Dysfunctional APPL1-Mediated Epigenetic Regulation in Diabetic Vascular Injury.

BACKGROUND: APN (adiponectin) and APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) are potent vasculoprotective molecules, and their deficiency (eg, hypoadiponectinemia) contributes to diabetic vascular complications. However, the molecular mechanisms that govern their vasculoprotective genes as well as their alteration by diabetes remain unknown. METHODS: Diabetic medium-cultured rat aortic endothelial cells, mouse aortic endothelial cells from high-fat-diet animals, and diabetic human aortic endothelial cells were used for molecular/cellular investigations. The in vivo concept-prove demonstration was conducted using diabetic vascular injury and diabetic hindlimb ischemia models. RESULTS: In vivo animal experiments showed that APN replenishment caused APPL1 nuclear translocation, resulting in an interaction with HDAC (histone deacetylase) 2, which inhibited HDAC2 activity and increased H3Kac27 levels. Based on transcriptionome pathway-specific real-time polymerase chain reaction profiling and bioinformatics analysis, Angpt1 (angiopoietin 1), Ocln (occludin), and Cav1 (caveolin 1) were found to be the top 3 vasculoprotective genes suppressed by diabetes and rescued by APN in an APPL1-dependent manner. APN reverses diabetes-induced inhibition of Cav1 interaction with APPL1. APN-induced Cav1 expression was not affected by Angpt1 or Ocln deficiency, whereas APN-induced APPL1 nuclear translocation or upregulation of Angpt1/Ocln expression was abolished in the absence of Cav1 both in vivo and in vitro, suggesting Cav1 is upstream molecule of Angpt1/Ocln in response to APN administration. Chromatin immunoprecipitation-qPCR (quantitative polymerase chain reaction) demonstrated that APN caused significant enrichment of H3K27ac in Angpt1 and Ocln promoter region, an effect blocked by APPL1/Cav1 knockdown or HDAC2 overexpression. The protective effects of APN on the vascular system were attenuated by overexpression of HDAC2 and abolished by knocking out APPL1 or Cav1. The double knockdown of ANGPT1/OCLN blunted APN vascular protection both in vitro and in vivo. Furthermore, in diabetic human endothelial cells, HDAC2 activity is increased, H3 acetylation is decreased, and ANGPT1/OCLN expression is reduced, suggesting that the findings have important translational implications. CONCLUSIONS: Hypoadiponectinemia and dysregulation of APPL1-mediated epigenetic regulation are novel mechanisms leading to diabetes-induced suppression of vasculoprotective gene expression. Diabetes-induced pathological vascular remodeling may be prevented by interventions promoting APPL1 nuclear translocation and inhibiting HDAC2.

Adiponectin-mediated promotion of CD44 suppresses diabetic vascular inflammatory effects.

While adiponectin (APN) was known to significantly abolish the diabetic endothelial inflammatory response, the specific mechanisms have yet to be elucidated. Aortic vascular tissues from mice fed normal and high-fat diets (HFD) were analyzed by transcriptome analysis. GO functional annotation showed that APN inhibited vascular endothelial inflammation in an APPL1-dependent manner. We confirmed that activation of the Wnt/ß-catenin signaling plays a key role in APN-mediated anti-inflammation. Mechanistically, APN promoted APPL1/reptin complex formation and ß-catenin nuclear translocation. Simultaneously, we identified APN promoted the expression of CD44 by activating TCF/LEF in an APPL1-mediated manner. Clinically, the serum levels of APN and CD44 were decreased in diabetes; the levels of these two proteins were positively correlated. Functionally, treatment with CD44 C-terminal polypeptides protected diabetes-induced vascular endothelial inflammation in vivo. Collectively, we provided a roadmap for APN-inhibited vascular inflammatory effects and CD44 might represent potential targets against the diabetic endothelial inflammatory effect.

Effect of divalent cations on electrochemiluminescence of metal-organic frameworks in bioassay.

The structural characteristics of electrochemiluminescent (ECL) microreticula enabled flexible designs for probing specific molecules. However, bioanalysts paid little attention to the impact of concomitant electrolytic carriers on ECL responsiveness of these grids. Our previous finding confirmed the collisional quenching of ECL radiative secondary building units from polarized Br- and I-. To further address this concern, herein typical cationic commonplaces including Na+, K+, Ca2+, … in buffer plus regular transition metals - their influences upon the ECL performance of a well-defined zinc porphyrin-organic framework (ZnPOF) were inspected in a one-by-one manner. Except for Na+/K+, a dozen of divalent metal chlorides exerted an adverse effect in the form of Stern-Volmer quenching on the ECL brightness, which was illuminated to be cation channeling in open voids of ZnPOFs and bonding with O2-reactive sites as exemplified by the model Ca2+ via systematic compositional investigation. Following this principle, a simplistic Ca2+-sensitive sensor was developed for quantitative evaluation of health-care calcium supplements with high precision. Above all, this work highlighted the non-negligible interference from those Mn + requisites to the susceptible MOF-based ECL, which should be paid extra attention in bioassays and mechanistic analyses.

Deletion of large-conductance calcium-activated potassium channels promotes vascular remodelling through the CTRP7-mediated PI3K/Akt signaling pathway.

