A 2D mathematical model for tumor angiogenesis: The roles of certain cells in the extra cellular matrix.

We present a 2D mathematical model of tumor angiogenesis which is an extension of the 1D model originally presented in Levine et al. (2000) [1]. Our model is connected to that 1D model by some transmission and boundary conditions which carry certain cells, the endothelials, pericytes and macrophages from the vessel wall into the extra cellular matrix. In our extended model we also include a mechanism for the action of anti-angiogenic factors such as angiostatin. We present numerical simulations in which we obtain a very good "qualitative agreement" with the time of the onset of vascularization of tumors and with the fact that the capillary tip growth accelerates as it approaches the "tumor".

Vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFß), angiostatin, and endostatin are increased in radiotherapy-induced gastrointestinal toxicity.

PURPOSE: Radiotherapy-induced gut toxicity (RIGT) is a debilitating effect of radiotherapy for cancer, often resulting in significant diarrhea and pain. Previous studies have highlighted roles of the intestinal microvasculature and matrix metalloproteinases (MMPs) in the development of RIGT. We hypothesized vascular mediators would be significantly altered in a dark agouti (DA) rat model of RIGT. Additionally, we aimed to assess the effect of MMP-2 and -9 inhibition on the response of tumor-associated microvascular endothelial cells (TAMECs) to radiation. METHODS: DA rats were administered 2.5 Gy abdominal irradiation (3 times/week over 6 weeks). Vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFß), von Willebrand factor (VWF), angiostatin, and endostatin expression was assessed at 3, 6, and 15 weeks. Additionally, DA rat mammary adenocarcinoma tumor-associated microvascular endothelial cells (TAMECs) were used to assess the effects of radiation (12 Gy) and the MMP inhibitor SB-3CT on MMP, VEGF, and TGFß expression, and cell viability. RESULTS: VEGF mRNA expression was significantly increased in the colon at week 15 (p = .0012), and TGFß mRNA expression was significantly increased in both the jejunum and colon at week 3 (p = .0280 and p = .0310, respectively). Endostatin immunostaining was significantly increased at week 3 (p = .0046), and angiostatin at 3 and 6 weeks (p = .0022 and p = .0135, respectively). MMP-2 and -9 mRNA and total protein levels were significantly increased following irradiation of TAMECs. Although this increase was significantly attenuated by SB-3CT, it did not significantly alter endothelial cell viability or VEGF and TGFß mRNA expression. CONCLUSIONS: Findings of this study support the involvement of VEGF, TGFß, angiostatin, endostatin, and MMP-2 in the pathobiology of RIGT. However, the relationship between these mediators is complex and needs further investigation to improve understanding of their therapeutic potential in RIGT.

Endogenous angiogenesis inhibitors prevent adaptive capillary growth in left ventricular pressure overload hypertrophy.

BACKGROUND: In left ventricular (LV) pressure-overload hypertrophy, lack of adaptive capillary growth contributes to progression to failure. Remodeling of the hypertrophied myocardium requires proteolysis of the extracellular matrix (ECM) carried out by matrix metalloproteinases (MMPs). MMPs, specifically MMP-9, are known to cleave ECM components to generate angiogenesis inhibitors (angiostatin, endostatin, tumstatin). We hypothesize that MMP-9 releases antiangiogenic factors during compensated and decompensated hypertrophy, which results in lack of adaptive capillary growth. METHODS: Newborn rabbits underwent aortic banding. Myocardial tissue from age-matched and banded animals at compensated (4 weeks) and decompensated hypertrophy (7 weeks), as identified by serial echocardiography, was analyzed by immunoblotting for angiostatin, endostatin, and tumstatin. MMP-9 activity was determined by zymography. A cell-permeable, potent, selective MMP-9 inhibitor was administered intrapericardially to animals with hypertrophied hearts and tissue was analyzed. RESULTS: MMP-9 is activated in hypertrophied myocardium versus in control hearts (22 ± 2 versus 16 ± 1; p = 0.04), which results in significantly increased levels of angiostatin (115 ± 10 versus 86 ± 7; p = 0.02), endostatin (33 ± 1 versus 28 ± 1; p = 0.006), and tumstatin (35 ± 6 versus 17 ± 4; p = 0.04). Zymography confirms inhibition of MMP-9 (hypertrophy + MMP-9 inhibitor, 14 ± 0.6 versus hypertrophy + vehicle, 17 ± 1; p = 0.01) and angiostatin, endostatin, and tumstatin are down-regulated, accompanied by up-regulation of capillary density (hypertrophy + MMP-9 inhibitor, 2.99 ± 0.07 versus hypertrophy + vehicle, 2.7 ± 0.05; p = 0.002). CONCLUSIONS: Up-regulation of angiogenesis inhibitors prevents adaptive capillary growth in pressure-overload hypertrophied myocardium. Therapeutic interventions aimed at inhibition of angiogenesis inhibitors are useful in maintaining capillary density and thereby preventing heart failure.

