Microscopic polyangiitis plasma-derived exosomal miR-1287-5p induces endothelial inflammatory injury and neutrophil adhesion by targeting CBL.

Background: An inflammatory environment around the vessel wall caused by leukocyte infiltration is one of the characteristic histopathological features of microscopic polyangiitis (MPA); however, the pathogenic mechanisms are not fully understood. Studies have found that circulating microRNA (miRNA) can be used as potential biomarkers for the diagnosis and classification of anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitides (AAV), and the E3 ubiquitin ligase casitas B-lineage lymphoma (CBL) seems to be associated with inflammation. In addition, evidence indicates that miRNA can be tracked into exosomes and transferred into recipient cells to mediate the process of vascular endothelial injury. Herein, we aimed to identify the profiles of exosomal miRNA, and determine the effect of exosomal miR-1287-5p and its target gene CBL on vascular endothelial cells in MPA. Method: We isolated plasma exosomes from patients with MPA (MPA-exo) and healthy controls (HC-exo) by ultracentrifugation and conducted exosome small-RNA sequencing to screen differential miRNA expression in MPA-exo (n = 3) compared to HC-exo (n = 3). We measured the expression levels of miR-1303, miR-1287-5p, and miR-129-1-3p using quantitative reverse transcription-polymerase chain reaction (qRT-PCR, n = 6) and performed dual luciferase reporter gene assays to confirm the downstream target gene of miR-1287-5p. In addition, we treated human umbilical vein endothelial cell (HUVEC) with MPA-exo, or transfected them with miR-1287-5p mimic/inhibitor or with CBL-siRNA/CBL-siRNA+ miR-1287-5p inhibitor. After cell culture, we evaluated the effects on vascular endothelial cells by examining the mRNA levels of IL-6, IL-8, MCP-1, ICAM-1 and E-selectin using qRT-PCR and performed neutrophil adhesion assay with haematoxylin staining. Result: Transmission electron microscopy, Western blot and nanoparticle tracking analysis showed that we successfully purified exosomes and MPA-exo could be absorbed into HUVEC. We screened a total of 1,077 miRNA by sequencing and observed a high abundance of miR-1287-5p in the exosomes obtained from MPA plasma. The dual luciferase reporter assay identified CBL as a downstream target gene of miR-1287-5p, and the results revealed that MPA-exo decreased CBL protein expression in HUVEC. In addition, treatment with MPA-exo, up-regulating miR-1287-5p or silencing of CBL in HUVEC significantly increased the mRNA expression of inflammatory factors (including IL-6, IL-8, and MCP-1) and adhesion molecules (including ICAM-1 and E-selection) and promoted the adhesion of neutrophils to HUVEC. However, down-regulating miR-1287-5p had the opposite effect. Conclusion: Our study revealed that MPA-exo was involved in the intercellular transfer of miR-1287-5p and subsequently promote the development of acute endothelial injury in MPA. MiR-1287-5p and CBL agonists may be promising therapeutic approach for MPA-induced vascular inflammatory injury.

Association of ATG7 gene polymorphisms with microscopic polyangiitis in Chinese individuals.

Microscopic polyangiitis (MPA) is a type of antineutrophil cytoplasmic antibody (ANCA)-related vasculitis. Autophagy-related gene 7 (ATG7) protects against complicated disorder states in model organisms, but the way ATG7 dysfunction contributes to MPA remains elusive. This investigation assessed the impacts of ATG7 single-nucleotide polymorphisms (SNPs) on microscopic polyangiitis (MPA) in China. A total of 211 controls and 214 MPA patients were recruited and analyzed. Polymerase chain reaction (PCR) and high-throughput sequencing were adopted to detect the ATG7 SNPs (rs75492008, rs2594966, rs6442260 and rs8154), and stratification analysis, different genetic models and differences in allele and genotype frequencies were evaluated. Haplotype evaluation was performed after linkage disequilibrium (LD) analyses, and interactions between alleles were assessed. Generalized multifactor dimensionality reduction (GMDR) was adopted to analyze SNP-SNP interactions among the four ATG7 SNPs and phosphatidylinositol-4, 5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and unc-nc-like autophagy activating kinase 1 (ULK1) SNPs previously studied by our team. Relationships between ATG7 polymorphisms, disease activity biomarkers and therapeutic effects in MPA were analyzed. Sex stratification analysis of the rs2594966 GG genotype with codominant and recessive models showed OR=3.42, 95% CI [1.19-9.80], P=0.041 and OR=3.31, 95% CI [1.23-8.90], P=0.012, respectively. Haplotype G-G-C-T was related to an increased MPA risk (OR=1.5, 95% CI [0.999-2.266], P=0.029). Permutation testing of GMDR suggested that ATG7 rs6442260 and rs8154, PIK3CA rs1607237, and ULK1 rs4964879 might interact with each other in MPA development (P<0.05). Among 214 MPA patients, 79 available complete follow-up clinical datasets were gathered from September 2009 to October 2020, showing that rs75492008 and rs4964879 affect the correlation between C-reactive protein (CRP) and the erythrocyte sedimentation rate (ESR) in MPA activity. Patients with rs8154 TT and rs1607237 CC genotypes had better clinical treatment effects (P<0.05). Gene polymorphisms may be related to MPA in China, exhibiting correlation with MPA activity indicators, treatment and prognosis.

