Reversal of Pulmonary Hypertension in a Human-Like Model: Therapeutic Targeting of Endothelial DHFR.

BACKGROUND: Pulmonary hypertension (PH) is a progressive disorder characterized by remodeling of the pulmonary vasculature and elevated mean pulmonary arterial pressure, resulting in right heart failure. METHODS: Here, we show that direct targeting of the endothelium to uncouple eNOS (endothelial nitric oxide synthase) with DAHP (2,4-diamino 6-hydroxypyrimidine; an inhibitor of GTP cyclohydrolase 1, the rate-limiting synthetic enzyme for the critical eNOS cofactor tetrahydrobiopterin) induces human-like, time-dependent progression of PH phenotypes in mice. RESULTS: Critical phenotypic features include progressive elevation in mean pulmonary arterial pressure, right ventricular systolic blood pressure, and right ventricle (RV)/left ventricle plus septum (LV+S) weight ratio; extensive vascular remodeling of pulmonary arterioles with increased medial thickness/perivascular collagen deposition and increased expression of PCNA (proliferative cell nuclear antigen) and alpha-actin; markedly increased total and mitochondrial superoxide production, substantially reduced tetrahydrobiopterin and nitric oxide bioavailabilities; and formation of an array of human-like vascular lesions. Intriguingly, novel in-house generated endothelial-specific dihydrofolate reductase (DHFR) transgenic mice (tg-EC-DHFR) were completely protected from the pathophysiological and molecular features of PH upon DAHP treatment or hypoxia exposure. Furthermore, DHFR overexpression with a pCMV-DHFR plasmid transfection in mice after initiation of DAHP treatment completely reversed PH phenotypes. DHFR knockout mice spontaneously developed PH at baseline and had no additional deterioration in response to hypoxia, indicating an intrinsic role of DHFR deficiency in causing PH. RNA-sequencing experiments indicated great similarity in gene regulation profiles between the DAHP model and human patients with PH. CONCLUSIONS: Taken together, these results establish a novel human-like murine model of PH that has long been lacking in the field, which can be broadly used for future mechanistic and translational studies. These data also indicate that targeting endothelial DHFR deficiency represents a novel and robust therapeutic strategy for the treatment of PH.

KDM4B protects against obesity and metabolic dysfunction.

Although significant progress has been made in understanding epigenetic regulation of in vitro adipogenesis, the physiological functions of epigenetic regulators in metabolism and their roles in obesity remain largely elusive. Here, we report that KDM4B (lysine demethylase 4B) in adipose tissues plays a critical role in energy balance, oxidation, lipolysis, and thermogenesis. Loss of KDM4B in mice resulted in obesity associated with reduced energy expenditure and impaired adaptive thermogenesis. Obesity in KDM4B-deficient mice was accompanied by hyperlipidemia, insulin resistance, and pathological changes in the liver and pancreas. Adipocyte-specific deletion of Kdm4b revealed that the adipose tissues were the main sites for KDM4B antiobesity effects. KDM4B directly controlled the expression of multiple metabolic genes, including Ppargc1a and Ppara Collectively, our studies identify KDM4B as an essential epigenetic factor for the regulation of metabolic health and maintaining normal body weight in mice. KDM4B may provide a therapeutic target for treatment of obesity.

Protein Phosphotyrosine Phosphatase 1B (PTP1B) in Calpain-dependent Feedback Regulation of Vascular Endothelial Growth Factor Receptor (VEGFR2) in Endothelial Cells: IMPLICATIONS IN VEGF-DEPENDENT ANGIOGENESIS AND DIABETIC WOUND HEALING.

The VEGF/VEGFR2/Akt/eNOS/NO pathway is essential to VEGF-induced angiogenesis. We have previously discovered a novel role of calpain in mediating VEGF-induced PI3K/AMPK/Akt/eNOS activation through Ezrin. Here, we sought to identify possible feedback regulation of VEGFR2 by calpain via its substrate protein phosphotyrosine phosphatase 1B (PTP1B), and the relevance of this pathway to VEGF-induced angiogenesis, especially in diabetic wound healing. Overexpression of PTP1B inhibited VEGF-induced VEGFR2 and Akt phosphorylation in bovine aortic endothelial cells, while PTP1B siRNA increased both, implicating negative regulation of VEGFR2 by PTP1B. Calpain inhibitor ALLN induced VEGFR2 activation, which can be completely blocked by PTP1B overexpression. Calpain activation induced by overexpression or Ca/A23187 resulted in PTP1B cleavage, which can be blocked by ALLN. Moreover, calpain activation inhibited VEGF-induced VEGFR2 phosphorylation, which can be restored by PTP1B siRNA. These data implicate calpain/PTP1B negative feedback regulation of VEGFR2, in addition to the primary signaling pathway of VEGF/VEGFR2/calpain/PI3K/AMPK/Akt/eNOS. We next examined a potential role of PTP1B in VEGF-induced angiogenesis. Endothelial cells transfected with PTP1B siRNA showed faster wound closure in response to VEGF. Aortic discs isolated from PTP1B siRNA-transfected mice also had augmented endothelial outgrowth. Importantly, PTP1B inhibition and/or calpain overexpression significantly accelerated wound healing in STZ-induced diabetic mice. In conclusion, our data for the first time demonstrate a calpain/PTP1B/VEGFR2 negative feedback loop in the regulation of VEGF-induced angiogenesis. Modulation of local PTP1B and/or calpain activities may prove beneficial in the treatment of impaired wound healing in diabetes.

