Functional role of vitronectin in breast cancer.

Breast Cancer is the most common form of cancer in women worldwide, impacting nearly 2.1 million women each year. Identification of new biomarkers could be key for early diagnosis and detection. Vitronectin, a glycoprotein that is abundantly found in serum, extracellular matrix, and bone, binds to integrin αvß3, and promotes cell adhesion and migration. Current studies indicate that patients with amplified vitronectin levels have lower survival rates than patients without amplified vitronectin levels. In this study, we focused on the role of vitronectin in breast cancer survival and its functional role as a non-invasive biomarker for early stage and stage specific breast cancer detection. To confirm that the expression of vitronectin is amplified in breast cancer, a total of 240 serum samples (n = 240), 200 from breast cancer patients and 40 controls were analyzed using the Reverse Phase Protein Array (RPPA) technique. Of the 240 samples, 120 samples were of African American (AA) descent, while the other 120 were of White American (WA) descent. Data indicated that there were some possible racial disparities in vitronectin levels and, differences also seen in the recurrent patient samples. Next, we tried to uncover the underlying mechanism which plays a critical role in vitronectin expression. The cellular data from four different breast cancer cell lines- MCF7, MDA-MB-231, MDA-MB-468, and HCC1599 indicated that the PI3K/AKT axis is modulating the expression of vitronectin. We believe that vitronectin concentration levels are involved and connected to the metastasis of breast cancer in certain patients, specifically based on recurrence or ethnicity, which is detrimental for poor prognosis. Therefore, in this current study we showed that the serum vitronectin levels could be an early marker for the breast cancer survival and we also determine the cellular signaling factors which modulate the expression and concentration of vitronectin.

Vitronectin from brain pericytes promotes adult forebrain neurogenesis by stimulating CNTF.

Vitronectin (VTN) is a glycoprotein in the blood and affects hemostasis. VTN is also present in the extracellular matrix of various organs but little is known about its function in healthy adult tissues. We show, in adult mice, that VTN is uniquely expressed by approximately half of the pericytes of subventricular zone (SVZ) where neurogenesis continues throughout life. Intracerebral VTN antibody injection or VTN knockout reduced neurogenesis as well as expression of pro-neurogenic CNTF, and anti-neurogenic LIF and IL-6. Conversely, injections of VTN, or plasma from VTN+/+, but not VTN-/- mice, increased these cytokines. VTN promoted SVZ neurogenesis when LIF and IL-6 were suppressed by co-administration of a gp130 inhibitor. Unexpectedly, VTN inhibited FAK signaling and VTN-/- mice had increased FAK signaling in the SVZ. Further, an FAK inhibitor or VTN increased CNTF expression, but not in conditional astrocytic FAK knockout mice, suggesting that VTN increases CNTF through FAK inhibition in astrocytes. These results identify a novel role of pericyte-derived VTN in the brain, where it regulates SVZ neurogenesis through co-expression of CNTF, LIF and IL-6. VTN-integrin-FAK and gp130 signaling may provide novel targets to induce neurogenesis for cell replacement therapies.

BPIFB1 (LPLUNC1) inhibits migration and invasion of nasopharyngeal carcinoma by interacting with VTN and VIM.

