Angiogenic Growth Research Articles

Preparation and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(D,L-lactic-co-glycolic acid) microspheres using a double emulsion/solvent evaporation technique

Biodegradable Poly(lactic-co-glycolic acid; PLGA), microspheres encapsulating the angiogenic protein recombinant human vascular endothelial growth factor (rhVEGF) were formed to achieve VEGF release in a sustained manner. These microspheres are a promising delivery system which can be used for therapeutic angiogenesis. The PLGA microspheres incorporating two different initial loading amounts of rhVEGF have been prepared by a modified water-in-oil-in-water (w/o/w) double emulsion/solvent evaporation technique. The microspheres have been characterized by particle size distribution, environmental scanning electron microscopy (ESEM), light microscopy, encapsulation efficiency and their degradation was studied in vitro. The rhVEGF released from microspheres was quantified by the competitive enzyme-linked immunosorbent assay (ELISA) and human umbilical vein endothelial cell (HUVEC) proliferation assay was used to assess biological activity of the released VEGF. The microspheres were spherical with diameters of 10–60 µm and the encapsulation efficiency was between 46% and 60%. The release kinetics of rhVEGF was studied for two different amounts: 5 µg VEGF (V5) and 50 µg VEGF (V50) per 500 mg starting polymer. The total protein (VEGF:BSA) release increased up to 4 weeks for two rhVEGF concentrations. The ELISA results showed that the burst release for V5 and V50 microspheres were 4 and 27 ng/mL, respectively. For V5, the microspheres showed an initial burst release, followed by a higher steady-state release until 14 days. VEGF release increased up to 2 weeks for V50 microsphere. HUVEC proliferation assay showed that endothelial cells responded to bioactive VEGF by proliferating and migrating.

Abstract A53: Antiangiogenic activity of GER, a derivative of sweet leaf tea extract, is mediated by the downregulation of bFGF and VEGF

Abstract Mounting evidence indicates that angiogenesis is essential for the proliferation and survival of number of malignant diseases, thus inhibition of angiogenesis might be an effective therapeutic strategy. Antiangiogenic agent that targets both bFGF and VEGF (VEGFR) are considered to be effective adjuncts to the treatment of solid tumors or cancer preventive. Chinese sweet leaf tea, a hot water extract of Rubus suavissimus S. Lee (Rosaceae) (RUS) has been reported to have antiangiogenic activities evidenced by its ability to inhibit the angiogenic initiation and growth of human placental vein tissue (Liu et al., Phytother, Res. 20: 806-813, 2006) and experimental corneal neovascularization (Hakan, O.F., et al, Pharmaceu. Biol. 45: 44-47, 2007). We reported that this particular extract has the ability to inhibit the endothelial tubule formation, migration, and VEGF (VEGFR) of the HUVEC cells (Proc. Am. Assoc. Cancer Res, 1432, 2009). Here we investigated the anti-angiogenic activity of GER, a combination of gallic acid, ellagic acid, and rubusoside purified from the RUS and the relevant molecular mechanisms using HUVEC cells. Our preliminary data demonstrated that GER (5 to 25 µg/ml) markedly inhibited migration of HUVEC cells compared to the vehicle control. This inhibitory effect was concentration-dependent. At a similar concentration, the endothelial tubule formation was almost completely inhibited. When HUVEC cells were treated with GER (5 to 20 µg/ml), the expression of both bFGF and VEGF were markedly inhibited even at the lowest starting concentration. These inhibitory effects were also concentration-dependent. To further confirm the involvement of VEGF pathway in GER-induced anti-angiogenic activity, the protein level of tyrosine kinase receptor VEGFR2 (Flk-1/KDR) was examined. Intriguingly, the expression of Flk-1 was significantly suppressed in HUVEC cells treated with GER (40 µg/ml) by 54.1% ± 2.3% in comparison to that of vehicle control. In contrast, VEGFR2 kinase activity and protein levels of VEGFR1 remained unchanged compared to that of the vehicle control. These results suggest that GER derived from the sweet leaf tea extract may be completely responsible for the observed anti-angiogenic effect of the sweet leaf tea reported previously by down-regulating proangiogenic factors bFGF and VEGF. The chemopreventive effect of GER and its parent extract RUS on the development of human pancreatic carcinoma (L3.6pl cells) in mouse orthotopic model is currently under investigation and will be reported. This study was supported by NIH grant 1R21 AT002882-01A2. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A53.

