Effects of ghrelin on homocysteine-induced dysfunction and inflammatory response in rat cardiac microvascular endothelial cells

Ghrelin is a well-characterized hormone that has protective effects on endothelial cells. Elevated HCY (homocysteine) can be a cardiovascular risk factor, but it is not known whether ghrelin can inhibit HCY-induced dysfunction and inflammatory response in rat CMECs (cardiac microvascular endothelial cells). We found that HCY treatment for 24 h inhibited proliferation and NO (nitric oxide) secretion, but with increased cell apoptosis and secretion of cytokines in CMECs. In contrast, ghrelin pretreatment significantly improved proliferation and NO secretion, and inhibited cell apoptosis and secretion of cytokines in HCY-induced CMECs. In addition, Western blot assay showed that NF-κB (nuclear factor κB) and cleaved-caspase 3 expression were elevated, and PCNA (proliferating cell nuclear antigen) and eNOS (endothelial nitric oxide synthase) expression were decreased after treatment with HCY, which was significantly reversed by pretreatment with ghrelin. The data suggest that ghrelin inhibits HCY-induced CMEC dysfunction and inflammatory response, probably mediated by inhibition of NF-κB activation.     

NADPH oxidase 4 promotes cardiac microvascular angiogenesis after hypoxia/reoxygenation in vitro

Microvascular endothelial cell dysfunction plays a key role in myocardial ischemia/reperfusion (I/R) injury, wherein reactive oxygen species (ROS)-dependent signaling is intensively involved. However, the roles of the various ROS sources remain unclear. This study sought to investigate the role of NADPH oxidase 4 (Nox4) in the cardiac microvascular endothelium in response to I/R injury. Adult rat cardiac microvascular endothelial cells (CMECs) were isolated and subjected to hypoxia/reoxygenation (H/R). Our results showed that Nox4 was highly expressed in CMECs, was significantly increased at both mRNA and protein levels after H/R injury, and contributed to H/R-stimulated increase in Nox activity and ROS generation. Downregulation of Nox4 by small interfering RNA transfection did not affect cell viability or ROS production under normoxia, but exacerbated H/R injury as evidenced by increased apoptosis and inhibited cell survival, migration, and angiogenesis after H/R. Nox4 inhibition also increased prolyl hydroxylase 2 (PHD2) expression and blocked H/R-induced increases in HIF-1α and VEGF expression. Pretreatment with DMOG, a specific competitive PHD inhibitor, upregulated HIF-1α and VEGF expression and significantly reversed Nox4 knockdown-induced injury. However, Nox2 was scarcely expressed and played a minimal role in CMEC survival and angiogenesis after H/R, though a modest upregulation of Nox2 was observed. In conclusion, this study demonstrated a previously unrecognized protective role of Nox4, a ROS-generating enzyme and the major Nox isoform in CMECs, against H/R injury by inhibiting apoptosis and promoting migration and angiogenesis via a PHD2-dependent upregulation of HIF-1/VEGF proangiogenic signaling.     

Effects of miR-33a-5P on ABCA1/G1-Mediated Cholesterol Efflux under Inflammatory Stress in THP-1 Macrophages

The present study is to investigate whether inflammatory cytokines inhibit ABCA1/ABCG1-mediated cholesterol efflux by regulating miR-33a-5P in THP-1 macrophages. We used interleukin-6 and tumor necrosis factor-alpha in the presence or absence of native low density lipoprotein (LDL) to stimulate THP-1 macrophages. THP-1 macrophages were infected by either control lentivirus vectors or lentivirus encoding miR-33a-5P or antisense miR-33a-5P. The effects of inflammatory cytokines, miR-33a-5P and antisense miR-33a-5P on intracellular lipids accumulation and intracellular cholesterol contents were assessed by oil red O staining and quantitative intracellular cholesterol assay. ApoA-I-mediated cholesterol efflux was examined using the fluorescent sterol (BODIPY-cholesterol). The gene and protein expressions of the molecules involved in cholesterol trafficking were examined using quantitative real-time polymerase chain reaction and Western blotting. Inflammatory cytokines or miR-33a-5P increased intracellular lipid accumulation and decreased apoA-I-mediated cholesterol efflux via decreasing the expression of ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. However, antisense miR-33a-5P reversed the effects of inflammatory cytokines on intracellular lipid accumulation, cholesterol efflux, and the expression of miR-33a-5P, ABCA1 and ABCG1 in the absence or presence of LDL in THP-1 macrophages. This study indicated that inflammatory cytokines inhibited ABCA1/ABCG1-mediated cholesterol efflux by up-regulating miR-33a-5P in THP-1 macrophages.     

