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Abstract Alzheimer's Disease (AD) is a progressive neurodegenerative disease caused by the deposition of Aβ aggregates or neurofibrillary tangles. AD patients are primarily diagnosed with the concurrent development of several cardiovascular dysfunctions. While few studies have indicated the presence of intramyocardial Aβ aggregates, none of the studies have performed detailed analyses for pathomechanism of cardiac dysfunction in AD patients. This manuscript used aged APPSWE/PS1 Tg and littermate age-matched wildtype (Wt) mice to characterize cardiac dysfunction and analyze associated pathophysiology. Detailed assessment of cardiac functional parameters demonstrated the development of diastolic dysfunction in APPSWE/PS1 Tg hearts compared to Wt hearts. Muscle function evaluation showed functional impairment (decreased exercise tolerance and muscle strength) in APPSWE/PS1 Tg mice. Biochemical and histochemical analysis revealed Aβ aggregate accumulation in APPSWE/PS1 Tg mice myocardium. APPSWE/PS1 Tg mice hearts also demonstrated histopathological remodeling (increased collagen deposition and myocyte cross-sectional area). Additionally, APPSWE/PS1 Tg hearts showed altered mitochondrial dynamics, reduced antioxidant protein levels, and impaired mitochondrial proteostasis compared to Wt mice. APPSWE/PS1 Tg hearts also developed mitochondrial dysfunction with decreased OXPHOS and PDH protein complex expressions, altered ETC complex dynamics, decreased complex activities, and reduced mitochondrial respiration. Our results indicated that Aβ aggregates in APPSWE/PS1 Tg hearts are associated with defects in mitochondrial respiration and complex activities, which may collectively lead to cardiac diastolic dysfunction and myocardial pathological remodeling.

BackgroundPulmonary arterial hypertension (PAH) is a rare, chronic, progressive form of pulmonary hypertension in which increased arterial pressure causes remodeling of the arterial system and is associated with heart failure. Methamphetamine is a stimulant that has recently become a focus in PAH research, but the recent trends and demographics of this cohort of patients are not known. The study aimed to analyze the overall trends and demographics of PAH patients with and without concurrent methamphetamine usage.MethodsThe study used the National Inpatient Sample (NIS), Healthcare Cost and Utilization Project (HCUP), and Agency for Healthcare Research and Quality (AHRQ) from 2008 to 2020 to calculate nationally weighted estimates for both conditions by ICD-9 and ICD-10 diagnosis codes. We used several statistical measures, including descriptive statistics with design-based chi-square and t-tests, trend analysis with Cochran-Armitage test, generalized linear models, and other data preprocessing measures.ResultsA significant increase was evident in patients with pulmonary arterial hypertension (PAH) and concurrent methamphetamine use (9.2-fold). Most of the hospitalized patients were males (59.16%), aged 41–64 (45.77%), White (68.64%), from the West (53.09%), with Medicaid (50.48%), and with median income

Abstract Alzheimer’s disease (AD), a neurodegenerative disease that mostly affects the elderly, slowly impairs memory, cognition, and daily tasks. AD has long been one of the most debilitating chronic neurological disorders, affecting mostly people over 65. In this study, we investigated the use of Vision Transformer (ViT) for Magnetic Resonance Image processing in the context of AD diagnosis. ViT was utilized to extract features from MRIs, map them to a feature sequence, perform sequence modeling to maintain interdependencies, and classify features using a time series transformer. The proposed model was evaluated using ADNI T1-weighted MRIs for binary and multiclass classification. Two data collections, Complete 1Yr 1.5T and Complete 3Yr 3T, from the ADNI database were used for training and testing. A random split approach was used, allocating 60% for training and 20% for testing and validation, resulting in sample sizes of (211, 70, 70) and (1378, 458, 458), respectively. The performance of our proposed model was compared to various deep learning models, including CNN with BiL-STM and ViT with Bi-LSTM. The suggested technique diagnoses AD with high accuracy (99.048% for binary and 99.014% for multiclass classification), precision, recall, and F-score. Our proposed method offers researchers an approach to more efficient early clinical diagnosis and interventions.

BackgroundPeripheral artery disease (PAD) is on the rise worldwide, ranking as the third leading cause of atherosclerosis-related morbidity; much less is known about its trends in hospitalizations among methamphetamine and cocaine users.ObjectivesWe aim to evaluate the overall trend in the prevalence of hospital admission for PAD with or without the use of stimulant abuse (methamphetamine and cocaine) across the United States. Additionally, we evaluated the PAD-related hospitalizations trend stratified by age, race, sex, and geographic location.MethodsWe used the National Inpatient Sample (NIS) database from 2008 to 2020. The Cochran Armitage trend test was used to compare the trend between groups. Multivariate logistic regression was used to examine adjusted odds for PAD and CLI hospitalizations among methamphetamine and cocaine users.ResultsBetween 2008 and 2020, PAD-related hospitalizations showed an increasing trend in Hispanics, African Americans, and western states, while a decreasing trend in southern and Midwestern states (p-trend 64 years, southern and western states (p-trend 64 years, and CLI-related amputations declined for women, White patient population, age group >40, and all regions (p-trend

Sigmar1 is a ubiquitously expressed, multifunctional protein known for its cardioprotective roles in cardiovascular diseases. While accumulating evidence indicate a critical role of Sigmar1 in cardiac biology, its physiological function in the vasculature remains unknown. In this study, we characterized the expression of Sigmar1 in the vascular wall and assessed its physiological function in the vascular system using global Sigmar1 knockout (Sigmar1−/−) mice. We determined the expression of Sigmar1 in the vascular tissue using immunostaining and biochemical experiments in both human and mouse blood vessels. Deletion of Sigmar1 globally in mice (Sigmar1−/−) led to blood vessel wall reorganizations characterized by nuclei disarray of vascular smooth muscle cells, altered organizations of elastic lamina, and higher collagen fibers deposition in and around the arteries compared to wildtype littermate controls (Wt). Vascular function was assessed in mice using non-invasive time-transit method of aortic stiffness measurement and flow-mediated dilation (FMD) of the left femoral artery. Sigmar1−/− mice showed a notable increase in arterial stiffness in the abdominal aorta and failed to increase the vessel diameter in response to reactive-hyperemia compared to Wt. This was consistent with reduced plasma and tissue nitric-oxide bioavailability (NOx) and decreased phosphorylation of endothelial nitric oxide synthase (eNOS) in the aorta of Sigmar1−/− mice. Ultrastructural analysis by transmission electron microscopy (TEM) of aorta sections showed accumulation of elongated shaped mitochondria in both vascular smooth muscle and endothelial cells of Sigmar1−/− mice. In accordance, decreased mitochondrial respirometry parameters were found in ex-vivo aortic rings from Sigmar1 deficient mice compared to Wt controls. These data indicate a potential role of Sigmar1 in maintaining vascular homeostasis.

