Angiogenesis Inhibitor TNP-470 Research Articles

Heat shock transcription factor 1 protects heart after pressure overload through promoting myocardial angiogenesis

BackgroundHeat shock transcription factor 1 (HSF1) plays an important role in exercise-induced cardiac hypertrophy. However, its role in pressure overload-induced cardiac hypertrophy is not completely clear. We here elucidate whether...

Heat shock transcription factor 1 protects heart after pressure overload through promoting myocardial angiogenesis in male mice

Heat shock transcription factor 1 (HSF1) plays an important role not only in excise-induced cardiac hypertrophy but also in protection against pressure overload-induced cardiac dysfunction. However, the mechanism is not completely understood. We here elucidate the potential mechanisms by which HSF1 protects against pressure overload-induced cardiac remodeling and dysfunction. A sustained constriction of transverse aorta (TAC) was imposed to HSF1 transgenic (TG), knockout (KO) and their littermate wild type (WT) male mice. Four weeks later, adaptive responses to TAC, such as cardiac hypertrophy, contractility and angiogenesis evaluated by echocardiography, catheterization, coronary perfusion pressure and immunohistochemistry were well preserved in TG but not in KO compared with WT mice. An angiogenesis inhibitor TNP-470 abrogated all these adaptive responses in TG mice, while cardiac transfection of VEGF with angiopoietin-1 rescued the broken heart in KO mice. In response to TAC, p53 was downregulated and hypoxia-inducing transcription factor-1 (HIF-1) was upregulated not only in the heart but also in the cultured cardiac endothelial cells (EC) of TG mice as compared to WT mice whereas these changes became opposite in KO mice. A small interfering RNA (siRNA) of HIF-1 but not a p53 gene impaired the adaptive responses of the heart and EC in TG mice, and a siRNA of p53 but not a HIF-1 gene significantly reversed the heart and EC disorders in KO mice after TAC. We conclude that HSF1 promotes cardiac angiogenesis through suppression of p53 and subsequent upregulation of HIF-1 in endothelial cells during chronic pressure overload, leading to the maintenance of cardiac adaptation.

Estrogen Rescues Preexisting Severe Pulmonary Hypertension in Rats

Pulmonary hypertension (PH) is characterized by progressive increase in pulmonary artery pressure leading to right ventricular (RV) hypertrophy, RV failure, and death. Current treatments only temporarily reduce severity of the disease, and an ideal therapy is still lacking. Estrogen pretreatment has been shown to attenuate development of PH. Because PH is not often diagnosed early, we examined if estrogen can rescue preexisting advanced PH. PH was induced in male rats with monocrotaline (60 mg/kg). At Day 21, rats were either treated with 17-β estradiol or estrogen (E2, 42.5 μg/kg/d), estrogen receptor-β agonist (diarylpropionitrile, 850 μg/kg/d), or estrogen receptor α-agonist (4,4',4"-[4-Propyl-(1H)-pyrazole-1,3,5-triyl] trisphenol, 850 μg/kg/d) for 10 days or left untreated to develop RV failure. Serial echocardiography, cardiac catheterization, immunohistochemistry, Western blot, and real-time polymerase chain reaction were performed. Estrogen therapy prevented progression of PH to RV failure and restored lung and RV structure and function. This restoration was maintained even after removal of estrogen at Day 30, resulting in 100% survival at Day 42. Estradiol treatment restored the loss of blood vessels in the lungs and RV. In the presence of angiogenesis inhibitor TNP-470 (30 mg/kg) or estrogen receptor-β antagonist (PHTPP, 850 μg/kg/d), estrogen failed to rescue PH. Estrogen receptor-β selective agonist was as effective as estrogen in rescuing PH. Estrogen rescues preexisting severe PH in rats by restoring lung and RV structure and function that are maintained even after removal of estrogen. Estrogen-induced rescue of PH is associated with stimulation of cardiopulmonary neoangiogenesis, suppression of inflammation, fibrosis, and RV hypertrophy. Furthermore, estrogen rescue is likely mediated through estrogen receptor-β.

