Inhibition Of Neovascularization Research Articles

Effect of sorafenib on des-γ-carboxyprothrombin secretion by a human hepatocellular carcinoma cell line.

Patients with hepatocellular carcinoma (HCC) who respond to sorafenib have been reported to exhibit an increase in the level of des-γ-carboxyprothrombin (DCP) in the blood, subsequent to the initiation of sorafenib treatment. In the present study, the levels of secretion of DCP and DCP with more γ-carboxyglutamic residues (NX-DCP) and the effects of hypoxic conditions were examined in 13 liver cancer cell lines, and the presence of vitamin K and sorafenib, in the KYN-2 cell line, which resulted in confirmed DCP and NX-DCP secretion. DCP, NX-DCP and prothrombin secretion were confirmed in 2/13 cell lines, KYN-2 and KIM-1. The level of secretions increased under hypoxic conditions. The addition of vitamin K suppressed cell proliferation, and DCP expression decreased to below detectable levels, however the level of prothrombin expression increased. Sorafenib treatment increased the level of apoptosis and suppressed cell proliferation, and decreased DCP and NX-DCP. In contrast, levels of prothrombin and vascular endothelial growth factor (VEGF) expression exhibited a slight increase. When the same experiment was conducted under hypoxic conditions, DCP secretion significantly decreased in the presence of sorafenib. The level of DCP secretion increased by several fold in the sorafenib-treated and non-treated cells compared with the normoxic conditions. Prothrombin and VEGF values with normoxic conditions remained almost similar with hypoxic conditions. Under hypoxic conditions, NX-DCP significantly decreased below the control values for the first 48 h subsequent to sorafenib treatment, but significantly increased at 72 h. In vivo experiments demonstrated that sorafenib inhibited angiogenesis and tumor proliferation, but the levels of DCP and NX-DCP did not differ significantly from the controls. These findings indicate that the suppression of neovascularization by sorafenib promotes blood vessel ischemia, producing hypoxic conditions whereby vitamin K uptake and utilization efficiency is reduced.

Protective Effect of Perindopril on Tumor Progression and Angiogenesis in Animal Model of Breast Cancer

Studies have shown that the renin angiotensin system via angiogenesis is involved in tumor development. Therefore, objective of the present study was to examine the effect of perindopril on tumor growth and angiogenesis in animal models of breast cancer. In the present study, the effect of perindopril on tumor development of mammary gland cancer induced by 7,12-dimethylbenz[a]anthracene, mouse tumor xenograft and corneal micropocket model has been investigated. Anti-angiogenic effect by chick yolk sac membrane assay has also been studied. In the present study, it has been found that perindopril produced a significant inhibition of tumor growth, in DMBA induced breast cancer. Treatment also produced significant suppression of cancer biomarkers such as lactate dehydrogenase, gamma glutamyl transferase and inflammatory markers such as C-reactive protein, erythrocyte sedimentation rate. Histopathological analysis also showed that perindopril was able to inhibit tumor development by the inhibition of hyperplastic lesions. Perindopril produced significant inhibition of tumor growth, in a mouse xenograft model and caused inhibition of neovascularization in the corneal micropocket model. In chick yolk sac membrane assay, perindopril showed inhibition of vascular growth and reduced blood vessel formation. Therefore, perindopril is widely used in clinical practice, may represent a neo-adjuvant therapy for treatment of breast cancer.

Triplet therapy with afatinib, cetuximab, and bevacizumab induces deep remission in lung cancer cells harboring EGFR T790M invivo.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have changed the treatment strategy for EGFR‐mutant lung cancers; however, resistance usually occurs due to a secondary mutation, T790M, in EGFR. Combination therapy using afatinib and cetuximab has had good results in lung tumors harboring EGFR T790M mutations in clinical trials. The effect of bevacizumab, an antivascular endothelial growth factor (VEGF) antibody, combined with EGFR‐TKIs has also been investigated. We hypothesized that the dose of afatinib and cetuximab could be reduced by combination with bevacizumab and that the triplet therapy may result in better tumor inhibition with tolerable toxicity. Using a xenograft mouse model with H1975‐harboring EGFRL 858R+T790M and RPC‐9‐harboring EGFR 19DEL+T790M, we tested the efficacy of the triplet therapy with a modified dose of afatinib, cetuximab, and bevacizumab, and compared this therapy to single and double therapies. Triplet therapy with afatinib, cetuximab, and bevacizumab induced pathological complete remission in xenograft tumors with H1975 and RPC‐9 cells versus tumors treated with single or double therapies. We saw no body weight loss in the mice. The triple therapy induced a significant reduction in CD31‐positive vascular endothelial cells and increased cleaved caspase‐3‐positive cells in the tumors. This suggests that one mechanism underlying the deep remission could be suppression of neovascularization and induction of apoptosis by intensive inhibition of driver oncoproteins and VEGF. These results highlight the potential of afatinib, cetuximab, and bevacizumab to induce deep remission in tumors harboring EGFRT 790M mutations. Therefore, clinical trials of this combination therapy are warranted.

