Cardiovascular Protective Effects of NP-6A4, a Drug with the FDA Designation for Pediatric Cardiomyopathy, in Female Rats with Obesity and Pre-Diabetes.

BACKGROUND: Obese and pre-diabetic women have a higher risk for cardiovascular death than age-matched men with the same symptoms, and there are no effective treatments. We reported that obese and pre-diabetic female Zucker Diabetic Fatty (ZDF-F) rats recapitulate metabolic and cardiac pathology of young obese and pre-diabetic women and exhibit suppression of cardio-reparative AT2R. Here, we investigated whether NP-6A4, a new AT2R agonist with the FDA designation for pediatric cardiomyopathy, mitigate heart disease in ZDF-F rats by restoring AT2R expression. METHODS: ZDF-F rats on a high-fat diet (to induce hyperglycemia) were treated with saline, NP-6A4 (10 mg/kg/day), or NP-6A4 + PD123319 (AT2R-specific antagonist, 5 mg/kg/day) for 4 weeks (n = 21). Cardiac functions, structure, and signaling were assessed by echocardiography, histology, immunohistochemistry, immunoblotting, and cardiac proteome analysis. RESULTS: NP-6A4 treatment attenuated cardiac dysfunction, microvascular damage (-625%) and cardiomyocyte hypertrophy (-263%), and increased capillary density (200%) and AT2R expression (240%) (p < 0.05). NP-6A4 activated a new 8-protein autophagy network and increased autophagy marker LC3-II but suppressed autophagy receptor p62 and autophagy inhibitor Rubicon. Co-treatment with AT2R antagonist PD123319 suppressed NP-6A4's protective effects, confirming that NP-6A4 acts through AT2R. NP-6A4-AT2R-induced cardioprotection was independent of changes in body weight, hyperglycemia, hyperinsulinemia, or blood pressure. CONCLUSIONS: Cardiac autophagy impairment underlies heart disease induced by obesity and pre-diabetes, and there are no drugs to re-activate autophagy. We propose that NP-6A4 can be an effective drug to reactivate cardiac autophagy and treat obesity- and pre-diabetes-induced heart disease, particularly for young and obese women.

Biomarkers selection for population normalization in SARS-CoV-2 wastewater-based epidemiology.

Wastewater-based epidemiology (WBE) has been one of the most cost-effective approaches to track the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels in the communities since the coronavirus disease 2019 (COVID-19) outbreak in 2020. Normalizing SARS-CoV-2 concentrations by the population biomarkers in wastewater is critical for interpreting the viral loads, comparing the epidemiological trends among the sewersheds, and identifying the vulnerable communities. In this study, five population biomarkers, pepper mild mottle virus (PMMoV), creatinine (CRE), 5-hydroxyindoleacetic acid (5-HIAA), caffeine (CAF) and its metabolite paraxanthine (PARA) were investigated and validated for their utility in normalizing the SARS-CoV-2 loads through two normalizing approaches using the data from 64 wastewater treatment plants (WWTPs) in Missouri. Their utility in assessing the real-time population contributing to the wastewater was also evaluated. The best performing candidate was further tested for its capacity for improving correlation between normalized SARS-CoV-2 loads and the clinical cases reported in the City of Columbia, Missouri, a university town with a constantly fluctuating population. Our results showed that, except CRE, the direct and indirect normalization approaches using biomarkers allow accounting for the changes in wastewater dilution and differences in relative human waste input over time regardless flow volume and population of the given WWTP. Among selected biomarkers, PARA is the most reliable population biomarker in determining the SARS-CoV-2 load per capita due to its high accuracy, low variability, and high temporal consistency to reflect the change in population dynamics and dilution in wastewater. It also demonstrated its excellent utility for real-time assessment of the population contributing to the wastewater. In addition, the viral loads normalized by the PARA-estimated population significantly improved the correlation (rho=0.5878, p < 0.05) between SARS-CoV-2 load per capita and case numbers per capita. This chemical biomarker complements the current normalization scheme recommended by CDC and helps us understand the size, distribution, and dynamics of local populations for forecasting the prevalence of SARS-CoV2 within each sewershed.

Identification and quantification of bioactive compounds suppressing SARS-CoV-2 signals in wastewater-based epidemiology surveillance.

