Bovine Capillary Endothelial Cells Research Articles

Abstract 2848: Preclinical activity of OM-RCA-01, a humanized anti-FGFR1 antibody, in renal cell carcinoma (RCC)

Abstract Background: Fibroblast growth factor (FGF) receptor 1 (FGFR1) is a potential therapeutic target for treatment of metastatic RCC. Early we reported that anti-FGFR1 monoclonal antibody inhibited ligand-induced phosphorylation of FGFR1, activation of MAP kinase and Akt downstream signaling pathways in FGFR1 expressing endothelial (bovine adrenal cortex capillary endothelial cells) and human RCC (Caki-1) lines (Tsimafeyeu et al. AACR/JCA joint conference 2010). Today we investigated the preclinical activity of OM-RCA-01, a novel therapeutic humanized anti-FGFR1 antibody with high affinity (Kd of 1.59 nM), in RCC. Methods: To assess the effect of anti-FGFR1 antibody on FGF-mediated signaling, the human renal carcinoma Caki-1 FGFR1-expressing cells were incubated (0.5% FBS) and were dosed with OM-RCA-01 at 100, 10, and 1 mcg/ml. Control wells were left untreated. Three hours after dosing, basic FGF was added at a concentration of 50 ng/ml. Additional control wells were treated with OM-RCA-01 without FGF-stimulation. Cell growth inhibition was determined using Promega's Cell Titer-Glo® assay. Charles River female NCr nu/nu mice (6-12 weeks of age) were set up with 1 mm3 Caki-1 tumor fragments sc in flank. Tumor sizes were measured in a blind fashion twice a week with a vernier caliper. Mice with established tumors (an average size of 80 - 120 mg) were randomly divided into vehicle, non-specific IgG or OM-RCA-01 groups per 10 animals in group. Animals were treated with antibody using different doses (1 or 10 mg/kg). Endpoint was significant differences in tumor growth delay. Results: In vitro study showed that basic FGF significantly increased proliferation of the human FGFR1-expressing renal carcinoma cells (P=0.011). No effect of basic FGF on cell line proliferation was observed when the cells were incubated with OM-RCA-01 antibody at any concentrations up to 100 mcg/kg in comparison with FGF-untreated control (P=0.855). In vivo, the tumors in untreated mice or mice treated with non-specific IgG continued their aggressive growth to reach the size of 2000 cm3, at which point the mice were killed. In contrast, treatment with OM-RCA-01 not only significant arrested further growth of the tumors (P=0.006) but also demonstrated differences in tumor volume compared with vehicle already on Day 13. A similar anti-tumor activity of OM-RCA-01 was observed when the antibody was given in low (1 mg/kg) or high (10 mg/kg) doses (P=0.917). Conclusions: High-affinity humanized anti-FGFR1 antibody OM-RCA-01 suppressed ligand-induced proliferation of human RCC cells in vitro. OM-RCA-01 has significant early anti-tumor efficacy in Caki-1 xenograft model. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2848. doi:1538-7445.AM2012-2848

Effects of dynein on microtubule mechanics and centrosome positioning

To determine forces on intracellular microtubules, we measured shape changes of individual microtubules following laser severing in bovine capillary endothelial cells. Surprisingly, regions near newly created minus ends increased in curvature following severing, whereas regions near new microtubule plus ends depolymerized without any observable change in shape. With dynein inhibited, regions near severed minus ends straightened rapidly following severing. These observations suggest that dynein exerts a pulling force on the microtubule that buckles the newly created minus end. Moreover, the lack of any observable straightening suggests that dynein prevents lateral motion of microtubules. To explain these results, we developed a model for intracellular microtubule mechanics that predicts the enhanced buckling at the minus end of a severed microtubule. Our results show that microtubule shapes reflect a dynamic force balance in which dynein motor and friction forces dominate elastic forces arising from bending moments. A centrosomal array of microtubules subjected to dynein pulling forces and resisted by dynein friction is predicted to center on the experimentally observed time scale, with or without the pushing forces derived from microtubule buckling at the cell periphery.

