The Anti-senescence Effect Of Calcitonin Gene-related Peptide On Endothelial Progenitor Cells In Hypertension And The Underlying Mechanisms

Chapter 1Expression of calcitonin gene-related peptide and klotho in endothelial progenitor cellsBACKGROUNDEndothelial progenitor cells(EPCs),a stem cell population,can be recruited from the bone marrow,mobilized to the circulation and differentiated into endothelial cells.There is growing evidence that many cytokines,some of which are secreted by EPCs itself,play an important role in the maintenance and regulation of EPC function.The reports from others and ours showed that endothelial cells were able to synthesize and secrete CGRP,which was supposed to participate in the protection of endothelial function.As an effective endogenous vasodilator,CGRP has been demonstrated to be involved in multiple cardiovascular protections.Klotho(K1),a novel endogenous"anti-aging"protein,has been reported to protect against endothelial dysfunction through its effects of anti-apoptosis and anti-oxidation.Based on the evidence for an essential role of EPCs in the maintenance of endothelial function and its ability of autocrine/paracrine action,in the present study,we aimed to explore whether EPCs are able to express CGRP and K1. METHODSEPCs were isolated by density gradient centrifugation from human cord blood or peripheral blood mononuclear cells,and cultured in EBM-2 supplemented with EGM-2 Single-Quots.Adherent EPCs were characterized by dual staining for acetylated low-density lipoprotein (ac-LDL) and ulex europaeus agglutinin-1(UEA-1),and by flow cytometry detecting the stem cell marker CD 133 and CD34 or by immunofluorescent analysis for endothelial marker proteins(vascular endothelial growth factor receptor-2[VEGFR2],CD31,CD144 and von Willebrand factor[vWF]).The expressions of CGRPⅠ,CGRPⅡand K1 mRNA were examined by RT-PCR and verified by DNA sequence.The protein expression(both inside the cell and released into the medium) were determined by radioimmunoassay(RIA) or ELISA.We also measured the time courses of both CGRPⅠand K1 mRNA expressions at different days of EPCs cultivation(3,7,10,14 and 21). The locations of CGRP and K1 protein inside EPCs were evaluated by laser scanning confocal microscopy.RESULTSThe present study demonstrated that:(1) Cord or peripheral blood mononuclear cells cultivated with EGM-2 generated EPCs as expected, which were characterized by typical morphology and phenotype;(2) EPCs expressed CGRP(ⅠandⅡ),of which the predominant subtype was CGRPⅠ;EPCs also expressed klotho(including membrane and secreted forms),and expression of the secreted form was dominated;(3) The early stage of EPCs,rather than the late ones,were the major population that produce and release CGRP and K1,and in the period of EPC differentiation,similar changes were found in the mRNA expressions of both CGRPⅠand K1.CONCLUSIONEPCs(mainly early stage of EPCs) synthesize and secrete both CGRP(mainly CGRPⅠ) and klotho(mainly the secreted form),and the mRNA expressions of CGRPⅠand klotho are positively correlated. Chapter 2Calcitonin gene-related peptide and senescence of endothelial progenitor cells in hypertensionBACKGROUNDThere were reports that both in experimental models of hypertension and in patients with hypertension,the quantity of EPCs were reduced and their functions were impaired,suggesting that the dysfunction of EPCs are closely associated with endothelial dysfunction in hypertension. Clinical and experimental data showed that EPCs senescence was accelerated in hypertension,which resulted in the dysfunction of EPCs.It has been suggested that the alterations of internal vasoactive substances like AngⅡ,rather than elevated blood pressure itself,may account for EPCs dysfunction under hypertensive conditions.CGRP,a principal transmitter of sensory nerves,has been suggested to be strongly associated with the development of hypertension.Our previous studies showed that the synthesis and release of CGRP were significantly decreased in hypertensive patients and animals,and pharmacological