VEGF is upregulated by hypoxia-induced mitogenic factor via the PI-3K/Akt-NF-κB signaling pathway

BACKGROUND: Hypoxia-induced mitogenic factor (HIMF) is developmentally regulated and plays an important role in lung pathogenesis. We initially found that HIMF promotes vascular tubule formation in a matrigel plug model. In this study, we investigated the mechanisms which HIMF enhances expression of vascular endothelial growth factor (VEGF) in lung tissues and epithelial cells. METHODS: Recombinant HIMF protein was intratracheally instilled into adult mouse lungs, VEGF expression was examined by immunohistochemical staining and Western blot. The promoter-luciferase reporter assay, RT-PCR, and Western blot were performed to examine the effects of HIMF on VEGF expression in mouse lung epithelial cell line MLE-12. The activation of NF-kappa B (NF-κB) and phosphorylation of Akt, IKK and IκBα were examined by luciferase assay and Western blot, respectively. RESULTS: Intratracheal instillation of HIMF protein resulted in significant increase of VEGF, mainly localized to airway epithelial and alveolar type II cells. Deletion of NF-κB binding sites within VEGF promoter abolished HIMF-induced VEGF expression in MLE-12 cells, suggesting that activation of NF-κB is essential for VEGF upregulation induced by HIMF. Stimulation of lung epithelial cells by HIMF resulted in phosphorylation of IKK and IκBα, leading to activation of NF-κB. In addition, HIMF strongly induced Akt phosphorylation, and suppression of Akt activation by specific inhibitors and dominant negative mutants for PI-3K, and IKK or IκBα blocked HIMF-induced NF-κB activation and attenuated HIMF-induced VEGF production. CONCLUSION: These results suggest that HIMF enhances VEGF production in mouse lung epithelial cells in a PI-3K/Akt-NF-κB signaling pathway-dependent manner, and may play critical roles in pulmonary angiogenesis

A peptide corresponding to the neuropilin-1-binding site on VEGF165 induces apoptosis of neuropilin-1-expressing breast tumour cells

There is increasing evidence that vascular endothelial growth factor (VEGF) has autocrine as well as paracrine functions in tumour biology. Vascular endothelial growth factor-mediated cell survival signalling occurs via the classical tyrosine kinase receptors Flt-1, KDR/Flk-1 and the more novel neuropilin (NP) receptors, NP-1 and NP-2. A 24-mer peptide, which binds to neuropilin-1, induced apoptosis of murine and human breast carcinoma cells, whereas a peptide directed against KDR had no effect. Both anti-NP1 and anti-KDR peptides induced endothelial cell apoptosis. Confocal microscopy using 5-(6)-carboxyfluorescein-labelled peptides showed that anti-NP1 bound to both tumour and endothelial cells, whereas anti-KDR bound endothelial cells only. This study demonstrates that NP-1 plays an essential role in autocrine antiapoptotic signalling by VEGF in tumour cells and that NP1-blockade induces tumour cell and endothelial cell apoptosis. Specific peptides can therefore be used to target both autocrine (tumour cells) and paracrine (endothelial cells) signalling by VEGF

Co-expression of vascular endothelial growth factor (VEGF) and its receptors (flk-1 and flt-1) in hormone-induced mammary cancer in the Noble rat

Vascular endothelial growth factor (VEGF) is recognized to play a predominant role in breast cancer prognosis. The action of VEGF is mediated by two high-affinity receptors with ligand-stimulated tyrosine kinase activity: VEGFR-1/flt-1 and VEGFR-2/flk-1, which are expressed mainly in vascular endothelial cells. To the best of our knowledge, no previous studies on the expression of these receptors in breast cancer cells has been made. We have established a new animal model for breast cancer, using a combination of 17β-oestradiol and testosterone as ‘carcinogens’. Taking advantage of the animal model, we have demonstrated that mammary cancer cells expressed not only high levels of VEGF but also, surprisingly, its receptors (flt-1 and flk-1) in mammary cancer cells. Intense reactivities to VEGF, flt-1 and flk-1 were observed in mammary cancer cells, especially in invasive mammary carcinoma. Western blot analysis confirmed the increase in flk-1 and flt-1 proteins in induced mammary cancers. Based on these observations, we hypothesize that in mammary cancer, VEGF regulates, in addition to endothelial proliferation and angiogenesis, also growth of cancer cells by an autocrine mechanism mediated through its receptors. To further verify this hypothesis, we investigated the correlation between cellular proliferation and the expression of VEGF, flt-1 and flk-1. Using double-labelling immunocytochemistry, we have shown a correlation between high VEGF activity and Ki-67 expression. The Ki-67 indices in the areas of strong and weak VEGF reactivities were 58.3% and 3.7% respectively. Similarly, there was also a correlation of strong flk-1 and Ki-67 reactivity. The Ki-67 indices for areas of strong and weak flk-1 reactivities were 53.9% and 3.1% respectively. On the other hand, there was a reverse correlation between flt-1 and Ki-67 activities. These results indicate that overexpression of VEGF and flk-1 is correlated with high Ki-67 index. The data, therefore, suggest that VEGF may act as an autocrine growth factor for mammary cancer cells in vivo and this autocrine regulatory role may be mediated through flk-1. The present study is the first report showing that VEGF may act as a growth stimulator for mammary cancer cells. © 1999 Cancer Research Campaig

