Regulation Of Angiogenic Factors Research Articles

Biomaterial-assisted strategies to improve islet graft revascularization and transplant outcomes.

Islet transplantation holds significant promise as a curative approach for type 1 diabetes (T1D). However, the transition of islet transplantation from the experimental phase to widespread clinical implementation has not occurred yet. One major hurdle in this field is the challenge of insufficient vascularization and subsequent early loss of transplanted islets, especially in non-intraportal transplantation sites. The establishment of a fully functional vascular system following transplantation is crucial for the survival and secretion function of islet grafts. This vascular network not only ensures the delivery of oxygen and nutrients, but also plays a critical role in insulin release and the timely removal of metabolic waste from the grafts. This review summarizes recent advances in effective strategies to improve graft revascularization and enhance islet survival. These advancements include the local release and regulation of angiogenic factors (e.g., vascular endothelial growth factor, VEGF), co-transplantation of vascular fragments, and pre-vascularization of the graft site. These innovative approaches pave the way for the development of effective islet transplantation therapies for individuals with T1D.

Seasonal and temporal variation in the placenta during melatonin supplementation in a bovine compromised pregnancy model.

Compromised pregnancies result in a poorly functioning placenta restricting the amount of oxygen and nutrient supply to the fetus resulting in intrauterine growth restriction (IUGR). Supplementing dietary melatonin during a compromised pregnancy increased uteroplacental blood flow and prevented IUGR in a seasonal-dependent manner. The objectives were to evaluate seasonal melatonin-mediated changes in temporal alterations of the bovine placental vascularity and transcript abundance of clock genes, angiogenic factors, and nutrient sensing genes in 54 underfed pregnant Brangus heifers (Fall, n = 29; Summer, n = 25). At day 160 of gestation, heifers were assigned to treatments consisting of adequately fed (ADQ-CON; 100% NRC; n = 13), nutrient restricted (RES-CON; 60% NRC; n = 13), and ADQ or RES supplemented with 20 mg/d of melatonin (ADQ-MEL, n = 13; RES-MEL, n = 15). The animals were fed daily at 0900 hours until day 240 where Cesarean sections were performed in the morning (0500 hours) or afternoon (1300 hours) for placentome collections. In both seasons, we observed a temporal alteration of the core clock genes in the cotyledonary tissue in a season-dependent manner. In the fall, ARNTL, CLOCK, NR1D1, and RORA transcript abundance were decreased (P ≤ 0.05) in the afternoon compared to the morning; whereas in the summer, ARNTL, PER2, and RORA expression were increased (P ≤ 0.05) in the afternoon. Interestingly, in both seasons, there was a concomitant temporal increase (P ≤ 0.05) of cotyledonary blood vessel perfusion and caruncular melatonin receptor 1A transcript abundance. Melatonin supplementation did not alter the melatonin receptor 1A transcript abundance (P > 0.05), however, in the summer, melatonin supplementation increased cotyledonary VEGFA, CRY1, and RORA (P ≤ 0.05) transcript abundance. In addition, during the summer the placentomes from underfed dams had increased average capillary size and HIF1α transcript abundance compared to those adequately fed (P ≤ 0.05). In conclusion, these data indicate increased cotyledonary blood vessel size and blood distribution after feeding to better facilitate nutrient transport. Interestingly, the maternal nutritional plane appears to play a crucial role in regulating the bovine placental circadian clock. Based on these findings, the regulation of angiogenic factors and clock genes in the bovine placenta appears to be an underlying mechanism of the therapeutic effect of dietary melatonin supplementation in the summer.

Sulfated Hyaluronan Alters Endothelial Cell Activation in Vitro by Controlling the Biological Activity of the Angiogenic Factors Vascular Endothelial Growth Factor-A and Tissue Inhibitor of Metalloproteinase-3

