Role Of Dipeptidyl Peptidase Ⅳ Inhibition/glucagon-like Peptide-1 In Experimental Pulmonary Hypertension In Rats

Dipeptidyl peptidase IV(DPP-4),also recognized as a surface marker(CD26)of T-cell,is well known for its role in glucose homeostasis,and DPP-4 inhibiton(DPP-4i)exhibits multiple actions in cardiovascular diseases,such as improving the left ventricular(LV)function,preventing cardiovascular remodeling,enhancing endothelium-dependent relaxation of mesenteric arteries,attenuating neointima formation after vascular injury,and reducing systolic pressure.Glucagon-like peptide-1(GLP-1),a pancreatic source hormone,can be cleavage by DPP-4,thereafter,turn into inactive form.Apart from its role in glucose homeostasis,GLP-1 has also be demonstrated to be involved in the regulation of cardiovascular disorders,such as improving endothelial function,reducing the proliferation of vascular smooth muscle cells,inhibiting aptosis of cardiomyocytes,attenuating inflammatory response,relaxating arteries,and reducing blood pressure.GLP-1 has been demonstrated to mediate the role of DPP-4i in different disorders including glucose homeostasis and cardiovascular diseases.However,the effect and the underlying mechanism of DPP-4i on pulmonary hypertension(PH)remains unclear.The doubt that whether GLP-1 mediates the protective effect of DPP-4i on PH needs to be explored.PartⅠ.The effect of DPP-4 inhibition on pulmonary hypertension and the underlying mechanism AIM: This study aims to explore the distribution of DPP-4 in lung tissues both from human beings and rats,and,to investigate the effect of DPP-4i on pulmonary arterial remodeling in rats with PH and the potential mechanisms underlying.METHODS: Male Wistar rats were injected intraperitoneally with monocrotaline(MCT,60 mg/kg,i.p.)with/without DPP-4 inhibitor(Sitagliptin,SG)at different dose(20,40,and 80 mg/kg,gavage).Four weeks later,rats were sacrificed for hemodynamic measurement;morphological analysis by HE,gomori aldehyde fuchsin(GAF)staining,masson trichrome staining(MTS),picrosirius red(PSR)staining,toluidine blue(TB)staining and immunohistochemical staining with primary antibody against DPP-4,smooth muscle alpha actin(α-SMA),tryptase,CD68,proliferating cell nuclear antigen(PCNA),caspase-3,and CD31;as well as examination of identified molecules using western blot and RT-qPCR.Beisides,pulmonary fibrosis-related PH were established with an intratracheal administration of bleomycin hydrochloride(4 U/kg).In addition,rats were housed in a 10% hypoxia chamber for 4 weeks in which the fraction of oxygen was maintained at 10% to establish hypoxia-related PH.In vitro,human pulmonary arterial smooth muscle cells(PASMCs)were employed to explore the underlying mechanism by cell proliferation and migration assay.RESULTS: Our results showed that DPP-4 was expressed in epithelial cells,endothelial cells,smooth muscle cells,and inflammatory cells in lung tissues from both rat and human being.DPP-4i(Sitagliptin)attenuated the right ventricular systolic pressure(RVSP),right ventricle remodeling,hypertrophy of pulmonary arterial medial layer,inflammatory cell infiltration,and endothelial–mesenchymal transition(EndMT)in monocrotaline(MCT)-induced PH.Similarly,DPP-4i also alleviated bleomycin-and chronic hypoxia-induced PH in rats.In cultured human pulmonary arterial smooth muscle cells(PASMCs),DPP-4i could up-regulated the expresseion of phosphatase and tensin homolog deleted on chromosome ten(PTEN),reduce the transduction of AKT/MAPK signaling,thereafter,inhibit platelet derived growth factor(PDGF)-BB-induced proliferation and migration,which was abolished by PTEN knockout.CONCLUSION: These results demonstrate that DPP-4 inhibition alleviates pulmonary arterial remodeling in experimental PH partly by inhibiting proliferation and migration of PASMCs via the regulation of PTEN-AKT/MAPK signaling.Collectively,our data suggest that DPP-4 might be a promising target for treating PH.PartⅡ.The effect of DPP-4 inhibition on pulmonary hypertension and the underlying mechanism AIM: This study aims to explore the effct of GLP-1 on PH resulted from MCT,bleomycin,and chronic hypoxia.in lung tissues both from human beings and rats,and,and to determine whether GLP-1/GLP-1R mediated the protective role of DPP-4i on PH and the potential mechanisms underlying.METHODS: Male Wistar rats,injected intraperitoneally with monocrotaline(MCT,60 mg/kg,i.p.),were given DPP-4 inhibitor(Sitagliptin,SG,80 mg/kg,gavage)administration with/without GLP-1 receptor(GLP-1R)antagonist(Exendin 3,Ex-3,40 μg/kg/d,intraperitoneal injection).Meanwhile,other MCT-treated rats were given GLP-1R agonist(liraglutide,0.1,0.2,0.4,0.8 mg/kg/d,subcutaneously injection).Four weeks later,rats were sacrificed for hemodynamic measurement;morphological analysis by HE,GAF staining,MTS,PSR staining,TB staining and immunohistochemical staining with primary antibody against α-SMA,CD68,and CD31;as well as examination of identified molecules using western blot and RT-qPCR.Beisides,pulmonary fibrosis-related PH were established with an intratracheal administration of bleomycin hydrochloride(4 U/kg).In addition,rats were housed in a 10% hypoxia chamber for 4 weeks in which the fraction of oxygen was maintained at 10% to establish hypoxia-related PH.In vitro,endothelial-to-mesenchymal transition(EndMT)in human umbilical vein endothelial cells(HUVECs),resulted from transforming growth factor-β(TGF-β1,5 ng/ml)+ interleukin-1 beta(IL-1β,5 ng/ml),were employed to explore the underlying mechanism.RESULTS: Our results showed that sitagliptin successfully attenuated the right ventricular systolic pressure(RVSP),hypertrophy of pulmonary arterial medial layer,inflammatory cell infiltration,and endothelial–mesenchymal transition(EndMT)in monocrotaline(MCT)-induced PH.The aforementioned effects were abolished by the supplement with GLP-1R antagonist(Ex-3).GLP-1R agonist(liraglutide)effectively the right ventricular systolic pressure(RVSP),hypertrophy of pulmonary arterial medial layer,inflammatory cell infiltration,and endothelial–mesenchymal transition(EndMT)in monocrotaline(MCT)-induced PH.Similarly,liraglutide also alleviated bleomycin-and chronic hypoxia-induced PH in rats.In cultured HUVECs,liraglutide reduced the percent of cells undergoing TGF-β1&IL-1β-induced EndMT,inhibited the expression of mesenchymal markers(Vimentin,α-SMA),recovered the expression of endothelial markers(vascular endothelial cadherin,VE-cadherin;zonula occludens-1,ZO-1),and,decreased the phosphorylation of smad3,p42 and p38.The aforementioned effects were abolished by Ex-3.CONCLUSION: These results demonstrate that GLP-1 could directly and effectively alleviate pulmonary arterial remodeling in three different experimental PH,and mediated the effect of DPP-4i on PH.The action in PH might be attributed to its inhibition role in TGF-β1&IL-1β-induced EndMT via blocking Smad-dependent and-independent signaling.Collectively,our data suggest GLP-1/GLP-1R mediates the inhibition role of DPP-4i in PH,and might be a promising target for treating PH.