Suppression of peritoneal tumorigenesis by placenta-derived mesenchymal stem cells expressing endostatin on colorectal cancer.

MSCs-based therapy for cancer is a relatively new but rapidly growing area of research. Human term placenta, an attractive source of MSCs (PMSCs), appears to have great advantage due to its easy access without invasive procedures, its lack of ethical issues and its high-throughput and young age. In the present study, we isolated MSCs from placenta and characterized their morphology and differentiation capacities. We next investigated the underlying antitumor effects and the potential mechanism of PMSCs to express endostatin using adenoviral transduction (Ad-Endo) in a colorectal peritoneal carcinomatosis (CRPC) mouse model. For in vitro experiments, the migratory potential of Ad-Endo-PMSCs towards tumor cells was demonstrated using a double-chamber assay, and the anti-angiogenesis ability of endostatin from engineered PMSCs was evaluated using the tube formation assay. For the in vivo study, mice harboring CT26 colorectal cancer indicated a significant reduction in tumor nodules and a prolongation of survival following Ad-Endo-PMSCs therapy. These observations were associated with significantly decreased tumor cell proliferation and blood vessel counts as well as increased tumor cell apoptosis. These data suggested the potential of PMSCs-based gene therapy for the targeted delivery of therapeutic proteins in cancer.

Experimental studies on treatment of pancreatic cancer with double-regulated duplicative adenovirus AdTPHre-hEndo carrying human endostatin gene.

BACKGROUND: Gene-virus targeted therapy is a promising new method of treating pancreatic cancer. To increase the efficacy and decrease the side-effect, we constructed a conditionally replicative adenovirus (CRAd) expressing human endostatin, with a human Telomoerase Reverse Transcriptase (hTERT) promoter for the regulation of the early stage of adenovirus expression of gene E1a and a Hypoxia Response Element (HRE) promoter to regulate the gene E1b. METHODS: A gene recombination technique was adopted to construct and generate the adenovirus AdTPHre-hEndo. Pancreatic cancer cells were studied both in vitro and in vivo. Western blotting was adopted to observe the expressions of protein E1A and E1B; duplication assay was applied to observe the selective duplication capability of recombinant cells. MTT assay was applied to measure the lethal effects of virus on pancreatic cancer cells, and ELISA was adopted to detect the human endostatin gene expression. A pancreatic cancer transplantation tumor model of nude mice was constructed to observe the antitumor effects of the virus. RESULTS: Double-regulated duplicative adenovirus AdTPHre-hEndo genes were successfully constructed. Duplication and lethal assays proved that AdTPHre-hEndo could replicate specifically in pancreatic cancer cells and kill them. The endostatin expression in a cultured supernatant from tumor cells was significantly higher than that obtained from non-duplicative adenovirus vectors carrying that gene. The animal experiment demonstrated that AdTPHre-hEndo has a high capability to limit pancreatic cancer growth. CONCLUSIONS: AdTPHre-hEndo has a special ability to duplicate and kill pancreatic cancer cells in in vitro and in vivo experiments, thus providing a new gene-virus-based treatment system for pancreatic cancer.

Molecular cloning, expression and purification of recombinant soluble mouse endostatin as an anti-angiogenic protein in Escherichia coli.

Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni-NTA (Ni(2+)-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.

Endostatin, an inhibitor of angiogenesis, decreases after bidirectional superior cavopulmonary anastamosis.

