Study On The Molecular Mechanisms Of Vascular Endothelial Cell Apoptosis And Angiogenesis By Using A Novel Small Molecule Compound

BACKGROUND AND OBJECTIVEVascular endothelial cells(VECs)play important roles in vascular development. Numerous studies show that dysregulation of VEC apoptosis leads to vascular dysfunction and is involved in various inflammatory and degenerative diseases.These severe diseases impair our health seriously.At present,how to prevent and cure these diseases is a problem to solve.Angiogenesis refers to the formation of new capillaries from preexisting vessels. Angiogenesis plays essential roles in physiological processes such as embryonic development and in pathologic conditions(e.g.,tumor growth and atherosclerosis). The process of angiogenesis consists of several signal transductions,which include proliferation,survival,migration,extracellular matrix degradation,differentiation and morphogenesis.Angiogenesis has close relationship with endothelial cell apoptosis. Inhibition of VEC apoptosis providing VEC survival is thought to be an essential issue during vessel formation.The molecular mechanisms associating VEC apoptosis with angiogenesis are not elucidated clearly now.The use of small cell-permeable molecules to effect biological phenomena,also known as chemical genetics,has made a significant impact in diverse areas of biological medicine.The utilization of chemical genetics may discover key factors involved in apoptosis and angiogenesis.Therefore,it can provide experimental evidences to illustrate issues mentioned above.Our previous study confirmed that human umbilical vein endothelial cell(HUVEC)apoptosis was induced by deprived of serum and FGF-2.We synthesized a series of 2,3-dihydro-3-substituted-1,4-benzoxazine derivatives and found that 6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine(ABO)could obviously elevate VEC viability in the absence of serum and FGF-2.Consequently we presume that ABO might be an inhibitor of VEC apoptosis as well as a promoter of angiogenesis.In this study,we aim to elucidate the role of ABO in VEC apoptosis and angiogenesis as well as the molecular mechanisms associating endothelial cell apoptosis with angiogenesis utilizing small molecule compound,and provide new insights into the clinical prevention and therapy for blood vessel degenerative diseases.It is well known that redox plays important roles in cell apoptosis and angiogenesis. The components involved in redox include mitochondria,reactive oxygen species (ROS),NADPH oxidase,superoxide dismutase(SOD),nitric oxide(NO)and endothelial nitric oxide synthase(eNOS)etc.Accordingly,we first investigate the effect of ABO on signal factors involved in redox.Then,we study the role of ABO in other components closely related to apoptosis and angiogenesis(e.g., Phosphatidylcholine-specific phospholipase C(PC-PLC),P53,integrinβ₄,nuclear factorκB(NF-κB)and H-ras).CONTENTS1.Study of ABO on inhibiting vascular endothelial cell apoptosis induced by deprived of serum and FGF-22.Study of ABO on promoting angiogenesis3.Study of molecular mechanisms on ABO inhibiting VEC apoptosis and promoting angiogenesisMETHODS1.Vascular endothelial cell culture:HUVECs were obtained as described previously [Jaffe et al.,1973].2.Cell apoptosis measurement: 2.1 The cell viability was determined by MTT-assay2.2 Observation of cell morphological changes by phase contrast microscope2.3 Analysis of nuclear fragmentation and chromatin condensation by acridine orange staining combined with laser scan confocol microscope2.4 TUNEL assay to determine the apoptotic rate3.Angiogenesis assay in vitro:capillary-like tube formation on Matrigel as described previously[Kureishi Y et al,2000]4.Angiogenesis assay in vivo:chick embryo chorioallantoic membrane(CAM) method as described previously[Ribatti et al,1997]5.Cell migration assay in vitro:monolayer cell wound healing assay as described previously[Bürk,1973;Vasvari et al,2007]6.Mitochondrial membrane potential(MMP)assay:fluorescent probe(TMRM) combined with laser scan confocol microscope as described previously[Falchi et al,2005]7.ROS assay:fluorescent probe(DCHF)combined with laser