Cooperative Roles of Hepatocyte Growth Factor and Plasminogen Activator in Tubular Morphogenesis by Human Microvascular Endothelial Cells

Epidermal growth factor (EGF) or transforming growth factor-α (TGF-α) stimulated cell migration, chemotaxis, and the expression of tissue-type plasminogen activator (t-PA) in human omental microvascular endothelial (HOME) cells. Hepatocyte growth factor (HGF) stimulated cell proliferation, but had a negligible stimulatory effect on cell migration, the expression of t-PA and tube-like formation into collagen gel in HOME cells. Basic fibroblast growth factor stimulated cell proliferation, cell migration, tubulogenesis and the expression of urokinase-type plasminogen activator (u-PA) in bovine aortic endothelial (BAE) cells. HOME and BAE cells had both high- and low-affinity receptors for HGF. In BAE cells, u-PA activity and tube-like structures in collagen gel were induced in the presence of HGF alone. In contrast, in HOME cells, t-PA activity and tube-like structures were induced in the presence of TGF-a alone, but not in the presence of HGF alone. However, we observed a marked induction of tube formation by HOME cells when both t-PA and HGF were added simultaneously. In the model system for tumor angiogenesis, when HOME cells were co-cultured with a renal cancer cell line, KPK13, tube-like structures were induced in the presence of HGF: KPK13 cells expressed large amounts of t-PA mRNA. Our present study suggested that HGF in concert with active t-PA could be angiogenic in HOME cells.     

Study of puupehenone and related compounds as inhibitors of angiogenesis

Puupehenone, a sesquiterpene produced by certain sponges, was selected in the course of a blind screening for new potential inhibitors of angiogenesis. In our study, we compare the potential anti-angiogenic activities of puupehenone and another 11 related compounds that were either natural products from marine origin or their synthetic derivatives. The effects of these compounds were determined with cell growth and differentiation assays on bovine aorta endothelial cells. Our results show that these compounds are weak inhibitors to cell growth and are not selective for endothelial cells. However, contrary to cell growth, the differentiation of endothelial cells into tubular structures was completely inhibited by 7 of these compounds at concentrations equal or lower than 3 microM. Three of these compounds, isozonarol, 8-epipuupehedione and 8 epi-9,11-dihydropuupehedione, completely inhibited the in vivo angiogenesis in the CAM assay at doses equal or lower than 30 nmol/egg. Further characterisation showed that these 3 terpenes also inhibited endothelial cell production of urokinase and invasion. One compound (8-epipuupehedione) inhibited endothelial cell migration in a dose-dependent manner. The anti-angiogenic properties of the selected compounds, the simplicity of their structures and the feasibility of their synthesis make them attractive drugs for further evaluation in the treatment of angiogenesis-related pathologies.     

1,4-Naphthoquinone is a potent inhibitor of human cancer cell growth and angiogenesis

Angiogenesis inhibitors are beneficial for the prevention and treatment of angiogenesis-dependent diseases including cancer. Vitamin K2 and K3, which are naphthoquinone derivatives, inhibit angiogenesis. We examined the anti-cancer and anti-angiogenic effects of naphthoquinones and its structurally related compounds. Among these 13 compounds, 1,4-naphthoquinone strongly inhibited both human colon cancer cell (HCT116) growth and angiogenesis. To clarify the anti-angiogenic mechanism, the effects of 1,4-naphthoquinone on human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and chemotaxis were examined. Consequently, 1,4-naphthoquinone inhibited HUVEC functions. These results suggest that 1,4-naphthoquinone may be useful to cancer therapy.     

In vivo and in vitro evaluation of erianin, a novel anti-angiogenic agent

This study evaluated the anti-angiogenic activities of erianin in vivo and in vitro. Erianin, a natural product from Dendrobium chrysotoxum, caused moderate growth delay in xenografted human hepatoma Bel7402 and melanoma A375 and induced significant vascular shutdown within 4 h of administering 100 mg/kg of the drug. Erianin also displayed potent anti-angiogenic activities in vitro: it abrogated spontaneous or basic fibroblast growth factor-induced neovascularisation in chick embryo; it inhibited proliferation of human umbilical vein endothelial cells (EC(50) 34.1+/-12.7 nM), disrupted endothelial tube formation, and abolished migration across collagen and adhesion to fibronectin. Erianin also exerted selective inhibition toward endothelial cells, and quiescent endothelium showed more resistance than in proliferative and tumour conditions. In a cytoskeletal study, erianin depolymerised both F-actin and beta-tubulin, more significantly in proliferating endothelial cells than in confluent cells. In conclusion, erianin caused extensive tumour necrosis, growth delay and rapid vascular shutdown in hepatoma and melanoma models; it inhibited angiogenesis in vivo and in vitro and induced endothelial cytoskeletal disorganisation. These findings suggest that erianin has the therapeutic potential to inhibit angiogenesis in vivo and in vitro.     