Beta-35 is a transferrin-derived inhibitor of angiogenesis and tumor growth

An angiogenesis inhibitor named Beta-35 has been identified and purified from the conditioned medium of mouse pancreatic β cells tumor cells. Beta-35 has a molecular weight of 35kDa and inhibits DNA synthesis of bovine capillary endothelial cells at a half-maximal concentration of approximately 5nM. It shows anti-angiogenic activity in the chick embryo chorioallantoic membrane at a dose of about 1μg/embryo. Amino acid microsequencing and mass spectrometric analysis of the purified protein demonstrate that Beta-35 contains the first 314 residues of the N-terminal sequence of bovine transferrin. We have cloned and expressed this protein in Escherichia coli using the corresponding gene segment of Beta-35 contained in the cDNA of human transferrin. The recombinant protein of Beta-35 shows significant anti-tumor activity at a dose of 5mg/kg/day against human pancreatic cancer or melanoma implanted subcutaneously in SCID mice.

Ultra-rapid activation of TRPV4 ion channels by mechanical forces applied to cell surface β1 integrins

Integrins are ubiquitous transmembrane mechanoreceptors that elicit changes in intracellular biochemistry in response to mechanical force application, but these alterations generally proceed over seconds to minutes. Stress-sensitive ion channels represent another class of mechanoreceptors that are activated much more rapidly (within msec), and recent findings suggest that calcium influx through Transient Receptor Potential Vanilloid-4 (TRPV4) channels expressed in the plasma membrane of bovine capillary endothelial cells is required for mechanical strain-induced changes in focal adhesion assembly, cell orientation and directional migration. However, whether mechanically stretching a cell's extracellular matrix (ECM) adhesions might directly activate cell surface ion channels remains unknown. Here we show that forces applied to beta1 integrins result in ultra-rapid (within 4 msec) activation of calcium influx through TRPV4 channels. The TRPV4 channels were specifically activated by mechanical strain in the cytoskeletal backbone of the focal adhesion, and not by deformation of the lipid bilayer or submembranous cortical cytoskeleton alone. This early-immediate calcium signaling response required the distal region of the beta1 integrin cytoplasmic tail that contains a binding site for the integrin-associated transmembrane CD98 protein, and external force application to CD98 within focal adhesions activated the same ultra-rapid calcium signaling response. Local direct strain-dependent activation of TRPV4 channels mediated by force transfer from integrins and CD98 may therefore enable compartmentalization of calcium signaling within focal adhesions that is critical for mechanical control of many cell behaviors that underlie cell and tissue development.

Increased transendothelial permeability of anti-cancer agent carboplatin with the aid of hyperbaric oxygenation

1. The objective was to investigate the transport of an anticancer agent carboplatin across the blood–brain barrier in combination with hyperbaric oxygenation treatment. An in vitro well-validated model of bovine brain capillary endothelial cells was used.2. A transendothelial transport of doxorubicin, a known P-glycoprotein substrate, was enhanced 1.5-fold by verapamil for 2-h incubation at 37°C. A transendothelial permeability coefficient of carboplatin (1.29 × 10−3 cm min−1) was also increased 1.8-fold by verapamil.3. Under the hyperbaric oxygenation conditions (at 0.2 MPa for the first 10 min), the transendothelial transport for 2 h of doxorubicin and carboplatin were increased 1.3- to 1.8-fold by hyperbaric oxygenation, like the suppressive effects of verapamil on P-gp function, without increase of the transport of lucifer yellow, a non P-glycoprotein substrate.4. Combination of hyperbaric oxygenation treatment and verapamil could not further increase the permeability coefficients of these drugs that were already enhanced by either treatment, implying the P-glycoprotein-mediated carboplatin efflux transport similarly as doxorubicin.5. Together with our reported high efficacy of carboplatin combined with hyperbaric oxygenation therapy on brain tumours, the present results suggest that carboplatin could be transported by P-glycoprotein, but that this efflux mechanism would be reduced by the hyperbaric oxygenation with the consequences of clinical efficacy.

Effect of Synthetic Peptides Derived from Human Apo(a) on Cell Proliferation, VEGF Production, and Angiogenesis in Cancer Cells and Vascular Endothelial Cells.