Fibroblast growth factor-2 drives and maintains progressive corneal neovascularization following HSV-1 infection.

Herpes simplex virus type 1 (HSV-1) infection of the cornea induces VEGF-A-dependent lymphangiogenesis that continues to develop well beyond the resolution of infection. Inflammatory leukocytes infiltrate the cornea and have been implicated to be essential for corneal neovascularization, an important clinically relevant manifestation of stromal keratitis. Here, we report that cornea infiltrating leukocytes including neutrophils and T cells do not have a significant role in corneal neovascularization past virus clearance. Antibody mediated depletion of these cells did not impact lymphatic or blood vessel genesis. Multiple pro-angiogenic factors including IL-6, angiopoietin-2, HGF, FGF-2, VEGF-A, and MMP-9 were expressed within the cornea following virus clearance. A single bolus of dexamethasone (DEX) at day 10 pi resulted in suppression of blood vessel genesis and regression of lymphatic vessels at day 21 pi compared to control-treated mice. Whereas IL-6 neutralization had a modest impact on hemangiogenesis (day 14–21 pi) and lymphangiogenesis (day 21 pi) in a time-dependent fashion, neutralization of FGF-2 had a more pronounced effect on the suppression of neovascularization (blood and lymphatic vessels) in a time-dependent, leukocyte-independent manner. Furthermore, FGF-2 neutralization suppressed the expression of all pro-angiogenic factors measured and preserved visual acuity.

Antiangiogenic activity of a bevacizumab-loaded polyurethane device in animal neovascularization models

To evaluate the antiangiogenic activity of bevacizumab-loaded polyurethane using two animal models of neovascularization. The percentage of blood vessels was evaluated in a chicken chorioallantoic membrane model (n=42) and in the rabbit cornea (n=24) with neovascularization induced by alkali injury. In each model, the animals were randomly divided into the groups treated with the bevacizumab-loaded polyurethane device, phosphate-buffered-saline (negative control) and bevacizumab commercial solution (positive control). Clinical examination, as well as histopathological and immunohistochemical evaluation, were performed in the rabbit eyes. Microvascular density in hot spot areas was determined in semi-thin sections of corneal tissue by hematoxylin-eosin staining and factor VIII immunohistochemistry. Immunohistochemical analysis was also performed to evaluate VEGF expression. In the evaluated models, the use of bevacizumab (Avastin®) and the bevacizumab-loaded polyurethane device led to similar results with regard to inhibition of neovascularization. In the chorioallantoic membrane model, the bevacizumab-loaded polyurethane device reduced angiogenesis by 50.27% when compared to the negative control group. In the rabbit model of corneal neovascularization, the mean density of vessels/field was reduced by 46.87% on analysis of factor VIII immunohistochemistry photos in the bevacizumab-loaded polyurethane device group as compared to the negative control (PBS) sections. In both models, no significant difference could be identified between the bevacizumab-loaded polyurethane device and the positive control group, leading to similar results with regard to inhibition of neovascularization. The present study shows that the bevacizumab-loaded polyurethane device may release bevacizumab and inhibit neovascularization similarly to commercial bevacizumab solution in the short-term.

Abstract P6-12-17: H-ferritin allows nanometronomic treatment of breast cancer with doxorubicin preventing drug resistance and circumventing cardiotoxicity

Abstract Chemotherapeutic treatment of breast cancer is based on maximum tolerated dose (MTD) approach.1 However, advanced stage tumors are not effectively eradicated by MTD owing to suboptimal drug targeting, onset of therapeutic resistance and neoangiogenesis. In contrast, “metronomic” chemotherapy is based on frequent drug administrationsat lower doses, resulting in neovascularization inhibition and induction of tumor dormancy.1,2 However, several limiting factors remain for LDM in order to displace MTD treatments in clinical practice, including 1) low drug accumulation at tumor site,2 2) controversial effectiveness against chemoresistance in advanced metastatic cancers, and 3) acquired resistance after prolonged treatment. Recent advances in nanotechnology could offer groundbreaking solutions to improve the effectiveness of LDM chemotherapy, by taking advantage of the unique targeting efficiency of engineered nanocarriers.3 Here, we propose a new concept of “nanometronomic” chemotherapy, exploiting the H-ferritin (HFn)-mediated targeted delivery of doxorubicin (DOX)in an aggressive and metastatic breast cancer mouse model with DOX-inducible chemoresistance. HFn nanocages naturally target cancer cells4 owing to its affinity for transferrin receptor 1. HFn-DOX was recently demonstrated to overcome chemoresistance by actively promoting DOX nuclear translocation in vitro5,6 and was tested as a MTD treatment on a DOX-sensitive tumor model with encouraging results.7 We find that LDM administration of HFn-DOX strongly improves the antitumor potential of DOX chemotherapy arresting the tumor progression. Indeed, in vitro and in vivo results demonstrate that HFn nanocages mediate the nuclear delivery of DOX and increase DOX accumulation both in tumor tissue and in cancer cell nuclei, resulting in increased efficacy. Moreover, we find that HFn-DOX antitumor effect is attributable to multiple nanodrug actions beyond cell killing, including inhibition of tumor angiogenesis and avoidance of chemoresistance. Otherwise, although an even better reduction of tumor progression was achieved with liposomal DOX (pl-DOX) a five-fold increase in MDR-1-positive cells has been displayed, suggesting that liposomal DOX is not suitable in view of a protracted metronomic treatment, due to the onset of chemoresistance. Multiparametric assessment of heart tissues, including histology, ultrastructural analysis of tissue morphology, and measurement of markers of reactive oxygen species and hepatic/renal conditions, provided evidence that metronomic HFn-DOX allowed us to overcome cardiotoxicity contrary to what is observed with DOX and pl-DOX. Our results suggest that HFn-DOX has tremendous potential for the development of “nanometronomic” chemotherapy toward safe and tailored oncological treatments. 1. Kareva I, et al. Cancer Lett 2015; 358: 100. 2. Kerbel RS. Cancer Res Treat 2007; 39: 150. 3. Cruz-Munoz W, et al. Angiogenesis 2014; 7: 661. 4. Corsi F, Mazzucchelli S. Ther Deliv. 2016; 7: 149. 5. Bellini M, et al.. J Controlled Rel 2014;196: 184. 6. Zhang L, et al. Adv. Healthcare Mat. 2015; 4: 1305. 7. Liang M, et al. Proc Natl Acad Sci USA 2014; 111: 14900. Citation Format: Mazzucchelli S, Fiandra L, Bellini M, Truffi M, Rizzuto MA, Sorrentino L, Longhi E, Nebuloni M, Prosperi D, Corsi F. H-ferritin allows nanometronomic treatment of breast cancer with doxorubicin preventing drug resistance and circumventing cardiotoxicity [abstract]. In: Proceedings of the Thirty-Ninth Annual CTRC-AACR San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-17.