Prdx2 Inhibits Macrophage Lipid Accumulation Through ROS-NF-κB-miR-33a-ABCA1 Pathway

Previous studies have unraveled that peroxiredoxin 2 (Prdx2) inhibits atherogenesis in mice, whereas its role in macrophage lipid accumulation or the underlying mechanisms remain unknown. THP-1 monocyte-derived foam cells were transfected with Prdx2-overexpressing plasmid vectors (pcDNA3.1-Prdx2) or Prdx2 siRNA. The expression of ABCA1, NF-κB p65 and miR-33a were detected by RT-PCR and Western blotting. Percentage of cholesterol efflux was evaluated by liquid scintillation counting. Cellular lipid droplets were assessed using Oil Red O staining. Intracellular cholesterol contents were measured using high performance liquid chromatography (HPLC). Furthermore, cells were pre-treated with NF-κB inhibitor PDTC and/or miR-33a inhibitor, followed by detection of the indices above. The results showed that overexpression of Prdx2 in THP-1 monocyte-derived foam cells significantly increased ABCA1 expression and the percentage of ［³H］-cholesterol efflux to apoA-1 (P＜0.05), whereas NF-κB p65 and miR-33a levels as well as lipid accumulation were decreased (P＜0.05). After pre-treatment with PDTC and/or miR-33a inhibitor, these effects were more obvious (P＜0.05). In contrast, silencing of Prdx2 significantly diminished ABCA1 expression and increased NF-κB p65 and miR-33a levels. At last, we found that Prdx2 overexpression obviously down-regulated the ROS level in THP-1 monocyte-derived foam cells. Altogether, Prdx2 promotes macrophage cholesterol efflux and inhibits intracellular lipid accumulation through the ROS-NF-κB-miR-33a-ABCA1 pathway.     

MicroRNA-199a-5p aggravates angiotensin II–induced vascular smooth muscle cell senescence by targeting Sirtuin-1 in abdominal aortic aneurysm

Vascular smooth muscle cells (VSMCs) senescence contributes to abdominal aortic aneurysm (AAA) formation although the underlying mechanisms remain unclear. This study aimed to investigate the role of miR-199a-5p in regulating VSMC senescence in AAA. VSMC senescence was determined by a senescence-associated β-galactosidase (SA-β-gal) assay. RT-PCR and Western blotting were performed to measure miRNA and protein level, respectively. The generation of reactive oxygen species (ROS) was evaluated by H2DCFDA staining. Dual-luciferase reporter assay was used to validate the target gene of miR-199a-5p. VSMCs exhibited increased senescence in AAA tissue relative to healthy aortic tissue from control donors. Compared with VSMCs isolated from control donors (control-VSMCs), those derived from patients with AAA (AAA-VSMCs) exhibited increased cellular senescence and ROS production. Angiotensin II (Ang II) induced VSMC senescence by promoting ROS generation. The level of miR-199a-5p expression was upregulated in the plasma from AAA patients and Ang II–treated VSMCs. Mechanistically, Ang II treatment significantly elevated miR-199a-5p level, thereby stimulating ROS generation by repressing Sirt1 and consequent VSMC senescence. Nevertheless, Ang II–induced VSMC senescence was partially attenuated by a miR-199a-5p inhibitor or Sirt1 activator. Our study revealed that miR-199a-5p aggravates Ang II–induced VSMC senescence by targeting Sirt1 and that miR-199a-5p is a potential therapeutic target for AAA.     