Stimulation of Cardiac Neoangiogenesis by Estrogen Therapy is One of the Key Mechanisms in Reversing Advanced Heart Failure

Estrogen(E2) has been shown to regulate angiogenesis in different tissues, but it is still not known if E2 could stimulate angiogenesis in the heart. Recently, we showed that E2 rescues advanced heart failure by reversing the myocardial contractile deficiency and restoring ejection fraction from ∼30% to ∼55%. Here we examined whether stimulation of angiogenesis in the heart is a mechanism involved in the E2-induced rescue of HF. Trans-aortic constriction(TAC) procedure was used to induce HF. Once the ejection fraction(EF) reached ∼30%, one group of mice was sacrificed and the other two groups were treated with E2(30 μg/kg/day, n=16), or E2 plus the angiogenesis inhibitor TNP-470(TNP, 30 mg/kg, n=4) for 10 days. Serial echocardiography, real-time PCR and immunocytochemistry were performed. RT-PCR showed that the transcript levels of two markers of angiogenesis, vascular endothelial growth factor(VEGF) and hypoxia-inducible factor-1a(HIF1a), were ∼10 fold downregulated in HF(0.17±0.06 for VEGF and 0.26±0.01 for HiF1a; normalized to CTRL). E2 treatment was not only able to reverse VEGF and HIF1a transcript level downregulation observed in HF, but to even upregulate both transcripts 3 fold higher than in healthy controls(3.24±0.1 for VEGF and 3.16±0.09 for HIF1a). Quantification of capillary density also revealed that E2 therapy not only completely reversed the loss of capillaries in HF, but significantly enhanced capillary density by ∼ 4 fold compared to HF(2.83±0.14 in E2 vs. 0.66±0.07 in HF, normalized to CTRL). Interestingly, E2 failed to rescue HF in the presence of TNP-470 (E2+TNP group) as EF (29.3±2.1%) was not significantly improved after 10 days of therapy. The capillary density of HF mice also did not improve in E2+TNP group(0.53±0.07). These data strongly support the vital role of angiogenesis in the rescue action of E2.

Mechanistic Insights into Reversal of Pulmonary Hypertension by Estrogen Therapy

Severe pulmonary hypertension(PH) leads to right-ventricular failure(RVF) and death. Estrogen(E2) pretreatment has been shown to attenuate development of PH. Here, we tested the hypothesis that E2 may also reverse advanced PH and investigated its underlying mechanisms. Male rats were treated with monocrotaline(MCT,60 mg/kg) to induce PH(n=36). At day-21 when PH had established, one group was euthanized(PH, n=8), and 5 other groups for 10 days received either i) E2 (0.017 mg/day, n=12) ii) E2 plus angiogenesis inhibitor TNP-470 (1.2 mg/day, n=3) iii) ERb-agonist DPN(0.34 mg/day, n=3) iv) ERa-agonist PPT(0.34 mg/day, n=3) or v) left untreated(RVF, n=7). Saline-treated rats served as controls(CTRL, n=6). Echocardiography, cardiac catheterization, RT-PCR, Western-blot and immunocytochemistry were performed. MCT-group developed severe PH at day21 as RV-pressure (RVP) increased from 31±2 in CTRL to 69±2 mmHg. E2 reversed PH(RVP=38±2mmHg, P<0.05) completely resulting in full recovery. Interestingly, E2 failed to rescue PH when applied together with TNP. VGEF protein in RV and lungs was reduced significantly ∼5fold in PH and E2 increased VEGF significantly ∼10 fold. Reduced RV capillary-density in PH was restored fully by E2. E2 reduced cardiopulmonary inflammation as IL-6 transcript upregulation in PH(∼22 fold in lung, ∼7 folds in RV) was partially reversed by E2. Lungs showed 4-fold increase in ED1-inflammatory cells in PH and E2 reversed this increase. Lung ERb-transcripts, but not ERa, were significantly reduced 6-fold in PH and were restored by E2. ERb protein in lung and RV was also significantly reduced ∼2 fold in PH and E2 restored ERb. ERb agonist DPN was as effective as E2 to rescue PH (RVP=34±1 mmHg), whereas PPT was not as effective. In conclusion, E2 rescues PH by stimulating cardiopulmonary neoangiogenesis and suppressing inflammation mainly via ERb.