Regenerative potential of lipofilling on skin aging

Background Features of skin aging are progressive cell and extracellular matrix atrophy. Subcutaneous fat plays a key role in it. Recent studies have proved that the vascular-stromal component of adipose tissue is a rich reserve of regenerative cell precursos able to release angiogenic and antiapoptotic growth factors that stimulate cell differentiation [1]. Thanks to the presence of stromal stem cells, the adipose tissue can now be considered an ideal biologic product to biostimulate and correct defects of the face and body. The authors present their experience with the application of lipofilling to rejuvenate the skin envelope of the face and the hands.

W1826 Increased Mucosa-Associated Diffusely Adherent E.Coli in Crohn's and Colon Cancer: Possible Role in Pathogenesis

Introduction Mucosa-associated adherent invasive Escherichia coli (AIEC) are found in colorectal cancer (CRC) and Crohn9s disease (IBD).1 They invade intestinal epithelial cells, replicate in macrophages and interact with epithelial cells in vitro to promote proinflammatory cytokine release and NFkB activation.2 The adherent-invasive phenotype strongly correlates with the ability to agglutinate red blood cells (RBCs). We suspect that mucosa-associated E coli may also play a role in CRC pathogenesis by activation of key cellular pathways. Our aim here was to identify and characterise adherence mechanisms for our colonic AIEC isolates. Methods A fosmid library was constructed containing 30–60 kb DNA fragments from E coli HM358, a CRC mucosa-associated isolate. Clones were screened for the ability to haemagglutinate RBCs. Agglutinating positive (HA+) clones were further assessed for their ability to adhere to epithelial Hep2 cells, to replicate within macrophages and for their ability to translocate across M-cells. They were then subjected to 454 DNA sequencing. Subsequently, 280 E coli isolates from IBD, CRC and healthy control patients were assessed for the presence of a gene cluster identified from sequence analysis. Results Of the 964 fosmid clones constructed, 8 displayed a strong HA+ phenotype. Sequence data demonstrated all possessed a diffuse adherence gene cluster ( afaA-E ). HA+ positive clones were diffusely adherent to Hep2 cells and showed marked ability to replicate within macrophages when compared to HA- clones (p afa/dr in E coli promotes translocation across M cells, a key portal of mucosal entry. E coli containing afa/dr were more commonly isolated from IBD and CRC patients (57% afa positive) than from healthy controls (28% afa positive, p 2 test). Conclusion Afa/dr E coli are more commonly isolated from IBD and CRC patients than from healthy controls and have properties, including epithelial invasion and replication in macrophages, that overlap with AIEC. Other studies have shown that adherent afa/dr E coli play a key role in the upregulation of angiogenic vascular endothelial growth factor (VEGF) in the intestinal epithelium3 and they may thus have a role in pathogenesis of CRC as well as IBD.

