Neuronal Nitric Oxide Synthase Regulates Cerebellar Parallel Fiber Slow EPSC in Purkinje Neurons by Modulating STIM1-Gated TRPC3-Containing Channels.

Responding to burst stimulation of parallel fibers (PFs), cerebellar Purkinje neurons (PNs) generate a convolved synaptic response displaying a fast excitatory postsynaptic current (EPSCFast) followed by a slow EPSC (EPSCSlow). The latter is companied with a rise of intracellular Ca2+ and critical for motor coordination. The genesis of EPSCSlow in PNs results from activation of metabotropic type 1 glutamate receptor (mGluR1), oligomerization of stromal interaction molecule 1 (STIM1) on the membrane of endoplasmic reticulum (ER) and opening of transient receptor potential canonical 3 (TRPC3) channels on the plasma membrane. Neuronal nitric oxide synthase (nNOS) is abundantly expressed in PFs and granule neurons (GNs), catalyzing the production of nitric oxide (NO) hence regulating PF-PN synaptic function. We recently found that nNOS/NO regulates the morphological development of PNs through mGluR1-regulated Ca2+-dependent mechanism. This study investigated the role of nNOS/NO in regulating EPSCSlow. Electrophysiological analyses showed that EPSCSlow in cerebellar slices of nNOS knockout (nNOS-/-) mice was significantly larger than that in wildtype (WT) mice. Activation of mGluR1 in cultured PNs from nNOS-/- mice evoked larger TRPC3-channel mediated currents and intracellular Ca2+ rise than that in PNs from WT mice. In addition, nNOS inhibitor and NO-donor increased and decreased, respectively, the TRPC3-current and Ca2+ rise in PNs. Moreover, the NO-donor effectively decreased TRPC3 currents in HEK293 cells expressing WT STIM1, but not cells expressing a STIM1 with cysteine mutants. These novel findings indicate that nNOS/NO inhibits TRPC3-containig channel mediated cation influx during EPSCSlow, at least in part, by S-nitrosylation of STIM1.

Annexin A5 in Patients With Severe COVID-19 Disease: A Single-Center, Randomized, Double-Blind, Placebo-Controlled Feasibility Trial.

OBJECTIVES: To evaluate the study design and feasibility of drug administration and safety in a randomized clinical trial of recombinant human annexin A5 (SY-005), a constitutively expressed protein with anti-inflammatory, antiapoptotic, and anticoagulant properties, in patients with severe coronavirus disease 2019 (COVID-19). DESIGN: Double-blind, randomized clinical trial. SETTING: Two ICUs at an academic medical center. PATIENTS/SUBJECTS: Adults admitted to the ICU with a confirmed diagnosis of COVID-19 and requiring ventilatory or vasopressor support. INTERVENTIONS: SY-005, a recombinant human annexin A5, at 50 or 100 µg/kg IV every 12 hours for 7 days. MEASUREMENTS AND MAIN RESULTS: We enrolled 18 of the 55 eligible patients (33%) between April 21, 2021, and February 3, 2022. We administered 82% (196/238) of the anticipated doses of study medication and 86% (169/196) were given within 1 hour of the scheduled time. There were no drug-related serious adverse events. We captured 100% of the data that would be required for measuring clinical outcomes in a phase 2 or 3 trial. LIMITATIONS: The small sample size was a result of decreasing admissions of patients with COVID-19, which triggered a stopping rule for the trial. CONCLUSIONS: Although enrollment was low, administration of SY-005 to critically ill patients with COVID-19 every 12 hours for up to 7 days was feasible and safe. Further clinical trials of annexin A5 for the treatment of COVID-19 are warranted. Given reduction of severe COVID-19 disease, future studies should explore the safety and effectiveness of SY-005 use in non-COVID-related sepsis.

Annexin A5 Inhibits Endothelial Inflammation Induced by Lipopolysaccharide-Activated Platelets and Microvesicles via Phosphatidylserine Binding.

