The Soluble VEGF Receptor SFlt-1 Contributes To Impaired Neovascularization In Aged Mice And Clinical Significance Of Serum Cathepsin K Levels In Patients With Chronic Heart Failure

Chapter One:The Soluble VEGF Receptor sFlt-1 Contributes to Impaired Neovascularization in Aged MiceThe morbidity of limb ischemic disease is being more and more increased with the growth in the living standard,which more lead to the aging society.Aging has been shown to impair the mammalian body’s ability to form new blood vessels under ischemic pathological conditions,which causes a diminished capacity for tissue regeneration.In a wide range of mammals,the age-associated vascular failing and decline in revascularization are characterized by endothelial dysfunction and decreases in the numbers and intrinsic function of bone-marrow(BM)-derived endothelial progenitor cells(EPCs),a shift in an unbalance between vascular cell apoptosis and proliferation,and changes in the extracellular microenvironment(e.g.,alterations in growth factors,inflammatory cytokines,and oxidative stress).In 1989,vascular endothelial growth factor(VEGF)has been discovered as a specific factor to regulate angiogenesis.VEGF family contains seven members,VEGF-A,VEGF-B,PIGF,VEGF-C,VEGF-D,VEGF-E and VEGF-F.Their receptors,VEGFR-1,2,3 and Neuropilins.VEGF exerts it own physiological function via the binding to its receptor.VEGF regulates vascular permeability.It is known that VEGFR-1 is widespreadly expressed on not only endothelial cells but also hematopoietic stem cells,osteoblasts,placental trophoblastic cells and Monocyte-macrophages.VEGFR-1 has high affinity with VEGF,VEGF-B and PIGF.Clinical and labolatory studies showed that the soluble VEGFR-1(sFlt-1)as a part of the extracellular VEGFR-1 exhibits anti-angiogenic property.VEGFR1 has been shown to be increased in agiogenesis process under hypoxia.Previosy clinical study have clearly demonstrated that pregnant women with preeclampsia had increased levels of plasma sFlt-1 and decreased levels of PIGF and VEGF.It is well known that biological hypoxia/ischemia usually cause increases in the expression of proangiogenic growth factors such as Vascular endothelial growth factor(VEGF)and its receptor(VEGFR)activation(including Fltl and Flkl),which then initiate tubulogenesis from preexisting vessels by inducing vascular cellular events(including proliferation,migration,and invasion)and vascular lumen maturation.Accumulating evidence show that VEGF and VEGFR splicing can produce antiangiogenic actions under various pathological conditions.sFtll,also known as a VEGF antagonist,is a splice variant of the VEGF receptor lacking the cytoplasmic and transmenbrane domains.Experimental and clinical studies have led to a number of important observations that contribute to our understanding of an inhibitory splice variant of sFltl.For example,by binding and occupying the VEGFR,sFlt1 disturbs VEGF occupation and subsequent growth signal transduction in cultured cells.It has been reported that sFltl resulted in endothelial dysfunction and angiogenic actions in the pathogenesis of preeclampsia.Clinical study figured out to changes in plasma sFltl in metabolic disorder and atherosclerosis-based coronary artery disease that have relevance for the therapy and angiogenesis in these conditions.A single recent vascular biological study demonstrated that Wnt5/SC35 activation contributed to impaired vascularization in peripheral artery disease in humans and animals.Furthermore,a non-canonical Wnt-sFltl signal pathway has been shown to negatively regulate angiogenesis in myeloid cells.However,the role of Wnt5/SC35-sFlt-1 axis in angiogenesis and neovascularization in response to ischemic stress in aged animals and humas are largely unknown.Objective:The mechanism by which angiogenesis declines with aging is not fully understood.Soluble vascular endothelial growth factor receptor 1(VEGFR1)form(sFltl)contributes to endothelial dysfunction in pathological conditions.However,the roles of sFltl in ischemia-induced neovascularizationof aged animals have not been investigated.Methods:Here,we used histological and biological approaches to explore the effects of sFlt-1 in impaired neovascualrization in resposne to ischema in aged mice.The medhods includes as flow:Mouse hindlimb ischemic model and blood flow