Abstract 1715: Leptin regulates the IL-1 system in breast cancer

Abstract Leptin and IL-1 are inflammatory cytokines promoting breast cancer angiogenesis that are often linked to worse prognosis. We hypothesize the leptin angiogenic effects in mammary tumor (MT) may be linked to, or regulated, in part by IL-1 signaling, therefore, leptin and IL-1 may have synergistic functions in MT progression. To test this hypothesis leptin-dose (0-6.2 nM) and time-course (0-48h) effects on levels of IL-1 system components (IL-1α, IL-1β, IL-1Ra and IL-1R tI) were investigated in mouse MT-4T1 cells. To determine the contribution of specific leptin-activated signaling pathways on IL-1 regulation several kinase inhibitors AG490 (JAK2/STAT3), PD98059 (MEK/MAPK/ERK1/2), Wortmannin (PI-3K/AKT1), Rapamycin (mToR), Go6976 (PKC-Ca dependent), SB203580 (p38 MAP kinase) and SP600125 (JNK) were used and IL-1 system component levels were determined by ELISA and Western blot. To determine if leptin could regulate its own receptor (long isoform, OB-Rb and all isoforms) flow cytometry was used. Our results show that basal levels of IL-1α were 14 fold higher than IL-1β. Remarkably, leptin up-regulates all IL-1 system components and all OB-R isoforms showing the highest effects at 1.2nM/24h. JAK2/STAT3 and MAPK/ERK 1/2 were essential for leptin upregulation of IL-1 system. In contrast, PI-3K/AKT1, PKC and p38 kinases were mainly linked to leptin regulation of IL-1 receptor-related proteins (IL-1R tI and IL-1Ra). JNK and mTOR were related to leptin-mediated induction of IL-1R tI but were not involved on IL-1α or IL-1Ra regulation. Interestingly, leptin-mediated activation of mTOR appears to be a negative regulator of IL-1α but acts as an inductor of IL-1Ra. In conclusion, leptin is an important regulator of IL-1 system in MT. In turn, IL-1 signaling can promote VEGF expression and tumor macrophage recruitment that could indirectly enhance leptin-mediated angiogenic effects and MT growth. This work was supported in part by NIH/NCI 1SC1CA138658-02; NIH/UAB Breast SPORE Career Development Award; BC 504370 Susan G. Komen Foundation for the Cure and the Georgia Cancer Coalition Distinguished Cancer Scholar Award (to RRGP). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1715.

Abstract LB-371: Altered vascular endothelial growth factor mRNA stability may lead to cancer development following immunosuppressive therapy

Abstract The development of cancer is an increasing and major problem in immunosuppressed patients, particularly after solid organ transplantation. In part, the increased cancer risk is associated with the use of immunosuppressive agents, especially calcineurin inhibitors (CNIs). The CNIs are used to prevent inflammatory diseases and allograft rejection. However, little is known about the mechanism(s) underlying their ability to promote the development and recurrence of cancer. We have recently demonstrated that CNI can mediate the transcriptional activation of the angiogenic cytokine vascular endothelial growth factor (VEGF) and promote a rapid progression of human renal cancer. In contrast, the mTOR inhibitor rapamycin (RAPA), which is thought to have anti-cancer effects, has been reported to decrease VEGF expression. Here, we investigated whether the CNI cyclosporine (CsA) and RAPA could alter the mRNA stability of VEGF in 786-0 and Caki-1 renal cancer cells. By using actinomycin-D, we observed that CsA increased, while RAPA decreased the VEGF mRNA stability as observed by real time PCR. It is established that the mRNA-binding protein HuR may play a critical role in VEGF mRNA stability. By using HuR-siRNA, we found that the knockdown of HuR significantly decreased the CNI-induced VEGF mRNA stability. By Western blot analysis, it has been observed that CNI treatment induced the translocation of HuR from the nucleus to the cytoplasm; CNI also induced the association between HuR and protein kinase C (PKC)-δ, and promoted the phosphorylation of HuR. Interestingly, there was no significant change in the cytoplasmic translocation of HuR following RAPA treatment. Finally, we found that the inhibition of PKC-δ using a dominant-negative plasmid significantly decreased the CsA-induced cytoplasmic translocation of HuR and the VEGF mRNA stability. Together, we suggest that targeting the pathways that promote the transcription as well as the mRNA stability (post-transcriptional modification) of VEGF in response to CNI might serve as novel therapeutics for the prevention and treatment of cancer in immunosuppressed patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-371.