induction of CGRP expression and release could lower blood pressure and reverse vascular remodeling as well as improve endothelial function.Others showed that CGRP or adrenomedullin(AM, a member of CGRP superfamily) gene transfection could enhance the therapeutic effects and animal survival in EPCs transplantation.As mentioned before,EPCs were able to express CGRP,we thus hypothesized that the secreted CGRP from EPCs,along with the circulating CGRP,might act as an autocrine/paracrine factor to regulate EPCs function,and the reduced level of CGRP might contribute to the accelerated EPCs senescence in hypertension.As an anti-aging protein with anti-oxidative property,klotho(K1) plays important role in both regulating cellular senescence and maintaining endothelial function.It has also been demonstrated that K1 knockout mice exhibit impaired ischemia-induced angiogenesis and vasculogenesis,which is associated with a decrease in EPCs number and function.We therefore speculated that the plasma level of klotho protein might be reduced in hypertension and might be associated with the accelerated senescence of EPCs.Since the alterations of CGRP and klotho in EPCs were consistent, we thus carried out in vivo and in vitro experiments to determine the relation between them and their roles in EPCs senescence under hypertensive conditions.METHODSPartⅠ:Clinical studyWe recruited 20 patients with newly diagnosed essential hypertension and 20 age-and sex-matched healthy controls.In these subjects,we took peripheral blood samples to determine plasma levels of CGRP(RIA) and klotho(ELISA),to isolate EPCs for examining senescence-associated(β-galactosidase(SA-β-gal) and telomerase activities as well as mRNA expressions of CGRP and K1(real-time PCR). PartⅡ:Animal experimentTwenty 16-week-old male spontaneous hypertensive rats(SHR) and 20 weight- and age-matched male Wistar-Kyoto rats(WKY/Izm) were used to collect blood for determining plasma levels of CGRP(RIA) and klotho(ELISA) and to isolate EPCs for measuring SA-β-gal and telomerase activities as well as mRNA expressions of CGRP and K1 (real-time PCR).PartⅢ:In vitro experimentHuman cord blood samples were collected for isolation and cultivation of EPCs.The EPCs senescence model were established by incubation with AngⅡ(10_-7 M) for 48 h.The anti - senescence effects of CGRP were determined by giving exogenous CGRP(10_-8- 10^-6 M) or by over-expression of CGRP ?.EPCs senescence was evaluated by examining SA-β-gal and telomerase activities.The mRNA expressions (real-time PCR) of CGRP and K1 as well as their protein levels(RIA or western blot) were also measured.Furthermore,silence of K1 expression by miR RNA interference to determine whether the anti - senescence effect of CGRP on EPCs was through regulating K1 production.RESULTS(1) In patients with essential hypertension,the plasma levels of both CGRP and klotho were reduced;the SA-β-gal activity of peripheral blood-derived EPCs was elevated and the telomerase activity was reduced;the mRNA expressions of both CGRP and K1 in EPCs were down-regulated. (2) In spontaneous hypertensive rats(SHR),the plasma levels of both CGRP and klotho were reduced;the SA-β-gal activity of peripheral blood-derived EPCs was elevated and the telomerase activity was reduced.The mRNA expressions of both CGRP and K1 in EPCs were down-regulated.(3) AngⅡtreatment elevated SA-β-gal activity accompanied by the reduced telomerase activity and K1 expression in EPCs,which were reversed by administration of exogenous CGRP in a concentration -dependent manner or by over-expression of CGRPⅠ.The effect of CGRP was abolished in the presence of CGRP_8-37,a selective antagonist of CGRP receptor.