Recent SARS-CoV-2 wastewater-based epidemiology (WBE) surveillance have documented a positive correlation between the number of COVID-19 patients in a sewershed and the level of viral genetic material in the wastewater. Efforts have been made to use the wastewater SARS-CoV-2 viral load to predict the infected population within each sewershed using a multivariable regression approach. However, reported clear and sustained variability in SARS-CoV-2 viral load among treatment facilities receiving industrial wastewater have made clinical prediction challenging. Several classes of molecules released by regional industries and manufacturing facilities, particularly the food processing industry, can significantly suppress the SARS-CoV-2 signals in wastewater by breaking down the lipid-bilayer of the membranes. Therefore, a systematic ranking process in conjugation with metabolomic analysis was developed to identify the wastewater treatment facilities exhibiting SARS-CoV-2 suppression and identify and quantify the chemicals suppressing the SARS-COV-2 signals. By ranking the viral load per diagnosed case among the sewersheds, we successfully identified the wastewater treatment facilities in Missouri, USA that exhibit SARS-CoV-2 suppression (significantly lower than 5 × 1011 gene copies/reported case) and determined their suppression rates. Through both untargeted global chemical profiling and targeted analysis of wastewater samples, 40 compounds were identified as candidates of SARS-CoV-2 signal suppressors. Among these compounds, 14 had higher concentrations in wastewater treatment facilities that exhibited SARS-CoV-2 signal suppression compared to the unsuppressed control facilities. Stepwise regression analyses indicated that 4-nonylphenol, palmitelaidic acid, sodium oleate, and polyethylene glycol dioleate are positively correlated with SARS-CoV-2 signal suppression rates. Suppression activities were further confirmed by incubation studies, and the suppression kinetics for each bioactive compound were determined. According to the results of these experiments, bioactive molecules in wastewater can significantly reduce the stability of SARS-CoV-2 genetic marker signals. Based on the concentrations of these chemical suppressors, a correction factor could be developed to achieve more reliable and unbiased surveillance results for wastewater treatment facilities that receive wastewater from similar industries.

Biomarkers Selection for Population Normalization in SARS-CoV-2 Wastewater-based Epidemiology.

Wastewater-based epidemiology (WBE) has been one of the most cost-effective approaches to track the SARS-CoV-2 levels in the communities since the COVID-19 outbreak in 2020. Normalizing SARS-CoV-2 concentrations by the population biomarkers in wastewater can be critical for interpreting the viral loads, comparing the epidemiological trends among the sewersheds, and identifying the vulnerable communities. In this study, five population biomarkers, pepper mild mottle virus (pMMoV), creatinine (CRE), 5-hydroxyindoleacetic acid (5-HIAA), caffeine (CAF) and its metabolite paraxanthine (PARA) were investigated for their utility in normalizing the SARS-CoV-2 loads through developed direct and indirect approaches. Their utility in assessing the real-time population contributing to the wastewater was also evaluated. The best performed candidate was further tested for its capacity for improving correlation between normalized SARS-CoV-2 loads and the clinical cases reported in the City of Columbia, Missouri, a university town with a constantly fluctuated population. Our results showed that, except CRE, the direct and indirect normalization approaches using biomarkers allow accounting for the changes in wastewater dilution and differences in relative human waste input over time regardless flow volume and population at any given WWTP. Among selected biomarkers, PARA is the most reliable population biomarker in determining the SARS-CoV-2 load per capita due to its high accuracy, low variability, and high temporal consistency to reflect the change in population dynamics and dilution in wastewater. It also demonstrated its excellent utility for real-time assessment of the population contributing to the wastewater. In addition, the viral loads normalized by the PARA-estimated population significantly improved the correlation ( rho =0.5878, p <0.05) between SARS-CoV-2 load per capita and case numbers per capita. This chemical biomarker offers an excellent alternative to the currently CDC-recommended pMMoV genetic biomarker to help us understand the size, distribution, and dynamics of local populations for forecasting the prevalence of SARS-CoV2 within each sewershed. HIGHLIGHT bullet points: The paraxanthine (PARA), the metabolite of the caffeine, is a more reliable population biomarker in SARS-CoV-2 wastewater-based epidemiology studies than the currently recommended pMMoV genetic marker.SARS-CoV-2 load per capita could be directly normalized using the regression functions derived from correlation between paraxanthine and population without flowrate and population data.Normalizing SARS-CoV-2 levels with the chemical marker PARA significantly improved the correlation between viral loads per capita and case numbers per capita.The chemical marker PARA demonstrated its excellent utility for real-time assessment of the population contributing to the wastewater.

Defining biological and biophysical properties of SARS-CoV-2 genetic material in wastewater.

