Nanocurcumin Reduces High Glucose and Particulate Matter-Induced Endothelial Inflammation: Mitochondrial Function and Involvement of miR-221/222.

Purpose: Particulate matter (PM) 2.5, harmful air pollutants, and diabetes are associated with high morbidity and mortality from cardiovascular disease (CVD). However, the molecular mechanisms underlying the combined effects of PM and diabetes on CVD remain unclear. Methods: Endothelial cells (ECs) treated with high glucose (HG) and PM mimic hyperglycemia and air pollutant exposure in CVD. Endothelial inflammation was evaluated by Western blot and immunofluorescence of ICAM-1 expression and monocyte adhesion. The mechanisms underlying endothelial inflammation were elucidated through MitoSOX Red analysis, JC-1 staining, MitoTracker analysis, and Western blot analysis of mitochondrial fission-related, autophagy-related, and mitophagy-related proteins. Furthermore. nanocurcumin (NCur) pretreatment was used to test if it has a protective effect. Results: ECs under co-exposure to HG and PM increased ICAM-1 expression and monocyte adhesion, whereas NCur pretreatment attenuated these changes and improved endothelial inflammation. PM exposure increased mitochondrial ROS levels, worsened mitochondrial membrane potential, promoted mitochondrial fission, induced mitophagy, and aggravated inflammation in HG-treated ECs, while NCur reversed these changes. Also, HG and PM-induced endothelial inflammation is through the JNK signaling pathway and miR-221/222 specifically targeting ICAM-1 and BNIP3. PM exposure also aggravated mitochondrial ROS levels, mitochondrial fission, mitophagy, and endothelial inflammation in STZ-induced hyperglycemic mice, whereas NCur attenuated these changes. Conclusion: This study elucidated the mechanisms underlying HG and PM-induced endothelial inflammation in vitro and in vivo. HG and PM treatment increased mitochondrial ROS, mitochondrial fission, and mitophagy in ECs, whereas NCur reversed these conditions. In addition, miR-221/222 plays a role in the amelioration of endothelial inflammation through targeting Bnip3 and ICAM-1, and NCur pretreatment can modulate miR-221/222 levels. Therefore, NCur may be a promising approach to intervene in diabetes and air pollution-induced CVD.

Combined exposure to fine particulate matter and high glucose aggravates endothelial damage by increasing inflammation and mitophagy: the involvement of vitamin D.

BACKGROUND: Cardiovascular diseases (CVDs) are related to particulate matter (PM2.5) exposure. Researchers have not clearly determined whether hyperglycemia, a hallmark of diabetes, exacerbates PM2.5-induced endothelial damage. Thus, this study aimed to investigate the combined effects of PM2.5 and high glucose on endothelial damage. RESULTS: Here, we treated human umbilical vein endothelial cells (HUVECs) with 30 mM high glucose and 50 µg/mL PM (HG + PM) to simulate endothelial cells exposed to hyperglycemia and air pollution. First, we showed that HUVECs exposed to PM under high glucose conditions exhibited significant increases in cell damage and apoptosis compared with HUVECs exposed to PM or HG alone. In addition, PM significantly increased the production of reactive oxygen species (ROS) in HUVECs and mitochondria treated with HG and decreased the expression of superoxide dismutase 1 (SOD1), a free radical scavenging enzyme. The coexposure group exhibited significantly increased ROS production in cells and mitochondria, a lower mitochondrial membrane potential, and increased levels of the autophagy-related proteins p62, microtubule-associated protein 1 light chain 3ß (LC3B), and mitophagy-related protein BCL2 interacting protein 3 (Bnip3). Moreover, autophagosome-like structures were observed in the HG + PM group using transmission electron microscopy. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were also increased through the JNK/p38 signaling pathway in the HG + PM group. As a ROS scavenger, vitamin D treatment effectively protected cells under HG and PM conditions by increasing cell viability, reducing mitochondrial ROS production, and suppressing the formation of mitophagy and inflammation. Furthermore, diabetes was induced in mice by administering streptozotocin (STZ). Mice were treated with PM by intratracheal injection. Vitamin D effectively alleviated oxidative stress, mitophagy, and inflammation in the aortas of mice treated with STZ and PM. CONCLUSION: Taken together, simultaneous exposure to PM and high glucose exerts significant harmful effects on endothelial cells by inducing ROS production, mitophagy, and inflammation, while vitamin D reverses these effects.

Vitamin D3 decreases TNF-α-induced inflammation in lung epithelial cells through a reduction in mitochondrial fission and mitophagy.