Malignant Progression and Blockade of Angiogenesis in a Murine Transgenic Model of Neuroblastoma

Targeted expression of MYCN to the neural crest [under control of the rat tyrosine hydroxylase (TH) promoter] causes neuroblastoma in transgenic mice (TH-MYCN) and is a well-established model for this disease. Because high levels of MYCN are associated with enhanced tumor angiogenesis and poor clinical outcome in neuroblastoma, we serially characterized malignant progression, angiogenesis, and sensitivity to angiogenic blockade in tumors from these animals. Tumor cells were proliferative, secreted high levels of the angiogenic ligand vascular endothelial growth factor (VEGF), and recruited a complex vasculature expressing the angiogenic markers VEGF-R2, alpha-SMA, and matrix metalloproteinases MMP-2 and MMP-9, all of which are also expressed in human disease. Treatment of established murine tumors with the angiogenesis inhibitor TNP-470 caused near-complete ablation, with reduced proliferation, enhanced apoptosis, and vasculature disruption. Because TNP-470 has been associated with neurotoxicity, we tested the recently described water-soluble HPMA copolymer-TNP-470 conjugate (caplostatin), which showed comparable efficacy and was well tolerated without weight loss or neurotoxicity as measured by rotarod testing. This study highlights the importance of angiogenesis inhibition in a spontaneous murine tumor with native tumor-microenvironment interactions, validates the use of mice transgenic for TH-MYCN as a model for therapy in this common pediatric tumor, and supports further clinical development of caplostatin as an antiangiogenic therapy in childhood neuroblastoma.

Antiangiogenesis and Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Targeting as Part of a Combined-Modality Approach to the Treatment of Cancer

Antiangiogenesis and Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor Targeting as Part of a Combined-Modality Approach to the Treatment of Cancer

Synergistic Antitumor Effect of an Angiogenesis Inhibitor (TNP-470) and Tumor Necrosis Factor in Mice

We investigated the potentiation of combination therapy using tumor necrosis factor (TNF) with TNP-470, a potent angiogenesis inhibitor. We evaluated the antitumor effect in vivo against subcutaneous (s.c.) MC38 mouse colon adenocarcinoma tumors in C57BL/6 mice. The mice were treated with a single bolus injection via the tail vein of 3 or 8 microg rhTNF in 0.5% bovine serum albumin/normal saline (BSA/NS), or with 0.5% BSA/NS alone as a control, with or without TNP-470 pretreatment, given as 30 or 60 mg/kg x 2 days, s.c. DNA synthesis in human umbilical endothelial cells (HUVEC) was assessed by [(3)H]thymidine uptake after incubation with TNF, with or without TNP-470. The antitumor effect of TNP-470 pretreatment combined with 3 microg recombinant human (rh) TNF injection resulted in an 80% reduction of tumor volume compared with the control. This was significantly better than that induced by 3 microg rhTNF alone (P < 0.005). DNA synthesis in HUVEC was inhibited by TNF with TNP-470 in a dose-dependent manner, but there was no enhanced effect against MC38 in vitro. These results suggest that the combination of the angiogenesis inhibitor TNP-470 and TNF might have a synergistic antitumor effect on solid tumors in vivo.

Angiogenesis inhibitor TNP-470 prevents implanted liver metastases after partial hepatectomy in an experimental model without impairing wound healing.

The ability of the angiogenesis inhibitor TNP-470 to prevent liver metastasis after partial hepatectomy, and whether TNP-470 impairs liver regeneration or skin wound healing, was evaluated. Following the injection of VX2 carcinoma cells into the portal vein of rabbits, half of the animals underwent resection of the middle hepatic lobe (hepatectomized group) and half did not (non-hepatectomized group). TNP-470 (50 mg) was infused continuously into the portal vein in both groups for 7 days, while controls received only water. The hepatectomized TNP-470-treated group had significantly fewer tumours (mean(s.e.m.) 23.3(12.3)) than the hepatectomized control group (123.7(24.4)). There was no significant difference in the 5-bromo-2'-deoxyuridine labelling index of regenerated hepatocytes between the TNP-470-treated and control groups. Wound healing in TNP-470-treated animals was not impaired. Intraportal infusion of TNP-470 prevents the recurrence of liver metastasis after partial hepatectomy without impairing healing or liver regeneration.

