Endogenous Angiogenic Inhibitor Research Articles

BIOMEDICAL APPLICATION OF K5 PLASMINOGEN FRAGMENT

Aim. Plasminogen kringle 5 is an endogenous angiogenic inhibitor. The purpose of the present review was to highlight the potential biomedical application of kringle 5 in the regulation of angiogenesis and tumor growth. Methods. Angiogenesis is a complex process that involves endothelial cell proliferation, migration, basement membrane degradation, and neovessel organization. Since the uncontrolled growth of new blood vessels causes the progression of many common diseases, first of all, oncological diseases, autoimmune disorders, neovascular damage of the eye, the use of angiostatins can be a promising pharmacotherapeutic approach to the prevention and adjuvant therapy of these pathological conditions. The advantages of angiostatins application are their non-toxicity even at high doses, non-immunogenicity, lack of tolerance of target cells to their action. Angiostatins comprise a group of kringle-containing proteolytically-derived fragments of plasminogen/plasmin, which act as potent inhibitory mediators of endothelial proliferation and migration. Among all known angiostatin species, isolated K5 plasminogen fragment was shown to display the most potent inhibitory activity against proliferation of endothelial cells via triggering multiple signaling pathways, which lead to cell death and resulting angiogenesis suppression. Results. Current literature data suggest that in addition to expressed and highly specific cytotoxicity in relation to endotheliocytes and some types of tumor cells, the kringle domain 5 of human plasminogen has other advantages as an antiangiogenic and antitumor regulator, including its specific inhibitory activity, which affects only activated, proliferating endothelial cells, and therefore is non-toxic to other types of normal cells. As an endogenous protein, which is formed in the human organism, K5 does not provoke an immune response. K5 as a small polypeptide molecule with a stable structure can be obtained as a recombinant protein in E. coli cells, and can also be used in pharmacokinetic systems of targeted delivery and sustained release. Conclusions. The prospect of successful use of K5 as a therapeutic agent to manage pathological processes associated with dysregulation of angiogenesis makes it necessary to develop and improve methods of its production and to further test its plausible pleiotropic biological activities.

Thoracic perfusion of lobaplatin combined with endostar for treating malignant pleural effusions: A meta-analysis and systematic review.

Introduction:Lobaplatin is a new platinum-based cytotoxic chemotherapeutic agent. Endostar is an endogenous angiogenic inhibitor with implicated anti-tumor activity. This study was to investigate the efficacy and safety of thoracic perfusion of lobaplatin combined with endostar in the treatment of malignant pleural effusions (MPE).Methods:We searched the databases of Pubmed, the Cochrane Library, Embase, WanFang Data, and CNKI to select the studies regarding the efficacy and safety of lobaplatin combined with endostar to treat MPE. A total of 10[3–12] randomized controlled trials with 651 patients were included.Results:The objective response rate (P < .001, odds ratio = 4.08) and disease control rate (P < .001, odds ratio = 3.69) of lobaplatin combined with endostar were significantly higher than lobaplatin alone. In addition, lobaplatin combined with endostar remarkably promoted the quality of life of patients (P < .001, odds ratio = 3.93) compared with lobaplatin alone. Lobaplatin combined with endostar also promoted the quality of life of patients (P < .05, odds ratio = 2.56) compared with cisplatin combined with endostar. At the same time, the leukopenia rate (P < .05, odds ratio = .40) and the incidence of nausea and vomiting (P < .05, odds ratio = .38) of lobaplatin combined with endostar were significantly lower than that of cisplatin combined with endostar.Conclusions:The efficacy of lobaplatin combined with endostar was superior to lobaplatin alone. The safety was higher than cisplatin combined with endostar through thoracic perfusion in treating MPE, which indicated that lobaplatin combined with endostar could be the effective agent for controlling MPE.