Several pathologic conditions such as rheumatoid arthritis, ocular neovascularization, cancer, or atherosclerosis are often associated with abnormal angiogenesis, which requires innovative biomaterial-based treatment options to control the activity of angiogenic factors. Here, we studied how sulfated hyaluronan (sHA) and oversulfated chondroitin sulfate derivatives as potential components of functional biomaterials modulate vascular endothelial growth factor-A (VEGF-A) signaling and endothelial cell activity in vitro. Tissue inhibitor of metalloproteinase-3 (TIMP-3), an effective angiogenesis inhibitor, exerts its activity by competing with VEGF-A for binding to VEGF receptor-2 (VEGFR-2). However, even though TIMP-3 and VEGF-A are known to interact with glycosaminoglycans (GAGs), the potential role and mechanism by which GAGs alter the VEGF-A/TIMP-3 regulated VEGFR-2 signaling remains unclear. Combining surface plasmon resonance, immunobiochemical analysis, and molecular modeling, we demonstrate the simultaneous binding of VEGF-A and TIMP-3 to sHA-coated surfaces and identified a novel mechanism by which sulfated GAG derivatives control angiogenesis: GAG derivatives block the binding of VEGF-A and TIMP-3 to VEGFR-2 thereby reducing their biological activity in a defined, sulfation-dependent manner. This effect was stronger for sulfated GAG derivatives than for native GAGs. The simultaneous formation of TIMP-3/sHA complexes partially rescues the sHA inhibited VEGF-A/VEGFR-2 signaling and endothelial cell activation. These results provide novel insights into the regulation of angiogenic factors by GAG derivatives and highlight the potential of sHA derivatives for the treatment of diseases associated with increased VEGF-A and VEGFR-2 levels.

Regulation of angiogenic factors by the PI3K/Akt pathway in A549 lung cancer cells under hypoxic conditions.

The aim of the present study was to investigate the influence of hypoxia and PI3K inhibition on angiogenic factors in A549 lung cancer cells. A549 cells were treated with the PI3K inhibitor LY294002 under normoxic and hypoxic conditions. Untreated cells were used as the control group and those treated by the inhibitor, as the suppression group. The cells were further divided based on normoxic or hypoxic conditions and named: Normoxic control group, normoxic suppression group, hypoxic control group and hypoxic suppression group. Expression levels of hypoxia-inducible factor (HIF)-1α and AKT1 mRNA in all groups were determined by reverse transcriptase-quantitative polymerase chain reaction and concentrations of vascular endothelial growth factor (VEGF), angiotensin II (ANG-II), transforming growth factor (TGF)-α/β1, and tumor necrosis factor (TNF)-α in the culture supernatant were measured by enzyme-linked immunosorbent assay. The expression levels of HIF-1α and AKT1 mRNA in the hypoxic control group were higher than those in the normoxic control group and the expression levels of HIF-1α and AKT1 mRNA in the hypoxic control group were higher than those in the hypoxic suppression group. Compared to the normoxic control and normoxic suppression groups, the concentrations of VEGF and TNF-α in supernatant were higher in the hypoxic control and hypoxic suppression groups, respectively. However, TGF-α and TGF-β1 demonstrated the opposite trend of the aforementioned factors. The concentration of ANG-II in the hypoxic suppression group was higher than that in the normoxic suppression group. In addition, compared to the normoxic control group and hypoxic control group, the concentrations of VEGF and TGF-β1 in supernatant were lower in the normoxic suppression group and in the hypoxic suppression group, respectively. In conclusion, the results of the present study suggest that hypoxia can stimulate A549 lung cancer cells to secrete VEGF and TNF-α and inhibit TGF-α and TGF-β1. The ability of A549 cells to secrete VEGF and TGF-β1 is regulated by PI3K/Akt, and ANG-II expression may be regulated by the PI3K/Akt pathway under hypoxic condition.

Inhibition of chemokine (C-X-C motif) receptor four (CXCR4) at the fetal-maternal interface during early gestation in sheep: alterations in expression of chemokines, angiogenic factors and their receptors.

