Enhancement of LncRNA-HFRL expression induces cardiomyocyte inflammation, proliferation, and fibrosis via the sequestering of miR-149-5p-mediated collagen 22A inhibition.

Background: Long non-coding ribonucleic acids (lncRNAs) are believed to play crucial roles in cardiovascular diseases; however, details of the underlying mechanisms by which this occurs remain unclear. Methods: A mouse heart failure (HF) model was established using isoproterenol (ISO), and confirmed by immunostaining and echocardiography. RNA-sequencing was performed to screen the differential lncRNA expression profiles and heart failure relative lncRNA (HFRL) was selected as the target which was validated by quantitative real-time polymerase chain reaction (qRT-PCR). In HL-1 cells, the cardiac function, inflammatory, and fibrosis-related genes expression changes were examined by qRT-PCR after silencing of HFRL by lentivirus. Meanwhile, Cell Counting Kit-8 (CCK-8) assays were used to detect the effects of HFRL on the cell proliferation and viability. Reactive oxygen species (ROS) assays were also used to explore the role of HFRL in oxidative damage. Next, bioinformatics analysis was conducted to predict the potential binding microRNAs (mmu-miR-149-5p) to HFRL, which was confirmed by RNA-pulldown assays. The target gene of miR-149-5p was also predicted and further validated by Dual-luciferase reporter assays, qRT-PCR, and western blot. To investigate the synergistic regulatory effect of HFRL and miR-149-5p, HL-1 cells were infected with the lentivirus of HFRL with or without simultaneous knockdown of miR-149-5p. Then, qRT-PCR and western blot were used to examine cardiac function, inflammatory, and fibrosis-related gene expression changes, respectively. In HL-1 cells, CCK-8 assays were performed to detect the proliferation and viability. ROS assays were used to explore the oxidative damage. Results: The administration of ISO induced mice fibrosis, inflammation, and HF. The in-vitro results showed that knockdown of HFRL suppressed cardiomyocyte proliferation and viability, attenuated inflammatory, cardiac function, and fibrosis-related gene expression, and promoted oxidative damage. HFRL was found to bind to mmu-miR-149-5p and inversely target the 3'-untranscripted region of the collagen 22A1 gene. Thus, HFRL affected cardiomyocyte inflammation, proliferation, viability, oxidative damage, and pro-fibrotic function via sequestration to miR-149-5p. Conclusions: The HFRL/miR-149-5p axis plays an important role in regulating cardiac inflammation, proliferation, and fibrosis via a synergistic effect, which suggests that HFRL might be a novel target for HF.

Lack of the myotendinous junction marker col22a1 results in posture and locomotion disabilities in zebrafish.

The myotendinous junction (MTJ) is essential for the integrity of the musculoskeletal unit. Here, we show that gene ablation of the MTJ marker col22a1 in zebrafish results in MTJ dysfunction but with variable degrees of expression and distinct phenotypic classes. While most individuals reach adulthood with no overt muscle phenotype (class 1), a subset of the progeny displays severe movement impairment and die before metamorphosis (class 2). Yet all mutants display muscle weakness due to ineffective muscle force transmission that is ultimately detrimental for class-specific locomotion-related functions. Movement impairment at the critical stage of swimming postural learning causes class 2 larval death by compromising food intake. In class 1 adults, intensive exercise is required to uncover a decline in muscle performance, accompanied by higher energy demand and mitochondrial adaptation. This study underscores COL22A1 as a candidate gene for myopathies associated with dysfunctional force transmission and anticipates a phenotypically heterogeneous disease.

Are COL22A1 Gene Polymorphisms rs11784270 and rs6577958 Associated with Susceptibility to a Non-Contact Anterior Cruciate Ligament Injury in Polish Athletes?

Understanding the risk factors and etiology of ACL ruptures (anterior cruciate ligament) is crucial due to the injury's high occurrence, significant financial cost to the healthcare sector, and clinical consequences. In this study, we investigated the hypothesis that rs11784270 A/C and rs6577958 C/T SNPs (single gene polymorphism) within COL22A1 are associated with ACL ruptures (ACLR) in Polish soccer players. Methods: 228 athletes with ACLR (157 male, age 26 ± 4, 71 female, age 26 ± 6) and 202 control athletes (117 male, age 26 ± 6, 85 female, age 29 ± 2) engaged in the study. The buccal cell swabs were genotyped using TaqMan® pre-designed SNP genotyping assays, following the manufacturer's recommendations. The R program and SNPassoc package were used to determine the genotype and allele frequency distributions under the various inheritance models (co-dominant, dominant, recessive, and over-dominant). Further, p-values of <0.05 were considered statistically significant. We found no association between the analyzed polymorphisms and the risk of non-contact ACL ruptures in any of the studied models. Although the genetic variants investigated in this study were not associated with the risk of non-contact ACL ruptures, we assumed that the COL22A1 gene remains a candidate for further investigations in musculoskeletal injuries.

Decreased Trabecular Bone Mass in Col22a1-Deficient Mice.

The bone matrix is constantly remodeled by the coordinated activities of bone-forming osteoblasts and bone-resorbing osteoclasts. Whereas type I collagen is the most abundant bone matrix protein, there are several other proteins present, some of them specifically produced by osteoblasts. In a genome-wide expression screening for osteoblast differentiation markers we have previously identified two collagen-encoding genes with unknown function in bone remodeling. Here we show that one of them, Col22a1, is predominantly expressed in bone, cultured osteoblasts, but not in osteoclasts. Based on this specific expression pattern we generated a Col22a1-deficient mouse model, which was analyzed for skeletal defects by µCT, undecalcified histology and bone-specific histomorphometry. We observed that Col22a1-deficient mice display trabecular osteopenia, accompanied by significantly increased osteoclast numbers per bone surface. In contrast, cortical bone parameters, osteoblastogenesis or bone formation were unaffected by the absence of Col22a1. Likewise, primary osteoblasts from Col22a1-deficient mice did not display a cell-autonomous defect, and they did not show altered expression of Rankl or Opg, two key regulators of osteoclastogenesis. Taken together, we provide the first evidence for a physiological function of Col22a1 in bone remodeling, although the molecular mechanisms explaining the indirect influence of Col22a1 deficiency on osteoclasts remain to be identified.

eQTL variants in COL22A1 are associated with muscle injury in athletes.