SARS-CoV-2 genetic material has been detected in raw wastewater around the world throughout the COVID-19 pandemic and has served as a useful tool for monitoring community levels of SARS-CoV-2 infections. SARS-CoV-2 genetic material is highly detectable in a patient's feces and the household wastewater for several days before and after a positive COVID-19 qPCR test from throat or sputum samples. Here, we characterize genetic material collected from raw wastewater samples and determine recovery efficiency during a concentration process. We find that pasteurization of raw wastewater samples did not reduce SARS-CoV-2 signal if RNA is extracted immediately after pasteurization. On the contrary, we find that signal decreased by approximately half when RNA was extracted 24-36 h post-pasteurization and ~90% when freeze-thawed prior to concentration. As a matrix control, we use an engineered enveloped RNA virus. Surprisingly, after concentration, the recovery of SARS-CoV-2 signal is consistently higher than the recovery of the control virus leading us to question the nature of the SARS-CoV-2 genetic material detected in wastewater. We see no significant difference in signal after different 24-hour temperature changes; however, treatment with detergent decreases signal ~100-fold. Furthermore, the density of the samples is comparable to enveloped retrovirus particles, yet, interestingly, when raw wastewater samples were used to inoculate cells, no cytopathic effects were seen indicating that wastewater samples do not contain infectious SARS-CoV-2. Together, this suggests that wastewater contains fully intact enveloped particles.

Suppression of Inflammatory Cardiac Cytokine Network in Rats with Untreated Obesity and Pre-Diabetes by AT2 Receptor Agonist NP-6A4.

Obesity affects over 42% of the United States population and exacerbates heart disease, the leading cause of death in men and women. Obesity also increases pro-inflammatory cytokines that cause chronic tissue damage to vital organs. The standard-of-care does not sufficiently attenuate these inflammatory sequelae. Angiotensin II receptor AT2R is an anti-inflammatory and cardiovascular protective molecule; however, AT2R agonists are not used in the clinic to treat heart disease. NP-6A4 is a new AT2R peptide agonist with an FDA orphan drug designation for pediatric cardiomyopathy. NP-6A4 increases AT2R expression (mRNA and protein) and nitric oxide generation in human cardiovascular cells. AT2R-antagonist PD123319 and AT2RSiRNA suppress NP-6A4-effects indicating that NP-6A4 acts through AT2R. To determine whether NP-6A4 would mitigate cardiac damage from chronic inflammation induced by untreated obesity, we investigated the effects of 2-weeks NP-6A4 treatment (1.8 mg/kg delivered subcutaneously) on cardiac pathology of male Zucker obese (ZO) rats that display obesity, pre-diabetes and cardiac dysfunction. NP-6A4 attenuated cardiac diastolic and systolic dysfunction, cardiac fibrosis and cardiomyocyte hypertrophy, but increased myocardial capillary density. NP-6A4 treatment suppressed tubulointerstitial injury marker urinary ß-NAG, and liver injury marker alkaline phosphatase in serum. These protective effects of NP-6A4 occurred in the presence of obesity, hyperinsulinemia, hyperglycemia, and hyperlipidemia, and without modulating blood pressure. NP-6A4 increased expression of AT2R (consistent with human cells) and cardioprotective erythropoietin (EPO) and Notch1 in ZO rat heart, but suppressed nineteen inflammatory cytokines. Cardiac miRNA profiling and in silico analysis showed that NP-6A4 activated a unique miRNA network that may regulate expression of AT2R, EPO, Notch1 and inflammatory cytokines, and mitigate cardiac pathology. Seventeen pro-inflammatory and pro-fibrotic cytokines that increase during lethal cytokine storms caused by infections such as COVID-19 were among the cytokines suppressed by NP-6A4 treatment in ZO rat heart. Thus, NP-6A4 activates a novel anti-inflammatory network comprised of 21 proteins in the heart that was not reported previously. Since NP-6A4's unique mode of action suppresses pro-inflammatory cytokine network and attenuates myocardial damage, it can be an ideal adjuvant drug with other anti-glycemic, anti-hypertensive, standard-of-care drugs to protect the heart tissues from pro-inflammatory and pro-fibrotic cytokine attack induced by obesity.

AT2R agonist NP-6A4 mitigates aortic stiffness and proteolytic activity in mouse model of aneurysm.

Clinical and experimental studies show that angiotensin II (AngII) promotes vascular pathology via activation of AngII type 1 receptors (AT1Rs). We recently reported that NP-6A4, a selective peptide agonist for AngII type 2 receptor (AT2R), exerts protective effects on human vascular cells subjected to serum starvation or doxorubicin exposure. In this study, we investigated whether NP-6A4-induced AT2R activation could mitigate AngII-induced abdominal aortic aneurism (AAA) using AngII-treated Apoe-/- mice. Male Apoe-/- mice were infused with AngII (1 µg/kg/min) by implanting osmotic pumps subcutaneously for 28 days. A subset of mice was pre-treated subcutaneously with NP-6A4 (2.5 mg/kg/day) or vehicle for 14 days prior to AngII, and treatments were continued for 28 days. NP-6A4 significantly reduced aortic stiffness of the abdominal aorta induced by AngII as determined by ultrasound functional analyses and histochemical analyses. NP-6A4 also increased nitric oxide bioavailability in aortic tissues and suppressed AngII-induced increases in monocyte chemotactic protein-1, osteopontin and proteolytic activity of the aorta. However, NP-6A4 did not affect maximal intraluminal aortic diameter or AAA incidences significantly. These data suggest that the effects of AT2R agonist on vascular pathologies are selective, affecting the aortic stiffness and proteolytic activity without affecting the size of AAA.