Nanometronomic treatment of 4T1 breast cancer with nanocaged doxorubicin prevents drug resistance and circumvents cardiotoxicity.

Chemotherapeutic treatment of breast cancer is based on maximum tolerated dose (MTD) approach. However, advanced stage tumors are not effectively eradicated by MTD owing to suboptimal drug targeting, onset of therapeutic resistance and neoangiogenesis. In contrast, “metronomic” chemotherapy is based on frequent drug administrations at lower doses, resulting in neovascularization inhibition and induction of tumor dormancy. Here we show the potential of H-ferritin (HFn)-mediated targeted nanodelivery of metronomic doxorubicin (DOX) in the setting of a highly aggressive and metastatic 4T1 breast cancer mouse model with DOX-inducible expression of chemoresistance. We find that HFn-DOX administered at repeated doses of 1.24 mg kg−1 strongly improves the antitumor potential of DOX chemotherapy arresting the tumor progression. We find that such a potent antitumor effect is attributable to multiple nanodrug actions beyond cell killing, including inhibition of tumor angiogenesis and avoidance of chemoresistance. Multiparametric assessment of heart tissues, including histology, ultrastructural analysis of tissue morphology, and measurement of markers of reactive oxygen species and hepatic/renal conditions, provided evidence that metronomic HFn-DOX allowed us to overcome cardiotoxicity. Our results suggest that HFn-DOX has tremendous potential for the development of “nanometronomic” chemotherapy toward safe and tailored oncological treatments.