Angiogenesis, the formation of new blood vessels from the existing vascular bed, has been described as one of the hallmarks of cancer, and plays an essential role in tumor growth, invasion, and metastasis. The development of specific anti-angiogenic agents is an attractive therapeutic approach for the treatment of cancer. Apolipoprotein, Apo(a), is a large glycoprotein with multiple kringle structures, which play an important role in regulating the intermolecular reaction in the body. The evidence shows that Apo(a) has anti-angiogenic activity on vascular endothelial cells and inhibits tumor growth in mice. The 38th kringle on Apo(a) has been found to be homologous to the kringle 5 of plasminogen, a known endogenous anti-angiogenic kringle structure. According to our preliminary study, the 38th kringle of Apo(a) had an inhibitory effect on bovine capillary endothelial (BCE) cells. Seven peptides of the 38th kringle on Apo(a) were synthesized for screening of anti-angiogenic activity. Two synthetic peptides (P4 and P5) based on the 38th kringle structure sequence of Apo(a) were found to inhibit the proliferation of BCE cells. In the current study, we examined the effects of P4 and P5 synthetic peptides on cell proliferation by MTT assay, and VEGF production by ELIAS assay in human prostate cancer cells (C4-2B, LNCap, and PC3), and/or vascular endothelial cells such as human aortic endothelial cells (HAEC), human umbilical vein endothelial cell (HUVEC), and mouse microvascular endothelial cells (MVEC) after 2 days of incubation. The results are shown in the Table 1 and 2 below.Table 1Effect of synthetic peptides on cell proliferation in cancer and vascular endothelial cells (%)P4P5HAEC64.7±16.462.4±12.0HUVEC59.4±3.771.7±1.2MVEC76.9±6.175.8±5.6C4-2B64.1±5.160.9±4.3LNCap77.9±2.951.8±1.6PC375.8±3.475.4±3.6Cell proliferation assay was conducted by MTT methos. The % of cell proliferation in peptide treatment groups was compared to the cells without peptide treatmentTable 2Effect of synthetic peptides on VEGF production in cancer cells (pg/mL)ControlP4P5C4-2B905.0±87.7694.1±99.1720.1±106.2LNCap1116.5±191.6717.9±100.1794.3±119.7PC3902.6±52.0743.2±123.7721.3±92.7Our data indicate that these synthetic peptides inhibited the cell proliferation and VEGF production. We also examined the effect of P4 and P5 peptides on angiogenesis in HAEC and MVEC cells after 10h of incubation by Matrigel tube formation assay. The data indicate that angiogenesis declined by 40–50% when P4 or P5 was added to the cell culture. All these data suggest that P4 and P5, synthetic peptides derived from the 38th Kringle of Apo(a) have anti-proliferative and anti-angiogenic effects, which may potentially be applicable for cancer treatment. Further studies are in progress in order to define the mechanisms action of these peptides.

Indole-3-carbinol (I3C) exhibits inhibitory and preventive effects on prostate tumors in mice

Prostate cancer (PC) is the most commonly diagnosed malignancy for men in Western countries. Research showed that cruciferous vegetables containing indole derivatives were involved in cancer prevention. This study was designed to investigate the effect of indole-3-carbinol (I3C) in cell lines and on PC tumor growth in mice when given as a therapeutic and as a preventive treatment. The effect in vitro of 13C on the viability, proliferation and apoptosis of mouse PC cell line TRAMP-C2 and on bovine capillary endothelial (BCE) cells was examined using MTT, BrdU and FACS analyses. The effect of I3C (20 mg/kg body weight) as both a therapeutic and a preventive treatment on the growth of PC cells, inoculated subcutaneously in C57BL/6 mice, was evaluated using tumor volume measurements and immunohistochemistry. I3C decreased the proliferation rate in 3-folds (staining to Ki-67), and promoted apoptosis (staining with caspase 3). I3C, injected intraperitonially (I.P.), significantly inhibited the tumor growth (a 78% decrease in tumor volume) and affected the angiogenesis process by decreasing the microvessel density (CD31 endothelial marker) and complexity. I3C has a significant inhibitory effect on PC cells in vitro and in vivo, and offers a potential usage as both preventive and therapeutic agent for humans.