Alteration of angiogenic patterns on B16BL6 melanoma development promoted in Matrigel

Because the progression and metastasis of solid tumors depend on their local microcirculation, we sought to characterize tumor angiogenesis three dimensionally in a highly metastatic mouse melanoma model, B16BL6 (B16), injected with Matrigel into the subcutis in the skin on the back of syngeneic C57BL/6 mice. We found that B16 with Matrigel grew significantly faster than B16 alone and had altered tumor angiogenesis. Tumor vessels apparently grew vigorously in the opposite direction of the tumor without invading the tumor mass until at least day 10 of injection. In addition, vascular branching resulted not only from sprouting as was seen in B16 without Matrigel but also from vascular splitting, either because of compression from outside the vessels or from septum formation by endothelial cells. This phenomenon was characteristic of B16 cells, but not of other tumor cells, including Lewis lung carcinoma and ASH-1 hybridoma cell lines, both of which were tested under the same conditions. The reduction in various angiogenic factors in Matrigel did not affect the angiogenic patterns and tumor growth. We hypothesize that tumor vessels may vigorously alter their angiogenic patterns in response to the local microenvironment.

TGF Presence During IgE-dependent Sensitization Primes Mast Cells for Higher VEGF Production After Fc RI Activation

Binding of monomeric Immunoglobulin E (IgE) to the high affinity IgE receptor (FcεRI) on mast cells induces a sensitization process which increases cell survival, augments membrane receptor expression and diminishes activation threshold. Although IgE-dependent sensitization is fundamental for allergic reactions, little is known about the influence of locally produced mediators on the outcome of a posterior allergen challenge. Since Transforming Growth Factorβ (TGFβ) is an important immunomodulator present in most of the tissues where mast cells reside, we decided to analyze the consequences of TGFβ exposure during the sensitization step of mast cells on a posterior IgE-antigen stimulation. Bone Marrow-derived Mast Cells (BMMCs) were sensitized with IgE in the presence or absence of TGFβ. Then, antigen was added and the secretion of the angiogenic cytokine Vascular Endothelial Growth Factor (VEGF) was determined. BMMCs sensitized with IgE+TGFβ showed an increased antigen-induced VEGF secretion compared to those sensitized with IgE alone. Sensitization with IgE+TGFβ did not modify membrane FcεRI receptor expression neither altered antigen-induced degranulation of the cells. Although both IgE and IgE+TGFβ sensitized cells showed an increase in VEGF mRNA stabilization after antigen addition, VEGF mRNA half-life was longer in IgE+TGFβ sensitized cells. p38 MAPK inhibitor SB202196 blocked VEGF mRNA stabilization after antigen addition specially on IgE+TGFβ sensitized cells. These findings suggest that TGFβ presence during the sensitization phase of mast cells can induce modifications to the FcεRI signal transduction system, provoking increased VEGF mRNA stabilization and protein secretion after IgE-antigen stimulation through a p38 MAPK-dependent mechanism.

A Novel Assay to Assess the Effectiveness of Antiangiogenic Drugs in Human Breast Cancer

Many cytotoxic drugs maintain antiangiogenic properties, but there are no human, tumor-based assays to evaluate their antiangiogenic potential. We used a fibrin-thrombin clot-based angiogenesis model to evaluate the angiogenic response of human breast cancer to various cytotoxic agents commonly used in its treatment. Fragments of freshly harvested human breast tumors were embedded in fibrin-thrombin clots and treated with five drugs: adriamycin, taxol, 5-fluorouracil (5-FU), methotrexate, and vincristine. Each treatment group included a mean of 28 fragments (range 16-60). A total of four tumors were tested. Tumor fragments were tested with a single dose of each reagent. Angiogenic initiation, angiogenic growth, and overall angiogenic effect were determined for each treatment group using a previously validated scale. All four breast cancer specimens tested developed an angiogenic response, sprouting neovessels in vitro in a time-dependent fashion (r = 0.84, P = 0.0007). Taxol statistically inhibited angiogenesis in all four specimens with decreases in the mean angiogenic initiation, angiogenic growth, and overall effect that were 69%, 81%, and 94% of control values, respectively. Vincristine and 5-FU inhibited the mean overall angiogenic effect by 89% and 82% compared with control, respectively. Adriamycin inhibited overall effect 49%. Methotrexate was less effective. Freshly harvested breast cancer specimens develop an angiogenic response in a fibrin-thrombin clot-based angiogenesis model and respond to treatment with antineoplastic/antiangiogenic drugs. The antiangiogenic potential of commonly used breast cancer drugs varied among individual tumors. Data obtained from this model is unique and might potentially be used to further enhance the efficacy of cytotoxic regimens and individualize patient therapy.

