Qilin Yu

Loss of methyl-CpG-binding domain protein 2 enhances endothelial angiogenesis and protects mice against hind-limb ischemic injury.

Background— Despite intensive investigation, how DNA methylation influences endothelial function remains poorly understood. We used methyl-CpG–binding domain protein 2 (MBD2), an interpreter for DNA methylome–encoded information, to dissect the impact of DNA methylation on endothelial function in both physiological and pathophysiological states. Methods and Results— Human umbilical vein endothelial cells under normal conditions express moderate levels of MBD2, but knockdown of MBD2 by siRNA significantly enhanced angiogenesis and provided protection against H2O2-induced apoptosis. Remarkably, Mbd2−/− mice were protected against hind-limb ischemia evidenced by the significant improvement in perfusion recovery, along with increased capillary and arteriole formation. Loss of MBD2 activated endothelial survival and proangiogenic signals downstream of vascular endothelial growth factor signaling characterized by an increase in endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor receptor 2 expression, along with enhanced extracellular signal-regulated kinase 1/2 activation and BCL-2 expression. Mechanistic studies confirmed the methylation of CpG elements in the eNOS and vascular endothelial growth factor receptor 2 promoter. MBD2 binds to these methylated CpG elements and suppresses eNOS promoter activity. On ischemic insult, key endothelial genes such as eNOS and vascular endothelial growth factor receptor 2 undergo a DNA methylation turnover, and MBD2 interprets the changes of DNA methylation to suppress their expressions. Moreover, MBD2 modulation of eNOS expression is likely confined to endothelial cells because nonendothelial cells such as splenocytes fail to express eNOS after loss of MBD2. Conclusions— We provided direct evidence supporting that DNA methylation regulates endothelial function, which forms the molecular basis for understanding how environmental insults (epigenetic factor) affect the genome to modify disease susceptibility. Because MBD2 itself does not affect the methylation of DNA and is dispensable for normal physiology in mice, it could be a viable epigenetic target for modulating endothelial function in disease states.

Loss of Dicer Exacerbates Cyclophosphamide-Induced Bladder Overactivity by Enhancing Purinergic Signaling

microRNAs (miRNAs) have regulated the expression and function of genes implicated in many pathological settings, but their impact on the pathoetiological characteristics of overactive bladder (OAB) largely remains unknown. We have generated a mouse model in which adult mice can be induced for detrusor deletion of Dicer, an enzyme essential for miRNA processing. Targeted deletion of Dicer did not lead to a significant change for detrusor functionality under physiological conditions; however, loss of Dicer exacerbated cyclophosphamide-induced OAB, manifested by the higher severity of altered detrusor contractile force and sensitivity, abnormal urodynamics, and enhanced macrophage infiltration. Mechanistic studies revealed that loss of Dicer may impair the expression of miRNAs that are capable of targeting P2x mRNAs. As a result, mice deficient in Dicer manifest enhanced P2X expression in the detrusor on cyclophosphamide treatment, predisposing to the increased risk for OAB development. More important, studies using bladder biopsy samples of patients with OAB also demonstrated similar results as those found in animals. Taken together, our results suggest that miRNAs modulate OAB susceptibility by regulating purinergic signaling, in which the pathogenic insult induces the expression of miRNAs capable of targeting P2X mRNAs to suppress OAB symptoms.

MBD2 regulates TH17 differentiation and experimental autoimmune encephalomyelitis by controlling the homeostasis of T-bet/Hlx axis.