Effects of D- and L-Glucose and Mannitol on Retinal Capillary Cells: Inhibition by Nanomolar Aminoguanidine

Hyperglycemia may contribute directly to pericyte loss and capillary leakage in early diabetic retinopathy. To elucidate relative contributions of glycation, glycoxidation, sugar autoxidation, osmotic stress and metabolic effects in glucose-mediated capillary damage, we tested the effects of D-glucose, L-glucose, mannitol and the potentially protective effects of aminoguanidine on cultured bovine retinal capillary pericytes and endothelial cells. Media (containing 5 mM D-glucose) were supplemented to increase the concentration of each sugar by 5, 10, or 20 mM. Subconfluent pericytes and endothelial cells were exposed to the supplemented media in the presence or absence of aminoguanidine (1 nM-100 µM) for three days. Cell counts, viability and protein were determined. For both cell types, all three sugars produced concentration-dependent decreases in cell counts and protein content (p

Effect of benfotiamine on bovine retinal capillary endothelial cells cultured in high concentration of glucose

目的 研究苯磷硫胺对体外高糖环境中培养的牛视网膜毛细血管内皮细胞的生长及细胞凋亡的影响.方法 采用MTT法观察不同浓度的苯磷硫胺对高糖培养的牛视网膜毛细血管内皮细胞增生的影响,采用流式细胞分析技术观察选定浓度的苯磷硫胺对高糖环境中培养的牛视网膜毛细血管内皮细胞的凋亡的影响.结果 MTT法显示,一定浓度的苯磷硫胺可以降低高糖对牛视网膜毛细血管内皮细胞生长的抑制作用,且呈浓度相关性.在浓度为75 μmol/L时OD值差异有统计学意义(P＜0.01).流式细胞分析技术显示,75 μmol/L的苯磷硫胺可明显减少牛视网膜毛细血管内皮细胞的凋亡.结论 一定浓度的苯磷硫胺对体外高糖环境中培养的牛视网膜毛细血管内皮细胞具有明显的保护作用,其机制可能在于苯磷硫胺能减少细胞凋亡。

Recombinant human prothrombin kringle-2 inhibits B16F10 melanoma metastasis through inhibition of neovascularization and reduction of matrix metalloproteinase expression

Angiogenesis, a multi-step process which involves endothelial cell proliferation, adhesion, migration, and basement membrane (BM) degradation, is essential for tumor metastasis. Here we show that recombinant human prothrombin kringle-2 (rk-2) inhibited bovine capillary endothelial cell migration with an IC(50) (concentration for half maximal inhibition) of 38 nM and inhibited adhesion to extracellular matrix (ECM) proteins. Because tumor metastasis requires angiogenesis, we examined whether rk-2 could inhibit metastases induced by injection of B16F10 melanoma cells into mice. The results revealed that the metastatic tumors in mouse lung were markedly decreased in a dose-dependent manner and acute lung injury induced by B16F10 melanoma metastasis was diminished by systemic rk-2 treatment. In immunohistochemical analysis, rk-2 reduced expression of vascular endothelial growth factor, which is a potent angiogenic activator and neovascularization in the mouse lung. Also, rk-2 diminished the expression of matrix metalloproteinase-2 and -9 in the mouse lung which induces tumor metastasis and angiogenesis. These data suggest that inhibition of B16F10 melanoma metastasis by rk-2 was caused by inhibition of neovascularization and reduction of matrix metalloproteinase expression.

Liposome-mediated gene transfer of K1-5 suppresses tumor development and improves the prognosis of hepatocellular carcinoma in mice

It has been reported that kringle 1-5 (K1-5) has a potent and specific antiangiogenic activity. In the present study, we investigated the antitumor effect of gene transfer of K1-5 for hepatocellular carcinoma in mice. Inhibitory effect by the media of Cos-1 cells containing K1-5 on bovine capillary endothelial (BCE) cell proliferation was evaluated by a tetrazolium-based assay. For tumor growth, intrahepatic metastasis, and survival studies, intravenous injection of liposome-K1-5 cDNA complexes was performed to nude mice implanted with three hepatoma cell lines into the liver. Production of K1-5 was investigated by immunohistochemistry and Western blotting. The number of vessels in the tumor was counted in 0.125 mm2 fields. Expression of vascular endothelial growth factor (VEGF) and angiopoietin (Ang)-1 and -2 in tumors was investigated by Western blotting. Serum ALT levels and body weight of the mice were measured. Proliferation of BCE cells was inhibited by 44% in the media containing K1-5. Gene transfer of K1-5 suppressed tumor growth of the three hepatoma cell lines, respectively. In the K1-5-treated group, survival period was prolonged and the number of intrahepatic metastases was reduced. Expression of K1-5 protein was detected on hepatoma cells and hepatocytes. The number of vessels in tumor tissues was decreased by K1-5 transfection. Expression of angiopoietin-2 in tumor tissues was suppressed by K1-5 transfection. Serum ALT levels and body weight of mice were not influenced by K1-5 transfection. These findings suggest that antiangiogenic gene therapy with K1-5 cDNA will be a safe and effective strategy to suppress the growth of hepatocellular carcinoma.