scan confocol microscope8.NADPH oxidase activity assay as described[Li et al,2002]9.SOD activity assay:SOD detection kit10.NO production assay:NO detection kit11.eNOS activity assay:eNOS detection kit12.PC-PLC activity assay as described[Wu et al.,1997]13.Analysis of cellular distribution and expression of proteins:1)Examination of the changes in P53 and NF-κB protein levels and distributions by immunocytochemistry combined with laser scan confocol microscope2)Analysis the levels of P53,integrinβ₄ and H-ras by Western blot assayRESULTS1.ABO inhibits vascular endothelial cell apoptosis induced by deprived of serum and FGF-2 At 24 h,ABO(50-200μM)significantly increased VEC viability(Fig.2,p＜0.05). The optimized concentration is 50μM and the following studies use 50μM ABO to treat VECs.At 12 and 24 h,under phase microscope,the number of apoptotic bodies decreased obviously with treatment of ABO(Fig.3);AO staining showed that ABO inhibited nuclear fragmentation as well as chromatin condensation(Fig.4).TUNEL assay showed that the apoptotic rate decreased obviously when VECs were exposed to ABOfor 24 h(Fig.5,p＜0.05).2.ABO promotes angiogenesis2.1 Capillary-like tube formation assay on Matrigel in vitro showed that in the absent of serum and FGF-2,ABO significantly promote angiogenesis in a time-dependent manner(24-96 h)(Fig.6,p＜0.01);in the presence of FGF-2(70ng/mL),ABO and FGF-2 could synergistically facilitate angiogenesis(Fig.7);in the presence of serum (20%,v/v),ABO could not promote angiogenesis(Fig.8);in the presence of serum (20%,v/v)and FGF-2(70ng/mL),ABO facilitated angiogenesis at the late cultural stage(Fig.9).2.2 CAM assay in vivo showed that ABO(20nmol/100μL)promoted the formation of vascular net(Fig.10,p＜0.05).2.3 Monolayer cell wound healing assay in vitro showed that ABO(50μM)obviously promoted cell migration in a time-dependent manner(Fig.11,p＜0.01).3.Molecular mechanism of ABO inhibiting VEC apoptosis and promoting angiogenesis3.1 At 12 and 24 h,ABO significantly decreased mitochondrial membrane potential (Fig.12 & Fig.13,p＜0.05)and cellular ROS level(Fig.14 & Fig.15,p＜0.05).3.2 At 12 h,ABO significantly decreased the activities of NADPH oxidase(Fig.16, p＜0.05)and PC-PLC(Fig.19,p＜0.05).ABO has no effect on SOD activity(Fig.17, P＞0.05).3.3 At 12 h,ABO(50μM)significantly increased the activity of eNOS(Fig.18B, p＜0.05)and NO production(Fig.18A,p＜0.05).At 6 and 24 h,there is no difference of eNOS activity and NO production between control group and ABO treatment group(Fig.18). 3.4 Immunocytochemistry assay showed that NF-κB nuclear translocation was not observed when VECs were exposed to ABO for 6 and 12 h(Fig.23);at 24 h,ABO obviously depressed P53 expression and blocked P53 nuclear translocation(Fig.20, p＜0.05).3.5 Western Blot assay showed that at 24 h,ABO significantly inhibited P53(Fig.21B, p＜0.05)and integrinβ₄ expression(Fig.22B,p＜0.05),but ABO have no effect on P53 andβ₄ expression at 12 h(Fig.21A & Fig.22A,p＞0.05).At 12 and 24 h,ABO obviously depressed H-ras expression(Fig.24,p＜0.05).CONCLUSION1.ABO significantly inhibited vascular endothelial cell apoptosis induced by deprived of serum and FGF-2.2.ABO effectively promoted angiogenesis.3.In the absence of serum and FGF-2,ABO could maintain mitochondrial functions through stabilizing MMP and preventing hyperpolarization.Moreover,ABO decreased activity of NADPH oxidase.Accordingly,ABO could effectively inhibit VEC apoptosis and promote migration as well as angiogenesis due to depressing high level of cellular ROS.In addition,ABO further enhanced angiogenesis via increasing eNOS activity and NO production.4.ROS had close relationship with PC-PLC and P53,and all of them were associated with mitochondria.ABO probably effected relative factors involved in this signal transduction pathway,that is,controlling ROS levels,depressing PC-PLC activity as well as the expression of P53,integrinβ₄ and H-ras,to inhibit VEC apoptosis induced by deprived of serum and FGF-2 and promote angiogenesis.Our data suggested that ABO was a very useful agent for insights into various questions of angiogenesis and might have a therapeutic potential in vascular diseases.