Sepsis is caused by a dysregulated immune response to infection and is a leading cause of mortality globally. To date, no specific therapeutics are available to treat the underlying septic response. We and others have shown that recombinant human annexin A5 (Anx5) treatment inhibits pro-inflammatory cytokine production and improves survival in rodent sepsis models. During sepsis, activated platelets release microvesicles (MVs) with externalization of phosphatidylserine to which Anx5 binds with high affinity. We hypothesized that recombinant human Anx5 blocks the pro-inflammatory response induced by activated platelets and MVs in vascular endothelial cells under septic conditions via phosphatidylserine binding. Our data show that treatment with wildtype Anx5 reduced the expression of inflammatory cytokines and adhesion molecules induced by lipopolysaccharide (LPS)-activated platelets or MVs in endothelial cells (p < 0.01), which was not observed with Anx5 mutant deficient in phosphatidylserine binding. In addition, wildtype Anx5 treatment, but not Anx5 mutant, improved trans-endothelial electrical resistance (p < 0.05) and reduced monocyte (p < 0.001) and platelet (p < 0.001) adhesion to vascular endothelial cells in septic conditions. In conclusion, recombinant human Anx5 inhibits endothelial inflammation induced by activated platelets and MVs in septic conditions via phosphatidylserine binding, which may contribute to its anti-inflammatory effects in the treatment of sepsis.

Loss of fatty acid binding protein 3 ameliorates lipopolysaccharide-induced inflammation and endothelial dysfunction.

Circulating fatty acid-binding protein 3 (FABP3) is an effective biomarker of myocardial injury and peripheral artery disease (PAD). The endothelium, which forms the inner most layer of every blood vessel, is exposed to higher levels of FABP3 in PAD or following myocardial injury, but the pathophysiological role of endothelial FABP3, the effect of FABP3 exposure on endothelial cells, and related mechanisms are unknown. Here, we aimed to evaluate the pathophysiological role of endothelial FABP3 and related mechanisms in vitro. Our molecular and functional in vitro analyses show that (1) FABP3 is basally expressed in endothelial cells; (2) inflammatory stress in the form of lipopolysaccharide (LPS) upregulated endothelial FABP3 expression; (3) loss of endogenous FABP3 protected endothelial cells against LPS-induced endothelial dysfunction; however, exogenous FABP3 exposure exacerbated LPS-induced inflammation; (4) loss of endogenous FABP3 protected against LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling pathways. Together, these findings suggest that gain-of endothelial FABP3 exacerbates, whereas loss-of endothelial FABP3 inhibits LPS-induced endothelial dysfunction by promoting cell survival and anti-inflammatory and pro-angiogenic signaling. We propose that an increased circulating FABP3 in myocardial injury or PAD patients may be detrimental to endothelial function, and therefore, therapies aimed at inhibiting FABP3 may improve endothelial function in diseased states.

Novel circRNA-miRNA-mRNA networks regulated by maternal exercise in fetal hearts of pregestational diabetes.

BACKGROUND: Maternal exercise lowers the incidence of congenital heart defects (CHDs) induced by pregestational diabetes. However, the molecular mechanisms underlying the beneficial effects of maternal exercise remain unclear. The present study aimed to identify circular RNA (circRNA), microRNA (miRNA) and mRNA networks that are regulated by maternal exercise in fetal hearts of pregestational diabetes. METHODS: Pregestational diabetes was induced in adult C57BL/6 female mice by streptozotocin. The expression profiles of circRNAs, miRNAs and mRNAs in E10.5 fetal hearts of offspring of control and diabetic mothers with or without exercise were analyzed using next generation sequencing. circRNA-miRNA-mRNA networks in fetal hearts were mapped and key candidate transcripts were verified by qPCR analysis. RESULTS: Pregestational diabetes dysregulated the expression of 206 circRNAs, 66 miRNAs and 391 mRNAs in fetal hearts. Maternal exercise differentially regulated 188 circRNAs, 57 miRNAs and 506 mRNAs in fetal hearts of offspring of pregestational diabetes. A total of 5 circRNAs, 12 miRNAs, and 28 mRNAs were incorporated into a final maternal exercise-associated regulatory network in fetal hearts of offspring of maternal diabetes. Notably, maternal exercise normalized the dysregulated circ_0003226/circ_0015638/miR-351-5p and circ_0002768/miR-3102-3p.2-3p pairs in fetal hearts of pregestational diabetes. CONCLUSION: Maternal exercise reverses the dysregulated circ_0003226/circ_0015638/miR-351-5p and circ_0002768/miR-3102-3p.2-3p pairs, and partially normalizes circRNA, miRNA, and mRNA expression profiles in fetal hearts of pregestational diabetes. These findings shed new light on the potential mechanisms of the beneficial effects of maternal exercise on the developing heart in diabetic pregnancies.

