Hypoxia/reoxygenation: a dynamic regulator of lysyl oxidase-facilitated breast cancer migration

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up-regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (beta-aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX-dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor-1 (HIF-1alpha) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF-1alpha could not up-regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co-localization with hypoxic regions (defined by HIF-1alpha expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide-mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency.     

Indirubin-3'-monoxime, a derivative of a chinese antileukemia medicine, inhibits angiogenesis

Although the antiangiogenic activity of indirubin‐3‐monoxime (I3M), a derivative of a Chinese anti‐leukemia medicine, has been demonstrated using transgenic zebrafish, the detail molecular mechanism has not been elicited. To further establish its role in antiangiogenic activity, we tested its potential against human umbilical vein endothelial cells (HUVECs) and the in vivo Matrigel plug model was applied to evaluate new vessel formation. We also investigated the molecular mechanisms of I3M‐induced antiangiogenic effects in HUVECs. We found that I3M significantly inhibited HUVEC proliferation (2.5–20 µM), migration (2.5–20 µM), and tube formation (10–20 µM) in HUVECs. The number of microvessels growing from the aortic rings was suppressed by I3M treatment. Moreover, I3M suppressed neovascularization in Matrigel plugs in mice. The underlying antiangiogenic mechanism of I3M was correlated with down‐regulation of the vascular endothelial growth factor receptor‐2 activation, at least a part. These findings emphasize the potential use of I3M in pathological situations involving stimulated angiogenesis, such as tumor development. J. Cell. Biochem. 112: 1384–1391, 2011. © 2011 Wiley‐Liss, Inc.     

Endogenous reduction of miR‐185 accelerates cardiac function recovery in mice following myocardial infarction via targeting of cathepsin K

Angiogenesis is critical for re‐establishing the blood supply to the surviving myocardium after myocardial infarction (MI) in patients with acute coronary syndrome (ACS). MicroRNAs are recognised as important epigenetic regulators of endothelial function. The aim of this study was to determine the roles of microRNAs in angiogenesis. Eighteen circulating microRNAs including miR‐185‐5p were differently expressed in plasma from patients with ACS by high‐throughput RNA sequencing. The expressional levels of miR‐185‐5p were dramatically reduced in hearts isolated from mice following MI and cultured human umbilical vein endothelial cells (HUVECs) under hypoxia, as determined by fluorescence in situ hybridisation and quantitative RT‐PCR. Evidence from computational prediction and luciferase reporter gene activity indicated that cathepsin K (CatK) mRNA is a target of miR‐185‐5p. In HUVECs, miR‐185‐5p mimics inhibited cell proliferations, migrations and tube formations under hypoxia, while miR‐185‐5p inhibitors performed the opposites. Further, the inhibitory effects of miR‐185‐5p up‐regulation on cellular functions of HUVECs were abolished by CatK gene overexpression, and adenovirus‐mediated CatK gene silencing ablated these enhancive effects in HUVECs under hypoxia. In vivo studies indicated that gain‐function of miR‐185‐5p by agomir infusion down‐regulated CatK gene expression, impaired angiogenesis and delayed the recovery of cardiac functions in mice following MI. These actions of miR‐185‐5p agonists were mirrored by in vivo knockdown of CatK in mice with MI. Endogenous reductions of miR‐185‐5p in endothelial cells induced by hypoxia increase CatK gene expression to promote angiogenesis and to accelerate the recovery of cardiac function in mice following MI.     

Lysyl oxidase regulates actin filament formation through the p130(Cas)/Crk/DOCK180 signaling complex