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure.

BACKGROUND: Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. METHODS AND RESULTS: Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. CONCLUSIONS: BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

Endothelial cell calpain as a critical modulator of angiogenesis.

Calpains are a family of calcium-dependent non-lysosomal cysteine proteases. In particular, calpains residing in the endothelial cells play important roles in angiogenesis. It has been shown that calpain activity can be increased in endothelial cells by growth factors, primarily vascular endothelial growth factor (VEGF). VEGF/VEGFR2 induces calpain 2 dependent activation of PI3K/AMPK/Akt/eNOS pathway, and consequent nitric oxide production and physiological angiogenesis. Under pathological conditions such as tumor angiogenesis, endothelial calpains can be activated by hypoxia. This review focuses on the molecular regulatory mechanisms of calpain activation, and the newly identified mechanistic roles and downstream signaling events of calpains in physiological angiogenesis, and in the conditions of pathological tumor angiogenesis and diabetic wound healing, as well as retinopathy and atherosclerosis that are also associated with an increase in calpain activity. Further discussed include the differential strategies of modulating angiogenesis through manipulating calpain expression/activity in different pathological settings. Targeted limitation of angiogenesis in cancer and targeted promotion of angiogenesis in diabetic wound healing via modulations of calpains and calpain-dependent signaling mechanisms are of significant translational potential. Emerging strategies of tissue-specific targeting, environment-dependent targeting, and genome-targeted editing may turn out to be effective regimens for targeted manipulation of angiogenesis through calpain pathways, for differential treatments including both attenuation of tumor angiogenesis and potentiation of diabetic angiogenesis.

Netrin-1 improves post-injury cardiac function in vivo via DCC/NO-dependent preservation of mitochondrial integrity, while attenuating autophagy.

Reperfusion injury of the heart is a severe complication of angioplasty treatment of acute myocardial ischemia, for which no therapeutics are currently available. The present study aimed to identify whether and how a novel protein, netrin-1, induces cardioprotection in vivo during ischemia/reperfusion (I/R) injury. Wild type (WT) C57BL6/J mice were subjected to a 30 min coronary occlusion followed by a 24h reperfusion with vehicle (normal saline), netrin-1, UO126 (MEK1/2 inhibitor), PTIO (nitric oxide/NO scavenger), netrin-1/UO126 or netrin-1/PTIO intraventricularly. Some were injected of netrin-1 via tail vein. Netrin-1 at 5µg/kg induced a substantial reduction in infarct size (19.7 ± 5.0% from 41.3 ± 1.8% in the controls), and markedly improved cardiac function as measured by ejection fraction and fractional shortening from echocardiography. Experiments with mice deficient in netrin-1 receptor DCC (deleted in colorectal cancer, DCC+/-), or reperfusion with netrin-1/UO126 or netrin-1/PTIO, attenuated the protective effects of netrin-1, implicating intermediate roles of DCC, ERK1/2 and NO. Netrin-1 induced phosphorylation of ERK1/2 and eNOS was abolished in DCC+/-mice. Electron spin resonance (ESR) determination of NO production from isolated left ventricles demonstrated that netrin-1 improves NO bioavailability, which was attenuated by UO126 or in DCC+/-mice, suggesting upstream roles of DCC and ERK1/2 in NO production. Netrin-1 further reduced mitochondrial swelling and mitochondrial superoxide production, which was absent when co-treated with PTIO or UO126, or in DCC+/-mice, indicating critical roles of DCC, ERK1/2 and NO in preserving mitochondrial integrity. In a permanent coronary ligation model of myocardial infarction (MI) to assess post-MI remodeling, netrin-1 abolished the marked increase in autophagy. In summary, our data demonstrate robust cardioprotective effect of netrin-1 in vivo, as shown by reduced infarct size and improved cardiac function. Mechanistically, this protection is mediated by netrin-1 receptor DCC, and NO dependent preservation of mitochondria. This work clearly establishes a therapeutic potential of netrin-1 for acute treatment of MI, perhaps also for chronic post-MI remodeling. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.