Unlike genetic alterations, epigenetic modifications are reversible and amenable to pharmacological interventions, which make them appealing targets for clinical therapy. However, little is known about epigenetic regulation in experimental autoimmune encephalomyelitis (EAE). Here we demonstrated that methyl-CpG-binding domain protein 2 (MBD2), an epigenetic regulator, controls autoimmunity and EAE through T-bet/Hlx. Tbx21 and Hlx underwent a DNA methylation turnover upon polarizations and a unique methylation pattern was essential for TH17 development. Loss of Mbd2 resulted in a defect for reading the information encoded by this methylation turnover, which disrupted the homeostasis of T-bet/Hlx axis and suppressed TH17 differentiation. DNA demethylation induced similar effect on helper T cell differentiation. Therefore, Mbd2(-/-) mice were completely protected from EAE. Pathogenic splenocytes isolated from wild-type mice challenged with MOG35-55 could adoptively transfer disease to Mbd2(-/-) mice. In addition, Mbd2(-/-) mice reconstituted with unstimulated wild-type splenocytes developed EAE as wild-type mice did. These data would provide novel insights into epigenetic regulation of EAE.

Chop Deficiency Protects Mice Against Bleomycin-induced Pulmonary Fibrosis by Attenuating M2 Macrophage Production

C/EBP homologous protein (Chop) has been shown to have altered expression in patients with idiopathic pulmonary fibrosis (IPF), but its exact role in IPF pathoaetiology has not been fully addressed. Studies conducted in patients with IPF and Chop(-/-) mice have dissected the role of Chop and endoplasmic reticulum (ER) stress in pulmonary fibrosis pathogenesis. The effect of Chop deficiency on macrophage polarization and related signalling pathways were investigated to identify the underlying mechanisms. Patients with IPF and mice with bleomycin (BLM)-induced pulmonary fibrosis were affected by the altered Chop expression and ER stress. In particular, Chop deficiency protected mice against BLM-induced lung injury and fibrosis. Loss of Chop significantly attenuated transforming growth factor β (TGF-β) production and reduced M2 macrophage infiltration in the lung following BLM induction. Mechanistic studies showed that Chop deficiency repressed the M2 program in macrophages, which then attenuated TGF-β secretion. Specifically, loss of Chop promoted the expression of suppressors of cytokine signaling 1 and suppressors of cytokine signaling 3, and through which Chop deficiency repressed signal transducer and activator of transcription 6/peroxisome proliferator-activated receptor gamma signaling, the essential pathway for the M2 program in macrophages. Together, our data support the idea that Chop and ER stress are implicated in IPF pathoaetiology, involving at least the induction and differentiation of M2 macrophages.

Loss of Jak2 impairs endothelial function by attenuating Raf-1/MEK1/Sp-1 signaling along with altered eNOS activities.

A number of inhibitors have been used to dissect the functional relevance of Jak2 in endothelial homeostasis, with disparate results. Given that Jak2 deficiency leads to embryonic lethality, the exact role of Jak2 in the regulation of postnatal endothelial function is yet to be fully elucidated. We generated a model in which Jak2 deficiency can be induced by tamoxifen in adult mice. Loss of Jak2 significantly impaired endothelium-dependent response capacity for vasodilators. Matrigel plug assays indicated a notable decrease in endothelial angiogenic function in Jak2-deficient mice. Studies in a hindlimb ischemic model indicated that Jak2 activity is likely to be a prerequisite for prompt perfusion recovery, based on the concordance of temporal changes in Jak2 expression during the course of ischemic injury and perfusion recovery. A remarkable delay in perfusion recovery, along with reduced capillary and arteriole formation, was observed in Jak2-deficient mice. Antibody array studies indicated that loss of Jak2 led to repressed eNOS expression. In mechanistic studies, Jak2 deficiency attenuated Raf-1/MEK1 signaling, which then reduced activity of Sp-1, an essential transcription factor responsible for eNOS expression. These data are important not only for understanding the exact role that Jak2 plays in endothelial homeostasis, but also for assessing Jak2-based therapeutic strategies in a variety of clinical settings.

Chop deficiency prevents UUO-induced renal fibrosis by attenuating fibrotic signals originated from Hmgb1/TLR4/NFκB/IL-1β signaling.

