Addition of High Molecular Weight Hyaluronic Acid to Fibroblast-Like Stromal Cells Modulates Endogenous Hyaluronic Acid Metabolism and Enhances Proteolytic Processing and Secretion of Versican

Abstract

We have examined the effect of exogenous linear chain high molecular weight hyaluronic acid (HMW HA) on endogenously synthesized hyaluronic acid (HA) and associated binding proteins in primary cultures of fibroblast-like stromal cells that were obtained by collagenase digestion of the murine peripatellar fat pad. The cultures were expanded in DMEM that was supplemented with fetal bovine serum and basic fibroblast growth factor (bFGF) then exposed to macrophage-colony-stimulating factor (MCSF) to induce macrophage properties, before activation of inflammatory pathways using E. coli lipopolysaccharide (LPS). Under all culture conditions, a significant amount of endogenously synthesized HA localized in LAMP1-positive lysosomal vesicles. However, this intracellular pool was depleted after the addition of exogenous HMW HA and was accompanied by enhanced proteolytic processing and secretion of de novo synthesized versican, much of which was associated with endosomal compartments. No changes were detected in synthesis, secretion, or proteolytic processing of aggrecan or lubricin (PRG4). The addition of HMW HA also modulated a range of LPS-affected genes in the TLR signaling and phagocytosis pathways, as well as endogenous HA metabolism genes, such as Has1, Hyal1, Hyal2, and Tmem2. However, there was no evidence for association of endogenous or exogenous HMW HA with cell surface CD44, TLR2 or TLR4 protein, suggesting that its physiochemical effects on pericelluar pH and/or ionic strength might be the primary modulators of signal transduction and vesicular trafficking by this cell type. We discuss the implications of these findings in terms of a potential in vivo effect of therapeutically applied HMW HA on the modification of osteoarthritis-related joint pathologies, such as pro-inflammatory and degradative responses of multipotent mesenchymal cells residing in the synovial membrane, the underlying adipose tissue, and the articular cartilage surface.

Development and Characterization of a Topically Deliverable Prophylactic Against Oxidative Damage in Cochlear Cells

“…Our data (MTS assay and HA-BODIPY cellular tracking) indicate that HA and HAM are co-localizing with the mitochondria and potentially with mitochondrial-lysosomal complexes (Todkar et al, 2017). In other tissues, HA has been reported to localize in the lysosome (Xue et al, 2020); therefore, further investigation of the exact location of HAM in cellular structures would be warranted, especially given that the amount of information available on the role of HA in cochlear structures and hearing is extremely scarce. We were unable to pinpoint the exact mechanism of protection, although we excluded the involvement of the PPP pathway, or at least the involvement of the PPP-associated NADP/NADPH ratio (Kuehne et al, 2015).…”

Exosomes from dysfunctional chondrocytes affect osteoarthritis in Sprague-Dawley rats through FTO-dependent regulation of PIK3R5 mRNA stability

“…In a previous study, it was shown that macrophages with different polarization types have different effects on the development of OA. 3,20 After co-culturing primary chondrocytes with macrophages, it was strikingly found that ECM degradation in chondrocytes was significantly reduced (4h, first graph: both p < 0.001; 4h, second graph: shScr vs shONACT-#1: p = 0.002, shScr vs shONACT-#2: p < 0.001; 4i, first graph: shScr vs shONACT-#1: p = 0.001, shScr vs shONACT-#2: p < 0.001; 4i, second graph: shScr vs shONACT-#1: p = 0.010, shScr vs shONACT-#2: p = 0.006; 4j, first graph: shScr vs shONACT-#1: p = 0.002, shScr vs shONACT-#2: p < 0.001; 4j, second graph: shScr vs shONACT-#1: p = 0.007, shScr vs shONACT-#2: p= 0.002; 4k, first graph: both p < 0.001; 4k, second graph: shScr vs shONACT-#1: p < 0.002, shScr vs shONACT-#2: p < 0.001; all one-way ANOVA) after co-culturing with macrophages with shOANCT, and the percentage of apoptosis was significantly reduced (shScr vs shONACT-#1: p = 0.002, shScr vs shONACT-#2: p < 0.001; one-way ANOVA) (Figures 4h to 4l). Knockdown of OANCT relieves symptoms in rats with OA.…”

New Insights From Single-Cell Sequencing Data: Synovial Fibroblasts and Synovial Macrophages in Rheumatoid Arthritis

“…CD55+ fibroblasts locate to the intimal lining and are responsible for synovial fluid formation and turnover. Of note, hyaluronan synthase 1 (HAS1) ( 53 , 54 ), lubricant PRG4 ( 55 ) and DNASE1L3 were highly expressed. Previous studies have shown that lubricin/proteomeglycan-4 (PRG4) is a mucus glycoprotein secreted by synovial fibroblasts and superficial chondrocytes, which has a variety of homeostasis effects in the joint and play an anti-inflammatory role by combining with TLR2 and TLR4 ( 56 ).…”