We have previously demonstrated that lysyl oxidase (LOX) is expressed in invasive breast cancer cells compared to poorly invasive cells. Additionally, we have recently shown that LOX regulates cell migration, a key step in the invasion process, through a hydrogen peroxide-dependent mechanism involving the focal adhesion kinase (FAK)/Src signaling complex. Here we further elucidate the role of LOX in cell motility/migration by examining the role of LOX in actin filament polymerization. We demonstrate that inhibition of LOX leads to an increase in phalloidin staining, directly associated with an increase in actin stress fiber formation. This increase in staining was confirmed by activity assays showing an increase in Rho activity with decreased LOX activity. Additionally, Rac and Cdc42 activity decreased with the reduction in LOX activity. Taken together, these data demonstrate a loss of a motogenic phenotype with decreased LOX activity. Finally, in order to elucidate the mechanism by which LOX regulates actin polymerization, we have demonstrated that LOX facilitates p130(Cas) phosphorylation, which allows for the binding to CAS related kinase (Crk) and formation of the p130(Cas)/Crk/DOCK180 signaling complex. Formation of this complex leads to an increase in Rac-GTP, which decreases actin stress fiber formation and increases formation of lamellipodium. These data demonstrate that LOX regulates cell motility/migration through changes in actin filament polymerization, which involve the regulation of the p130(Cas)/Crk/DOCK180 signaling pathway. Elucidating the role of LOX in the regulation of cell motility will allow the development of more effective therapeutic strategies to treat invasive/metastatic breast cancer.     

Biphasic regulation of H2O2 on angiogenesis implicated NADPH oxidase

ROS (reactive oxygen species) take an important signalling role in angiogenesis. Although there are several ways to produce ROS in cells, multicomponent non‐phagocytic NADPH oxidase is an important source of ROS that contribute to angiogenesis. In the present work, we examined the effects of H₂O₂ on angiogenesis including proliferation and migration in HUVECs (human umbilical vein endothelial cells), new vessel formation in chicken embryo CAM (chorioallantoic membrane) and endothelial cell apoptosis, which is closely related to anti‐angiogenesis. Our results showed that H₂O₂ dose‐dependently increased the generation of O₂ ^? (superoxide anion) in HUVECs, which was suppressed by DPI (diphenylene iodonium) and APO (apocynin), two inhibitors of NADPH oxidase. H₂O₂ at low concentrations (10 µM) stimulated cell proliferation and migration, but at higher concentrations, inhibited both. Similarly, H₂O₂ at 4 nmol/cm² strongly induced new vessel formation in CAM, while it suppressed at high concentrations (higher than 4 nmol/cm²). Also, H₂O₂ (200～500 µM) could stimulate apoptosis in HUVECs. All the effects of H₂O₂ on angiogenesis could be suppressed by NADPH oxidase inhibitors, which suggests that NADPH oxidase acts downstream of H₂O₂ to produce O₂ ^? and then to regulate angiogenesis. In summary, our results suggest that H₂O₂ as well as O₂ ^? mediated by NADPH oxidase have biphasic effects on angiogenesis in vitro and in vivo.     

Effect of lysyl oxidase (LOX) on corpus cavernous fibrosis caused by ischaemic priapism

Penile fibrosis caused by ischemic priapism (IP) adversely affects patients’ erectile function. We explored the role of lysyl oxidase (LOX) in rat and human penes after ischemic priapism (IP) to verify the effects of anti‐LOX in relieving penile fibrosis and preventing erectile dysfunction caused by IP in rats. Seventy‐two rats were randomly divided into six groups: control group, control + β‐aminopropionitrile (BAPN) group, 9 hrs group, 9 hrs + BAPN group, 24 hrs group, and 24 hrs + BAPN group. β‐aminopropionitrile (BAPN), a specific inhibitor of LOX, was administered in the drinking water. At 1 week and 4 weeks, half of the rats in each group were randomly selected for the experiment. Compared to the control group, the erectile function of IP rats was significantly decreased while the expression of LOX in the corpus cavernosum was significantly up‐regulated in both 9 and 24 hrs group. Proliferated fibroblasts, decreased corpus cavernosum smooth muscle cells/collagen ratios, destroyed endothelial continuity, deposited abnormal collagen and disorganized fibers were observed in IP rats. The relative content of collage I and III was not obviously different among the groups. β‐aminopropionitrile (BAPN) could effectively improve the structure and erectile function of the penis, and enhance recovery. The data in this study suggests that LOX may play an important role in the fibrosis of corpus cavernosum after IP and anti‐LOX may be a novel target for patients suffering with IP.     