Previous work has shown an association between vitamin D3 deficiency and an increased risk for acquiring various inflammatory diseases. Vitamin D3 can reduce morbidity and mortality in these patients via different mechanisms. Lung inflammation is an important event in the initiation and development of respiratory disorders. However, the anti-inflammatory effects of vitamin D3 and the underlying mechanisms remained to be determined. The purpose of this study was to examine the effects and mechanisms of action of vitamin D3 (Vit. D) on the expression of intercellular adhesion molecule-1 (ICAM-1) in vitro and in vivo with or without tumor necrosis factor α (TNF-α) treatment. Pretreatment with Vit. D reduced the expression of ICAM-1 and leukocyte adhesion in TNF-α-treated A549 cells. TNF-α increased the accumulation of mitochondrial reactive oxygen species (mtROS), while Vit. D reduced this effect. Pretreatment with Vit. D attenuated TNF-α-induced mitochondrial fission, as shown by the increased expression of mitochondrial fission factor (Mff), phosphorylated dynamin-related protein 1 (p-DRP1), and mitophagy-related proteins (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3, Bnip3) in A549 cells. Inhibition of DRP1 or Mff significantly decreased ICAM-1 expression. In addition, we found that Vit. D decreased TNF-α-induced ICAM-1 expression, mitochondrial fission, and mitophagy via the AKT and NF-κB pathways. Moreover, ICAM-1 expression, mitochondrial fission, and mitophagy were increased in the lung tissues of TNF-α-treated mice, while Vit. D supplementation reduced these effects. In this study, we elucidated the mechanisms by which Vit. D reduces the expression of adhesion molecules in models of airway inflammation. Vit. D might be served as a novel therapeutic agent for the targeting of epithelial activation in lung inflammation. Graphical Headlights: • The expression of DRP1 and Mff, mitochondrial fission-related proteins, was increased in TNF-α-treated A549 cells. • The expression of Bnip3 and LC3B, mitophagy-related proteins, was increased in TNF-α-treated A549 cells. • Vit. D pretreatment decreased TNF-α-induced inflammation through the reduction of mitochondrial fission and mitophagy in A549 cells.

Conditioned medium from adipose-derived stem cells attenuates ischemia/reperfusion-induced cardiac injury through the microRNA-221/222/PUMA/ETS-1 pathway.

Rationale: Cardiovascular diseases, such as myocardial infarction (MI), are the leading causes of death worldwide. Reperfusion therapy is the common standard treatment for MI. However, myocardial ischemia/reperfusion (I/R) causes cardiomyocyte injury, including apoptosis and fibrosis. We aimed to investigate the effects of conditioned medium from adipose-derived stem cells (ADSC-CM) on apoptosis and fibrosis in I/R-treated hearts and hypoxia/reoxygenation (H/R)-treated cardiomyocytes and the underlying mechanisms. Methods: ADSC-CM was collected from ADSCs. The effects of intramuscular injection of ADSC-CM on cardiac function, cardiac apoptosis, and fibrosis examined by echocardiography, Evans blue/TTC staining, TUNEL assay, and Masson's trichrome staining in I/R-treated mice. We also examined the effects of ADSC-CM on apoptosis and fibrosis in H/R-treated H9c2 cells by annexin V/PI flow cytometry, TUNEL assay, and immunocytochemistry. Results: ADSC-CM treatment significantly reduced heart damage and fibrosis of I/R-treated mice and H/R-treated cardiomyocytes. In addition, the expression of apoptosis-related proteins, such as p53 upregulated modulator of apoptosis (PUMA), p-p53 and B-cell lymphoma 2 (BCL2), as well as the fibrosis-related proteins ETS-1, fibronectin and collagen 3, were significantly reduced by ADSC-CM treatment. Moreover, we demonstrated that ADSC-CM contains a large amount of miR-221/222, which can target and regulate PUMA or ETS-1 protein levels. Furthermore, the knockdown of PUMA and ETS-1 decreased the induction of apoptosis and fibrosis, respectively. MiR-221/222 overexpression achieved similar results. We also observed that cardiac I/R markedly increased apoptosis and fibrosis in miR-221/222 knockout (KO) mice, while ADSC-CM decreased these effects. The increased phosphorylation of p38 and NF-κB not only mediated myocardial apoptosis through the PUMA/p53/BCL2 pathway but also regulated fibrosis through the ETS-1/fibronectin/collagen 3 pathway. Conclusions: Overall, our results show that ADSC-CM attenuates cardiac apoptosis and fibrosis by reducing PUMA and ETS-1 expression, respectively. The protective effect is mediated via the miR-221/222/p38/NF-κB pathway.

MicroRNA-221/222 Mediates ADSC-Exosome-Induced Cardioprotection Against Ischemia/Reperfusion by Targeting PUMA and ETS-1.