Research progress of tumor angiogenesis inhibitors

Angiogenesis is one of the most important pathological characteristics in the development of tumor growth. Hence, antiangiogenesis is a hot topic in the field of cancer research. The current strategy for antiangiogenesis therapy of tumors is restoration of the angiogenic balance via either blockage of proangiogenic factors or application of angiogenic inhibitors. Endogenous angiogenic inhibitors show more promising prospects compared with proangiogenic factor antagonists. However, the underlying mechanisms of angiogenic inhibitors remain to be thoroughly elucidated. There are two types of endogenous angiogenic inhibitors. The first are hydrolyzed fragments of precursor proteins, such as plasminogen kringle 5 (K5), angiostatin/kringle 1–4, and endostatin, and the other are secreted proteins, such as pigment epithelium-derived factor (PEDF), kallikrein-binding protein (KBP/kallistatin), and antithrombin. Here we summarized research progress on the biological functions underlying mechanisms of tumor angiogenesis and application prospects of K5, PEDF, and KBP, so as to provide insights into the antiangiogenic therapies of tumor in the future.

Angio-3, a 10-residue peptide derived from human plasminogen kringle 3, suppresses tumor growth in mice via impeding both angiogenesis and vascular permeability.

Anti-angiogenesis therapy is an established therapeutic strategy for cancer. The endogenous angiogenic inhibitor angiostatin contains the first 3-4 kringle domains of plasminogen and inhibits both angiogenesis and vascular permeability. We present here a 10-residue peptide, Angio-3, derived from plasminogen kringle 3, which retains the functions of angiostatin in inhibiting both angiogenesis and vascular permeability. NMR studies indicate that Angio-3 holds a solution structure similar to the corresponding region of kringle 3. Mechanistically, Angio-3 inhibited both VEGF- and bFGF-induced angiogenesis by inhibiting EC proliferation and migration while inducing apoptosis. Inhibition of VEGF-induced vascular permeability results from its ability to impede VEGF-induced dissociation of adherens junction and tight junction proteins as well as the formation of actin stress fibers. When administered intravenously, Angio-3 inhibited subcutaneous breast cancer and melanoma growth by suppressing both tumor angiogenesis and intra-tumor vascular permeability. Hence, Angio-3 is a novel dual inhibitor of angiogenesis and vascular permeability. It is valuable as a lead peptide that can be further developed as therapeutics for diseases involving excessive angiogenesis and/or vascular permeability.

Kallistatin exerts anti-lymphangiogenic effects by inhibiting lymphatic endothelial cell proliferation, migration and tube formation.

Kallistatin has been recognized as an endogenous angiogenic inhibitor. However, its effects on lymphatic endothelial cells and lymphangiogenesis remain poorly understood. Lymphangiogenesis is involved in tumor metastasis via the lymphatic vasculature in various types of tumors. The aim of this study was to investigate the effects of kallistatin on lymphangiogenesis and the mechanism of action involved. Treatment with kallistatin recombinant protein or overexpression of kallistatin inhibited the proliferation, migration and tube formation of human lymphatic endothelial cells (hLECs), and induced apoptosis of hLECs. Furthermore, our results showed that the lymphatic vessel density (LVD) was reduced in lung and stomach sections from kallistatin-overexpressing transgenic mice. Treatment with kallistatin recombinant protein decreased the LVD in the implanted gastric xenograft tumors of nude mice. To the best of our knowledge, the present study is the first to demonstrate that kallistatin possesses anti-lymphangiogenic activity in vitro and in vivo. Moreover, kallistatin inhibited proliferation and migration of hLECs by reducing the phosphorylation of ERK and Akt, respectively. These findings suggested that kallistatin may be a promising agent that could be used to suppress cancer metastasis by inhibiting both angiogenesis and lymphangiogenesis.

Inhibition of integrin β3, a binding partner of kallistatin, leads to reduced viability, invasion and proliferation in NCI-H446 cells.