Clinical characteristics of a COVID-19 cohort treated at UCLA Ronald Reagan Medical Center during the breaking phase of the pandemic: A retrospective study.

To this date, COVID-19 remains an unresolved pandemic, and the impairment of redox homeostasis dictates the severity of clinical outcomes. Here we examined initial UCLA cohort of 440 COVID-19 patients hospitalized between March 1st and April 1st, 2020, representing the first wave of the pandemic. The mean age was 58.88 ± 21.12, among which males were significantly more than females (55.5 % vs. 44.5 %), most distinctively in age group of 50-69. The age groups of 50-69 (33.6 %) and ≥70 (34.8 %) dominated. The racial composition was in general agreement with Census data with slight under-representation of Hispanics and Asians, and over-representation of Caucasians. Smoking was a significant factor (28.8 % vs. 11.0 % in LA population), likewise for obesity (BMI ≥30) (37.4 % vs. 27.7 % in LA population). Patients suffering from obesity or BMI<18.5 checked into ICU at a significantly higher rate. A 74.5 % of the patients had comorbidities including diabetes, chronic kidney disease, chronic pulmonary disease, congestive heart failure and peripheral vascular disease. The levels of d-dimer were drastically upregulated (1159.5 ng/mL), indicating hypercoagulative state. Upregulated LDH (328 IU/L) indicated significant tissue damages. A distorted redox hemeostasis is a common trait associated with these risk factors and clinical markers. A quarter of the patients received antivirals, among which Remdesivir most prescribed (23.6 %). Majority received antithrombotics (75 %), and antibiotics. Upon admission, 67 patients were intubated or received CPR; 177 patients eventually received intensive care (40.2 %). While 290 were discharged alive, 10 remained hospitalized, 73 were transferred, and 36 died with 3 palliatively discharged. In summary, our data fully characterized a Californian cohort of COVID-19 at the breaking phase of the pandemic, indicating that population demographics, biophysical characters, comorbidities and molecular pathological parameters have significant impacts on the evolvement of a pandemic. These provide critical insights into effective management of COVID-19, and future break from another pathogen.

Perioperative Changes in Plasma Nitrite and IL-6 Levels Predict Postoperative Atrial Fibrillation (POAF) and Acute Kidney Injury (AKI) after Cardiac Surgery.

Background: Postoperative atrial fibrillation (POAF) and acute kidney injury (AKI) are common yet significant complications after cardiac surgery, with incidences of up to 40% for each. Here, we assessed plasma nitrite and serum interleukin-6 (IL-6) levels before and after cardiac surgery to quantify the extent to which oxidative stress and inflammation contribute to POAF and AKI occurrence. Methods: We prospectively enrolled 206 cardiac surgical patients. Plasma nitrite and serum IL-6 levels were determined preoperatively and at 24 h, 48 h and 72 h postoperatively. The patients had continuous EKG monitoring for occurrence of POAF, while daily serum creatinine was measured for determination of stage 1 + AKI. Results: Postoperatively, 78 (38%) patients experienced AF, and 47 (23%) patients experienced stage 1 + AKI. POAF analysis: Age, ACE-inhibitor use, valve surgery and percent change in baseline plasma nitrite at 24 h postoperatively were associated with POAF in multiple logistic regression analysis. The inclusion of this new biomarker significantly improved the POAF prediction model (AUC 0.77 for clinical risk factors alone, to AUC 0.81). AKI analysis: A history of diabetes mellitus was associated with AKI in multiple logistic regression analysis, and the addition of preoperative IL-6 levels improved the prediction model for AKI occurrence (AUC 0.69 to AUC 0.74). Conclusions: We previously observed selective upregulation of NADPH oxidase isoform 4 (NOX4) in patients with AF, a critical causal role of NOX4 for AF in zebrafish and a robust inhibitory effect of nitric oxide (NO) on NOX4. Our data innovatively demonstrate that a reduction in circulating nitrite levels, likely implicative of elevated NOX4-mediated oxidative stress, independently associates with POAF and improves POAF prediction, whereas the inclusion of circulating IL-6 levels improves the prediction model for AKI. Therefore, therapeutic strategies to mitigate these pathophysiological sequalae of surgical stress may reduce the incidence of severe postoperative complications of POAF and AKI.

Oxidative stress in atrial fibrillation: an emerging role of NADPH oxidase.