BACKGROUND: Bactericidal/Permeability-increasing-fold-containing family B member 1 (BPIFB1, previously termed LPLUNC1) is highly expressed in the nasopharynx, significantly downregulated in nasopharyngeal carcinoma (NPC), and associated with prognosis in NPC patients. Because metastasis represents the primary cause of NPC-related death, we explored the role of BPIFB1 in NPC migration and invasion. METHODS: The role of BPIFB1 in NPC metastasis was investigated in vitro and in vivo. A co-immunoprecipitation assay coupled with mass spectrometry was used to identify BPIFB1-binding proteins. Additionally, western blotting, immunofluorescence, and immunohistochemistry allowed assessment of the molecular mechanisms associated with BPIFB1-specific metastatic inhibition via vitronectin (VTN) and vimentin (VIM) interactions. RESULTS: Our results showed that BPIFB1 expression markedly inhibited NPC cell migration, invasion, and lung-metastatic abilities. Additionally, identification of two BPIFB1-interacting proteins, VTN and VIM, showed that BPIFB1 reduced VTN expression and the formation of a VTN-integrin αV complex in NPC cells, leading to inhibition of the FAK/Src/ERK signalling pathway. Moreover, BPIFB1 attenuated NPC cell migration and invasion by inhibiting VTN- or VIM-induced epithelial-mesenchymal transition. CONCLUSIONS: This study represents the first demonstration of BPIFB1 function in NPC migration, invasion, and lung metastasis. Our findings indicate that re-expression of BPIFB1 might represent a useful strategy for preventing and treating NPC.

Evaluation of Vitronectin Expression in Prostate Cancer and the Clinical Significance of the Association of Vitronectin Expression with Prostate Specific Antigen in Detecting Prostate Cancer.

PURPOSE: To detect the expression of vitronectin (VTN) in the tissues and blood serum of prostate cancer (PCa) patients, and evaluate its clinical significance and to evaluate the significance of the combined assay of VTN and prostate specific antigens (PSA) in PCa diagnosis. MATERIALS AND METHODS: To detect the expression of VTN as a potential marker for PCa diagnosis and prognosis, immunohistochemistry was performed on the tissues of 32 patients with metastatic PCa (PCaM), 34 patients with PCa without metastasis (PCa), and 41 patients with benign prostatic hyperplasia (BPH). The sera were then subjected to Western blot analysis. All cases were subsequently examined to determine the concentrations of PSA and VTN in the sera. The collected data were collated and analyzed. RESULTS: The positive expression rates of VTN in the tissues of the BPH and PCa groups (including PCa and PCaM groups) were 75.61% and 45.45%, respectively (P = .005). VTN was more highly expressed in the sera of the BPH patients (0.83 ± 0.07) than in the sera of the PCa patients (0.65 ± 0.06) (P < .05). It was also more highly expressed in the sera of the PCa patients than in the sera of the PCaM patients (0.35 ± 0.08) (P < .05). In the diagnosis of BPH and PCa, the Youden indexes of PSA detection, VTN detection, and combined detection were 0.2620, 0.3468, and 0.5635; the kappa values were 0.338, 0.304, and 0.448, respectively, and the areas under the receiver operating characteristic curve were 0.625, 0.673, and 0.703 (P < .05), respectively. CONCLUSION: VTN levels in sera may be used as a potential marker of PCa for the diagnosis and assessment of disease progression and metastasis. The combined detection of VTN and PSA in sera can be clinically applied in PCa diagnosis. .

Identification of outer membrane Porin D as a vitronectin-binding factor in cystic fibrosis clinical isolates of Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is a pathogen that frequently colonizes patients with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Several pathogens are known to bind vitronectin to increase their virulence. Vitronectin has been shown to enhance P. aeruginosa adhesion to host epithelial cells. METHODS: We screened clinical isolates from the airways of CF patients and from the bloodstream of patients with bacteremia for binding of vitronectin. Two-dimensional SDS-PAGE and a proteomic approach were used to identify vitronectin-receptors in P. aeruginosa. RESULTS: P. aeruginosa from the airways of CF patients (n=27) bound more vitronectin than bacteremic isolates (n=15, p=0.025). Porin D (OprD) was identified as a vitronectin-binding protein. A P. aeruginosa oprD transposon insertion mutant had a decreased binding to soluble and immobilized vitronectin (p≤0.001). CONCLUSIONS: P. aeruginosa isolates obtained from CF patients significantly bound vitronectin. Porin D was defined as a novel P. aeruginosa vitronectin-receptor, and we postulate that the Porin D-dependent interaction with vitronectin may be important for colonization.

Propolis modulates vitronectin, laminin, and heparan sulfate/heparin expression during experimental burn healing.