[Mechanism of inhibitory effect of angiostatin on plasminogen activation by its physiologic activators].

The influence of angiostatin K1-4.5--a fragment of the heavy chain of plasmin and a powerful inhibitor of angiogenesis--on kinetic parameters (k(Pg) and K(Pg)) of human Glu-plasminogen activation under the action of urokinase (uPA) not having affinity for fibrin and fibrin-specific tissue plasminogen activator (tPA) was investigated. Angiostatin does not affect the k(Pg) value, but increases the value K(Pg) urokinase plasminogen activation. A decrease in the k(Pg) value and an increase in the K(Pg) value were found for fibrin-stimulated plasminogen activation by tPA with increasing concentrations of angiostatin. The obtained results show that angiostatin is competitive inhibitor of the uPA activator activity, while it inhibits the activator activity of tPA by mixed type. Such an influence ofangiostatin on the kinetic constants ofthe urokinase plasminogen activation suggests that angiostatin dose dependent manner replaces plasminogen in the binary enzyme-substrate complex uPA-Pg. In case of fibrin-stimulated plasminogen activation by tPA, both zymogen and tPA are bound to fibrin with formation of the effective triple tPA-Pg-fibrin complex. Angiostatin replaces plasminogen both from the fibrin surface and from the enzyme-substrate tPA-Pg complex that leads to a decrease in k(Pg) and an increase in K(Pg) of plasminogen activation. Inhibition constants by angioststin (Ki) of plasminogen-activator activities of uPA and tPA determined by Dixon method were found to be 0.59 +/- 0.04 and 0.12 +/- 0.05 microM, respectively.

Elevated platelet angiostatin and circulating endothelial microfragments in idiopathic pulmonary arterial hypertension: a preliminary study.

INTRODUCTION: Idiopathic pulmonary arterial hypertension (IPAH) is characterized by pulmonary arteriolar narrowing and degeneration associated with in situ thrombosis. We hypothesized that microvascular endothelial injury and apoptosis may be an initiating mechanism in IPAH. Endothelial apoptosis generates endothelial microfragments (EMF), which can activate platelets. Platelets release both VEGF and angiostatin, which depending the balance can inhibit or induce endothelial apoptosis, respectively. MATERIALS AND METHODS: We measured EMFs from blood of IPAH patients as index of endothelial cell apoptosis/injury and levels of pro- and anti- EC apoptotic factors found in platelets. EMFs and platelets in blood samples from control subjects and patients with IPAH were measured using a 4-color flow cytometry protocol, and platelet levels of VEGF and angiostatin were determined by ELISAs and immunoblotting. RESULTS: Compared to controls, IPAH patients exhibited higher numbers of circulating EMFs and more activated/apoptotic platelets. IPAH patients also exhibited higher levels of platelet angiostatin; however, no significant difference was detected in platelet VEGF levels between the two groups. CONCLUSIONS: These results are consistent with an increase in EC dysfunction in patients with IPAH, possibly contributing to the progression of IPAH and its associated thrombosis.

