Shuai Wang

TRPV1 activation exacerbates hypoxia/reoxygenation-induced apoptosis in H9C2 cells via calcium overload and mitochondrial dysfunction.

Transient potential receptor vanilloid 1 (TRPV1) channels, which are expressed on sensory neurons, elicit cardioprotective effects during ischemia reperfusion injury by stimulating the release of neuropeptides, namely calcitonin gene-related peptide (CGRP) and substance P (SP). Recent studies show that TRPV1 channels are also expressed on cardiomyocytes and can exacerbate air pollutant-induced apoptosis. However, whether these channels present on cardiomyocytes directly modulate cell death and survival pathways during hypoxia/reoxygenation (H/R) injury remains unclear. In the present study, we investigated the role of TRPV1 in H/R induced apoptosis of H9C2 cardiomyocytes. We demonstrated that TRPV1 was indeed expressed in H9C2 cells, and activated by H/R injury. Although neuropeptide release caused by TRPV1 activation on sensory neurons elicits a cardioprotective effect, we found that capsaicin (CAP; a TRPV1 agonist) treatment of H9C2 cells paradoxically enhanced the level of apoptosis by increasing intracellular calcium and mitochondrial superoxide levels, attenuating mitochondrial membrane potential, and inhibiting mitochondrial biogenesis (measured by the expression of ATP synthase β). In contrast, treatment of cells with capsazepine (CPZ; a TRPV1 antagonist) or TRPV1 siRNA attenuated H/R induced-apoptosis. Furthermore, CAP and CPZ treatment revealed a similar effect on cell viability and mitochondrial superoxide production in primary cardiomyocytes. Finally, using both CGRP8–37 (a CGRP receptor antagonist) and RP67580 (a SP receptor antagonist) to exclude the confounding effects of neuropeptides, we confirmed aforementioned detrimental effects as TRPV1−/− mouse hearts exhibited improved cardiac function during ischemia/reperfusion. In summary, direct activation of TRPV1 in myocytes exacerbates H/R-induced apoptosis, likely through calcium overload and associated mitochondrial dysfunction. Our study provides a novel understanding of the role of myocyte TRPV1 channels in ischemia/reperfusion injury that sharply contrasts with its known extracardiac neuronal effects.

Wnt1 Participates in Inflammation Induced by Lipopolysaccharide Through Upregulating Scavenger Receptor A and NF-kB

The study investigated the role of wnt1 in the inflammatory response initiated by lipolysaccharide (LPS), and analyzed the association between wnt1, NF-KB, and inflammatory factors. THP-1 cells were activated with phorbol-12-myristate-13-acetate (PMA) and treated with LPS to induce inflammation. THP-1 cells were transfected with wnt1siRNA and overexpression plasmid to explore the relationship among wnt1, SRA, and NF-KB. Inhibitor of β-catenin and siRNA of FZD1were used to investigate the signaling events involved in SRA activation induced by wnt1. Levels of NF-kB protein and inflammatory cytokines were assessed followingwnt1 siRNA and LPS treatment. PMA activation and LPS treatment of THP-1 cells increased wnt1 protein levels. Wnt1 promoted SRA expression through activation of canonical wnt pathway. Wnt1 increased NF-kB protein levels and enhanced the secretion of IL-6, TNF-α, and iNOS through binding to SRA. These findings suggest that wnt1 increased SRA and NF-kB protein levels and participated in the inflammatory response.

Wnt1 positively regulates CD36 expression via TCF4 and PPAR-γ in macrophages.

Background: Scavenger receptors including CD36 control the phagocytosis of oxidized low-density lipoprotein and play an important role in macrophage physiology, but the underlying molecular mechanism by which CD36 is regulated in macrophages or during macrophage differentiation from monocytes remains to be determined. Methods: Here, we investigated the relationship between Wnt1 and CD36 during macrophage differentiation. CD36 was suppressed following knockdown of Wnt1 by siRNA, while it was increased by ectopic overexpression of Wnt1 in macrophages. Using a β-catenin inhibitor, peroxisome proliferator-activated receptor gamma (PPAR-γ) siRNA, and transcription factor 4 (TCF4) siRNA, we demonstrated that Wnt1 regulates the expression of CD36 through TCF4 and PPAR-γ. Co-immunoprecipitation, chromatin immunoprecipitation, and immunofluorescence experiments showed that β-catenin interacted with PPAR-γ and that PPAR-γ and TCF4 colocalized in the nucleus. Furthermore, Pax3 regulated Wnt1 via binding to the first binding site in the Wnt1 promoter. Results: Our study demonstrated that during macrophage differentiation from monocytes, Wnt1 promotes CD36 expression via activation of PPAR-γ and TCF4. Conclusions: Our findings suggest that Wnt1 plays an important role in macrophage physiology via activation of the canonical Wnt pathway.