Initiation of Acute Graft-Versus-Host Disease By Angiogenesis

Angiogenesis and inflammation are two closely related processes and inhibition of angiogenesis can ameliorate inflammatory diseases by reducing the recruitment of tissue infiltrating leukocytes. However, there is limited evidence on initial mechanisms of both processes and it is unknown if angiogenesis contributes to initiation of inflammation or is a mere consequence of inflammation.Recent research showed that graft-versus-host disease (GVHD) is associated to angiogenesis making the endothelium a potential new therapeutic target during allogeneic stem cell transplantation (bone marrow transplantation, allo-BMT).We studied kinetics and quantity of angiogenesis and leukocyte infiltration in murine acute GVHD models (LP/J→B6, B6→Balb/c, B6→BDF and 129S2/Sv→B6) by assessing CD31, CD4, CD8, CD11b by immune staining and FACS analysis. In GVHD target organs colon, skin and liver, we found significant increased vascular density and endothelial cell (EC) number but no leukocyte infiltration as early as day+2 after BMT (Fig. 1a,b). Leukocyte infiltration occurred secondary to angiogenesis at day+7 or later. No angiogenesis and inflammation occurred in the non-acute GVHD target organs joints, cardiac and skeletal muscle (Fig. 1c) suggesting a previously unrecognized role of the endothelium in GVHD organ tropism. Similar analyses in a standard model for inflammatory bowel disease, the 3% dextran sulfate sodium-induced (DSS) colitis, confirmed that angiogenesis preceded leukocyte infiltration in colon (d+1 vs. d+3) implicating that the significance of our results surpasses the field of transplantation biology.We aimed at identifying pathways that are relevant for initiation of angiogenesis during GVHD and first investigated the Vegfa/VEGFR1+2 axis. Based on our previous observation demonstrating a reduction of GVHD due to inhibition of neovascularization by anti-VE-cadherin antibodies (Penack et al, JNCI 2010), we performed therapeutic interventions with monoclonal blocking antibodies. Anti-VEGFR1+2 (DC101+MFI, Imclone) and anti-Vegfa (B20-4.1.1, Genentech) did not lead to GVHD reduction (Fig. 1d). Furthermore, we found no consistent upregulation of Vegfa and VEGFR2 expression levels in GVHD target organs during initiation of GVHD or later time points (Fig. 1e) suggesting that the Vefga/VEGFR2 pathway is not a major mechanism for initiation of angiogenesis in GVHD target organs.We next assessed known endothelial activation signs and performed qPCR analysis of adhesion molecules Icam1, Vcam1, E- and P-selectin (Fif. 1f) as well as immune staining of MHC-II (Fig. 1g). We confirmed previous knowledge that endothelial cells upregulate adhesion molecules and MHC-II during established GVHD. In sharp contrast, adhesion molecules and MHC-II were downregulated or not changed during the initiation of angiogenesis at day+2. This suggests that alternative pathways are important during initial angiogenesis in early GVHD.To identify alternative pathways during initial angiogenesis in an unbiased approach, we performed microarray analyses of FACS-sorted colon ECs (Fig. 2a) and LC-MS/MS proteome analyses of MACS-isolated liver ECs (Fig. 2c) at day+2 after allo-BMT vs. syn-BMT. Strikingly, we detected substantial metabolic and cytoskeletal changes in ECs of allo-BMT recipients (Fig. 2b,d). Recently, metabolic processes have been shown to be associated with cytoskeletal remodeling during EC migration and angiogenesis (Uebelhoer et al., J Vasc Res 2016). Accordingly, measuring single-cell mechanical characteristics with Real-Time deformabiliy cytometry (Otto et al., Nature Methods 2015) revealed profoundly higher deformation (D = 0.0547 ± 0.0011) and overall softening of ECs from allo-BMT recipients (Fig. 2e) indicating to enhanced EC migration and proliferation.We demonstrate that angiogenesis initiates GVHD in target organs and is restricted to target organs. Our findings implicate that endothelial biology plays a major role in GVHD organ tropism and disease development. We revealed novel genes and proteins regulating migration and proliferation of EC in initial angiogenesis during GVHD. Our study amends the knowledge on the interplay between the vasculature and inflammation opening a new window to develop therapeutic strategies targeting the endothelium. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The therapeutic effect of bevacizumab on plaque neovascularization in a rabbit model of atherosclerosis during contrast-enhanced ultrasonography.

The purpose of the study was to assess the therapeutic effect of the angiogenesis inhibitor bevacizumab on plaques of various stages in rabbit models using contrast-enhanced ultrasonography (CEUS). Abdominal aortic atherosclerosis was induced in 55 rabbits. Thirty-six randomly selected rabbits were divided into 2 groups according to the timing of the bevacizumab injection: an early-stage plaque group (Group ESP) and a later-stage plaque group (Group LSP). The remainder were considered the control group. Standard ultrasonography and CEUS imaging of the abdominal aorta were performed. The animals were euthanized after CEUS, and plaque specimens were harvested for histological staining of CD31. The control group exhibited a substantially higher enhanced intensity, a higher ratio of enhanced intensity in the plaque to that in the lumen, and an increased number of CD31-positive microvessels in the plaque sections than Groups ESP and LSP (P < 0.05 for all). A higher enhanced intensity (P = 0.044), a higher ratio of enhanced intensity in the plaque to that in the lumen (P = 0.023) and more CD31-positive microvessels in the plaque sections (P = 0.006) were found in Group LSP than in Group ESP. Bevacizumab demonstrated more advanced inhibition of neovascularization in early-stage plaques in rabbits.

Abstract 4667: pCR induced by triplet therapy with low-dose afatinib, cetuximab and bevcizumab in lung cancer cells harboring EGFR T790M

