Study On The Anti-angiogenesis Effect And The Underlying Mechanisms Of Pristimerin On Synovium In Adjuvant-induced Arthritis Rats

Rheumatoid arthritis(RA),a chronic systemic inflammatory disease,which is characterized by proliferation of synovial cells,resulting in the formation of pannus that invades cartilage and bone;in this manner,joint damage is exacerbated.Although the etiology of RA remains unclear,angiogenesis plays a pivotal role in the development of this disease.Angiogenesis,a highly regulated process,is the growth of new blood vessels from existing ones,which maintain inflammatory state by transporting inflammatory cells to the synovium and by supplying nutrients to the pannus;this process is an essential event in RA and may be performed by stimulating proliferation,migration,and tube formation of endothelial cells.Therefore,inhibition of angiogenesis is probably viable and effective for RA treatment.Angiogenesis is a complex process regulated by growth factors and cytokines.Among these factors and cytokines,vascular endothelial growth factor(VEGF)is a key regulator in angiogenesis.VEGF is implicated in biological activities,such as stimulating endothelial cell proliferation,migration,and tube formation,after this factor binds to VEGFR2[kinase domain region or fetal liver kinase 1(flk-1)],one of the primary receptors in angiogenesis.VEGFR2 is upregulated at sites where VEGF,which is produced in the synovium in response to pro-inflammatory cytokines,is upregulated.The binding of VEGF to VEGFR2 leads to the activation of diverse intracellular signaling molecules,including extracellular signal-regulated kinases(ERKs),phosphoinositide 3-kinase/AKT kinase,and p38 mitogen-activated protein kinase(p38-MAPK).Thus,the effect of VEGFR2 interactions with intracellular signaling molecules involved in angiogenesis should be investigated to help elucidate the pathogenesis of RA.Celastrus aculeatus Merr.(Celastrus),a traditional Chinese herbal medicine belonging to Celastraceae,has been used in China for centuries to treat various rheumatoid diseases.We previously reported that Celastrus exhibits anti-inflammatory activity and reduces the severity of clinical arthritis and bone destruction in a rat adjuvant-induced arthritis(AA)model of human RA.Celastraceae plants contain a bioactive natural triterpenoid quinine methide called pristimerin.Pristimerin is implicated in biological activities,such as antimicrobial,antiperoxidation,and anti-inflammatory effects;pristimerin can also inhibit tumor angiogenesis during cancer treatment.These findings have provided a basis for further studies on anti-angiogenic potential and related mechanisms of pristimerin to treat synovial angiogenesis in RA.Objectives:1.To study the effect of pristimerin on anti-angiogenesis.2.To investigate the anti-angiogenesis effect of pristimerin on synovium in adjuvant-induced arthritis rats.3.To explore the underlying mechanisms of pristimerin on anti-angiogenesis.Methods:1.The effect of pristimerin on proliferation,migration,invasion and tube formation of HFLS-RA and HUVECsVascular endothelial growth factor VEGF-induced HUVECs proliferation was evaluated by MTT to determine the viability of HFLS-RA and HUVECs.HFLS-RA migration was assessed by wound-healing and chemotaxis migration assays.HUVECs migration was also examined by wound-healing migration assay.HUVECs invasion was then detected using Transwell chambers.A matrigel tube formation assay was performed to evaluate capillary tube formation of HUVECs.2.The effect of pristimerin on rat aortic ring assayThe thoracic aortas rings were culturet 9 d,microvessel growth was quantified using a microscopic camera system at 100×magnification by manually counting the number of microvessels sprouting from rat aortic rings.With three parallel wells for each group in each time,three independent assays were performed.3.The effect of pristimerin on intersegmental vascular development of zebrafishWe observed and compared the number of Intersegmental vessel(ISV)and vessel sprouts and the inhibition rate of vessel length of the zebrafish after pristimerin treated with different concentration from 6 to 30 hours past fertilization(hpf).4.The effect of synovium angiogenesis in AA rats by pristimerinInduction and evaluation of rats with adjuvant arthritis(AA):SD rats were immunized subcutaneously at the base of the tail with Mtb.The rats were examined once daily and graded in terms of signs observed since the onset of arthritis.The volume of inflamed hind paws was determined by using the small animal limbs swelling instrument.The sections were stained with hematoxylin and eosin to evaluate arthropathological and histological characteristics.The polyclonal antibody recognizing CD34 was used as a microvessel marker to determine vessel density in synovial membrane tissues of inflamed joints.ELISA was applied to detect TNF-a,Ang-1,and MMP-9 expression in sera and VEGF,VEGFR2,and p-VEGFR2 in synovial tissues by western blot of rats.5.The effect of pristimerin on the