Pulmonary arteriovenous malformations (PAVMs) are a common source of morbidity after bidirectional superior cavopulmonary anastomosis (Glenn). The diversion of hepatic venous effluent away from the pulmonary circulation after Glenn appears to play a significant role in the pathogenesis of PAVMs. Although the liver is known to produce factors that regulate vascular development, specific hepatic inhibitors of angiogenesis have not been described in the post-Glenn population. Endostatin, produced from its precursor collagen XVIII, is a potent inhibitor of angiogenesis produced by the liver. This study aimed to investigate the hypothesis that endostatin levels decrease in patients after Glenn. Levels of endostatin and its precursor, long-type collagen XVIII, were determined by enzyme-linked immunoassay and immunoprecipitation, respectively, for serum samples from 38 patients undergoing Glenn, total cavopulmonary anastomosis (Fontan), or biventricular repair of cardiac defects. Samples were obtained before surgery and 24 h afterward. In patients undergoing a bidirectional Glenn procedure, endostatin levels decreased after surgery (n = 17; 4.42 vs 3.34 ng/ml; p < 0.001), and long type-collagen XVIII levels increased by 200 % (n = 10; p = 0.0001). However, endostatin levels did not change after surgery in patients undergoing Fontan (n = 13) or biventricular repair (n = 8). In patients undergoing Fontan, long-type collagen XVIII increased by 18 % (p < 0.01), whereas in control subjects, the levels were unchanged. These data suggest that the diversion of hepatic blood flow away from the pulmonary circulation in patients after the Glenn procedure inhibits endostatin production from collagen XVIII, resulting in decreased circulating serum endostatin levels. A decrease in endostatin may promote angiogenesis. The mechanism whereby the pulmonary circulation processes endostatin and its potential role in the pathogenesis of PAVMs warrant further study.

Increased endogenous H2S generation by CBS, CSE, and 3MST gene therapy improves ex vivo renovascular relaxation in hyperhomocysteinemia.

Hydrogen sulfide (H(2)S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H(2)S is produced as a metabolite of homocysteine (Hcy) by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H(2)S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H(2)S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H(2)S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H(2)S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H(2)S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.

Preliminary study on the effects of carbon dioxide and nitrogen pneumoperitoneums on endometriotic lesions.

OBJECTIVE: To determine the effects of carbon dioxide (CO(2)) and nitrogen (N(2)) pneumoperitoneums on endometriosis (EMs) lesions. METHODS: Female Wistar rats were randomized into the following 3 groups: CO(2) (N = 20), N(2) (N = 22) and air pneumoperitoneums (N = 9). After 5 weeks of establishment models, do the pneumoperitoneums. Then measure the size of EMs lesions and the related factors of serum and tissue after 1, 2, and 4 weeks of pneumoperitoneums. RESULTS: (1) One week after the pneumoperitoneum was established, the EMs lesions in the CO(2) group were largest in volume, whereas at 4 weeks the EMs lesions in the CO(2) group were smaller than the N(2) group. (2) The level of ICAM-1 and TIMP-2 of serum in CO(2) and N(2) group after 2 weeks of pneumoperitoneum were higher than air group. (3) The expression of CD44v6, ICAM-1, MMP-2 and VEGF of tissue in CO(2) and N(2) group after 1, 2 and 4 weeks of pneumoperitoneum were lower than air group, TIMP-2 and ENS were higher than air group. CONCLUSION: After a CO(2) pneumoperitoneum, EMs lesions were reduced in volume, suggesting an inhibitory effect on EMs lesions.

Host-derived MMP-13 exhibits a protective role in lung metastasis of melanoma cells by local endostatin production.

BACKGROUND: Although matrix metalloproteinases (MMPs) are implicated in tumourigenesis and cancer progression, the role of MMP-13 in melanoma cell metastases is poorly understood. METHODS: Lung metastases of mouse melanoma B16BL6 cells were analysed in MMP-13 knockout (KO) and wild-type (WT) mice after intravenous injection. The mRNA and protein expression of MMP-13 in lung tissues was analysed by RT-PCR, real-time PCR, immunoblotting and immunohistochemistry. The expression of SDF-1α, CXCR4 and endostatin, and effects of endostatin to cultured melanoma cells and lung metastases were also studied. RESULTS: Lung metastases of B16BL6 cells were significantly higher by 2.5-5.7-fold in MMP-13 KO mice than in WT mice. The expression of MMP-13 in WT mouse lung tissue was stimulated on day 1 after intravenous injection of the melanoma cells and MMP-13 was immunolocalised to vascular endothelial cells in the lungs. Endostatin formation, but not degradation of SDF-1α, in the lung tissue was associated with reduced lung metastasis in WT mice. Endostatin significantly inhibited migration of B16BL6 cells in monolayer wounding assay and remarkably suppressed Matrigel invasion and transendothelial invasion of the cells. In addition, lung metastases of melanoma cells in MMP-13 KO mice were reduced by intraperitoneal administration of endostatin. CONCLUSION: Our results suggest that MMP-13 is overproduced by endothelial cells in the lungs with melanoma cells and has a protective role in lung metastasis by local generation of endostatin.