Empagliflozin reduces high glucose-induced oxidative stress and miR-21-dependent TRAF3IP2 induction and RECK suppression, and inhibits human renal proximal tubular epithelial cell migration and epithelial-to-mesenchymal transition.

Proximal tubular epithelial cells (PTEC) in the S1 segment of the kidney abundantly express sodium-glucose co-transporters (SGLT) that play a critical role in whole body glucose homeostasis. We recently reported suppression of RECK (Reversion Inducing Cysteine Rich Protein with Kazal Motifs), a membrane anchored endogenous MMP inhibitor and anti-fibrotic mediator, in the kidneys of db/db mice, a model of diabetic kidney disease (DKD), as well as in high glucose (HG) treated human kidney proximal tubule cells (HK-2). We further demonstrated that empagliflozin (EMPA), an SGLT2 inhibitor, reversed these effects. Little is known regarding the mechanisms underlying RECK suppression under hyperglycemic conditions, and its rescue by EMPA. Consistent with our previous studies, HG (25 mM) suppressed RECK expression in HK-2 cells. Further mechanistic investigations revealed that HG induced superoxide and hydrogen peroxide generation, oxidative stress-dependent TRAF3IP2 upregulation, NF-κB and p38 MAPK activation, inflammatory cytokine expression (IL-1ß, IL-6, TNF-α, and MCP-1), miR-21 induction, MMP2 activation, and RECK suppression. Moreover, RECK gain-of-function inhibited HG-induced MMP2 activation and HK-2 cell migration. Similar to HG, advanced glycation end products (AGE) induced TRAF3IP2 and suppressed RECK, effects that were inhibited by EMPA. Importantly, EMPA treatment ameliorated all of these deleterious effects, and inhibited epithelial-to-mesenchymal transition (EMT) and HK-2 cell migration. Collectively, these findings indicate that hyperglycemia and associated AGE suppress RECK expression via oxidative stress/TRAF3IP2/NF-κB and p38 MAPK/miR-21 induction. Furthermore, these results suggest that interventions aimed at restoring RECK or inhibiting SGLT2 have the potential to treat kidney inflammatory response/fibrosis and nephropathy under chronic hyperglycemic conditions, such as DKD.

APR-246 alone and in combination with a phosphatidylserine-targeting antibody inhibits lung metastasis of human triple-negative breast cancer cells in nude mice.

BACKGROUND: Approximately 15-20% of all human breast cancers are classified as triple-negative because they lack estrogen and progesterone receptors and Her-2-neu, which are commonly targeted by chemotherapeutic drugs. New treatment strategies are therefore urgently needed to combat triple-negative breast cancers (TNBCs). Almost 80% of the triple-negative tumors express mutant p53 (mtp5), a functionally defective tumor suppressor protein. Whereas wild-type p53 (wtp53) promotes cell-cycle arrest and apoptosis and inhibits vascular endothelial growth factor-dependent angiogenesis, mtp53 fails to regulate these functions, resulting in tumor vascularization, growth, resistance to chemotherapy, and metastasis. Restoration of p53 function is therefore a promising drug-targeted strategy for suppressing TNBC metastasis. METHODS: APR-246 is a small-molecule drug that reactivates mtp53, thereby restoring p53 function. In this study, we sought to determine whether administration of APR-246, either alone or in combination with 2aG4, an antibody that targets phosphatidylserine residues on tumor blood vessels and disrupts tumor vasculature, effectively inhibits stem cell-like characteristics of tumor cells and migration in vitro, and metastasis of human mtp53-expressing TNBC cells to the lungs in mouse models. RESULTS: APR-246 reduced both the stem cell-like properties and migration of TNBC cells in vitro. In mouse models, administration of either APR-246 or 2aG4 reduced metastasis of TNBC cells to the lungs; a combination of the two diminished lung metastasis to the same extent as either agent alone. Combination treatment significantly reduced the incidence of lung metastasis compared either single agent alone. CONCLUSION: Metastasis of human mtp53-expressing TNBC cells to the lungs of nude mice is inhibited by the treatment that combines activation of mtp53 with targeting of phosphatidylserine residues on tumor blood vessels. We contend therefore that our findings strongly support the use of combination treatment involving mtp53 activation and immunotherapy in patients with TNBC.

