The Experimental Study Of Connective Tissue Growth Factor And Its Receptor Integrin α_νβ₃in Pathogenesis Of Hypertrophic Scar

【Background】Hypertrophic scar is a fibro-proliferative disorder after skin tissue injuries. Theactivation, proliferation, collagen synthesis or secretion and differentiation of fibroblasts,are main factors contributing to scar. Recent reports have demonstrated that CCN2, whichplays an important role in the functional regulation of fibroblasts as a cofactor of TGF-β1,is considered to be one of strong pro-fibrotic factors. Thus, elucidation of the role and themolecular mechanism of CCN2during the formation of hypertrophic scars, might providenew clues to the pathogenesis of hypertrophic scars. Integrin α_νβ₃is one of CCN2receptors. Previous studies have also shown that Integrin α_νβ₃play a major role in severalprocesses related to cell proliferation, differentiation, migration, survival and apoptosis.MAPKs signal, which is one of important pathways that regulate the cell signalingtransduction, has been reported to might be involved in wound healing and fibrosisregulated by CCN2. However, its specific effects might be tissue and organ dependent.Besides, the underlying molecular mechanisms are still unknown. Especially, the effects ofα_νβ₃and MAPKs signal in the trans-differentiation of hypertrophic scar derived fibroblastsare poorly understood. 【Purposes】1. To verify the regulation mechanism of CCN2receptor integrin α_νβ₃in thehypertrophic scars.2. To elucidate the specific effects of MAPK signaling in the CCN2regulation ofhypertrophic scars and the scar model in the rabbit ear.【Contents】1. Measure the expression levels of integrin ανintegrin β3in the hypertrophic scarsusing real-time PCR.2. Hypertrophic scar fibroblast transdifferentiation is induced by CCN2. Real-timePCR and immunoblotting were employed to detect the expression changes of the markersof fibroblasts trans-differentiationand integrin α_νβ₃in hypertrophic scar derived fibroblast.Detect the expression levels of molecular markers of hypertrophic scar fibroblaststransition using LM609which is integrin α_νβ₃specificity blocker. Observe the contractionfunction of the hypertrophic scar fibroblasts by three dimensional collagen gel assays.3. Hypertrophic scar fibroblasts were stimulated by CCN2. Western blot is used toobserve the activation of the MAPK signaling pathway. Detect the expression levels ofhypertrophic scar fibroblasts markers after using LM609. Measure the expression levels ofα-SMA, collagenâ…  and collagen â…¢ in normal adult fibroblasts after CCN2or jointstimulation with TGF-β1.Measure the expression levels of phosphorylation of ERK andJNK expression in hypertrophic scars. Construction rabbit ear hypertrophic scar model toobserve the role of CCN2and MAPK signaling pathway.【Results】1. The expression levels of integrin αv, integrin β3increased significantly inhypertrophic scar tissues, compared with normal skin tissues. Similarly, the expressionlevels of integrin αv, integrinβ3were also up-regulated in hypertrophic scar fibroblasts.2. After CCN2stimulation, the expression of α-SMA, collagenâ…  and collagen â…¢increased significantly. The expression of integrin αvand integrin β3mRNA was alsoup-regulated. LM609inhibited the CCN2induced expression of α-SMA, collagenâ…  and collagen â…¢. The three dimensional collagen gel assays showed that the contractility ofhypertrophic scar fibroblasts was inhibited by LM609.3. CCN2stimulation induced a rapid elevation in phosphorylated ERK, p38, and JNKin hypertrophic scar fibroblasts. The maximum phosphorylation of ERK and JNKoccurred15min after CCN2stimulation, while the maximum phosphorylation of p38occurred30min after CCN2stimulation. PD98059, SB203580, and SP600125signifcantly inhibited CCN2-induced phosphorylation of ERK, p38and JNK,respectively.4. The result shows that the treatment withPD98059signifcantly inhibited collagen ImRNA expression. However, SP600125and SB203580had no evident effects onCCN2-induced expression of collagen I mRNA. Similarly, the expression of collagen Iprotein signifcantly decreased in the presence of PD98059, whereas SP600125andSB203580had minimal effect on CCN2-induced collagen I expression. In addition,compared with control, CCN2signifcantly induced collagen I secretion. SP600125andPD98059signifcantly inhibited the secretion of collagen I, which was stimulated byCCN2. However, SB203580had no effect on CCN2-induced collagen I secretion.5. The expression level of a-SMA elevated signifcantly after CCN2treatment. Thiselevation was inhibited in the presence of SB203580, SP600125or PD98059. Thetreatment with PD98059signifcantly inhibited a-SMA expression. Immunocytochemistryalso showed that the expression of a-SMA by CCN2decreased signifcantly after thepresence of SP600125or PD98059. However, SB203580had no effect on CCN2-induceda-SMA expression.6. Our data showed that compared with the control, TGF-β1and CCN2, but not CCN2alone, markedly stimulated a-SMA, collagen I and collagen III expression, while thetreatment with SB431542signifcantly inhibited TGF-β1and CCN2-induced expression ofa-SMA, collagen I and collagen III. Moreover, when used alone, SB431542couldinhibited collagen I and collagen III as compared with control.7. The phosphorylation level of ERK and JNK was measured by Western blot andimmunohistochemistry. The expression level of p-ERK and p-JNK was markedly up-regulated in hypertrophic scar, compared with normal skin tissue. Similarly,immunohistochemistry also demonstrated that the p-ERK and p-JNK were activated inhypertrophic scar.8. In vivo experiments, a rabbit ear hypertrophic scar model was establishedsuccessfully. Compared with control, successive administration of SP600125or PD98059group signifcantly inhibited scar hyperplasia. Masson’s trichrome staining of scar tissuerevealed more features typical of collagen fbers in scar tissue in the control groupcompared with unwounded dermal tissue. The collagen bundles were more abundant,denser, thicker, and disorganized. On the other hand, the collagen fbers in the groupstreated with CCN2+SP600125, CCN2+PD98059, PD98059and SP600125were fewer,thinner, and more regularly arranged.【Conclusion】1. Compared with normal skin tissues and normal adult fibroblasts, the expressionlevels of integrin αv,integrin β3increased significantly in hypertrophic scar tissues andhypertrophic scar derived fibroblasts.2. CCN2can induce hypertrophic scar fibroblasts transdifferentiation, and the CCN2receptor integrin α_νβ₃play an important role in this process.3. CCN2can significantly activate the MAPK signaling pathway. Exogenousinhibition of ERK1/2and JNK signaling inhibits the expression levels of α-SMA, collagenI and collagen III in hypertrophic scar fibroblasts induced by CCN2.4. In vivo experiments, we successfully build a rabbit ear hypertrophic scar model.Exogenous inhibition of ERK1/2and JNK signaling can significantly inhibit scarformation.