Angiostatin regulates the expression of antiangiogenic and proapoptotic pathways via targeted inhibition of mitochondrial proteins.

Angiostatin, a proteolytic fragment of plasminogen, is a potent endogenous antiangiogenic agent. The molecular mechanisms governing angiostatin's antiangiogenic and antitumor effects are not well understood. Here, we report the identification of mitochondrial compartment as the ultimate target of angiostatin. After internalization of angiostatin into the cell, at least 2 proteins within the mitochondria bind this molecule: malate dehydrogenase, a member of Krebs cycle, and adenosine triphosphate synthase. In vitro and in vivo studies revealed differential regulation of key prosurvival and angiogenesis-related proteins in angiostatin-treated tumors and tumor-endothelium. Angiostatin induced apoptosis via down-regulation of mitochondrial BCL-2. Angiostatin treatment led to down-regulation of c-Myc and elevated levels of another key antiangiogenic protein, thrombospondin-1, reinforcing its antitumor and antiangiogenic effects. Further evidence is provided for reduced recruitment and infiltration of bone marrow-derived macrophages in angiostatin-treated tumors. The observed effects of angiostatin were restricted to the tumor site and were not observed in other major organs of the mice, indicating unique tumor specific bioavailability. Together, our data suggest mitochondria as a novel target for antiangiogenic therapy and provide mechanistic insights to the antiangiogenic and antitumor effects of angiostatin.

Pathological role of angiostatin in heart failure: an endogenous inhibitor of mesenchymal stem-cell activation.

OBJECTIVE: Recently, a clinical trial was initiated to evaluate the efficacy of transendocardial transplantation of autologous bone marrow-derived mesenchymal stem cells (MSC) for the treatment of heart failure (HF). Because some HF patient-derived sera did not induce proliferation of autologous MSC, the present study aimed to elucidate humoral factors in sera that attenuate MSC activation and to investigate the role of these humoral factors in the pathogenesis of HF. METHODS AND RESULTS: Inhibitory effects present in serum were analysed by culturing human MSC with sera from 10 HF patients (FS <25%, BNP >100 pg/ml) and four healthy control subjects. Among the patients, two sera from HF patients showed significant inhibitory activity on MSC proliferation. Protein array and ELISA analysis revealed that these sera contained high levels of angiostatin as well as the active form of matrix metalloproteinase (MMP)-9, which generates angiostatin. Angiostatin significantly inhibited the proliferation and migration of cultured human MSC and increased their apoptosis in a dose-dependent manner. In a rat HF model, serum levels of angiostatin and MMPs increased, but treatment with an MMP inhibitor suppressed these increases. CONCLUSIONS: The results suggest that angiostatin, which can attenuate the activity of MSC, might play a role in the progression of HF.

[Adenovirus mediated angiostatin gene therapy for ovarian cancer: experiment with nude mice].

OBJECTIVE: To built an expression vector of angiostatin (AG) gene with recombinated replication defective adenovirus and investigate the therapeutic effect of human AG gene on ovarian cancer. METHODS: (1) Human AG K (1 - 3) cDNA was inserted into the vector pShuttle to build the recombinant plasmid pShttle-AG (K1-3). pAdeno-X-AG (K1-3) was built by double-cut and recombinated pShttle-AG (K1-3) to vector pAdeno-X, and then recombinant adenovirus was finally prepared by transfection of pAdeno-X-AG (K1-3) into to the human embryo kidney cells of the line 293. (2) Human ovarian cancer cells of the line SKOV3 were inoculated subcutaneously into nude mice of the line BALB/c nu/nu to establish model of human ovarian cancer. Then the mice were randomly divided into 3 groups to be injected with Ad = AG (K1-3), Ad-LacZ, or phosphate buffered saline (PBS) around the cancer every 5 days. The tumor size was measured every 5 days to calculate the tumor volume and tumor inhibition rate. Three days after the last injection the mice were killed. The tumor tissues, livers, and kidneys of the mice underwent immunohistochemistry to calculate the microvessel density (MVD) and expression of vessel endothelial growth factor (VEGF) and AG. RESULTS: The tumor volume and weight of the Ad-AG (K1-3) group were significantly less than those of the PBS and Ad-LacZ groups (all P < 0.01), however, there were not significant difference between the latter two groups (both P > 0.05). The expression levels of CD34 and VEGF of the Ad-AG (K1-3) group were both significantly lower than those of the PBS and Ad-LacZ groups (all P < 0.01), however (both P > 0.05). CONCLUSION: Human angiostatin mediated by adenovirus suppresses the angiogenesis and the growth of human ovarian cancer in the nude mice model, which suggests that it is promising in clinical application.