Maternal vitamin D deficiency and developmental origins of health and disease (DOHaD).

Vitamin D is an essential nutrient that is metabolized in the body to generate an active metabolite (1,25(OH)2D) with hormone-like activity and highly diverse roles in cellular function. Vitamin D deficiency (VDD) is a prevalent but easily preventable nutritional disturbance. Emerging evidence demonstrates the importance of sufficient vitamin D concentrations during fetal life with deficiencies leading to long-term effects into adulthood. Here, we provide a detailed review and perspective of evidence for the role of maternal VDD in offspring long term health, particularly as it relates to Developmental Origins of Health and Disease (DOHaD). We focus on roles in neurobehavioral and cardiometabolic disorders in humans and highlight recent findings from zebrafish and rodent models that probe potential mechanisms linking early life VDD to later life health outcomes. Moreover, we explore evidence implicating epigenetic mechanisms as a mediator of this link. Gaps in our current understanding of how maternal VDD might result in deleterious offspring outcomes later in life are also addressed.

Deficiency of IL12p40 (Interleukin 12 p40) Promotes Ang II (Angiotensin II)-Induced Abdominal Aortic Aneurysm.

Objective- Abdominal aortic aneurysm is caused by the accumulation of inflammatory cells in the aortic wall. Our recent studies demonstrated that inhibition of Notch signaling attenuates abdominal aortic aneurysm formation by shifting the macrophage balance towards anti-inflammatory (M2) phenotype. Using IL12p40-/- (interleukin 12 p40) mice, we investigated the effects of M2-predominant macrophages on the development of abdominal aortic aneurysm. Approach and Results- Male (8-10 week-old) wild-type and IL12p40-/- mice (n=15) on C57BL/6 background were infused with Ang II (angiotensin II, 1000 ng/kg per minute) by implanting osmotic pumps subcutaneously for 28 days. In the IL12p40-/- mice, Ang II significantly increased the maximal intraluminal diameter (9/15) as determined by transabdominal ultrasound imaging. In addition, IL12p40-deletion significantly increased aortic stiffness in response to Ang II as measured by pulse wave velocity and atomic force microscopy. Histologically, IL12p40-/- mice exhibited increased maximal external diameter of aorta and aortic lesions associated with collagen deposition and increased elastin fragmentation compared with wild-type mice infused with Ang II. Mechanistically, IL12p40 deficiency by siRNA (small interfering RNA) augmented the Tgfß2-mediated Mmp2 expression in wild-type bone marrow-derived macrophages without affecting the expression of Mmp9. No such effects of IL12p40 deficiency on MMP2/MMP9 was observed in human aortic smooth muscle cells or fibroblasts. Depletion of macrophages in IL12p40-/- mice by clodronate liposomes significantly decreased the maximal external diameter of aorta and aortic stiffness in response to Ang II as determined by imaging and atomic force microscopy. Conclusions- IL12p40 depletion promotes the development of abdominal aortic aneurysm, in part, by facilitating recruitment of M2-like macrophages and potentiating aortic stiffness and fibrosis mediated by Tgfß2.

Cell-Specific Protective Signaling Induced by the Novel AT2R-Agonist NP-6A4 on Human Endothelial and Smooth Muscle Cells.

Cardiovascular disease incidence continues to rise and new treatment paradigms are warranted. We reported previously that activation of Angiotensin II receptor (encoded by the X-linked Agtr2 gene) by a new peptide agonist, NP-6A4, was more effective in protecting mouse cardiomyocyte HL-1 cells and human coronary artery vascular smooth muscle cells (hCAVSMCs) from acute nutrient deficiency than other drugs tested. To elucidate further the protective effects of NP-6A4 in human cells, we studied the effects of NP-6A4 treatment on functions of human coronary artery endothelial cells (hCAECs), and hCAVSMCs. In hCAVSMCs, NP-6A4 (1 µM) increased Agtr2 mRNA (sixfold, p < 0.05) after 12-h exposure, whereas in hCAECs, significant increase in Agtr2 mRNA (hCAECs: eightfold) was observed after prolonged exposure. Interestingly, NP-6A4 treatment (1 µM, 12 h) increased AT2R protein levels in all human cells tested. Pre-treatment with AT2R-antagonist PD123319 (20 µM) and anti-AT2R siRNA (1 µM) suppressed this effect. Thus, NP-6A4 activates a positive feedback loop for AT2R expression and signaling in hCAVSMCs and hCAECs. NP-6A4 (1-20 µM) increased cell index (CI) of hCAVSMCs as determined by real time cell analyzer (RTCA), indicating that high concentrations of NP-6A4 were not cytotoxic for hCAVSMCs, rather promoting better cell attachment and growth. Seahorse Extracellular Flux Assay revealed that NP-6A4 (1 µM) treatment for 7 days increased whole cell-based mitochondrial parameters of hCAVSMCs, specifically maximal respiration (p < 0.05), spare respiratory capacity (p < 0.05) and ATP production (p < 0.05). NP-6A4 (1 µM; 7 days) also suppressed Reactive Oxygen Species (ROS) in hCAVSMCs. Exposure to Doxorubicin (DOXO) (1 µM) increased ROS in hCAVSMCs and this effect was suppressed by NP-6A4 (1 µM). In hCAECs grown in complete medium, NP-6A4 (1 µM) and Ang II (1 µM) exerted similar changes in CI. Additionally, NP-6A4 (5 µM: 12 h) increased expression of eNOS (sixfold, p < 0.05) and generation of nitric oxide (1.3-fold, p < 0.05) in hCAECs and pre-treatment with PD123319 (20 µM) suppressed this effect partially (65%). Finally, NP-6A4 decreased phosphorylation of Jun-N-terminal kinase, implicated in apoptosis of ECs in atherosclerotic sites. Taken together, NP-6A4, through its ability to increase AT2R expression and signaling, exerts different cell-specific protective effects in human VSMCs and ECs.