analysis,Immunohistochemical analysis,Gene expression assays,Gelatin zymography,Immunoblotting.assay,Cell culture,Tubulogenesis assay,Cell migration,invasion,and proliferation assays,ELISA,BM-derived EPC and macrophage isolation and culture,Immunofluorescence,siRNA transfection protocol,and stalitical analysis(one-way analysis of variance(ANOVA)followed by Scheffe’s multiple-comparison post hoc test).Results1.Aging impairs angiogenesis in response to ischemiaAs compared with yong mice,the recovery of the ischemic/non-ischemic blood flow ratio in the aged mice remained impaired throughout the followup period.The aged mice had lower capillary density in not only non-ischemic but also ischemic muscles compared to the young mice,suggesting that aging impairs vascular regenerative capacity.2.Impact of aging on sFltl expression and the downstream signalingpathwayWe also observed that aged ischemic muscles had dramatically increased sFlt1 gene levels compared to those of the young mice.Consistent with the gene expression assay,immunostaining analysis revealed that sFlt-1+ staining signal(i.e.,splice isoforms sFlt-1[77 kDa]and sFlt-1 14[82 kDa])was markedly increased in the ischemic myofiber space of aged mice.In addition,immunofluorescence show that Flt-1 is expressed in the endothelial cells as well as infiltrated macrophages.The aged ischemic muscles had lower levels of p-VEGFR2 and p-Akt proteins compared to those of the young mice.3.Impact of aging on Wnt5a/11 and SC35 expressionsIschemic stress stimulated more Wnt5a protein expression in ischemic muscles of the aged mice compared to those of the young mice.Likewise,the aged ischemic muscles exhibited increased levels of Wnt5a mRNA.Similar to Wnt5a,the levels of Wnt11 gene were also higher in the aged mice.Wnt5a has been shown to upregulate SC35 in RAW24.7 cells.As anticipated,the aged ischemic muscles had dramatically higher expression of SC35 protein compared to the young muscles.However,although ischemic stress stimulated the expression of targeted Wnt family members(including Wnt3,Wnt3a,Wnt5b,Wnt7a,Wnt7b,Wnt8a,Wnt9b,WntlOa,and WntlOb)in both young and aged mice,aging did not affect these members in non-ischemic and ischemic muscles.4.Aging accelerates the inflammatory response in response to ischemiaThe levels of galactin-3 protein as well as TLR2 protein were increased in the ischemic muscles of aged mice compared with the young mice.Consistently,the present study’s immunochemical evaluation of ischemic and non-ischemic sections harvested on day 4 after surgery using mac-3 and CD45 antibodies revealed that higher numbers of leukocytes and macrophages were present in the extra-capillary space in ischemic muscles of the aged mice compared to those of the young mice.In addition,we observed that the levels of the gelatinolytic activities for MMP-2 and MMP-9 were significantly increased in ischemic tissues of the aged mice compared to the young mice.5.Aging impairs progenitor cell intrinsic functionsWe observed that the numbers of c-Kit+/CD31+ progenitor cells were decreased in the peripheral blood of the aged mice compared to the young mice.These results indicated that aging significantly impaired VEGF-A-induced c-Kit+ migration and invasion as well as proliferation.6.Aged BM-derived CD11+ cells exhibited antiangiogenic effects via the induction of sFltlin response to hypoxiaUnheated young cKit+ cells(Yc-Kit+Cs)-cultured medium(Yc-Kit+CM)and aged c-Kit+ cells(Ac-Kit+Cs)-cultured medium(AcKit+CM)had comparable stimulator effects.We observed that unheated aged CDllb+ cell(ACD11b+Cs)-cultured medium(ACD11b+CM)impaired HUVEC proliferation compared to the heated medium,whereas unheated young CD11b+ cell(YCD11b+Cs)-cultured medium(YCD11b+CM)stimulated HUVEC proliferation.YCD11b+CM enhanced VEGF-A-induced cell proliferation;this effect was abolished by replacement of the medium with ACD11b+CM.To explore these different effects,we determined the levels of the sFltl in four conditioned media.The ELISA results showed that BM-derived ACD11b+Cs had increased levels of sFltl protein in the conditioned medium in response to hypoxia compared to those of YCDllb+Cs.However,hypoxic stress had no effect on the sFltl protein production in the BM-derived Yc-Kit+Cs and Ac-Kit+Cs.YCD11b+CM stimulated HUVEC tubulogenesis compared to the VEGF positive