Nadan Wang

MicroRNA-663 Regulates Human Vascular Smooth Muscle Cell Phenotypic Switch and Vascular Neointimal Formation

Rationale: Abnormal phenotypic switch of vascular smooth muscle cell (VSMC) is a hallmark of vascular disorders such as atherosclerosis and restenosis after angioplasty. MicroRNAs (miRNAs) have emerged as important regulators for VSMC function, and we recently identified miR-663 as critical for controlling human aortic smooth muscle cell proliferation. Objective: To investigate whether miR-663 plays a role in human VSMC phenotypic switch and the development of neointima formation. Methods and Results: By using quantitative reverse-transcription polymerase chain reaction, we found that miR-663 was significantly downregulated in human aortic VSMCs on platelet-derived growth factor treatment, whereas expression was markedly increased during VSMC differentiation. Furthermore, we demonstrated that overexpression of miR-663 increased expression of VSMC differentiation marker genes, such as smooth muscle 22&agr;, smooth muscle &agr;-actin, calponin, and smooth muscle myosin heavy chain, and potently inhibited platelet-derived growth factor–induced VSMC proliferation and migration. We identified the transcription factor JunB and myosin light chain 9 as downstream targets of miR-663 in human VSMCs, because overexpression of miR-663 markedly inhibited expression of JunB and its downstream molecules, such as myosin light chain 9 and matrix metalloproteinase 9. Finally, we showed that adeno-miR-663 markedly suppressed the neointimal lesion formation by ≈50% in mice after vascular injury induced by carotid artery ligation, specifically via decreased JunB expression. Conclusions: These results indicate that miR-663 is a novel modulator of human VSMC phenotypic switch by targeting JunB/myosin light chain 9 expression. These findings suggest that targeting miR-663 or its specific downstream targets in human VSMCs may represent an attractive approach for the treatment of proliferative vascular diseases.

Obesity-induced adipokine imbalance impairs mouse pulmonary vascular endothelial function and primes the lung for injury

Obesity is a risk factor for the development of acute respiratory distress syndrome (ARDS) but mechanisms mediating this association are unknown. While obesity is known to impair systemic blood vessel function, and predisposes to systemic vascular diseases, its effects on the pulmonary circulation are largely unknown. We hypothesized that the chronic low grade inflammation of obesity impairs pulmonary vascular homeostasis and primes the lung for acute injury. The lung endothelium from obese mice expressed higher levels of leukocyte adhesion markers and lower levels of cell-cell junctional proteins when compared to lean mice. We tested whether systemic factors are responsible for these alterations in the pulmonary endothelium; treatment of primary lung endothelial cells with obese serum enhanced the expression of adhesion proteins and reduced the expression of endothelial junctional proteins when compared to lean serum. Alterations in pulmonary endothelial cells observed in obese mice were associated with enhanced susceptibility to LPS-induced lung injury. Restoring serum adiponectin levels reversed the effects of obesity on the lung endothelium and attenuated susceptibility to acute injury. Our work indicates that obesity impairs pulmonary vascular homeostasis and enhances susceptibility to acute injury and provides mechanistic insight into the increased prevalence of ARDS in obese humans.

Tissue-Specific Mitochondrial Decoding of Cytoplasmic Ca2+ Signals is Controlled by the Stoichiometry of MICU1/2 and MCU

SUMMARY Mitochondrial Ca2+ uptake through the Ca2+ uniporter supports cell functions, including oxidative metabolism, while meeting tissue-specific calcium signaling patterns and energy needs. The molecular mechanisms underlying tissue-specific control of the uniporter are unknown. Here, we investigated a possible role for tissue-specific stoichiometry between the Ca2+-sensing regulators (MICUs) and pore unit (MCU) of the uniporter. Low MICU1:MCU protein ratio lowered the [Ca2+] threshold for Ca2+ uptake and activation of oxidative metabolism but decreased the cooperativity of uniporter activation in heart and skeletal muscle compared to liver. In MICU1-overexpressing cells, MICU1 was pulled down by MCU proportionally to MICU1 overexpression, suggesting that MICU1:MCU protein ratio directly reflected their association. Overexpressing MICU1 in the heart increased MICU1:MCU ratio, leading to liver-like mitochondrial Ca2+ uptake phenotype and cardiac contractile dysfunction. Thus, the proportion of MICU1-free and MICU1-associated MCU controls these tissue-specific uniporter phenotypes and downstream Ca2+ tuning of oxidative metabolism.

Orphan Nuclear Receptor Nur77 Inhibits Cardiac Hypertrophic Response to Beta-Adrenergic Stimulation.