The role of bevacizumab in breast cancer

Neoangiogenesis and expression of the main angiogenic factor Vascular endothelial growth factor (VEGF) are critical steps already present at an early stage of breast cancer development. As tumor growth progresses other pro-angiogenic factors, including bFGF, TGFa and IL-8, are overexpressed and act in addition to VEGF. VEGF expression has been associated to poor prognosis and therapy outcome in breast cancer. Moreover, several studies have documented that HER-2 overexpression induces VEGF expression/secretion and neovascularization and that VEGF expression increases when tumors become resistant to treatment. On these bases the anti-VEGF drug Bevacizumab has been evaluated in the clinical setting in breast cancer patients. Recently, phase III studies have demonstrated that Bevacizumab in combination with taxanes produces a high rate of responses and increases the Progression Free survival of breast cancer patients. A large array of studies is currently ongoing with Bevacizumab in combination with chemotherapy and hormone-therapy in the metastatic, neoadjuvant and adjuvant setting.

Decreased expression of pigment epithelium derived factor (PEDF), an inhibitor of angiogenesis, in placentas of unexplained stillbirths

Normal placental vascular development depends upon the complex interactions between angiogenic inducers and inhibitors within the placenta. Alterations within the placental microenvironment can promote an imbalance in angiogenic mediators which may be associated with adverse perinatal outcomes. The purpose of this study was to investigate the placentas of infants with unexplained stillbirth as compared to live-born infants and to determine whether alterations in angiogenic inducer vascular endothelial growth factor (VEGF) or inhibitor pigment epithelium-derived factor (PEDF) are associated with altered angiogenesis, vascular remodeling and stillbirth. Placentas of 22 unexplained stillbirths and 44 age-matched live-born controls were scored for microvascular density (MVD), vasculopathy and microvascular permeability. A subset was scored for expression of angiogenic inducer VEGF and inhibitor pigment epithelium-derived factor. Stillborn placentas demonstrated higher MVD than controls (mean+SD: 116.6+/-46.3 v. 60.8+/-13.5, respectively, p<0.001). Vasculopathy was present in 10/22 (45%) stillbirths compared to 0/44 (0%) controls (p<0.001); increased vascular permeability was present in 15/22 (68%) cases and 5/44 (11%) controls (p<0.001). PEDF expression was significantly lower in stillborn placentas (1.7+/-0.3) than live-born controls (3.6+/-0.8, p<0.01) while VEGF expression was similar (3.3+/-0.7 v. 3.7+/-0.4, respectively, p>0.05). In conclusion, we found that unexplained stillbirth is associated with loss of angiogenic inhibitor PEDF, vasculopathy and heightened angiogenesis in the placenta.

Evaluation of Transforming Growth Factor and Vascular Endothelial Growth Factor Polymorphisms in Taiwan Chinese Patients with Pterygium