Anti-tumor effect of radiation response by combined treatment with angiogenesis inhibitor, TNP-470, in oral squamous cell carcinoma

Blocking angiogenesis may enhance conventional anticancer treatments such as radiation therapy. In this study, we examined the effects of the angiogenesis inhibitor TNP-470 on human OSCC cell lines HSC2 and KB, with combining radiation therapy in the nude mouse. We evaluated cell-induced neovascularization with dorsal air sac assay, and selected two cells (HSC2: low, KB: high) with different level of cell-induced angiogenesis. The angiogenesis inhibitor TNP-470 was given 30 mg/kg s.c. daily on day 1-5, and irradiation, 8 Gy x 1, was administered on day 1 each week for 3 weeks. Significant inhibition of tumor growth relative to untreated controls was achieved in KB cells showing high induced angiogenesis with both TNP-470 (P < 0.01) and radiation (P < 0.01) and combining TNP-470 and radiation (P < 0.01). We saw little effect of TNP-470 either alone or in addition to the effect of radiation on the HSC2 cells showing low induced angiogenesis. These results suggested that TNP-470 significantly enhanced the effect of radiation on the cells with high neovascularization. These findings indicated that individual evaluation of each tumor neovascularization potential will be important before deciding the anti-angiogenesis treatment.

Endostatin binds biglycan and LDL and interferes with LDL retention to the subendothelial matrix during atherosclerosis

Retention of lipoproteins to proteoglycans in the subendothelial matrix (SEM) is an early event in atherosclerosis. We recently reported that collagen XVIII and its proteolytically released fragment endostatin (ES) are differentially depleted in blood vessels affected by atherosclerosis. Loss of collagen XVIII/ES in atherosclerosis-prone mice enhanced plaque neovascularization and increased the vascular permeability to lipids by distinct mechanisms. Impaired endothelial barrier function increased the influx of lipoproteins across the endothelium; however, we hypothesized that enhanced retention might be a second mechanism leading to the increased lipid content in atheromas lacking collagen XVIII. We now demonstrate a novel property of ES that binds both the matrix proteoglycan biglycan and LDL and interferes with LDL retention to biglycan and to SEM. A peptide encompassing the alpha coil in the ES crystal structure mediates the major blocking effect of ES on LDL retention. ES inhibits the macrophage uptake of biglycan-associated LDL indirectly by interfering with LDL retention to biglycan, but it has no direct effect on the macrophage uptake of native or modified lipoproteins. Thus, loss of ES in advanced atheromas enhances lipoprotein retention in SEM. Our data reveal a third protective role of this vascular basement membrane component during atherosclerosis.

TNP-470 fails to block the onset of angiogenesis and early tumor establishment in an intravital minimal disease model

The angiogenesis inhibitor TNP-470 (AGM-1470) has shown encouraging results in animal models of established tumors. However, results of recent clinical trials using TNP-470 have been disappointing. Since little is known about the effects of TNP-470 at the minimal disease stage, we analyzed the effects of TNP-470 on the early stages of tumor establishment. Twenty thousand green fluorescent protein (GFP)-transfected murine CT-26 (colonic carcinoma) or Panc-02-H0 (pancreatic adenocarcinoma) cells were inoculated in dorsal skin-fold chambers in BALB/c or C57BL6 mice. Tumor area and microvessel density (MVD) were quantified by intravital microscopy (IVM). Body weight was also monitored. Effects were compared with those in a conventional model involving subcutaneous (s.c.) inoculation of 10(6) tumor cells, followed by measurement of tumor volume, endogenous plasma VEGF/endostatin (ELISA) and proliferation/apoptosis/microvessel density (Ki-67/TUNEL/CD-34). TNP-470 was injected s.c. over the 10-day experimental period (30 mg/kg every 2 days [n=6] to 100 mg/kg/day [n=5 dorsal skin-fold chamber model, n=4 s.c. tumor model]). At 30 mg/kg/every second day neither CT-26 nor PANC-02-H0 tumors were inhibited in neither of the two models. TNP-470 dosage was escalated in CT-26-bearing animals until an antiangiogenic effect could be observed. In the IVM model, only TNP-470 100 mg/kg/day reduced MVD (P=0.006), but failed to block the onset of angiogenesis and tumor area increase. Body weight decreased by 25% (P<0.05). In the subcutaneous tumor model, tumor growth was reduced (P=0.045) but not blocked, while vascular endothelial growth factor (VEGF)/endostatin synthesis and Ki67/TUNEL/CD-34 were not significantly affected. While capable of reducing tumor growth in a conventional model, treatment with TNP-470 does not block the onset of angiogenesis and tumor establishment in a model of minimal disease. When used as a single agent TNP-470 does not control minimal tumor disease in experimental colonic carcinoma.