Angiostatin K1-3 induces E-selectin via AP1 and Ets1: a mediator for anti-angiogenic action of K1-3.

BACKGROUND: Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1-3 included. We previously showed that K1-3 was the most potent angiostatin to induce E-selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1-3-induced E-selectin expression and investigate the role of E-selectin in the anti-angiogenic action of K1-3. METHODS AND RESULTS: Quantitative real time RT-PCR and Western blotting analyses confirmed a time-dependent increase of E-selectin mRNA and protein induced by K1-3. Subcellular fractionation and immunofluorescence microscopy showed the co-localization of K1-3-induced E-selectin with caveolin 1 (Cav1) in lipid rafts in which E-selectin may behave as a signaling receptor. Promoter-driven reporter assays and site-directed mutagenesis showed that K1-3 induced E-selectin expression via promoter activation and AP1 and Ets-1 binding sites in the proximal E-selectin promoter were required for E-selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1-3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E-selectin by K1-3. A modulatory role of E-selectin in the anti-angiogenic action of K1-3 was manifested by both overexpression and knockdown of E-selectin followed by cell proliferation assay. CONCLUSIONS: We show that K1-3 induced E-selectin expression via AP1 and Ets-1 binding to the proximal E-selectin promoter (-356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E-selectin as a novel target for the anti-angiogenic therapy.

Neutrophil apoptosis: selective regulation by different ligands of integrin alphaMbeta2.

Neutrophils undergo spontaneous apoptosis, but their survival can be extended during inflammatory responses. alpha(M)beta(2) is reported either to delay or accelerate neutrophil apoptosis, but the mechanisms by which this integrin can support such diametrically opposed responses are poorly understood. The abilities of closely related alpha(M)beta(2) ligands, plasminogen and angiostatin, derived from plasminogen, as well as fibrinogen and its two derivative alpha(M)beta(2) recognition peptides, P1 and P2-C, differed markedly in their effects on neutrophil apoptosis. Plasminogen, fibrinogen, and P2-C suppressed apoptosis via activation of Akt and ERK1/2 kinases, while angiostatin and P1 failed to activate these prosurvival pathways and did not prevent neutrophil apoptosis. Using cells transfected with alpha(M)beta(2) or its individual alpha(M) or beta(2) subunits, and purified receptors and its constituent chains, we show that engagement of both subunits with prosurvival ligands is essential for induction of the prosurvival response. Hence, engagement of a single integrin by closely related ligands can induce distinct signaling pathways, which can elicit distinct cellular responses.

Mouse macrophage metalloelastase generates angiostatin from plasminogen and suppresses tumor angiogenesis in murine colon cancer.