Pterygium is an invasive and highly vascularized growth, thought to arise from activated and proliferating limbal epithelial stem cells. Epidemiologic studies have found the increase of active angiogenic and epithelial growth factors in pterygia, and implicated that these molecules could be involved directly or indirectly in the pathogenesis of pterygia as causative factors. The aim of this study was to investigate the association of polymorphisms of transforming growth factor (TGF) and vascular endothelial growth factor (VEGF) with pterygium. A total of 133 pterygium patients and 105 volunteers without pterygium were enrolled in this study. Polymerase chain reaction based restriction fragment length polymorphism analysis was used to resolve the TGF-Beta1-509 and VEGF-460 genotypes. There was no significant difference in the allele frequency or genotype of TGF-Beta1-509 or VEGF-460 between total pterygium and the control group. No interaction between TGF-Beta1-509 and VEGF-460 was found either. These results indicate that TGF-Beta1-509 and VEGF-460 polymorphisms were not highly associated with the pathology of pterygium. However, it may still be worthwhile to continue to search for angiogenic gene polymorphisms in order to predict the development of pterygium.

Update on Clinical Data with Regimens Inhibiting Angiogenesis and Epidermal Growth Factor Receptor for Patients with Newly Diagnosed Metastatic Colorectal Cancer

As a result of the development of novel chemotherapeutic drugs and targeted biologic agents, the treatment of colon cancer has changed significantly over the past 10 years. Today, we have more active agents to use in colon cancer than ever before. The better understanding underlying the pathogenesis of this disease at the molecular level has allowed us to take advantage of 2 key pathways, the angiogenic and epidermal growth factor (EGF) signaling pathways. The combination of traditional chemotherapy drugs with agents that inhibit these pathways has led to a significant improvement in survival. At present, patients with metastatic colon cancer routinely achieve a median survival time > 2 years. The numerous agents available have made the choice of initial treatment more difficult for a newly diagnosed patient. Herein, we review the 2 main molecular targets of biologic therapy in colon cancer and examine the clinical evidence for regimens that inhibit angiogenesis and EGF receptor alone or in combination for newly diagnosed metastatic colorectal cancer.

ERK and Smads: Getting together for angiogenic sprouting

See article by Zhou et al. [8] (pages 390–399) in this issue. Sprouting of new blood vessels from preexisting capillaries is a multi-faceted and highly orchestrated process that is dependent on a finely tuned balance between pro-angiogenic and anti-angiogenic microenvironmental clues [1]. Perturbations in the equilibrium between pro-angiogenic and anti-angiogenic factors can lead to excessive or defective vascularization [1,2]. Inputs from the microenvironment impinge upon receptive endothelial cells to initiate a multi-step response characterized by degradation of the basement membrane, sprout formation, proliferation, migration along the sprouts and organization into a tubular network of vascular-like structures that ultimately, with the recruitment of mural cells, acquire the anatomical and functional characteristics of mature blood vessels [1]. Microenvironmental clues originating from angiogenic protein and lipid growth factors, tissue oxygenation, adjacent cells, or components of the extracellular matrix are processed by the endothelial cells via intracellular and intercellular signaling pathways that are temporally and spatially segregated to coordinate the initiation, progression and completion of each stage of the angiogenic response [1,3,4]. These signaling pathways, rather than following a linear path, are organized via a cascade of signaling molecules, scaffolding proteins and multi-domain adaptor proteins into highly interconnected networks that provide endothelial cells with a myriad of opportunities for signal integration and crosstalk [5–7]. … *Signal Transduction Laboratory, Methodist Research Institute, Clarian Health Partners, 1800 N. Capitol Blvd, Noyes Bldg, Room E504, Indianapolis, IN, 46202, USA. Tel.: +1 317 962 6891; fax: +1 317 962 9369. mrizzo{at}clarian.org