Renal fibrosis, particularly tubulointerstitial fibrosis is considered to be the final manifestation of almost all chronic kidney diseases (CKDs). Herein we demonstrated evidence that CHOP-related ER stress is associated with the development of renal fibrosis in both CKD patients and unilateral ureteral obstruction (UUO)-induced animals, and specifically, mice deficient in Chop were protected from UUO-induced renal fibrosis. Mechanistic studies revealed that loss of Chop protected tubular cells from UUO-induced apoptosis and secondary necrosis along with attenuated Hmgb1 passive release and active secretion. As a result, Chop deficiency suppressed Hmgb1/TLR4/NFκB signaling, which then repressed UUO-induced IL-1β production. Consequently, the IL-1β downstream Erk1/2 activity and its related c-Jun transcriptional activity were reduced, leading to attenuated production of TGF-β1 following UUO insult. It was further noted that reduced IL-1β production also inhibited UUO-induced PI3K/AKT signaling, and both of which ultimately protected mice from UUO-induced renal fibrosis. Together, our data support that suppression of CHOP expression could be a viable therapeutic strategy to prevent renal fibrosis in patients with CKDs.

Aloperine protects mice against ischemia reperfusion (IR)-induced renal injury by regulating PI3K/AKT/mTOR signaling and AP-1 activity.

Aloperine is a quinolizidine alkaloid extracted from the leaves of Sophora plants. It has been recognized with the potential to treat inflammatory and allergic diseases as well as tumors. In this report, we demonstrate that pretreatment with aloperine provided protection for mice against ischemia-reperfusion (IR)-induced acute renal injury as manifested by the attenuated inflammatory infiltration, reduced tubular apoptosis, and well-preserved renal function. Mechanistic studies revealed that aloperine selectively repressed IL-1β and IFN-γ expression by regulating PI3K/Akt/mTOR signaling and NF-κB transcriptional activity. However, aloperine did not show a perceptible impact on IL-6 and TGF-β expression and the related Jak2/Stat3 signaling. It was also noted that aloperine regulates AP-1 activity, through which it not only enhances SOD expression to increase reactive oxygen species (ROS) detoxification but also promotes the expression of antiapoptotic Bcl-2, thereby preventing tubular cells from IR-induced apoptosis. Collectively, our data suggest that administration of aloperine prior to IR insults, such as renal transplantation, could be a viable approach to prevent IR-induced injuries.

Modulation of the Protein Kinase Cδ Interaction with the “d” Subunit of F1F0-ATP Synthase in Neonatal Cardiac Myocytes DEVELOPMENT OF CELL-PERMEABLE, MITOCHONDRIALLY TARGETED INHIBITOR AND FACILITATOR PEPTIDES

The F1F0-ATP synthase provides ∼90% of cardiac ATP, yet little is known regarding its regulation under normal or pathological conditions. Previously, we demonstrated that protein kinase Cδ (PKCδ) inhibits F1F0 activity via an interaction with the “d” subunit of F1F0-ATP synthase (dF1F0) in neonatal cardiac myocytes (NCMs) (Nguyen, T., Ogbi, M., and Johnson, J. A. (2008) J. Biol. Chem. 283, 29831–29840). We have now identified a dF1F0-derived peptide (NH2-2AGRKLALKTIDWVSF16-COOH) that inhibits PKCδ binding to dF1F0 in overlay assays. We have also identified a second dF1F0-derived peptide (NH2-111RVREYEKQLEKIKNMI126-COOH) that facilitates PKCδ binding to dF1F0. Incubation of NCMs with versions of these peptides containing HIV-Tat protein transduction and mammalian mitochondrial targeting sequences resulted in their delivery into mitochondria. Preincubation of NCMs, with 10 nm extracellular concentrations of the mitochondrially targeted PKCδ-dF1F0 interaction inhibitor, decreased 100 nm 4β-phorbol 12-myristate 13-acetate (4β-PMA)-induced co-immunoprecipitation of PKCδ with dF1F0 by 50 ± 15% and abolished the 30 nm 4β-PMA-induced inhibition of F1F0-ATPase activity. A scrambled sequence (inactive) peptide, which contained HIV-Tat and mitochondrial targeting sequences, was without effect. In contrast, the cell-permeable, mitochondrially targeted PKCδ-dF1F0 facilitator peptide by itself induced the PKCδ-dF1F0 co-immunoprecipitation and inhibited F1F0-ATPase activity. In in vitro PKC add-back experiments, the PKCδ-F1F0 inhibitor blocked PKCδ-mediated inhibition of F1F0-ATPase activity, whereas the facilitator induced inhibition. We have developed the first cell-permeable, mitochondrially targeted modulators of the PKCδ-dF1F0 interaction in NCMs. These novel peptides will improve our understanding of cardiac F1F0 regulation and may have potential as therapeutics to attenuate cardiac injury.