Pharmacokinetics of recombinant human annexin A5 (SY-005) in patients with severe COVID-19.

Objective: Annexin A5 is a phosphatidylserine binding protein with anti-inflammatory, anticoagulant and anti-apoptotic properties. Preclinical studies have shown that annexin A5 inhibits pro-inflammatory responses and improves organ function and survival in rodent models of sepsis. This clinical trial aimed to evaluate the pharmacokinetic (PK) properties of the recombinant human annexin A5 (SY-005) in severe COVID-19. Methods: This was a pilot randomized, double-blind, placebo-controlled trial. Severe COVID-19 patients were randomly assigned to receive intravenous 50 µg/kg (low dose, n = 3), 100 µg/kg (high dose, n = 5) of SY-005 or placebo (n = 5) every 12 h for 7 days. Plasma SY-005 levels were assessed using enzyme-linked immunosorbent assay (ELISA) and the PK parameters were determined using non-compartmental analysis. Results: All patients treated with SY-005 had a normal baseline estimated glomerular filtration rate (eGFR, 104-125 mL/min/1.73 m2). Both low and high doses of SY-005 were cleared within 6 h after intravenous administration. Plasma maximum concentrations (Cmax), half-life, clearance and volume distribution of low and high doses of SY-005 were 402.4 and 848.9 ng/mL, 0.92 and 0.96 h, 7.52 and 15.19 L/h, and 9.98 and 20.79 L, respectively. Daily pre-dose circulating annexin A5 levels were not significantly different when SY-005 was administered at the low or the high dose 12-h intervals. There was no significant effect on activated partial thromboplastin time (aPTT) or INR (international normalized ratio of prothrombin time) during 7 days of SY-005 treatment. Conclusion: SY-005 doses of 50 and 100 µg/kg were detectable and subsequently cleared from the plasma in severe COVID-19 patients with normal baseline renal function. There was no significant plasma SY-005 accumulation 6 h after drug administration and coagulation was not altered during 7 days of treatment. Clinical trials Registration: This study was registered with ClinicalTrials.gov (NCT04748757, first posted on 10 February 2021).

Maternal Delta-9-Tetrahydrocannabinol Exposure Induces Abnormalities of the Developing Heart in Mice.