ABSTRACT The orphan nuclear receptor Nur77 plays critical roles in cardiovascular diseases, and its expression is markedly induced in the heart after beta-adrenergic receptor (β-AR) activation. However, the functional significance of Nur77 in β-AR signaling in the heart remains unclear. By using Northern blot, Western blot, and immunofluorescent staining assays, we showed that Nur77 expression was markedly upregulated in cardiomyocytes in response to multiple hypertrophic stimuli, including isoproterenol (ISO), phenylephrine (PE), and endothelin-1 (ET-1). In a time- and dose-dependent manner, ISO increases Nur77 expression in the nuclei of cardiomyocytes. Overexpression of Nur77 markedly inhibited ISO-induced cardiac hypertrophy by inducing nuclear translocation of Nur77 in cardiomyocytes. Furthermore, cardiac overexpression of Nur77 by intramyocardial injection of Ad-Nur77 substantially inhibited cardiac hypertrophy and ameliorated cardiac dysfunction after chronic infusion of ISO in mice. Mechanistically, we demonstrated that Nur77 functionally interacts with NFATc3 and GATA4 and inhibits their transcriptional activities, which are critical for the development of cardiac hypertrophy. These results demonstrate for the first time that Nur77 is a novel negative regulator for the β-AR-induced cardiac hypertrophy through inhibiting the NFATc3 and GATA4 transcriptional pathways. Targeting Nur77 may represent a potentially novel therapeutic strategy for preventing cardiac hypertrophy and heart failure.

Induction of Nur77 by hyperoside inhibits vascular smooth muscle cell proliferation and neointimal formation.

Nur77 is an orphan nuclear receptor that belongs to the nuclear receptor 4A (NR4A) subfamily, which has been implicated in a variety of biological events, such as cell apoptosis, proliferation, inflammation, and metabolism. Activation of Nur77 has recently been shown to be beneficial for the treatment of cardiovascular and metabolic diseases. The purpose of this study is to identify novel natural Nur77 activators and investigate their roles in preventing vascular diseases. By measuring Nur77 expression using quantitative RT-PCR, we screened active ingredients extracted from Chinese herb medicines with beneficial cardiovascular effects. Hyperoside (quercetin 3-D-galactoside) was identified as one of the potent activators for inducing Nur77 expression and activating its transcriptional activity in vascular smooth muscle cells (VSMCs). We demonstrated that hyperoside, in a time and dose dependent manner, markedly increased the expression of Nur77 in rat VSMCs, with an EC50 of ∼0.83 μM. Mechanistically, we found that hyperoside significantly increased the phosphorylation of ERK1/2 MAP kinase and its downstream target cAMP response element-binding protein (CREB), both of which contributed to the hyperoside-induced Nur77 expression in rat VSMCs. Moreover, through activation of Nur77 receptor, hyperoside markedly inhibited both vascular smooth muscle cell proliferation in vitro and the carotid artery ligation-induced neointimal formation in vivo. These findings demonstrate that hyperoside is a potent natural activator of Nur77 receptor, which can be potentially used for prevention and treatment of occlusive vascular diseases.

Bitter Taste Receptor Agonists Mitigate Features of Allergic Asthma in Mice

Asthma is characterized by airway inflammation, mucus secretion, remodeling and hyperresponsiveness (AHR). Recent research has established the bronchodilatory effect of bitter taste receptor (TAS2R) agonists in various models. Comprehensive pre-clinical studies aimed at establishing effectiveness of TAS2R agonists in disease models are lacking. Here we aimed to determine the effect of TAS2R agonists on features of asthma. Further, we elucidated a mechanism by which TAS2R agonists mitigate features of asthma. Asthma was induced in mice using intranasal house dust mite or aerosol ova-albumin challenge, and chloroquine or quinine were tested in both prophylactic and treatment models. Allergen challenge resulted in airway inflammation as evidenced by increased immune cells infiltration and release of cytokines and chemokines in the lungs, which were significantly attenuated in TAS2R agonists treated mice. TAS2R agonists attenuated features of airway remodeling including smooth muscle mass, extracellular matrix deposition and pro-fibrotic signaling, and also prevented mucus accumulation and development of AHR in mice. Mechanistic studies using human neutrophils demonstrated that inhibition of immune cell chemotaxis is a key mechanism by which TAS2R agonists blocked allergic airway inflammation and exerted anti-asthma effects. Our comprehensive studies establish the effectiveness of TAS2R agonists in mitigating multiple features of allergic asthma.

