the developing stage being equivalent to 18-year-old human. GR mRNA and protein abundance was determined with real time qRT-PCR and Western blot. GR gene promoter methylation and binding of transcription factors were measured with methylated DNA immunoprecipitation (MeDIP) and Chromatin immunoprecipitation (ChIP). The results showed that antenatal hypoxia significantly decreased the expression of GR mRNA and protein in the hearts of adult male offspring, but not in females, which is ascribed to the differential changes of alternative exon1 mRNA variants of GR gene in male and female hearts in response to prenatal hypoxia. In addition, the downregulation of GR expression in the male heart was correlated with increased methylation levels of CpG dinucleotides in promoters of exon 14, 15, 16, 17, and 110, which resulted in a decrease in the binding of their transcription factors. Thus, the study reveals that antenatal hypoxia results in a reprogramming and long-term change in GR gene expression in the heart by hypermethylation of GR promoter in a sex-differential pattern, which provides a novel mechanism regarding the increased vulnerability of heart later in life with exposure of prenatal hypoxia