Assessment of type 2 diabetes risk conferred by SNPs rs2241766 and rs1501299 in the ADIPOQ gene, a case/control study combined with meta-analyses

We conducted a case/control study to assess the impact of two SNPs, rs2241766 and rs1501299 within the ADIPOQ gene, on type 2 diabetes (T2D) susceptibility in a Chinese Han dataset (741 cases and 902 controls). SNP rs2241766 was found significantly associated with T2D risk in the additive model, dominant model and recessive model. A marginal association was detected for SNP rs1501299 in the additive model and recessive model after Bonferroni correction, and haplotype analysis provided additional evidence supporting the association between these two SNPs and T2D risk. A meta-analysis including 29 published datasets along with current dataset was next carried out to further confirm the association. In consistent with our case/control results, rs2241766 showed a significant association with T2D in the dominant model and additive model, and the association between rs1501299 and T2D was also characterized in the homozygote model, dominant model, recessive model, and additive model. Of note, the association became much stronger in East Asians after exclusion of ethnic stratification. Together, our data support that the rs2241766 and rs1501299 polymorphisms within the ADIPOQ gene confer genetic susceptibility for type 2 diabetes, especially in the Chinese Han population.

Role of C/EBP homologous protein and endoplasmic reticulum stress in asthma exacerbation by regulating the IL-4/signal transducer and activator of transcription 6/transcription factor EC/IL-4 receptor α positive feedback loop in M2 macrophages

Background: C/EBP homologous protein (Chop), a marker of endoplasmic reticulum (ER) stress, exhibits aberrant expression patterns during asthma development. However, its exact role in asthma pathogenesis is not fully understood. Objectives: We aimed to determine the function and mechanism of Chop in the pathogenesis of allergic asthma in patients and animals. Methods: Studies were conducted in asthmatic patients and Chop−/− mice to dissect the role of Chop and ER stress in asthma pathogenesis. An ovalbumin (OVA)–induced allergic airway inflammation model was used to address the effect of Chop deficiency on asthma development. Next, the effect of Chop deficiency on macrophage polarization and related signaling pathways was investigated to demonstrate the underlying mechanisms. Results: Asthmatic patients and mice after OVA induction exhibited aberrant Chop expression along with ER stress. Specifically, Chop was noted to be specifically overexpressed in macrophages, and mice deficient in Chop were protected from OVA‐induced allergic airway inflammation, as manifested by attenuated airway inflammation, remodeling, and hyperresponsiveness. Chop was found to exacerbate allergic airway inflammation by enhancing M2 programming in macrophages. Mechanistic studies characterized an IL‐4/signal transducer and activator of transcription 6/transcription factor EC (Tfec)/IL‐4 receptor &agr; positive feedback regulatory loop, in which IL‐4 induces Chop expression, which then promotes signal transducer and activator of transcription 6 signaling to transcribe Tfec expression. Finally, Tfec transcribes IL‐4 receptor &agr; expression to promote M2 programming in macrophages. Conclusions: Chop and ER stress are implicated in asthma pathogenesis, which involves regulation of M2 programming in macrophages.