Direct comparison of four adeno-associated virus serotypes in mediating the production of antiangiogenic proteins in mouse muscle.

To determine the adeno-associated virus (AAV) serotype that most efficiently mediates muscle expression of antiangiogenic proteins, we injected four different serotype (1, 2, 7, and 8) AAV vectors encoding mouse endostatin (mEnd) or human soluble FLK-1 (hsFLK-1) into a quadriceps muscle of C57BL/6 mice. The highest plasma levels of therapeutic protein were observed in AAV8-injected mice (8 > 7 > 1 > 2). Sustained expression of mEnd was detected for 6 months, whereas concentrations of hsFLK-1 declined to the background level within 2 weeks caused by neutralizing anti-hsFLK-1 antibody. These data demonstrate that AAV8 (mEnd) serotype is the most efficient mediator for protein expression.

[Suppression of colon cancer in murine model by recombinant human endostatin adenovirus and cisplatin].

OBJECTIVE: To investigate the efficacy, side effect and anti-tumor mechanism of recombinant human endostatin adenovirus (Ad-hE) and cisplatin on murine colon cancer. METHODS: Mice with CT26 colon cancer were randomly divided into 5 groups, being given Ad-hE via tail vein, cisplatin (Cis) intraperitoneally, Ad-hE plus cisplatin (Ad-hE+Cis), empty adenovirus (Ad-N), and saline (NS), respectively. The therapeutic effect and side effect of the treatments and the angiogenesis and apoptosis of tumor cells were observed. RESULTS: Ad-hE + Cis suppressed the growth of lung metastatic tumor and prolonged survival time of the murine CT26 colon cancer model. The anti-tumor activity was associated with decreased microvessel density and increased apoptosis of tumor cells. CONCLUSION: Recombinant human endostatin adenovirus enhances the anti-tumor activity of cisplatin without increasing toxicity.

[Antitumor effect by combination of shRNA interfering plasmid targeting PKM2 with recombinant endostatin].

OBJECTIVE: To investigate the enhancement effect of the combination of shRNA interfering plasmid targeting PKM2 with recombinant Endostatin in the treatment of lung cancer. METHODS: Twenty five BABL/nu/nu mice bearing A549 lung cancer were divided into 5 groups (NS control, psh-Control, psh-PKM2 treated group, Endostar treated group, psh-PKM2+Endostar treated group) and treated with shRNA interfering plasmid targeting PKM2 and recombinant Endostatin respectively or in combination. The expression of PKM2 in A549 detected with immunofluorescent assay. The interference effect of psh-PKM2 was determined by Western blot. The tumor volume, microvessel density (MVD), apoptosis index (AI) and side effects were observed. RESULTS: The combination treatment of RNA interfering plasmid targeting PKM2 with recombinant Endostatin inhibited tumor growth obviously (P < 0.05); The combination group revealed a decreased MVD and an increased AI (P < 0.05). CONCLUSION: The combination of shRNA interfering plasmid targeting PKM2 with recombinant Endostatin might enhance anti-tumor effect by increasing the apoptosis of the cancer cell.

Systemic administration of mesenchymal stem cells loaded with a novel oncolytic adenovirus carrying IL-24/endostatin enhances glioma therapy.