Chemokine (C-X-C motif) ligand 12 (CXCL12) and its receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), are involved in significant biological processes associated with early pregnancy including increasing trophoblast invasion and stimulating placental vascularization. To further elucidate functions of CXCL12-CXCR4 signaling during early gestation, our objective was to inhibit CXCR4 in vivo using a CXCR4 antagonist, AMD3100. We hypothesized that inhibition of CXCR4 would negatively affect chemokine and angiogenic factor regulation imperative for placental development in sheep. Osmotic pumps containing PBS (control) or AMD3100 (CXCR4 antagonist) were surgically installed ipsilateral to the corpus luteum on d 12 of gestation and administered treatments directly into the uterine lumen. Maternal (caruncle and intercaruncle) and fetal membrane tissues were collected on d 23 of gestation and mRNA and protein expression were analyzed for vascular endothelial growth factor (VEGF), kinase insert domain receptor (KDR), fms related tyrosine kinase 1 (FLT1), fibroblast growth factor 2 (FGF2), angiopoietin 1 (ANGPT1), hypoxia inducible factor 1 ɑ subunit (HIF1A), CXCL12, and its corresponding receptors (CXCR4 and CXCR7). Immunohistochemical procedures were performed for analysis of CXCL12 and cell proliferation. In caruncle tissue ipsilateral to the pump, mRNA for KDR, ANGPT1, HIF1A, and CXCL12 increased (P < 0.05) in treated ewes compared to control, whereas caruncle tissue contralateral to the pump had increased expression (P < 0.05) of KDR, and CXCL12 in treated ewes. In fetal membrane, CXCR4 mRNA and protein decreased (P < 0.05), while VEGF protein decreased (P < 0.05) in caruncle and fetal membrane tissue from treated ewes. Results from this study highlight the importance of CXCL12-CXCR4 signaling at the fetal-maternal interface. Inhibiting this axis may disrupt typical regulation of angiogenic factors needed for placental development and embryo growth.

Abstract 2526: Regulation of angiogenic factors in glioblastoma progression

Abstract Background &amp; Purpose: Glioblastoma is the most malignant primary brain tumor in humans and has a very low survival rate. The aggressiveness of this cancer can be attributed to the presence of glioblastoma stem cells (GSC), which are highly invasive and therapy resistant. Increased aggressiveness of GSCs is attributed to the release of angiogenic factors, which promotes the formation of new blood vessels that enhances tumor cell survival and proliferation. The neovascularization also provides a scaffold for tumor cells to migrate, enabling metastasis. Therefore, identifying the altered expression of angiogenic factors is of clinical importance. In this study, we examined the changes in the expression of pro-angiogenic factors in GSCs as compared to the original tumor from which they were derived. We also determined if differentiation of GSC towards a less invasive phenotype decreased the levels of these pro-angiogenic factors. Methods: GBM tumor cells were grown in complete media containing 10% Fetal Calf Serum (FCS) and GSCs were grown in media containing B27, heparin, EGF, and FGF. GSCs were differentiated by growing them in media containing 10% FCS and B27 with vitamin A, for about one week. We examined the expression of a panel of pro-angiogenic factors in GSCs (33-GSC) and in the original tumor cells (33-T) from which GSCs were derived. We then examined if differentiation of 33-GSC decreased the levels of these pro-angiogenic factors. Changes in protein levels were evaluated by immunofluorescence staining. The mRNA levels were quantitated using real time Q-PCR. Results: Immunofluorescence staining showed that the GSCs express higher levels of pro-angiogenic factors examined (11 out of 16). GSCs were successfully differentiated into astrocytes as indicated by morphology and GFAP expression. The differentiation of GSCs into astrocytes was sufficient to cause decreased expression of VEGF and c-MYC. Our data showed 27 fold increase in VEGF mRNA levels, 63 fold increase in MMP2 mRNA levels, 11 fold increase in MMP9 mRNA levels, and 2 fold increase in c-MYC mRNA levels in GSCs compared to the tumor line, indicating the regulation of these pro-angiogenic factors at transcriptional level. Conclusions: Increased expression of pro-angiogenic factors in GSCs may render these cells to be more aggressive and invasive, and suggests that differential expression of angiogenic factors in GSC cells may play an important role in the GBM progression, and therapy resistance. Citation Format: Carolina Larrain, Umadevi V. Wesley, Paul Clark, John S. Kuo, Robert Dempsey. Regulation of angiogenic factors in glioblastoma progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2526.

Melatonin Regulates Angiogenic Factors under Hypoxia in Breast Cancer Cell Lines.

Angiogenesis is the process of new blood vessel formation, regulated by a number of pro- and antiangiogenic factors and usually begins in response to hypoxia. Exogenous administration of melatonin has shown numerous anti-tumor effects and appears to inhibit tumor angiogenesis. However, many factors involved in the anti-angiogenic effect of melatonin are still under investigation. Here, we evaluate the effects of melatonin on cell viability and expression of angiogenic factors in MCF-7 and MDA-MB-231 breast cancer cells under hypoxic conditions. Cell viability was investigated by MTT and gene and protein expression of the hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF-A) were verified by qPCR and immunocytochemistry after melatonin treatment (1 mM) under hypoxic conditions. Additionally, a protein array with 20 different cytokines/factors was performed on tumor cell lysates. The results showed that 1 mM of melatonin reduced the viability of MCF-7 and MDA-MB-231 cells (p < .05). This treatment also decreased both gene and protein expression of HIF-1α and VEGF-A under hypoxic conditions (p < .05). Among the proteins evaluated by protein array, melatonin treatment during hypoxia reduced VEGF-C, VEGFR receptors (VEGFR2 and VEGFR3), matrix metalloproteinase 9 (MMP9) and Angiogenin in MCF-7 cells. In MDA-MB-231 cells, a significant decrease was observed in VEGFR2, epidermal growth factor receptor (EGFR) and Angiogenin (p < .05). Taken together, these results showed that melatonin acts in the regulation of angiogenic factors in breast tumor cells and suggests an anti-angiogenic activity, particularly under hypoxic conditions.