Cis-Diamminedichloroplatinum-resistant cell lines derived from human epithelial ovarian carcinoma express increased susceptibility to angiogenesis inhibitor TNP-470

Objective. To elucidate the efficacy of TNP-470 on ovarian carcinomas by using cis-diamminedichloroplatinum (CDDP)-resistant cell lines. Methods. The susceptibility of human ovarian carcinoma-derived cell lines and its resistant cell lines against cis-diamminedichloroplatinum (CDDP) to angiogenesis inhibitor, TNP-470, were analyzed using three human cultured cell lines derived from ovarian carcinoma (TYK, KF-92, and Nakajima) and each CDDP-resistant cell line (TYK-R, TYK-R′, KF/ra, KF/rb, Nakajima-S1, and Nakajima-S2). Results. TNP-470 revealed suppression of thymidine incorporation by all of the nine cell lines linearly dependent on the concentration of TNP-470. Significant suppression was not observed for either uridine or leucine incorporation by all nine cell lines. To elucidate the site of each cell line, in which TNP-470 revealed the antitumor effect, the incorporation of 3H-TNP-470 by cultured cells or by DNA extracted from cultured cells was examined in the cell lines, and the ratio of 3H-TNP-470 incorporation by DNA to 3H-TNP-470 incorporation by cultured cells ranged from 2.3% to 4.4% in three parent cell lines. The ratio in the CDDP-resistant cell lines ranged from 11.0% to 46.7%. The ability of TNP-470 to inhibit neoplastic growth in vivo was evaluated using KF-92, KF/ra, KF/rb, Nakajima, Nakajima-S1, and Nakajima-S2. Concerning KF-92, KF/ra, and KF/rb, 30 mg/kg of TNP-470 significantly suppressed the tumorigenicity of KF-92, 10 and 30 mg/kg of TNP-470 suppressed the tumorigenicity of KF/ra, and 30 mg/kg of TNP-470 suppressed the tumorigenicity of KF/rb. Concerning Nakajima, Nakajima-S1, and Nakajima-S2, TNP-470 revealed no inhibitory effect on the tumorigenicity of Nakajima. Contrary, significant inhibition was observed when 30 mg/kg of TNP-470 was used to the CDDP-resistant cell lines Nakajima-S1 and Nakajima-S2. Conclusion. These results suggest that the clinical application of TNP-470 may be one of the possible treatments for the CDDP-resistant ovarian carcinomas.

Angiogenesis Inhibitor TNP-470 Can Suppress Hepatocellular Carcinoma Growth Without Retarding Liver Regeneration After Partial Hepatectomy

We investigated the suppressive effect of the angiogenesis inhibitor TNP-470 on accelerated hepatocellular carcinoma (HCC) growth in the regenerating liver. After 70% partial hepatectomy (PH), AH-130 cells were injected into the portal vein of Donryu rats. A control group was given the vehicle only, and the treated group was given 10 mg/kg TNP-470 subcutaneously every second day, from 24 h after tumor implantation, seven times. On day 14, tumor growth was evaluated by the number of foci on the liver surface, liver weight, and the microvessel density of the tumor. The number of foci was significantly less in the treated group (116.5 +/- 103.1) than in the control group (319.3 +/- 223.1) ( P < 0.05), as was microvessel density, which was 31.3 +/- 14.0/mm2 in the treated group and 61.2 +/- 18.9/mm2 in the control group ( P < 0.05). The liver tended to weigh less in the treated group (12.15 +/- 1.28 g) than in the control group (15.22 +/- 5.35 g). We also assessed whether TNP-470 retards liver regeneration. Seven days after 70% PH, the liver weight in the treated group was similar to that in the control group. Total bilirubin, serum glutamic oxaloacetic transaminase, and serum glutamic pyruvic transaminase were not higher in the treated group than in the control group. TNP-470 can suppress HCC growth without retarding liver regeneration after PH.