Oncolytic adenovirus-mediated gene therapy shows promise for cancer treatment; however, the systemic delivery of oncolytic adenovirus to tumors remains challenging. Recently, mesenchymal stem cells (MSCs) have emerged as potential vehicles for improving delivery. Yet, because the oncolytic adenovirus replicates in MSCs, balancing MSC viability with viral load is key to achieving optimal therapeutic effect. We thus developed an all-in-one Tet-on system that can regulate replication of oncolytic adenovirus. Then, we loaded the novel oncolytic adenovirus carrying interleukin (IL)-24 and/or Endostatin in human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) for glioma therapy. In vitro assays demonstrated that this novel oncolytic adenovirus could efficiently replicate and kill glioma cells while sparing normal cells. Moreover, doxycycline effectively regulated oncolytic adenovirus replication in the hUCB-MSCs. The doxycycline induction group with dual expression of IL-24 and Endostatin exhibited significantly greater antitumor effects than other groups in a xenograft model of glioma. Thus, this strategy for systemic delivery of oncolytic adenovirus with its oncolytic activity controlled by a Tet-on system is a promising method for achieving antitumor efficacy in glioma, especially for metastatic tumors.

Endostatin gene therapy enhances the efficacy of IL-2 in suppressing metastatic renal cell carcinoma in mice.

We investigated whether the administration of IL-2 combined with endostatin gene therapy was able to produce additive or even synergistic immunomodulatory activity in a mouse model of metastatic renal carcinoma. Renca cells were injected into the tail vein of BALB/c mice. After 24 h, the animals were randomly divided into four groups (5 mice/group). One group of mice was the control, the second group received treatment with 100,000 UI of Recombinant IL-2 (Proleukin, Chiron) twice a day, 1 day per week during 2 weeks (IL-2), the third group received treatment with a subcutaneous inoculation of 3.6 x 10(6) endostatin-producing cells, and the fourth group received both therapies (IL-2 + ES). Mice were treated for 2 weeks. In the survival studies, 10 mice/group daily, mice were monitored daily until they died. The presence of metastases led to a twofold increase in endostatin levels. Subcutaneous inoculation of NIH/3T3-LendSN cells resulted in a 2.75 and 2.78-fold increase in endostatin levels in the ES and IL-2 + ES group, respectively. At the end of the study, there was a significant decrease in lung wet weight, lung nodules area, and microvascular area (MVA) in all treated groups compared with the control group (P < 0.001). The significant difference in lung wet weight and lung nodules area between groups IL-2 and IL-2 + ES revealed a synergistic antitumor effect of the combined treatment (P < 0.05). The IL-2 + ES therapy Kaplan-Meier survival curves showed that the probability of survival was significantly higher for mice treated with the combined therapy (log-rank test, P = 0.0028). Conjugated therapy caused an increase in the infiltration of CD4, CD8 and CD49b lymphocytes. An increase in the amount of CD8 cells (P < 0.01) was observed when animals received both ES and IL-2, suggesting an additive effect of ES over IL-2 treatment. A synergistic effect of ES on the infiltration of CD4 (P < 0.001) and CD49b cells (P < 0.01) was also observed over the effect of IL-2. Here, we show that ES led to an increase in CD4 T helper cells as well as cytotoxic lymphocytes, such as NK cells and CD8 cells, within tumors of IL-2 treated mice. This means that ES plays a role in supporting the actions of T cells.

[Antitumor effect by combination of recombinant endostatin adenovirus with carboplatin].

OBJECTIVE: To investigate the antitumor effect of recombinant Endostatin adenovirus (Ad-E) with carboplatin in mice with Lewis lung cancer. METHODS: C57BL/6 mice bearing Lewis lung cancer received Ad-E and carboplatin respectively or in combination. The tumor volume, tumor net weight, survival time and side effects were observed. Histological analysis was conducted to assess microvessel density (MVD) within tumor tissue and apoptotic cells were identified using the TUNEL assay. RESULTS: Compared with the control groups, the combination treatment significantly suppressed the tumor growth and prolonged survival time of the mice without obvious side-effects. The histological analysis revealed the combination treatment led to a decreased MVD and increased percent apoptosis in LL/2 tumors. CONCLUSION: Our studies indicate that a combination of Ad-E with carboplatin can present better antitumor effects without obvious side-effect.