Previous studies showed that the mouse macrophage metalloelastase (MME) generates the angiogenesis inhibitor angiostatin from plasminogen in vitro. This study aimed to determine whether tumor cells engineered with MME could generate angiostatin and suppress tumor angiogenesis in vivo. Murine CT-26 colon cancer cells stably transfected with MME were inoculated subcutaneously. A radioisotope tracer, immunoblotting, immunofluorescence and immunohistochemistry were used to explore the pathway of the angiostatin generation. The results showed that tumors derived from MME-transfected cells demonstrated a less microvessel density compared with control tumors derived from vector-transfected and non-transfected cells (P<0.001). The expression of vascular endothelial growth factor (VEGF) was significantly lower in the MME-transfected group compared with that of the controls. The growth of MME-transfected tumors was significantly retarded compared with the control tumors (P<0.001). Western blot analysis, using a specific anti-mouse plasminogen (1-4 Kringle) antibody, demonstrated two strong immunoreactive bands (38- and 35-kDa) in MME-transfected tumors. gamma-ray counting data demonstrated that plasminogen cleavage occurred mostly in tumors formed by cells forced to express. We concluded that MME was demonstrated to be an efficient angiostatin-producing MMP and its presence was negatively correlated with the growth of colon cancer in tumor-bearing mice. These findings provide direct evidence that MME generates angiostatin in tumor-bearing mice and the therapeutic application of MME against tumors.

Angiostatin inhibits monocyte/macrophage migration via disruption of actin cytoskeleton.

In light of the involvement of tumor-associated macrophages (TAM) in the promotion of tumor growth and metastasis, strategies to prevent TAM recruitment within the tumor microenvironment are currently under investigation. The recent observation that angiostatin reduces macrophage infiltration in an atherosclerosis model prompted our laboratory to further explore the use of human plasminogen angiostatin (hK1-3) protein as a macrophage modulatory agent. We demonstrate that hK1-3 blocks migration of murine peritoneal macrophages (91% decrease, P<0.00005) and human monocytes (85% decrease, P<0.05) in vitro. Cell viability of hK1-3-treated cells is not affected, as determined by fluorochrome-labeled inhibitors of caspase-propidium iodide (FLICA/PI) flow cytometry analysis. Furthermore, confocal microscopy of phalloidin-stained cells reveals that hK1-3 leads to disruption of actin filopodia/lamellipodia in human monocytes and induces distinct podosome accumulation in mature differentiated macrophages. Paradoxically, we observed a 3.5-fold increase in secretion and a 3- to 5.5-fold increase in gelatinolytic activity of macrophage-produced matrix metalloproteinase-9, which we suggest is a cellular response to compensate for the dominant static effect of hK1-3 on actin. We also demonstrate that hK1-3 induces the phosphorylation of extracellular signal-regulated kinase (ERK1/2) in human monocytes. hK1-3-mediated macrophage immobilization has the potential to be exploited therapeutically in pathological conditions associated with cellular hypoxia, such as cancer and atherosclerosis.

Antiangiogenic therapy enhances the efficacy of transcatheter arterial embolization for hepatocellular carcinomas.

Transcatheter arterial embolization (TAE) is a well-established technique for unresectable hepatic malignancies. However, TAE can temporally halt the growth of hepatic tumors by blocking their arterial supply, but often tumors rapidly develop collateral blood vessels via angiogenesis. We have previously demonstrated that intraportal delivery of adeno-associated viral particles expressing angiostatin leads to long-term expression of angiostatin capable of inhibiting angiogenesis and suppressing the outgrowth of tumors in the liver. Thus, we hypothesize that adeno-associated virus (AAV)-mediated antiangiogenic therapy could enhance the efficacy of TAE to combat liver cancers. To achieve this objective, we engineered a recombinant AAV vector encoding rat angiostatin. Intraportal delivery of this vector led to long term and stable transgenic expression of angiostatin locally in rat hepatocytes and suppressed the growth of CBRH7919 hepatocellular carcinomas established in rat livers by inhibiting formation of neovessels. Although TAE therapy led to necrosis of liver tumors and suppressed their growth, the neovessels of tumors were rapidly reformed 3 weeks after TAE. However, intraportal injection of AAV-angiostatin inhibited the angiogenesis stimulated by TAE, synergized with TAE in suppressing growth of tumors established in livers and prolonged the survival of rats. In conclusion, these encouraging results warrant future investigation of the use of antiangiogenic therapy for enhancing the therapeutic efficacy of TAE to treat unresectable liver cancers.