Endothelial dysfunction and endothelial activation are common early events in vascular diseases and can arise from mitochondrial dysfunction. Neurogranin (Ng) is a 17kD protein well known to regulate intracellular Ca2+-calmodulin (CaM) complex signaling, and its dysfunction is significantly implicated in brain aging and neurodegenerative diseases. We found that Ng is also expressed in human aortic endothelial cells (HAECs), and depleting Ng promotes Ca2+-CaM complex-dependent endothelial activation and redox imbalances. Endothelial-specific Ng knockout (Cre-CDH5-Ngf/f) mice demonstrate a significant delay in the flow-mediated dilation (FMD) response. Therefore, it is critical to characterize how endothelial Ng expression regulates reactive oxygen species (ROS) generation and affects cardiovascular disease. Label-free quantification proteomics identified that mitochondrial dysfunction and the oxidative phosphorylation pathway are significantly changed in the aorta of Cre-CDH5-Ngf/f mice. We found that a significant amount of Ng is expressed in the mitochondrial fraction of HAECs using western blotting and colocalized with the mitochondrial marker, COX IV, using immunofluorescence staining. Seahorse assay demonstrated that a lack of Ng decreases mitochondrial respiration. Treatment with MitoEbselen significantly restores the oxygen consumption rate in Ng knockdown cells. With the RoGFP-Orp1 approach, we identified that Ng knockdown increases mitochondrial-specific hydrogen peroxide (H2O2) production, and MitoEbselen treatment significantly reduced mitochondrial ROS (mtROS) levels in Ng knockdown cells. These results suggest that Ng plays a significant role in mtROS production. We discovered that MitoEbselen treatment also rescues decreased eNOS expression and nitric oxide (NO) levels in Ng knockdown cells, which implicates the critical role of Ng in mtROS-NO balance in the endothelial cells.

Hydropersulfide and hydropolysulfide metabolites are increasingly important reactive sulfur species (RSS) regulating numerous cellular redox dependent functions. Intracellular production of these species is known to occur through RSS interactions or through translational mechanisms involving cysteinyl t-RNA synthetases. However, regulation of these species under cell stress conditions, such as hypoxia, that are known to modulate RSS remain poorly understood. Here we define an important mechanism of increased persulfide and polysulfide production involving cystathionine gamma lyase (CSE) phosphorylation at serine 346 and threonine 355 in a substrate specific manner, under acute hypoxic conditions. Hypoxic phosphorylation of CSE occurs in an AMP kinase dependent manner increasing enzyme activity involving unique inter- and intramolecular interactions within the tetramer. Importantly, both cellular hypoxia and tissue ischemia result in AMP Kinase dependent CSE phosphorylation that regulates blood flow in ischemic tissues. Our findings reveal hypoxia molecular signaling pathways regulating CSE dependent persulfide and polysulfide production impacting tissue and cellular response to stress.

Amyotrophic lateral sclerosis (ALS) is a complex systemic disease that primarily involves motor neuron dysfunction and skeletal muscle atrophy. One commonly used mouse model to study ALS was generated by transgenic expression of a mutant form of human superoxide dismutase 1 (SOD1) gene harboring a single amino acid substitution of glycine to alanine at codon 93 (G93A*SOD1). Although mutant-SOD1 is ubiquitously expressed in G93A*SOD1 mice, a detailed analysis of the skeletal muscle expression pattern of the mutant protein and the resultant muscle pathology were never performed. Using different skeletal muscles isolated from G93A*SOD1 mice, we extensively characterized the pathological sequelae of histological, molecular, ultrastructural, and biochemical alterations. Muscle atrophy in G93A*SOD1 mice was associated with increased and differential expression of mutant-SOD1 across myofibers and increased MuRF1 protein level. In addition, high collagen deposition and myopathic changes sections accompanied the reduced muscle strength in the G93A*SOD1 mice. Furthermore, all the muscles in G93A*SOD1 mice showed altered protein levels associated with different signaling pathways, including inflammation, mitochondrial membrane transport, mitochondrial lipid uptake, and antioxidant enzymes. In addition, the mutant-SOD1 protein was found in the mitochondrial fraction in the muscles from G93A*SOD1 mice, which was accompanied by vacuolized and abnormal mitochondria, altered OXPHOS and PDH complex protein levels, and defects in mitochondrial respiration. Overall, we reported the pathological sequelae observed in the skeletal muscles of G93A*SOD1 mice resulting from the whole-body mutant-SOD1 protein expression.

Abstract Background Recent single-center reports have suggested that community-acquired bacteremic co-infection in the context of Coronavirus disease 2019 (COVID-19) may be an important driver of mortality; however, these reports have not been validated with a multicenter, demographically diverse, cohort study with data spanning the pandemic. Methods In this multicenter, retrospective cohort study, inpatient encounters were assessed for COVID-19 with community-acquired bacteremic co-infection using 48-h post-admission blood cultures and grouped by: (1) confirmed co-infection [recovery of bacterial pathogen], (2) suspected co-infection [negative culture with ≥ 2 antimicrobials administered], and (3) no evidence of co-infection [no culture]. The primary outcomes were in-hospital mortality, ICU admission, and mechanical ventilation. COVID-19 bacterial co-infection risk factors and impact on primary outcomes were determined using multivariate logistic regressions and expressed as adjusted odds ratios with 95% confidence intervals (Cohort, OR 95% CI, Wald test p value). Results The studied cohorts included 13,781 COVID-19 inpatient encounters from 2020 to 2022 in the University of Alabama at Birmingham (UAB, n = 4075) and Ochsner Louisiana State University Health—Shreveport (OLHS, n = 9706) cohorts with confirmed (2.5%), suspected (46%), or no community-acquired bacterial co-infection (51.5%) and a comparison cohort consisting of 99,170 inpatient encounters from 2010 to 2019 (UAB pre-COVID-19 pandemic cohort). Significantly increased likelihood of COVID-19 bacterial co-infection was observed in patients with elevated ≥ 15 neutrophil-to-lymphocyte ratio (UAB: 1.95 [1.21–3.07]; OLHS: 3.65 [2.66–5.05], p