Bone morphogenetic proteins in tumour associated angiogenesis and implication in cancer therapies

Bone morphogenetic protein (BMP) belongs to transforming growth factor-β superfamily. To date, more than 20 BMPs have been identified in humans. BMPs play a critical role in embryonic and postnatal development, and also in maintaining homeostasis in different organs and tissues by regulating cell differentiation, proliferation, survival and motility. They play important roles in the development and progression of certain malignancies, including prostate cancer, breast cancer, lung cancer, etc. Recently, more evidence shows that BMPs are also involved in tumour associated angiogenesis. For example BMP can either directly regulate the functions of vascular endothelial cells or indirectly influence the angiogenesis via regulation of angiogenic factors, such as vascular endothelial growth factor (VEGF). Such crosstalk can also be reflected in the interaction with other angiogenic factors, like hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF). All these factors are involved in the orchestration of the angiogenic process during tumour development and progression. Review of the relevant studies will provide a comprehensive prospective on current understanding and shed light on the corresponding therapeutic opportunity.

Rapamycin inhibits oxidative and angiogenic mediators in diabetic retinopathy

ObjectiveTo evaluate the role of rapamycin in the prevention of diabetic oxidative stress and the regulation of angiogenic factors. DesignExperimental animal study. MethodsDiabetes was induced in 20 adult male Wistar rats by a single intraperitoneal administration of streptozotocin (60 mg/kg). Rats were randomly assigned into diabetic and rapamycin groups (n = 10). Ten healthy normal adult male rats of same age formed the control group. All groups were followed for 3 months. Rapamycin group received 1 mg/kg rapamycin via orogastric gavage during the last 4 weeks. At the end of 12 weeks, rats were sacrificed and biochemical oxidative stress markers (malondialdehyde and nitrotyrosine), together with vascular endothelial growth factor, hypoxia-inducible factor-1α, and pigment epithelium-derived factor, were measured in the retina. Blood biochemical analyses were also done. ResultsIn the diabetic group, retinal malondialdehyde and nitrotyrosine levels were increased in comparison with control and rapamycin groups (p < 0.05). Rapamycin suppressed oxidative stress and showed a beneficial effect. It also decreased all angiomodulator cytokines compared with the diabetic group (p < 0.05). Correspondingly, rapamycin also decreased plasma malondialdehyde levels compared with the diabetic group (p = 0.037). ConclusionsRapamycin may have a protective role against diabetes-induced oxidative retinal injury and may decrease angiomodulator cytokines.

Retinal vessel pathologies in a rat model of periventricular leucomalazia

AbstractPurpose Alternating oxygen levels during delivery and supplemental oxygen therapies can cause pathological vascularization of the retina (retinopathy of prematurity, ROP) and/or loss of oligodendrocytes in the periventricular area of the brain with subsequent white matter damage (periventricular leukomalacia (PVL). The aim of this study was to examine retinal vessel pathologies in the standard PVL rat model in order to identify uniform damage pathways.Methods Ischemia was induced in post natal day (P) 6 Long Evans rats with unilateral (left side) carotid ligation (UCL) and exposure to different oxygen concentrations. In total, four different groups were examined: (A) hypoxia/ischemia (UCL + 6% O2, 1 h), (B) hyperoxia (80%O2, 24 h), (C) hypoxia/ischemia + hyperoxia (UCL + 6% O2, 1 h + 80% O2, 24h), (D) normoxia. Morphological analysis of vessel development was performed at P11 and P21. Quantitative PCR was performed at P7, P11, and P21 (VEGF, HIF1a, EPOR, TNFa, iNOS and BMP‐9).Results Distinct retardation in deeper vascular plexus development was observed most prominent in left eyes of groups A and C. Diameters of big retinal arteries were significantly increased in central retinae at P21 and showed retardation in vascular remodeling. Left retinae of groups A and C at P11 displayed reduced capillary free zones (CFZ) and an increase in the number of branching points (BP). Quantitative gene expression analysis showed down regulation of angiogenic factors at P7.Conclusion This is the first report on concurring damage to the retina that was evaluated in a model of white matter injury in the neonatal brain. We observed mild damage to the vessel development and maturation, which is the basis for a model for moderate forms of ROP.