A truncated form of CD9-partner 1 (CD9P-1), GS-168AT2, potently inhibits in vivo tumour-induced angiogenesis and tumour growth

Background:

Tetraspanins are transmembrane proteins known to contribute to angiogenesis. CD9 partner-1 (CD9P-1/EWI-F), a glycosylated type 1 transmembrane immunoglobulin, is a member of the tetraspanin web, but its role in angiogenesis remains to be elucidated.

Methods:

We measured the expression of CD9P-1 under angiogenic and angiostatic conditions, and the influence of its knockdown onto capillary structures formation by human endothelial cells (hECs). A truncated form of CDP-1, GS-168AT2, was produced and challenged vs hEC proliferation, migration and capillaries'' formation. Its association with CD9P-1, CD9, CD81 and CD151 and the expressions of these later at hEC surface were analysed. Finally, its effects onto in vivo tumour-induced angiogenesis and tumour growth were investigated.

Results:

Vascular endothelial growth factor (VEGF)-induced capillary tube-like formation was inhibited by tumour necrosis factor α and was associated with a rise in CD9P-1 mRNA expression (P<0.05); accordingly, knockdown of CD9P-1 inhibited VEGF-dependent in vitro angiogenesis. GS-168AT2 dose-dependently inhibited in vitro angiogenesis, hEC migration and proliferation (P<0.05). Co-precipitation experiments suggest that GS-168AT2 corresponds to the sequence by which CD9P-1 physiologically associates with CD81. GS-168AT2 induced the depletion of CD151, CD9 and CD9P-1 from hEC surface, correlating with GS-168AT2 degradation. Finally, in vivo injections of GS-168AT2 inhibited tumour-associated angiogenesis by 53.4±9.5% (P=0.03), and reduced tumour growth of Calu 6 tumour xenografts by 73.9±16.4% (P=0.007) without bodyweight loss.

Conclusion:

The truncated form of CD9P-1, GS-168AT2, potently inhibits angiogenesis and cell migration by at least the downregulation of CD151 and CD9, which provides the first evidences for the central role of CD9P-1 in tumour-associated angiogenesis and tumour growth.     

Rosmarinic acid inhibits angiogenesis and its mechanism of action in vitro

Rosmarinic acid (RA), a water-soluble polyphenolic compound with anti-oxidative and anti-inflammatory activities, inhibited several important steps of angiogenesis including proliferation, migration, adhesion and tube formation of human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. RA also reduced intracellular reactive oxygen species (ROS) level, H2O2-dependent VEGF expression and IL-8 release of endothelial cells. These findings suggested that the anti-angiogenic potential of RA might be related to its anti-oxidative activity, which further resulted in the inhibition of ROS-associated VEGF expression and IL-8 release.     

EGCG对肿瘤生物抑制机制影响的探讨

目的:探讨表没食子儿茶素没食子酸酯(EGCG)通过抑制血管内皮生长因子(VEGF)的血管生成效应减少肿瘤生长和血管生成。方法:MTT法检测内皮细胞生长、Transwell检测内皮细胞迁移,同时检测内皮细胞体外小管形成情况及Matrigel胶塞体内实验检测体内血管生成情况;建立异位胃癌裸鼠模型,检测肿瘤生长及肿瘤组织微血管密度,明确EGCG对肿瘤生长和血管生成的抑制作用及其机制。结果:体外实验显示,随着EGCG处理时间和剂量的增加,VEGF诱导生长的内皮细胞数呈时间和剂量依赖性地减少;随着EGCG剂量的增加,VEGF诱导迁移的内皮细胞数和形成的小管样结构也剂量依赖性地减少,差异有统计学意义,P<0.05;Matrigel胶塞体内实验也显示EGCG抑制VEGF诱导的胶塞血管化;动物实验显示治疗组肿瘤生长缓慢,生长曲线明显低于对照组,平均肿瘤抑制率为60.4%,P<0.01;治疗组肿瘤组织微血管密度显著降低,P<0.01。结论:EGCG可以抑制VEGF诱导的血管生成,从而抑制肿瘤生长和血管生成。     

