lncRNA VELRP Modulates Pulmonary Arterial Smooth Muscle Cell Proliferation and Promotes Vascular Remodeling in Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension is a devastating vascular disorder characterized by extensive pulmonary vascular remodeling, ultimately leading to right ventricular failure and death. Activation of PDGF (platelet-derived growth factor) signaling promotes the hyperproliferation of pulmonary arterial smooth muscle cells (PASMCs), thus contributing to the pulmonary vascular remodeling. However, the molecular mechanisms that govern hyperproliferation of PASMCs induced by PDGF remain largely unknown, including the contribution of long noncoding RNAs (lncRNAs). In this study, we aimed to identify a novel lncRNA regulated by PDGF implicated in PASMC proliferation in pulmonary vascular remodeling. METHODS: RNA-sequencing analysis was conducted to identify a novel lncRNA named vessel-enriched lncRNA regulated by PDGF-BB (VELRP). Functional investigations of VELRP were performed using knockdown and overexpression strategies along with RNA sequencing. Validation of the function and potential mechanisms of VELRP were performed through Western blot, RNA immunoprecipitation, and chromatin immunoprecipitation assays. RESULTS: We identified a novel vessel-enriched lncRNA with an increased response to PDGF-BB stimulus. VELRP was identified as an evolutionarily conserved RNA molecules. Modulation of VELRP in PASMCs significantly altered cell proliferation. Mechanistically, VELRP enhances trimethylation of H3K4 by interacting with WDR5 (WD repeat-containing protein 5), leading to increased expression of CDK (cyclin-dependent kinase) 1, CDK2, and CDK4 and consequent hyperproliferation of PASMCs. The pathological relevance of VELRP upregulation in pulmonary artery was confirmed using rat pulmonary hypertension models in vivo, as well as in PASMCs from patients with idiopathic pulmonary arterial hypertension patients. Specific knockdown of VELRP in smooth muscle cells using adeno-associated virus type 9 SM22α (smooth muscle protein 22α) promoter-shRNA-mediated silencing of VELRP resulted in a significant decrease in right ventricular systolic pressure and vascular remodeling in rat pulmonary hypertension model. CONCLUSIONS: VELRP, as an lncRNA upregulated by PDGF-BB, mediates PASMC proliferation via WDR5/CDK signaling. In vivo studies demonstrate that targeted intervention of VELRP in smooth muscle cells can prevent the development of pulmonary hypertension.

N6-methyladenosine-driven miR-143/145-KLF4 circuit orchestrates the phenotypic switch of pulmonary artery smooth muscle cells.

Pulmonary hypertension (PH) is characterized by vascular remodeling predominantly driven by a phenotypic switching in pulmonary artery smooth muscle cells (PASMCs). However, the underlying mechanisms for this phenotypic alteration remain incompletely understood. Here, we identified that RNA methyltransferase METTL3 is significantly elevated in the lungs of hypoxic PH (HPH) mice and rats, as well as in the pulmonary arteries (PAs) of HPH rats. Targeted deletion of Mettl3 in smooth muscle cells exacerbated hemodynamic consequences of hypoxia-induced PH and accelerated pulmonary vascular remodeling in vivo. Additionally, the absence of METTL3 markedly induced phenotypic switching in PASMCs in vitro. Mechanistically, METTL3 depletion attenuated m6A modification and hindered the processing of pri-miR-143/145, leading to a downregulation of miR-143-3p and miR-145-5p. Inhibition of hnRNPA2B1, an m6A mediator involved in miRNA maturation, similarly resulted in a significant reduction of miR-143-3p and miR-145-5p. We demonstrated that miR-145-5p targets Krüppel-like factor 4 (KLF4) and miR-143-3p targets fascin actin-bundling protein 1 (FSCN1) in PASMCs. The decrease of miR-145-5p subsequently induced an upregulation of KLF4, which in turn suppressed miR-143/145 transcription, establishing a positive feedback circuit between KLF4 and miR-143/145. This regulatory circuit facilitates the persistent suppression of contractile marker genes, thereby sustaining PASMC phenotypic switch. Collectively, hypoxia-induced upregulation of METTL3, along with m6A mediated regulation of miR-143/145, might serve as a protective mechanism against phenotypic switch of PASMCs. Our results highlight a potential therapeutic strategy targeting m6A modified miR-143/145-KLF4 loop in the treatment of PH.