OBJECTIVE: This study was aimed at assessing the dynamics of vitronectin (VN), laminin (LN), and heparan sulfate/heparin (HS/HP) content changes during experimental burn healing. METHODS: VN, LN, and HS/HP were isolated and purified from normal and injured skin of domestic pigs, on the 3rd, 5th, 10th, 15th, and 21st days following thermal damage. The wounds were treated with apitherapeutic agent (propolis), silver sulfadiazine (SSD), physiological salt solution, and propolis vehicle. VN and LN were quantified using an immunoenzymatic assay and HS/HP was estimated by densitometric analysis. RESULTS: Propolis treatment stimulated significant increases in VN, LN, and HS/HP contents during the initial phase of study, followed by a reduction in the estimated extracellular matrix molecules. Similar patterns, although less extreme, were observed after treatment with SSD. CONCLUSIONS: The beneficial effects of propolis on experimental wounds make it a potential apitherapeutic agent in topical burn management.

Targeting ανß3 and ανß5 inhibits photon-induced hypermigration of malignant glioma cells.

BACKGROUND: Sublethal photon irradiation was recently suspected to increase tumor cell motility and promote locoregional recurrence of disease. This study was set up to describe mechanisms underlying increased glioma cell migration through photon irradiation and to analyse the modifiability of photon-altered glioma cell motility by integrin inhibition. METHODS: Eight µm pore size membranes were coated with vitronectin (VN), collagen I and collagen IV. U87 and Ln229 glioma cells were analysed in migration experiments with and without radiotherapy (RT), serum stimulation and addition of monoclonal antibodies directed to human integrins ανß3 and ανß5. Quantitative FACS analysis of integrins was performed in U87 and Ln229 glioma cells following RT. Statistical analysis was performed using Student's t-test. RESULTS: Glioma cell migration is serum-dependent and can be increased by photon RT which leads to enhanced expression of Vn receptor integrins. Blocking of either ανß3 or ανß5 integrins by antibodies inhibits Vn-based migration of both untreated and photon-irradiated glioma cells. CONCLUSIONS: Peripheral glioma cells are at risk of attraction into the adjacent healthy brain by serum components leaking through the blood brain barrier (BBB). Radiation therapy is associated with upregulation of Vn receptor integrins and enhanced glioma cell migration at sublethal doses. This effect can be inhibited by specific integrin blockade. Future therapeutical benefit may be derived from pharmacological integrin inhibition in combination with photon irradiation.

Association of vitronectin and plasminogen activator inhibitor-1 levels with the risk of metabolic syndrome and type 2 diabetes mellitus. Results from the D.E.S.I.R. prospective cohort.

It was the objective of this study to investigate the relation between vitronectin and plasminogen activator inhibitor (PAI)-1 plasma levels with nine-year incidences of the metabolic syndrome (MetS) and of type 2 diabetes mellitus (T2DM). Baseline plasma concentrations of vitronectin and PAI-1 were measured in 627 healthy participants from the prospective D.E.S.I.R. cohort who subsequently developed MetS (n = 487) and T2DM (n = 182) over a nine-year follow-up (42 presented both) and who were matched with two healthy control subjects each by use of a nested case-control design. Parameters composing the MetS explained about 20% of plasma vitronectin levels. An increase of one standard deviation of vitronectin was associated with increased risks of both the MetS (odds ratio [OR] = 1.21 [1.07 - 1.37], p = 0.003) and T2DM (OR = 1.24 [1.01 - 1.53], p = 0.045). Corresponding ORs for PAI-1 levels were 1.46 [1.27 - 1.68] (p<10(-4)) and 1.40 [1.14 - 1.72] (p = 0.0012). However, the effects of vitronectin and PAI-1 levels on outcomes were not independent. The vitronectin-MetS association was restricted to individuals with low to modest PAI-1 levels (OR = 1.33 [1.14 - 1.54], p = 0.0003) while no association was observed in individuals with high PAI-1 levels (OR = 0.87 [0.68 - 1.10], p = 0.24), the test for interaction being highly significant (p = 0.0009). In conclusion, baseline plasma vitronectin is a marker of incident MetS at nine years. Its predictive ability for MetS and T2DM should not be assessed independently of PAI-1 levels.