Downregulation of adenine nucleotide translocator 1 exacerbates tumor necrosis factor-α-mediated cardiac inflammatory responses.

Inflammation contributes significantly to cardiac dysfunction. Although the initial phase of inflammation is essential for repair and healing, excessive proinflammatory cytokines are detrimental to the heart. We found that adenine nucleotide translocator isoform-1 (ANT1) protein levels were significantly decreased in the inflamed heart of C57BL/6 mice following cecal ligation and puncture. To understand the molecular mechanisms involved, we performed small-interfering RNA-mediated knockdown of ANT1 and studied tumor necrosis factor-α (TNFα)-induced inflammatory responses in myocardium-derived H9c2 cells and cardiomyocytes. ANT1 knockdown significantly increased swollen mitochondria and mitochondrial reactive oxygen species, concomitant with increased TNFα-induced NF-κB reporter gene activity and interleukin-6 and TNFα expression. A mitochondrial-targeted antioxidant mito-TEMPO attenuated TNFα-induced mitochondrial reactive oxygen species, NF-κB reporter gene activity, and cytokine expression in ANT1 knockdown cells. Interestingly, TNFα or lipopolysaccharide (LPS) treatment significantly decreased ANT1 protein levels, suggesting a feed-forward regulation of proinflammatory cytokine expression activated by ANT1 downregulation. These data suggest that ANT1 downregulation contributes to cardiac inflammation post-cecal ligation and puncture. Preventing ANT1 downregulation could provide a novel molecular target to temper cardiac inflammation.

PD-1 Modulates Radiation-Induced Cardiac Toxicity through Cytotoxic T Lymphocytes

Introduction: Combined immune checkpoint blockade has led to rare autoimmune complications, such as fatal myocarditis. Recent approvals of several anti–programmed death 1 (anti–PD‐1) drugs for lung cancer treatment prompted ongoing clinical trials that directly combine PD‐1 inhibitors with thoracic radiotherapy for locally advanced lung cancer. Overlapping toxicities from either modality have the potential to increase the risk for radiation‐induced cardiotoxicity (RICT), which is well documented among patients with Hodgkins disease and breast cancer. Methods: To investigate cardiotoxicity without the compounding pulmonary toxicity from thoracic radiotherapy, we developed a technique to deliver cardiac irradiation (CIR) in a mouse model concurrently with PD‐1 blockade to determine the presence of cardiac toxicity by using physiological testing and mortality as end points along with histological analysis. Results: We observed an acute mortality of 30% within 2 weeks after CIR plus anti–PD‐1 antibody compared with 0% from CIR plus immunoglobulin G (p = 0.023). Physiological testing demonstrated a reduced left ventricular ejection fraction (p < 0.01) by echocardiogram. Tissue analyses revealed increased immune cell infiltrates within cardiac tissue. Depletion of CD8‐positive lymphocytes with anti‐CD8 antibody reversed the acute mortality, suggesting that the toxicity is CD8‐positive cell–mediated. To validate these findings using a clinically relevant fractionated radiotherapy regimen, we repeated the study by delivering five daily fractions of 6 Gy. Similar mortality, cardiac dysfunction, and histological changes were observed in mice receiving fractionated radiotherapy with concurrent anti–PD‐1 therapy. Conclusions: This study provides strong preclinical evidence that radiation‐induced cardiotoxicity is modulated by the PD‐1 axis and that PD‐1 blockade should be administered with careful radiotherapy planning with an effort of reducing cardiac dose.

Pharmacological Inhibition of PD-1 Exacerbates Radiation-Induced Cardiac Toxicity Through Cytotoxic T Cell–Mediated Myocarditis

Pharmacological Inhibition of PD-1 Exacerbates Radiation-Induced Cardiac Toxicity Through Cytotoxic T CelleMediated Myocarditis S. Du, L. Zhou, G. Shukla, N. Wang, L. Yang, X. Ma, Y. Wang, A.P. Dicker, Y. Lu, and B. Lu; Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, PA, Department of Thoracic Oncology, Cancer Center, West China Hospital, Sichuan University, Chengdu, China, Sidney Kimmel Medical College at Thomas Jefferson University, Sidney Kimmel Cancer Center, Philadelphia, PA, Center for Translational Medicine, Department of Medicine, Thomas Jefferson University, Philadelphia, PA, Shanghai Cancer Center, Shanghai, China, Emergency Medicine, Thomas Jefferson University, Philadelphia, PA, Department of Radiation Oncology, Thomas Jefferson University, Philadelphia, PA, Department of Thoracic Oncology, Cancer Center and State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, China