Effects of angiogenesis inhibitor TNP-470 on the development of uterine adenomyosis in mice

Objective To investigate the effects of angiogenesis inhibitor TNP-470 on uterine microvessels in mice. Pituitary grafting frequently induced uterine adenomyosis. Design In vivo experimental study. Setting Department of Biological Sciences, University of Tokyo and Medical Research Institute, Tokyo Medical and Dental University. Animal(s) SHN mice, which are known to develop uterine adenomyosis spontaneously, and also very soon after pituitary grafting. Intervention(s) Immunohistochemical study on uterine blood vessels using an antibody to von Willebrand factor in pituitary gland-implanted mice with or without TNP-470. Main outcome measure(s) Reduced incidence of uterine adenomyosis. Result(s) Twelve of 15 mice developed uterine adenomyosis with dilated blood vessels, but none of the TNP-470-treated mice with shrunken microvessels. The number of bromodeoxyuridine immunoreactive cells and activities of thymidylate synthase and thymidine kinase in uterine tissues were markedly reduced in TNP-470-treated mice. Conclusion(s) TNP-470, a potent inhibitor of the development of vascular endothelium, reduced the development of endometrial blood vessels resulting in a lowered incidence of uterine adenomyosis induced by pituitary grafting in mice, and reduced the increase in S-phase cells and enzyme activity for pyrimidine nucleotide synthesis.

Angiogenesis inhibitor TNP-470 (AGM-1470) suppresses vascular smooth muscle cell proliferation after balloon injury in rats

Background. Although TNP-470, a synthetic analog of fumagillin, may inhibit vascular intimal hyperplasia, the effects of TNP-470 on smooth muscle cell (SMC) proliferation have not been demonstrated in vivo. The aim of this study was to confirm the effect of TNP-470 on vascular SMC proliferation using a rat carotid artery balloon injury model. Materials and methods. Rats were treated with vehicle or with TNP-470 at low dosage (10 mg/kg), medium dosage (20 mg/kg), or high dosage (40 mg/kg). The animals received subcutaneous injections of materials three times a week from the day following balloon injury. All rats were sacrificed at 2 weeks after injury. The ratio of intimal-to-medial cross-sectional areas (I/M ratio) and the PCNA labeling index was calculated for each group. The DNA synthesis of cultured SMCs was also evaluated using [ 3H]thymidine incorporation assays. Smooth muscle cells were stimulated with basic fibroblast growth factor and TNP-470 (0.01–100 ng/ml) were added. Results. The inhibition of intimal hyperplasia increased in a dose-dependent manner. TNP-470 also decreased PCNA expression in the neointima and inhibited DNA synthesis of cultured SMCs. Conclusion. TNP-470 may be useful in the prevention of vascular intimal hyperplasia.

Endothelial-directed hepatic regeneration after partial hepatectomy.

To determine the role of the microvascular endothelium in the regulation of regenerating liver mass after partial hepatectomy. Angiogenesis is critical for both pathologic and physiologic processes. The ability of certain tissues, such as the liver, kidney, and spleen, to regenerate after injury is poorly understood. The liver will regenerate to its normal mass within 8 days of surgical excision. Because the authors have previously shown that the endothelial cell regulates tumor mass, we hypothesized that normal adult organ mass is also controlled by the endothelial cell. Two-thirds partial hepatectomy was performed in 7- to 8-week-old C57 BL/6 mice, followed by systemic treatment with either the angiogenesis stimulator basic fibroblast growth factor (bFGF) (1 microg/g/d intraperitoneal) or the angiogenesis inhibitor TNP-470 (30 mg/kg/qod subcutaneous). Groups of three mice were then euthanized on postoperative days 2, 4, 6, and 8, and the livers were weighed and analyzed by immunohistochemistry. bFGF accelerated hepatic regeneration by 42%, 19%, 16%, and 16% on postoperative days 2, 4, 6, and 8, respectively. TNP-470 inhibited hepatic regeneration by 46%, 74%, 67%, and 64% on postoperative days 2, 4, 6, and 8, respectively. Immunohistochemistry revealed that bFGF and TNP-470 primarily affected the endothelial compartment. Specifically, bFGF increased endothelial proliferation and decreased endothelial apoptosis. TNP-470, in contrast, inhibited endothelial cell proliferation. The cessation of the regenerative process correlated with a decrease in endothelial proliferation and an increase in endothelial apoptosis. The systemic administration of angiogenesis agents modulates the regeneration of hepatic mass primarily by affecting endothelial cell proliferation or apoptosis. Endothelial cell apoptosis is associated with the cessation of the regenerative process in control mice. These results suggest that the endothelial cell is one of the key mediators of regenerating adult tissue mass in this partial hepatectomy model.