Resveratrol and quercetin inhibit angiogenesis in vitro

Resveratrol and quercetin are polyphenolic compounds found in grapes, red wine and other food products. In this study, we examined the effect of resveratrol and quercetin on the inhibition of angiogenesis in vitro. Resveratrol and quercetin inhibited the growth of bovine aorta endothelial (BAE) cells in a concentration-dependent manner (6-100 microM).The migration of BAE was obviously inhibited by resveratrol and weakly inhibited by quercetin. When the lengths of all tubes constructed in the 3-D culture system with or without resveratrol or quercetin were measured, resveratrol was found to inhibit the tube formation of BAE cells in a concentration-dependent manner (6-100 microM). On the other hand, quercetin at concentrations above 100 microM significantly inhibited the tube formation of vascular endothelial cells. From these results, we suggest that resveratrol and quercetin may prove useful in the development of useful therapeutic agents or preventive food factors for tumor angiogenesis.     

Angiostatic activities of medroxyprogesterone acetate and its analogues

The effects of medroxyprogesterone acetate (MPA) (1) and related compounds (II-VI) upon angiogenesis induced by basic fibroblast growth factor (bFGF) or transforming growth factor-α (TGF-α) were investigated using a rabbit cornea system for assay of angiogenesis. Dexamethasone (Dex) was used as a positive control. The MPA analogues tested were 6,6′-dehydro MPA (II), megestrol acetate (III), l-dehydromegestrol acetate (IV), melengestrol acetate (V), and l-dehydromelengestrol acetate (VI). The inhibitory activities of these steroids using bFGF were in the order: Dex = MPA = (VI) = (V) > (IV) > (III). Steroid (II) was inactive. 5α-dihydrotestosterone was weakly active, while estradiol-17β and progesterone were inactive. The angiostatic activity of MPA was completely abolished by mefipristone (RU 486) which showed no anti-angiogenic activity in this assay. With TGF-α, the order of angiostatic activities was Dex = (VI) > (IV) > (III) > (V). Steroid (II) was again inactive. Dex, MPA, and all the MPA analogues except steroid (II) markedly inhibited the activity of plasminogen activator secreted by cultured calf pulmonary artery endothelial cells, but did not inhibit growth of these cells. The binding affinities of MPA and its analogues to glucocorticoid, progesterone and androgen receptors were determined, but were found not to be correlated with their angiostatic activities.     

15(S)-hydroxyeicosatetraenoic acid induces angiogenesis via activation of PI3K-Akt-mTOR-S6K1 signaling

To determine whether the lipoxygenase metabolites of arachidonic acid, 5(S)-, 12(S)-, and 15(S)-hydroxyeicosatetraenoic acids [5(S)-HETE, 12(S)-HETE, and 15(S)-HETE, respectively] are angiogenic, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All three HETEs stimulated HDMVEC tube formation and migration. Because 15(S)-HETE was found to be more potent than 5(S)-HETE and 12(S)-HETE in HDMVEC tube formation, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 15(S)-HETE stimulated Akt and S6K1 phosphorylation in HDMVEC in a time-dependent manner. Wortmannin and LY294002, two specific inhibitors of phosphatidylinositol 3-kinase (PI3K), blocked both Akt and S6K1 phosphorylation, whereas rapamycin, a specific inhibitor of Akt downstream effector, mammalian target of rapamycin (mTOR), suppressed only S6K1 phosphorylation induced by 15(S)-HETE suggesting that this eicosanoid activates the PI3K-Akt-mTOR-S6K1 signaling in HDMVEC. Wortmannin, LY294002, and rapamycin also inhibited 15(S)-HETE-induced HDMVEC tube formation and migration. In addition, all three HETEs stimulated angiogenesis as measured by in vivo Matrigel plug assay with 15(S)-HETE being more potent. Pharmacologic inhibition of PI3K-Akt-mTOR-S6K1 signaling completely suppressed 15(S)-HETE-induced in vivo angiogenesis. Consistent with these observations, adenoviral-mediated expression of dominant-negative Akt also blocked 15(S)-HETE-induced HDMVEC tube formation and migration and in vivo angiogenesis. Together, these results show for the first time that 15(S)-HETE stimulates angiogenesis via activation of PI3K-Akt-mTOR-S6K1 signaling.     