Cross-talk between macrophages and atrial myocytes in atrial fibrillation

Increased macrophage accumulation occurs in the atria of patients with atrial fibrillation (AF). However, the phenotype and functions of the macrophages in AF remain unclear. We investigated the macrophage-atrial myocyte interaction in AF patients and found that the increased macrophages were mainly pro-inflammatory macrophages (iNOS+, Arg1−). Tachypacing of HL-1 atrial myocytes also led to pro-inflammatory macrophage polarization. In addition, lipopolysaccharide (LPS)-stimulated pro-inflammatory macrophages-induced atrial electrical remodeling, evidenced by increased AF incidence and decreased atrial effective refractory period and L-type calcium currents (ICa-L) in both canine and mouse AF models. Depletion of macrophages relieved LPS-induced atrial electrical remodeling, confirming the role of pro-inflammatory macrophages in the pathogenesis of AF. We also found that the effect of LPS-stimulated macrophages on atrial myocytes was mediated by secretion of interleukin 1 beta (IL-1β), which inhibited atrial myocyte quaking protein (QKI) expression. IL-1β knockout in macrophages restored the LPS-stimulated macrophage-induced inhibition of QKI and CACNA1C (α1C subunit of L-type calcium channel) in atrial myocytes. Meanwhile, QKI overexpression in atrial myocytes restored the LPS-stimulated macrophage-induced electrical remodeling through enhanced binding of QKI to CACNA1C mRNA, which upregulated the expression of CACNA1C as well as ICa-L. In contrast, QKI knockout inhibited CACNA1C expression. Finally, using transcription factor activation profiling plate array and chromatin immunoprecipitation, we revealed that special AT-rich sequence binding protein 1 activated QKI transcription. Taken together, our study uncovered the functional interaction between macrophages and atrial myocytes in AF. AF induced pro-inflammatory macrophage polarization while pro-inflammatory macrophages exacerbated atrial electrical remodeling by secreting IL-1β, further inhibiting QKI expression in atrial myocytes, which contributed to ICa-L downregulation. Our study demonstrates a novel molecular mechanism underlying the pathogenesis and progression of AF and suggests that QKI is a potential therapeutic target.

Upregulation of miRNA-155 expression by OxLDL in dendritic cells involves JAK1/2 kinase and transcription factors YY1 and MYB

Dendritic cells (DCs) have been implicated in the pathogenesis of atherosclerosis (AS). Whereas the pathogenic role of oxidized low-density lipoprotein (oxLDL) in the development and progression of AS has been recognized previously, the contribution of microRNA-155 (miR-155) to AS was previously not fully understood. It had also been noted that miR-155 levels were upregulated by oxLDL in various cell types under different (patho)physiological conditions, but its underlying mechanisms had not been examiend in detail. Thus, in the present study, we observed that oxLDL treatment increased miR-155 expression in DCs, and transfecting DCs with siRNA against scavenger receptor A (SRA) revealed that repression of SRA attenuated this upregulation. We also noted that miR-155 negatively regulated SRA expression by suppressing the JNK pathway. Furthermore, we noted that Yin Yang 1 (YY1) and V-Myb avian myeloblastosis viral oncogene homolog (MYB), which were also upregulated by oxLDL, directly bound to the cognate sequences of the promoter region of miR-155 to activate its transcription. In addition, using SP600125, a specific inhibitor for c-Jun N-terminal kinase (JNK) signaling, we demonstrated that JNK signaling was involved in the miR‑155-mediated suppression of SRA expression. Thus, in the present study we uncovered the molecular mechanism through which miR-155 expression is regulated by oxLDL, and we also identified a negative feedback loop, miR‑155-JNK-SRA-miR-155. Our findings thus provide novel insights into the regulatory network underlying the expression and activity of miR-155 in DCs.