N6-methyladenosine modification of KLF2 may contribute to endothelial-to-mesenchymal transition in pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a progressive disease characterized by pulmonary vascular remodeling. Increasing evidence indicates that endothelial-to-mesenchymal transition (EndMT) in pulmonary artery endothelial cells (PAECs) is a pivotal trigger initiating this remodeling. However, the regulatory mechanisms underlying EndMT in PH are still not fully understood. METHODS: Cytokine-induced hPAECs were assessed using RNA methylation quantification, qRT-PCR, and western blotting to determine the involvement of N6-methyladenosine (m6A) methylation in EndMT. Lentivirus-mediated silencing, overexpression, tube formation, and wound healing assays were utilized to investigate the function of METTL3 in EndMT. Endothelial-specific gene knockout, hemodynamic measurement, and immunostaining were performed to explore the roles of METTL3 in pulmonary vascular remodeling and PH. RNA-seq, RNA Immunoprecipitation-based qPCR, mRNA stability assay, m6A mutation, and dual-luciferase assays were employed to elucidate the mechanisms of RNA methylation in EndMT. RESULTS: The global levels of m6A and METTL3 expression were found to decrease in TNF-α- and TGF-ß1-induced EndMT in human PAECs (hPAECs). METTL3 inhibition led to reduced endothelial markers (CD31 and VE-cadherin) and increased mesenchymal markers (SM22 and N-cadherin) as well as EndMT-related transcription factors (Snail, Zeb1, Zeb2, and Slug). The endothelial-specific knockout of Mettl3 promoted EndMT and exacerbated pulmonary vascular remodeling and hypoxia-induced PH (HPH) in mice. Mechanistically, METTL3-mediated m6A modification of kruppel-like factor 2 (KLF2) plays a crucial role in the EndMT process. KLF2 overexpression increased CD31 and VE-cadherin levels while decreasing SM22, N-cadherin, and EndMT-related transcription factors, thereby mitigating EndMT in PH. Mutations in the m6A site of KLF2 mRNA compromise KLF2 expression, subsequently diminishing its protective effect against EndMT. Furthermore, KLF2 modulates SM22 expression through direct binding to its promoter. CONCLUSIONS: Our findings unveil a novel METTL3/KLF2 pathway critical for protecting hPAECs against EndMT, highlighting a promising avenue for therapeutic investigation in PH.

Cavity-Directed Synthesis of Labile Polyoxometalates for Catalysis in Confined Spaces.

The artificial microenvironments inside coordination cages have gained significant attention for performing enzyme-like catalytic reactions by facilitating the formation of labile and complex molecules through a "ship-in-a-bottle" approach. Despite many fascinating examples, this approach remains scarcely explored in the context of synthesizing metallic clusters such as polyoxometalates (POMs). The development of innovative approaches to control and influence the speciation of POMs in aqueous solutions would greatly advance their applicability and could ultimately lead to the formation of elusive clusters that cannot be synthesized by using traditional methods. In this study, we employ host-guest stabilization within a coordination cage to enable a novel cavity-directed synthesis of labile POMs in aqueous solutions under mild conditions. The elusive Lindqvist [M6O19]2- (M=Mo or W) POMs were successfully synthesized at room temperature via the condensation of molybdate or tungstate building blocks within the confined cavity of a robust and water-soluble Pt6L4(NO3)12 coordination cage. Importantly, the encapsulation of these POMs enhances their stability in water, rendering them efficient catalysts for environmentally friendly and selective sulfoxidation reactions using H2O2 as a green oxidant in a pure aqueous medium. The approach developed in this paper offers a means to synthesize and stabilize the otherwise unstable metal-oxo clusters in water, which can broaden the scope of their applications.

Correlation between oxygenation status of extra-synovial tissue of wrist and disease activity in rheumatoid arthritis: a photoacoustic imaging study.

OBJECTIVES: Rheumatoid arthritis (RA) is characterized by hypoxia in the synovial tissue. While photoacoustic imaging (PA) offers a method to evaluate tissue oxygenation in RA patients, studies exploring the link between extra-synovial tissue of wrist oxygenation and disease activity remain scarce. We aimed to assess synovial oxygenation in RA patients using a multimodal photoacoustic-ultrasound (PA/US) imaging system and establish its correlation with disease activity. METHODS: A retrospective study was conducted on 111 patients with RA and 72 healthy controls from 2022 to 2023. Dual-wavelength PA imaging quantified oxygen saturation (So2) levels in the synovial membrane and peri-wrist region. Oxygenation states were categorised as hyperoxia, intermediate oxygenation, and hypoxia based on So2 values. The association between oxygenation levels and the clinical disease activity index was evaluated using a one-way analysis of variance, complemented by the Kruskal-Wallis test with Bonferroni adjustment. RESULTS: Of the patients with RA, 39 exhibited hyperoxia, 24 had intermediate oxygenation, and 48 had hypoxia in the wrist extra-synovial tissue. All of the control participants exhibited the hyperoxia status. Oxygenation levels in patients with RA correlated with clinical metrics. Patients with intermediate oxygenation had a lower disease activity index compared with those with hypoxia and hyperoxia. CONCLUSION: A significant correlation exists between wrist extra-synovial tissue oxygenation and disease activity in patients with RA.