control,whereas it was suppressed by ACD11b+CM.Likewise,YCD11b+CM accelerated the VEGF-induced HUVEC tubulogenesis,whereas the beneficial effects of VEGF-A and YCD11b+CM were abolished by the addition of ACD11b+CM.7.Up-regulation of Wnt5a/SC35 axis is responsible for the down-stream sFltl release and decreased VEGFR2/Aktsignaling-related EC and EPC angiogenic actionsWnt5a silencing suppressed not only its mRNA but also down-stream SC35 protein expression and sFlt1 release into culture medium of ACD11b+Cs under hypoxia.As compared with siContconditioned concentrated ACD11b+CM,siWnt5aconditioned concentrated ACD11b+CM markedly promoted the VEGF-induced phosphorylation of VEGFR2 and Akt in HUVECs.Likewise,siWnt5a-conditioned ACD11b+CM ameliorated HUVEC tubulogenic action in response to VEGF.On the other hand,unheated ACD11b+CM suppressed VEGFinduced young EPC-like c-Kit+ cell proliferation as compared with unheated ACD11b+CM.Moreover,siWnt5a-conditioned ACD11b+CM exhibited a stimulatory effect on EPC tubulogenesis.Conclusion1)Aging impaired ischemia-induced blood flow recovery accompanied with the increasing of plasma and ischemic muscle soluble Flt-1 levels;2)The ischemic muscles of aged mice had decreased levels of p-VEGFR2 and p-Akt and increased levels of Wnt5a and SC35 expressions as well as increased numbers of infiltrated inflammatory cells and matrix metalloproteinase-9 activity;3)Hypoxic stress stimulated sFltl expression in cultured ACDllb+Cs of aged BM,and this effect was reversed by siWnt5a treatment;4)The cultured medium of aged mouse BM-derived ACD11b+Cs under hypoxic conditions suppressed HUVEC and EPC angiogenic responses in response to VEGF;these effects were ameliorated by siWnt5a-conditioned ACD11b+CM.Thus,aging can impair neovascularization in response to hypoxia through the VEGFR2/Akt signaling inactivation in ECs and ECPs that is mediated by Wnt5a/SC35 activated macrophages-derived sFltl production in advanced ageChapter Two:Clinical Significance of Serum Cathepsin K Levels in Patients with Chronic Heart FailureCysteinyl cathepsins are papain family members of the cysteine protease superfamily.In humans,11 members have been identified-cathepsins B,C,F,H,K,L,O,S,V,W,and X-all of which share a conserved active three-dimensional pocket formed with cysteine,histidine,and asparagine residues.Cathepsin contains a signaling peptide,proregion,heavy chain,and light chain Cathepsins destined for the lysosome are further processed in the Golgi apparatus by modification of mannose residues to mannose-6-phosphate(m6p).Following this modification,cathepsins are relocated to the acidic compartments,lysosomes and endosomes,through either the mannose-6-phosphate receptor-dependent or-independent pathways,where the enzymes are activated by different proteases,such as pepsin,neutrophil elastase,cathepsin D and various cysteine proteases to function in unwanted substrate.Recent studies indicated that cathepsins as a nontraditional role can be involved in the intracellular signaling pathways and cellular functions to contribute to the neovascularization and cardiovascular remodeling.Cathepsin K(Cat K)is a member of the papain family.It was first found in macrophages and osteoclasts and was later also identified to be highly expressed in the vascular smooth muscle cells of atherosclerotic lesions and inflammatory cells.In addition to degrading collagen,Cat K degrades elastin and fibrin,and plays a critical role in bone remodeling,vascular regeneration,and the formation and development of atherosclerosis.Hua and colleagues reported that CatK knockout partly reversed the impaired cardiomyocyte contractility and dysregulated calcium handling associated with high-fat diet.In H9c2 myoblasts,they also observed that silencing of CatK inhibited palmitic acid-induced impairment of myocyte contractility,indicating that CatK cant myocardial contraction ability.In generally,elevated SERCA2a has been postulated to reduce cardiac hypertrophy.However,contrary to this notion,the authors found an upregulation of SERCA2a and phospholamban after high-fat feeding.More importantly,CatK knockout resulted in elevated SERCA2a and phospholamban and the ratio of phospholamban to SERCA2a.Although the significance of these findings are difficult to explain in the context of improved contractility,the elevated