Cardiovascular disease is a major health problem in industrialized and developing countries and is the leading cause of death and disability. Myocardial ischemia/reperfusion (I/R) causes cardiomyocyte damage such as apoptosis and hypertrophy. The purpose of this study was to investigate the effects of exosomes from adipose-derived stem cells (ADSC-Exo) on hearts from I/R mice and to explore the underlying mechanisms. ADSC-Exo significantly decreased I/R-induced cardiomyocyte apoptosis and hypertrophy, as detected by TdT-mediated dUTP nick end-labeling (TUNEL) and wheat germ agglutinin (WGA) staining, respectively. In addition, the expression of apoptosis-related proteins p-p53 and PUMA and hypertrophy-related proteins ETS-1 and ANP were significantly reduced in the cardiomyocytes of ADSC-Exo-treated I/R mice compared to those of control mice. Both PUMA and ETS-1 are reported to be target genes for miR-221/222. I/R operation significantly reduced miR-221/222 expression, while ADSC-Exo treatment increased miR-221/222 expression, as detected by RT-qPCR. We also observed that cardiac I/R operation markedly increased cell apoptosis and hypertrophy in miR-221/222 knockout (KO) mice, while ADSC-Exo reduced the effects of I/R operation. Furthermore, ADSC-Exo protected H9c2 cardiomyocytes from H2O2-induced damage by reducing apoptosis and hypertrophy in vitro. H2O2 treatment significantly reduced miR-221/222 expression, while ADSC-Exo treatment reversed this effect in H9c2 cells. ADSC-Exo treatment decreased H2O2-induced PUMA and ETS-1 expression. Compared with control treatment, I/R treatment significantly reduced p-AKT and increased p-p65, while ADSC-Exo and miR-221/222 mimics attenuated these effects. The AKT activator SC79 and p65 inhibitor Bay 11-7082 reduced H2O2-induced cell apoptosis and hypertrophy. Based on these findings, ADSC-Exo prevents cardiac I/R injury through the miR-221/miR-222/PUMA/ETS-1 pathway. Therefore, ADSC-Exo is an effective inhibitor of I/R-induced heart injury.

Vitamin D Attenuates Ischemia/Reperfusion-Induced Cardiac Injury by Reducing Mitochondrial Fission and Mitophagy.

Myocardial infarction is the leading cause of morbidity and mortality worldwide. Although myocardial reperfusion after ischemia (I/R) is an effective method to save ischemic myocardium, it can cause adverse reactions, including increased oxidative stress and cardiomyocyte apoptosis. Mitochondrial fission and mitophagy are essential factors for mitochondrial quality control, but whether they play key roles in cardiac I/R injury remains unknown. New pharmacological or molecular interventions to alleviate reperfusion injury are currently considered desirable therapies. Vitamin D3 (Vit D3) regulates cardiovascular function, but its physiological role in I/R-exposed hearts, especially its effects on mitochondrial homeostasis, remains unclear. An in vitro hypoxia/reoxygenation (H/R) model was established in H9c2 cells to simulate myocardial I/R injury. H/R treatment significantly reduced H9c2 cell viability, increased apoptosis, and activated caspase 3. In addition, H/R treatment increased mitochondrial fission, as manifested by increased expression of phosphorylated dynein-related protein 1 (p-Drp1) and mitochondrial fission factor (Mff) as well as increased mitochondrial translocation of Drp1. Treatment with the mitochondrial reactive oxygen species scavenger MitoTEMPO increased cell viability and decreased mitochondrial fission. H/R conditions elicited excessive mitophagy, as indicated by increased expression of BCL2-interacting protein 3 (BNIP3) and light chain (LC3BII/I) and increased formation of autolysosomes. In contrast, Vit D3 reversed these effects. In a mouse model of I/R, apoptosis, mitochondrial fission, and mitophagy were induced. Vit D3 treatment mitigated apoptosis, mitochondrial fission, mitophagy, and myocardial ultrastructural abnormalities. The results indicate that Vit D3 exerts cardioprotective effects against I/R cardiac injury by protecting mitochondrial structural and functional integrity and reducing mitophagy.

Particulate matters increase epithelial-mesenchymal transition and lung fibrosis through the ETS-1/NF-κB-dependent pathway in lung epithelial cells.