Metabolic profiling of patients with different idiopathic inflammatory myopathy subtypes reveals potential biomarkers in plasma.

Idiopathic inflammatory myopathy (IIM) are heterogeneous autoimmune diseases that primarily affect the proximal muscles. IIM subtypes include dermatomyositis (DM), polymyositis (PM), and anti-synthetase syndrome (ASS). Metabolic disturbances may cause irreversible structural damage to muscle fibers in patients with IIM. However, the metabolite profile of patients with different IIM subtypes remains elusive. To investigate metabolic alterations and identify patients with different IIM subtypes, we comprehensively profiled plasma metabolomics of 46 DM, 13 PM, 12 ASS patients, and 30 healthy controls (HCs) using UHPLC-Q Exactive HF mass spectrometer. Multiple statistical analyses and random forest were used to discover differential metabolites and potential biomarkers. We found that tryptophan metabolism, phenylalanine and tyrosine metabolism, fatty acid biosynthesis, beta-oxidation of very long chain fatty acids, alpha-linolenic acid and linoleic acid metabolism, steroidogenesis, bile acid biosynthesis, purine metabolism, and caffeine metabolism are all enriched in the DM, PM, and ASS groups. We also found that different subtypes of IIM have their unique metabolic pathways. We constructed three models (five metabolites) to identify DM, PM, ASS from HC in the discovery and validation sets. Five to seven metabolites can distinguish DM from PM, DM from ASS, and PM from ASS. A panel of seven metabolites can identify anti-melanoma differentiation-associated gene 5 positive (MDA5 +) DM with high accuracy in the discovery and validation sets. Our results provide potential biomarkers for diagnosing different subtypes of IIM and a better understanding of the underlying mechanisms of IIM.

Decreased AGGF1 facilitates the progression of placenta accreta spectrum via mediating the P53 signaling pathway under the regulation of miR-1296-5p.

Placenta accreta spectrum (PAS), an emerging health issue worldwide, is the major causative factor of maternal morbidity and mortality in modern obstetrics, but limited studies have contributed to our understanding of the molecular biology of PAS. This study addressed the expression of AGGF1 and its specific role in the etiology of PAS. The expression of AGGF1 in the placentas of PAS was determined by quantitative PCR, western blot and immunohistochemistry. CCK-8 assay, wound healing assay, Transwell invasion assay and flow cytometry assay were performed to monitor cell proliferation, migration, invasion and apoptosis. The interaction between miR-1296-5p and AGGF1 was detected by dual-luciferase reporter gene assay. Results showed that the mRNA and protein expression of AGGF1 was decremented in placental tissues of PAS patients, compared with samples from women with placenta previa and normal pregnant women. Downregulation of AGGF1 promoted cell proliferation, invasion and migration, inhibited apoptosis in vitro, decreased P53 and Bax expression, and simultaneously increased Bcl-2 expression, whereas overexpression of AGGF1 had the opposite results. Additionally, the dual-luciferase assay confirmed AGGF1 as a target gene of miR-1296-5p in placental tissues of PAS. Particularly, miR-1296-5p fostered HTR8/SVneo cell proliferation, invasion, repression of apoptosis and regulation of P53 signaling axis by downregulating AGGF1 expression. Collectively, our study accentuated that downregulation of placental AGGF1 promoted trophoblast over-invasion by mediating the P53 signaling pathway under the regulation of miR-1296-5p.

Clinical Significance of Different Profiles of anti-Ro Antibodies in Connective Tissue Diseases.