BackgroundKallistatin is a serine proteinase inhibitor and heparin-binding protein. It is considered an endogenous angiogenic inhibitor. In addition, multiple studies demonstrated that kallistatin directly inhibits cancer cell growth. However, the molecular mechanisms underlying these effects remain unclear.MethodsPull-down, immunoprecipitation, and immunoblotting were used for binding experiments. To elucidate the mechanisms, integrin β3 knockdown (siRNA) or blockage (antibody treatment) on the cell surface of small the cell lung cancer NCI-H446 cell line was used.ResultsInterestingly, kallistatin was capable of binding integrin β3 on the cell surface of NCI-H446 cells. Meanwhile, integrin β3 knockdown or blockage resulted in loss of antitumor activities induced by kallistatin. Furthermore, kallistatin suppressed tyrosine phosphorylation of integrin β3 and its downstream signaling pathways, including FAK/-Src, AKT and Erk/MAPK. Viability, proliferation and migration of NCI-H446 cells were inhibited by kallistatin, with Bcl-2 and Grb2 downregulation, and Bax, cleaved caspase-9 and caspase 3 upregulation.ConclusionsThese findings reveal a novel role for kallistatin in preventing small cell lung cancer growth and mobility, by direct interaction with integrin β3, leading to blockade of the related signaling pathway.

Thoracic perfusion of recombinant human endostatin (Endostar) combined with chemotherapeutic agents versus chemotherapeutic agents alone for treating malignant pleural effusions: a systematic evaluation and meta-analysis.

BackgroundEndostar is a new endogenous angiogenic inhibitor with implicated anti-tumor activity. This study was to investigate whether thoracic perfusion of Endostar could be used to control malignant pleural effusions (MPE).MethodsWe searched the databases of MEDLINE, Web of Science, EMBASE, Goggle, Cochrance Library and CNKI to select the studies regarding the efficacy of Endostar to treat MPE. A total of 13 randomised controlled trials (RCTs) with 1066 patients were included.ResultsThe overall response rate (ORR) (P < 0.001; odds ratio = 3.58) and disease control rate (DCR) (P < 0.001; odds ratio = 2.97) of Endostar combined with chemotherapeutic agents were significantly higher than those of chemotherapeutic agents alone. In addition, Endostar combined treatment remarkably promoted quality of life (QOL) of patients (P < 0.001; odds ratio = 3.04) compared with that of chemotherapeutic agents alone. Moreover, Endostar combined treatment did not have an impact on the incidence of adverse reactions (AEs) (P < 0.05).ConclusionsThe efficacy of Endostar combined chemotherapeutic agents was superior to chemotherapeutic agents alone through thoracic perfusion in treating MPE, which indicated that Endostar could be an effective agent for controlling MPE.

Abstract 3372: Semaphorin 3A normalizes the tumor vasculature and impairs tumor progression in a Nrp-1-independent manner

Abstract It is widely described that tumor vessel normalization, occurring in response to certain anti-angiogenic therapies, represents a remarkably advantageous anti-cancer strategy (1). We have demonstrated that Semaphorin 3A (Sema3A), an axon guidance cue part of class 3 semaphorins family, is an endogenous angiogenic inhibitor able to efficiently impair tumor progression, prolong the survival and normalize the tumor vasculature in different mouse models of spontaneous tumorigenesis (2). Moreover, we recently showed that Sema3A, by extending the normalization window and abrogating tumor hypoxia, overcame the resistance to the anti-angiogenic therapy inhibiting metastasis dissemination (3). Stemming from these findings we sought to investigate the molecular mechanisms of vessel normalization and metastasis inhibition induced by Sema3A. Interestingly, by confocal microscope and western blot analysis, in a co-culture systems of human endothelial cells (ECs) and pericytes grown in contact, we observed that Sema3A dramatically down-modulated its receptor Nrp-1 in both cell types, with the consequent over-expression of PDGF-B and Ang-1, known to promote vessel maturation. Moreover, a wide screening of different genes and pathways modulated in the ECs/pericyte co-cultures revealed that the most modulated was the HGF/Met pathway. In fact, we observed that c-Met phosphorylation was impaired in FACS-sorted ECs co-cultured with human pericytes, compared to ECs grown as single layer. To better investigate the specific role of Sema3A in modulating HGF/Met activation in vessels, we detected a strong inhibition of HGF-induced Met phosphorylation in Nrp-1 silenced ECs induced by Sema3A, suggesting that this semaphorin could directly interfere with Met signaling. Notably, Sema3A impaired HGF-induced Met phosphorylation, not only in ECs, but also in several Nrp-1-silenced gastric, lung and pancreatic tumor cell lines, inducing apoptosis and blocking the invasiveness. Finally, treating an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC) with adeno-associate virus (AAV)-8 expressing Sema3A, we observed a strong inhibition of tumor growth, a dramatic reduction of liver metastasis and a normalized and perfused tumor vessels phenotype. Remarkably, we found that Sema3A strongly and specifically inhibited Met activation in both tumor cells and vessels, in parallel to a down-modulation of Nrp-1. We conclude that Sema3A normalizes the tumor vasculature and blocks cancer progression in a Nrp-1-independent manner, in part by inhibiting HGF/Met pathway.