The roles of chemokine CXCL12 in embryonic and brain tumor angiogenesis

The formation of blood vessels in embryos and tumors are different processes but under the control of common molecular mechanisms. Chemokine CXCL12 involved in both embryonic and tumor angiogenesis. In this review, we summarize recent advances in understanding the roles of CXCL12 in brain tumor angiogenesis/vasculogenesis. CXCL12 and its cognate receptors are abnormally induced in brain tumors, in particular in tumor cells and endothelium. Pathologically enhanced CXCL12 signaling may promote the formation of new vessels through recruiting circulating endothelial progenitor cells or directly enhancing the migration/growth of endothelial cells. Therefore, CXCL12 signaling represents an important mechanism that regulates brain tumor angiogenesis/vasculogenesis and may provide potential targets for anti-angiogenic therapy in malignant gliomas.     

二氢杨梅素通过ERK/VEGFA/VEGFR2通路对体内外胃癌血管生成的影响

背景与目的：二氢杨梅素（dihydromyricetin,DHM）通过抑制细胞周期、促进细胞凋亡和抑制新生血管生成等机制发挥抗肿瘤作用。探讨DHM对胃癌的抗癌作用,并研究其可能作用机制。方法：不同剂量DHM作用体外人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）和胃癌细胞系MKN28后,采用细胞计数试剂盒（cell counting kit-8,CCK-8）法筛选无毒剂量的DHM,Transwell小室实验测定细胞侵袭;小管形成实验测定HUVEC体外血管生成能力;采用蛋白质印迹法（Western blot）检测血管内皮生长因子A（vascular endothelial growth factor A, VEGFA）、phospho-血管内皮生长因子受体（vascular endothelial growth factor receptor, VEGFR）2、细胞外信号调节激酶（extracellular signal-regulated kinase,ERK）、phospho-c-Jun氨基端激酶（c-Jun NH2-terminal kinase,JNK）和phospho-p38表达水平;基质胶塞实验测定DHM对体内血管形成的影响;通过皮下肿瘤细胞注射法建立MKN28裸鼠异种移植瘤模型,观察DHM给药对体内胃癌生长的影响,Ki-67增殖指数及CD34、VEGFA在移植瘤组织中的表达通过免疫组织化学法测定。结果：（0.5~2.5 μmol/L）的DHM对HUVEC和MKN28细胞生长无明显抑制作用,该浓度范围的DHM呈浓度依赖性地抑制HUVEC侵袭和小管形成,VEGFA（20 μg/L）可明显逆转DHM对HUVEC侵袭和小管形成的抑制效果;DHM处理可导致MKN28细胞中VEGFA和phospho-ERK表达呈剂量依赖性下降,而对p-p38和p-JNK的表达无明显影响;DHM处理可导致HUVEC中phospho-VEGFR2表达呈剂量依赖性下降;低剂量（30 mg/kg）DHM对裸鼠无明显的不良反应,但可抑制体内血管形成,同时有效延缓体内胃癌生长,体内肿瘤组织中的CD34和VEGFA的表达受到DHM的显著下调作用,但低剂量DHM对Ki-67无明显作用。结论：低剂量的DHM可有效抑制体内外血管生成和体内胃癌生长,该作用至少部分是通过ERK/VEGFA/VEGFR2信号通路来实现的。     

A quantitative assay using basement membrane extracts to study tumor angiogenesis in vivo