Objective: Anti-Ro60 and anti-Ro52 antibodies are associated with different connective tissue diseases (CTDs). However, the clinical significance of anti-Ro antibodies is not always consistent among different global regions. The aim of this study was to investigate the clinical characteristics of patients with anti-Ro antibodies. Methods: A total of 1596 inpatients with anti-Ro antibodies were included in the study. Demographic, clinical, and serological data were compared between individuals with different profiles of anti-Ro antibodies: patients with anti-Ro52 antibodies alone, patients with anti-Ro60 antibodies alone, and patients with combined anti-Ro52 and anti-Ro60 antibodies. Results: Of the 1596 patients, 1362 (85.3%) were female, the mean age was 45.5 years, and systemic lupus erythematosus (SLE) (46.0%) and Sjogren's syndrome (SS) (19.0%) were the most common CTD diagnoses. Among the patients with anti-Ro52 antibodies alone, idiopathic inflammatory myopathy (18.8%) and SLE (17.6%) were the most common CTD diagnoses. The coexistent autoantibodies of this group were significantly lower compared with those of the other two groups, while the presence of anti-Jo1 antibodies were significantly higher compared with those of the other two groups (3.7% vs. 0.6% vs. 1.9%, p = 0.029). In addition, the patients with isolated anti-Ro52 antibodies were more likely to suffer from interstitial lung disease (35.5% vs. 11.3% vs. 13.7%, p < 10-4) and pulmonary arterial hypertension (10.1% vs. 5.3% vs. 3.6%, p = 0.001) compared with the other two groups of patients. Compared with patients with isolated anti-Ro52 or anti-Ro60 antibodies, the patients with combined anti-Ro52 and anti-Ro60 antibodies were more likely to suffer from xerophthalmia and xerostomia. Furthermore, hypocomplementemia, hyperglobulinemia, and proteinuria were particularly prevalent in patients with anti-Ro60 antibodies. Conclusion: Different profiles of anti-Ro antibodies were significantly associated with clinical phenotypic features in CTDs, indicating the potential diagnostic and prognostic value of these antibodies in clinical practice.

Clinical significance of anti-rheumatoid arthritis 33 antibody in patients with systemic lupus erythematosus.

Although anti-rheumatoid arthritis (RA) 33 antibodies have been reported to be present in various connective tissue diseases (CTDs), the clinical significance of anti-RA33 in CTDs is still obscure. This study was performed to explore the clinical significance of anti-RA33 in CTDs, especially systemic lupus erythematosus (SLE). A total of 565 patients with positive anti-nuclear antibodies who had been tested for anti-RA33 were included in this study and were further classified into RA33-positive and RA33-negative groups. The association between anti-RA33 and the clinical features of CTDs was examined. Receiver operating characteristic (ROC) analysis was performed to explore the diagnostic value of anti-RA33 in SLE and SLE-related organ involvement. The results showed that SLE was the most common disease in CTD patients positive for anti-RA33 (48.8%). Compared with the RA33-negative group, higher proportions of SLE-associated antibodies and SLE patients with a high disease activity as well as lower levels of serum complement components were observed in the RA33-positive group (all p < 0.05). Furthermore, CTD patients with positive anti-RA33 were more likely to suffer from mucocutaneous and hematological involvement as well as interstitial lung disease (all p < 0.05). ROC analysis revealed an area under the curve value of 0.634 (95% confidence interval: 0.587-0.681) for anti-RA33 in the diagnosis of SLE, with a specificity and sensitivity of 92.9% and 13.5%, respectively. Taken together, this study reveals a significant association between anti-RA33 and the clinical features of CTDs, especially SLE, indicating a potential clinical significance of anti-RA33 in the management of SLE.

Proteomics Analysis of Plasma-Derived Exosomes Unveils the Aberrant Complement and Coagulation Cascades in Dermatomyositis/Polymyositis.

Dermatomyositis and polymyositis (DM/PM) are systemic autoimmune diseases characterized by proximal muscle weakness. The underlying pathogenetic mechanism of this disease remains under-researched. Here, using proteomics analysis, a great overlap of differentially expressed plasma exosomal proteins involved in the complement and coagulation cascade pathway, including FGA, FGB, FGG, C1QB, C1QC, and VWF, was identified in DM/PM patients versus healthy controls. Correlation analysis showed that the expression levels of complement-associated proteins (C1QB and C1QC) correlated positively with CRP, ESR, and platelet count. ROC curve analysis demonstrated that complement and coagulation cascade-associated proteins could be strong predictors for DM/PM. In addition, we also identified several other proteins that were differentially expressed in DM and PM. The selected candidate proteins were further validated by parallel reaction monitoring (PRM) and enzyme-linked immunosorbent assay (ELISA). Together, our findings indicate that these exosome-derived proteins might participate in microvascular damage in DM/PM through the activation of the complement and coagulation cascade pathway and function as biomarkers for the clinical diagnosis of DM/PM.