The Involvement Of HDAC In The Keloid Formation

Keloid is a pathological wound healing response to cutaneous injury andcharacterized by fibroblastic proliferation and accumulation of extracellular matrix.Clinically, keloids have proven to be very resistant to treatment. Despite the relativelyhigh prevalence of keloids in the general population, the mechanisms underlying keloidformation are only partially understood. Recent studies indicate that histone deacetylase（HDAC）activity is also associated with the development and progression of somechronic diseases characterized by fibrosis. And inhibition of HDAC activity of fibroblastsby a number of structurally divergent classes of HDAC inhibitors causes significantinhibition of extracellular matrix synthesis. Furthermore, HDAC inhibitors have been shown to induce arrest, differentiation, and/or apoptosis of proliferating cells. So wehypothesize that increased activity of HDAC may be associated with keloid fibroblastproliferation and accumulation of extracellular matrix. In this study, we will research ofclass I HDAC（HDAC1and HDAC2）expressions in keloid fibroblasts, and throughrespectively in vivo and in vitro experimental intervention with HDAC inhibitor TSA, toclarify how class I HDACs are involved in keloid fibroblasts proliferation and collagensecretion. Our study will open up the possibility of testing HDAC inhibitors for clinicalprevention and treatment of keloids.Experiment1The study of HDAC1and HDAC2expressions in keloid fibroblastsPurpose: To investigate HDAC1and HDAC2expressions in keloid fibroblasts andnormal fibroblasts. To provide new pathogenetic mechanisms for keloid formation andnew treatment point.Methods: We cultured keloid fibroblasts and normal skin fibroblasts. Then, we researchedthe expressions of HDAC1and HDAC2in keloid fibroblasts and normal fibroblasts byRT-PCR and western blot.Results: The increase of HDAC1and HDAC2mRNA expressions were observed inkeloid fibroblasts compared to normal fibroblasts. These results were also confirmed bywestern blot analysis. Western blot analysis showed that the expressions of HDAC1andHDAC2protein were markedly up-regulated in keloid fibroblasts compared to normalfibroblasts.Conclusion: Our study confirmed that expressions of HDAC1and HDAC2in thekeloid-derived fibroblasts were significantly higher. Enhanced HDAC1and HDAC2activity, which makes the original gene expression balance has been broken, leading toabnormal cell proliferation, apoptosis and fibrosis and other biological characteristics.HDAC1and HDAC2activity may be a new pathogenetic mechanism for keloidformation. Experiment2Histone deacetylase inhibitor TSA induces apoptosis of keloidfibroblastsPurpose: As TSA could induce growth arrest and apoptosis of many kind of cells, the aimof this study is to determine whether HDAC inhibitor trichostatin A（TSA）could induceapoptosis of proliferating keloid fibroblasts and inhibit the expressions of HDAC1andHDAC2.Methods: We first studied the effect of escalating doses（200nmol/L,400nmol/L,600nmol/L or800nmol/L）of TSA on keloid fibroblasts viability using MTT proliferationassays. Then, to further investigate the inhibitory effect of TSA on proliferation of keloidfibroblasts, we stained cells using Hoechst staining after600nmol/L TSA treatment, andflow cytometry analysis was performed as a second independent method. Furthermore,after treatment with600nmol/L TSA for48h, we researched the expressions of HDAC1and HDAC2protein expressions in keloid fibroblasts by western blot.Results: HDAC inhibitor TSA inhibited the growth of keloid fibroblasts indose-dependent and time-dependent manner monitored using MTT proliferation assays.Lower TSA doses of200nmol/L were well tolerated with preserved cell viability relativeto control. A statistically significant decrease in cell viability was observed at aconcentration of TSA（600nmol/L and800nmol/L）after incubation for12h, compared tocontrol. Hoechst staining and flow cytometry analysis showed significantly moreapoptotic cells were observed in600nmol/L TSA treated group compared to controls.Furthermore, treatment with TSA abrogated HDAC1and HDAC2expressionsdramatically. Western blot analysis showed that treatment with TSA for48h, theexpressions of HDAC1and HDAC2were markedly decreased in keloid fibroblasts.Conclusion: On the basis of our in vitro evidence we can state that TSA could inhibitproliferation and induce apoptosis of keloid fibroblasts with the decreased expressions ofHDAC1and HDAC2.Experiment3Histone deacetylase inhibitor TSA inhibits collagen synthesis of keloid fibroblastsPurpose: The aim of this study is to determine whether HDAC inhibitor TSA could causeabrogation of TGF-β induced collagen synthesis in keloid fibroblasts.Methods: We researched the expressions of collagen I in keloid fibroblasts treatment with600nmol/L TSA or/and5ng/ml TGF-β1by RT-PCR and western blot.Results: The addition of5ng/ml TGF-β1up-regulated collagen I mRNA expressioncompared to controls by RT-PCR analysis. But incubation with600nmol/L TSA, theincrease of collagen I mRNA expression induced by TGF-β1was reduced by50%compared to controls. Furthermore, treatment with TSA alone abrogated collagen I mRNAexpression dramatically. These results were confirmed by western blot analysis. Westernblot analysis showed that treatment with TSA, the expression of collagen I protein inducedby TGF-β1was markedly decreased.Conclusion: On the basis of our in vitro evidence we can state that TSA inhibition ofECM may be an appropriate therapeutic strategy for the management of keloid. Our studyopens up the possibility of testing HDAC inhibitor, an agent already in clinical trials forthe treatment of many tumors, for clinical keloid treatment use.Experiment4Histone deacetylase inhibitor TSA reduces hypertrophic scarring in arabbit ear modelPurpose: To test the ability of HDAC inhibitor TSA to reduce hypertrophic scar formationin a rabbit ear model, which had been confirmed to have potent anti-fibrogenic effects andsuppress collagen synthesis of skin fibroblasts.Methods: We had developed a reliable model in rabbit that resulted in hypertrophicscarring. Four1cm full-thickness circular wounds were made on each ear. After thewounds re-epithelialized,0.02%TSA was intradermally injected to the wounds in thetreatment group. Collagen I and fibronectin expressions were detected by RT-PCR andwestern blot at postoperative day（POD）23. Scar hypertrophy was quantified by measurement of the scar elevation index（SEI）at POD45.Results: Compared to the control group, the injection of TSA led to much morenormal-looking of scars in the rabbit ear. SEI at POD45was significantly decreased afterinjections of TSA versus untreated scars（1.45±0.09vs.2.07±0.10）. Further more, weconfirmed the decreased expression of collagen I and fibronectin at POD23（after beingtreated with TSA for one week）in the treated scars compared to the control by RT-PCRand western blot analysis.Conclusions: The introduction of TSA can result in the decreased formation ofhypertrophic scars in a rabbit ear model, which is corroborated by evidence of decreasedcollagen I and fibronectin synthesis.