Multiple sclerosis (MS) is a leading cause of neurodegenerative disability in younger individuals. When diagnosed early, MS can be managed more effectively, stabilizing clinical symptoms and delaying disease progression. The identification of specific serum biomarkers for early-stage MS could facilitate more successful treatment of this condition. Because MS is an inflammatory disease, we assessed changes in enzymes of the endothelial hydrogen sulfide (H2S) pathway in response to inflammatory cytokines. Blotting analysis was conducted to detect Cystathionine γ-lyase (CSE), Cystathionine beta synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (MST) in human brain microvascular endothelial apical and basolateral microparticles (MPs) and cells following exposure to tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). CSE was increased in MPs and cells by exposure to TNF-α/IFN-γ; CBS was elevated in apical MPs but not in cells or basolateral MPs; MST was not significantly affected by cytokine exposure. To test how our findings relate to MS patients, we evaluated levels of CSE, CBS, and MST in serum samples from healthy control and MS patients. We found significantly decreased levels of CBS and MST (p = 0.0004, 0.009) in MS serum samples, whereas serum levels of CSE were marginally increased (p = 0.06). These observations support increased CSE and lower CBS and MST expression being associated with the vascular inflammation in MS. These changes in endothelial-derived sulfide enzymes at sites of inflammation in the brain may help to explain sulfide-dependent changes in vascular dysfunction/neuroinflammation underlying MS. These findings further support the use of serum samples to assess enzymatic biomarkers derived from circulating MPs. For example, “liquid biopsy” can be an important tool for allowing early diagnosis of MS, prior to the advanced progression of neurodegeneration associated with this disease.

Emerging evidence indicates that vascular stress is an important contributor to the pathophysiology of Alzheimer's disease and related dementias (ADRD). Hydrogen sulfide (H2S) and its metabolites (acid-labile (e.g., iron-sulfur clusters) and bound (e.g., per-, poly-) sulfides) have been shown to modulate both vascular and neuronal homeostasis. We recently reported that elevated plasma sulfides were associated with cognitive dysfunction and measures of microvascular disease in ADRD. Here we extend our previous work to show associations between elevated sulfides and magnetic resonance-based metrics of brain atrophy and white matter integrity. Elevated bound sulfides were associated with decreased grey matter volume, while increased acid labile sulfides were associated with decreased white matter integrity and greater ventricular volume. These findings are consistent with alterations in sulfide metabolism in ADRD which may represent maladaptive responses to oxidative stress.

Sigma1 receptor protein (Sigmar1) is a small, multifunctional molecular chaperone protein ubiquitously expressed in almost all body tissues. This protein has previously shown its cardioprotective roles in rodent models of cardiac hypertrophy, heart failure, and ischemia-reperfusion injury. Extensive literature also suggested its protective functions in several central nervous system disorders. Sigmar1’s molecular functions in the pulmonary system remained unknown. Therefore, we aimed to determine the expression of Sigmar1 in the lungs. We also examined whether Sigmar1 ablation results in histological, ultrastructural, and biochemical changes associated with lung pathology over aging in mice. In the current study, we first confirmed the presence of Sigmar1 protein in human and mouse lungs using immunohistochemistry and immunostaining. We used the Sigmar1 global knockout mouse (Sigmar1−/−) to determine the pathophysiological role of Sigmar1 in lungs over aging. The histological staining of lung sections showed altered alveolar structures, higher immune cells infiltration, and upregulation of inflammatory markers (such as pNFκB) in Sigmar1−/− mice compared to wildtype (Wt) littermate control mice (Wt). This indicates higher pulmonary inflammation resulting from Sigmar1 deficiency in mice, which was associated with increased pulmonary fibrosis. The protein levels of some fibrotic markers, fibronectin, and pSMAD2 Ser 245/250/255 and Ser 465/467, were also elevated in mice lungs in the absence of Sigmar1 compared to Wt. The ultrastructural analysis of lungs in Wt mice showed numerous multilamellar bodies of different sizes with densely packed lipid lamellae and mitochondria with a dark matrix and dense cristae. In contrast, the Sigmar1−/− mice lung tissues showed altered multilamellar body structures in alveolar epithelial type-II pneumocytes with partial loss of lipid lamellae structures in the lamellar bodies. This was further associated with higher protein levels of all four surfactant proteins, SFTP-A, SFTP-B, SFTP-C, and SFTP-D, in the Sigmar1−/− mice lungs. This is the first study showing Sigmar1’s expression pattern in human and mouse lungs and its association with lung pathophysiology. Our findings suggest that Sigmar1 deficiency leads to increased pulmonary inflammation, higher pulmonary fibrosis, alterations of the multilamellar body stuructures, and elevated levels of lung surfactant proteins.

Background: Following traumatic bone loss or removal of bone tumors, the failure of bone allograft transplantation for large bone defect repair remains a significant problem in orthopedics. Therefore, new strategies that can efficiently enhance allograft healing and long-term incorporation are critically needed. Method: In this study, we first injected Notch-activating Jagged1 peptide to mice and then isolated bone marrow tissues and cells for proliferation and differentiation assays. Femur bone allograft surgery was also performed in Jagged1 pre-treated mice, and bone defect healing process were monitored by histology, Micro-CT and biomechanical testing. Result: Our results showed that Jagged1 therapeutic injection is sufficient to maximally activate Notch and promote bone marrow stromal cell proliferation in vivo, while no effects on bone structure were observed. More importantly, Jagged1 pre-treatment significantly promoted bone callus formation and increased bone mechanical strength during allograft healing in a femur bone defect mouse model. Conclusion: This study reveals that Notch in vivo activation can be induced by injection of Jagged1 peptide for expansion of local native stromal cells that will significantly enhance bone callus formation. The Translational potential of this Article: The clinical uses of this therapeutic strategy would be immediately applicable for chronic long bone defect repair. More importantly, this devised strategy for expansion of endogenous BMSCs can also be applied to enhance other tissue and organ repair.