(4) Silence of K1 expression by miR RNAi could also abolish the anti -senescence effect of CGRP on EPCs.CONCLUSIONThe accelerated EPCs senescence in hypertension is related to the reduction of CGRP,which may work as an endogenous protective substance to counteract the oxidative stress-induced EPCs senescence in hypertension through increasing the production of anti-aging protein klotho. Chapter 3Anti-senescence effects of rutaecarpine on endothelial progenitor cells in hypertension BACKGROUNDWe have demonstrated that accelerated EPCs senescence induced by the increased oxidative stress under hypertensive conditions is related to the reduction of CGRP,while administration of exogenous CGRP or CGRPⅠover-expression could effectively inhibit EPCs senescence induced by AngⅡ.It is therefore likely that increase the production of CGRP could be a novel way to improve EPCs function through inhibiting senescence in hypertension.Though CGRP exerts extensive cardiovascular actions such as relaxing vessels,protecting endothelial function and inhibiting smooth muscle cell proliferation,its inconvenience of intravenous administration and short half-life in plasma limits its therapeutic application in the hypertensive patients.To our excitement,investigators have found that some drugs can stimulate the synthesis and release of endogenous CGRP. It has been well documented that activation of vanilloid receptor subtype 1(VR1,or transient receptor potential vanilloid 1,TRPV1),will lead to the synthesis and release of CGRP,suggesting that activation of TRPV1 might be an effective way to prevent the development of hypertension. Rutaecarpine(RUT),a major quinazolinocarboline alkaloid isolated from Wu-Chu-Yu,has been demonstrated to exert positive inotropic and chronotropic effects on isolated guinea pig atrium,which can be abolished by TRPV1 antagonist capsazepine(CAPZ) or CGRP_8-37, indicating that RUT can stimulate the synthesis and release of CGRP via activation of TRPV1.Our recent work has shown that the depressor and vasodilator effects of rutaecarpine are related to stimulation of endogenous CGRP synthesis and release via activation of TRPV1 on the capsaicin-sensitive sensory nerves.Besides existence in the terminals and cell bodies of sensory neurons,TRPV1 was also presented in endothelial cells.Accordingly,we presumed that RUT might have an inhibitory effect on EPCs senescence in hypertension though stimulating CGRP production via TRPV1.The aim of the present study was to test the hypothesis in both spontaneous hypertensive animals and cultured EPCs.METHODSPartⅠ:Animal experimentRats were divided into 6 groups:(1) the wild-type Wistar-Kyoto rats (WKY/Izm);(2) spontaneous hypertensive rats(SHR);(3) +RUT(10 mg/kg/day);(4) +RUT(20 mg/kg/day);(5) +RUT(40 mg/kg/day);(6) +LOS(30 mg/kg/day),losartan as a positive drug.All rats were treated by intragastric administration for 2 weeks. Blood was collected for determination of plasma levels of CGRP (RIA) and klotho(ELISA);EPCs were isolated from both blood and bone marrow(femur) to detect SA-β-gal and telomerase activities as well as mRNA expressions of CGRP and K1(real-time PCR).PartⅡ:in vitro experimentHuman cord blood samples were collected for isolation and cultivation of EPCs.The EPCs senescence model were established by incubation with AngⅡ(10-7 M) for 48 h.The anti - senescence effect of RUT were determined by giving RUT(10^-7-10^-5 M) for 1 h.EPCs senescence was evaluated by examining SA-β-gal and telomerase activities,and mRNA expressions(real-time PCR) of CGRP and K1 as well as their protein levels(RIA or western blot).RESULTS(1) In SHR,the plasma levels of both CGRP and klotho were reduced;the SA-β-gal activity of peripheral blood-derived EPCs was elevated and the telomerase activity was reduced.The mRNA expressions of both CGRP and K1 in EPCs were down-regulated.These changes were reversed by administration of RUT.(2) Both mRNA and protein of TRPV1 were expressed in EPCs.(3) AngⅡtreatment elevated SA-β-gal activity accompanied by the reduced telomerase activity and K1 expression in EPCs,which were reversed by administration of exogenous RUT in a concentration -dependent manner.The effect of RUT was abolished in the presence of CAPZ,the antagonist of TRPV1.CONCLUSIONRUT exerts anti-senescence effect on EPCs in hypertension is through stimulating the production of CGRP via TRPV1,and the underlying mechanism is related to up-regulation of K1 expression.