Introduction: Cannabis is increasingly being consumed by pregnant women for recreational purposes as well as for its antiemetic and anxiolytic effects despite limited studies on its safety during pregnancy. Importantly, phytocannabinoids found in cannabis can pass through the placenta and enter the fetal circulation. Recent reports suggest gestational cannabis use is associated with negative fetal outcomes, including fetal growth restriction and perinatal intensive care, however, the effects of delta-9-tetrahydrocannabinol (THC) on fetal heart development remains to be elucidated. Materials and Methods: We aimed to determine the outcomes of maternal THC exposure on fetal heart development in mice by administering 0, 5, or 10 mg/kg/day of THC orally to C57BL/6 dams starting at embryonic day (E)3.5. Offspring were collected at E12.5 for molecular analysis, at E17.5 to analyze cardiac morphology or at postnatal day (PND)21 to assess heart function. Results: Maternal THC exposure in E17.5 fetuses resulted in an array of cardiac abnormalities with an incidence of 44% and 55% in the 5 and 10 mg/kg treatment groups, respectively. Maternal THC exposure in offspring resulted in ventricular septal defect, higher semilunar valve volume relative to orifice ratio, and higher myocardial wall thickness. Notably, cell proliferation within the ventricular myocardium was increased, and expression of multiple cardiac transcription factors was downregulated in THC-exposed E12.5 fetuses. Furthermore, heart function was compromised with lower left ventricular ejection fraction, fractional shortening, and cardiac output in PND21 pups exposed to THC compared to controls. Discussion: The results show that maternal THC exposure during gestation induces myocardial hyperplasia and semilunar valve thickening in the fetal heart and postnatal cardiac dysfunction. Our study suggests that maternal cannabis consumption may induce abnormalities in the developing heart and cardiac dysfunction in postnatal life.

Inhibition of intraflagellar transport protein-88 promotes epithelial-to-mesenchymal transition and reduces cardiac remodeling post-myocardial infarction.

The epicardium is a potential source of cardiac progenitors to support reparative angiogenesis after myocardial infarction (MI) through epithelial-to-mesenchymal transition (EMT). Primary cilia are recognized as hubs of cellular signaling, and their presence can alter downstream pathways to modulate EMT. The present study aimed to examine the effects of inhibiting intraflagellar transport protein-88 (Ift88), a protein vital to ciliary assembly, on epicardial EMT and cardiac remodeling post-MI. Epicardium derived cells (EPDCs) were cultured from E13.5 heart explants and treated with adenoviral vector encoding short-hairpin RNA against the mouse Ift88 (Ad-shIft88) to disassemble the primary cilium. Effects of Ad-shIft88 on epicardial EMT and cardiac remodeling were examined in mice post-MI. Our results show that Ad-shIft88 enhanced EMT of cultured EPDCs. In adult mice, intra-myocardial administration of Ad-shIft88 increased the number of Wilms tumor 1 (Wt1) positive cells in the epicardium and myocardium, promoted expression of genes associated with epicardial EMT, and enhanced capillary and arteriolar densities post-MI. Additionally, intra-myocardial Ad-shIft88 treatment attenuated cardiac hypertrophy and improved myocardial function three weeks post-MI. In conclusion, knockdown of Ift88 improves epicardial EMT, neovascularization and cardiac remodeling in the ischemic heart. Our study highlights the primary cilium as a potential therapeutic target post-MI.

Maternal nicotine exposure induces congenital heart defects in the offspring of mice.

Maternal cigarette smoking is a risk factor for congenital heart defects (CHDs). Nicotine replacement therapies are often offered to pregnant women following failed attempts of smoking cessation. However, the impact of nicotine on embryonic heart development is not well understood. In the present study, the effects of maternal nicotine exposure (MNE) during pregnancy on foetal heart morphogenesis were studied. Adult female mice were treated with nicotine using subcutaneous osmotic pumps at 0.75 or 1.5 mg/kg/day and subsequently bred with male mice. Our results show that MNE dose-dependently increased CHDs in foetal mice. CHDs included atrial and ventricular septal defects, double outlet right ventricle, unguarded tricuspid orifice, hypoplastic left ventricle, thickened aortic and pulmonary valves, and ventricular hypertrophy. MNE also significantly reduced coronary artery size and vessel abundance in foetal hearts. Moreover, MNE resulted in higher levels of oxidative stress and altered the expression of key cardiogenic regulators in the developing heart. Nicotine exposure reduced epicardial-to-mesenchymal transition in foetal hearts. In conclusion, MNE induces CHDs and coronary artery malformation in mice. These findings provide insight into the adverse outcomes of foetuses by MNE during pregnancy.

Therapeutic Potential of Annexins in Sepsis and COVID-19.