The recent rise in illicit use of methamphetamine (METH), a highly addictive psychostimulant, is a huge health care burden due to its central and peripheral toxic effects. Mounting clinical studies have noted that METH use in humans is associated with the development of cardiomyopathy; however, preclinical studies and animal models to dissect detailed molecular mechanisms of METH-associated cardiomyopathy development are scarce. The present study utilized a unique very long-access binge and crash procedure of METH self-administration to characterize the sequelae of pathological alterations that occur with METH-associated cardiomyopathy. Rats were allowed to intravenously self-administer METH for 96 h continuous weekly sessions over 8 weeks. Cardiac function, histochemistry, ultrastructure, and biochemical experiments were performed 24 h after the cessation of drug administration. Voluntary METH self-administration induced pathological cardiac remodeling as indicated by cardiomyocyte hypertrophy, myocyte disarray, interstitial and perivascular fibrosis accompanied by compromised cardiac systolic function. Ultrastructural examination and native gel electrophoresis revealed altered mitochondrial morphology and reduced mitochondrial oxidative phosphorylation (OXPHOS) supercomplexes (SCs) stability and assembly in METH exposed hearts. Redox-sensitive assays revealed significantly attenuated mitochondrial respiratory complex activities with a compensatory increase in pyruvate dehydrogenase (PDH) activity reminiscent of metabolic remodeling. Increased autophagy flux and increased mitochondrial antioxidant protein level was observed in METH exposed heart. Treatment with mitoTEMPO reduced the autophagy level indicating the involvement of mitochondrial dysfunction in the adaptive activation of autophagy in METH exposed hearts. Altogether, we have reported a novel METH-associated cardiomyopathy model using voluntary drug seeking behavior. Our studies indicated that METH self-administration profoundly affects mitochondrial ultrastructure, OXPHOS SCs assembly and redox activity accompanied by increased PDH activity that may underlie observed cardiac dysfunction.

Methamphetamine (METH) is an addictive illicit drug used worldwide that causes significant damage to blood vessels resulting in cardiovascular dysfunction. Recent studies highlight increased prevalence of cardiovascular disease (CVD) and associated complications including hypertension, vasospasm, left ventricular hypertrophy, and coronary artery disease in younger populations due to METH use. Here we report that METH administration in a mouse model of ‘binge and crash’ decreases cardiovascular function via cystathionine gamma lyase (CSE), hydrogen sulfide (H2S), nitric oxide (NO) (CSE/H2S/NO) dependent pathway. METH significantly reduced H2S and NO bioavailability in plasma and skeletal muscle tissues co-incident with a significant reduction in flow-mediated vasodilation (FMD) and blood flow velocity revealing endothelial dysfunction. METH administration also reduced cardiac ejection fraction (EF) and fractional shortening (FS) associated with increased tissue and perivascular fibrosis. Importantly, METH treatment selectively decreased CSE expression and sulfide bioavailability along with reduced eNOS phosphorylation and NO levels. Exogenous sulfide therapy or endothelial CSE transgenic overexpression corrected cardiovascular and associated pathological responses due to METH implicating a central molecular regulatory pathway for tissue pathology. These findings reveal that therapeutic intervention targeting CSE/H2S bioavailability may be useful in attenuating METH mediated cardiovascular disease.

Hydrogen sulfide (H2S) is a gasotransmitter that exerts a multitude of functions in both physiologic and pathophysiologic processes. H2S-synthesizing enzymes are increased in a variety of human malignancies, including colon, prostate, breast, renal, urothelial, ovarian, oral squamous cell, and thyroid cancers. In cancer, H2S promotes tumor growth, cellular and mitochondrial bioenergetics, migration, invasion, angiogenesis, tumor blood flow, metastasis, epithelia–mesenchymal transition, DNA repair, protein sulfhydration, and chemotherapy resistance Additionally, in some malignancies, increased H2S-synthesizing enzyme expression correlates with a worse prognosis and a higher tumor stage. Here we review the role of H2S in cancer, with an emphasis on the molecular mechanisms by which H2S promotes cancer development, progression, dedifferentiation, and metastasis.

Mental stress is a risk factor for myocardial infarction in women. The central hypothesis of this study is that restraint stress induces sex-specific changes in gene expression in the heart, which leads to an intensified response to ischemia/reperfusion injury due to the development of a pro-oxidative environment in female hearts. We challenged male and female C57BL/6 mice in a restraint stress model to mimic the effects of mental stress. Exposure to restraint stress led to sex differences in the expression of genes involved in cardiac hypertrophy, inflammation, and iron-dependent cell death (ferroptosis). Among those genes, we identified tumor protein p53 and cyclin-dependent kinase inhibitor 1A (p21), which have established controversial roles in ferroptosis. The exacerbated response to I/R injury in restraint-stressed females correlated with downregulation of p53 and nuclear factor erythroid 2–related factor 2 (Nrf2, a master regulator of the antioxidant response system-ARE). S-female hearts also showed increased superoxide levels, lipid peroxidation, and prostaglandin-endoperoxide synthase 2 (Ptgs2) expression (a hallmark of ferroptosis) compared with those of their male counterparts. Our study is the first to test the sex-specific impact of restraint stress on the heart in the setting of I/R and its outcome.

The brain is sensitive to oxygen deprivation. Here, the authors show in experimental animals that sensitivity to hypoxia is inversely related to the level of sulfide:quinone oxidoreductast (SQOR) and the capacity to catabolize sulfide in the brain.