Atrial fibrillation (AF) is the most common cardiac arrhythmia. Patients with AF have up to seven-fold higher risk of suffering from ischemic stroke. Better understanding of etiologies of AF and its thromboembolic complications are required for improved patient care, as current anti-arrhythmic therapies have limited efficacy and off target effects. Accumulating evidence has implicated a potential role of oxidative stress in the pathogenesis of AF. Excessive production of reactive oxygen species (ROS) is likely involved in the structural and electrical remodeling of the heart, contributing to fibrosis and thrombosis. In particular, NADPH oxidase (NOX) has emerged as a potential enzymatic source for ROS production in AF based on growing evidence from clinical and animal studies. Indeed, NOX can be activated by known upstream triggers of AF such as angiotensin II and atrial stretch. In addition, treatments such as statins, antioxidants, ACEI or AT1RB have been shown to prevent post-operative AF; among which ACEI/AT1RB and statins can attenuate NOX activity. On the other hand, detailed molecular mechanisms by which specific NOX isoform(s) are involved in the pathogenesis of AF and the extent to which activation of NOX plays a causal role in AF development remains to be determined. The current review discusses causes and consequences of oxidative stress in AF with a special focus on the emerging role of NOX pathways.

Endothelium-specific sepiapterin reductase deficiency in DOCA-salt hypertension.

The endothelial nitric oxide synthase (eNOS) requires tetrahydrobiopterin (H(4)B) as a cofactor and, in its absence, produces superoxide (O(2)(·-)) rather than nitric oxide (NO(·)), a condition referred to as eNOS uncoupling. DOCA-salt-induced hypertension is associated with H(4)B oxidation and uncoupling of eNOS. The present study investigated whether administration of sepiapterin or H(4)B recouples eNOS in DOCA-salt hypertension. Bioavailable NO(·) detected by electron spin resonance was markedly reduced in aortas of DOCA-salt hypertensive mice. Preincubation with sepiapterin (10 µmol/l for 30 min) failed to improve NO(·) bioavailability in hypertensive aortas while it augmented NO(·) production from control vessels, implicating a hypertension-associated deficiency in sepiapterin reductase (SPR), the rate-limiting enzyme for sepiapterin conversion to H(4)B. Indeed, a decreased SPR expression was observed in aortic endothelial cells, but not in endothelium-denuded aortic remains, implicating an endothelium-specific SPR deficiency. Administration of hypertensive aortas with H(4)B (10 µmol/l, 30 min) partially restored vascular NO(·) production. Combined administration of H(4)B and the NADPH oxidase inhibitor apocynin (100 µmol/l, 30 min) fully restored NO(·) bioavailability while reducing O(2)(·-) production. In angiotensin II-induced hypertension, however, aortic endothelial SPR expression was not affected. In summary, administration of sepiapterin is not effective in recoupling eNOS in DOCA-salt hypertension, due to an endothelium-specific loss in SPR, whereas coadministration of H(4)B and apocynin is highly efficient in recoupling eNOS. This is consistent with our previous observations that in angiotensin II hypertension, endothelial deficiency in dihydrofolate reductase is alternatively responsible for uncoupling of eNOS. Taken together, these data indicate that strategies specifically targeting at different H(4)B metabolic enzymes might be necessary in restoring eNOS function in different types of hypertension.

Cardiac vulnerability to ischemia/reperfusion injury drastically increases in late pregnancy.

Although the murine late pregnant (LP) heart is speculated to be a better functioning heart during physiological conditions, the susceptibility of LP hearts to I/R injury is still unknown. The aims of this study were to investigate the cardiac vulnerability of LP rodents to ischemia/reperfusion (I/R) injury and to explore its underlying mechanisms. In vivo female rat hearts [non-pregnant (NP) or LP] or ex vivo Langendorff-perfused mouse hearts were subjected to I/R. The infarct size was approximately fourfold larger in LP animals compared with NP both in vivo and ex vivo. The heart functional recovery was extremely poor in LP mice compared with NP (~10% recovery in LP vs. 80% recovery in NP at the end of reperfusion, P < 0.01). Interestingly, the poor functional recovery and the larger infarct size in LP were partially restored one day post-partum and almost fully restored 1 week post-partum to their corresponding NP levels. Mitochondrial respiratory function and the threshold for opening of the mitochondrial permeability transition pore were significantly lower in LP compared with NP when they both were subjected to myocardial I/R injury [Respiratory control ratio = 1.9 ± 0.1 vs. 4.0 ± 0.5 in NP, P < 0.05; calcium retention capacity (CRC) = 167 ± 10 vs. 233 ± 18 nmol/mg protein in NP, P < 0.01]. Cardiac reactive oxygen species (ROS) generation, as well mitochondrial superoxide production, was approximately twofold higher in LP compared with NP following I/R. The phosphorylation levels of Akt, ERK1/2, and STAT3, but not GSK3ß, were significantly reduced in the hearts from LP subjected to I/R. In conclusion, increased mitochondrial ROS generation, decreased CRC as well as impaired activation of Akt/ERK/STAT3 at reperfusion are the possible underlying mechanisms for higher vulnerability of LP hearts to I/R.