Sepsis is a continuing problem in modern healthcare, with a relatively high prevalence, and a significant mortality rate worldwide. Currently, no specific anti-sepsis treatment exists despite decades of research on developing potential therapies. Annexins are molecules that show efficacy in preclinical models of sepsis but have not been investigated as a potential therapy in patients with sepsis. Human annexins play important roles in cell membrane dynamics, as well as mediation of systemic effects. Most notably, annexins are highly involved in anti-inflammatory processes, adaptive immunity, modulation of coagulation and fibrinolysis, as well as protective shielding of cells from phagocytosis. These discoveries led to the development of analogous peptides which mimic their physiological function, and investigation into the potential of using the annexins and their analogous peptides as therapeutic agents in conditions where inflammation and coagulation play a large role in the pathophysiology. In numerous studies, treatment with recombinant human annexins and annexin analogue peptides have consistently found positive outcomes in animal models of sepsis, myocardial infarction, and ischemia reperfusion injury. Annexins A1 and A5 improve organ function and reduce mortality in animal sepsis models, inhibit inflammatory processes, reduce inflammatory mediator release, and protect against ischemic injury. The mechanisms of action and demonstrated efficacy of annexins in animal models support development of annexins and their analogues for the treatment of sepsis. The effects of annexin A5 on inflammation and platelet activation may be particularly beneficial in disease caused by SARS-CoV-2 infection. Safety and efficacy of recombinant human annexin A5 are currently being studied in clinical trials in sepsis and severe COVID-19 patients.

Myocardium-Specific Deletion of Rac1 Causes Ventricular Noncompaction and Outflow Tract Defects.

BACKGROUND: Left ventricular noncompaction (LVNC) is a cardiomyopathy that can lead to arrhythmias, embolic events and heart failure. Despite our current knowledge of cardiac development, the mechanisms underlying noncompaction of the ventricular myocardium are still poorly understood. The small GTPase Rac1 acts as a crucial regulator of numerous developmental events. The present study aimed to investigate the cardiomyocyte specific role of Rac1 in embryonic heart development. METHODS AND RESULTS: The Nkx2.5-Cre transgenic mice were crossed with Rac1f/f mice to generate mice with a cardiomyocyte specific deletion of Rac1 (Rac1Nkx2.5) during heart development. Embryonic Rac1Nkx2.5 hearts at E12.5-E18.5 were collected for histological analysis. Overall, Rac1Nkx2.5 hearts displayed a bifid apex, along with hypertrabeculation and a thin compact myocardium. Rac1Nkx2.5 hearts also exhibited ventricular septal defects (VSDs) and double outlet right ventricle (DORV) or overriding aorta. Cardiomyocytes had a rounded morphology and were highly disorganized, and the myocardial expression of Scrib, a planar cell polarity protein, was reduced in Rac1Nkx2.5 hearts. In addition, cell proliferation rate was significantly decreased in the Rac1Nkx2.5 ventricular myocardium at E9.5. CONCLUSIONS: Rac1 deficiency in the myocardium impairs cardiomyocyte elongation and organization, and proliferative growth of the heart. A spectrum of CHDs arises in Rac1Nkx2.5 hearts, implicating Rac1 signaling in the ventricular myocardium as a crucial regulator of OFT alignment, along with compact myocardium growth and development.

NOX2 Is Critical to Endocardial to Mesenchymal Transition and Heart Development.