Dual sources of vitronectin in the human lower urinary tract: synthesis by urothelium vs. extravasation from the bloodstream.

Vitronectin (VN), secreted into the bloodstream by liver hepatocytes, is known to anchor epithelial cells to basement membranes through interactions with cell surface integrin receptors. We report here that VN is also synthesized by urothelial cells of urothelium in vivo and in vitro. In situ hybridization, dideoxy sequencing, immunohistochemistry, and ELISA of urothelial cell mRNA, cDNA, tissue, and protein extracts demonstrated that the VN gene is active in vivo and in vitro. The expression of VN by urothelium is hypothesized to constitute one of several pathways that anchor basal cells to an underlying substratum and explains why urothelial cells adhere to glass and propagate under serum-free conditions. Therefore, two sources of VN in the human urinary bladder are recognized: 1) localized synthesis by urothelial cells and 2) extravasation of liver VN through fenestrated capillaries. When human plasma was fractionated by denaturing heparin affinity chromatography, VN was isolated in a biologically active form that supported rapid spreading of urothelial cells in vitro under serum-free conditions. This activity was inhibited by the matricellular protein SPARC via direct binding of VN to SPARC through a Ca(+2)-dependent mechanism. A novel form of VN, isolated from the same heparin affinity chromatography column and designated as the VN(c) chromatomer, also supported cell spreading but failed to interact with SPARC. Therefore, the steady-state balance among urothelial cells, their extracellular milieu, and matricellular proteins constitutes a principal mechanism by which urothelia are anchored to an underlying substrata in the face of constant bladder cycling.

Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells.

Although adipose tissue is an expandable and readily attainable source of proliferating, multipotent stem cells, its potential for use in regenerative medicine has not been extensively explored. Here we report that adult human and mouse adipose-derived stem cells can be reprogrammed to induced pluripotent stem (iPS) cells with substantially higher efficiencies than those reported for human and mouse fibroblasts. Unexpectedly, both human and mouse iPS cells can be obtained in feeder-free conditions. We discovered that adipose-derived stem cells intrinsically express high levels of pluripotency factors such as basic FGF, TGFbeta, fibronectin, and vitronectin and can serve as feeders for both autologous and heterologous pluripotent cells. These results demonstrate a great potential for adipose-derived cells in regenerative therapeutics and as a model for studying the molecular mechanisms of feeder-free iPS generation and maintenance.

Role of sperm alphavbeta3 integrin in mouse fertilization.

Oocyte integrins have been described as essential for fertilization. But this concept has been challenged by deletion experiments. Recently, we have shown that sperm integrin alpha6beta1 plays a determinant role in mouse gamete interaction. In this study, we demonstrate the presence of alphavbeta3 integrin by Western blot and immunofluorescence on the sperm membrane. Oocytes and/or sperm preincubations with anti-alphav or anti-beta3 antibodies were performed before in vitro fertilization on cumulus-intact and zona-free egg assays. We observed inhibitory effects on the fusion process mostly by means of sperm function. An antibody directed against vitronectin inhibited gametes fusion, whereas the presence of exogenous vitronectin increased its efficiency. We suggest that vitronectin (on multimeric forms) can play a first nonspecific link corresponding to loosely bound spermatozoa to oocyte and that this link could be mediated by means of oocyte proteoglycans or integrins, and sperm alphavbeta3 integrin.

Increased vitronectin production by complement-stimulated human retinal pigment epithelial cells.