Abstract A16: Semaphorin 3A normalizes the tumor vasculature and impairs cancer progression in a Nrp-1-independent manner

Abstract It is widely described that tumor vessel normalization, occurring in response to certain anti-angiogenic therapies, represents a remarkably advantageous anti-cancer strategy (1). We have demonstrated that Semaphorin 3A (Sema3A), an axon guidance cue part of class 3 semaphorins family, is an endogenous angiogenic inhibitor able to efficiently impair tumor progression, prolong the survival and normalize the tumor vasculature in different mouse models of spontaneous tumorigenesis (2). Moreover, we recently showed that Sema3A, by extending the normalization window and abrogating tumor hypoxia, overcame the resistance to the anti-angiogenic therapy inhibiting metastasis dissemination (3). Stemming from these findings we sought to investigate the molecular mechanisms of vessel normalization and metastasis inhibition induced by Sema3A. Interestingly, by confocal microscope and western blot analysis, in a co-culture systems of human endothelial cells (ECs) and pericytes grown in contact, we observed that Sema3A dramatically down-modulated its receptor Nrp-1 in both cell types, with the consequent over-expression of PDGF-B and Ang-1, known to promote vessel maturation. Moreover, a wide screening of different genes and pathways modulated in the ECs/pericyte co-cultures, revealed that the most modulated was the HGF/Met pathway. In fact, we observed that c-Met phosphorylation was impaired in FACS-sorted ECs co-cultured with human pericytes, compared to ECs grown as single layer. To better investigate the specific role of Sema3A in modulating HGF/Met activation in vessels, we detected a strong inhibition of HGF-induced Met phosphorylation in Nrp-1 silenced ECs induced by Sema3A, suggesting that this semaphorin could directly interfere with Met signaling. Finally, treating an orthotopic mouse model of pancreatic ductal adenocarcinoma (PDAC) with adeno-associate virus (AAV)-8 expressing Sema3A, we observed a strong inhibition of tumor growth, a dramatic reduction of liver metastasis and a normalized and perfused tumor vessels phenotype. Remarkably, we found that Sema3A strongly and specifically inhibited Met activation in both tumor cells and vessels, in parallel to a down-modulation of Nrp-1 in normalized vasculature. We conclude that Sema3A normalizes the tumor vasculature and blocks cancer progression by acting on Nrp-1 and HGF/Met pathway.

The anticancer potential of thrombospondin-1 by inhibiting angiogenesis and stroma reaction during cervical carcinogenesis

Abstract Tumor growth is angiogenesis dependent. Angiogenic switch (the acquisition of an angiogenic phenotype) is essential for cervical carcinogenesis. Thrombospondin-1 (TSP-1) is an endogenous angiogenic inhibitor with multiple functional domains and interacting receptors. The disruption of TSP-1 fence (the expression in basal epithelia) occurred concordantly during the transition from low-grade squamous intraepithelial lesion into high-grade squamous intraepithelial lesion. This concordance suggests that TSP-1 plays a role in the regulation of angiogenic switch during cervical carcinogenesis. Tumor vasculature as a therapeutic target offers a paradigm shift for anticancer therapy. Endothelial cells do not appear to acquire resistance during antiangiogenic therapy. Low-and-frequent dose “metronic” chemotherapy is found to be antiangiogenic, which is more effective in targeting tumor endothelia than traditional large, single bolus doses. Meanwhile, the invasion process of cancers is associated with stroma reaction, which is characterized by fibroblasts' activation. In addition to the well-known angiogenesis inhibitor, TSP-1 also has a novel role of blocking activated fibroblasts (myofibroblasts) from invading cancer. Activated fibroblasts during stroma reaction could be used as an efficient drug delivery system to prevent or slow the local growth of cancer cells. Elucidation of the mechanism by which fibroblasts are recruited into cancer stroma could lead to new insights into not only the mechanisms of cancer progression but also strategies for cancer treatment. A better understanding of stromal contributions to cancer progression will likely result in the identification of new therapeutics targeting the stroma.