Tumoristatic effects of endostatin in prostate cancer is dependent on androgen receptor status.

BACKGROUND: Although anti-angiogenic therapy is a promising new line of therapy for prostate cancer, we recently reported that stable expression of endostatin arrested the progression of prostate cancer to poorly differentiated state and distant metastasis in TRAMP mice. However, the same therapy failed to provide any benefit when given either during or after the onset of metastatic switch. The present study determined the possible mechanisms behind the selective advantage of endostatin therapy in early-stage disease. METHODS: Angiogenesis-related gene expression analysis was performed to identify target genes and molecular pathways involved in the therapy effects. Based on the results from in vivo studies, and recapitulation of the in vivo data in vitro using tumorigenic and non-tumorigenic human prostate cancer cells that are either androgen-sensitive or androgen-independent, analyses of possible mechanisms of the selective advantage of early treatment were performed using assays for cell proliferation, apoptosis, migration, and cell signaling. The identified mechanisms were further confirmed in vivo. RESULTS: Results indicated that cells with high androgen receptor (AR) expression were more sensitive to endostatin treatment than androgen-independent cells with low or no AR expression. Endostatin was found to significantly downregulate the expression of growth factors, receptor tyrosine kinases, proteases, and AR both in vitro and in vivo only when the cells express high-levels of AR. Cell proliferation was not influenced by endostatin treatment but migration was significantly affected only in androgen-sensitive cells. Targeted downregulation of AR prior to endostatin treatment in androgen-sensitive cells and overexpression of AR in androgen-independent cells indicated that the effect of endostatin via AR downregulation is mediated by a non-genotropic mechanism on Ras and RhoA pathways, and independently of AR on MAPK/ERK pathway. CONCLUSIONS: These data indicate that systemically stable endostatin expression delays the onset of metastatic switch by acting on multiple pathways involving AR.

Mesalamine restores angiogenic balance in experimental ulcerative colitis by reducing expression of endostatin and angiostatin: novel molecular mechanism for therapeutic action of mesalamine.

Mesalamine (5-aminosalicylate acid, 5-ASA) is an effective treatment for ulcerative colitis (UC). The mechanisms of its actions are not fully understood. Because angiogenesis is critical for healing UC, we examined whether 5-ASA alters the angiogenic balance between angiogenic factors [e.g., vascular endothelial growth factor (VEGF)] and antiangiogenic factors (e.g., endostatin and angiostatin) in the colon in experimental UC. Rats were treated with saline or 5-ASA (100 mg/kg) twice daily and euthanized 3 or 7 days after iodoacetamide-induced UC. Clinical signs (e.g., lethargy, diarrhea) and UC lesions were measured. Expression of VEGF, endostatin, angiostatin, tissue necrosis factor alpha (TNF-alpha), and matrix metalloproteinases (MMPs) 2 and 9 was determined by Western blots, enzyme-linked immunosorbent assay, and zymography in the distal colon. 5-ASA treatment reduced lethargy and diarrhea and significantly decreased colonic lesions (by approximately 50%) compared with saline treatment in UC (both, P < 0.05). 5-ASA did not reverse the increased levels of VEGF, but it significantly reduced expression of endostatin and angiostatin in UC compared with vehicle treatment (both, P < 0.05). Furthermore, 5-ASA treatment significantly diminished increased activity of TNF-alpha and MMP9 in UC. This is the first demonstration that 5-ASA treatment reverses an imbalance between the angiogenic factor VEGF and antiangiogenic factors endostatin and angiostatin in experimental UC. The effect of 5-ASA in UC may be caused by the down-regulation of expression of endostatin and angiostatin by modulation of MMP2 and MMP9 via inhibition of TNFalpha. The inhibition of antiangiogenic factors may represent a novel molecular mechanism of the therapeutic action of 5-ASA.

