Study On The Activation Effects And Mechanism(s) Of Angiotensin â…¡ On Fibroblasts Of Hypertrophic Scar

BackgroundHypertrophic scar(HS)is an unique pathological phenomenon in human beings resulting from an excessive response to a cutaneous injury .Nowdays,the pathogenesis of HS remains unclear.In skin,the fibroblasts comprise at least 40% to 60% of the cell population,and are thought to be responsible for the increased matrix production and subsequent formation of hypertrophic scars and keloids after injury,most likely under the stimulation of persistently high levels of fibrogenic cytokines.Recent studies have documented the existence of complete renin-angiotensin system in human skin. Angiotensin II(Ang II),a key peptide of the RAS,can promote cell proliferation and collagen synthesis of skin-derived fibroblasts,which indicates that Ang II may participate in HS formation. Ang II type I receptor's antagonists have been used to treat hypertension for years.Some investigators also attempted to use them to treat miocardial hypertrophy and liver fibrosis and get satisfactory effects.The study on the effects and mechanism(s) of Ang II in HS will provide new insight into the pathgenesis of hypertrophic scar.ObjectiveThis study was designed to compare the expression of Ang II receptor type I (AT1)and type II(AT2) in normal skin(NS) and HS,and then to investigate the effects of Ang II and its receptor antagonists on proliferation,activation,extracellular matrix synthesis and TGF-β1 expresssion of fibroblast derived from HS. We also tried to explore the activation of JAK2/STAT1,STAT3 signaling pathway induced by Ang II and the expression change of TGF-β1 and FN mRNA after JAK2 was blocked.Methods1.HS and NS tissue were collected and immunohistochemistry was used to determine the expression of AT1 and AT2;Fibroblasts were freshly isolated from normal skin and hypertrophic scars and were cultured. The expression of angiotensin II receptors protein and mRNA in fibroblast was detected by Western blot and RT-PCR respectively.2. Cell counting,MTT assay,3H-thymidine incorporation(3H -TdR) were used to study the effects of Ang II on the proliferation and activity of cultured fibroblasts.Collagen synthesis of fibroblast was determined by [3H]-proline incorporation.3. The RT-PCR and Western blot were performed to detect the mRNA and protein of procollagen I,procollagen III,fibronectin(FN) and TGF-β1 ,respectively.4.The phosphorylation level of JAK2,STAT1,STAT3 were used to evaluate the effects of Ang II on the activation of JAK2/STAT1,STAT3 signaling pathway by immunoblotting.Moreover, the expression of p-STAT1 and p-STAT3 in fibroblast nuclear was also determined . The change of TGF-β1 expression was detected after JAK2 was blocked by AG490.Results1. AT1 and AT2 receptors were expressed in both NS and HS tissue ,and also in the fibroblasts from NS and HS.They were upregulated in human HS tissue and the fibroblast of HS,especially AT1 receptor.The expression of mRNA and protein of AT1 in fibroblast of HS was 3.1 and 2.3 fold vs NS, respectively.2. Ang II induced the proliferation and activation of fibroblast from HS through AT1 receptor.3. Ang II can promote the accumulation of extracelluar matrix sucah as procollagen I,procollagen III and FN, and also induce the synthesis of TGF-β1 of fibroblast .AT1 antagonist Losartan can inhibit this effect.4. AG490,an JAK2 inhibitor can partly decrease the expression of TGF-β1and FN mRNA of fibroblast which was stimulated by Ang II.5. Ang II increased JAK2,STAT1 and STAT3 phosphorylation levels,especially p-STAT3.In addition, Ang II also promoted p-STAT3 and p-STAT1 transducting into nuclear.The Ang II-induced JAK2/STAT1,STAT3 cascade was markedly inhibited by Losartan,an AT1 receptor-specific antagonist, and AG490, an JAK2 inhibitor,whereas PD123319,an AT2 receptor blocker, had no effects on this activation.Conclusions1. Skin is one of the target organs of Ang II and fibroblast is the target cell of Ang II.2. Most effects of Ang II on fibroblast are mediated by the AT1 receptor,such as cell proliferation,cell activation,accumulation of extracellular matrix and synthesis of TGF-β1.All the above effects can be inhibited by AT1-specific antagonist.3. Ang II activates JAK2/STAT1,STAT3 signaling pathway through phosphorylating of JAK2,STAT1 and STAT3,and this effect is also induced by AT1 receptor. In HS fibroblast, Ang II can activation JAK2,which is the upstream signal molecule of STAT1 and STAT3.4. AT1 receptor-mediated activation of fibroblast is partially via the JAK2 signal transduction pathway,at least.