The myotendinous junction (MTJ) is at high risk of muscle injury, and collagen XXII is strictly expressed at tissue junctions, specifically at the MTJ. We investigated the hypothesis that single-nucleotide polymorphisms (SNPs) related to collagen type XXII α-1 chain gene (COL22A1) mRNA expression are associated with susceptibility to muscle injury in athletes. History of muscle injury was assessed in 3,320 Japanese athletes using a questionnaire, and two expression quantitative trait loci (eQTL) SNPs for COL22A1 (rs11784270 A/C and rs6577958 T/C) were analyzed using the TaqMan SNP Genotyping Assay. rs11784270 [odds ratio (OR) = 1.80, 95% confidence interval (CI) = 1.27-2.62, P = 0.0006] and rs6577958 (OR = 1.45, 95% CI = 1.10-1.94, P = 0.0083) were significantly associated with muscle injury under A and T allele additive genetic models, respectively. These results suggest that the expression level of COL22A1 at the MTJ influences muscle injury risk in athletes.

Lgr5 and Col22a1 Mark Progenitor Cells in the Lineage toward Juvenile Articular Chondrocytes.

The synovial joint forms from a pool of progenitor cells in the future region of the joint, the interzone. Expression of Gdf5 and Wnt9a has been used to mark the earliest cellular processes in the formation of the interzone and the progenitor cells. However, lineage specification and progression toward the different tissues of the joint are not well understood. Here, by lineage-tracing studies we identify a population of Lgr5+ interzone cells that contribute to the formation of cruciate ligaments, synovial membrane, and articular chondrocytes of the joint. This finding is supported by single-cell transcriptome analyses. We show that Col22a1, a marker of early articular chondrocytes, is co-expressed with Lgr5+ cells prior to cavitation as an important lineage marker specifying the progression toward articular chondrocytes. Lgr5+ cells contribute to the repair of a joint defect with the re-establishment of a Col22a1-expressing superficial layer.

Comparison of the Transcriptomes and Proteomes of Serum Exosomes from Marek's Disease Virus-Vaccinated and Protected and Lymphoma-Bearing Chickens.

Marek's disease virus (MDV) is the causative agent of Marek's disease (MD), a complex pathology of chickens characterized by paralysis, immunosuppression, and T-cell lymphomagenesis. MD is controlled in poultry production via vaccines administered in ovo or at hatch, and these confer protection against lymphoma formation, but not superinfection by MDV field strains. Despite vaccine-induced humoral and cell-mediated immune responses, mechanisms eliciting systemic protection remain unclear. Here we report the contents of serum exosomes to assess their possible roles as indicators of systemic immunity, and alternatively, tumor formation. We examined the RNA and protein content of serum exosomes from CVI988 (Rispens)-vaccinated and protected chickens (VEX), and unvaccinated tumor-bearing chickens (TEX), via deep-sequencing and mass spectrometry, respectively. Bioinformatic analyses of microRNAs (miRNAs) and predicted miRNA targets indicated a greater abundance of tumor suppressor miRNAs in VEX compared to TEX. Conversely, oncomiRs originating from cellular (miRs 106a-363) and MDV miRNA clusters were more abundant in TEX compared to VEX. Most notably, mRNAs mapping to the entire MDV genome were identified in VEX, while mRNAs mapping to the repeats flanking the unique long (IRL/TRL) were identified in TEX. These data suggest that long-term systemic vaccine-induced immune responses may be mediated at the level of VEX which transfer viral mRNAs to antigen presenting cells systemically. Proteomic analyses of these exosomes suggested potential biomarkers for VEX and TEX. These data provide important putative insight into MDV-mediated immune suppression and vaccine responses, as well as potential serum biomarkers for MD protection and susceptibility.

A Human Skin Model Recapitulates Systemic Sclerosis Dermal Fibrosis and Identifies COL22A1 as a TGFß Early Response Gene that Mediates Fibroblast to Myofibroblast Transition.

: Systemic sclerosis (SSc) is a complex multi-system autoimmune disease characterized by immune dysregulation, vasculopathy, and organ fibrosis. Skin fibrosis causes high morbidity and impaired quality of life in affected individuals. Animal models do not fully recapitulate the human disease. Thus, there is a critical need to identify ex vivo models for the dermal fibrosis characteristic of SSc. We identified genes regulated by the pro-fibrotic factor TGFß in human skin maintained in organ culture. The molecular signature of human skin overlapped with that which was identified in SSc patient biopsies, suggesting that this model recapitulates the dermal fibrosis characteristic of the human disease. We further characterized the regulation and functional impact of a previously unreported gene in the setting of dermal fibrosis, COL22A1, and show that silencing COL22A1 significantly reduced TGFß-induced ACTA2 expression. COL22A1 expression was significantly increased in dermal fibroblasts from patients with SSc. In summary, we identified the molecular fingerprint of TGFß in human skin and demonstrated that COL22A1 is associated with the pathogenesis of fibrosis in SSc as an early response gene that may have important implications for fibroblast activation. Further, this model will provide a critical tool with direct relevance to human disease to facilitate the assessment of potential therapies for fibrosis.