PURPOSE: A variation in the complement factor H gene was associated with an enhanced risk to develop especially early age-related macular degeneration. Drusen and basal laminar deposits are hallmarks of this AMD manifestation that contain vitronectin as a major component. In this study, the correlation between complement stimulation and vitronectin production of retinal pigment epithelial (RPE) cells was investigated. METHODS: ARPE-19 cells, a permanent cell line of human RPE cells, were supplemented with and without human complement competent serum in medium with and without heat inactivated fetal calf serum. The cells were examined in situ for their vitronectin production as an effective inhibitor of alternatively activated complement by immunohistochemistry. Semi-quantitative RT-PCR and Western blots were performed to analyze vitronectin mRNA and protein. RESULTS: A strong immunohistochemical staining for vitronectin was observed after complement supplementation. The enhanced production of this complement inactivator by ARPE-19 cells was confirmed by Western blot, whereas the expression analysis revealed unaltered mRNA amounts. CONCLUSIONS: A stimulation of RPE cells with complement resulted in an upregulated production of vitronectin. This may support the concept of a protective mechanism, since vitronectin is the major inhibitor of complement activated by the alternative pathway. On the other hand, this increased vitronectin production after complement stimulation may contribute to focal or diffuse deposits in Bruch's membrane, as observed in early AMD.

[The regulation of mast cell migration. Part 1: cell adhesion molecules]. / Regulacja migracji komórek tucznych. Czesc 1: czasteczki adhezji miedzykomórkowej.

Mast cells take part in multiple pathological processes, in some of which mast cell accumulation is central to pathogenesis. They are also vital factors in many physiological reactions. Therefore it seems to be of great importance to understand the mechanisms underlying mast cell migration into and within tissues. There are many factors that regulate the migration of mast cell progenitors from the blood into tissues and the migration of mature mast cells within tissues, leading to the rapid local accumulation that occurs in diverse pathological conditions. Without any doubt, cell-surface adhesion molecules are central to the migratory process, as they facilitate the binding of cells to extracellular matrix (ECM) proteins. Immature and mature mast cells express different adhesion molecules, especially integrins, that are involved in mast cell adhesion to such ECM proteins as laminin, fibronectin, vitronectin, and collagens. The expression of adhesion molecules alters during mast cell development and maturation. What is more, mast cell adhesion molecule expression and mast cell adhesion to ECM proteins may be regulated by some cytokines.

Deoxyribonucleic acid methyl transferases 3a and 3b associate with the nuclear orphan receptor COUP-TFI during gene activation.

Transcriptional activation of silent genes can require the erasure of epigenetic marks such as DNA methylation at CpGs (cytosine-guanine dinucleotide). Active demethylation events have been observed, and associated processes are repeatedly suspected to involve DNA glycosylases such as mCpG binding domain protein 4, thymine DNA glycosylase (TDG), Demeter, and repressor of silencing 1. A complete characterization of the molecular mechanisms occurring in metazoan is nonetheless awaited. Here, we report that activation of the endogenous vitronectin gene in P19 cells by the nuclear receptor chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is observed in parallel with the recruitment of TDG and p68 RNA helicase, two components of a putative demethylation complex. Interestingly, when activated, the vitronectin gene was loaded with DNA methyltransferases 3a and 3b (Dnmt3a/b), and a strand-biased decrease in CpG methylation was detected. Dnmt3a was further found to associate with COUP-TFI and TDG in vivo, and cotransfection experiments demonstrated that Dnmt3a/b can enhance COUP-TFI-mediated activation of a methylated reporter gene. These results suggest that Dnmt3a/b could cooperate with the orphan receptor COUP-TFI to regulate transcription of the vitronectin gene.

Changes in glycosylation of vitronectin modulate multimerization and collagen binding during liver regeneration.