Lower circulating levels of glycine are consistently reported in association with cardiovascular disease (CVD), but the causative role and therapeutic potential of glycine in atherosclerosis, the underlying cause of most CVDs, remain to be established. Here, following the identification of reduced circulating glycine in patients with significant coronary artery disease (sCAD), we investigated a causative role of glycine in atherosclerosis by modulating glycine availability in atheroprone mice. We further evaluated the atheroprotective potential of DT-109, a recently identified glycine-based compound with dual lipid/glucose-lowering properties. Glycine deficiency enhanced, while glycine supplementation attenuated, atherosclerosis development in apolipoprotein E-deficient (Apoe−/−) mice. DT-109 treatment showed the most significant atheroprotective effects and lowered atherosclerosis in the whole aortic tree and aortic sinus concomitant with reduced superoxide. In Apoe−/− mice with established atherosclerosis, DT-109 treatment significantly reduced atherosclerosis and aortic superoxide independent of lipid-lowering effects. Targeted metabolomics and kinetics studies revealed that DT-109 induces glutathione formation in mononuclear cells. In bone marrow-derived macrophages (BMDMs), glycine and DT-109 attenuated superoxide formation induced by glycine deficiency. This was abolished in BMDMs from glutamate-cysteine ligase modifier subunit-deficient (Gclm−/-) mice in which glutathione biosynthesis is impaired. Metabolic flux and carbon tracing experiments revealed that glycine deficiency inhibits glutathione formation in BMDMs while glycine-based treatment induces de novo glutathione biosynthesis. Through a combination of studies in patients with CAD, in vivo studies using atherosclerotic mice and in vitro studies using macrophages, we demonstrated a causative role of glycine in atherosclerosis and identified glycine-based treatment as an approach to mitigate atherosclerosis through antioxidant effects mediated by induction of glutathione biosynthesis.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is involved in a global outbreak affecting millions of people who manifest a variety of symptoms. Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is increasingly associated with cardiovascular complications requiring hospitalizations; however, the mechanisms underlying these complications remain unknown. Nitric oxide (NO) and hydrogen sulfide (H2S) are gasotransmitters that regulate key cardiovascular functions. Methods: Blood samples were obtained from 68 COVID-19 patients and 33 controls and NO and H2S metabolites were assessed. H2S and NO levels were compared between cases and controls in the entire study population and subgroups based on race. The availability of gasotransmitters was examined based on severity and outcome of COVID-19 infection. The performance of H2S and NO levels in predicting COVID-19 infection was also analyzed. Multivariable regression analysis was performed to identify the effects of traditional determinants of gasotransmitters on NO and H2S levels in the patients with COVID-19 infection. Results: Significantly reduced NO and H2S levels were observed in both Caucasian and African American COVID-19 patients compared to healthy controls. COVID-19 patients who died had significantly higher NO and H2S levels compared to COVID-19 patients who survived. Receiver-operating characteristic analysis of NO and H2S metabolites in the study population showed free sulfide levels to be highly predictive of COVID-19 infection based on reduced availability. Traditional determinants of gasotransmitters, namely age, race, sex, diabetes, and hypertension had no effect on NO and H2S levels in COVID-19 patients. Conclusion: These observations provide the first insight into the role of NO and H2S in COVID-19 infection, where their low availability may be a result of reduced synthesis secondary to endotheliitis, or increased consumption from scavenging of reactive oxygen species.

Recent evidence has revealed that exposing cells to exogenous H 2 S or inhibiting cellular H 2 S synthesis can modulate cell cycle checkpoints, DNA damage and repair, and the expression of proteins involved in the maintenance of genomic stability, all suggesting that H 2 S plays an important role in the DNA damage response (DDR). Here we review the role of H 2 S in the DRR and maintenance of genomic stability. Treatment of various cell types with pharmacologic H 2 S donors or cellular H 2 S synthesis inhibitors modulate the G 1 checkpoint, inhibition of DNA synthesis, and cause p21, and p53 induction. Moreover, in some cell models H 2 S exposure induces PARP-1 and g-H2AX foci formation, increases PCNA, CHK2, Ku70, Ku80, and DNA polymerase-d protein expression, and maintains mitochondrial genomic stability. Our group has also revealed that H 2 S bioavailability and the ATR kinase regulate each other with ATR inhibition lowering cellular H 2 S concentrations, whereas intracellular H 2 S concentrations regulate ATR kinase activity via ATR serine 435 phosphorylation. In summary, these findings have many implications for the DDR, for cancer chemotherapy, and fundamental biochemical metabolic pathways involving H 2 S.

Oxidative stress drives the pathogenesis of atrial fibrillation (AF), the most common arrhythmia. In the cardiovascular system, cystathionine γ-lyase (CSE) serves as the primary enzyme producing hydrogen sulfide (H2S), a mammalian gasotransmitter that reduces oxidative stress. Using a case control study design in patients with and without AF and a mouse model of CSE knockout (CSE-KO), we evaluated the role of H2S in the etiology of AF. Patients with AF (n = 51) had significantly reduced plasma acid labile sulfide levels compared to patients without AF (n = 65). In addition, patients with persistent AF (n = 25) showed lower plasma free sulfide levels compared to patients with paroxysmal AF (n = 26). Consistent with an important role for H2S in AF, CSE-KO mice had decreased atrial sulfide levels, increased atrial superoxide levels, and enhanced propensity for induced persistent AF compared to wild type (WT) mice. Rescuing H2S signaling in CSE-KO mice by Diallyl trisulfide (DATS) supplementation or reconstitution with endothelial cell specific CSE over-expression significantly reduced atrial superoxide, increased sulfide levels, and lowered AF inducibility. Lastly, low H2S levels in CSE KO mice was associated with atrial electrical remodeling including longer effective refractory periods, slower conduction velocity, increased myocyte calcium sparks, and increased myocyte action potential duration that were reversed by DATS supplementation or endothelial CSE overexpression. Our findings demonstrate an important role of CSE and H2S bioavailability in regulating electrical remodeling and susceptibility to AF.

