RHEB gene therapy maintains the chondrogenic characteristics and protects cartilage tissue from degenerative damage during experimental murine osteoarthritis

Abstract

Objective: Osteoarthritis (OA) is characterized by cartilage degeneration resulting from hypertrophic changes in chondrocytes caused by altered gene expression. The involvement of Ras homolog enriched in brain (RHEB) in OA regulation is unclear. Methods: Human knee articular cartilage samples-were analyzed for structural and biological changes by histology, immunohistochemistry, real time PCR and western blotting. OA-mouse model developed by surgical destabilization of the medial meniscus (DMM) were treated with adenovirus harboring Rheb gene to analyze onset and progression of OA. Histological scoring, immunohistochemistry, and TUNEL assay was performed to assess cartilage damage across the entire joint. Results: Human and mouse OA cartilage is degenerated and has markedly reduced levels of RHEB. Human OA-degenerated chondrocytes (DC) exhibited a fibroblastic phenotype and 80 % of degenerative cartilage were senescent, with higher levels of reactive oxygen species (ROS). Gene expression analysis of DC revealed almost no COL2A1 expression and reduced SOX9 and RHEB expression. Transient transfection of RHEB rescued the DC phenotype and reduced senescence and ROS levels markedly. RHEB overexpression also increased COL2A1 and SOX9 expression. In an OA-mouse model, the Rheb protein level decreased as the severity of OA increased. Ectopic expression of Rheb using adenovirus in mouse-OA cartilage suppressed surgically-induced OA pathogenesis accompanied by modulation of Adamts5, Mmp 13, Col 10, and Col2a1 expression. Rheb induction significantly reduced apoptosis in OA-cartilage. Conclusion: RHEB plays an important role in maintaining the chondrogenic characteristics of chondrocytes, and has potential in preventing progression of OA in the destabilize the medial meniscus (DMM) mouse model of OA.

SPRY4 acts as an indicator of osteoarthritis severity and regulates chondrocyte hypertrophy and ECM protease expression

“…1b , expression levels of chondrogenic marker genes ( ACAN, SOX9 , and COL2 ) were lower, whereas the expression levels of hypertrophic marker genes ( RUNX2 and MMP13 ) were higher in the DCs than in the HCs. These results support those of previous studies 25 , 26 . Interestingly, the SPRY4 protein expression level was notably lower in degenerated cartilage than in healthy cartilage (Fig.…”

“…2014-096). Healthy and degenerated cartilage tissues were separated based on a previously described protocol 26 . Briefly, the white-colored, intact, abundant hyaline cartilage tissues were classified as relatively healthy tissue, whereas the red-colored, collapsed, fibrous cartilage tissues were classified as relatively degenerated tissue.…”

“…Chondrocytes and cartilage samples were fixed with 4% (w/v) paraformaldehyde (PFA; Biosesang, Seongnam, Korea). In the case of cartilage, samples were decalcified in a decalcification solution (BBC Biochemical, WA) for 1 week and processed as described previously 26 . After fixation, whole chondrocytes and cartilage specimens were paraffinized and hydrated before staining.…”

“…Eight-week-old healthy, male Sprague-Dawley (SD) rats (Orient-bio, Seongnam, Korea) were anesthetized with zoletil (50 mg/kg) and rompun (10 mg/kg). OA was induced by dissecting the medial meniscus to initiate degeneration and collapse of the right knee joint of the hind limb for the OA-induced group ( n = 4), as described previously 26 . Rats in the control group ( n = 4) were surgically treated the same without dissecting the medial meniscus.…”

Proteomic Analysis of Red Ginseng on Prolonging the Life Span of Male Drosophila melanogaster

“…The mTOR pathway remained relatively balanced and was not overactivated. In addition, Rheb can reduce reactive oxygen species (ROS) and oxidative damage independently of the mTOR pathway (Ashraf et al, 2019). On feeding D. melanogaster RG, Rheb may reduce the oxidative damage and prolong the life span.…”