Elucidating the mechanisms and factors regulating multimerization is biologically important in order to modulate the biological activities of functional proteins, especially adhesive proteins in the extracellular matrix (ECM). Vitronectin (VN) is a multifunctional glycoprotein present in plasma and ECM. Linkage of cellular adhesion and fibrinolysis by VN plays an essential role during tissue remodeling. Our previous study determined that the collagen-binding activity of VN was markedly enhanced with the decreased glycosylation during liver regeneration. This study demonstrated how alternations of glycans modulate the biological activity of VN. Human and rat VNs were used because of their similarities in structure and activities. The binding affinity of human VN to immobilized collagen was shown to be higher at pH 4.5 than at 7.5, at 37 degrees C than at 4 degrees C. Sedimentation velocity studies indicated that the greater the multimerization of human VN, the better it bound to collagen. The results indicate that the collagen binding of VN was modulated through its multimerization. Stepwise trimming of glycan with various exoglycosidases increased both the multimer size and the collagen binding of human VN, indicating that they are modulated by changes in glycosylation. The multimer sizes of VN purified from plasma of partially hepatectomized (PH) rats and sham-operated (SH) rats increased by about 45 and 31%, respectively, compared with those of nonoperated (NO) rats. In accordance with this, PH-VN exhibited remarkably enhanced collagen binding than SH-VN and NO-VN on surface plasmon resonance. In the PH rat sera, the multimer VN was increased in both amount and size compared with those in SH- and NO-sera. The results demonstrate that glycan alterations during tissue remodeling induce increased multimerization state to enhance the biological activity of VN.

Chimeric vitronectin:insulin-like growth factor proteins enhance cell growth and migration through co-activation of receptors.

Complexes comprised of IGF-I, IGF-binding proteins and the ECM protein vitronectin (VN) stimulate cell migration and growth and can replace the requirement for serum for the ex vivo expansion of cells, as well as promote wound healing in vivo. Moreover, the activity of the complexes is dependent on co-activation of the IGF-I receptor and VN-binding integrins. In view of this we sought to develop chimeric proteins able to recapitulate the action of the multiprotein complex within a single molecular species. We report here the production of two recombinant chimeric proteins, incorporating domains of VN linked to IGF-I, which mimic the functions of the complex. Further, the activity of the chimeric proteins is dependent on co-activation of the IGF-I- and VN-binding cell surface receptors. Clearly the use of chimeras that mimic the activity of growth factor:ECM complexes, such as these, offer manufacturing advantages that ultimately will facilitate translation to cost-effective therapies.

Induction of vitronectin and integrin alphav in the retina after optic nerve injury.

PURPOSE: Vitronectin is a secreted glycoprotein present in blood plasma and is present in the extracellular matrix of many tissues. It was found in the retinal cDNA library that contains genes whose expression is upregulated after optic nerve injury in a previous study. The purpose of this study was to assess the temporal and spatial changes in expression of vitronectin and integrin alphav in the retina following optic nerve injury. METHODS: Adult Balb/c mice underwent crush of the optic nerve in one eye only. RT-PCR was used to determine the temporal expression of vitronectin mRNA in the retina after injury. In addition, expression at the protein level in the retina and the optic nerve of vitronectin and its major receptor subunit, integrin alphav, was analyzed by immunohistochemistry. RESULTS: Upregulation of vitronectin mRNA in the retina was detected at one day after injury, peaking at three days, and maintained up to one week. An elevated expression of vitronectin protein was also observed in the inner retina, optic nerve head, and the optic nerve after nerve crush. In the inner retina, the increased expression of vitronectin was found in retinal ganglion cells (RGCs) and its surrounding extracellular matrix. Expression of integrin alphav was also increased in the RGC layer and in the glial cells of the nerve head and the crush site. CONCLUSIONS: As vitronectin is an extracellular protein that can support cell attachment and promote neurite extension, elevated expression of vitronectin and its receptor may facilitate axonal regeneration following injury. We propose that treatment sustaining secretion of endogenous vitronectin or direct application of exogenous vitronectin may be a method to augment regeneration of the severed optic axons.

Expression of angiostatin, integrin alphavbeta3, and vitronectin in human lungs in sepsis.