NADPH oxidases (NOX) are a major source of reactive oxygen species (ROS) production in the heart. ROS signaling regulates gene expression, cell proliferation, apoptosis, and migration. However, the role of NOX2 in embryonic heart development remains elusive. We hypothesized that deficiency of Nox2 disrupts endocardial to mesenchymal transition (EndMT) and results in congenital septal and valvular defects. Our data show that 34% of Nox2-/- neonatal mice had various congenital heart defects (CHDs) including atrial septal defects (ASD), ventricular septal defects (VSD), atrioventricular canal defects (AVCD), and malformation of atrioventricular and aortic valves. Notably, Nox2-/- embryonic hearts show abnormal development of the endocardial cushion as evidenced by decreased cell proliferation and an increased rate of apoptosis. Additionally, Nox2 deficiency disrupted EndMT of atrioventricular cushion explants ex vivo. Furthermore, treatment with N-acetylcysteine (NAC) to reduce ROS levels in the wild-type endocardial cushion explants decreased the number of cells undergoing EndMT. Importantly, deficiency of Nox2 was associated with reduced expression of Gata4, Tgfß2, Bmp2, Bmp4, and Snail1, which are critical to endocardial cushion and valvoseptal development. We conclude that NOX2 is critical to EndMT, endocardial cushion cell proliferation, and normal embryonic heart development.

Synergistic stabilization by nitrosoglutathione-induced thiol modifications in the stromal interaction molecule-2 luminal domain suppresses basal and store operated calcium entry.

Stromal interaction molecule-1 and -2 (STIM1/2) are endoplasmic reticulum (ER) membrane-inserted calcium (Ca2+) sensing proteins that, together with Orai1-composed Ca2+ channels on the plasma membrane (PM), regulate intracellular Ca2+ levels. Recent evidence suggests that S-nitrosylation of the luminal STIM1 Cys residues inhibits store operated Ca2+ entry (SOCE). However, the effects of thiol modifications on STIM2 during nitrosative stress and their role in regulating basal Ca2+ levels remain unknown. Here, we demonstrate that the nitric oxide (NO) donor nitrosoglutathione (GSNO) thermodynamically stabilizes the STIM2 Ca2+ sensing region in a Cys-specific manner. We uncovered a remarkable synergism in this stabilization involving the three luminal Cys of STIM2, which is unique to this paralog. S-Nitrosylation causes structural perturbations that converge on the face of the EF-hand and sterile α motif (EF-SAM) domain, implicated in unfolding-coupled activation. In HEK293T cells, enhanced free basal cytosolic Ca2+ and SOCE mediated by STIM2 overexpression could be attenuated by GSNO or mutation of the modifiable Cys located in the luminal domain. Collectively, we identify the Cys residues within the N-terminal region of STIM2 as modifiable targets during nitrosative stress that can profoundly and cooperatively affect basal Ca2+ and SOCE regulation.

Sapropterin reduces coronary artery malformation in offspring of pregestational diabetes mice.

Endothelial nitric oxide synthase (eNOS) and oxidative stress are critical to embryonic coronary artery development. Maternal diabetes increases oxidative stress and reduces eNOS activity in the fetal heart. Sapropterin (Kuvan®) is an orally active, synthetic form of tetrahydrobiopterin (BH4) and a co-factor for eNOS with antioxidant properties. The aim of the present study was to examine the effects of sapropterin on fetal coronary artery development during pregestational diabetes in mice. Diabetes was induced by streptozotocin to adult female C57BL/6 mice. Sapropterin (10 mg/kg/day) was orally administered to pregnant mice from E0.5 to E18.5. Fetal hearts were collected at E18.5 for coronary artery morphological analysis. Sapropterin treatment to diabetic dams reduced the incidence of coronary artery malformation in offspring from 50.0% to 20.6%. Decreases in coronary artery luminal diameter, volume and abundance in fetal hearts from diabetic mothers, were prevented by sapropterin treatment. Maternal diabetes reduced epicardial epithelial-to-mesenchymal transition (EMT) and expression of transcription and growth factors critical to coronary artery development including hypoxia-inducible factor 1a (Hif1a), Snail1, Slug, ß-catenin, retinaldehyde dehydrogenase 2 (Aldh1a2), basic fibroblast growth factor (bFGF) and vascular endothelial group factor receptor 2 (Vegfr2) in E12.5 hearts. Additionally, eNOS phosphorylation was lower while oxidative stress was higher in E12.5 hearts from maternal diabetes. Notably, these abnormalities were all restored to normal levels after sapropterin treatment. In conclusion, sapropterin treatment increases eNOS activity, lowers oxidative stress and reduces coronary artery malformation in offspring of pregestational diabetes. Sapropterin may have therapeutic potential in preventing coronary artery malformation in maternal diabetes.