Restoration of circPSMC3 sensitizes gefitinib-resistant esophageal squamous cell carcinoma cells to gefitinib by regulating miR-10a-5p/PTEN axis

Circular RNAs (circRNAs) has been shown to play an important role in the progression of various cancers. However, the function and underlying mechanisms of circRNAs affecting chemotherapy resistance in esophageal squamous cell carcinoma (ESCC) remain largely unknown. In this study, we used gefitinib-resistant (GR) ESCC cells to investigate the function of circPSMC3 and clarify the underlying mechanism in chemotherapy resistance in ESCC. The results suggested that circPSMC3 expression was downregulated, but miR-10a-5p was upregulated in ESCC tissues and cells, as well as in GR ESCC cells. CircPSMC3 overexpression increased the sensitivity of ESCC cells to gefitinib, as indicated by reduced half maximal inhibitory concentration value, increased apoptosis rate and cleaved caspase-3 protein expression. CircPSMC3 directly interacted with miR-10a-5p and inhibited the expression of miR-10a-5p. Phosphatase and tensin homolog (PTEN) was a direct target of miR-10a-5p and circPSMC3 promoted PTEN expression via decreasing miR-10a-5p level. Moreover, the effect of circPSMC3 on resistance of GR ESCC cells to gefitinib was remarkably reduced by restoration of miR-10a-5p and downregultion of PTEN. Taken together, these observations suggested that upregulation of circPSMC3 overcame resistance of GR ESCC cells to gefitinib by modulating the miR-10a-5p/PTEN axis, which provide a new therapeutic strategy for overcoming gefitinib resistance in ESCC.     

An antagonism between the AKT and beta-adrenergic signaling pathways mediated through their reciprocal effects on miR-199a-5p

We have recently reported that downregulation of miR-199a-5p is necessary and sufficient for inducing upregulation of its targets, including hypoxia-inducible factor-1alpha (Hif-1α) and Sirt1, during hypoxia preconditioning (HPC). Conversely, others and we have reported that miR-199a-5p is upregulated during cardiac hypertrophy. Thus, the objective of this study was to delineate the signaling pathways that regulate the expression of miR-199a-5p and its targets, and their role in myocyte survival during hypoxia. Since HPC is mediated through activation of the AKT pathway, we questioned if AKT is sufficient for inducing downregulation of miR-199a-5p. Our present study shows that overexpression of a constitutively active AKT (caAKT) induced 70% reduction in miR-199a-5p and was associated with a robust increase in HiF-1α (10 ± 2 fold) and Sirt1 (4 ± 0.8 fold) that was reversed by overexpression of miR-199a-5p. Similarly, insulin receptor-stimulated activation of the AKT pathway induced downregulation of miR-199a-5p and upregulation of its targets. In contrast, β-adrenergic receptor (βAR) activation in vitro and in vivo, induced 1.8–3.5-fold increase in miR-199a-5p. Accordingly, we predicted that βAR would antagonize AKT-induced, miR-199a-5p-dependent, upregulation of Hif-1α and Sirt1. Indeed, pre-treating the myocytes with isoproterenol before applying HPC, caAKT, or insulin resulted in 87 ± 3%, 75 ± 15%, and 100% reductions in Hif-1α expression, respectively, and sensitized the cells to hypoxic injury. Thus, activation of beta-adrenergic signaling counteracts the survival effects of the AKT pathway via upregulating miR-199a-5p.     

Ionizing radiation-inducible microRNA miR-193a-3p induces apoptosis by directly targeting Mcl-1

The functions of microRNAs (miRNAs) as either oncogenes or tumor suppressors in regulating cancer-related events have been established. We analyzed the alterations in the miRNA expression profile of the glioma cell line U-251 caused by ionizing radiation (IR) by using an miRNA array and identified several miRNAs whose expression was significantly affected by IR. Among the IR-responsive miRNAs, we further examined the function of miR-193a-3p, which exhibited the most significant growth-inhibiting effect. miR-193a-3p was observed to induce apoptosis in both U-251 and HeLa cells. We also demonstrated that miR-193a-3p induces the accumulation of intracellular reactive oxygen species (ROS) and DNA damage as determined by the level of γH2AX and by performing the comet assay. The induction of both apoptosis and DNA damage by miR-193a-3p was blocked by antioxidant treatment, indicating the crucial role of ROS in the action of miR-193a-3p. Among the putative target proteins, the expression of Mcl-1, an anti-apoptotic Bcl-2 family member, decreased because of miR-193a-3p transfection. A reporter assay using a luciferase construct containing the 3′-untranslated region of Mcl-1 confirmed that Mcl-1 is a direct target of miR-193a-3p. Down-regulation of Mcl-1 by siRNA transfection closely mimicked the outcome of miR-193a-3p transfection showing increased ROS, DNA damage, cytochrome c release, and apoptosis. Ectopic expression of Mcl-1 suppressed the pro-apoptotic action of miR-193a-3p, suggesting that Mcl-1 depletion is critical for miR-193a-3p induced apoptosis. Collectively, our results suggest a novel function for miR-193a-3p and its potential application in cancer therapy.     