Background The mutated α‐B‐Crystallin (CryABR120G) mouse model of desmin‐related myopathy (DRM) shows an age‐dependent onset of pathologic cardiac remodeling and progression of heart failure. CryABR120G expression in cardiomyocytes affects the mitochondrial spatial organization within the myofibrils, but the molecular perturbation within the mitochondria in the relation of the overall course of the proteotoxic disease remains unclear. Methods and Results CryABR120G mice show an accumulation of electron‐dense aggregates and myofibrillar degeneration associated with the development of cardiac dysfunction. Though extensive studies demonstrated that these altered ultrastructural changes cause cardiac contractility impairment, the molecular mechanism of cardiomyocyte death remains elusive. Here, we explore early pathological processes within the mitochondria contributing to the contractile dysfunction and determine the pathogenic basis for the heart failure observed in the CryABR120G mice. In the present study, we report that the CryABR120G mice transgenic hearts undergo altered mitochondrial dynamics associated with increased level of dynamin‐related protein 1 and decreased level of optic atrophy type 1 as well as mitofusin 1 over the disease process. In association with these changes, an altered level of the components of mitochondrial oxidative phosphorylation and pyruvate dehydrogenase complex regulatory proteins occurs before the manifestation of pathologic adverse remodeling in the CryABR120G hearts. Mitochondria isolated from CryABR120G transgenic hearts without visible pathology show decreased electron transport chain complex activities and mitochondrial respiration. Taken together, we demonstrated the involvement of mitochondria in the pathologic remodeling and progression of DRM‐associated cellular dysfunction. Conclusions Mitochondrial dysfunction in the form of altered mitochondrial dynamics, oxidative phosphorylation and pyruvate dehydrogenase complex proteins level, abnormal electron transport chain complex activities, and mitochondrial respiration are evident on the CryABR120G hearts before the onset of detectable pathologies and development of cardiac contractile dysfunction.

Mitochondria are highly dynamic organelles that constantly undergo fission and fusion events to adapt to changes in the cellular environment. Aberrant mitochondrial fission has been associated with several types of cardiovascular dysfunction; inhibition of pathologically aberrant mitochondrial fission has been shown to be cardioprotective. Pathological fission is mediated by the excessive activation of GTPase dynamin-related protein 1 (Drp1), making it an attractive therapeutic target in numerous cardiovascular diseases. Mitochondrial division inhibitor (mdivi-1) is widely used small molecule reported to inhibit Drp1-dependent fission, elongate mitochondria, and mitigate injury. The purpose of our study was to understand the pleiotropic effects of mdivi-1 on mitochondrial dynamics, mitochondrial respiration, electron transport activities, and macro-autophagy. In this study, we found that mdivi-1 treatment decreased Drp1 expression, proteolytically cleaved L-OPA1, and altered the expression of OXPHOS complex proteins, resulting in increased superoxide production. The altered expression of OXPHOS complex proteins may be directly associated with decreased Drp1 expression, as Drp1 siRNA knockdown in cardiomyocytes showed similar effects. Results from an autophagy flux assay showed that mdivi-1 induced impaired autophagy flux that could be restored by Atg7 overexpression, suggesting that mdivi-1 mediated inhibition of macro-autophagy in cardiomyocytes. Treatment with mdivi-1 resulted in increased expression of p62, which is required for Atg7 overexpression-induced rescue of mdivi-1-mediated impaired autophagy flux. In addition, mdivi-1-dependent proteolytic processing of L-OPA1 was associated with increased mitochondrial superoxide production and altered expression of mitochondrial serine/proteases. Overall, the novel pleiotropic effect of mdivi-1 in cardiomyocytes included proteolytically cleaved L-OPA1, altered expression of OXPHOS complex proteins, and increased superoxide production, which together resulted in defects in mitochondrial respiration and inhibition of macro-autophagy.

Endothelial nitric oxide (NO) is a critical mediator of vascular function and vascular remodeling. NO is produced by endothelial nitric oxide synthase (eNOS), which is activated by calcium (Ca2+)-dependent and Ca2+-independent pathways. Here, we report that neurogranin (Ng), which regulates Ca2+-calmodulin (CaM) signaling in the brain, is uniquely expressed in endothelial cells (EC) of human and mouse vasculature, and is also required for eNOS regulation. To test the role of Ng in eNOS activation, Ng knockdown in human aortic endothelial cells (HAEC) was performed using Ng SiRNA along with Ng knockout (Ng −/−) in mice. Depletion of Ng expression decreased eNOS activity in HAEC and NO production in mice. We show that Ng expression was decreased by short-term laminar flow and long-them oscillating flow shear stress, and that Ng siRNA with shear stress decreased eNOS expression as well as eNOS phosphorylation at S1177. We further reveled that lack of Ng expression decreases both AKT-dependent eNOS phosphorylation, NF-κB-mediated eNOS expression, and promotes endothelial activation. Our findings also indicate that Ng modulates Ca2+-dependent calcineurin (CaN) activity, which suppresses Ca2+-independent AKT-dependent eNOS signaling. Moreover, deletion of Ng in mice also reduced eNOS activity and caused endothelial dysfunction in flow-mediated dilation experiments. Our results demonstrate that Ng plays a crucial role in Ca2+-CaM-dependent eNOS regulation and contributes to vascular remodeling, which is important for the pathophysiology of cardiovascular disease.

Cardiovascular disease is the leading cause of death and disability worldwide with increased oxidative stress and reduced NO bioavailability serving as key risk factors. For decades, elevation in protein abundance and enzymatic activity of xanthine oxidoreductase (XOR) under hypoxic/inflammatory conditions has been associated with organ damage and vascular dysfunction. Recent reports have challenged this dogma by identifying a beneficial function for XOR, under similar hypoxic/acidic conditions, whereby XOR catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO) through poorly defined mechanisms. We previously reported that hydrogen sulfide (H2S/sulfide) confers significant vascular benefit under these same conditions via NO2- mediated mechanisms independent of nitric oxide synthase (NOS). Here we report for the first time the convergence of H2S, XOR, and nitrite to form a concerted triad for NO generation. Specifically, hypoxic endothelial cells show a dose-dependent, sulfide and polysulfide (diallyl trisulfide (DATS)-induced, NOS-independent NO2- reduction to NO that is dependent upon the enzymatic activity of XOR. Interestingly, nitrite reduction to NO was found to be slower and more sustained with DATS compared to H2S. Capacity for sulfide/polysulfide to produce an XOR-dependent impact on NO generation translates to salutary actions in vivo as DATS administration in cystathionine-γ-lyase (CSE) knockout mice significantly improved hindlimb ischemia blood flow post ligation, while the XOR-specific inhibitor, febuxostat (Febx), abrogated this benefit. Moreover, flow-mediated vasodilation (FMD) in CSE knockout mice following administration of DATS resulted in greater than 4-fold enhancement in femoral artery dilation while co-treatment with Febx completely completely abrogated this effect. Together, these results identify XOR as a focal point of convergence between sulfide- and nitrite-mediated signaling, as well as affirm the critical need to reexamine current dogma regarding inhibition of XOR in the context of vascular dysfunction.