Role of Lysyl Oxidase Propeptide in Secretion and Enzyme Activity

Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX‐PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full‐length LOX cDNA (pre‐pro‐LOX), the N‐glycosylation null pre‐[N/Q]pro‐LOX cDNA and the deletion mutant pre‐LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N‐terminal propeptide sequence. The results show that glycosylation of the LOX‐PP is not required for secretion and extracellular processing of pro‐LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX‐PP for pro‐LOX exit from the ER and is the first to highlight the influence of LOX‐PP glycosylation on LOX enzyme activity. J. Cell. Biochem. 111: 1231–1243, 2010. © 2010 Wiley‐Liss, Inc.     

MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion independent of its topoisomerase II inhibition

The macrolide compound MFTZ‐1 has been identified as a novel topoisomerase II (Top2) inhibitor with potent in vitro and in vivo anti‐tumour activities. In this study, we further examined the effects of MFTZ‐1 on hypoxia‐inducible factor‐1α (HIF‐1α) accumulation, vascular endothelial growth factor (VEGF) secretion and angiogenesis. MFTZ‐1 reduced HIF‐1α accumulation driven by hypoxia or growth factors in human cancer cells. Mechanistic studies revealed that MFTZ‐1 did not affect the degradation of HIF‐1α protein or the level of HIF‐1α mRNA. By contrast, MFTZ‐1 apparently inhibited constitutive and inducible activation of both phosphatidylinositol‐3‐kinase (PI3K)‐Akt and p42/p44 mitogen‐activated protein kinase (MAPK) pathways. Further studies revealed that MFTZ‐1 abrogated the HIF‐1α‐driven increase in VEGF mRNA and protein secretion. MFTZ‐1 also lowered the basal level of VEGF secretion. The results reveal an important feature that MFTZ‐1 can reduce constitutive, HIF‐1α‐independent VEGF secretion and concurrently antagonize inducible, HIF‐1α‐dependent VEGF secretion. Moreover, MFTZ‐1 disrupted tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by hypoxia with low‐concentration serum or by serum at normoxia, and inhibited HUVECs migration at normoxia. MFTZ‐1 also prevented microvessel outgrowth from rat aortic ring. These data reflect the potent anti‐angiogenesis of MFTZ‐1 under different conditions. Furthermore, using specific small interfering RNA targeting Top2α or Top2‐defective HL60/MX2 cells, we showed that MFTZ‐1 affected HIF‐1α accumulation and HUVECs tube formation irrelevant to its Top2 inhibition. Taken together, our data collectively reveal that MFTZ‐1 reduces constitutive and inducible HIF‐1α accumulation and VEGF secretion possibly via PI3K‐Akt and MAPK pathways, eliciting anti‐angiogenesis independently of its Top2 inhibition.     

转化生长因子β通过Smad4非依赖的方式促进赖氨酰氧化酶的表达

目的:研究血管平滑肌细胞内转化生长因子β(TGF-β)信号怎样调节赖氨酰氧化酶(LOX)的表达。方法:分离小鼠原代血管平滑肌细胞,用携带不同Smad4干扰序列的慢病毒感染原代细胞,建立Smad4敲低稳定细胞系;分别用TGF-β刺激原代细胞或对照和Smad4敲低细胞,利用免疫印迹及Real-time PCR技术检测Smad4敲低后LOX的表达改变。结果:获得了Smad4敲低的细胞;TGF-β能诱导LOX表达,且在Smad4敲低的细胞内,LOX升高更明显。结论:TGF-β通过Smad4非依赖的方式促进LOX的表达。     

Hemokinins modulate endothelium function and promote angiogenesis through neurokinin-1 receptor

Substance P as a member of tachykinin family plays an important role in angiogenesis. Hemokinins (HKs) have been identified as new members of substance P-like peptides of tachykinin family. However, the effects of HKs on endothelial cells and angiogenesis have not been studied. For the first time, here we demonstrated that r/mHK-1, hHK-1 and hHK(4-11) dose-dependently stimulated the proliferation, migration, adhesion and tube formation of freshly isolated human umbilical vein endothelial cells (HUVECs), and further exhibited in vivo angiogenic effects in chick embryo chorioallantoic membrane model. The angiogenic effects of HKs were inhibited by the selective antagonist of neurokinin-1 rather than neurokinin-2 receptor. Mechanistically, HKs activated ERK1/2 phosphorylation, stimulated nitric oxide production, and upregulated the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) in HUVECs. Taken together, our data suggest that HKs emerge as pivotal endogenous regulators of angiogenesis and represent potential targets for the intervention of angiogenesis in different pathological conditions given their specific peripheral distribution.     