Gene Transfer of Kringle 1–5 Suppresses Tumor Development and Improves Prognosis of Mice With Hepatocellular Carcinoma

Recent studies indicate that kringle 1-5 has a potent and specific antiangiogenic activity. Here, we investigated the antitumor effect of kringle 1-5 gene transfer on hepatocellular carcinoma in mice. The inhibitory effect of kringle 1-5 protein on proliferation of bovine capillary endothelial cells was evaluated by a tetrazolium-based assay. To study tumor growth, intrahepatic metastasis, and survival, liposome/kringle 1-5 complementary DNA complexes were injected intravenously in nude mice preimplanted with 1 of 3 hepatoma cell lines into the liver. Production of kringle 1-5 was tested by immunohistochemistry and Western blotting. Intratumoral vessel density was quantified. Expression of vascular endothelial growth factor, angiopoietin-1, and angiopoietin-2 in tumors was examined by Western blotting. Serum alanine aminotransferase and alpha-fetoprotein levels and body weights were measured. Proliferation of bovine capillary endothelial cells was inhibited by purified kringle 1-5 in a dose-dependent manner. Gene transfer of kringle 1-5 caused a significant reduction in vessel density with suppression of tumor growth of the 3 hepatoma cell lines and serum alpha-fetoprotein levels, prolonged the survival period, and reduced the number of intrahepatic metastases. Among the analyzed angiogenic factors, kringle 1-5 reduced angiopoietin-2 expression levels. Expression of kringle 1-5 protein was detected on hepatoma cells and hepatocytes in the liver. However, it did not alter serum alanine aminotransferase levels and body weights, suggesting kringle 1-5 lacks severe side effects. Antiangiogenic gene therapy with kringle 1-5 complementary DNA is a promising safe and effective strategy for suppression of growth of hepatocellular carcinoma.

Structure–activity relationships of the human prothrombin kringle-2 peptide derivative NSA9: anti-proliferative activity and cellular internalization

The human prothrombin kringle-2 protein inhibits angiogenesis and LLC (Lewis lung carcinoma) growth and metastasis in mice. Additionally, the NSA9 peptide (NSAVQLVEN) derived from human prothrombin kringle-2 has been reported to inhibit the proliferation of BCE (bovine capillary endothelial) cells and CAM (chorioallantoic membrane) angiogenesis. In the present study, we examined the structure-activity relationships of the NSA9 peptide in inhibiting the proliferation of endothelial cells lines e.g. BCE and HUVE (human umbilical vein endothelial). N- or C-terminal truncated derivatives and reverse sequence analogues of NSA9 were prepared and their anti-proliferative activities were assessed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay. This cell proliferation assay demonstrated that both the N-terminal region and sequence orientation of NSA9 are important for inhibiting the proliferation of endothelial cells. In particular 2 C-terminal truncation derivatives of NSA9 [NSA7 (NSAVQLV) and NSA8 (NSAVQLVE)] inhibited cellular proliferation to a greater extent than did NSA9. The heptapeptide NSA7, was found to be more potent than NSA9 in inhibiting CAM angiogenesis, and tubular formation and migration of HUVE cells. In addition NSA9, NSA8 and NSA7 peptides exhibited considerable inhibitory effects on the proliferation of tumour cells such as B16F10 (murine melanoma), LLC and L929 (murine fibroblast). Also, cellular internalization studies demonstrated that NSA7 was internalized into both endothelial and tumour cells more easily than was NSA9. In conclusion, these results suggest that NSA7, residing within the full sequence of NSA9, contains the required sequence for anti-proliferative activity and cellular internalization.