Cell surface F1Fo ATP synthase: a new paradigm?

The mitochondrial F1Fo adenosine triphosphate (ATP) synthase is one of the most thoroughly studied enzyme complexes known. Yet, a number of new observations suggesting that the enzyme is also located on the cell surface necessitate further investigation. While the mitochondrial synthase utilizes the proton gradient generated by oxidative phosphorylation to power ATP synthesis, the cell surface synthase has instead been implicated in numerous activities, including the mediation of intracellular pH, cellular response to antiangiogenic agents, and cholesterol homeostasis. Intriguingly, a common thread uniting these various models of cell surface ATP synthase functions is the apparently caveolar distribution of the enzyme. Recent studies concerning the cell surface ATP synthase manifest applications in the regulation of serum cholesterol levels, cellular proliferation and antitumor strategies. This review addresses the expression, interactions, functions, and consequences of inhibition of cell surface ATP synthase, an enzyme now displaying a shift in paradigm, as well as of location.

Expression of angiostatin cDNA in human gallbladder carcinoma cell line GBC-SD and its effect on endothelial proliferation and growth.

AIM: To explore the influence of angiostatin up-regulation on the biologic behavior of gallbladder carcinoma cells in vitro and in vitro, and the potential value of angiostatin gene therapy for gallbladder carcinoma. METHODS: A eukaryotic expression vector of pcDNA3.1(+) containing murine angiostatin was constructed and identified by restriction endonuclease digestion and sequencing. The recombinant vector pcDNA3.1-angiostatin was transfected into human gallbladder carcinoma cell line GBC-SD with Lipofectamine 2000, and paralleled with the vector and mock control. The resistant clone was screened by G418 filtration. Angiostatin transcription and protein expression were examined by RT-PCR, immunofluorescence and Western-blot. The supernatant was collected to treat endothelial cells. Cell proliferation and growth in vitro were observed under microscope. RESULTS: Murine angiostatin cDNA was successfully cloned into the eukaryotic expression vector pcDNA3.1 (+). After 14 d of transfection and selection with G418, macroscopic resistant cell cloning was formed in the experimental group transfected with pcDNA 3.1(+)-angiostatin and vector control. But untreated cells died in the mock control. Angiostatin was detected by RT-PCR and protein expression was detected in the experimental group by immunofluorescence and Western-blot. Cell proliferation and growth in vitro in the three groups were observed respectively under microscope. No significant difference was observed in the growth speed of GBC-SD cells between groups that were transfected with and without angiostatin. After treatment with supernatant, significant differences were observed in endothelial cell (ECV-304) growth in vitro. The cell proliferation and growth were inhibited. CONCLUSION: Angiostatin does not directly inhibit human gallbladder carcinoma cell proliferation and growth in vitro, but the secretion of angiostatin inhabits endothelial cell proliferation and growth.

p130-angiomotin associates to actin and controls endothelial cell shape.

Angiomotin, an 80 kDa protein expressed in endothelial cells, promotes cell migration and invasion, and stabilizes tube formation in vitro. Angiomotin belongs to a new protein family with two additional members, Amotl-1 and Amotl-2, which are characterized by conserved coiled-coil domains and C-terminal PDZ binding motifs. Here, we report the identification of a 130 kDa splice isoform of angiomotin that is expressed in different cell types including vascular endothelial cells, as well as cytotrophoblasts of the placenta. p130-Angiomotin consists of a cytoplasmic N-terminal extension that mediates its association with F-actin. Transfection of p130-angiomotin into endothelial cells induces actin fiber formation and changes cell shape. The p130-angiomotin protein remained associated with actin after destabilization of actin fibers with cytochalasin B. In contrast to p80-angiomotin, p130-angiomotin does not promote cell migration and did not respond to angiostatin. We propose that p80- and p130-angiomotin play coordinating roles in tube formation by affecting cell migration and cell shape, respectively.