Lysyl oxidase propeptide sensitizes pancreatic and breast cancer cells to doxorubicin‐induced apoptosis

RAS mutations or its activation by upstream receptor tyrosine kinases are frequently associated with poor response of carcinomas to chemotherapy. The 18 kDa propeptide domain of lysyl oxidase (LOX‐PP) released from the secreted precursor protein (Pro‐LOX) has been shown to inhibit RAS signaling and the transformed phenotype of breast, pancreatic, lung, and prostate cancer cells in culture, and formation of tumors by Her‐2/neu‐driven breast cancer cells in a mouse xenograft model. Here, we tested the effects of LOX‐PP on MIA PaCa‐2 pancreatic cancer cells, driven by mutant RAS. In MIA PaCa‐2 cells in culture, LOX‐PP attenuated the ERK and AKT activities and decreased the levels of the NF‐κB p65 and RelB subunits and cyclin D1, which are activated by RAS signaling. In mouse xenograft growth, LOX‐PP reduced growth of tumors by these pancreatic cancer cells, and the nuclear levels of the p65 NF‐κB subunit and cyclin D1 proteins. While biological agents attenuate tumor growth when used alone, often they have additive or synergistic effects when used in combination with chemotherapeutic agents. Thus, we next tested the hypotheses that LOX‐PP sensitizes pancreatic and breast cancer cells to the chemotherapeutic agent doxorubicin. Purified LOX‐PP enhanced the cytotoxic effects of doxorubicin in pancreatic and breast cancer cells, as judged by ATP production, Cell Death ELISA assays, caspase 3 activation, PARP cleavage, and Annexin V staining. Thus, LOX‐PP potentiates the cytotoxicity of doxorubicin on breast and pancreatic cancer cells, warranting additional studies with a broader spectrum of current cancer treatment modalities. J. Cell. Biochem. 111: 1160–1168, 2010. © 2010 Wiley‐Liss, Inc.     

AMP-activated protein kinase (AMPK) signaling in endothelial cells is essential for angiogenesis in response to hypoxic stress

AMP-activated protein kinase (AMPK) is a stress-activated protein kinase that is regulated by hypoxia and other cellular stresses that result in diminished cellular ATP levels. Here, we investigated whether AMPK signaling in endothelial cells has a role in regulating angiogenesis. Hypoxia induced the activating phosphorylation of AMPK in human umbilical vein endothelial cells (HUVECs), and AMPK activation was required for the maintenance of pro-angiogenic Akt signaling under these conditions. Suppression of AMPK signaling inhibited both HUVEC migration to VEGF and in vitro differentiation into tube-like structures in hypoxic, but not normoxic cultures. Dominant-negative AMPK also inhibited in vivo angiogenesis in Matrigel plugs that were implanted subcutaneously in mice. These data identify AMPK signaling as a new regulator of angiogenesis that is specifically required for endothelial cell migration and differentiation under conditions of hypoxia. As such, endothelial AMPK signaling may be a critical determinant of blood vessel recruitment to tissues that are subjected to ischemic stress.     

