Mechanism Of Histone Deacetylase -4 Treatment Of Osteoarthritis

BackgroundOsteoarthritis (OA) is the most prevalent disease in the elderly, associated with the irreversible degeneration of articular cartilage. Currently, no therapeutic methods exist for Prevention of articular cartilage degradation or treatment of its damage due to the precise pathogenetic mechanism of the OA remaining unknown. When osteoarthritis develops, articular cartilage chondrocytes undergo chondrocyte hypertrophic changes recapitulate some of the differentiation processes that occur in embryogenesis. Genetic modifications that stimulate chondrocyte hypertrophic changes are frequently associated with a higher incidence of OA or accelerated OA development. However, some previous studies have reported inhibition of chondrocyte hypertrophy is a promising approach for preventing or treating OA.Histone deacetylase4(HDAC4) belongs to class II HDACs modulating cell growth and differentiation by governing chromatin structure and repressing the activity of specific transcription factors. Genetic studies using knockout mice have demonstrated that overexpression of HDAC4in proliferating chondrocytes in vivo inhibits chondrocyte hypertrophy while knockout of HDAC4results in premature ossification of developing bones due to ectopic and early onset chondrocyte hypertrophic. This remarkable phenotype was originally attributed to the ability of HDAC4to bind and inhibit the activity of Runx2, a transcription factor necessary for chondrocyte hypertrophy during endochondral bone formation. Chondrocyte hypertrophic differentiation plays a critical role in endogenous bone formation. During endogenous bone development, specific biomarker characterized specific-stage chondrocyte phenotype. For example, the biomarkers of proliferating chondrocyte are Sry-related high-mobility-group box9(Sox9) or Collagen-2, prehypertrophic chondrocyte is Indian hedgehog (Ihh) and Hypertrophic chondrocyte are metalloproteinase-13(MMP-13) and Typex.These data, above mentioned, suggested:1), the significant mechanism in articular cartilage degeneration when OA occurred is chondrocyte hypertrophic change;2), Inhibition of articular chondrocyte hypertrophic change is a effective method to attenuate cartilage detruction in related to OA;3), when OA occurred, articular chondrocyte hypertrophic change is similar to chondrocyte differentiation in the process of endogenous bone formation;4), HDAC4do inhibit chondrocyte differentiation by decreasing Runx2transcriptional activity in endogenous bone development, but It is unclear what role of HDAC4in O A process.To detect whether HDAC4could prevent and attenuate articular cartilage degeneration in the process of OA, therefore, we try to carry out my experiment including three steps as followed. One step is collecting human knee joint cartilage samples and detecting trends of change of HDAC4, Runx2, Typex and MMP-13with destruction of cartilage. The second step is constructing adenovirus vector expressing human HDAC4and furthermore detecting whether Ad-HDAC4could successfully transduce human HDAC4gene to rat chondrtocyte in vivo and vitro, and express target protein. The third step is rat OA model is induced by the anterior cruciate ligament transection (ACLT), and injected Ad-HDAC4intraarticularly at1week postsurgery. All rats were raised for three monthes and the whole knee joints were extracted for result analysis.Materials and methods(1) Cartilage samples from the tibia obtained during total knee arthroplasty were divided into OA cartilage from the more affected compartment (usually medial) and "relatively normal" cartilage from the uninvolved compartment (usually lateral)[N=15,10females,5males, age68.5±9.6(mean±standard deviation (SD)), range57-81]. Age-matched normal cartilage samples were also obtained from knee joint undergoing amputation [N=7, six males, age58.8±13.6(mean±SD), range47-65].(2) Crtilage samples each group were sectioned at a thickness of6-um. Safranin-O staining was performed and the severity of cartilage damage was assessed using the modified Mankin grading system.(3) We firstly compared the HDAC4, Runx2, MMP-13and Typex levels between relative normal and OA regions from identical cartilage by western blot analysis. In this way, each individual acted as his/her own control. We additionally compared HDAC4, Runx2, MMP-13and Typex levels in normal and osteoarthritic human cartilage obtained from different individuals. Furthermore, we compared expression of HDAC4in the OA model induced by anterior cruciate ligament transsection (ACLT) and normal samples (n=10) from rat by RT-PCR analysis.