Angiostatin is directly cytotoxic to tumor cells at low extracellular pH: a mechanism dependent on cell surface-associated ATP synthase.

Angiostatin, a proteolytic fragment of plasminogen, is a potent angiogenesis inhibitor able to suppress tumor growth and metastasis through inhibition of endothelial cell proliferation and migration. Previously, we showed that angiostatin binds and inhibits F(1)F(o) ATP synthase on the endothelial cell surface and that anti-ATP synthase antibodies reduce endothelial cell proliferation. ATP synthase also occurs on the extracellular surface of a variety of cancer cells, where its function is as yet unknown. Here, we report that ATP synthase is present and active on the tumor cell surface, and angiostatin, or antibody directed against the catalytic beta-subunit of ATP synthase, inhibits the activity of the synthase. We show that tumor cell surface ATP synthase is more active at low extracellular pH (pH(e)). Low pH(e) is a unique characteristic of the tumor microenvironment. Although the mechanism of action of angiostatin has not been fully elucidated, angiostatin treatment in combination with acidosis decreases the intracellular pH (pH(i)) of endothelial cells, leading to cell death. We also find that, at low pH(e), angiostatin and anti-beta-subunit antibody induce intracellular acidification of A549 cells, as well as a direct cytotoxicity that is absent in tumor cells with low levels of extracellular ATP synthase. These results establish angiostatin as an antitumorigenic and antiangiogenic agent through a mechanism implicating tumor cell surface ATP synthase. Furthermore, these data provide evidence that extracellular ATP synthase plays a role in regulating pH(i) in cells challenged by acidosis.

A comparison of antiangiogenic therapies for the prevention of liver metastases.

Angiogenesis is essential for solid tumor growth. Although successful antiangiogenic therapies have been demonstrated in animal models, a systematic comparison of the efficacy of different antiangiogenic factors has not been described in the hepatic environment. To address this issue, CT26 murine colon carcinoma cells were transfected with retroviral vectors encoding murine endostatin (mEndostatin), human angiostatin (hAngiostatin), murine-soluble vascular endothelial growth factor receptor-2, (msFlk-1), or murine-soluble Tie2 (msTie2). The transfected cells were then subjected to another round of transfection with a luciferase cDNA-encoding retroviral vector. Expression of these putative antiangiogenic proteins inhibited the proliferation of human umbilical vein endothelial cells in vitro but not tumor cells. To examine effects on tumor growth in vivo, modified cells were delivered via intrasplenic injection into BALB/c mice to induce liver metastases. Tumor burden was measured weekly by bioluminescence. Growth of hepatic metastases in vivo was significantly reduced in mice that were administered cells expressing msTie2 (76% reduction compared with control cells 21 days after intrasplenic inoculation; P < 0.05). Similar results were observed with cells that expressed msFlk-1 and hAngiostatin. However, expression of mEndostatin had no significant effect on the growth of liver metastases compared with control animals. These findings indicate that multiple antiangiogenic pathways are necessary for the growth of hepatic metastases, and each of these pathways is a potential clinically relevant antiangiogenic target for the treatment of this disease.

Review of selected patents for cancer therapy targeting tumor angiogenesis.

Targeting tumor angiogenesis to treat cancer has been the focus of intense research in recent decades. The resulting increase in our knowledge of cancer biology has lead to the development of several new classes of investigational agents that inhibit the angiogenic process. While many clinical trials on antiangiogenic compounds have had disappointing results, the recent approval of the first effective drug targeting tumor vessels has revived interest in further drug development for angiogenesis inhibitors. Of the plethora of new patents for antiangiogenic compounds, only a few describe compounds that will become effective and non-toxic therapies for patients with cancer. This review examines representative patents related to cancer angiogenesis in the context of our current knowledge of the biological processes leading to tumor vascularization and explores the future of multi-targeted therapy and nanoformulation technology in the field of angiogenic therapy.