Regulation of decidualization and angiogenesis in the human endometrium: mini review.

The human endometrium is a dynamic tissue that undergoes regular cycles of menstruation, menstrual repair, proliferation and secretory differentiation in response to hypoxia and the female sex hormones. We identified new target genes that are regulated by progesterone during the decidualization of human endometrial stromal cells (ESC), including interleukin-15 (IL-15), fibulin-1 (FBLN-1), and heart and neural crest derivatives expressed transcript 2 (HAND2). IL-15 is deeply involved in the hormonal control of the human endometrium by progesterone and may be important in embryo implantation. FBLN-1 has been shown to be an important extracellular matrix that mediates progesterone action in human ESC differentiation toward implantation. Moreover, progestin-induced HAND2 is a transcription factor that contributes to the increased levels of FBLN-1 in human ESC. Several mediators, including vascular endothelial growth factor (VEGF), angiopoietin (ANGPT) and stromal cell-derived factor 1 (SDF-1), regulate human endometrial angiogenesis. Hypoxia increased the expression of VEGF and decreased the expression of SDF-1 in ESCs. Furthermore, hypoxia reduced ANGPT1 levels in ESC; however, ANGPT2 levels were unaffected. Estradiol simultaneously induced the expressions of VEGF and SDF-1, suppressing ANGPT1 production. Therefore, hypoxia and estradiol caused an increase in the ANGPT2/ANGPT1 ratio. Hypoxia and female sex hormones are involved in the regulation of angiogenic factors in an independent manner in human ESC. Analysis of the process of decidualization and angiogenesis in the human endometrium would provide useful information for the fields of reproductive biology, regenerative medicine and tissue engineering.

Regulation of hepatocyte growth factor expression by NF-κB and PPARγ in adipose tissue

Hepatocyte growth factor (HGF) is expressed as an angiogenic factor in adipose tissue. However, the molecular mechanism of Hgf expression remains largely unknown in the tissue. We addressed the issue by studying Hgf expression in adipocytes and macrophages. Hgf was expressed more in the stromal-vascular fraction than the adipocyte fraction. The expression was fivefold more in macrophages than the stromal-vascular faction and was reduced by 50% after macrophage deletion in adipose tissue. The expression was reduced by differentiation in adipocytes and by tumor necrosis factor-α or lipopolysaccharide treatment in macrophages. The expression was suppressed by nuclear factor (NF)-κB in C57BL/6 mice with NF-κB p65 overexpression under the aP2 gene promoter (aP2-p65 mice) but enhanced by inactivation of NF-κB p65 in mouse embryonic fibroblasts. The Hgf gene promoter was suppressed by p65 overexpression, which blocked peroxisome proliferator-activated receptor-γ (PPARγ) interaction with RNA polymerase II. The p65 activity was abolished by knockdown of histone deacetylase 3. Hgf expression was upregulated by hypoxia in vitro and in vivo. Compared with vascular endothelial growth factor (Vegf), which was predominately expressed in mature adipocytes, Hgf was mainly expressed in nonadipocytes, suggesting that Hgf and Vegf may have different cell sources in adipose tissue. In mechanism, Hgf expression is inhibited by NF-κB through suppression of PPARγ function in the Hgf gene promoter. Both Hgf and Vegf are induced by hypoxia. The study provides a molecular mechanism for the difference of inflammation and hypoxia in the regulation of angiogenic factors.