We describe a quantitative assay for assessing tumor angiogenesis in vivo using basement membrane extracts (Matrigel). Nude mice were injected s.c. with liquid Matrigel mixed with HT1080 human fibrosarcoma cells. Since Matrigel rapidly forms a solid gel at body temperature, the gel containing tumor cells can be removed immediately and then processed for histological studies. Tumor angiogenesis was monitored quantitatively by measuring both the number and the total area of neovessels present in the gels using an image analyzer, which could be achieved approximately 72 hr later. Furthermore, HT1080 cell-conditioned medium, which may contain various tumor-derived factors, promoted the basement membrane degradation, migration, proliferation and tube formation of endothelial cells in vitro, as did Matrigel, although to a lesser extent. In addition, Northern blot analysis demonstrated that the amount of vascular endothelial growth factor (VEGF) mRNA in HT1080 cells was much higher than that in human fibroblasts or NIH3T3 cells. Our results suggest that angiogenesis observed in our assay may be due to the synergic effects of tumor angiogenic factors such as VEGF, and Matrigel. The advantages of our assay are: 1) it is possible to assess early angiogenesis quantitatively; and 2) this assay may be applicable for screening anti-angiogenic therapeutic agents to be used against human neoplasms. © 1996 Wiley-Liss, Inc.     

Anti-metastatic and anti-angiogenic activities of a new matrix metalloproteinase inhibitor,TN-6b

We investigated the anti-metastatic and anti-angiogenic effects of TN-6b, a new broad-spectrum inhibitor of matrix metalloproteinases (MMPs), against Lewis lung carcinoma (LLC) and hepatic sinusoidal endothelial (HSE) cells. TN-6b potently inhibited the activities of MMP-2 and -9 secreted by LLC and HSE cells in a zymogram assay. TN-6b, at non-cytotoxic concentrations, caused a marked inhibition of invasion and migration of LLC, and tube-like formation of HSE cells. In contrast, TN-6d, an inactive enantiomer of TN-6b, did not inhibit the invasion and tube-like formation. Daily subcutaneous (s.c.) administration of TN-6b at doses of 30 and 60 mg/kg in mice resulted in a potent inhibition of tumour-induced angiogenesis of B16 melanomas and lymph node metastasis of LLC cells. In conclusion, TN-6b effectively inhibited lymph node metastasis of LLC cells through its anti-invasive and anti-angiogenic properties. These findings suggest that the MMP inhibition correlates well with its anti-angiogenic and anti-metastatic efficacy and TN-6b has the therapeutic potential to inhibit angiogenesis and metastasis in vivo and in vitro.     

Anti-angiogenic efficacy of grape seed extract in endothelial cells

The present study is focused on the investigation of in vitro angiogenic potential of grape seed extract (GSE). Human umbilical vein endothelial cells (HUVEC) in culture were used to assess the effect of GSE on proliferation, survival, matrix metalloproteinases (MMPs) secretion and capillary tube formation. Our data show that GSE significantly inhibited cell growth (< or =91%, P<0.001) and cell viability (< or =64%, P<0.005) of HUVEC. Further studies by BrdU incorporation and annexin V staining showed that GSE strongly inhibits DNA synthesis (< or =76%, P<0.001) and induces apoptotic cell death (< or =42.8% versus control 2.6%, P<0.05) in HUVEC, respectively. Similar GSE treatment decreased secreted levels of MMP-2 from HUVEC. GSE also inhibited capillary tube formation on Matrigel by endothelial cells in a dose-dependent manner. These findings suggest that GSE possesses an anti-angiogenic potential, which is associated with its antiproliferative, proapoptotic and inhibition of MMP-2 secretion in endothelial cells. Further studies are warranted to evaluate the in vivo anti-angiogenic efficacy of GSE for its possible usefulness in the inhibition of tumor angiogenesis.     

Anti-angiogenic effects of a nutrient mixture on human umbilical vein endothelial cells