MiR-216a-5p protects 16HBE cells from H2O2-induced oxidative stress through targeting HMGB1/NF-kB pathway

Asthma is a complex, chronic inflammatory disorder of the bronchial tree, and can affect patients of all ages including children. High mobility group box 1 (HMGB1) has been proved as a therapeutic target in children with asthma, and was predicted to be the target gene of microRNA-216a-5p (miR-216a-5p). The present study aimed to investigate the function of miR-216a-5p in asthma by creating a human bronchial epithelial cell (16HBE) injury model using H?O?. A significantly elevation of HMGB1 protein expression and a reduction of miR-216a-5p expression were observed in children with asthma as well as in H?O? stimulated 16HBE cells. Dual luciferase reporter assays confirmed the target reaction between HMGB1 and miR-216a-5p. MiR-216a-5p repressed HMGB1 protein expression in H?O? induced 16HBE cells. Moreover, miR-216a-5p inhibited H?O? induced cell injury by elevating cell proliferation and decreasing cell apoptosis in 16HBE cells. Furthermore, miR-216a-5p repressed NF-kB pathway activation in H?O? induced 16HBE cells. In conclusion, these results suggested that miR-216a-5p functions as a negative regulator of H?O? induced 16HBE cell injury through targeting HMGB1/NF-kB pathway, provided a potential therapeutic target for asthma.     

miR-34a-5p was involved in chronic intermittent hypoxia-induced autophagy of human coronary artery endothelial cells via Bcl-2/beclin 1 signal transduction pathway

Autophagy refers to the genetically regulated process to regulate the survival and death of cells, which is conserved in evolution. Typically, autophagy exerts a vital part under physiopathological conditions. Whether autophagy can be resulted from chronic intermittent hypoxia (CIH), a prominent characteristic of obstructive sleep apnea-hypopnea syndrome (OSAHS), remains to be investigated. Furthermore, microRNAs (miRNAs) can serve as the regulating factors in a variety of benign and malignant diseases; nonetheless, it remains to be fully illustrated about the way by which miRNAs modulate autophagy. According to our results, for human coronary artery endothelial cells (HCAECs), CIH increased the expression of autophagy-associated proteins, which depended on the concentration and time; besides, it could promote autophagic vacuole (AV) formation. In addition, CIH could activate beclin 1, which was dependent on dose and time. In HCAECs, microRNA-34a-5p (miR-34a-5p) was overexpressed after exposed to CIH, and its target protein B-cell lymphoma 2 (Bcl-2) was downregulated. Moreover, inhibiting miR-34a-5p increased Bcl-2 and p62 expression, while downregulating beclin 1, Vps34, Atg5, and LC3 levels, implying the role of miR-34a-5p in CIH-induced autophagy. Moreover, exogenous upregulation of Bcl-2 could block miR-34a-5p influence on CIH-induced autophagy through suppressing beclin 1 expression. Additionally, beclin 1 could enhance the autophagy induced by CIH. In conclusion, overexpression of miR-34a-5p activated beclin 1 through Bcl-2 inhibition in CIH and participated in CIH-induced autophagy.     

miR-200a mediates protection of thymosin β-4 in cardiac microvascular endothelial cells as a novel mechanism under hypoxia-reoxygenation injury