Effect of angiogenesis inhibitor TNP-470 on the growth, blood flow, and microvessel density in xenografts of human uterine carcinosarcoma in nude mice

Objective Carcinosarcoma is the most aggressive neoplasm of among the known uterine malignancies. Most tumors show lymph–vascular space invasion and are clinically resistant to any chemotherapeutic drug currently used as well as any radiotherapy. This is the first study to investigate a novel therapeutic approach using an angiogenesis inhibitor TNP-470, a synthetic analogue of fumagillin, for human uterine carcinosarcoma in vivo. Methods The growth-inhibitory and anti-angiogenic effects of TNP-470 were examined after inoculating a human uterine carcinosarcoma cell line, FU-MMT-1, in nude mice. Intratumoral blood flow was evaluated weekly by color Doppler ultrasound (CDU) after the xenografts measurably developed during the period of treatment. The microvessel density (MVD) in TNP-470-treated xenografts was also immunohistochemically examined. Results TNP-470 significantly reduced the volume as well as the weight of the xenografts when this therapy was started 3 weeks (Day 21) after the inoculation of FU-MMT-1, in comparison to the controls. Neither weight loss nor ataxia was observed in any mice of this therapy. A main feeding artery for the xenograft was detected by CDU in all mice treated in this study. However, no significant difference in either the vessel density visualized by CDU or MVD between the TNP-470-treated xenografts and controls was observed. Conclusion These results suggest that TNP-470 may inhibit the growth of human uterine carcinosarcoma in vivo. We speculate that TNP-470 may be a useful agent for adjuvant therapy in patients with advanced or recurrent uterine carcinosarcomas. However, a further evaluation in molecular level of the anti-angiogenic effect of TNP-470 against this tumor in vivo is thus called for.

Antitumor activity of the angiogenesis inhibitor TNP-470 on murine lymphoma/leukemia cells in vivo and in vitro.

The aim of this study was to evaluate the effects of an angiogenesis inhibitor in a non-immunocompromised setting in which transplanted tumor cells home and expand in a manner mimicking the original tumor in the donor. We used a novel animal model for T-cell lymphoma/leukemia (TLL) to test the antitumor effect of TNP-470, a well-established angiogenesis inhibitor. Cells from spontaneously arisen TLL tumors were transferred to syngenic recipients. The mice were treated with TNP-470 (30 mg/kg) or vehicle every other day for 2 weeks. Mice were sacrificed on day 15 after transfer, and body and organ weights were measured. Cell cycle and morphologic analysis was performed on cells and/or sections from selected organs. The cytotoxic effect of TNP-470 was assayed in vitro using the TLL-M and HL-60 cell lines. TNP-470 treatment significantly reduced total tumor load and tumor mass in specific organs infiltrated with lymphoma/leukemia. This was associated with an increased apoptosis in these organs. We also observed side effects of TNP-470 not previously reported, such as diminished extramedullary erythropoiesis and disrupted liver morphology. In vitro TLL-M cells were resistant to cytotoxic effects of moderate doses of TNP-470. TNP-470 treatment has a beneficial effect on tumor load in the TLL transfer model, most likely caused by the antiangiogenic effect of TNP-470. This is supported by the observation of increased apoptosis in infiltrated organs. The TLL transfer model is well suited for further studies of combinations with TNP-470 or other angiogenesis inhibitors and cytotoxic drugs.

Preparation of poly-lactic acid microspheres containing the angiogenesis inhibitor TNP-470 with medium-chain triglyceride and the in vitro evaluation of release profiles

TNP-470 (AGM-1470, 6-0-(N-chloroacetylcarbamoyl)-fumagillol), a derivative of fumagillin, is a promising angiogenesis inhibitor. However, as TNP-470 is very unstable in in vitro and in vivo, it has been difficult to verify its pharmacological efficacy in the clinical medicine. The preparation of a drug delivery system (DDS) in a microsphere form was studied for the stable inclusion and controlled release of TNP-470. Medium-chain triglyceride (MCTG) as an effective stabilizer and poly-lactic acid (PLA) as a biodegradable carrier were used for this purpose. The release of TNP-470 from the MCTG containing DDS continued for approximately 2 weeks, while the release of TNP-470 from the one without MCTG stopped after only 5 days. It was proved that TNP-470 could be released much more stable for much longer period from the MCTG containing DDS compared to the one without DDS.