The Effect And Mechanism Of Activated Hepatic Stellate Cells Promoting Angiogenesis Of Hepatocellular Carcinoma

IntroductionPrimary hepatocellular carcinoma (HCC) is a common malignant tumor and asignificant public health concern worldwide. Despite the availability of numeroustreatment options for patients with HCC, a high rate of recurrence and metastasisresults in a low five-year survival rate for this fatal disease. Thus, there is an urgentneed to advance our understanding of the mechanism for HCC recurrence andmetastasis and to improve the efficacy of the current treatment strategies.HCC, likemany tumors, has a rich blood supply. HCC relies on the formation of blood vesselsfor growth and metastasis. The formation of new vessels in the tumor is controlledsimultaneously by pro-and anti-angiogenic factors. The most important of severalpro-angiogenic factors is vascular endothelial growth factor (VEGF). Theup-regulation of VEGF expression in tumor tissue has been reported to be associatedwith poor prognosis in several cancers, including HCC.Recently, many studies have provided strong evidence that the interaction between tumor cells and their microenvironment, consisting of stromal cells and theextracellular matrix (ECM), contributes to cancer progression. In the majority oftumors, an abnormal network of stromal cells, growth factors, cytokines andchemokines is critical for the induction of angiogenesis. In HCC, VEGF has beenreported to be produced by both HCC cells and stromal cells.The majority of HCC patients have a history of chronic liver disease, and thepresence of liver cirrhosis is closely associated with the development of HCC. Hepaticstellate cells activation is a fundamental step in the development of liver fibrosis andultimately cirrhosis. In addition to these actions, several recent studies have indicatedthat activated hepatic stellate cells (aHSC) infiltrate the liver tumor stroma andbecome one of the most important stromal cell types in the liver tumor environment.Clinical evidence has revealed that the presence of peritumoral aHSC correlates withthe recurrence of HCC. Although some data have suggested an important contributionof aHSC to HCC proliferation and metastasis, the exact molecular interactions thatoccur between these two cell types are unknown. It has been reported that aHSC caninteract with HCC cells in a paracrine manner. In the process of liver fibrosis andportal hypertension, aHSC have been reported to synthesize pro-angiogenic factors,such as VEGF, angiopoietin-1and several metalloproteinases. In experimentallyinduced liver metastasis, aHSC is pro-angiogenic during the progression of livercancer. These studies suggest that aHSC may contribute to the angiogenicrequirement of HCC proliferation and metastasis. However, the mechanisms remainunclear. Platelet-derived growth factor (PDGF) is a potent mitogen for mesenchymal cellsthat is synthesized, stored and released by many cell types, including tumor cells.PDGF is a dimeric glycoprotein that is composed of two A chains (PDGF-AA), two Bchains (PDGF-BB) or a combination of the two (PDGF-AB). In colorectal livermetastasis, tumor cell-derived PDGF-BB promotes tumor growth via agrowth-promoting effect on aHSC. PDGF-BB also affects the angiogenic propertiesof aHSC during liver fibrosis. However, in liver cancer, the function of PDGF-BB inaHSC has not been well characterized.From these observations, we speculate that HCC-derived factors may influenceaHSC to regulate angiogenesis. Our study aimed to explore this mechanism. Weanalyzed the correlation between aHSC and angiogenesis in HCC tissue, and in vitroparacrine effects of secreted, HCC-derived PDGF-BB on the pro-angiogenic geneexpression of aHSC. Our results revealed a pro-angiogenic role for aHSC on theproliferation and metastasis of HCC cells.Materials and methodsCell CultureThe liver cancer cell line HepG2was purchased from the animal centerlaboratory at Sun Yat-sen University. The activated hepatic stellate cell line LX-2wasobtained from the American Type Culture Collection (ATCC, Manassas, US). Allcells were routinely cultured in high-glucose Dulbecco’s modified eagle medium(DMEM)(Gibco BRL, Grand Island, NY,US) supplemented with10%fetal bovine serum (FBS)(Hyclone, Australia) at37°C with5%CO2and95%air in a humidifiedincubator. An indirect co-culture of LX-2and HepG2cells was assembled usingTranswell membranes (24mm diameter,0.4μm pore size, Corning Costar, Acton,MA). Approximately1×104LX-2cells were placed in the lower chamber, and1×103HepG2cells were placed on the membrane insert. The co-cultures were maintainedfor72h.Collection of Conditioned Medium (CM)To prepare the conditioned medium, the cells were washed twice with serum-freeDMEM one day after being seeded into T75flasks (1×106cells). The cells wereincubated with serum-free DMEM for24h. Simultaneously, serum-free DMEM wasplaced in cell-free culture flasks under the same conditions to serve as a control. Forinhibition experiments, CM from HepG2cells was preincubated with a