Recombinant human prothrombin kringle-2 induces bovine capillary endothelial cell cycle arrest at G0–G1 phase through inhibition of cyclin D1/CDK4 complex: Modulation of reactive oxygen species generation and up-regulation of cyclin-dependent kinase inhibitors

Prothrombin is a plasma glycoprotein involved in blood coagulation and, as we have previously reported, prothrombin kringles inhibit BCE (bovine capillary endothelial) cell proliferation. To reveal the mechanism, we investigated the influence of rk-2 (recombinant human prothrombin kringle-2) on the BCE cell cycle progression and ROS (reactive oxygen species) generation using FACS (fluorescence-activated cell sorter) analysis. Cell cycle analysis showed a decrease of G(1) phase cells in cells treated with bFGF (basic fibroblast growth factor) and an increase in cells treated with rk-2, as compared with the control cells. But, the portion of the S phase was reversed. In Western blot analysis, bFGF induced cytoplasmic translocation of p21(Waf1/Cip1) and p27(Kip1) and phosphorylation of p27(Kip1) but rk-2 treatment inhibited translocation of p21(Waf1/Cip1) and p27(Kip1) from nucleus to cytoplasm and phosphorylation of p27(Kip1). Also, rk-2 induced up-regulation of p53 and nuclear p21(Waf1/Cip1) and inhibited the cyclin D1/CDK4 (cyclin-dependent kinase 4) complex. The ROS level of rk-2-treated BCE cells was increased 2-fold when compared with the control, but treatment with NAC (N-Acetyl-L: -cysteine), an anti-oxidant, decreased ROS generation about 55% as compared with the rk-2 treatment. NAC treatment also restored cell cycle progression inhibited by rk-2 and down-regulated p53 and nuclear p21(Waf1/Cip1) expression induced by rk-2.These data suggest that rk-2 induces the BCE cell cycle arrest at G(0)-G(1) phase through inhibition of the cyclin D1/CDK4 complex caused by increase of ROS generation and nuclear cyclin-dependent kinase inhibitors.

The magnitude and time-dependence of the apoptotic response of normal and malignant cells subjected to ionizing radiation versus hyperthermia

Purpose: The purpose of the study was to examine the optimal time of exposure and dose of heat and ionizing radiation that results in the killing of human cancer cells in vitro via apoptosis vs. necrosis.Materials and methods: Human mammary carcinoma, colorectal carcinoma and normal bovine capillary endothelial (BCE) cell lines were subjected to 20 Gy ionizing radiation and 6, 12, 24, and 72 h later assessed for apoptosis using detection of apoptotic bodies and caspase assays. Necrosis was detected by loss of cells from the surface and lactate dehydrogenase (LDH) release. The colorectal carcinoma cells were subjected to hyperthermia using temperatures ranging from 39 – 44°C for 5, 15 or 45 min. exposures and at varying times post-treatment, apoptosis and necrosis were measured.Results: In response to ionizing radiation, none of the cells underwent necrosis and some cell types apoptosed 24 and 72 h posttreatment. The colorectal cancer cells exhibited a steady increase of apoptosis at 6, 12, and 24 h. When these cells were exposed to 40°C for 5 min, caspases increased within 6 h and a significant fraction (50%) of cells apoptosed. If the time of exposure to 40°C was increased to 15 or 45 min, 80% and 100% of the dying cells apoptosed, respectively. A temperature of 39°C did not cause cell death even after 45 min exposures. If heat was elevated to 42 or 44°C, increased necrosis was observed with a corresponding decrease in apoptosis.Conclusions: These studies reveal time and temperature dependent in vitro cell responses to ionizing radiation and water-bath hyperthermia.