Matrix metalloproteinases (MMPs) have been recognized as key players in the degradation of the extracellular matrix (ECM) by migration and proliferation of endothelial cells and their subsequent invasion of the underlying stroma. The prevention of ECM degradation through the inhibition of MMP activity has been shown to be a promising therapeutic approach to block the invasion that occurs during angiogenesis. In previous studies, we demonstrated the anti-tumor effect of a nutrient mixture (NM) containing ascorbic acid, lysine, proline, green tea extract, arginine, N-acetyl cysteine, selenium, copper and manganese on various tumor cell lines in vivo and in vitro. The aim of the present study was to determine whether this mixture has anti-angiogenic effects on human umbilical vein endothelial cells (HUVECs). At near confluence, the HUVEC cell cultures were tested with NM at 0, 10, 50, 100, 500, and 1000 microg/ml in triplicate at each dose for proliferation, migration, MMP expression, and invasion. Cell proliferation was evaluated by MTT assay, invasion potential by Matrigel invasion, MMP expression by gelatinase zymography, and cell migration by a 2 mm-wide scratch in plates. For tube formation, HUVECs were cultured in previously polymerized Matrigel. NM inhibited HUVEC migration, MMP expression and invasion through Matrigel in a dose-dependent manner. Zymography showed a dose-dependent inhibition of MMP-2 expression with virtual total inhibition at a 500 microg/ml concentration. Invasion through Matrigel was totally inhibited at 500 microg/ml NM. NM reduced cell migration by scratch test in a dose-dependent fashion with total inhibition at a 500 microg/ml concentration. NM also inhibited the tube formation of HUVECs, but did not significantly inhibit cell proliferation. These results together with our earlier findings suggest that NM is a relatively non-toxic formulation with anti-angiogenic effects, such as inhibiting vascular tube formation and endothelial cell invasion and migration.     

Atorvastatin shows antitumor effect in hepatocellular carcinoma development by inhibiting angiogenesis via TGF-β1/pERK signaling pathway

Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins may reduce HCC incidence. Its antitumor activities may be mediated by disrupting several hepatocarcinogenic pathways. To evaluate in vivo and in vitro the antiproliferative and antiangiogenic action of atorvastatin (AT) in the development of HCC as well as its mechanisms of action. In vivo model: hexachlorobenzene (HCB) was used to promote the development of HCC in Balb/C nude mice. Number of hepatic tumor, liver cell proliferation parameters (proliferating cell nuclear antigen, PCNA), angiogenesis, and VEGF levels were analyzed. In vitro model: Hep-G2 and Ea-hy926 cells were used to evaluate the effect of different doses of AT on HCB induced cell proliferation, migration, and vasculogenesis and to analyze proliferative parameters. In vivo: AT prevented liver growth and tumor development and inhibited PCNA, TGF-β1, and pERK levels increase. AT prevented skin blood vessel formation. In vitro, AT prevented cell proliferation and migration as well as tubular formation in the endothelial cell line by inhibiting the MAPK ERK pathway. We were able to demonstrate the potential AT antiproliferative and antiangiogenic effects in an HCC model and the involvement of TGF-β1 and pERK pathways.     

Morphine promotes angiogenesis by activating PI3K/Akt/HIF-1α pathway and upregulating VEGF in hepatocellular carcinoma

BackgroundHepatocellular carcinoma (HCC) is characterized by the neo-angiogenesis induced by tumor and adjacent cells. It is a leading cancer-related cause of death. Morphine has effects on angiogenesis with pro-angiogenic or anti-angiogenic phonotypes. This study explores the function of morphine on cancer cell growth, angiogenesis and the underlying mechanism in HCC.MethodsMorphine was used to treat BEL-7402 or HCC-LM3 cells and human umbilical vein endothelial cells (HUVECs) were subsequently incubated in the conditioned media (CM) of HCC cells. The potential effects of cell proliferation, migration and tube formation of CM-treated HUVECs were investigated. Furthermore, the angiogenesis regulated factors of VEGFA, PIGF, ANG-1, ANG-2, FGF-1 and FGF-2 were assessed. siRNA and LY294002 were further used to explore the mechanism mediating the effects of morphine on the angiogenesis pathway. The neovascularization effect by morphine was confirmed through the use of human HCC cancer heterotopic mouse model in vivo.ResultsA significantly increased cell proliferation, migration, and tube formation effect of HUVECs induced by the CM from HCC cell lines treated with morphine was observed. More VEGFA secretion in CM from LM3 or BEL-7402 cell lines was found than the controls (P=0.03 and P=0.027, respectively). VEGFA knock-down could significantly reverse cell proliferation, migration and tube formation induced by the CM from HCC cell lines with morphine treatment. Further molecular experiments indicated that VEGFA secretion was activated by morphine potentially through the PI3K/Akt/HIF-1α pathway. Morphine-induced neovascularization was also observed by the IHC of CD31 and VEGFA.ConclusionsMorphine promotes angiogenesis in hepatocellular carcinoma possibly through the activation of the PI3K/Akt/HIF-1α pathway and VEGFA stimulation.