expression of signaling molecules in VEGF-induced HUVECsVEGFR2,p-VEGFR2 and its downstream PI3K-AKT-mOTR,ERK1/2,JNK,and p38 signaling molecules in VEGF-induced HUVECs were analyzed by western blot.Results:1.Pristimerin inhibits the viability of HFLS-RA and HUVECsThe results showed that pristimerin inhibited cell viability in a dose-and time-dependent manner when cells were cultured in a normal cell culture medium.VEGF stimulation increased the number of HUVECs to approximately 1.5-fold.Moreover,0.125,0.25,and 0.50 μM of pristimerin markedly inhibited VEGF-mediated HUVECs viability.2.Pristimerin inhibits VEGF-induced HFLS-RA migrationTreated with pristimerin,HFLS-RA displayed less migration after the cells were stimulated with VEGF.Transwell chambers results showed that HFLS-RA migrated toward VFGF,but this migration was suppressed by pristimerin.3.Pristimerin inhibits VEGF-induced HUVECs migration,invasion and tube formationThe results showed that pristimerin completely inhibited VEGF-induced HUVECs migration in a concentration-dependent manner;pristimerin significantly inhibited VEGF-induced HUVECs migration.Numerous HUVECs migrated to the underside of the filters in the Transwell chambers after these cells were stimulated with VEGF.However,the number of invasive cells was significantly reduced in the presence of pristimerin.Robust and complete tubular-like structures were formed in the presence of VEGF when HUVECs were plated onto matrigel.Pristimerin disrupted tube formation in a concentration-dependent manner compared with the tube formation of HUVECs induced by VEGF.4.Pristimerin suppresses VEGF-induced vessel sprout formation ex vivoVEGF significantly triggered endothelial cell migration and microvessel sprouting,leading to the formation of a complex microvessel network emerging from the aortic rings and growing outward;by contrast,pristimerin inhibited microvessel sprouting in a dose-dependent manner.5.Pristimerin suppresses intersegmental vascular development of zebrafishStudy results showed that nimbidiol suppress ISV development in each pristimerin-treated group,and compared with the control group,the number of ISV decreased markedly in the experimental group.While,at the same time,the number of vessel sprouts and inhibition rate increased.6.Pristimerin reduces AA rats Arthritis severity scores and back metatarsalArthritis severity scores and back metatarsal volume of vehicle-treated rats significantly increased compared with those of control rats;this increase continued until the peak phase(on 21 day)of the disease was reached,while pristimerin inhibited the increase in arthritis scores and back metatarsal volume of AA rats.Likewise,the severity of AA in pristimerin-treated rats was significantly reduced;however,macroscopic signs of arthritis,such as erythema and/or swelling,were markedly observed in vehicle-treated rats.7.Pristimerin decreases severity of arthritis in AA ratsSynovial mononuclear cell infiltrations,synovial membrane hyperplasia,pannus formation,and cartilage damage were significantly reduced in the joints of pristimerin-treated rats compared with vehicle control rats.8.Pristimerin inhibits synovium angiogenesis in AA ratsCD34 positive was hardly observed in the synovium of normal control rats,whereas a strong CD34 positive was found in the synovial tissue of vehicle-treated rats.Furthermore,the microvessel density of pristimerin-treated rats was less than that of model control rats.9.Pristimerin reduces the expression of proangiogenic mediators in AA ratsAng-1 and MMP-9 secretion in pristimerin-treated rats was significantly inhibited compared with that in vehicle-treated controls.VEGF and the activation of VEGFR2 in pristimerin-treated rats were also inhibited.10.Pristimerin decreases autophosphorylation of VEGFR2 in VEGF-induced HUVECsPristimerin decreased VEGF-induced HUVECs tyrosine autophosphorylation of VEGFR2 in a dose-dependent manner but not the total of VEGFR2;this result suggested that the prevention of VEGFR2 autophosphorylation contributed to the inhibitory effect of pristimerin on VEGF-induced angiogenesis.11.Pristimerin attenuates the activation of VEGFR-2-mediated signaling pathway in VEGF-induced HUVECsThe results show that the expression levels of p-PI3K,p-AKT,p-mTOR,p-ERKl/2,p-JNK,and p-p38 were markedly increased by VEGF treatment in HUVECs,suggesting that the effect of pristimerin on VEGF-induced angiogenesis was inhibited by the downregulation of VEGFR2-mediatedConclusions:1.Pristimerin can inhibit VEGF-induced HUVECs proliferation,migration,and tube formation in vitro,as well as VEGF-induced HFLS-RA migration.2.Pristimerin suppresses intersegmental vascular development of zebrafish and VEGF-induced vessel sprout formation ex vivo.3.Pristimerin can suppress synovium angiogenesis in AA rats;therefore,pristimerin could exhibit potent anti-angiogenic activity against arthritis.4.Pristimerin may be block VEGFR-2 autophosphorylation and downregulated the activation of PI3K-AKT--mTOR,ERK1/2,JNK,and p38 in VEGF-induced HUVECs.