Diabetic Cardiomyopathy: Impact of Biological Sex on Disease Development and Molecular Signatures.

Diabetic cardiomyopathy refers to a unique set of heart-specific pathological variables induced by hyperglycemia and insulin resistance. Given that cardiovascular disease (CVD) is the leading cause of death in the world, and type 2 diabetes incidence continues to rise, understanding the complex interplay between these two morbidities and developing novel therapeutic strategies is vital. Two hallmark characteristics specific to diabetic cardiomyopathy are diastolic dysfunction and cardiac structural mal-adaptations, arising from cardiac cellular responses to the complex toxicity induced by hyperglycemia with or without hyperinsulinemia. While type 2 diabetes is more prevalent in men compared to women, cardiovascular risk is higher in diabetic women than in diabetic men, suggesting that diabetic women take a steeper path to cardiomyopathy and heart failure. Accumulating evidence from randomized clinical trials indicate that although pre-menopausal women have lower risk of CVDs, compared to age-matched men, this advantage is lost in diabetic pre-menopausal women, which suggests estrogen availability does not protect from increased cardiovascular risk. Notably, few human studies have assessed molecular and cellular mechanisms regarding similarities and differences in the progression of diabetic cardiomyopathy in men versus women. Additionally, most pre-clinical rodent studies fail to include female animals, leaving a void in available data to truly understand the impact of biological sex differences in diabetes-induced dysfunction of cardiovascular cells. Elegant reviews in the past have discussed in detail the roles of estrogen-mediated signaling in cardiovascular protection, sex differences associated with telomerase activity in the heart, and cardiac responses to exercise. In this review, we focus on the emerging cellular and molecular markers that define sex differences in diabetic cardiomyopathy based on the recent clinical and pre-clinical evidence. We also discuss miR-208a, MED13, and AT2R, which may provide new therapeutic targets with hopes to develop novel treatment paradigms to treat diabetic cardiomyopathy uniquely between men and women.

Comparison of Cardiac miRNA Transcriptomes Induced by Diabetes and Rapamycin Treatment and Identification of a Rapamycin-Associated Cardiac MicroRNA Signature.

Rapamycin (Rap), an inhibitor of mTORC1, reduces obesity and improves lifespan in mice. However, hyperglycemia and lipid disorders are adverse side effects in patients receiving Rap treatment. We previously reported that diabetes induces pansuppression of cardiac cytokines in Zucker obese rats (ZO-C). Rap treatment (750 µg/kg/day for 12 weeks) reduced their obesity and cardiac fibrosis significantly; however, it increased their hyperglycemia and did not improve their cardiac diastolic parameters. Moreover, Rap treatment of healthy Zucker lean rats (ZL-C) induced cardiac fibrosis. Rap-induced changes in ZL-C's cardiac cytokine profile shared similarities with that of diabetes-induced ZO-C. Therefore, we hypothesized that the cardiac microRNA transcriptome induced by diabetes and Rap treatment could share similarities. Here, we compared the cardiac miRNA transcriptome of ZL-C to ZO-C, Rap-treated ZL (ZL-Rap), and ZO (ZO-Rap). We report that 80% of diabetes-induced miRNA transcriptome (40 differentially expressed miRNAs by minimum 1.5-fold in ZO-C versus ZL-C; p ≤ 0.05) is similar to 47% of Rap-induced miRNA transcriptome in ZL (68 differentially expressed miRNAs by minimum 1.5-fold in ZL-Rap versus ZL-C; p ≤ 0.05). This remarkable similarity between diabetes-induced and Rap-induced cardiac microRNA transcriptome underscores the role of miRNAs in Rap-induced insulin resistance. We also show that Rap treatment altered the expression of the same 17 miRNAs in ZL and ZO hearts indicating that these 17 miRNAs comprise a unique Rap-induced cardiac miRNA signature. Interestingly, only four miRNAs were significantly differentially expressed between ZO-C and ZO-Rap, indicating that, unlike the nondiabetic heart, Rap did not substantially change the miRNA transcriptome in the diabetic heart. In silico analyses showed that (a) mRNA-miRNA interactions exist between differentially expressed cardiac cytokines and miRNAs, (b) human orthologs of rat miRNAs that are strongly correlated with cardiac fibrosis may modulate profibrotic TGF-ß signaling, and (c) changes in miRNA transcriptome caused by diabetes or Rap treatment include cardioprotective miRNAs indicating a concurrent activation of an adaptive mechanism to protect the heart in conditions that exacerbate diabetes.