Dependence of cathepsin L-induced coronary endothelial dysfunction upon activation of NAD(P)H oxidase.

Cathepsin L is a cysteine protease that can generate endogenous endostatin in vascular and epithelial basement membranes and importantly participates in a variety of pathophysiological processes. The present study was designed to determine whether this cathepsin L-derived endogenous endostatin alters endothelium-dependent vasodilator responses in coronary arteries via NAD(P)H oxidase activation. In isolated and perfused small bovine coronary arteries, administration of cathepsin L (200 ng/ml) markedly attenuated endothelium-dependent vasodilator responses to bradykinin or A23187 by 56.16+/-9.58% and 68.95+/-10.32%, respectively. This inhibitory effect of cathepsin L on endothelium-dependent vasodilator responses could be significantly reversed by pre-incubation of the arteries with O(2)(-) scavenger, Tiron, or neutralizing anti-endostatin antibody. By fluorescent ELISA assay, cathepsin L dose-dependently increased endostatin production in coronary arteries. In situ high-speed dual wavelength switching fluorescent microscopic imaging showed that cathepsin L decreased bradykinin- and A23187-induced NO levels in the intact endothelium, but it had no effect on Ca(2+) response to these vasodilators. This cathepsin L-induced reduction of NO was restored by the pretreatment of an anti-endostatin antibody. Electron spin resonance (ESR) analysis demonstrated that cathepsin L increased O(2)(-) production which could be markedly attenuated by the NAD(P)H oxidase inhibitors, apocynin or anti-endostatin antibody. It is concluded that endostatin could be endogenously produced in coronary arteries when cathepsin L is increased and that this cathepsin L-derived endostatin, if excessive, may result in endothelial dysfunction through enhanced production of O(2)(-) due to NAD(P)H oxidase activation.

A pharmacologically based multiscale mathematical model of angiogenesis and its use in investigating the efficacy of a new cancer treatment strategy.

Tumor angiogenesis is the process by which new blood vessels are formed and enhance the oxygenation and growth of tumors. As angiogenesis is recognized as being a critical event in cancer development, considerable efforts have been made to identify inhibitors of this process. Cytostatic treatments that target the molecular events of the angiogenesis process have been developed, and have met with some success. However, it is usually difficult to preclinically assess the effectiveness of targeted therapies, and apparently promising compounds sometimes fail in clinical trials. We have developed a multiscale mathematical model of angiogenesis and tumor growth. At the molecular level, the model focuses on molecular competition between pro- and anti-angiogenic substances modeled on the basis of pharmacological laws. At the tissue scale, the model uses partial differential equations to describe the spatio-temporal changes in cancer cells during three stages of the cell cycle, as well as those of the endothelial cells that constitute the blood vessel walls. This model is used to qualitatively assess how efficient endostatin gene therapy is. Endostatin is an anti-angiogenic endogenous substance. The gene therapy entails overexpressing endostatin in the tumor and in the surrounding tissue. Simulations show that there is a critical treatment dose below which increasing the duration of treatment leads to a loss of efficacy. This theoretical model may be useful to evaluate the efficacy of therapies targeting angiogenesis, and could therefore contribute to designing prospective clinical trials.

Induction of endostatin expression in meniscal fibrochondrocytes by co-culture with endothelial cells.