Abstract Background. EGFR T790M plays a major role on the resistance for 1st or 2nd-generation EGFR-TKIs (e.g. erlotinib, afatinib) in lung tumors with activating EGFR mutations. 3rd generation EGFR-TKIs (e.g. AZD9291) show an excellent response in lung tumors with EGFR T790M, while the acquired resistance is inevitable (NEJM 2015). The combination of afatinib and cetuximab also induced significant response in the lung cancers in clinical trial (Cancer Discov. 2014). However, there is clearly still room for improvement in the combination therapy. Bevacizumab, a key drug in lung cancer treatment, is thought to have a wide variety of effects, including anti-vascularization and improved drug delivery. Thus, we hypothesized that the dosage of afatinib and cetuximab could be reduced by combining with bevacizumab, and that the triple therapy might produce better tumor inhibition with tolerable toxicity. Methods. Two lung cancer cell lines, H1975, with EGFR L858R and T790M, and RPC-9, with EGFR exon 19 deletion and T790M, were injected subcutaneously into nude mice as xenograft models. Mice were divided into four groups 10 days after tumor cell inoculation (vehicle, afatinib, afatinib + cetuximab, afatinib + cetuximab + bevcacizumab). Compared with previous reports (afatinib 25 mg/kg, 5 times/week, cetuximab 1 mg/mouse, every 3 days; J. Clin. Invest. 2009), the low dose of afatinib (10 mg/kg, 5 times/week; i.e., 60% less drug) or cetuximab (0.1 mg/mouse, once/week; i.e., 90% less drug) were administered to the mice for 1 month. Bevacizumab was injected at 2 mg/kg twice/week. Following the therapies, the mice were observed for 1 month without treatment. Results. Surprisingly, the triple therapy induced a pathological complete response (pCR) in H1975 and RPC-9 cell xenograft tumors; in contrast, the tumors treated with single or double therapy were inhibited only partially. AZD9291 monotherapy did not reproduce pCR. Furthermore pCR was maintained during the observation period in the triple therapy group. There was no body weight loss in any group. Frozen tumors were obtained from RPC-9 xenograft models after 1 week treatment with each regimen. Expression levels of pEGFR, pAKT, and pERK were decreased in the triple therapy group. CD31-positive blood vessels and Ki-67-positive cells in tumors with triple therapy were reduced significantly versus the tumors in other groups. Cleaved caspase-3 expression in the tumors with triple therapy was positive in a higher number of tumor cells than in the other groups. There were no difference of afatinib concentration among the tumors. Conclusions. We demonstrated that a low-dose of afatinib and cetuximab combined with bevacizumab induced pCR in EGFR T790M mutated cell xenograft tumors with no obvious adverse events. We suggest that the suppression of neovascularization and induction of apoptosis may play important roles in the triple therapy. This project is supported by KAKEN(No26893155) Citation Format: Kadoaki Ohashi, Kenichiro Kudo, Eiki Ichihara, Hiroe Kayatani, Hisao Higo, Yuka Kato, Yasuko Kurata, Daisuke Minami, Takashi Ninomiya, Toshio Kubo, Katsuyuki Hotta, Mitsune Tanimoto, Katsuyuki Kiura. pCR induced by triplet therapy with low-dose afatinib, cetuximab and bevcizumab in lung cancer cells harboring EGFR T790M. [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 4667.

Antiangiogenic and Antitumor Activities of Aflibercept, a Soluble VEGF Receptor-1 and -2, in a Mouse Model of Hepatocellular Carcinoma

BACKGROUND & AIM: Aflibercept known as ziv-aflibercept in the United States is a soluble decoy receptor of both vascular endothelial growth factor (VEGF) receptor-1 and -2 known to inhibit the binding of VEGF and placental growth factor (PlGF) to VEGF receptor-1 and -2. Here, we analyzed the mechanisms of the antitumor effects of aflibercept in mouse hepatoma models. METHODS: In in vitro studies, we determined the effects of aflibercept on human umbilical vein cell (HUVEC) proliferation and bone marrow (BM) cell differentiation to endothelial progenitor cells (EPCs). In in vivo experiments, aflibercept was injected intraperitoneally in hepatoma cell tumor-bearing mice, and its inhibitory effects on tumor growth and BM cell migration to tumor tissues were evaluated. RESULTS: Aflibercept suppressed phosphorylation of VEGF receptor-1 and -2 in HUVEC and dose-dependently inhibited VEGF-induced HUVEC proliferation. It suppressed the differentiation of BM cells to EPCs and migration of BM cells to tumor tissues. It also suppressed tumor growth and prolonged survival time of tumor-bearing mice without side effects. In tumor tissues, aflibercept upregulated the expression of hypoxia inducible factor1-α, VEGF, PlGF, fibroblast growth factor-2, platelet derived growth factor-BB, and transforming growth factor-α and reduced microvascular density. It also reduced sinusoidal density in noncancerous liver tissues. CONCLUSIONS: Our results demonstrated potent antitumor activity for aflibercept in a mouse model of hepatocellular carcinoma. These effects were mediated through inhibition of neovascularization, caused by inhibition of endothelial cell proliferation, EPC differentiation, and BM cell migration to tumor tissues.

Intraperitoneal Perfusion Therapy of Endostar Combined with Platinum Chemotherapy for Malignant Serous Effusions: A Meta-analysis.

Malignant serous effusions (MSE) are one complication in patients with advanced cancer. Endostar is a new anti-tumor drug targeting vessels which exerts potent inhibition of neovascularization. This study aimed to systematically evaluate the efficacy and safety of intraperitoneal perfusion therapy of Endostar combined with platinum chemotherapy for malignant serous effusions (MSE). Randomized controlled trials (RCTs) on intraperitoneal perfusion therapy of Endostar combined with platinum chemotherapy for malignant serous effusions were searched in the electronic data of PubMed, EMBASE, Web of Science, CNKI, VIP, CBM and WanFang. The quality of RCTs was evaluated by two independent researchers and a meta-analysis was performed using RevMan 5.3 software. The total of 25 RCTs included in the meta-analysis covered 1,253 patients, and all literature quality was evaluated as "B" grade. The meta-analysis showed that Endostar combined with platinum had an advantage over platinum alone in terms of response rate of effusions (76% vs 48%, RR=1.63, 95%CI: 1.50-1.78, P<0.00001) and improvement rate in quality of life (69% vs 44%, RR=1.57, 95%CI: 1.42-1.74, P<0.00001). As for safety, there was no significant difference between the two groups in the incidences of nausea and vomiting (35% vs 34%, RR=1.01, 95%CI: 0.87-1.18, P=0.88), leucopenia (38% vs 38%, RR=1, 95%CI: 0.87-1.15, P=0.99), and renal impairment (18% vs 20%, RR=0.86, 95%CI: 0.43-1.74, P=0.68). Endostar combined with platinum by intraperitoneal perfusion is effective for malignant serous effusions, and patient quality of life is significantly improved without the incidence of adverse reactions being obviously increased.