Abstract 190: The anti-cancer potential of thrombospondin-1 by inhibiting angiogenesis and stroma reaction during cervical carcinogenesis

Abstract Introduction: Thrombospondin-1 (TSP-1) is an endogenous angiogenic inhibitor with different biological functions on various cell types. The invasion process of cancers is associated with stroma reaction, which is characterized by fibroblasts activation. Our study was to investigate the role of TSP-1 in angiogenic switch and tumor stroma reaction, which is mainly characterized by fibroblast activation to create a permissive microenvironment for tumor progression. Material and Methods: Immunohistochemistry was used to check TSP-1 expression and the stroma makers for human surgical specimens. Subcutaneous tumors were generated by inoculating 107 SiHa-vector or SiHa-TSP-1 cells into SCID mice. Matrigel multi-cellular co-culture invasion assay was used to detect the inhibitory ability of TSP-1 on the invasive ability of activated fibroblasts (NIH-3T3) into tumor cells cluster. Results: The disruption of TSP-1 fence (the expression in basal epithelia) in human surgical specimens occurred concordantly during the transition from low grade squamous intraepithelial lesion (SIL) into high SIL. Meanwhile it was accompanied by an emergence in the upregulation of stroma markers, α-SMA and desmin. Transfection of SiHa cervical cancer cells with a plasmid expressing the TSP-1 exhibited anti-angiogenic activity in vitro, and resulted in reduced tumor growth in SCID mice. It was accompanied by a decrease in tumor vascularization and lower expressions of α-SMA and desmin, as compared with vector controls. Transfection with TSP-1 and purified TSP-1 added to NIH3T3 cells did not alter the protein levels of α-SMA and desmin, but significantly inhibited matrix metalloprotease-2 (MMP-2) activity. Transforming growth factor β (TGF-β), which is a major factor in the fibroblast activation, increased α-SMA and desmin expression, and the ability of cell migration and invasion in NIH3T3 cells. The increased migration ability and the invasive ability into tumor cluster of TGF-β-treated-NIH3T3 cells were dose-dependently inhibited by TSP-1. In contrast, transfection and exogenous addition of TSP-1 in SiHa cells has little effect on the invasive ability of the NIH3T3 cells. Discussion: Our findings suggest that TSP-1 play an inhibitory role in the angiogenic switch during cervical carcinogenesis. Meanwhile, TSP-1 can inhibit tumor stroma reaction that could be attributed to the blockage of activated fibroblasts from invading cancer cells. Our current study demonstrates that TSP-1 plays a broader role in tumor cell biology including the ability to block activated fibroblast (myofibroblasts) migration and invasion, in addition to its well-known anti-angiogenic effects. Citation Format: Ming-Ping Wu, Li-Wha Wu, Cheng-Yang Chou. The anti-cancer potential of thrombospondin-1 by inhibiting angiogenesis and stroma reaction during cervical carcinogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 190. doi:10.1158/1538-7445.AM2014-190

Recombinant Human Kallistatin Inhibits Angiogenesis by Blocking VEGF Signaling Pathway

Kallistatin has been recognized as an endogenous angiogenic inhibitor. However, the underlying molecular mechanism remains poorly understood. Taking it into account that vascular endothelial growth factor (VEGF) has been implicated in all aspects of normal and pathological vasculogenesis and angiogenesis. In this study, we investigated whether VEGF signaling pathway was impacted by the anti-angiogenic effect of recombinant human kallistatin (rhKal). We found that the rhKal inhibited proliferation as well as induced apoptosis of cultured human umbilical vein endothelial cells (HUVECs) in both concentration- and time-dependent manners. The rhKal also suppressed the VEGF-induced migration and tube formation of HUVECs. Furthermore, our data revealed that the rhKal suppressed the VEGF165-stimulated tyrosine phosphorylation of VEGFR-2 as well as its downstream signal molecular activation. The inhibition of receptor phosphorylation was correlated with a decrease in VEGF-triggered phosphorylation of angiogenesis signal molecules AKT and ERK, but not stress-related JNK. Taken together, these findings added the knowledge for us to understand the anti-angiogenic mechanism of kallistatin, which suggested that the rhKal could be worth as a candidate compound for further development for the purpose of anti-angiogenic therapies.