Angiostatin, integrin alphavbeta3, and vitronectin play important roles in inflammation. However, there is very little information on expression of these molecules in the lungs of humans with sepsis. Therefore, as a first step to eventually study the function of these molecules, the authors conducted an immunohistochemical study to evaluate their expression in lungs of normal (N = 8) and sepsis patients (N = 8). In normal lungs, angiostatin expression was minimal in the alveolar septa and alveolar macrophages, and absent in large blood vessels, bronchioles, and interstitium. In sepsis patients, the staining was intense in the septa, neutrophils, alveolar macrophages, and large blood vessels. Integrin alphavbeta3 staining was observed in occasional bronchiolar epithelial cells and a few alveolar macrophages in the normal lungs. The integrin was expressed extensively and intensely in bronchiolar epithelium and alveolar macrophages, and with lesser intensity in large blood vessels in inflamed lungs. Compared to the normal lung, vitronectin expression was increased in alveolar macrophages and in vascular smooth muscles in inflamed lungs. These data show cell-specific increase in the expression of integrin alphavbeta3, angiostatin, and vitronectin in inflamed lungs of sepsis patients. Because all these molecules can have significant influence on inflammation, the data reported in this manuscript create a need for further investigation.

Expression, production, and characterization of full-length vitronectin in Escherichia coli.

Vitronectin (VN) is one of the primary adhesive proteins in serum and serves to promote the attachment and spreading of a wide variety of cell types to tissue culture plastic. In this study, the pGEX2t expression vector was used to express full-length human VN as a GST-tagged fusion protein in Escherichia coli. GST/VN production was induced with IPTG and the protein was found to localize to inclusion bodies. The inclusion bodies were isolated from cell lysates, washed once with 2 M urea and Triton X-100, and then solubilized with 8 M urea in the presence of a reducing compound. Solubilized GST/VN was purified by heparin affinity chromatography and refolded by dialysis against phosphate buffered saline. Approximately 40 mg of GST/VN was recovered from 1L of bacterial culture. Purified GST/VN migrated at the predicted molecular mass on SDS-PAGE and was recognized by both anti-GST and anti-VN antibodies. GST/VN bound to heparin and promoted cell adhesion, spreading, and growth to a similar extent as that observed with plasma-derived VN. As such, the production of recombinant VN in bacteria represents a rapid and convenient method to produce large quantities of VN for cellular studies.

Heavy ion irradiation inhibits in vitro angiogenesis even at sublethal dose.

Angiogenesis is essential for tumor growth and metastasis. Because endothelial cells are genetically stable, they rarely acquire resistance to anticancer modalities, and could, thus, be a suitable target for radiation therapy. Heavy ion radiation therapy has attracted attention as an effective modality for cancer therapy because of its highly lethal effects, but the effects of heavy ion irradiation on in vitro cell function associated with angiogenesis have not been reported. Our study found that in vitro angiogenesis was inhibited by high linear energy transfer carbon ion irradiation even at sublethal dose (0.1 Gy). ECV304 and HUVEC human umbilical vascular endothelial cells were irradiated with 290 MeV carbon ion beams of approximately 110 keV/ micro m or 4 MV X-ray of approximately 1 keV/ micro m. Their adhesiveness and migration to vitronectin or osteopontin were inhibited, and capillary-like tube structures in three-dimensional culture were destroyed after carbon ion irradiation concomitant with the inhibition of matrix metalloproteinase-2 activity, down-regulation of alphaVbeta3 integrin, which is one of the adhesion molecules, slight up-regulation of membrane type1- matrix metalloproteinase, and significant up-regulation of tissue inhibitor of metalloproteinase-2. On the other hand, sublethal X-ray irradiation promoted migration of endothelial cells, and the capillary-like tube structure in three-dimensional culture progressed even after 16 Gy irradiation. These results provide an implication that heavy ion beam therapy could be superior to conventional photon beam therapy in preventive effects on in vitro angiogenesis even at sublethal dose, and might inhibit angiogenesis in vivo.