Inhibition of a vascular ocular tumor growth by IL-12 gene transfer

Ocular tumors such as retinoblastoma and uveal melanoma have devastating effects on vision. Patients with uveal melanoma also have low 5-year survival rates, thus new therapeutic modalities are necessary. As both retinoblastoma and uveal melanoma are highly vascular, we tested application of a gene transduction approach with a potent TH1 cytokine also endowed with strong anti-angiogenic activity, Interleukin-12 (IL-12). Gene transfer into murine 99E1 uveal melanoma-like cells, while having no effects on growth in vitro, essentially blocked subcutaneous tumor growth in vivo without evident signs of toxicity. Orthotopic intraocular injection resulted in invasive tumors that destroyed ocular architecture by the control cells while the IL-12 transduced cells rarely formed tumors. Histological analysis revealed highly invasive and angiogenic tumor growth in the controls and poorly vascularized tumors in the presence of IL-12. The tumor repression effect could be reproduced by a systemic anti-angiogenic effect, where controlateral injection of IL-12 expressing cells strongly repressed growth in tumors formed by parental 99E1 cells. This was associated with significantly lowered tumor vessel densities, a trend toward lower VEGF levels in the lesion, and significantly decreased NK cells in the parental tumors exposed to systemic IL-12. Taken together, our data suggest that IL-12 gene transfer can provide anti-angiogenic effects without toxicity and may be particularly suited for therapy of vascularized ocular tumors.

The Trouble with Having a Big Heart

When the heart is put under stress, it adapts by increasing in size, a process known as cardiac hypertrophy. After prolonged periods, however, heart failure ensues. Sano et al . examined the contribution that angiogenesis (the growth of new blood vessels) makes to the development of hypertrophy and cardiac failure. They established a model of hypertrophy by introducing a severe transverse aorta constriction (TAC) in mice. The resultant increase in pressure caused a gradual development of hypertrophy over 14 days, with an accompanying increase in the number of blood vessels in the heart, followed by a second phase (days 14 to 28), during which the continued pressure led to systolic dysfunction. Increased amounts of messenger RNA and protein for the angiogenic factor vascular endothelial growth factor (VEGF) were present in the TAC heart, peaking on day 14 and sharply decreasing by day 28. Hypertrophy results in ischemia (insufficient oxygen) in the myocardium, and the authors found that both the production and activity of hypoxia-inducible factor (HIF)-1α (a transcription factor that induces angiogenic factors under ischemic conditions) were stimulated in the hearts of TAC mice from days 3 to 14, returning to basal levels by day 28. The tumor suppressor p53 is known to bind to and promote the degradation of HIF-1α, and elevated amounts of p53 in the myocardium were observed 14 days after TAC. Producing TAC in p53-deficient mice resulted in higher HIF-1α activity, amounts of VEGF, and numbers of blood vessels than in wild-type mice, leading to better systolic function. This study establishes p53 as an important regulator of cardiac hypertrophy by inhibiting angiogenesis. M. Sano, T. Minamino, H. Toko, H. Miyauchi, M. Orimo, Y. Qin, H. Akazawa, K. Tateno, Y. Kayama, M. Harada, I. Shimizu, T. Asahara, H. Hamada, S. Tomita, J. D. Molkentin, Y. Zou, I. Komuro, p53-induced inhibition of Hif-1 causes cardiac dysfunction during pressure overload. Nature 446 , 444-448 (2007). [PubMed]

Gliostatin/thymidine phosphorylase-regulated vascular endothelial growth-factor production in human fibroblast-like synoviocytes

Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities. The purpose of this study was to elucidate whether GLS/TP is involved in the regulation of the angiogenic cytokine vascular endothelial growth factor (VEGF) in rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLSs) from patients with RA were cultured and stimulated with recombinant human GLS (rHuGLS) and interleukin (IL)-1beta. Immunohistochemistry showed that GLS/TP and VEGF were detectable in the synovial lining cells. In cultured FLSs, both VEGF mRNA and protein levels were markedly increased by rHuIL-1beta treatment. rHuGLS increased VEGF mRNA expression in a dose-dependent manner. We detected high concentrations of VEGF165 protein in culture supernatants from FLSs treated with rHuGLS (300 ng/ml), which were comparable to GLS levels found in synovial fluid of RA patients. These findings indicate that GLS/TP and VEGF have synergistic effects on angiogenesis in rheumatoid synovitis, and that GLS/TP has a role in regulating VEGF.