Inhibition of XO or NOX attenuates diethylstilbestrol-induced endothelial nitric oxide deficiency without affecting its effects on LNCaP cell invasion and apoptosis.

Oestrogen protects cardiovascular health partially via an up-regulation of NO• (NO radical) production. Its synthetic analogue DES (diethylstilbestrol), used as a potent androgen deprivation therapy for patients with prostate cancer, is however associated with high incidence of thromboembolic events. Exposure of BAECs (bovine aortic endothelial cells) to pharmacologically relevant dosage (12.5 µmol/l, 24 h) of DES resulted in a marked reduction in endothelial NO• bioavailability determined by ESR (electron spin resonance), while 17ß-oestradiol instead increased NO• production as expected. Intriguingly, endothelial O(2)•- (superoxide anion) production was up-regulated by DES in vitro and in vivo, which was, however, attenuated by the ER (oestrogen receptor) antagonist ICI 182780, the XO (xanthine oxidase) inhibitor oxypurinol or the NOX (NADPH oxidase) inhibitor NSC23766. These agents also restored NO• production. DES alone in a cell-free system did not produce any ESR-sound O(2)•- signal. Of note, eNOS (endothelial NO synthase) mRNA and protein remained unchanged in response to DES. These results suggest that receptor-dependent activation of XO or NOX, and subsequent production of O(2)•-, mediate DES-induced NO• deficiency. This could represent a previously unrecognized mechanism that is responsible for cardiovascular complications of DES administration. Importantly, DES-induced suppression of LNCaP cell invasion and apoptosis were not affected by XO or NOX inhibitor. Therefore combinatorial therapy of DES and XO/NOX inhibitor may prove to be an innovative and useful therapeutic option in eliminating cardiovascular complications of DES, while preserving its anti-cancer effects, benefiting patients with advanced cancer who do not respond well to any other treatments but DES.

Intralipid, a clinically safe compound, protects the heart against ischemia-reperfusion injury more efficiently than cyclosporine-A.

BACKGROUND: We have recently shown that postischemic administration of intralipid protects the heart against ischemia-reperfusion injury. Here we compared the cardioprotective effects of intralipid with cyclosporine-A, a potent inhibitor of the mitochondrial permeability transition pore opening. METHODS: In vivo rat hearts or isolated Langendorff-perfused mouse hearts were subjected to ischemia followed by reperfusion with intralipid (0.5%, 1% and 2% ex-vivo, and 20% in vivo), cyclosporine-A (0.2 µM, 0.8 µM, and 1.5 µM ex- vivo and 10 mg/kg in vivo), or vehicle. The hemodynamic function, infarct size, calcium retention capacity, mitochondrial superoxide production, and phosphorylation levels of protein kinase B (Akt)/glycogen synthase kinase-3ß (GSK-3ß) were measured. The values are mean ± SEM. RESULTS: Administration of intralipid at reperfusion significantly reduced myocardial infarct size compared with cyclosporine-A in vivo (infarct size/area at risk)%: 22.9 ± 2.5% vs. 35.2 ± 3.5%; P = 0.030, n = 7/group). Postischemic administration of intralipid at its optimal dose (1%) was more effective than cyclosporine-A (0.8 µM) in protecting the ex vivo heart against ischemia-reperfusion injury, as the rate pressure product at the end of reperfusion was significantly higher (mmHg · beats/min: 12,740 ± 675 [n = 7] vs. 9,203 ± 10,781 [n = 5], P = 0.024), and the infarct size was markedly smaller (17.3 ± 2.9 [n = 7] vs. 29.2 ± 2.7 [n = 5], P = 0.014). Intralipid was as efficient as cyclosporine-A in inhibiting the mitochondrial permeability transition pore opening (calcium retention capacity = 280 ± 8.2 vs. 260.3 ± 2.9 nmol/mg mitochondria protein in cyclosporine-A, P = 0.454, n = 6) and in reducing cardiac mitochondrial superoxide production. Unlike intralipid, which increased phosphorylation of Akt (6-fold) and GSK-3ß (5-fold), cyclosporine-A had no effect on the activation of these prosurvival kinases. CONCLUSIONS: Although intralipid inhibits the opening of the mitochondrial permeability transition pore as efficiently as cyclosporine-A, intralipid is more effective in reducing the infarct size and improving the cardiac functional recovery.

Novel and robust treatment of pulmonary hypertension with netrin-1 and netrin-1-derived small peptides.