Say NO to ROS: Their Roles in Embryonic Heart Development and Pathogenesis of Congenital Heart Defects in Maternal Diabetes.

Congenital heart defects (CHDs) are the most prevalent and serious birth defect, occurring in 1% of all live births. Pregestational maternal diabetes is a known risk factor for the development of CHDs, elevating the risk in the child by more than four-fold. As the prevalence of diabetes rapidly rises among women of childbearing age, there is a need to investigate the mechanisms and potential preventative strategies for these defects. In experimental animal models of pregestational diabetes induced-CHDs, upwards of 50% of offspring display congenital malformations of the heart, including septal, valvular, and outflow tract defects. Specifically, the imbalance of nitric oxide (NO) and reactive oxygen species (ROS) signaling is a major driver of the development of CHDs in offspring of mice with pregestational diabetes. NO from endothelial nitric oxide synthase (eNOS) is crucial to cardiogenesis, regulating various cellular and molecular processes. In fact, deficiency in eNOS results in CHDs and coronary artery malformation. Embryonic hearts from diabetic dams exhibit eNOS uncoupling and oxidative stress. Maternal treatment with sapropterin, a cofactor of eNOS, and antioxidants such as N-acetylcysteine, vitamin E, and glutathione as well as maternal exercise have been shown to improve eNOS function, reduce oxidative stress, and lower the incidence CHDs in the offspring of mice with pregestational diabetes. This review summarizes recent data on pregestational diabetes-induced CHDs, and offers insights into the important roles of NO and ROS in embryonic heart development and pathogenesis of CHDs in maternal diabetes.

Maternal voluntary exercise mitigates oxidative stress and incidence of congenital heart defects in pre-gestational diabetes.

Women with pre-gestational diabetes have a higher risk of producing children with congenital heart defects (CHDs), caused predominantly by hyperglycemia-induced oxidative stress. In this study, we evaluated if exercise during pregnancy could mitigate oxidative stress and reduce the incidence of CHDs in the offspring of diabetic mice. Female mice were treated with streptozotocin to induce pre-gestational diabetes, then mated with healthy males to produce offspring. They were also given access to running wheels 1 week before mating and allowed to exercise voluntarily until E18.5. Heart morphology, gene expression, and oxidative stress were assessed in foetal hearts. Maternal voluntary exercise results in a significantly lower incidence of CHDs from 59.5% to 25%. Additionally, diabetes-induced defects in coronary artery and capillary morphogenesis were also lower with exercise. Myocardial cell proliferation and epithelial-mesenchymal transition at E12.5 was significantly lower with pre-gestational diabetes which was mitigated with maternal exercise. Cardiac gene expression of Notch1, Snail1, Gata4 and Cyclin D1 was significantly higher in the embryos of diabetic mice that exercised compared to the non-exercised group. Furthermore, maternal exercise produced lower reactive oxygen species (ROS) and oxidative stress in the foetal heart. In conclusion, maternal exercise mitigates ROS and oxidative damage in the foetal heart, and results in a lower incidence of CHDs in the offspring of pre-gestational diabetes. Exercise may be an effective intervention to compliment clinical management and further minimize CHD risk in mothers with diabetes.

Binder-Free Graphene/Silver Nanowire Gel-Like Composite with Tunable Properties and Multifunctional Applications.