Mechanisms of corneal neovascularization and modern options for its suppression

Quite a number of pathological factors exist that can disturb the balance between anti-angiogenic and proangiogenic mechanisms, thus causing vascularization of the cornea. The neovessels are immature, ill-formed, and show increased permeability, which is dangerous of corneal edema, lipid deposition, and opacification. Moreover, as known, corneal neovascularization (CNV; preexisting or postoperative) may contribute to immune response against the transplant. Suppression of neovascularization is able to decrease the risk of corneal transplant rejection. In order to identify the principal strategy for struggling against CNV, we should first get a better understanding of its etiology, pathogenesis, and role in transplant immunity as well as mechanisms of action of available treatment methods.

Association of TIMP3 expression with vessel density, macrophage infiltration and prognosis in human malignant melanoma

AimsSeveral anti-tumour properties have been ascribed to the tissue inhibitor of matrix metalloproteinases-3 (TIMP3) gene, including inhibition of neovascularisation in tumour xenografts. Reduced protein expression has been linked to promoter hypermethylation and allelic loss of heterozygosity in various human malignancies. In melanoma-positive lymph nodes from patients, we evaluated the association between TIMP3 expression, vessel density, macrophage infiltration and potential correlations with disease-free survival (DFS) and overall survival (OS). Patients and methodsTIMP3 expression was analysed by immunohistochemistry (IHC) in melanoma lymph node biopsies of stage III melanoma patients (n = 43). Blood vessel density and macrophage infiltration were quantitatively assessed and correlation with TIMP3 expression was investigated. Methylation status of the gene promoter was determined using methylation-specific polymerase chain reaction (MSP). Protein expression and promoter methylation status were investigated for associations with DFS and OS. ResultsReduced expression of TIMP3, as determined by IHC, was observed in 74% of the cases (32 in 43). A significant inverse correlation was observed between TIMP3 expression and vessel density (p = 0.031). Correlation between TIMP3 expression and macrophage infiltration was not statistically significant (p = 0.369). MSP analysis revealed methylation of the gene promoter in 18% (7 in 38) of the analysed cases. No differences in OS and DFS were observed between cases with high and low TIMP3 expression. Gene promoter methylation was significantly associated with both poor 5-year DFS (p = 0.024) and OS (p = 0.034). ConclusionsOur data indicate that TIMP3 is a dominant negative regulator of angiogenesis in cutaneous melanoma and gene silencing by promoter methylation is associated with poor outcome.

Curcumin Inhibits Non-Small Cell Lung Cancer Cells Metastasis through the Adiponectin/NF-κb/MMPs Signaling Pathway.

Adipose tissue is now considered as an endocrine organ involved in metabolic and inflammatory reactions. Adiponectin, a 244–amino acid peptide hormone, is associated with insulin resistance and carcinogenesis. Curcumin (diferuloylmethane) is the principal curcuminoid of the popular Indian spice, turmeric. Curcumin possesses antitumor effects, including the inhibition of neovascularization and regulation of cell cycle and apoptosis. However, the effects of adiponectin and curcumin on non-small cell lung cancer (NSCLC) remain unclear. In this study, we evaluated the expression of adiponectin in paired tumors and normal lung tissues from 77 patients with NSCLC using real-time polymerase chain reaction, western blotting, and immunohistochemistry. Kaplan–Meier survival analysis showed that patients with low adiponectin expression ratio (<1) had significantly longer survival time than those with high expression ratio (>1) (p = 0.015). Curcumin inhibited the migratory and invasive ability of A549 cells via the inhibition of adiponectin expression by blocking the adiponectin receptor 1. Curcumin treatment also inhibited the in vivo tumor growth of A549 cells and adiponectin expression. These results suggest that adiponectin can be a prognostic indicator of NSCLC. The effect of curcumin in decreasing the migratory and invasive ability of A549 cells by inhibiting adiponectin expression is probably mediated through NF-κB/MMP pathways. Curcumin could be an important potential adjuvant therapeutic agent for lung cancer in the future.