Limited medical therapies have been implemented for the treatment of the devastating cardiorespiratory disease of pulmonary hypertension (PH) while none of which is sufficiently effective to stop or regress development of PH. We have previously shown that netrin-1, an axon-guiding protein during development, protects against ischemia reperfusion injury induced myocardial infarction via modest and stable production of nitric oxide (NO) and attenuation of oxidative stress. Since NO deficiency and oxidative stress-mediated vascular remodeling play important roles in the pathogenesis of PH, our present study investigated therapeutic effects on PH of netrin-1 and its derived small peptides. Infused into mice for 3 weeks during exposure to hypoxia, netrin-1 and netrin-1 derived small peptides V1, V2 or V3 substantially alleviated pathophysiological and molecular features of PH, as indicated by abrogated increases in mean pulmonary artery pressure (mPAP) and right ventricular systolic pressure (RVSP), attenuated right ventricular hypertrophy, diminished vascular remodeling of medial thickening and upregulation in smooth muscle alpha-actin (SMA) and proliferative cell nuclear antigen (PCNA), and alleviated perivascular and peribronchial fibrosis reflected by collagen deposition. NO bioavailability was substantially improved by treatment with netrin-1 and netrin-1 derived small peptides, while hypoxia induced increases in total superoxide production and eNOS uncoupling activity were all attenuated. These dual mechanisms of increasing NO bioavailability and decreasing oxidative stress at the same time, underlie robust protective effects on PH of netrin-1 and its derived small peptides, which are different from existing medications that primarily target NO signaling alone. Our data for the first time demonstrate intriguing findings that netrin-1 and netrin-1 derived small peptides can be used as novel and robust therapeutics for the treatment of PH.

Combination of folic acid with nifedipine is completely effective in attenuating aortic aneurysm formation as a novel oral medication.

Aortic aneurysms are prevalent and severe vascular diseases with high mortality from unpredicted ruptures, while the only treatment option is surgical correction of large aneurysms with considerable risk. We have shown that folic acid (FA) is highly effective in alleviating development of aneurysms although not sufficient to completely attenuate aneurysm formation. Here, we examined therapeutic effects on aneurysms of combining FA with Nifedipine as novel and potentially more effective oral medication. Oral administration with FA (15 mg/kg/day) significantly reduced incidence of AAA from 85.71% to 18.75% in Ang II-infused apolipoprotein E (apoE) null mice, while combination of FA with Nifedipine (1.5, 5.0 or 20 mg/kg/day) substantially and completely further reduced incidence of AAA to 12.5%, 11.76% and 0.00% respectively in a dose-dependent manner. The combinatory therapy substantially and completely further alleviated enlargement of abdominal aortas defined by ultrasound, vascular remodeling characterized by elastin degradation and adventitial hypertrophy, as well as aortic superoxide production and eNOS uncoupling activity also in a dose-dependent manner, with combination of FA with 20 mg/kg/day Nifedipine attenuating all of these features by 100% to control levels. Aortic NO and H4B bioavailabilities were also dose-dependently further improved by combining FA with Nifedipine. These data establish entirely innovative and robust therapeutic regime of FA combined with Nifedipine for the treatment of aortic aneurysms. The comminatory therapy can serve as a first-in-class and most effective oral medication for aortic aneurysms, which can be rapidly translated into clinical practice to revolutionize management of the devastating vascular diseases of aortic aneurysms known as silent killers.

Robust therapeutic effects on COVID-19 of novel small molecules: Alleviation of SARS-CoV-2 S protein induction of ACE2/TMPRSS2, NOX2/ROS, and MCP-1.

While new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) constantly emerge to prolong the pandemic of COVID-19, robust and safe therapeutics are in urgent need. During the previous and ongoing fight against the pandemic in China, Traditional Chinese Medicine (TCM) has proven to be markedly effective in treating COVID-19. Among active ingredients of TCM recipes, small molecules such as quercetin, glabridin, gallic acid, and chrysoeriol have been predicted to target viral receptor angiotensin-converting enzyme 2 (ACE2) via system pharmacology/molecular docking/visualization analyses. Of note, endothelial dysfunction induced by oxidative stress and inflammation represents a critical mediator of acute respiratory distress syndrome (ARDS) and multi-organ injuries in patients with COVID-19. Hence, in the present study, we examined whether quercetin, glabridin, gallic acide and chrysoeriol regulate viral receptors of ACE2 and transmembrane serine protease 2 (TMPRSS2), redox modulator NADPH oxidase isoform 2 (NOX2), and inflammatory protein of monocyte chemoattractant protein-1 (MCP-1) in endothelial cells to mediate therapeutic protection against COVID-19. Indeed, quercetin, glabridin, gallic acide and chrysoeriol completely attenuated SARS-CoV-2 spike protein (S protein)-induced upregulation in ACE2 protein expression in endothelial cells. In addition, these small molecules abolished S protein upregulation of cleaved/active form of TMPRSS2, while native TMPRSS2 was not significantly regulated. Moreover, these small molecules completely abrogated S protein-induced upregulation in NOX2 protein expression, which resulted in alleviated superoxide production, confirming their preventive efficacies against S protein-induced oxidative stress in endothelial cells. In addition, treatment with these small molecules abolished S protein induction of MCP-1 expression. Collectively, our findings for the first time demonstrate that these novel small molecules may be used as novel and robust therapeutic options for the treatment of patients with COVID-19, via effective attenuation of S protein induction of endothelial oxidative stress and inflammation.