(4) Adenovirus vector expressing human HDAC4was constructed in vitro and transfected rat chondrocyte. Efficiency of transfection were assessed by directly observing GFP expression in vitro and immunofluorescent staining in vivo, and expression of HDAC4was assessed by western blot analysis at48hours post transfection.(5) The method of intraarticular injection was evaluated by intraarticularly injected Indian ink.(6) Experimental OA was induced by anterior cruciate ligament transsection (ACLT) surgery, implemented as previously described by Gehua et al. Our intraarticular injection of HDAC4were processed according to Hsieh gene delivery protocol. All rats were randomly divided into five groups:ACLT (N=10), ACLT+Ad-GFP (N=10), ACLT+Ad-HDAC4-1(N=10), ACLT+Ad-HDAC4-3(N=10), and Sham (N=10). Experimental rats were injected intraarticularly with either AdHDAC4or AdGFP (2×108PFU), and Sham group has no any intraarticular injection treatment. Only once was acted as ACLT+Ad-HDAC4-1group and once per week for three consecutive weeks was acted as ACLT+Ad-HDAC4-3group1week after surgical operation.(7) all rats were intraperitoneal injected with BrdU (B5002, Sigma-Aldrich, St Louis, MO) dissolved in0.9%saline and0.007N NaOH at20mg/ml and once per day for two consecutive days and150mg/kg BrdU in total before sacrifice. At two monthes postsurgery, all rats were sacrificed for assessment including details as followed:1), Three rats each group were processed for gross morphology determined by Indian ink staining;2), Cartilage samples from three rats each group were processed for histopathologic slides and destruction was determined by safranin O/fast green staining.3), Total RNAs were extracted from cartilage samples three rats each group for RT-PCR analysis;4), Knee joint cartilage samples from three rats each group were processed for immunohistochemical (IHC) staining. Experimental data are displayed as means±st andard deviations and analyzed by one-way analysis of variance (ANOVA) post hoc tes t of LSD’s method. The statistical significance level of one-way ANOVA was set at P<0.05.ResultsWestern blot showed the level of HDAC4decreased, but Runx2, Typex and MMP-13increased with cartilage injury severity in human knee joint cartilage samples. The mRNA level of HDAC4in rat osteoarthritic-affected cartilage is significantly lower than that in normal rat cartilage. In vitro and vivo, rat articular cartilage was transfected by adenovirus expressing human HDAC4gene, which determined by GFP expression and immunofluorescent staining. Human HDAC4was successfully expressed by rat knee joint chondrocyte transfected by Ad-HDAC4, which determined by western blot annlysis. Gross morphology showed the cartilage destruction in both ACLT+Ad-GFP group and ACLT+Ad-HDAC4-3group has no difference, but ACLT+Ad-HDAC4-1group is decreased when compared to ACLT group, which was determined by Indian ink staining and Meachim Score.5-Bromo-2-deoxyUridine (BrDU) staining showed there are no difference in ratio positive cell (PC) of total cell (TC) between ACLT group and ACLT+Ad-GFP group, but the ratio PC/TC in both ACLT+Ad-HDAC4-1and. ACLT+Ad-HDAC4-3groups is higher than that in ACLT group. Meanwhile, the result of cell apoptosis is contrary to BrDU staining. IHC staining showed expression of Runx2, MMP-13and Typex in AClT group are no different from ACLT+Ad-GFP group, but the ratio of PC/TC in in both ACLT+Ad-HDAC4-1and ACLT+Ad-HDAC4-3groups is significantly decreased compared to AC1T group. Gene expression such as Aggrecan、Sox9、Col-2、Runx2、Typex、MMP-13、rat HDAC4and Ihh in ACLT group are similar to ACLT+Ad-GFP group. Gene expression including Aggrecan、Sox9、 Col-2、Runx2、Rat HDAC4and Ihh reach to peak in ACLT+Ad-HDAC4-1group, which is statistically different from ACLT group. Compared to ACLT group, the expression of Runx2, typex and MMP-13in both ACLT+Ad-HDAC4-1and ACLT+Ad-HDAC4-3groups is significantly decreased.Conclusions(1) Overexpression of HDAC4in articular cartilage chondrocyte could prevent and treat cartilage degeneration by promoting chondrocyte proliferation and inhibiting chondrocyte apoptosis and chondrocyte hypertrophic changes;(2) HDAC4is a target acted as gene or drug therapeutic articular cartilage destruction in rat OA model induced by anterior cruciate ligament transaction (ACLT).