Vascular endothelial growth factor and angiopoietin production by primate follicles during culture is a function of growth rate, gonadotrophin exposure and oxygen milieu

What is the time course of production of vascular endothelial growth factor-A (VEGF-A), angiopoietin (ANGPT)-1 and ANGPT-2 by primate follicles during encapsulated three-dimensional culture, and what conditions affect their production? Primate follicles produce VEGF-A and ANGPT-2 in vitro, particularly after developing to the antral stage, with VEGF production influenced by FSH concentration and O(2) tension. Folliculogenesis, i.e. the development of primordial follicles into mature, antral follicles, requires the creation of a vascular network in the follicle wall via a process called angiogenesis. Angiogenic factors including VEGFs and ANGPTs have documented roles in angiogenesis. However, direct studies on the production and regulation of angiogenic factors by individual, growing follicles are limited. Ovaries (n = 9 pairs) were obtained from rhesus macaques during the early follicular phase of the menstrual cycle (cycle days 1-4). Secondary (125-225 µm) follicles were isolated mechanically, encapsulated into alginate (0.25% w/v) and cultured for 40 days. Individual follicles were cultured in a 5 or 20% O(2) environment in alpha minimum essential medium supplemented with recombinant human (h) FSH. Half of the follicles had recombinant hLH added to the media from Days 30 to 40. Follicle diameters were measured weekly. Follicles were categorized at Week 5 as no-grow (NG; <250 μm in diameter), slow-grow (SG; 251-499 μm) and fast-grow (FG; >500 μm). VEGF-A, ANGPT-1 and -2 concentrations in media were measured by ELISA. VEGF concentrations were low throughout the culture for NG follicles. SG and FG follicles had detectable VEGF concentrations at Week 2, which continued to rise throughout culture. VEGF concentrations were distinct (P < 0.05) among all three follicle categories during Weeks 4 and 5. VEGF concentrations were higher (P < 0.05) in SG follicles in the presence of high/mid-dose FSH at 5% O(2). In contrast, there were no dose-dependent differences in VEGF production for FG follicles based on FSH concentrations or O(2) tension. At Week 5, follicles that produced metaphase II oocytes, following exposure to an ovulatory hCG dose, secreted higher concentrations of VEGF than those containing germinal vesicle-intact oocytes. Media concentrations of ANGPT-1 were low throughout culture for all three follicle categories. ANGPT-2 concentrations were low throughout culture for NG follicles. In contrast, ANGPT-2 concentrations of SG and FG follicles continued to rise from Weeks 1 to 4. During Weeks 2-4, ANGPT-2 concentrations in FG follicles were significantly higher than those of SG and NG follicles (P < 0.05). This study reports VEGF-A, ANGPT-1 and -2 production by in vitro-developed individual primate (macaque) follicles, that is limited to the interval from the secondary to small antral stage. After VEGF and ANGPT-1 assays, the limited remaining samples did not allow assessment of the independent effects of gonadotrophin and O(2) on the ANGPT-2 production by cultured follicles. Findings await translation to human follicles. The above findings provide novel information on the process of primate follicle maturation. We hypothesize that a symbiotic relationship between elevated concentrations of ANGPT-2 and VEGF allows FG antral follicles to excel in follicle maturation, e.g. by promoting its vascularization. Elevated ANGPT-2 may also offer possible insight into future oocyte quality as early as Week 2, compared with Week 4 for VEGF and follicle size. The study was funded by the following grants: NIH U54 RR024347/HD058294/PL1-EB008542 (Oncofertility Consortium), NIH U54-HD018185 (SCCPIR), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), NIH FIC TW/HD-00668, ONPRC 8P51OD011092. There are no conflicts of interest to declare.

Regulation of Angiogenic Factors in the Mouse Intrahippocampal Kainic Acid Model of Epileptogenesis (IN9-1.004)

Regulation of Angiogenic Factors in the Mouse Intrahippocampal Kainic Acid Model of Epileptogenesis (IN9-1.004)

Regulation of Angiogenic Factors in the Mouse Intrahippocampal Kainic Acid Model of Epileptogenesis (P02.210)

Regulation of Angiogenic Factors in the Mouse Intrahippocampal Kainic Acid Model of Epileptogenesis (P02.210)

Natural Products for Liver Diseases: Basic, Clinical, and Translational Research

Natural Products for Liver Diseases: Basic, Clinical, and Translational Research

Abstract C206: High antitumor complete response rate to Namitecan (ST1968) in preclinical model of pediatric sarcomas.