PDGF-BBneutralizing antibody at a concentration of10μg/ml as previously described.Expression Constructs and TransfectionThe expression construct for full-length human PDGF-BB was generated bycloning a PCR-amplified PDGF-BB cDNA fragment into the lentiviral vectorpCDH-CMV-MCS-EF1-copGFP, which allowed for stable transfection. Viruspackaging was performed in293T cells after cotransfection of the recombinantlentiviral expression plasmids and packaging plasmids (pGag/Pol, pRev and pVSV-G)using Lipofectamine2000(Invitrogen, Carlsbad, CA, US). Target cells were infected with the harvested virus for12h, and the original medium was replaced with freshmedium. The lentiviral transduction efficiency was monitored by Western blot.MTT AssayThe proliferation rate of LX-2cells was measured using an MTT assay. Briefly,LX-2cells were seeded in96-well plates at a density of2×104cells/well. After24h,conditioned medium was added to the wells, and the plates were incubated at37°C foran additional24h. MTT (50μl of5mg/ml) was added to the culture, which wasincubated for4h at37°C. The optical density was measured at490nm on a platereader. The absorbance values directly correlated with the number of proliferatingcells in the culture.Migration AssayTo determine whether the HepG2cells attracted the LX-2cells, a migrationassay was performed using Transwell chambers containing polycarbonate filters with8μm pores (Corning Costar, Acton, MA, US). The lower compartment containedconditioned medium. The LX-2cells were harvested, resuspended in DMEM withoutFBS at a density of5×104cells/ml and placed in the upper chamber. After incubationat37°C for24h, the filters were collected, and the cells that adhered to the lowersurface were fixed, stained and counted. The experiments were performed in triplicateand repeated three times with consistent results. Western Blot AnalysisCells were collected in phosphate-buffered saline (PBS) and lysed on ice for30min in RIPA lysis buffer. The protein content in the cell lysates was quantitated usingthe Bradford method. Equal amounts of protein lysate (30μg) were resolved on10%SDS polyacrylamide (SDS-PAGE) gels and electrophoretically transferred ontopolyvinylidine fluoride membranes. The membranes were blocked in5%nonfat driedmilk for1h. The blots were probed with the indicated primary antibodies (PDGF-BB,PDGFR-β or VEGF-A) overnight at4°C and then with the appropriate horseradishperoxidase-conjugated secondary antibody (1:5000) for1h at room temperature. Theblots were developed using enhanced chemiluminescence (ECL)(Santa Cruz, CA, US)and exposed to X-ray film. GAPDH was included as a loading control.Enzyme-Linked Immunosorbent Assay (ELISA)PDGF-BB is a secreted protein; therefore, the concentration of PDGF-BB in theculture medium was measured to estimate the expression level of PDGF-BB inHepG2cells. The amount of PDGF-BB in the HepG2supernatant was analyzed usinga commercial ELISA kit (eBioscience, San Diego, CA, US) according to themanufacturer’s instructions.Statistical AnalysisAll results are expressed as the mean±SE of at least three independentexperiments. Statistical analyses were performed using the SPSS statistical software for Microsoft Windows, version13.0(Professional Statistic, Chicago, IL). Atwo-tailed paired Student’s t-test and ANOVA was used to determine significancebetween the test and the control conditions. The criterion for significance was P<0.05for all comparisons.Results1. aHSC was found existed in the HCC stroma,the proportion of which was higherwhen HCC cells were poorly differentiated. The aHSC density was positivelycorrelated with HCC microvessel density.2. In vitro experiments showed that HCC cells increased the proliferation andmigration of aHSC.Paracrine effect of HCC cells was thought to play an importantrole.3. PDGF-BB is expressed in HCC cells and is secreted to act in a paracrine manner.HCC cells stimulate the PDGFR-β and VEGF-A expression of aHSC in a paracrinemanner. PDGF-BB secreted by HCC cells stimulates the pro-angiogenic properties ofHSCs.ConclusionThe present data indicate that aHSC exsites in the HCC stroma. The aHSCdensity was positively correlated with HCC microvessel density. These findingsindicate aHSC accumulates in the microenvironment of HCC and associated withtumor angiogenesis. We further showed that conditioned medium from HCC cellssignificantly increased the proliferation and migration of aHSC in vitro. This result indicates that HCC cells act in a paracrine manner to attract aHSC and encourage theiraccumulation within the HCC stroma.We also found that PDGF-BB was expressed inthe HepG2cells and PDGF-BB derived from HCC cells drives the VEGF-Aexpression of aHSC. The induced up-regulation of VEGF-A expression in aHSC byHCC cell-derived PDGF-BB may create a pro-angiogenic microenvironment thatencourages HCC angiogenesis. In conclusion, the results of this study provide severalnew insights into a new therapeutic approach of simultaneously targeting tumor cellsand aHSC to effectively prevent the development of HCC.