BACKGROUND: Particulate matters (PMs) in ambient air pollution are closely related to the incidence of respiratory diseases and decreased lung function. Our previous report demonstrated that PMs-induced oxidative stress increased the expression of proinflammatory intracellular adhesion molecule-1 (ICAM-1) through the IL-6/AKT/STAT3/NF-κB pathway in A549 cells. However, the role of O-PMs in epithelial-mesenchymal transition (EMT) development and pulmonary fibrosis and the related mechanisms have not been determined. The aim of this study was to investigate the effects of O-PMs on the pathogenesis of EMT and pulmonary fibrosis as well as the expression of ETS-1 and NF-κB p65, in vitro and in vivo. RESULTS: O-PMs treatment induced EMT development, fibronectin expression, and cell migration. O-PMs affected the expression of the EMT-related transcription factors NF-κB p65 and ETS-1. Interference with NF-κB p65 significantly decreased O-PMs-induced fibronectin expression. In addition, O-PMs affected the expression of fibronectin, E-cadherin, and vimentin through modulating ETS-1 expression. ATN-161, an antagonist of integrin α5ß1, decreased the expression of fibronectin and ETS-1 and EMT development. EMT development and the expression of fibronectin and ETS-1 were increased in the lung tissue of mice after exposure to PMs for 7 and 14 days. There was a significant correlation between fibronectin and ETS-1 expression in human pulmonary fibrosis tissue. CONCLUSION: O-PMs can induce EMT and fibronectin expression through the activation of transcription factors ETS-1 and NF-κB in A549 cells. PMs can induce EMT development and the expression of fibronectin and ETS-1 in mouse lung tissues. These findings suggest that the ETS-1 pathway could be a novel and alternative mechanism for EMT development and pulmonary fibrosis.

Interleukin-6 from Adipose-Derived Stem Cells Promotes Tissue Repair by the Increase of Cell Proliferation and Hair Follicles in Ischemia/Reperfusion-Treated Skin Flaps.

The most common postoperative complication after reconstructive surgery is flap necrosis. Adipose-derived stem cells (ADSCs) and their secretomes are reported to mediate skin repair. This study was designed to investigate whether conditioned media from ADSCs (ADSC-CM) protects ischemia/reperfusion- (I/R-) induced injury in skin flaps by promoting cell proliferation and increasing the number of hair follicles. The mouse flap model of ischemia was ligating the long thoracic vessels for 3 h, followed by blood reperfusion. ADSC-CM was administered to the flaps, and their survival was observed on postoperative day 5. ADSC-CM treatment led to a significant increase in cell proliferation and the number of hair follicles. IL-6 levels in the lysate and CM from ADSCs were significantly higher than those from Hs68 fibroblasts. Furthermore, a strong decrease in cell proliferation and the number of hair follicles was observed after treatment with IL-6-neutralizing antibodies or si-IL-6-ADSC. In addition, ADSC transplantation increased flap repair, cell proliferation, and hair follicle number in I/R injury of IL-6-knockout mice. In conclusion, IL-6 secreted from ADSCs promotes the survival of I/R-induced flaps by increasing cell proliferation and the number of hair follicles. ADSCs represent a promising therapy for preventing skin flap necrosis following reconstructive and plastic surgery.

PM2.5-induced oxidative stress increases intercellular adhesion molecule-1 expression in lung epithelial cells through the IL-6/AKT/STAT3/NF-κB-dependent pathway.

BACKGROUND: Epidemiological studies have shown that ambient air pollution is closely associated with increased respiratory inflammation and decreased lung function. Particulate matters (PMs) are major components of air pollution that damages lung cells. However, the mechanisms remain to be elucidated. This study examines the effects of PMs on intercellular adhesion molecule-1 (ICAM-1) expression and the related mechanisms in vitro and in vivo. RESULT: The cytotoxicity, reactive oxygen species (ROS) generation, and monocyte adherence to A549 cells were more severely affected by treatment with O-PMs (organic solvent-extractable fraction of SRM1649b) than with W-PMs (water-soluble fraction of SRM1649b). We observed a significant increase in ICAM-1 expression by O-PMs, but not W-PMs. O-PMs also induced the phosphorylation of AKT, p65, and STAT3. Pretreating A549 cells with N-acetyl cysteine (NAC), an antioxidant, attenuated O-PMs-induced ROS generation, the phosphorylation of the mentioned kinases, and the expression of ICAM-1. Furthermore, an AKT inhibitor (LY294002), NF-κB inhibitor (BAY11-7082), and STAT3 inhibitor (Stattic) significantly down-regulated O-PMs-induced ICAM-1 expression as well as the adhesion of U937 cells to epithelial cells. Interleukin-6 (IL-6) was the most significantly changed cytokine in O-PMs-treated A549 cells according to the analysis of the cytokine antibody array. The IL-6 receptor inhibitor tocilizumab (TCZ) and small interfering RNA for IL-6 significantly reduced ICAM-1 secretion and expression as well as the reduction of the AKT, p65, and STAT3 phosphorylation in O-PMs-treated A549 cells. In addition, the intratracheal instillation of PMs significantly increased the levels of the ICAM-1 and IL-6 in lung tissues and plasma in WT mice, but not in IL-6 knockout mice. Pre-administration of NAC attenuated those PMs-induced adverse effects in WT mice. Furthermore, patients with chronic obstructive pulmonary disease (COPD) had higher plasma levels of ICAM-1 and IL-6 compared to healthy subjects. CONCLUSION: These results suggest that PMs increase ICAM-1 expression in pulmonary epithelial cells in vitro and in vivo through the IL-6/AKT/STAT3/NF-κB signaling pathway.