Peptides encoded by exon 6 of VEGF inhibit endothelial cell biological responses and angiogenesis induced by VEGF

VEGF induces pathological angiogenesis and is an important target for the development of novel antiangiogenic molecules. In this study, we tested synthetic peptides based on the sequence of VEGF(189) for their ability to inhibit VEGF receptor binding and biological responses. We identified 12-amino acid peptides derived from exon 6 that inhibited VEGF binding to HUVECs, VEGF-stimulated ERK activation, and prostacyclin production. These peptides inhibited VEGF-induced mitogenesis, migration, and VEGF-dependent survival of endothelial cells, but caused no increase in apoptosis in the absence of VEGF. Exon 6-encoded peptides also caused a marked inhibition of VEGF-induced angiogenesis in vitro. Studies of effects of peptides on cross-linking of VEGF to its receptors and on binding of VEGF to porcine aortic endothelial cells expressing either KDR or neuropilin-1 showed that exon 6-encoded peptides effectively blocked the interaction of VEGF with both receptors. Exon 6-derived peptides caused release of bFGF from endothelial cells but inhibited bFGF-dependent ERK activation, cell proliferation and angiogenesis. Our findings indicate that VEGF exon 6-encoded peptides inhibit VEGF-induced angiogenesis, at least in part through inhibition of VEGF binding to KDR. In addition, exon 6-encoded peptides are also effective inhibitors of bFGF-mediated angiogenesis.     

Caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis through modulating GSK3β/β-catenin pathway

In this study, we investigate the anti‐cancer activity of caudatin in carcinomic human alveolar basal epithelial cell line A549 and anti‐angiogenic activity in human umbilical vein endothelial cells (HUVECs). We show that caudatin impairs the cell viability and induces G₀/G₁ phase arrest in A549 cells with a dose dependent manner. A549 cells, not HUVECs, dealing with caudatin exhibited typical characteristics of apoptosis, which were accompanied by activation of caspase‐3, caspase‐9 and Poly(ADP–Ribose) Polymerase (PARP). In addition, caudatin treatment resulted in a decrease of β‐catenin and increase of phosphorylation of β‐catenin, and inhibited phosphorylation levels of GSK3β (Ser 9) in A549 cells. Conditional medium of A549 cells‐induced or growth factors‐induced tube formation of HUVECs was markedly inhibited by caudatin treatment, which was associated with the inhibiting VEGF secretion from A549 cells by caudatin. Our findings suggest that caudatin inhibits carcinomic human alveolar basal epithelial cell growth and angiogenesis by targeting GSK3β/β‐catenin pathway and suppressing VEGF production. J. Cell. Biochem. 113: 3403–3410, 2012. © 2012 Wiley Periodicals, Inc.     

Hypoxia‐induced endothelial secretion of macrophage migration inhibitory factor and role in endothelial progenitor cell recruitment

Macrophage migration inhibitory factor (MIF) is a pleiotropic inflammatory cytokine that was recently identified as a non‐cognate ligand of the CXC‐family chemokine receptors 2 and 4 (CXCR2 and CXCR4). MIF is expressed and secreted from endothelial cells (ECs) following atherogenic stimulation, exhibits chemokine‐like properties and promotes the recruitment of leucocytes to atherogenic endothelium. CXCR4 expressed on endothelial progenitor cells (EPCs) and EC‐derived CXCL12, the cognate ligand of CXCR4, have been demonstrated to be critical when EPCs are recruited to ischemic tissues. Here we studied whether hypoxic stimulation triggers MIF secretion from ECs and whether the MIF/CXCR4 axis contributes to EPC recruitment. Exposure of human umbilical vein endothelial cells (HUVECs) and human aortic endothelial cells (HAoECs) to 1% hypoxia led to the specific release of substantial amounts of MIF. Hypoxia‐induced MIF release followed a biphasic behaviour. MIF secretion in the first phase peaked at 60 min. and was inhibited by glyburide, indicating that this MIF pool was secreted by a non‐classical mechanism and originated from pre‐formed MIF stores. Early hypoxia‐triggered MIF secretion was not inhibited by cycloheximide and echinomycin, inhibitors of general and hypoxia‐inducible factor (HIF)‐1α‐induced protein synthesis, respectively. A second phase of MIF secretion peaked around 8 hrs and was likely due to HIF‐1α‐induced de novo synthesis of MIF. To functionally investigate the role of hypoxia‐inducible secreted MIF on the recruitment of EPCs, we subjected human AcLDL⁺ KDR⁺ CD31⁺ EPCs to a chemotactic MIF gradient. MIF potently promoted EPC chemotaxis in a dose‐dependent bell‐shaped manner (peak: 10 ng/ml MIF). Importantly, EPC migration was induced by supernatants of hypoxia‐conditioned HUVECs, an effect that was completely abrogated by anti‐MIF‐ or anti‐CXCR4‐antibodies. Thus, hypoxia‐induced MIF secretion from ECs might play an important role in the recruitment and migration of EPCs to hypoxic tissues such as after ischemia‐induced myocardial damage.     