Flavored and Nicotine-Containing E-Cigarettes Induce Impaired Angiogenesis and Diabetic Wound Healing via Increased Endothelial Oxidative Stress and Reduced NO Bioavailability.

The prevalent use of electronic cigarettes (e-cigarettes) has increased exponentially in recent years, especially in youth who are attracted to flavored e-cigarettes. Indeed, e-cigarette or vaping product use-associated lung injury (EVALI) cases started to emerge in the United States in August 2019, resulting in 2807 hospitalized cases and 68 deaths as of 18 February 2020. In the present study, we investigated, for the first time, whether flavored and nicotine containing e-cigarettes induce endothelial dysfunction to result in impaired angiogenesis and wound healing particularly under diabetic condition. Nicotine containing e-cigarettes with various contents of nicotine (0, 1.2%, 2.4%), and flavored e-cigarettes of classic tobacco, mint, menthol, and vanilla or fruit from BLU (nicotine 2.4%) or JUUL (nicotine 3%), were used to treat endothelial cells in vitro and streptozotocin-induced diabetic mice in vivo. Endothelial cell superoxide production, determined by dihydroethidium (DHE) fluorescent imaging and electron spin resonance (ESR), was markedly increased by exposure to e-cigarette extract (e-CSE) in a nicotine-content dependent manner, while nitric oxide (NO) bioavailability detected by DAF-FM fluorescent imaging was substantially decreased. All of the different flavored e-cigarettes examined also showed significant effects in increasing superoxide production while diminishing NO bioavailability. Endothelial cell apoptosis evaluated by caspase 3 activity was markedly increased by exposure to e-CSE prepared from flavored and nicotine containing e-cigarettes. Endothelial monolayer wound assays revealed that nicotine-containing and flavored e-cigarettes induced impaired angiogenic wound repair of endothelial cell monolayers. Furthermore, vascular endothelial growth factor (VEGF) stimulated wound healing in diabetic mice was impaired by exposure to e-CSEs prepared from nicotine-containing and flavored e-cigarettes. Taken together, our data demonstrate for the first time that flavored and nicotine-containing e-cigarettes induce endothelial dysfunction through excessive ROS production, resulting in decreased NO bioavailability, increased endothelial cell apoptosis, and impairment in angiogenesis and wound healing, especially under diabetic condition. These responses of endothelial dysfunction likely underlie harmful effects of e-cigarettes in endothelial-rich organs, such as heart and lungs. These data also indicate that rigorous regulation on e-cigarette use should be enforced in diabetic and/or surgical patients to avoid severe consequences from impaired angiogenesis/wound healing.

An ezrin/calpain/PI3K/AMPK/eNOSs1179 signaling cascade mediating VEGF-dependent endothelial nitric oxide production.

Calpain was recently reported to mediate vascular endothelial growth factor (VEGF)-induced angiogenesis. In the present study, we investigated detailed molecular mechanisms. VEGF (100 ng/mL) induced a marked increase in endothelial cell production of NO(*), specifically detected by electron spin resonance. This response was abolished by inhibition of calpain with N-acetyl-leucyl-leucyl-norleucinal (ALLN) or Calpeptin. Both also diminished membrane-specific calpain activation by VEGF, which was intriguingly attenuated by silencing ezrin with RNA interference. A rapid membrane colocalization of calpain and ezrin occurred as short as 10 minutes after VEGF stimulation. AKT, AMP-dependent kinase (AMPK), and endothelial nitric oxide synthase (eNOS)(s1179) phosphorylations in VEGF-stimulated endothelial cells were markedly enhanced, which were however significantly attenuated by either ALLN, Calpeptin, or ezrin small interfering RNA, as well as by Wortmannin or compound C (respectively for phosphatidylinositol 3-kinase [PI3K] or AMPK). The latter 3 also abolished VEGF induction of NO(*). These data indicate that AMPK and AKT are both downstream of PI3K and that AKT activation is partially dependent on AMPK. The interrelationship between AMPK and AKT, although known to be individually important in mediating VEGF activation of eNOS, is clearly characterized. Furthermore, AMPK/AKT/eNOS(s1179) was found downstream of a calpain/ezrin membrane interaction. These data no doubt provide new insights into the long mystified signaling gap between VEGF receptors and PI3K/AKT or AMPK-dependent eNOS activation. In view of the well-established significance of VEGF-dependent angiogenesis, these findings might have broad and important implications in cardiovascular pathophysiology.