Thymosin β-4 (Tβ4) is a ubiquitous protein, which has been suggested to regulate multiple cell signal pathways and a variety of cellular functions. However, the role Tβ4 plays in the cardiac microvascular endothelial cells (CMECs) under myocardial ischemia/reperfusion injury is currently unknown. Here we investigated the effects of Tβ4 on hypoxia/reoxygenation (H/R) induced CMECs injury and its potential molecular mechanism. Cultured CMECs were positively identified by flow cytometry using antibody against CD31 and VWF/Factor VIII, which are constitutively expressed on the surface of CMECs. Then the reduced level of Tβ4 was detected in H/R-CMECs by a real-time quantitative polymerase chain reaction. To determine the effects of Tβ4 on H/R-CMECs, we transfected the overexpression or silence vector of Tβ4 into CMECs under H/R condition. Our results indicated that H/R treatment could reduce proliferation, increased apoptosis, adhesion, and reactive oxygen species (ROS) production in CMECs, which were attenuated by Tβ4 overexpression or aggravated by Tβ4 silencing, implying Tβ4 is able to promote CMECs against H/R-induced cell injury. Furthermore, the microRNA-200a (miR-200a) level was also increased by Tβ4 in H/R-CMECs or reduced by Tβ4 small interfering RNA. To investigated the mechanism of protective effects of Tβ4 on CMECs injury, the miR-200a inhibitor was transfected into H/R-CMECs. The results indicated that inhibition of miR-200a inversed the protection of Tβ4 on H/R-CMECs, specifically including cell proliferation, cell adhesion, cell apoptosis, and ROS production, as well as nuclear factor erythroid 2-related factor 2 (Nrf2) nuclear translocation. In conclusion, our results determined that Tβ4 attenuated H/R-induced CMECs injury by miR-200a-Nrf2 signaling.     

MiR-133a-3p inhibits scar formation in scalded mice and suppresses the proliferation and migration of scar derived-fibroblasts by targeting connective tissue growth factor

Excessive scar formation post burn injury can cause great pain to the patients. MiR-133a-3p has been demonstrated to be anti-fibrotic in some fibrosis-related diseases. However, its possible role in scar formation has not been elucidated yet. In present study, the effect of miR-133a-3p on scar formation was investigated in a scalded model of mice. Moreover, the function of miR-133a-3p on proliferation and migration of scar-derived fibroblasts (SFs) was studied in vitro. It was found that miR-133a-3p was dramatically downregulated in scar tissue of scalded mice. Upregulation of miR-133a-3p by miR-133a-3p agomir obviously inhibited the scar formation in scalded mice. Histological staining showed that upregulation of miR-133a-3p attenuated the excessive deposition of collagen in scar tissue of scalded mice. In vitro study showed that upregulation of miR-133a-3p effectively suppressed the proliferation and migration of SFs. Besides, upregulation of miR-133a-3p attenuated the protein levels of α-smooth muscle actin (α-SMA) and collagen I, indicating that miR-133a-3p could suppress the activation of SFs. The expression of connective tissue growth factor (CTGF), a critical mediator in cell proliferation, migration and extracellular matrix (ECM) synthesis, was also downregulated by the upregulation of miR-133a-3p. Luciferase reporter assay validated that CTGF was directly targeted by miR-133a-3p. In addition, overexpression of CTGF abolished the effect of miR-133a-3p on inhibiting the proliferation, migration and activation of SFs, indicating that miR-133a-3p functioned by targeting CTGF. Therefore, miR-133a-3p might be a promising target for treating pathological scars.     

Effect of miR-499a-5p on damage of cardiomyocyte induced by hypoxia-reoxygenation via downregulating CD38 protein

The aim is to investigate the mechanism of miR-499a-5p on the damage of cardiomyocyte induced by hypoxia/reoxygenation. The activity of lactate dehydrogenase (LDH), apoptosis rate and the expression of miR-499a-5p and cluster of differentiation 38 (CD38) in hypoxia-reoxygenation model cells were detected by LDH Cytotoxicity Assay Kit, flow cytometry, real-time polymerase chain reaction, and Western blot analysis, respectively. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p or silencing of CD38 in H9c2 cells. The target relationship between miR-499a-5p and CD38 was verified by Targetscan online prediction and dual-luciferase assay. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p and CD38. Apoptosis, the activity of LDH and the expression of CD38 were increased (P < .05) while expression of miR-499a-5p was decreased (P < .05) in hypoxia/reoxygenation model cells. Apoptosis and the activity of LDH in H9c2 cells after overexpression of miR-499a-5p or silence of CD38 were decreased (P < .05). The results of Targetscan online prediction and dual-luciferase assay indicated that CD38 was a potential target gene of miR-499a-5p. Overexpression of CD38 could reverse the inhibition of miR-499a-5p on LDH activity and apoptosis in H9c2 cells. miR-499a-5p could relief the injury of cardiomyocytes induced by hypoxia/reoxygenation via targeting CD38.