3-O-Acetyloleanolic acid exhibits anti-angiogenic effects and induces apoptosis in human umbilical vein endothelial cells

3-O-Acetyloleanolic acid, a pentacyclic triterpenoid isolated from cowpea seeds, inhibited proliferation, migration and tube formation of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. HUVECs. The induced apoptosis was characterized by detection of cell surface annexin V and sub-G1 populations. The number of cells immunostained with annexin V-fluorescein isothiocyanate increased after treatment with 3-O-acetyloleanolic acid. The sub-G1 cell populations were also increased in treated HUVECs. 3-O-Acetyloleanolic acid induced activation of caspase 3, a critical mediator of apoptosis signaling. It also significantly inhibited angiogenesis in an in vivo Matrigel plug assay. 3-O-Acetyloleanolic acid thus exhibits anti-angiogenic effects and induces apoptosis in HUVECs and the results suggest that it has a potential use for suppression of the tumor growth stimulated by angiogenesis.     

Rac1蛋白活化加速缺氧诱导的人血管内皮细胞衰老

为阐明Rac1蛋白在人脐静脉内皮细胞（human umbilical vein endothelial cells,HUVECs）衰老中的作用及分子机制,我们采用持续缺氧的方法诱导内皮细胞衰老,检测缺氧前后内皮细胞衰老标志基因SA-β-Gal和PAI-1的表达、细胞周期分布和细胞增殖情况,同时分析缺氧前后细胞内Rac1蛋白的表达.结果显示,持续缺氧96 h后,HUVECs体积变大,细胞浆内颗粒和空泡增多,SA-β-Gal活性明显增加,PAI-1基因表达升高,细胞发生G1期阻滞,细胞增殖受抑,活化型Rac1蛋白表达上调,提示持续缺氧诱导的内皮细胞衰老可能与Rac1蛋白的活化有关.为进一步明确内皮细胞衰老与Rac1蛋白的关系,应用逆转录病毒将持续活化型Rac1（V12Rac1）和主导抑制型Rac1（N17Rac1）基因分别瞬时感染HUVECs,比较三种HUVECs（HUVECs,V12Rac1-HUVECs,N17Rac1-HUVECs）缺氧后的衰老变化,并分析其下游调控分子--血清反应因子（serum response factor,SRF）的表达和定位变化.研究发现,缺氧培养V12Rac1-HUVECs 48 h即可引起细胞衰老,表现为SA-β-Gal活性明显增加,PAI-1基因表达升高,细胞出现明显的G1期阻滞并且细胞增殖受抑,其改变与缺氧96 h的HUVECs相似;而N17Rac1明显抑制缺氧引起的内皮细胞衰老发生.上述结果说明,Rac1蛋白活化可以加速缺氧诱导的内皮细胞衰老,而抑制Rac1蛋白的活性则可抑制缺氧诱导的内皮细胞衰老.为进一步研究Rac1蛋白引起内皮细胞衰老的机制,通过免疫荧光染色及Western blot分析检测三种细胞缺氧处理后SRF的表达,发现：与HUVECs细胞比较,V12Rac1引起缺氧48 h HUVECs核蛋白中SRF的表达明显下降,SRF入核转位受到明显抑制;而N17Rac1感染后,缺氧HUVECs细胞核蛋白中SRF表达明显增多.上述结果提示：缺氧状态下Rac1蛋白活化能够明显加速HUVECs衰老,而抑制Rac1蛋白活性则明显抑制缺氧诱导的HUVECs衰老,SRF蛋白的核转位活化参与了Rac1蛋白调控HUVECs衰老的发生.