A Phase II, Randomized, Double-blind, Placebo-controlled Multicenter Trial of Endostar in Patients With Metastatic Melanoma

Endostatin is a potent endogenous angiogenic inhibitor with implicated antitumor activity. However, efficacy of recombinant human endostatin (rhES) in clinical trials is controversial, and application of rhES in treatment of metastatic melanoma awaits further investigations. This phase II trial evaluated the efficacy and safety of a soluble and stable rhES (Endostar) plus dacarbazine in patients with metastatic melanomas that contains no mutations in c-kit and BRAF genes. A total of 110 patients received placebo plus dacarbazine (250 mg/m², n = 54) or Endostar (7.5 mg/m²) plus dacarbazine (250 mg/m², n = 56). The primary end points were progression-free survival (PFS) and overall survival (OS). Median PFS in the Endostar plus dacarbazine arm was 4.5 months versus 1.5 months in the placebo plus dacarbazine arm (hazard ratio (HR) = 0.578; P = 0.013). There were statistically significant improvements in OS (median, 12.0 months versus 8.0 months; HR, 0.522; P = 0.005) in favor of the Endostar plus dacarbazine arm. The regimen was generally well tolerated and had a manageable toxicity profile. Our trial suggests that Endostar plus dacarbazine is well tolerated in patients with metastatic melanoma harboring no genetic mutations popular for targeted therapy and yields a significant improvement in PFS and OS.

Angiogenesis and hypoxia in the kidney

Loss of glomerular function associated with the presence of tubulointerstitial lesions, which are characterized by peritubular capillary loss, is a common finding in progressive renal disorders. Dysregulated expression of angiogenic factors (such as vascular endothelial growth factor [VEGF] and angiopoietins) and endogenous angiogenic inhibitors (such as thrombospondin-1, angiostatin and endostatin) underlie these conditions and negatively influence the balance between capillary formation and regression, resulting in capillary rarefaction. Recent studies have provided unequivocal evidence for a pathogenic role of tubulointerstitial hypoxia and the involvement of hypoxia-inducible transcription factors in the advanced stages of chronic kidney disease. The mainstay of potential angiogenic therapies is the application of angiogenic factors with the primary aim of ameliorating reduced oxygenation in the ischaemic tubulointerstitium. However, this strategy is strongly associated with inflammation and changes in vascular permeability. For example, supraphysiological expression of VEGF results in glomerular expansion and proteinuria, whereas VEGF blockade using neutralizing antibodies can cause hypertension and thrombotic microangiopathy. These effects highlight the importance of tight regulation of angiogenic factors and inhibitors. Novel therapeutic approaches that target vascular maturation and normalization are now being developed to protect kidneys from capillary rarefaction and hypoxic injury.

Abstract 3270: Combination strategy to circumvent the acquired resistance to VEGF receptor tyrosine kinase inhibitor in M24met melanoma in mice