To realize macroscopic utilization of the excellent properties of graphene, various forms of graphene assemblies have been investigated. Among them, the gel form assemblies show great advantages because of their shapeable and self-healable properties and facile and simple manufacturing processes. For the conventional gel-formed graphene assemblies, a relatively large content of binders including hydrophilic polymers, celluloses, or/and amorphous inorganic materials is necessary in achieving the gelation. However, these binders are electrically nonconductive and electrochemically inactive, which would weaken the favorable functionalities of the composite, and the potential advantages of graphene cannot be fully utilized. Herein, a binder-free silver nanowire (Ag-NW)/reduced graphene oxide (rGO) gel-like composite is designed and successfully fabricated by employing the ultralong Ag-NWs to enhance the hierarchical synergistic effects. The fabrication technique is highly efficient and repeatable, and the obtained composite is flexible, stretchable, and self-healable. Furthermore, the overall properties of the composite can be easily adjusted in a wide range by controlling the mass ratio between Ag-NW and rGO, which makes it multipurpose and suitable in different applications. Several demonstrations have been carried out, and some special performances including linear strain sensing range and rapid transformation from wet to dry state are found in this unique composite. This binder-free structure could also be expanded to other material systems, which may offer a valuable inspiration for the development of functional devices based on the nanocomposite.

Ablation of both Cx40 and Panx1 results in similar cardiovascular phenotypes exhibited in Cx40 knockout mice.

Connexins (Cxs) and pannexins (Panxs) are highly regulated large-pore channel-forming proteins that participate in cellular communication via small molecular exchange with the extracellular microenvironment, or in the case of connexins, directly between cells. Given the putative functional overlap between single membrane-spanning connexin hemichannels and Panx channels, and cardiovascular system prevalence, we generated the first Cx40-/-Panx1-/- mouse with the anticipation that this genetic modification would lead to a severe cardiovascular phenotype. Mice null for both Cx40 and Panx1 produced litter sizes and adult growth progression similar to wild-type (WT), Cx40-/- and Panx1-/- mice. Akin to Cx40-/- mice, Cx40-/-Panx1-/- mice exhibited cardiac hypertrophy and elevated systolic, diastolic, and mean arterial blood pressure compared with WT and Panx1-/- mice; however assessment of left ventricular ejection fraction and fractional shortening revealed no evidence of cardiac dysfunction between groups. Furthermore, Cx40-/-, Panx1-/-, and Cx40-/-Panx1-/- mice demonstrated impaired endothelial-mediated vasodilation of aortic segments to increasing concentrations of methacholine (MCh) compared with WT, highlighting roles for both Cx40 and Panx1 in vascular endothelial cell (EC) function. Surprisingly, elevated kidney renin mRNA expression, plasma renin activity, and extraglomerular renin-producing cell populations found in Cx40-/- mice was further exaggerated in double knockout mice. Thus, while gestation and gross development were conserved in Cx40-/-Panx1-/- mice, they exhibit cardiac hypertrophy, hypertension, and impaired endothelial-mediated vasodilation that phenocopies Cx40-/- mice. Nevertheless, the augmented renin homeostasis observed in the double knockout mice suggests that both Cx40 and Panx1 may play an integrative role.

Bicuspid aortic valve formation: Nos3 mutation leads to abnormal lineage patterning of neural crest cells and the second heart field.

The bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, belongs to the most prevalent congenital heart diseases in the world, occurring in 0.5-2% of the general population. We aimed to understand how changes in early cellular contributions result in BAV formation and impact cardiovascular outflow tract development. Detailed 3D reconstructions, immunohistochemistry and morphometrics determined that, during valvulogenesis, the non-coronary leaflet separates from the parietal outflow tract cushion instead of originating from an intercalated cushion. Nos3-/- mice develop a BAV without a raphe as a result of incomplete separation of the parietal outflow tract cushion into the right and non-coronary leaflet. Genetic lineage tracing of endothelial, second heart field and neural crest cells revealed altered deposition of neural crest cells and second heart field cells within the parietal outflow tract cushion of Nos3-/- embryos. The abnormal cell lineage distributions also affected the positioning of the aortic and pulmonary valves at the orifice level. The results demonstrate that the development of the right and non-coronary leaflets are closely related. A small deviation in the distribution of neural crest and second heart field populations affects normal valve formation and results in the predominant right-non-type BAV in Nos3-/- mice.