Maternal vitamin D deficiency during pregnancy affects expression of adipogenic-regulating genes peroxisome proliferator-activated receptor gamma (PPARγ) and vitamin D receptor (VDR) in lean male mice offspring.

PURPOSE: Maternal vitamin D deficiency during pregnancy is a widespread issue that may have long-lasting consequences on offspring adiposity. We sought to determine how maternal vitamin D deficiency during the perinatal period would affect offspring adipose tissue development and gene expression. METHODS: Female C57BL/6 J mice were fed either a vitamin D deficient (VDD) or control diet from 4 weeks before pregnancy (periconception) until 7 days postparturition. Male offspring were weighed and euthanized at 75 days of age (early adult period), at which point serum was collected for biochemical analyses, and perigonadal and subcutaneous white adipose tissue (PGAT and SQAT, respectively) were excised, weighed, then flash-frozen for later histology and analyses of adipogenic gene expression. RESULTS: All adult male offspring were nonobese; there were no significant differences in body weight, adipose pad weight, or adipocyte size. However, VDD-exposed offspring had greater expression of the adipogenic-regulating genes peroxisome proliferator-activated receptor gamma (Pparg) and vitamin D receptor (Vdr). CONCLUSIONS: This study suggests that exposure to vitamin D deficiency during the perinatal period can directly affect genes involved in the development of adipose tissue in nonobese offspring. These novel findings invite further investigation into the mechanisms by which maternal vitamin D status during pregnancy affects adipose development and metabolic health of offspring.

Time Course of Vitamin D Depletion and Repletion in Reproductive-age Female C57BL/6 Mice.

The use of animal models in vitamin D deficiency (VDD) research, particularly in regard to maternal deficits, has increased dramatically, yet these studies may be confounded due to ill-conceived experimental timelines. We conducted 2 experiments to (1) characterize the time course of VDD induction and repletion and (2) explore the long-term consequences of VDD on calcium homeostasis and body composition in reproductive-age female mice. Eight-week-old female C57BL/6 mice were randomized to receive either a vitamin D sufficient (VDS) or VDD diet; serum was collected weekly. At week 4, VDD mice were switched to VDS diet, and serum was collected weekly until week 8. Another group of same-age female mice was maintained on VDD diet for 40 wk. Body weights and serum were collected every 2 wk until week 40, when body composition was measured by using echoMRI. Mice did not become VDD until week 3 of the VDD diet and, after decreasing slightly at 4 wk, serum 25-hydroxyvitamin D remained unchanged through 40 wk. Vitamin D repletion to 25-hydroxyvitamin D concentrations considered adequate by the Institute of Medicine took 2 to 3 wk. Prolonged VDD in mice was marked by hypocalcemia and hyperparathyroidism and led to proportional decreases in both lean and fat mass. These data provide guidance in the design of studies using mice as a maternal VDD model, especially those exploring its effects on the developmental origins of health and disease and highlight the importance of monitoring and controlling the calciotropic effects of diet-induced VDD. This study also shows that prolonged VDD in reproductive-age female C57BL/6 mice induces metabolically meaningful changes in absolute, but not relative, body composition.

In utero vitamin D deficiency predisposes offspring to long-term adverse adipose tissue effects.