Abstract Anti-angiogenic therapy targeting the tumor vasculature has demonstrated clinical benefits in certain types of tumors. However, many patients develop resistance and eventually progress on anti-angiogenic therapy. The mechanism of resistance is largely unknown and warrants extensively investigation. We developed a human xenograft tumor model (M24met/R) in mice that has acquired resistance to VEGF receptor tyrosine kinase inhibitors (VEGFR TKI), by chronically treating the parental M24met tumors (M24met/P) with PF-0337210 (20-40 mg/kg, QD, PO) and serially passaging resistant tumor fragments in nude mice. M24met/R tumors were also resistant to axitinib, a selective VEGFR TKI in late stage clinical development. Immunohistochemical (IHC) staining revealed higher degree of hypoxia and lower microvessel density in M24met/R tumors compared to parental tumors. Compared with those in M24met/P, vessels in M24met/R tumors appeared to be larger in diameter and more mature as shown by high incidence of co-localization between endothelial cells and perivascular cells (CD31+Desmin+). Microarray analysis of tumor lysates (human UA133 2.0, mouse 430 2.0 chip) revealed distinct gene expression profiles between M24met/R tumors and size-matched M24met/P tumors. Of note, several angiogenesis-related genes, including human VEGF, bFGF, integrin α5β1, IL-8, PDGF,CSF and murine VEGF, integrin α5β1, ALK1 (activin receptor-like kinase 1), IL-8, c-Met, IL-6 were up-regulated in M24met/R tumors, whereas murine VEGFR1 and 2, PROX1, PDGFR and endogenous angiogenic inhibitor TSP-1 were down-regulated in the resistant tumors. The above observations were verified with ELISA and/or IHC. Next, targeted combination approaches were applied in an attempt to circumvent M24met/R resistance to axitinib. Concurrent administration of axitinib plus either anti-IL-8 monoclonal antibody (mAb), c-Met inhibitor, anti-integrin α5 mAb or anti-ALK1 mAb significantly improved anti-tumor efficacy compared to single agents alone. IHC analyses showed that combinatorial therapy of anti-ALK1 plus axitinib reduced the amount of CD31+Desmin+ vessels compared to axitinib alone, suggesting that targeting ALK1 destroyed VEGFR TKI-resistant vessels. Together, our results suggested that multiple alternative angiogenic factors contributed to the acquired resistance of M24met/R melanoma to VEGFR TKI. Combination treatment using inhibitor of IL-8, c-Met, integrin α5β1 or ALK1 with VEGFR TKI may represent a new strategy to mitigate resistance to VEGFR TKI in the clinic. 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 3270. doi:10.1158/1538-7445.AM2011-3270

Inhibition of angiogenesis and the growth of murine colon cancer with recombinant human endostatin adenovirus

14634 Background: Inhibition of angiogenesis is a promising approach to treat cancer. Endostatin, the COOH-terminal proteolytic fragment of the basement membrane component collagen XVIII, represents one of the most potent endogenous angiogenic inhibitors. Recombinant human endostatin adenoviruses (Ad-hE), encapsulated with (Ad-hE/Lipo) and without liposome, were evaluated for their potential anti-tumor activity. Methods: MTT colorimetric assay was used to evaluate the inhibition of endostatin on human umbilical vein endothelial cells (HUVECs). An in vitro model was established for the observation of angiogenesis using primary culture of CT26 tumor tissues. Mice bearing CT26 colon cancer either by subcutaneous or lung metastatsis, were treated with different concentrations of Ad-hE or Ad-hE/Lipo by intratumor or vena caudalis injection respectively. The level of endostatin in blood was tested using ELISA. In addition, the in vivo anti- angiogenesis effect was analyzed using either immunohistochemical CD31 ...

Kallikrein-binding protein inhibits growth of gastric carcinoma by reducing vascular endothelial growth factor production and angiogenesis

Kallikrein-binding protein (KBP) has been identified as an endogenous angiogenic inhibitor. We previously showed that KBP inhibited rat retinal neovascularization by down-regulation of vascular endothelial growth factor (VEGF) in endothelial cells. However, its antiangiogenic potential for inhibition of gastric carcinoma and the effect on VEGF in tumor cells have not been elucidated. The present study was designed to investigate the effect of KBP on growth of gastric carcinoma and the possible molecular mechanism. Recombinant KBP dose dependently inhibited proliferation and induced apoptosis of endothelial cells, but no effect on proliferation and apoptosis of SGC-7,901 gastric carcinoma cells. I.p. injection of KBP resulted in growth inhibition of both heterotopic and orthotopic gastric carcinoma xenografts at 61.4% and 52.3%, respectively. Microvessel density in tumor tissues treated with KBP was significantly decreased, suggesting that KBP suppressed tumor growth by antiangiogenesis. The expression and release of VEGF, a major angiogenic stimulator, were down-regulated by KBP in SGC-7,901 cells and gastric carcinoma xenografts. RNA levels of VEGF in SGC-7,901 cells were also decreased by KBP, thus suggesting the regulation at the transcriptional level. Therefore, hypoxia-inducible factor 1alpha (HIF-1alpha), a crucial transcriptional factor for VEGF expression, was examined in SGC-7,901 cells treated by KBP. KBP reduced HIF-1alpha protein level and nuclear translocation, which may be responsible for the down-regulation of VEGF transcription. Down-regulation of VEGF expression and release in tumor cells through inhibiting HIF-1alpha, thus attenuating the paracrine effect of VEGF on endothelial cell proliferation and vascular permeability in tumor tissues, may represent a novel mechanism for the antiangiogenic and antitumor activity of KBP.