INTRODUCTION: The ultimate goal after meniscus damage is the preservation of the original meniscal tissue, which is often impossible due to the limited healing capacity of meniscal lesions, especially in the avascular zone. Factors produced by endothelial cells of meniscal vessels may contribute to better wound healing in vascularized zones. We therefore investigated the expression of different angiogenic factors, growth hormones and cytokines in human fibrochondrocytes and in fibrochondrocytes upon co-culture with endothelial cells, to examine mechanisms of repair of meniscal injury in more detail and to investigate the potential use of endothelial cells in co-cultures for autologous meniscal repair utilizing tissue engineering technology. MATERIALS AND METHODS: Gene expression of SMAD-4, iNOS, IL-1beta, VEGF, MMP-1, MMP-3, MMP-13, aggrecan, biglycan, vimentin, collagen-I, -II, -III, -IV, -VI, -X, -XVIII, angiopoietin-1, angiopoietin-2, and thrombostatin-1 were investigated in fibrochondrocytes in comparison to cells in co-culture with human umbilical vein endothelial cells (HUVEC). The expression of endostatin was enumerated in cell supernatants. A proliferation assay was used to investigate the mitotic activity of the cells. RESULTS: In presence of HUVEC, meniscal fibrochondrocytes expressed SMAD-4, iNOS, IL-1beta, VEGF, MMP-1, MMP-3, MMP-13, aggrecan, biglycan, vimentin, collagen-I, -II, -III, -VI, and -XVIII at rates comparable to cells without HUVECs. Note that the expression of endostatin was significantly higher in the co-culture when compared to the separate fibrochondrocyte cultures and the proliferation rate of endothelial cells was significantly decreased in co-culture. CONCLUSION: We conclude that the expression of the anti-angiogenic factor endostatin increased in the fibrochondrocytes. This may limit the regeneration capacities of meniscal injury in vivo.

Modulation of angiogenic phenotype alters tumorigenicity in rat ovarian epithelial cells.

Vascular endothelial growth factor (VEGF) expression correlates with microvessel density, stage, malignant ascites, metastasis, and survival in ovarian cancer. By transducing VEGF165 into a nontumorigenic rat ovarian surface epithelial cell line (ROSE199), we investigated the direct effect of an angiogenic phenotype on tumor development. The neu oncogene, which is overexpressed in >30% of ovarian cancers, was used in comparison. Neu-transfected ROSE199 cells showed phenotypic characteristics of transformation in vitro with an abundance of focus-forming units in monolayer cultures and anchorage-independent growth in soft agar. In contrast, VEGF-secreting ROSE199 cells (VR) retained normal morphology and in vitro growth characteristics (e.g., proliferation rate) compared with parental ROSE199 cells. Interestingly, injection of VR cells into athymic mice formed malignant ascites in 100% of the animals when injected into the peritoneum and developed vascularized tumors in 85% of the mice when injected s.c. Furthermore, blocking VEGF-mediated signaling by the Flk-1/KDR receptor kinase inhibitor SU5416 significantly inhibited the growth of VR tumors. To validate that the proangiogenic switch is responsible for tumor development, the angiogenic phenotype was balanced by the inducible coexpression of endostatin under the control of Tet-activated promoter. Coexpression of endostatin along with VEGF reversed the tumorigenic phenotype of VR cells. These studies show that alterations in the angiogenic characteristics of ovarian surface epithelium may play an important role in the etiology of ovarian cancer, and that inhibition of angiogenesis can be effective in the treatment of epithelial ovarian cancer.

Optimization of microencapsulated recombinant CHO cell growth, endostatin production, and stability of microcapsule in vivo.

Microencapsulation of recombinant cells secreting endostatin offers a promising approach to tumor gene therapy in which therapeutic protein is delivered in a sustainable and long-term fashion by encapsulated recombinant cells. However, the studies of cell growth and protein production in vivo are very limited. In this study, the effects of microencapsulation parameters on in vivo cell growth, endostatin production, and microcapsule stability after implantation in the peritoneal cavity of mice were for the first time investigated. Microcapsules with liquid core reached higher cell density and endostatin production at day 18 than microcapsules with solid core. There was no significant difference in stability whether the core of the microcapsule was solid or liquid. Decrease in microcapsule size increased the stability of microcapsule. The microcapsules kept intact in the peritoneal cavity of mice after 36 days of implantation when the microcapsules size was 240 microm in diameter, which gave rise to high endostatin production as well. The optimized microencapsulation conditions for in vivo implantation are liquid core and 240 microm in diameter. This study provides useful information for antiangiogenic gene therapy to tumors using microencapsulated recombinant cells.