Toll-Like Receptor 2 (TLR2) Knockout Abrogates Diabetic and Obese Phenotypes While Restoring Endothelial Function via Inhibition of NOX1.

We have previously demonstrated a novel role of bone morphogenic protein 4 (BMP4) in inducing NOX1-dependent endothelial nitric oxide synthase (eNOS) uncoupling, endothelial dysfunction, and inflammatory activation in type 2 diabetes mellitus (T2DM). However, how BMP4 activates NOX1 and whether targeting the new mechanistic pathway revealed is effective in preserving endothelial function in T2DM remains unclear. In this study, we observed that BMP4 induced a marked, time-dependent increase in physiological binding between TLR2 and NOX1 in aortic endothelial cells as well as increased binding of TLR2 to NOXO1. In TLR2 knockout (Tlr2 -/-) mice fed high-fat diet, body weight gain was significantly less compared with wild-type (WT) mice both in males and females. The high-fat diet-induced increases in fasting blood glucose levels, as well as in circulating insulin and leptin levels, were absent in Tlr2 -/- mice. High-fat feeding induced increases in overall fat mass, and in fat mass of different pockets were abrogated in Tlr2 -/- mice. Whereas energy intake was similar in high-fat-fed WT and Tlr2 -/- mice, TLR2 deficiency resulted in higher energy expenditure attributable to improved physical activity, which was accompanied by restored skeletal muscle mitochondrial function. In addition, TLR2 deficiency recoupled eNOS, reduced total superoxide production, improved H4B and NO bioavailabilities in aortas, and restored endothelium-dependent vasorelaxation. Collectively, our data strongly indicate that TLR2 plays important roles in the development of metabolic features of T2DM and its related endothelial/vascular dysfunction. Therefore, targeting TLR2 may represent a novel therapeutic strategy for T2DM, obesity, and cardiovascular complications via specific inhibition of NOX1.

Therapeutic application of estrogen for COVID-19: Attenuation of SARS-CoV-2 spike protein and IL-6 stimulated, ACE2-dependent NOX2 activation, ROS production and MCP-1 upregulation in endothelial cells.

The outbreak of COVID-19 has remained uncontained with urgent need for robust therapeutics. We have previously reported sex difference of COVID-19 for the first time indicating male predisposition. Males are more susceptible than females, and more often to develop into severe cases with higher mortality. This predisposition is potentially linked to higher prevalence of cigarette smoking. Nonetheless, we found for the first time that cigarette smoking extract (CSE) had no effect on angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) expression in endothelial cells. The otherwise observed worse outcomes in smokers is likely linked to baseline respiratory diseases associated with chronic smoking. Instead, we hypothesized that estrogen mediated protection might underlie lower morbidity, severity and mortality of COVID-19 in females. Of note, endothelial inflammation and barrier dysfunction are major mediators of disease progression, and development of acute respiratory distress syndrome (ARDS) and multi-organ failure in patients with COVID-19. Therefore, we investigated potential protective effects of estrogen on endothelial cells against oxidative stress induced by interleukin-6 (IL-6) and SARS-CoV-2 spike protein (S protein). Indeed, 17ß-estradiol completely reversed S protein-induced selective activation of NADPH oxidase isoform 2 (NOX2) and reactive oxygen species (ROS) production that are ACE2-dependent, as well as ACE2 upregulation and induction of pro-inflammatory gene monocyte chemoattractant protein-1 (MCP-1) in endothelial cells to effectively attenuate endothelial dysfunction. Effects of IL-6 on activating NOX2-dependent ROS production and upregulation of MCP-1 were also completely attenuated by 17ß-estradiol. Of note, co-treatment with CSE had no additional effects on S protein stimulated endothelial oxidative stress, confirming that current smoking status is likely unrelated to more severe disease in chronic smokers. These data indicate that estrogen can serve as a novel therapy for patients with COVID-19 via inhibition of initial viral responses and attenuation of cytokine storm induced endothelial dysfunction, to substantially alleviate morbidity, severity and mortality of the disease, especially in men and post-menopause women. Short-term administration of estrogen can therefore be readily applied to the clinical management of COVID-19 as a robust therapeutic option.