phospholambanto-SERCA2a ratio may partly explain the incre,ased resting Ca2+ in the myocytes from cathepsin K knockout mice.2013,the authors also demonstrated that pressure overload dampened cardiomyocyte contractile function along with decreased resting Ca2+ levels and delayed Ca2+ clearance,which were partly resolved by CatK knockout.In cultured H9c2 myoblast cells,silencing of CatK blunted it with decreased resting Ca2+ levels and delayed Ca2+ clearance,whereas CatK did not affect on blood pressure,indicating that CatK deletion-mediated beneficial effects may be due to cardiac specifically.In addition,CatK overexpression accelerated heart failure.Collectively,CatK activity appears to modulate cardiac hypertrophy.Recently,we observed that the patients with atrial fibrillation and coronary artery disease had increased levels of plasma CatK levels.However,it is unclear whether there is a relationship between plasma CatK levels and chronic heart failure(CHF).Thus,in this study,we have the first time to examine the changes of plasma CatK levels in CHF patients to identify the novel biomarker for CHF.Objective:Cysteinyl cathepsin K(CatK)is one of the most potent mammalian collagenases involved in cardiovascular disease.Here,we investigated the clinical predictive value of serum CatK levels in patients with chronic heart failure(CHF).Methods:We recruited 134 consecutive patients with CHF who were admitted to Yanbian University Hospital(Yanji,China)between March 2012 and March 2014 for the in-hospital treatment of decompensation of CHF.All enrolled patients had New York Heart Association(NYHA)functional class Ⅱ-Ⅳ and CHF with a reduced ejection fraction of ischemic(having myocardial infarction history),hypertension(diagnosis of primary hypertension),and idiopathic dilated cardiomyopathic etiologies.These CHF patients were taking standard medical therapeutics with diuretics,inotropic agents(e.g.,digoxin),statins,a β-blocker,angiotensin-converting enzyme inhibitors(ACEI)and/or angiotensin type 1 receptor blockers(ARBs).We divided the CHF patients into two groups by their left ventricular(LV)ejection fraction(LVEF)values:the 44 patients showing LVEF<40%(the "LowLVEF" group)and the 90 patients showing LVEF values 40%(the "highLVEF" group).Venous blood samples were obtained for chemical analysis after an overnight fast.Serum CatK levels were evaluated by ELISA kits in duplicate.The levels of NTproBNP,troponin I,LDL,HDL,hs-CRP,and hemoglobin A1c were studied.The LVPWT,IVST,LVDd,LVSd,LVEF,and LAD were calculated by a Sonos 2500 ultrasound system.Results:1.The clinical characteristics of the two patient groups:there were no significant between-group differences in the patients’ age or BMI values.2.Our linear regression analysis showed that the CatK levels correlated negatively with the LVEF(r =-0.4,P<0.001)and correlated positively with the LVDd(r = 0.2,P<0.01)and LVDs(r = 0.3,P<0.001)levels.There was also a significant correlation between CatK and LAD(r = 0.3,P<0.01).3.According to the NYHA functional classification,the 134 patients were distributed as follows:17 with NYHA class Ⅱ CHF,47 with NYHA class Ⅲ,and 70 with NYHA class IV.The LVDd and LVDs values derived from echocardiography were 55.9 ± 14.5 mm and 48.7 ± 11.9 mm,respectively,suggesting LV dilation.The IVST and LVPWT values were 9.3±1.5 mm and 12.5±2,1 mm,respectively.The LVEF was reduced to a mean value of 41.9 ± 8.3%.The plasma NTproBNP and CatK levels were 4024±4026 pg/mL and 51.6 ± 16.6 pg/mL,respectively,suggesting elevated neurohumoral factor and CatK.4.In the single logistic regression analysis,age,gender,hypertension,LVDd,LAD,and CatK were significantly associated with CHF.However,BMI,diabetes mellitus,hs-CRP,and troponin I were not associated with CHF.The multiple logistic regression analysis using age,gender,hypertension,LAD,LVDd,and CatK revealed that hypertension(odds ratio[OR]4.11;95%confidence interval[CI]1.08-15.69;P<0.05),LAD(OR 1.13;95%CI 1.01-1.25;P<0.05),LVDd(OR 1.20;95%CI,1.05-1.37;P<0.01),and the CatK(OR 0.90;95%CI 0.84-0.95;P<0.01)levels were significantly correlated with CHF.Conclusion1.The presence of elevated serum levels of CatK can serve as a novel biomarker of CHF;2.The monitoring of circulating CatK can be used as a noninvasive way of showing the mechanisms of cardiac remodeling and dysfunction in CHF.