Stem Cell Therapy for Corneal Regeneration Medicine and Contemporary Nanomedicine for Corneal Disorders

The ocular surface is the outermost part of the visual system that faces many extrinsic or intrinsic threats, such as chemical burn, infectious pathogens, thermal injury, Stevens-Johnson syndrome, ocular pemphegoid, and other autoimmune diseases. The cornea plays an important role in conducting light into the eyes and protecting intraocular structures. Several ocular surface diseases will lead to the neovascularization or conjunctivalization of corneal epithelium, leaving opacified optical media. It is believed that some corneal limbal cells may present stem cell-like properties and are capable of regenerating corneal epithelium. Therefore, cultivation of limbal cells and reconstruction of the ocular surface with these limbal cell grafts have attracted tremendous interest in the past few years. Currently, stem cells are found to potentiate regenerative medicine by their capability of differentiation into multiple lineage cells. Among these, the most common cell sources for clinical use are embryonic, adult, and induced stem cells. Different stem cells have varied specific advantages and limitations for in vivo and in vitro expansion. Other than ocular surface diseases, culture and transplantation of corneal endothelial cells is another major issue for corneal decompensation and awaits further studies to find out comprehensive solutions dealing with nonregenerative corneal endothelium. Recently, studies of in vitro endothelium culture and ρ-associated kinase (ROCK) inhibitor have gained encouraging results. Some clinical trials have already been finished and achieved remarkable vision recovery. Finally, nanotechnology has shown great improvement in ocular drug delivery systems during the past two decades. Strategies to reconstruct the ocular surface could combine with nanoparticles to facilitate wound healing, drug delivery, and even neovascularization inhibition. In this review article, we summarized the major advances of corneal limbal stem cells, limbal stem cell deficiency, corneal endothelial cell culture/transplantation, and application of nanotechnology on ocular surface reconstruction. We also illustrated potential applications of current knowledge for the future treatment of ocular surface diseases.

Abstract 1368: The impact of bevacizumab on combination low-dose afatinib and cetuximab therapy in lung cancer cells harboring activated EGFR mutations

Abstract Background. The combination of afatinib and cetuximab induced an unprecedented response in lung tumors harboring EGFR T790M in clinical trials (Cancer Discov. 2014). However, up to 20% of patients discontinued treatment due to adverse events, including skin rashes and diarrhea, and the median progression-free survival of the combination therapy was 4.7 months. There is clearly still room for improvement in the therapy. Bevacizumab, a key drug in lung cancer treatment, is thought to have a wide variety of effects, including anti-vascularization, normalization of tumor vasculature, and improved drug delivery. Thus, we hypothesized that the dosage of afatinib and cetuximab could be reduced by combining with bevacizumab, and that the triple therapy might produce better tumor inhibition with tolerable toxicity. Methods. Two lung cancer cell lines, H1975, with EGFR L858R and T790M, and RPC-9, with EGFR exon 19 deletion and T790M, were injected subcutaneously into nude mice as xenograft models. Mice were divided into four groups 10 days after tumor cell inoculation (vehicle, afatinib, afatinib + cetuximab, afatinib + cetuximab + bevcacizumab). Compared with previous reports (afatinib 25 mg/kg, 5 times/week, cetuximab 1 mg/mouse, every 3 days; J. Clin. Invest. 2009), the low dose of afatinib (10 mg/kg, 5 times/week; i.e., 60% less drug) or cetuximab (0.1 mg/mouse, once/week; i.e., 90% less drug) were administered to the mice for 1 month. Bevacizumab was injected at 2 mg/kg twice/week. Following the therapies, the mice were observed for 1 month without treatment. Results. Surprisingly, the triple therapy induced a pathological complete response (pCR) in H1975 and RPC-9 cell xenograft tumors; in contrast, the tumors treated with single or double therapy were inhibited only partially. Furthermore pCR was maintained during the observation period in the triple therapy group. There was no body weight loss in any group. Frozen tumors were obtained from RPC-9 xenograft models after 1 week treatment with each regimen. Expression levels of pEGFR, pAKT, and pERK were decreased in the triple therapy group. CD31-positive blood vessels and Ki-67-positive cells in tumors with triple therapy were reduced significantly versus the tumors in other groups. Cleaved caspase-3 expression in the tumors with triple therapy was positive in a higher number of tumor cells than in the other groups. Conclusions. We demonstrated that a low-dose of afatinib and cetuximab combined with bevacizumab induced pCR in EGFR T790M mutated cell xenograft tumors with no obvious adverse events. We suggest that the suppression of neovascularization and induction of apoptosis may play important roles in the triple therapy. Note: This abstract was not presented at the meeting. Citation Format: Kenichiro Kudo, Kadoaki Ohashi, Eiki Ichihara, Daisuke Minami, Hisao Kubo, Akiko Sato, Yuka Kato, Hideko Isozaki, Hiroe Kayatani, Tomoki Tamura, Mitsune Tanimoto, Katsuyuki Kiura. The impact of bevacizumab on combination low-dose afatinib and cetuximab therapy in lung cancer cells harboring activated EGFR mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1368. doi:10.1158/1538-7445.AM2015-1368

Dietary ω-3 polyunsaturated fatty acids decrease retinal neovascularization by adipose–endoplasmic reticulum stress reduction to increase adiponectin