Abstract Namitecan (ST1968), a new water-soluble camptothecin analogues, exhibits an improved pharmacological profile over conventional camptothecins. This molecule was selected for clinical development on the basis of preclinical evidence of superior efficacy than Topotecan or irinotecan and of its favorable therapeutic index. Namitecan has shown an high rate of sustained disease control in patients with solid tumors in Phase I, as predicted by preclinical data. Given the interest for the irinotecan treatment of pediatric tumors, this study was designed to explore the efficacy of Namitecan in preclinical models of pediatric sarcomas (TC-71, RH30, RD/TE671, A204 and U2OS cell lines). Namitecan showed an impressive antitumor efficacy in 4/5 of the tested xenograft sarcoma models at doses ranging 15–30 mg/kg given i.v. in a q4d×3w, as documented by the rate of complete tumor regression without evidence of tumor regrowth at the end of the experiments (around 120 days). The outstanding Namitecan activity against pediatric sarcoma xenografts is emphasized by the observation that complete tumor regression could be achieved also by the dose of 15 mg/kg (i.e., ½ MTD) allowing the use of well-tolerated effective doses in clinical therapy. Moreover, the i.v. administration of Namitecan resulted in a high and persistent drug accumulation in TC-71 tumor xenograft, which could account for the good therapeutic index. Since no correlation could be found between in vitro growth inhibitory activity and in vivo antitumor efficacy, it is likely that other in vivo effects may contribute to the drug antitumor efficacy. Indeed, in the RD/TE671 rhabdomyosarcoma model, we found that Namitecan activity was associated with a marked antiangiogenic effect, which was consistent with the down regulation of proangiogenic factors, including VEGF, bFGF and CCL-2, an angiogenesis chemokine mediator. Furthermore, we found that HIF-1α expression is strongly down regulated in our cellular models following treatment with Namitecan. These findings are in line with a supposed antiangiogenic activity of camptothecins likely to affect proliferation and migration of endothelial cells and down regulation of angiogenic factors and the known ability of Topoisomerase I inhibitors to inhibit the translation of HIF-1α protein which is a transcriptional activator of VEGF. In conclusion, the available evidence supports that inhibition of angiogenesis contributes to the responsiveness of pediatric sarcomas to Namitecan, according to the role of angiogenesis in malignant behavior of these tumor types. Thus, the impressive efficacy of Namitecan against pediatric sarcoma may reflect: i) direct cytotoxic effects on tumor cells, as a result of persistent tumor accumulation of the drug and high topo I expression, ii) inhibition of angiogenesis and iii) a favorable in vivo drug distribution. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C206.

Abstract LB-129: Loss of Rab25 expression promotes tumor formation and angiogenesis in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies worldwide and is particularly prevalent in some areas of Asian countries. With the advent of next-generation sequencing technologies, it is now possible for us to integrate and analyze large amounts of data at genome-wide scale, with the goal for a better understanding of cellular function and disease development. In order to elucidate the molecular mechanisms that drive tumorigenesis in ESCC, we employed a high-throughput sequencing technology named RNA-Seq (RNA sequencing) to identify dysregulated gene expression in 14 patient-derived ESCC or adjacent non-tumor (NT) clinical specimens of Chinese origin. The expression of Rab25, a family member of Ras-associated binding GTPases, was found to be downregulated in tumour tissues significantly. By both qPCR and immunohistochemistry analysis on a tissue microarray, expression of Rab25 was found to be significantly decreased in a larger cohort of ESCC tumors compared with NT counterparts. Patients with higher levels of Rab25 expression were also significantly associated with a better overall survival. Consistently, absent or downregulated Rab25 expression was also detected in all ESCC cell lines examined as compared to a pooled normal tissue control. Rab25 expression in ESCC cell lines could be restored by demethylation treatment with 5-aza-dC, suggesting Rab25 is silenced via epigenetic alterations. Rab25 functions as an important mediator of vesicle trafficking, regulating cell polarity and distribution of membrane proteins, which are important for the maintenance of normal epithelial lining of the esophagus. We hypothesized that loss of Rab25 expression could lead to dysregulation in the maintenance of this normal epithelium and thus result in tumor development. The tumor suppressive function of Rab25 and the tumorigenic effect of its loss were evaluated by lentiviral-based overexpression and shRNA-knockdown of Rab25 in ESCC cell lines. In vivo studies found Rab25 to suppress tumor formation and angiogenesis in nude mice. Rab25-knockdwon cells gave rise to significantly more and larger tumors as compared to negative control cells. Immunohistochemical staining of resected xenografts revealed an increase in microvessel density and tumor proliferation as shown by higher expressions of endothelial marker CD34 and proliferation marker PCNA, respectively, in Rab25-knockdown cells as compared to negative controls. Similarly, conditioned medium from Rab25-knockdown cells enhanced tube-forming capability of HUVECs, suggesting potential regulation of angiogenic factors by Rab25-mediated pathway. Work on the identification of potential angiogenic factors regulated by Rab25 is now currently underway using a human angiogenesis antibody array. In summary, Rab25 functions as an important tumor suppressor gene in ESCC development by regulating angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-129. doi:10.1158/1538-7445.AM2011-LB-129