It has been reported that pathological angiogenesis contributes to both experimental colitis and inflammatory bowel disease. Recently, we demonstrated that endothelial caveolin-1 plays a key role in the pathological angiogenesis of dextran sodium sulfate (DSS) colitis. However, the molecular mechanism of caveolin-1 regulation of endothelial function is unknown. In this study, we examined how the antennapedia- (AP-) conjugated caveolin-1 scaffolding domain (AP-Cav) modulates vascular endothelial growth factor- (VEGF-) dependent colon endothelial cell angiogenic responses, as seen during colitis. We used mouse colon endothelial cells and found that AP-Cav significantly inhibited VEGF-mediated bromodeoxyuridine (BrdU) incorporation into colon microvascular endothelial cells. AP-Cav significantly blunted VEGF-dependent extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation at 10 minutes and 2 hours after stimulation, compared with the AP control peptide. AP-Cav + VEGF-A treatment also significantly increased c-Jun N-terminal kinase (JNK) phosphorylation at 2 hours. AP-Cav + VEGF-A treatment significantly downregulated retinoblastoma (Rb) protein levels, upregulated cleaved caspase-3 protein levels at 4 hours, and induced apoptosis. Thus, our study suggests that disruption of endothelial caveolin-1 function via the AP-Cav diverts VEGF signaling responses away from endothelial cell proliferation and toward apoptosis through the inhibition of mitogen-activated protein (MAP) kinase signaling and the induction of JNK-associated apoptosis.

Joint repair: Growing new cartilage from placental stem cells Stem cells derived from placental tissue may help in treating damaged joints, thanks to an improved method for encouraging them to form cartilage. Placenta-derived stem cells are readily available, as the placenta is usually discarded after birth. These stem cells can easily be grown into cartilage tissue in a laboratory, but not when transplanted. Researchers led by Yufeng Dong (Louisiana State University Health Sciences Center, Shreveport, USA) and Xifu Shang (Anhui Provincial Hospital, Hefei, China) treated stem cells with a naturally occurring protein, JAG1, which blocks the signal that prevents them from forming cartilage. They found that JAG1 increased the stem cells’ capacity for cartilage formation. When JAG1-stimulated stem cells were injected into a mouse model of joint disease, cartilage formation was improved, and joint inflammation was reduced.

Insufficient hydrogen sulfide (H2S) has been implicated in Type 2 diabetic mellitus (T2DM) and hyperhomocysteinemia (HHcy)-related cardiovascular complications. We investigated the role of H2S in T2DM and HHcy-induced endothelial dysfunction in small mesenteric artery (SMA) of db/db mice fed a high methionine (HM) diet. HM diet (8 weeks) induced HHcy in both T2DM db/db mice and non-diabetic db/+ mice (total plasma Hcy: 48.4 and 31.3 µM, respectively), and aggravated the impaired endothelium-derived hyperpolarization factor (EDHF)-induced endothelium-dependent relaxation to acetylcholine (ACh), determined by the presence of eNOS inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME) and prostacyclin (PGI2) inhibitor indomethacin (INDO), in SMA from db/db mice but not that from db/+ mice. A non-selective Ca2+-active potassium channel (KCa) opener NS309 rescued T2DM/HHcy-impaired EDHF-mediated vascular relaxation to ACh. EDHF-induced relaxation to ACh was inhibited by a non-selective KCa blocker TEA and intermediate-conductance KCa blocker (IKCa) Tram-34, but not by small-conductance KCa (SKCa) blocker Apamin. HHcy potentiated the reduction of free sulfide, H2S and cystathionine γ-lyase protein, which converts L-cysteine to H2S, in SMA of db/db mice. Importantly, a stable H2S donor DATS diminished the enhanced O2- production in SMAs and lung endothelial cells of T2DM/HHcy mice. Antioxidant PEG-SOD and DATS improved T2DM/HHcy impaired relaxation to ACh. Moreover, HHcy increased hyperglycemia-induced IKCa tyrosine nitration in human micro-vascular endothelial cells. EDHF-induced vascular relaxation to L-cysteine was not altered, whereas such relaxation to NaHS was potentiated by HHcy in SMA of db/db mice which was abolished by ATP-sensitive potassium channel blocker Glycolamide but not by KCa blockers. Conclusions: Intermediate HHcy potentiated H2S reduction via CSE-downregulation in microvasculature of T2DM mice. H2S is justified as an EDHF. Insufficient H2S impaired EDHF-induced vascular relaxation via oxidative stress and IKCa inactivation in T2DM/HHcy mice. H2S therapy may be beneficial for prevention and treatment of micro-vascular complications in patients with T2DM and HHcy. Keywords: Hydrogen sulfide, Endothelial dysfunction, Micro-vasculature, T2DM, Calcium-activated potassium channel (KCa)

Hydrogen sulfide (H2S) has emerged as an important physiological and pathophysiological signaling molecule in the cardiovascular system influencing vascular tone, cytoprotective responses, redox reactions, vascular adaptation, and mitochondrial respiration. However, bioavailable levels of H2S in its various biochemical metabolite forms during clinical cardiovascular disease remain poorly understood. We performed a case-controlled study to quantify and compare the bioavailability of various biochemical forms of H2S in patients with and without cardiovascular disease (CVD). In our study, we used the reverse-phase high performance liquid chromatography monobromobimane assay to analytically measure bioavailable pools of H2S. Single nucleotide polymorphisms (SNPs) were also identified using DNA Pyrosequencing. We found that plasma acid labile sulfide levels were significantly reduced in Caucasian females with CVD compared with those without the disease. Conversely, plasma bound sulfane sulfur levels were significantly reduced in Caucasian males with CVD compared with those without the disease. Surprisingly, gender differences of H2S bioavailability were not observed in African Americans, although H2S bioavailability was significantly lower overall in this ethnic group compared to Caucasians. We also performed SNP analysis of H2S synthesizing enzymes and found a significant increase in cystathionine gamma-lyase (CTH) 1364 G-T allele frequency in patients with CVD compared to controls. Lastly, plasma H2S bioavailability was found to be predictive for cardiovascular disease in Caucasian subjects as determined by receiver operator characteristic analysis. These findings reveal that plasma H2S bioavailability could be considered a biomarker for CVD in an ethnic and gender manner. Cystathionine gamma-lyase 1346 G-T SNP might also contribute to the risk of cardiovascular disease development.