Reply from the Authors

Braet and Soon question the conclusions we have drawn in a recent review1.Ballermann B.J. Glomerular endothelial cell differentiation.Kidney Int. 2005; 67: 1668-1671Google Scholar about the relationship between caveolae and fenestrae in glomerular endothelium. They suggest that we were too hasty in stating that fenestrae do not represent fused caveolae. There is little doubt about the dynamic nature of fenestrae. Work with liver sinusoidal endothelial cells is particularly interesting because these cells retain very nice sieve plate structures in culture which can change rapidly by alterations in actin microfilament dynamics2.Braet F. Wisse E. Structural and functional aspects of liver sinusoidal endothelial cell fenestrae: A review.Comp Hepatol. 2002; 1: 1Google Scholar. Also, VEGF-A can induce endothelial fenestration. Conversely, interference with VEGF-A function in renal glomeruli, for instance, in preeclampsia3.Maynard S.E. Min J.Y. Merchan J. et al.Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia.J Clin Invest. 2003; 111: 649-658Google Scholar or with podocyte VEGF-A haploinsufficiency4.Eremina V. Sood M. Haigh J. et al.Glomerular-specific alterations of VEGF-A expression lead to distinct congenital and acquired renal diseases.J Clin Invest. 2003; 111: 707-716Google Scholar leads to an absence of glomerular fenestrae. Finally, VEGF-A seems to induce caveolin-1-containing vesicles to fuse and to form transendothelial cell channels5.Vasile E. Qu H. Dvorak H.F. et al.Caveolae and vesiculo-vacuolar organelles in bovine capillary endothelial cells cultured with VPF/VEGF on floating Matrigel-collagen gels.J Histochem Cytochem. 1999; 47: 159-167Google Scholar , 6.Yokomori H. Oda M. Yoshimura K. et al.Vascular endothelial growth factor increases fenestral permeability in hepatic sinusoidal endothelial cells.Liver Int. 2003; 23: 467-475Google Scholar. Such circumstantial evidence has suggested that fenestrae represent fused caveolae. However, in caveolin-1-deficient mice known to lack caveolae altogether7.Razani B. Engelman J.A. Wang X.B. et al.Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.J Biol Chem. 2001; 276: 38121-38138Google Scholar, we found glomerular endothelium with fenestrae that were normal in size and density, even though no caveolin-1 and no caveolae were observed at all8.Sorensson J. Fierlbeck W. Heider T. et al.Glomerular endothelial fenestrae in vivo are not formed from caveolae.J Am Soc Nephrol. 2002; 13: 2639-2647Google Scholar. In normal rat glomeruli examined for caveolin-1 localization by immunogold electron microscopy, fenestrated portions of glomerular endothelium were completely devoid of caveolin-1, similar to the report by Esser et al9.Esser S. Wolburg K. Wolburg H. et al.Vascular endothelial growth factor induces endothelial fenestrations in vitro.J Cell Biol. 1998; 140: 947-959Google Scholar for the choroid plexus endothelium. Now, our experiments leave open the possibility that fenestrae in other vascular beds could represent fused caveolae, and that caveolar precursor vesicles lacking caveolin-1 could fuse to form fenestrae. But if caveolae are defined as specialized plasma membrane invaginations stabilized by caveolins, then our findings in caveolin-deficient mice seem to prove that fenestrae in glomerular endothelium cannot represent fused caveolae.

The human prothrombin kringle-2 derived peptide, NSA9, is internalized into bovine capillary endothelial cells through endocytosis and energy-dependent pathways

Human prothrombin kringle-2 and its partial peptide, NSA9 (NSAVQLVEN), have been reported to have potent anti-angiogenic activities. Here, the internalization mechanism of NSA9 into bovine capillary endothelial (BCE) cells was examined using lactate dehydrogenase (LDH) release assay, fluorescence microscopy, and flow cytometry. LDH release assay results suggested that the integrity of the BCE cell membrane was unaffected by NSA9. Fluorescence microscopy indicated that internalized NSA9 was localized in the cytoplasm around the nucleus, and showed a punctuated fluorescence pattern, which is indicative of endocytic vesicles. Also, the cellular internalization of NSA9 is significantly inhibited by depletion of the cellular ATP pool, endocytosis inhibitors such as chloroquine and nocodazole, and incubation at low temperature (4 °C). In addition, the anti-proliferative activity of NSA9 against BCE cells was diminished in the presence of endocytosis or metabolic inhibitors. In conclusion, these results strongly suggest that NSA9 might exert its anti-proliferative activity through internalization into BCE cells by endocytosis and energy-dependent pathways.