Analysis of rs1864182 and rs1864183 variants in ATG10 gene and antineutrophil cytoplasmic autoantibody-associated vasculitis in Chinese Guangxi population.

OBJECTIVES: To investigate the association of autophagy-associated gene 10 (ATG10) gene polymorphisms (rs1864182 and rs1864183) with antineutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) in Chinese Guangxi population. METHODS: The single nucleotide polymorphisms (SNPs) of ATG10 rs1864182 and rs1864183 in 395 participants (195 AAVs and 200 healthy controls) were genotyped. Generalized multiple dimensionality reduction (GMDR) was used to analyze the SNP-SNP interactions among two SNPs of ATG10 gene and other SNPs of autophagy gene previously studied by our research team. RESULTS: In this study, we found that the two ATG10 SNPs were not associated with AAV risk in Chinese Guangxi population. However, there were statistically significant differences in the incidence of hemoptysis, hematuria, and proteinuria among the three genotypes of ATG10 rs1864182 and rs1864183 (p < 0.05). Moreover, permutation test of GMDR suggested that immunity-related GTPase M(IRGM) rs4958847, autophagy-associated gene 7 (ATG7) rs6442260, ATG7 rs2594966, ATG10 rs1864183, protein kinase B(AKT2) rs3730051, and AKT2 rs11552192 might interact with each other in the process of developing AAV (p < 0.05). CONCLUSIONS: Our results indicated that there existed no association between ATG10 SNPs and AAV, and SNP-SNP interactions among IRGM rs4958847, ATG7 rs6442260, ATG7 rs2594966, ATG10 rs1864183, AKT2 rs3730051, and AKT2 rs11552192 may confer AAV risk in the Chinese Guangxi population.

Gene polymorphisms in ULK1 and PIK3CA are associated with the risk of microscopic polyangiitis in the Guangxi Zhuang Autonomous Region in China.

BACKGROUND: Microscopic polyangiitis (MPA) is a systemic autoimmune disease characterized by inflammation of small- and medium-sized blood vessels. Autophagy-related protein polymorphisms are involved in autoimmune disease. The aim of this study was to evaluate the effects of single-nucleotide polymorphisms (SNPs) in the ULK1 and PIK3CA genes on the risk of MPA. METHOD: A total of 208 patients with MPA and 211 controls in the Guangxi Zhuang Autonomous Region were recruited and analyzed. The SNPs selected were detected by polymerase chain reaction and high-throughput sequencing. The differences in allele and genotype frequency, various genetic models, and stratification analyses were evaluated, haplotype evaluation was performed after linkage disequilibrium analysis, and the interaction between gene alleles was analyzed. RESULTS: A statistically significant difference was detected in the genotypic distribution of two SNPs between the two groups: ULK1 rs4964879 (p = 0.019) and PIK3CA rs1607237 (p = 0.002). The results of the genetic models revealed that ULK1 rs4964879 and rs9481 were statistically significantly associated with an increased risk of MPA, whereas PIK3CA rs1607237 was associated with a reduced risk. The association between SNPs and MPA risk was affected by age, sex, and ethnicity. The ULK1 haplotype (G-T-A-C-G-A) and PIK3CA haplotype (T-G) were associated with a reduced risk of MPA, while the PIK3CA haplotype (C-G) was associated with an increased risk. CONCLUSION: In this study, polymorphisms in the autophagy-related genes ULK1 and PIK3CA and their association with MPA were examined. The results showed that the polymorphisms in ULK1 (rs4964879 and rs9481) and PIK3CA (rs1607237) were significantly associated with MPA risk in the Guangxi population. However, the molecular mechanisms are still unclear; basic science research and studies with larger samples are needed to confirm our conclusions and explore the underlying mechanisms.