Biology subjects

Mobasheri, A.

Publications and source records attributed to Mobasheri, A..

4 recordsLinked to original sources

bioRxiv2023

Age and anatomical region related differences in vascularization of the porcine meniscus using micro-computed tomography imaging

Meniscal lesions in vascularized regions are known to regenerate while lack of vascular supply leads to poor healing. Here we developed and validated novel methodology for three-dimensional structural analysis of meniscal vascular structures with high-resolution micro-computed tomography ({micro}CT). We collected porcine medial menisci from 10 neonatal (not-developed meniscus, n-) and 10 adults (fully developed meniscus, a-). The menisci were cut into anatomical regions (anterior horn (n-AH & a-AH), central body (n-CB & a-CB), and posterior horn (n-PH & a-PH). Specimens were cut in half, fixed, and one specimen underwent critical point drying and {micro}CT imaging, while other specimen underwent immunohistochemistry and vascularity biomarker CD31 staining for validation of {micro}CT. Parameters describing vascular structures were calculated from {micro}CT. The vascular network in neonatal spread throughout meniscus, while in adult was limited to a few vessels in outer region, mostly on femoral side. a-AH, a-CB, and a-PH had three, five, and seven times greater vascular volume than neonate, respectively. Moreover, thickness of blood vessels, in three regions, was six times higher in adult than in newborn. Finally, a-PH appeared to have thicker blood vessels than both a-AH and a-CB. For the first time, critical point drying-based {micro}CT imaging allowed detailed three-dimensional visualization and quantitative analysis of vascularized meniscal structures. We showed more vascularity in neonatal menisci, while adult menisci had fewer and thicker vascularity especially limited to the femoral surface which is involved in load transmission response, thus suggesting how nutritional support in this area of the outer zone is more necessary.

biophysics↗

bioRxiv2020

Combining segmental bulk- and single-cell RNA-sequencing to define the chondrocyte gene expression signature in the murine knee joint

ObjectiveDue to the small size of the murine knee joint, extracting the chondrocyte transcriptome from articular cartilage (AC) is a major technical challenge. In this study, we demonstrate a new and pragmatic approach of combining bulk RNA-sequencing (RNA-seq) and single cell (sc)RNA-seq to address this problem. DesignWe propose a new cutting strategy of the murine femur which produces three segments with a predictable mixed cell populations, where one segment contains AC and growth plate (GP) chondrocytes, another contains GP chondrocytes, and the last segment contains only bone and bone marrow. We analysed the bulk RNA-seq of the different segments to find common and distinct genes between the segments. Then, the segment containing AC chondrocytes was digested and analysed via scRNA-seq. ResultsDifferential expression analysis using bulk RNA-seq identified 350 candidate chondrocyte gene in the AC segment. Gene set enrichment analysis of these genes revealed biological processes related- and non-related to chondrocytes, including, cartilage development (adj. p-value: 3.45E-17) and endochondral bone growth (adj. p-value 1.22E-4), respectively. ScRNA-seq of the AC segment found a cluster of 131 cells containing mainly chondrocytes. This cluster had 759 differentially expressed genes which enriched for extracellular matrix organisation (adj. p-value 7.76E-40) and other joint development processes. The intersection of the gene sets of bulk- and scRNA-seq contained 75 genes, where all but ten genes were previously implicated in cartilage homeostasis or osteoarthritis (OA) progression. ConclusionsOur approach has the potential to detect the scarce disease phenotypes of chondrocytes in murine OA models.

molecular biology↗

bioRxiv2020

Clusterin secretion is attenuated by pro-inflammatory cytokines in culture models of cartilage degradation

Proteomic studies have implicated clusterin as a potential biomarker of osteoarthritis (OA). However, there are two isoforms of clusterin with opposing functions, and their roles in OA have not previously been clarified. The secreted form of clusterin (sCLU) is a cytoprotective extracellular chaperone which prevents protein aggregation and enhances cell proliferation and viability, whereas nuclear clusterin (nCLU) acts as a pro-death signal. In this study, we focused on the role of sCLU and used established, pathophysiologically relevant, in vitro culture models to validate this potential biomarker of cartilage degradation. The secretome of equine cartilage explants, osteochondral biopsies and chondrocytes was analysed by western blotting for released sCLU, cartilage oligomeric protein (COMP) and matrix metalloproteinases (MMP) 3 and 13, following treatment with or without pro-inflammatory cytokines interleukin-1{beta} (IL-1{beta}) and tumour necrosis factor- (TNF-). The amount of sulphated glycosaminoglycans (sGAG) released into the medium was determined by dimethylmethylene blue (DMMB) analysis. Clusterin mRNA expression was quantified by real-time PCR. MMP-3, MMP-13, COMP and sGAG released from explants and osteochondral biopsies was elevated with cytokine treatment, confirming cartilage degradation in these models. Release of sCLU was attenuated with cytokine treatment in all three in vitro models. Expression of clusterin mRNA in cartilage explants and chondrocytes was down-regulated 7-days post cytokine stimulation. Cytokine stimulation attenuated expression and secretion of sCLU, therefore potentially limiting the cytoprotection which sCLU provides. These observations further implicate sCLU as having a role in OA, and diagnostic value as a potential biomarker for cartilage degradation.

molecular biology↗

bioRxiv2020

Pro-inflammatory cytokines drive deregulation of potassium channel expression in primary synovial fibroblasts

The synovium secretes synovial fluid, but is also richly innervated with nociceptors and acts as a gateway between avascular joint tissues and the circulatory system. Resident fibroblast-like synoviocytes (FLS) calcium-activated potassium channels (KCa) change in activity in arthritis models and this correlates with FLS activation. ObjectiveTo investigate this activation in an in vitro model of inflammatory arthritis; 72hr treatment with cytokines TNF and IL1{beta}. MethodsFLS cells were isolated from rat synovial membranes. We analysed global changes in FLS mRNA by RNA-sequencing and then focused on FLS ion channels genes and corresponding FLS electrophysiological phenotype, finally modelling data with Ingenuity Pathway Analysis (IPA) and MATLAB. ResultsIPA showed significant activation of inflammatory, osteoarthritic and calcium signalling canonical pathways by cytokines, and we identified ~200 channel gene transcripts. The large KCa (BK) channel consists of the pore forming Kcnma1 together with {beta}-subunits. Following cytokine treatment, a significant increase in Kcnma1 RNA abundance was detected by qPCR and changes in several ion channels were detected by RNA-sequencing, including a loss of BK channel {beta}-subunit expression Kcnmb1/2 and increase in Kcnmb3. In electrophysiological experiments, there was a decrease in overall current density at 20mV without change in chord conductance at this potential. ConclusionTNF and IL1{beta} treatment of FLS in vitro recapitulated several common features of inflammatory arthritis at the transcriptomic level, including increase in Kcnma1 and Kcnmb3 gene expression.

bioinformatics↗