Oxidized low‑density lipoprotein upregulates microRNA‑146a via JNK and NF‑κB signaling

Increasing evidence suggested the involvement of microRNA (miR)-146a in the pathogenesis of multiple diseases, including atherosclerosis, bacterial infection and cancer. However, the mechanism by which miR-146a is regulated in macrophages exposed to oxidized low-density lipoprotein (oxLDL) has remained elusive. The present study aimed to explore the molecular pathway of miR-146a regulation in response to oxLDL. Human THP-1 macrophages were pre-treated with small interfering RNA specific for scavenger receptors or with pharmacological inhibitors prior to oxLDL administration. A filter plate screening assay was performed to identify oxLDL-inducible transcription factors that bind to the miR-146a promoter. The exact binding sites were mapped by chromatin immunoprecipitation. The effects of miR-146a on markers of macrophage maturation were studied by flow cytometry. The results revealed that miR-146a expression was deceased when c-jun N-terminal kinase (JNK) or nuclear factor (NF)-κB signaling was inhibited. By forming a complex with c-jun, which was promoted by oxLDL, the NF-κB sub-unit p65 facilitated the binding of c-jun to the miR-146a promoter to trigger transcriptional activation. miR-146a negatively regulated macrophage maturation by reducing the expression of CD86 and CD80. The present study demonstrated that oxLDL positively regulates miR-146a via the JNK and NF-κB pathways in macrophages, and that miR-146a inhibits inflammatory activation.

Rspo2 suppresses CD36-mediated apoptosis in oxidized low density lipoprotein-induced macrophages

Oxidized low density lipoprotein (oxLDL)-induced apoptosis of macrophages contributes to the formation of atherosclerotic plaques. R-spondin 2 (Rspo2), a member of the cysteine-rich secreted proteins, has been shown to be involved in the oncogenesis of several types of cancer. It has also been found to be abundantly expressed among the four R-spondin members in macrophages. The present study was performed to determine whether Rspo2 is involved in the ox-LDL-induced apoptosis of macrophages. It was identified that Rspo2 inhibited oxLDL-induced apoptosis in the presence of endoplasmic reticulum (ER) stress activator using flow cytometry. In addition, Rspo2 was observed to suppress oxLDL-induced ER stress and reactive oxygen species production as demonstrated by western blotting. Furthermore, analysis of the role of Rspo2 in macrophage lipid uptake identified that Rspo2 negatively regulated the Dil-oxLDL uptake by inhibiting the expression of cluster of differentiation (CD)36, through the transcription factor, peroxisome proliferator-activated receptor (PPAR)-γ. The manipulation of Rspo2 had a direct effect on PPAR-γ nuclear translocation. In addition, chromatin immunoprecipitation analysis revealed that Rspo2 manipulation led to regulation of the direct binding between PPAR-γ and CD36. In conclusion, Rspo2 was found to have a negative regulatory effect during oxLDL-induced macrophage apoptosis by regulating lipid uptake.

Interruption of CD40 Pathway Improves Efficacy of Transplanted Endothelial Progenitor Cells in Monocrotaline Induced Pulmonary Arterial Hypertension.

Background/Aims: Transplantation of endothelial progenitor cells (EPCs) plays a therapeutic role in pulmonary arterial hypertension (PAH). Meanwhile, recruitment of progenitors has potential inflammatory effects and exaggerates vascular injury. CD40 pathway is identified as a major player in vascular inflammatory events. In this study, we investigated the role of CD40 pathway in regulating early outgrowth EPC functions, and searched for improvements in PAH cell therapy. Methods: EPCs were isolated from rat bone marrow and cultured for 7 days. After treatment with soluble CD40 ligand (sCD40L) for 24 hours, EPC migration, adhesion, proliferation, paracrine and vasculogenesis functions were tested. Rat PAH model was founded by subcutaneous injection of monocrotaline (MCT). Control EPCs or lentivirus vectors (Lv)-shRNA-CD40 EPCs were infused via tail vein at day 7, 14, and 21 after MCT injection. Therapeutic effects were evaluated at day 28. Results: sCD40L dose-dependently impaired EPC migration, adhesion, proliferation, and vasculogenesis functions. However, paracrine effects of soluble intercellular adhesion molecule-1, vascular endothelial growth factor and interleukin-6 were dose-dependently improved by sCD40L. Control EPC-derived conditioned medium protected endothelial cell in vitro vasculogenesis, while sCD40L-pretreated ones showed detrimental effects. After MCT injection, sCD40L levels in rat serum increased gradually. Other than in vitro results, benefits of both two EPC treatments were obvious, even taken at day 21. Benefits of control EPCs wore off over time, but those of Lv-shRNA-CD40 EPCs were more effective and enduring, as characterized by both ameliorated rat hemodynamic and reversed vascular remodeling. Furthermore, Lv-shRNA-CD40 EPCs integrated into endothelium better, rather than into adventitia and media. Conclusion: sCD40L impaired protective effects of EPCs. Traditional EPC treatments were limited in PAH, while interruption of CD40 pathway of transplanted cells could apparently improve the therapeutic efficacy.