Retinopathy of prematurity (ROP) is a vision-threatening disease in premature infants. Serum adiponectin (APN) concentrations positively correlate with postnatal growth and gestational age, important risk factors for ROP development. Dietary ω-3 (n-3) long-chain polyunsaturated fatty acids (ω-3 LCPUFAs) suppress ROP and oxygen-induced retinopathy (OIR) in a mouse model of human ROP, but the mechanism is not fully understood. We examined the role of APN in ROP development and whether circulating APN concentrations are increased by dietary ω-3 LCPUFAs to mediate the protective effect in ROP. Serum APN concentrations were correlated with ROP development and serum ω-3 LCPUFA concentrations in preterm infants. Mouse OIR was then used to determine whether ω-3 LCPUFA supplementation increases serum APN concentrations, which then suppress retinopathy. We found that in preterm infants, low serum APN concentrations positively correlate with ROP, and serum APN concentrations positively correlate with serum ω-3 LCPUFA concentrations. In mouse OIR, serum total APN and bioactive high-molecular-weight APN concentrations are increased by ω-3 LCPUFA feed. White adipose tissue, where APN is produced and assembled in the endoplasmic reticulum, is the major source of serum APN. In mouse OIR, adipose endoplasmic reticulum stress is increased, and APN production is suppressed. ω-3 LCPUFA feed in mice increases APN production by reducing adipose endoplasmic reticulum stress markers. Dietary ω-3 LCPUFA suppression of neovascularization is reduced from 70% to 10% with APN deficiency. APN receptors localize in the retina, particularly to pathologic neovessels. Our findings suggest that increasing APN by ω-3 LCPUFA supplementation in total parental nutrition for preterm infants may suppress ROP.

Quantitative comparison of mRNA level of corticotropin- releasing factor peptide family in normal kidney and corresponding clear cell renal cell carcinoma specimens.

466 Background: Urocortin (Ucn), a 40 amino acid peptide, belongs to the corticotropin releasing factor (CRF) family and exerts its actions mainly in the periphery through activation of two CRF receptors (CRFRs), CRFR1 and CRFR2 and a binding protein (CRFBP/CRHBP). Both receptors are G-protein coupled and binding of Ucn leads to activation of cAMP-dependent protein kinases via elevation of cAMP levels. Ucn and Urocortin III (UcnIII) are involved in conditions such as regulation of local inflammation, angiogenesis, and inhibition of proliferation. Suppression of neovascularization and inhibition of tumor cell cycling by Urocortins is modulated through CRFRs. Activation of CRFRs by e.g. Ucn inhibits angiogenesis via reduction of vascular endothelial growth factor (VEGF) and suppresses the cell proliferation. Here we characterized the whole CRF family on mRNA levels quantitatively comparing the normal and clear cell carcinoma of the kidney (ccRCC). Methods: In this study we measured the mRNA level of Ucn, UcnIII, CRFR1, CRFR2 and CRHBP in 78 RCC samples and paired histologically normal appearing tissues using quantitative PCR. Statistical analyses were carried out using univariate logistic regression analysis. Results: We found tumour specific down regulation of mRNA expression of UcnIII, CRFR1, CRFR2 and CRHBP in samples of RCCs with clear cell histology compared to paired normal tissues (P&lt;0.01 for all targets). Only Ucn did not show any expression difference between two groups (p=0.17). Conclusions: For the first time we showed the expression profile of the whole CRF peptide family in ccRCC compared to normal kidney on mRNA level. Our data underlines the malfunction of this actually strong protective and anticancer system in renal malignancy and shows the involvement of CRF system in carcinogenesis in kidney. More studies are necessary to find out the detailed roles of the CRF system in renal carcinoma.

A novel approach to port-wine stains.

ORIGINAL ARTICLE, p 669 Port‐wine stains (PWSs) are capillary vascular malformations that occur in 3 per 1000 live births. Selective photothermolysis of PWS blood vessels with the pulsed‐dye laser (PDL) is considered the gold standard of treatment. PDL is very effective at PWS clearance, with a low incidence of side‐effects.1 However, multiple treatments are required in part due to the body's normal wound healing response, which results in revascularization and regeneration of PWS blood vessels following laser exposure.2 PDL‐induced angiogenesis makes it very difficult to achieve complete clearance of PWSs. This has prompted investigation into combination therapy with antiangiogenic agents after laser irradiation to modulate the neovascularization skin response to laser damage and enhance the effects of laser therapy. Rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR), was initially investigated as a potential adjuvant therapy after laser irradiation. Rapamycin is thought to have antiangiogenic properties through downregulation of hypoxia‐inducible factor (HIF)‐1α, which leads to a decrease in vascular endothelial growth factor (VEGF) production and reduction in the stimulation of VEGF by vascular endothelial cells.3 4 Both systemic and topical formulations of rapamycin have shown promising inhibition of neovascularization in the skin after laser treatment.5 6 Topical formulations are preferable in order to obviate systemic side‐effects, assuming that an appropriate depth of penetration is achievable. Initial studies with rapamycin have led to the investigation of other antiangiogenic agents such as imiquimod, which activates toll‐like receptor 7 leading to activation of antiangiogenic cytokines and reduction of angiogenic stimulators.7