Abstract LB-58: Angiogenic signaling is upregulated in human oral squamous carcinoma cells with acquired resistance to the EGFR inhibitor gefitinib

Abstract Tyrosine kinase inhibitors (TKIs) have improved the therapeutic options for patients with EGFR-driven tumors; however initially responsive patients frequently relapse whilst on therapy. To investigate acquired resistance to EGFR TKIs we developed and characterized in vitro and in vivo models of gefitinib-resistance through chronic drug exposure of a squamous carcinoma cell line, CAL-27. The derived CALR subline showed 166-fold resistance to gefitinib compared with CALS cells (drug-sensitive controls). Constitutive levels of EGFR were unchanged but phospho-EGFR was reduced and phospho-AKT increased in CALR cells. No upregulation of MET or IGF-1R signalling pathways, previously reported in acquired resistance to EGFR TKIs, was seen. The growth rate and vascularisation of CALR xenografts was significantly greater than CALS, although in vitro proliferation rates were similar, observations recapitulated in a second, independently-derived gefitinib-resistant cell line. We thus hypothesised that the differential in vivo growth may be attributed to differences in angiogenic signalling between gefitinib-sensitive and -resistant tumors. To test this hypothesis we examined the expression of HIF1α (hypoxia-inducible factor 1α) a key mediator of tumor angiogenesis through transcriptional regulation of angiogenic factors, most notably VEGF-A. Here we present the novel finding that HIF1α is constitutively upregulated in gefitinib-resistant CALR cells in normoxia in vitro, and in xenograft tumors. This is accompanied by a concomitant increase in constitutive VEGF-A secretion from the resistant cells determined by ELISA, demonstrating that HIF1α is transcriptionally active. Multiplex antibody arrays (Proteome Profiler™, R&amp;D Systems) were used to assess the expression of 55 angiogenesis-related proteins in gefitinib-sensitive and -resistant cells. Membranes spotted with capture antibodies were incubated with lysates or mouse sera and signals from a chemiluminescent substrate were quantified by densitometry using ImageQuant software (Nonlinear Dynamics). We identified 22 angiogenic proteins differentially expressed between CALS and CALR cells in vitro, several of which are transcriptionally regulated by HIF1α. These include pro-angiogenic factors upregulated in the CALR cells such as VEGF-A (confirming ELISA data) whilst a number of anti-angiogenic proteins were down-regulated, two of which were independently identified in xenograft-bearing mouse sera. In summary, using our model of gefitinib-resistant squamous carcinoma we have identified HIF1α as a potential novel mediator of gefitinib resistance and, with the aid of novel antibody array technology, have identified angiogenesis-related molecules not previously reported to associate with EGFR TKI acquired resistance. Together these data support our hypothesis that increased signalling via the EGFR-HIF-AKT axis is associated with the acquisition of resistance to gefitinib in oral squamous carcinoma. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-58.

Oxygen-induced retinopathy: a model for vascular pathology in the retina

Ischaemic vascular disease in the retina may either leave retina permanently ischaemic with slow degradation of vision, or alternatively lead to proliferative vascular disease, which can also destroy vision. To investigate the molecular and cellular mechanisms that contribute to this pathology a mouse model has been studied extensively. The model is based on the exposure of mouse pups to hyperoxia during a phase when their retinal vasculature is still developing. This leads to capillary depletion, and upon return to room air, results in retinal ischaemia and proliferative vascular disease in the retinal vasculature (oxygen-induced retinopathy (OIR)). Numerous studies using this OIR model have revealed that the regulation of angiogenic factors and the influence of inflammatory cells play a pivotal role in the vascular pathogenesis. It has also been demonstrated in the OIR model that proliferative vascular disease is not the only possible outcome of ischaemia-induced angiogenesis in the retina, but that ischaemic areas in the retina can be revascularised with healthy blood vessels. Therefore, understanding the factors that control the balance between pathological and healthy angiogenesis in the OIR model may have important implications for human retinal ischaemic disease.