Role of cantharidin in the activation of IKKα/IκBα/NF‑κB pathway by inhibiting PP2A activity in cholangiocarcinoma cell lines

Cantharidin (CAN), a potent inhibitor of serine/threonine-protein phosphatase 2A (PP2A), is widely used in clinical practice, particularly in the treatment of advanced cancer due to its specific action on these types of cancer. In the present study, the inhibitory effect of CAN was examined in two cholangiocarcinoma cell lines (QBC939 and Hucc-t1). Following treatment with CAN, cell viability was effectively reduced in QBC939 and Hucc-t1 cells and normal human intrahepatic biliary epithelial cells. However, a slight increase in reactive oxygen species levels in QBC939 cells treated with CAN was observed post-treatment. CAN significantly inhibited cell migration and invasion in a dose-dependent manner. Western blot analysis demonstrated that the nuclear factor-κB (NF-κB) pathway was stimulated by CAN, which was confirmed by the upregulated phosphorylation levels of inhibitor of NF-κB kinase subunit α (IKKα) and NF-κB inhibitor α (IκBα) in cells, and an increased NF-κB p65 subunit level in the nucleus. The expression levels of 72 kDa type IV collagenase (MMP2) and matrix metalloproteinase 9 (MMP9) were downregulated by CAN. Notably, there was a negative association between MMP2 and MMP9 expression levels, and NF-κB p65, although NF-κB p65 regulates the expression of MMP2 and MMP9 and has a positive association with these proteins in various types of cancer. Notably, it was observed that CAN exerted specific inhibition on PP2A activity and thereby resulted in the activation of the IKKα/IκBα/NF-κB pathway. Therefore, CAN-induced cell inhibition maybe partially dependent on the activation of the IKKα/IκBα/NF-κB pathway. In conclusion, it was demonstrated that CAN selectively and effectively inhibited cholangiocarcinoma cell migration and invasion. The present study may provide a novel insight into the use of CAN as a therapeutic candidate in the treatment of cholangiocarcinoma.

Lentivirus‑mediated overexpression of CD97/ADGRE5 reverses dysregulated high glucose‑induced endothelial cell migration

Elevated blood glucose levels contribute to a series of complications in patients with diabetes mellitus, including chronic ulcers and accelerated atherosclerosis. Dysregulated endothelial migration induced by high glucose is important in vascular-associated complications. In the present study, cluster of differentiation (CD)97/adhesion G protein-coupled receptor 5 (ADGRE5), a member of the G protein-coupled receptor protein family, stimulated angiogenesis was investigated to determine its role in cell models of diabetes mellitus using lentivirus-mediated overexpression and siRNA transfection. The results revealed reduced expression in high glucose-treated human umbilical vein endothelial cells and in the endothelium of diabetic mice. Among the three CD97 isoforms, the majority of the expression of CD97 (EGF1,2,5) in the endothelial cells was regulated by high levels of glucose. Using stable lentivirus-mediated transfection and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology, the present study constructed CD97-overexpression and CD97-knockout endothelial cell lines. Migration assays showed that the lentivirus-mediated overexpression of CD97/ADGRE5 improved the inhibition of high glucose-induced endothelial cell migration. In addition, using cytoskeleton staining, it was found that CD97 promoted membrane ruffling and lamellipodia formation. Cell division cycle 42, a small GTP-binding protein, and its downstream factor, actin-related protein 2, were involved in CD97-induced actin reorganization in endothelial cells. Additionally, the use of transcription factor filter plate assays revealed that the nuclear translocation of signal transducer and activator of transcription 1 stimulated by high glucose contributed to the inhibited transcription of CD97. In conclusion, the present study established that the overexpression of CD97 improved high glucose-induced dysfunction of endothelial cell migration. These findings provide insight to assist in identifying therapeutic targets with potential to ameliorate certain vascular complications of diabetes.