The Effect Of Angiotensin-converting Enzyme Inhibitor (ACE), Captoprol On The Proliferation, Differentiation And Migration Of Human Epidermal Stem Cells: A Novel Function Of ACE In Skin

Background:The Renin-angiotensin-aldosterone system (RAS) plays a decisive role in theprogression of nephropathy in which angiotensin-converting enzyme (ACE) is the keyrate limiting enzyme. Therefore, angiotensin converting enzyme inhibitors have beenused as first-line antihypertensive drugs, widely used in the treatment ofcardiovascular and renal disease. However, a variety of angiotensin convertingenzyme inhibitors (ACEI)50%of the adverse reactions occurring in the skin, such asrash, urticaria, alopecia, and even severe skin reactions, the specific mechanism ofaction is not entirely clear. Increased risk of long-term treatment of people withdiabetes who take ACE inhibitors occurred foot ulcers and lower limb amputations.The epidermal stem cell (ESC) self-renewal of skin tissue is an important target organrepair and regeneration, and thus is of great significance to clarify the relationshipbetween them and so.Objective：The present studies were undertaken to investigate the effect angiotensin-convertingenzyme (ACE) inhibitor, captoprol on the epidermal regeneration of wound, exploredthe potential cellular and signaling mechanisms of ACE inhibitor’s effect on the reepithelialization of wound.Methods：By differential adhesion method to obtain human epidermal stem cells in vitro culture.The use of epidermal stem cell surface markers recognized by their identification;ESC surface expression assay of ACE. Of the captopril experimental and controlgroups: the experimental group containing different concentrations of captoprilepidermal stem cell culture medium (K-SFM), the control group was simply K-FSMmedium.(1) XTT cell enzyme-linked immunosorbent assay according to the ESC proliferationactivity and establish the optimal concentration of captopril;(2). Observed in vitro wound model captopril experimental group and the controlgroup epidermal stem cell migration;(3). K10expression by flow cytometry to observe the effect of captopril on ESCdifferentiation;(4) cell colony formation rate analysis and analysis of cell adhesion ability to observethe effect of captopril on the ESC;(5). flow cytometry effect of captopril on ESC apoptosis;(6). Elisa method to detect cell supernatant change ANG2and ANG1-7, and toexplore the role of ESC ACEI way.(7). Detected downstream substrates ANG Ⅱ AT1and AT2expression in ESCs,Grouping observed ANG Ⅱ, AT1blocker (Losartan), AT2blocker (PD123,319),ACEI+Losartan and ACEI+PD123,319in the epidermal stem cell proliferation, migration effect, to analyze the role of ACEI on ESC points.(8). Western blotting (Western blot) was observed joined ACE inhibitors extracellularsignal-regulated kinase before and after ESC (ERK), phosphorylated extracellularregulated kinase (phospho-ERK),(AKT),(phospho-AKT),(STAT1),(phospho-STAT1),(STAT3),(phospho-STAT3) changes in the signal path byanalyzing the ACE investigate biological function in maintaining the ESCs in the roleResults：(1). The cells were cultured in vitro β1-integrin and K19double labelingimmunofluorescence staining, cells were double labeled83.55%positive cells, whichESCs. Immunofluorescence staining and RT-PCR results show the expression of ACEon the ESC. Flow cytometry to detect cultured cells ACE positive rate of74.2%.(2).10-6mol/l captopril significantly inhibited the proliferation of ESCs (P <0.05),and reached a peak in the first five days. Captopril10-6mol/l significantly inhibitedcell migration capacity (P <0.05), inhibited adhesion capacity (P <0.05) and inhibitedcloning capacity (P <0.05).(3). Flow cytometry results showed that captopril:10-6mol/l did not affect theexpression of K10(P>0.05) and apoptosis (P>0.05).(4). Elisa assay epidermal stem cell supernatants after captopril treatment changes,ANG2expression differences between the experimental group and control group wasstatistically significant (P <0.05), while there was no significant difference (P>0.05)of ANG1-7expression.(5). RT-PCR results are shown ESC express AT1and AT2, XTT cell enzyme-linked immunosorbent assay ANG Ⅱ, Losartan, PD123,319, ACEI+Losartan and ACEI+PD123,319pairs ESCs proliferation outcome differences were statistically significant(P <0.05); vitro wound model observation ANG Ⅱ, Losartan, PD123,319, ACEI+Losartan and ACEI+PD123,319对ESCs migration results statistically significantdifference (P <0.05).(6). Western blotting (Western blot) observed ESCs phospho-ERK, ESCsphospho-STAT1, ESCs phospho-STAT3protein expression differences werestatistically significant (P <0.05) after joining ACE inhibitors.Conclusion：ANG2regulated by the concentration of ACE and AT1and AT2adjust the balancebetween work, which may be activated ESCs phospho-ERK, phospho-STAT1,phospho-STAT3signaling pathway ESC proliferation, migration and adhesion andthus affect the skin damage repair and self-renewal.