Hydrogen sulfide (H2S) is a gasotransmitter that exerts numerous physiologic and pathophysiologic effects. Recently, a role for H2S in DNA repair has been identified, where H2S modulates cell cycle checkpoint responses, the DNA damage response (DDR), and mitochondrial and nuclear genomic stability. In addition, several DNA repair proteins modulate cellular H2S concentrations and cellular sulfur metabolism and, in turn, are regulated by cellular H2S concentrations. Many DDR proteins are now pharmacologically inhibited in targeted cancer therapies. As H2S and the enzymes that synthesize it are increased in many human malignancies, it is likely that H2S synthesis inhibition by these therapies is an underappreciated aspect of these cancer treatments. Moreover, both H2S and DDR protein activities in cancer and cardiovascular diseases are becoming increasingly apparent, implicating a DDR–H2S signaling axis in these pathophysiologic processes. Taken together, H2S and DNA repair likely play a central and presently poorly understood role in both normal cellular function and a wide array of human pathophysiologic processes. Here, we review the role of H2S in DNA repair.

Glioblastoma (GBM) has a poor prognosis despite intensive treatment with surgery and chemoradiotherapy. Previous studies using dose-escalated radiotherapy have demonstrated improved survival; however, increased rates of radionecrosis have limited its use. Development of radiosensitizers could improve patient outcome. In the present study, we report the use of sodium sulfide (Na2S), a hydrogen sulfide (H2S) donor, to selectively kill GBM cells (T98G and U87) while sparing normal human cerebral microvascular endothelial cells (hCMEC/D3). Na2S also decreased mitochondrial respiration, increased oxidative stress and induced γH2AX foci and oxidative base damage in GBM cells. Since Na2S did not significantly alter T98G capacity to perform non-homologous end-joining or base excision repair, it is possible that GBM cell killing could be attributed to increased damage induction due to enhanced reactive oxygen species production. Interestingly, Na2S enhanced mitochondrial respiration, produced a more reducing environment and did not induce high levels of DNA damage in hCMEC/D3. Taken together, this data suggests involvement of mitochondrial respiration in Na2S toxicity in GBM cells. The fact that survival of LN-18 GBM cells lacking mitochondrial DNA (ρ0) was not altered by Na2S whereas the survival of LN-18 ρ+ cells was compromised supports this conclusion. When cells were treated with Na2S and photon or proton radiation, GBM cell killing was enhanced, which opens the possibility of H2S being a radiosensitizer. Therefore, this study provides the first evidence that H2S donors could be used in GBM therapy to potentiate radiation-induced killing. Keywords: Hydrogen sulfide, Glioblastoma, Ionizing radiation, DNA damage, DNA repair, Mitochondria, Reactive oxygen species

Hydrogen sulfide (H2S) is an important gaseous signaling molecule in the cardiovascular system. In addition to free H2S, H2S can be oxidized to polysulfide which can be biologically active. Since the impact of H2S on endothelial solute barrier function is not known, we sought to determine whether H2S and its various metabolites affect endothelial permeability. In vitro permeability was evaluated using albumin flux and transendothelial electrical resistance. Different H2S donors were used to examine the effects of exogenous H2S. To evaluate the role of endogenous H2S, mouse aortic endothelial cells (MAECs) were isolated from wild type mice and mice lacking cystathionine γ-lyase (CSE), a predominant source of H2S in endothelial cells. In vivo permeability was evaluated using the Miles assay. We observed that polysulfide donors induced rapid albumin flux across endothelium. Comparatively, free sulfide donors increased permeability only with higher concentrations and at later time points. Increased solute permeability was associated with disruption of endothelial junction proteins claudin 5 and VE-cadherin, along with enhanced actin stress fiber formation. Importantly, sulfide donors that increase permeability elicited a preferential increase in polysulfide levels within endothelium. Similarly, CSE deficient MAECs showed enhanced solute barrier function along with reduced endogenous bound sulfane sulfur. CSE siRNA knockdown also enhanced endothelial junction structures with increased claudin 5 protein expression. In vivo, CSE genetic deficiency significantly blunted VEGF induced hyperpermeability revealing an important role of the enzyme for barrier function. In summary, endothelial solute permeability is critically regulated via exogenous and endogenous sulfide bioavailability with a prominent role of polysulfides.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Nitric oxide (NO) and hydrogen sulfide (H2S) have emerged as dominant redox regulators of numerous aspects of cellular and physiological functions within several organ systems included cardiovascular, immune and neurological tissues. Recent studies have begun to reveal that these two gaseous molecules may have redundant or overlapping pathophysiological functions often involving similar molecular targets. However, it remains less clear when and how NO and H2S may interact under biological and disease processes. In this graphical review, we discuss the current understanding of NO and H2S interactions and how they may functionally influence each other and what this may mean for biology and mechanisms of disease.

Diabetes mellitus (DM) is a chronic metabolic disorder characterized by inappropriate hyperglycemia due to lack of or resistance to insulin. Patients with DM are frequently afflicted with ischemic vascular disease or wound healing defect. It is well known that type 2 DM causes amplification of the atherosclerotic process, endothelial cell dysfunction, glycosylation of extracellular matrix proteins, and vascular denervation. These complications ultimately lead to impairment of neovascularization and diabetic wound healing. Therapeutic angiogenesis remains an attractive treatment modality for chronic ischemic disorders including PAD and/or diabetic wound healing. Many experimental studies have identified better approaches for diabetic cardiovascular complications, however, successful clinical translation has been limited possibly due to the narrow therapeutic targets of these agents or the lack of rigorous evaluation of pathology and therapeutic mechanisms in experimental models of disease. This paper discusses the current body of evidence identifying endothelial dysfunction and impaired angiogenesis during diabetes.