The large-conductance calcium-activated potassium (BK) channel is a critical regulator and potential therapeutic target of vascular tone and architecture, and abnormal expression or dysfunction of this channel is linked to many vascular diseases. Vascular remodelling is the early pathological basis of severe vascular diseases. Delaying the progression of vascular remodelling can reduce cardiovascular events, but the pathogenesis remains unclear. To clarify the role of BK channels in vascular remodelling, we use rats with BK channel α subunit knockout (BK α ‒/‒). The results show that BK α ‒/‒ rats have smaller inner and outer diameters, thickened aortic walls, increased fibrosis, and disordered elastic fibers of the aortas compared with WT rats. When the expression and function of BK α are inhibited in human umbilical arterial smooth muscle cells (HUASMCs), the expressions of matrix metalloproteinase 2 (MMP2), MMP9, and interleukin-6 are enhanced, while the expressions of smooth muscle cell contractile phenotype proteins are reduced. RNA sequencing, bioinformatics analysis and qPCR verification show that C1q/tumor necrosis factor-related protein 7 ( CTRP7) is the downstream target gene. Furthermore, except for that of MMPs, a similar pattern of IL-6, smooth muscle cell contractile phenotype proteins expression trend is observed after CTRP7 knockdown. Moreover, knockdown of both BK α and CTRP7 in HUASMCs activates PI3K/Akt signaling. Additionally, CTRP7 is expressed in vascular smooth muscle cells (VSMCs), and BK α deficiency activates the PI3K/Akt pathway by reducing CTRP7 level. Therefore, we first show that BK channel deficiency leads to vascular remodelling. The BK channel and CTRP7 may serve as potential targets for the treatment of cardiovascular diseases.

Potential regulatory role of miRNA and mRNA link to metabolism affected by chronic intermittent hypoxia.

Aim: Intermittent hypoxia (IH) is the prominent feature of obstructive sleep apnea (OSA) pathophysiology, which is an in dependent risk factor of cardiovascular complications. The effects of IH on adipocyte metabolism were explored by high-throughput sequencing technology. Methods: Plasma was collected from OSA patients and control group to perform mRNA sequencing. 3T3-L1 cells were differentiated into adipocytes then subjected to a 5%-21% O2 hypoxic environment (IH) for 24 h. High-throughput sequencing method was used to determine differential mRNA and miRNA patterns in fat cells exposed to IH. We then performed Gene Ontology (GO) analysis, identified relevant KEGG pathways and miRNA-target-pathways. Results: Sequencing data showed that OSA affected the expression of 343 mRNAs in the plasma. At the same time, we found that IH affected the expression of 3034 mRNAs in the adipocytes. In addition, 68 differentially expressed mRNAs were overlapped in plasma from OSA patient and IH-induced adipocyte model. We observe that 68 differential genes could be connected to 49 reciprocally expressed miRNAs. We showed that IH significantly reduced the expression of miR-182-5p and miR-30c-2-3p. KEGG predicted that the function of expressed miR-182-5p and miR-30c-2-3p was enriched to AKT signaling pathway. Notably, IH activated PI3K/AKT pathway in fat cells. Conclusion: Our results demonstrated that IH might induce adipocyte metabolism by regulating miR-182-5p and miR-30c-2-3p.

Macrophage polarization is involved in liver fibrosis induced by ß 1-adrenoceptor autoantibody.

Accumulating evidence suggests that liver injury can be induced by the over-expression of ß 1-adrenergic receptors (ß 1-ARs). High titers of autoantibodies specific to ß 1-adrenergic receptors (ß 1-AA) are detected in the sera of heart failure patients, potentially playing agonist-like roles. However, the role of ß 1-AA in liver function has not been characterized. In this study, we collect the sera of primary biliary cholangitis (PBC) patients, a condition which easily develops into liver fibrosis, and analyze the relationship between PBC and ß 1-AA. A passive immunization model is established to assess the effect of ß 1-AA on the liver. Subsequently, the effect of ß 1-AA on macrophages is investigated in vitro. Results show that PBC patients have a high titer and ratio of ß 1-AA, compared to controls. Liver injury and fibrosis are induced by ß 1-AA. In vitro experiments with ROS probe demonstrate that ß 1-AA induces macrophages to produce ROS and secrete TNFα. These effects can be partially reversed by metoprolol, a blocker for ß 1-AR. Results from the transwell and phagocytosis assays show that ß 1-AA promotes macrophage migration and phagocytosis. FCM tests suggest that ß 1-AA induces the alteration of M1 rather than M2 markers in macrophages. Finally, the Annexin V/PI assay indicates that macrophage culture supernatants stimulated by ß 1-AA cause hepatocyte apoptosis. Overall, these results suggest that ß 1-AA is involved in PBC. The ß 1-AA-induced activation, phagocytosis and phenotypic modification of macrophages may play an important role in the development of hepatic fibrosis and injury.

[Research advances of occludin in vascular endothelial injury].

Endothelial tight junctions (TJs) serve as an important barrier in vascular endothelial structure and maintain vascular function homeostasis. Occludin, the most representative tight junction protein, is involved in sealing cell connections and maintaining the integrity and permeability of vascular endothelium. Recent studies have shown that alterations in the expression, distribution, and structure of endothelial TJs may lead to many related vascular diseases and pathologies (such as stroke, atherosclerosis, and pulmonary hypertension etc.). Here, we reviewed the research advances on the relationship between occludin and vascular endothelial injury, including the biological information of occludin, the signal pathways that occludin exerts the protective effect of vascular endothelium, and the relationship between occludin and vascular endothelial injury-related diseases.