Concave Pit-Containing Scaffold Surfaces Improve Stem Cell-Derived Osteoblast Performance and Lead to Significant Bone Tissue Formation

Scaffold surface features are thought to be important regulators of stem cell performance and endurance in tissue engineering applications, but details about these fundamental aspects of stem cell biology remain largely unclear.In the present study, smooth clinical-grade lactide-coglyolic acid 85:15 (PLGA) scaffolds were carved as membranes and treated with NMP (N-metil-pyrrolidone) to create controlled subtractive pits or microcavities. Scanning electron and confocal microscopy revealed that the NMP-treated membranes contained: (i) large microcavities of 80-120 microm in diameter and 40-100 microm in depth, which we termed primary; and (ii) smaller microcavities of 10-20 microm in diameter and 3-10 microm in depth located within the primary cavities, which we termed secondary. We asked whether a microcavity-rich scaffold had distinct bone-forming capabilities compared to a smooth one. To do so, mesenchymal stem cells derived from human dental pulp were seeded onto the two types of scaffold and monitored over time for cytoarchitectural characteristics, differentiation status and production of important factors, including bone morphogenetic protein-2 (BMP-2) and vascular endothelial growth factor (VEGF). We found that the microcavity-rich scaffold enhanced cell adhesion: the cells created intimate contact with secondary microcavities and were polarized. These cytological responses were not seen with the smooth-surface scaffold. Moreover, cells on the microcavity-rich scaffold released larger amounts of BMP-2 and VEGF into the culture medium and expressed higher alkaline phosphatase activity. When this type of scaffold was transplanted into rats, superior bone formation was elicited compared to cells seeded on the smooth scaffold.In conclusion, surface microcavities appear to support a more vigorous osteogenic response of stem cells and should be used in the design of therapeutic substrates to improve bone repair and bioengineering applications in the future

Endostatin inhibits VEGF-A induced osteoclastic bone resorption in vitro

BACKGROUND: Endostatin is a C-terminal fragment of collagen XVIII which is a component of basement membranes with the structural properties of both collagens and proteoglycans. Endostatin has a major role in angiogenesis which is intimately associated with bone development and remodeling. Signaling between the endothelial cells and the bone cells, for example, may have a role in recruitment of osteoclastic precursor cells. Our study aims at exploring a possibility that endostatin, either as a part of basement membrane or as a soluble molecule, may control osteoclastogenesis and osteoclastic bone resorption in vitro. METHODS: Rat pit formation assay was employed in order to examine the effect of endostatin alone or in combination with vascular endothelial growth factor-A (VEGF-A) on bone resorption in vitro. Effect of these agents on osteoclast differentiation in vitro was also tested. Osteoclastogenesis and the number of osteoclasts were followed by tartrate resistant acid phosphatase (TRACP) staining and resorption was evaluated by measuring the area of excavated pits. RESULTS: Endostatin inhibited the VEGF-A stimulated osteoclastic bone resorption, whereas endostatin alone had no effect on the basal resorption level in the absence of VEGF-A. In addition, endostatin could inhibit osteoclast differentiation in vitro independent of VEGF-A. CONCLUSION: Our in vitro data indicate that collagen XVIII/endostatin can suppress VEGF-A induced osteoclastic bone resorption to the basal level. Osteoclastogenesis is also inhibited by endostatin. The regulatory effect of endostatin, however, is not critical since endostatin alone does not modify the basal bone resorption