The fetal period represents an important window of susceptibility for later obesity and metabolic disease. Maternal vitamin D deficiency (VDD) during pregnancy is a global concern that may have long-lasting consequences on offspring metabolic health. We sought to determine whether a VDD in utero environment affects fetal adipose tissue development and offspring metabolic disease predisposition in adulthood. Furthermore, we sought to explore the extent to which the VDD intrauterine environment interacts with genetic background or postnatal environment to influence metabolic health. Eight-week-old P0 female C57BL/6J mice were fed either a VDD diet or sufficient diet (VDS) from four weeks before pregnancy (periconception) then bred to male Avy/a mice. Females were maintained on the diets throughout gestation. At weaning, Avy/a and a/a male F1 offspring were randomized to low-fat (LFD) or high-fat diet (HFD) until 19 weeks of age, at which point serum and adipose tissue were harvested for analyses. Mice born to VDD dams weighed less at weaning than offspring born to VDS dams but experienced rapid weight gain in the four weeks post weaning, and acquired a greater ratio of perigonadal (PGAT) to subcutaneous (SQAT) than control offspring. Additionally, these mice were more susceptible to HFD-induced adipocyte hypertrophy. Offspring of VDD dams also had greater expression of Pparg transcript. These novel findings demonstrate that in utero VDD, an easily correctable but highly prevalent health concern, predisposes offspring to long-term adipose tissue consequences and possible adverse metabolic health complications.

Associations between cytokines, endocrine stress response, and gastrointestinal symptoms in autism spectrum disorder.

Many children and adolescents with autism spectrum disorder (ASD) have significant gastrointestinal (GI) symptoms, but the etiology is currently unknown. Some individuals with ASD show altered reactivity to stress and altered immune markers relative to typically-developing individuals, particularly stress-responsive cytokines including tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6). Acute and chronic stress is associated with the onset and exacerbation of GI symptoms in those without ASD. The present study examined whether GI symptoms in ASD were associated with increases in cortisol, a stress-associated endocrine marker, and TNF-α and IL-6 in response to stress. As hypothesized, a greater amount of lower GI tract symptoms were significantly associated with post-stress cortisol concentration. The relationship between cortisol response to stress and GI functioning was greater for children who had a history of regressive autism. Exploratory analyses revealed significant correlations between cortisol response, intelligence, and inappropriate speech. In contrast, symptoms of the lower GI tract were not associated with levels of TNF-α or IL-6. Significant correlations were found, however, between TNF-α and IL-6 and irritability, socialization, and intelligence. These findings suggest that individuals with ASD and symptoms of the lower GI tract may have an increased response to stress, but this effect is not associated with concomitant changes in TNF-α and IL-6. The relationship between cortisol stress response and lower GI tract symptoms in children with regressive autism, as well as the relationships between cortisol, IL-6, and intelligence in ASD, warrant further investigation.

Vitamin D insufficiency and insulin resistance in obese adolescents.

Obese adolescents represent a particularly vulnerable group for vitamin D deficiency which appears to have negative consequences on insulin resistance and glucose homeostasis. Poor vitamin D status is also associated with future risk of type 2 diabetes and metabolic syndrome in the obese. The biological mechanisms by which vitamin D influences glycemic control in obesity are not well understood, but are thought to involve enhancement of peripheral/hepatic uptake of glucose, attenuation of inflammation and/or regulation of insulin synthesis/secretion by pancreatic ß cells. Related to the latter, recent data suggest that the active form of vitamin, 1,25-dihydroxyvitamin D, does not impact insulin release in healthy pancreatic islets; instead they require an environmental stressor such as inflammation or vitamin D deficiency to see an effect. To date, a number of observational studies exploring the relationship between the vitamin D status of obese adolescents and markers of glucose homeostasis have been published. Most, although not all, show significant associations between circulating 25-hydroxyvitamn D concentrations and insulin sensitivity/resistance indices. In interpreting the collective findings of these reports, significant considerations surface including the effects of pubertal status, vitamin D status, influence of parathyroid hormone status and the presence of nonalcoholic fatty liver disease. The few published clinical trials using vitamin D supplementation to improve insulin resistance and impaired glucose tolerance in obese adolescents have yielded beneficial effects. However, there is a need for more randomized controlled trials. Future investigations should involve larger sample sizes of obese adolescents with documented vitamin D deficiency, and careful selection of the dose, dosing regimen and achievement of target 25-hydroxyvitamn D serum concentrations. These trials should also include clamp-derived measures of in vivo sensitivity and ß-cell function to more fully characterize the effects of vitamin D replenishment on insulin resistance.

Vitamin D deficiency & childhood obesity: a tale of two epidemics.

Obesity and vitamin D deficiency represent two of the most wide-spread health concerns in the United States, especially among children. There is a well-established inverse relationship between vitamin D status and obesity; however, it is unknown as to whether vitamin D deficiency contributes to, or is a consequence of obesity. Based on available research, the positive effects of correcting hypovitaminosis D in obesity seem to be primarily related to its action on glycemic control.