Kallikrein-binding protein suppresses growth of hepatocellular carcinoma by anti-angiogenic activity

Effect of kallikrein-binding protein (KBP), an endogenous angiogenic inhibitor, on the growth of hepatocellular carcinoma and the possible mechanism were investigated. KBP inhibited proliferation and induced apoptosis of endothelial cells, but had no effect on the proliferation and apoptosis of hepatocarcinoma cell line HepG2. Intraperitoneal injection of KBP significantly suppressed the tumor growth and inhibited intratumoral neovascularization both in grafted hepatocarcinoma mice and xenografted hepatocarcinoma athymic mice. Moreover, KBP reduced expression of VEGF and HIF-1α nuclear translocation in HepG2 cells and xenografts. Down-regulation of VEGF in tumor cells through inhibiting HIF-1α may represent a novel mechanism for the anti-angiogenic and anti-tumor activity of KBP.

Spinal cord injury–induced expression of the antiangiogenic endostatin/collagen XVIII in areas of vascular remodelling

Spinal cord injury (SCI) induces the disruption of neural and vascular structures. In contrast to the emerging knowledge of mechanisms regulating the onset of the postinjury angiogenic response, little is known about counterregulatory signals. Using immunohistochemical methods, the authors investigated the expression of the endogenous angiogenic inhibitor endostatin/collagen XVIII during the tissue remodeling response to SCI. After SCI, endostatin/collagen XVIII+ cells accumulated at the lesion site, in pannecrotic regions (especially in areas of cavity formation), at the lesion margin/areas of ongoing secondary damage, and in perivascular Virchow-Robin spaces. In remote areas (> 0.75 cm from the epicenter) a more modest accumulation of endostatin/collagen XVIII+ cells was observed, especially in areas of pronounced Wallerian degeneration. The numbers of endostatin/collagen XVIII+ cells reached their maximum on Day 7 after SCI. The cell numbers remained elevated in both, the lesion and remote regions, compared with control spinal cords for 4 weeks afterwards. In addition to being predominantly confined to ED1+-activated microglia/macrophages within the pannecrotic lesion core, endostatin/collagen XVIII expression was frequently detected by the endothelium/vessel walls. Numbers of lesional endostatin/collagen XVIII+ endothelium/vessel walls were found to increase early by Day 1 postinjury, reaching their maximum on Day 3 and declining subsequently to enhanced (above control) levels 30 days after SCI. The authors detected that in comparison to the early expression of neoangiogenic factors, there was a postponed lesional expression of the antiangiogenic endostatin/collagen XVIII. Furthermore, the expression of endostatin/collagen XVIII was localized to areas of neovascular pruning and retraction (cavity formation). The expression of endostatin/collagen XVIII by macrophages in a "late" activated phagocytic mode suggests that this factor plays a role in counteracting the preceding "early" neoangiogenic response after SCI.

Ocular neovascularization: Implication of endogenous angiogenic inhibitors and potential therapy

Ocular neovascularization (NV) is the primary cause of blindness in a wide range of ocular diseases, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD). The exact mechanism underlying the pathogenesis of ocular NV is not yet well understood, and as a consequence, there is no satisfactory therapy for ocular NV. In the last 10 years, a number of studies provided increasing evidence demonstrating that the imbalance between angiogenic stimulating factors and angiogenic inhibitors is a major contributor to the angiogenesis induced by various insults, such as hypoxia or ischemia, inflammation and tumor. The angiogenic inhibitors alone or in combination with other existing therapies are, therefore, believed to be promising in the treatment of ocular NV in the near future. This article reviews recent progress in studies on the mechanisms and treatment of ocular NV, focusing on the implication and therapeutic potential of endogenous angiogenic inhibitors in ocular NV.