The place of VEGF inhibition in the current management of renal cell carcinoma

Vascular endothelial growth factor (VEGF) is overexpressed in around 80% of patients with clear cell carcinoma of the kidney owing to the inactivation of von Hippel Lindau gene activity. VEGF stimulates angiogenesis and acts as an autocrine growth factor. A number of different agents are now available which target VEGF and its signalling pathways. A significant body of evidence has accumulated demonstrating that antagonism of VEGF and its downstream pathways is clinically useful in a significant proportion of patients with metastatic clear cell carcinoma of the kidney. Enough data is now available to recommend that patients with metastatic clear cell carcinoma of the kidney should at some point during the course of their disease be offered entry into a clinical trial enabling exposure to a targeted inhibitor of VEGF or its signalling pathways. Assuming early clinical trial data is substantiated by ongoing registration studies, efforts should be made to minimise the time taken between licensing and general availability of these active agents

Investigating the Antiproliferative Activity of High Affinity DNA Aptamer on Cancer Cells

10.1371/journal.pone.0050964PLoS ONE81

Soluble perlecan domain i enhances vascular endothelial growth factor-165 activity and receptor phosphorylation in human bone marrow endothelial cells

<p>Abstract</p> <p>Background</p> <p>Immobilized recombinant perlecan domain I (PlnDI) binds and modulates the activity of heparin-binding growth factors, <it>in vitro</it>. However, activities for PlnDI, in solution, have not been reported. In this study, we assessed the ability of soluble forms to modulate vascular endothelial growth factor-165 (VEGF₁₆₅) enhanced capillary tube-like formation, and VEGF receptor-2 phosphorylation of human bone marrow endothelial cells, <it>in vitro</it>.</p> <p>Results</p> <p>In solution, PlnDI binds VEGF₁₆₅in a heparan sulfate and pH dependent manner. Capillary tube-like formation is enhanced by exogenous PlnDI; however, PlnDI/VEGF₁₆₅mixtures combine to enhance formation beyond that stimulated by either PlnDI or VEGF₁₆₅alone. PlnDI also stimulates VEGF receptor-2 phosphorylation, and mixtures of PlnDI/VEGF₁₆₅reduce the time required for peak VEGF receptor-2 phosphorylation (Tyr-951), and increase Akt phosphorylation. PlnDI binds both immobilized neuropilin-1 and VEGF receptor-2, but has a greater affinity for neuropilin-1. PlnDI binding to neuropilin-1, but not to VEGF receptor-2 is dependent upon the heparan sulfate chains adorning PlnDI. Interestingly, the presence of VEGF₁₆₅but not VEGF₁₂₁significantly enhances PlnDI binding to Neuropilin-1 and VEGF receptor-2.</p> <p>Conclusions</p> <p>Our observations suggest soluble forms of PlnDI are biologically active. Moreover, PlnDI heparan sulfate chains alone or together with VEGF₁₆₅can enhance VEGFR-2 signaling and angiogenic events, <it>in vitro</it>. We propose PlnDI liberated during basement membrane or extracellular matrix turnover may have similar activities, <it>in vivo</it>.</p

Expression patterns of angiogenic and lymphangiogenic factors in ductal breast carcinoma in situ

The objective of this study was to investigate expression of various growth factors associated with angiogenesis and lymphangiogenesis and of their receptors in ductal carcinomas in situ of the breast (DCIS). We studied protein expression of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF)-A, endothelin (ET)-1, and VEGF-C, and their receptors bFGF-R1, Flt-1, KDR, ETAR, ETBR, and Flt-4 immunohistochemically in 200 DCIS (pure DCIS: n=96; DCIS adjacent to an invasive component: n=104) using self-constructed tissue microarrays. Basic fibroblast growth factor-R1, VEGF-C, Flt-4, and ETAR were expressed in the tumour cells in the majority of cases, whereas bFGF and Flt-1 expression was rarely observed. VEGF-A, KDR, ET-1, and ETBR were variably expressed. The findings of VEGF-C and its receptor Flt-4 as lymphangiogenic factors being expressed in tumour cells of nearly all DCIS lesions and the observed expression of various angiogenic growth factors in most DCIS suggest that in situ carcinomas are capable of inducing angiogenesis and lymphangiogenesis. Moreover, we found a higher angiogenic activity in pure DCIS as compared to DCIS with concomitant invasive carcinoma. This association of angiogenic factors with pure DCIS was considerably more pronounced in the subgroup of non-high-grade DCIS (n=103) as compared with high-grade DCIS (n=94). Determination of these angiogenic markers may therefore facilitate discrimination between biologically different subgroups of DCIS and could help to identify a particularly angiogenic subset with a potentially higher probability of recurrence or of progression to invasiveness. For these DCIS, targeting angiogenesis may represent a feasible therapeutic approach for prevention of progression of DCIS to invasion