Bacillolysin MA, a Novel Bacterial Metalloproteinase That Produces Angiostatin-like Fragments from Plasminogen and Activates Protease Zymogens in the Coagulation and Fibrinolysis Systems

We isolated a novel protease that converts plasminogen to angiostatin-like fragments (BL-angiostatins) from a culture of Bacillus megaterium A9542 through a single-step chromatography on CM-cellulose. The protease, designated bacillolysin MA (BL-MA), belongs to a family of neutral metalloproteinases based on the nucleotide sequence of its gene. At an enzyme:substrate ratio of 1:540, BL-MA cleaved human plasminogen mainly at Ser441-Val442 to form BL-angiostatin and miniplasminogen with a K(m) of 3.0 +/- 0.8 microM and a k(cat) of 0.70 +/- 0.09 s(-1). The resulting BL-angiostatins inhibited the proliferation, migration, and tube formation of vascular endothelial cells at concentrations of 1-10 microg/ml. Although BL-MA failed to activate plasminogen, it increased urokinase-catalyzed activation of plasminogen caused by production of miniplasminogen, which is highly susceptible to activation. In addition, BL-MA was active in converting prourokinase, prothrombin, coagulation factor X, and protein C to their active forms. BL-MA enhanced both the clotting of human plasma and clot dissolution in the presence of prourokinase. Thus, BL-MA affects blood coagulation and fibrinolysis systems and can be used to produce angiostatin-like plasminogen fragments and active serine proteases of human plasma.

Inhibition of vessel permeability by TNP-470 and its polymer conjugate, caplostatin

Angiogenesis inhibitors, such as TNP-470 and the nontoxic HPMA copolymer-TNP-470 (caplostatin), are emerging as a class of anticancer drugs. We report that TNP-470 and caplostatin inhibit vascular hyperpermeability of tumor blood vessels as well as that induced in mouse skin by different mediators. Treatment with TNP-470 or angiostatin for 3 days was sufficient to reduce permeability of tumor blood vessels, delayed-type hypersensitivity, and pulmonary edema induced by IL-2. TNP-470 also inhibited VPF/VEGF-induced phosphorylation of VEGFR-2, calcium influx, and RhoA activation in endothelial cells. These results identify an activity of TNP-470, that of inhibiting vessel hyperpermeability. This activity likely contributes to TNP-470's antiangiogenic effect and suggests that caplostatin can be used in the treatment of cancer and inflammation.

The methods for determining the purity and in vitro or in vivo activity of recombinant human endostatin

In order to establish the methods of high-performance liquid chromatography (HPLC) for determining the purity of recombinant human endostatin (rhEndostatin) and in vitro or in vivo activity of rhEndostatin, two columns were firstly used in HPLC analysis for determining the purity of rhEndostatin, including Waters Symmetry 300TMC (4.6 mm × 250 4mm, 5 μm) and the SuperdexTM75 HR 10/30. Cell lines, bovine capillary endothelial cells (BCEs) or human umbilical vein endothelial cells (HUVECs) expression human vascular endothelial growth factor (hVEGF) were used in method MTT or LDH as substrate, respectively. The bioactivity in vivo was assayed by the anti-tumor proliferation rate in H22 liver tumor-bearing mice. The results showed that the retention time of rhEndostatin sample was stable at 19.066 min or 11.506 min in reverse phase HPLC (RP-HPLC) or gel filtering HPLC (GF-HPLC). The stableness, repeat and recovery rates were over 99% in both methods and there was no statistical difference between these two methods (p > 0.05). In nonserum culture medium, rhEndostatin can sensitively and stably inhibit the prolifera- tion of the HUVEC cells that were transfected with plasmid encoding hVEGF. LDH sub- strate methods is the most sensitive and stable method. The anti-tumor activity in H22 tumor-bearing mice was also highly repeatable and had an inhibition rate over 50% at 20 mg kg-1 weight. As a conclusion, the RP-HPLC and GF-HPLC set up in this paper are highly repeatable, accurate and sensitive for detecting the purity of rhEndostatin. The bioactivity of rhEndostatin can be measured through detection the proliferation-inhibition on HUVECs transfectants with hVEGF in vitro or on H22 liver tumor in vivo.