Thymidine Phosphorylase is Noncompetitively Inhibited by 5′-O-Trityl-Inosine (KIN59) and Related Compounds

We found that 5′-O-trityl-inosine (KIN59) inhibits recombinant bacterial (E. coli) and human thymidine phosphorylase (TPase) with an IC50 of 44 μM and 67 μM, respectively. In contrast to previously described TPase inhibitors, KIN59 does not compete with thymidine (dThd) at the pyrimidine nucleoside-binding site or with inorganic phosphate (Pi) at the phosphate-binding site of the enzyme. These findings are strongly suggestive for the presence of an allosteric binding site at the enzyme. TPase is identical to the angiogenic protein platelet-derived endothelial cell growth factor (PD-ECGF). As such, PD-ECGF stimulates angiogenesis in the chick chorioallantoic membrane (CAM) assay. This angiogenic response was completely inhibited by KIN59. Inosine did not inhibit the enzyme or the angiogenic effect of TPase, confirming that the 5′-O-trityl group in KIN59 is essential for the observed effect. Our observations indicate that allosteric sites in TPase may regulate its biological activity.

Neopterin as a Stimulator of Vascular Endothelial Growth Factor and Erythropoietin Gene Expression in an Ovarian Carcinoma Cell Line

Abstract Increases in serum neopterin concentrations are a regular finding in patients with ovarian cancer. The strong correlation between neopterin and tumor progression might be explained by a direct influence of the pteridine compound on tumor cell growth. In the present study we investigated whether neopterin affects the production of the angiogenic promotors vascular endothelial growth factor (VEGF) and erythropoietin (EPO) in the human ovarian carcinoma cell line OVCAR-3. VEGF and EPO gene expression were detected via RT-PCR. and a possible involvement of hypoxiainducible factor-1 was assessed by electrophoretic mobility shift assays. Neopterin amplified the hypoxia-induced VEGF and EPO gene expression as well as protein synthesis in a concentration-dependent manner. In addition, generation of both VEGF and EPO could be detected following incubation of cells with neopterin under normoxic conditions, too. Neopterin-induced synthesis of VEGF and EPO was preceeded by a stimulation of hypoxiainducible factor-1 uptake into the nucleus of OVCAR-3 cells. From these data we conclude that neopterin acts as a mediator of angiogenesis. Stimulation of VEGF and EPO generation in ovarian carcinoma cells may promote vascularization of the malignant tissue and thus tumor growth and development.

Myoblast transplantation for heart failure – From bench to bedside

Heart failure causes morbidity and mortality. Cell transplantation using skeletal muscle myoblast is promising for myocardial repair as it can regenerate and repair the injury. Skeletal myoblasts are unipotent progenitor cells that can be expanded and genetically modified to deliver angiogenic cytokines and growth factors to encourage angiomyogenesis. Myoblast transplantation inhibits ventricular remodelling, decreases left ventricular diastolic dimension, increases myocardial wall thickness and minimizes global ventricular dilatation in animals. Ongoing trials with skeletal myoblast transplantation show improvement in perfusion and metabolic activity. Time constraints and the problem of generating autologous skeletal myoblasts for every patient can be overcome if allogeneic skeletal myoblasts from healthy young donors can be made available. Myoblast transplantation is confronted with the problem of donor cell survival post-transplantation. Its safety and feasibility have been documented during animal and phase I studies. The only serious postoperative adverse event related to the procedure was ventricular arrhythmias. The results of phase I studies are still preliminary. Endpoint measurements highlight improvement in quality of life, reduced nitroglycerine consumption, enhanced exercise tolerance, improvement in NYHA Class and wall motion by echocardiography, and significantly reduced perfusion defects. Future directions include concerted collaborative efforts, strict inclusion and exclusion criteria, better establishment of target population. Further work needs to be done on the ideal cell type, optimal number of cells and route of administration. The most suitable time for cell transplantation after ischemic injury and optimal mode of cell delivery are evaluated. The use of cell-based techniques to assist with cardiac regeneration holds promise for the treatment of heart failure.