Günter Finkenzeller

Both v-Ha-Ras and v-Raf Stimulate Expression of the Vascular Endothelial Growth Factor in NIH 3T3 Cells

Stimulation of NIH 3T3 cells with platelet-derived growth factor (PDGF)-BB and 12-O-tetradecanoylphorbol-13-acetate (TPA) enhances vascular endothelial growth factor (VEGF) gene expression. To address the question of whether Ras and Raf are involved in the induction of VEGF gene expression by PDGF and TPA, we examined the effects of both factors on NIH 3T3 cells stably transfected with v-Ha-ras or v-raf. In serum-starved NIH 3T3 cells, only low levels of mRNA expression can be detected, whereas both ras and raf transformed cell lines express enhanced levels of a 4.3-kilobase VEGF transcript. Stimulation with PDGF or TPA resulted in increased VEGF mRNA in all cell lines, with highest levels found in the transformed cells. Immunofluorescence studies confirmed that the elevated VEGF mRNA expression correlated with enhanced protein levels. Positive immunofluorescence signals could be detected in v-Ha-ras or v-raf transformed cell lines but not in unstimulated NIH 3T3 cells. VEGF from conditioned medium of v-raf transformed NIH 3T3 cells was partially purified by chromatography on heparin-Sepharose. Biological activity of this VEGF protein was demonstrated by competition with binding of recombinant I-VEGF to human umbilical vein endothelial cells and by its ability to stimulate proliferation of these cells.

Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression

Stimulation of NIH3T3 cells with platelet-derived growth factor (PDGF)-BB enhances expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a key mediator of tumor angiogenesis. Here, we identified cis-acting VEGF promoter elements and trans-acting factors which are involved in PDGF-stimulated VEGF expression. By 5′-deletion and transient transfection analysis, a G+C-rich region at −85 to −50 of the human VEGF promoter was shown to be necessary and sufficient for both PDGF inducible and basal expression. The region contains three potential recognition sites for Sp1 transcription factors, which overlap with two Egr-1 sites. Mutations that abolish the ability of Sp1 to interact with the VEGF promoter element also abrogate expression induced by PDGF. Mutations of the potential Egr-1 binding sites did not affect PDGF responsiveness. Gel shift and antibody supershift analyses showed that Sp1 and Sp3 interact constitutively with the VEGF promoter element. Our data strongly suggest that enhanced VEGF gene expression in PDGF-induced NIH3T3 cells is mediated by Sp1 and/or Sp3 transcription factors bound to the −85 to −50 promoter region of the VEGF gene.

Inhibition of Vascular Endothelial Growth Factor Expression in HEC1A Endometrial Cancer Cells through Interactions of Estrogen Receptor α and Sp3 Proteins

Treatment of HEC1A endometrial cancer cells with 10 nm 17β-estradiol (E2) resulted in decreased vascular endothelial growth factor (VEGF) mRNA expression, and a similar response was observed using a construct, pVEGF1, containing a VEGF gene promoter insert from −2018 to +50. In HEC1A cells transiently transfected with pVEGF1 and a series of deletion plasmids, it was shown that E2-dependent down-regulation was dependent on wild-type estrogen receptor α (ERα) and reversed by the anti-estrogen ICI 182,780, and this response was not affected by progestins. Deletion analysis of the VEGF gene promoter identified an overlapping G/GC-rich site between −66 to −47 that was required for decreased transactivation by E2. Protein-DNA binding studies using electrophoretic mobility shift and DNA footprinting assays showed that both Sp1 and Sp3 proteins bound this region of the VEGF promoter. Coimmunoprecipitation and pull-down assays demonstrated that Sp3 and ERα proteins physically interact, and the interacting domains of both proteins are different from those previously observed for interactions between Sp1 and ERα proteins. Using a dominant negative form of Sp3 and transcriptional activation assays in Schneider SL-2 insect cells, it was confirmed that ERα-Sp3 interactions define a pathway for E2-mediated inhibition of gene expression, and this represents a new mechanism for decreased gene expression by E2.

Expression of the vascular endothelial growth factor gene is inhibited by p73

Recently, p73, a new member of the p53 family, has been cloned and mapped to chromosome 1p36, a region that is frequently deleted in a variety of human cancers. p73 can activate p53-responsive promoters and induce apoptosis when overexpressed in certain p53-deficient tumor cells. In contrast to p53, analysis of the p73 gene in several human solid tumors did not reveal loss of p73 expression or mutations in the p73 gene. However, transcriptional silencing of the p73 gene by hypermethylation of a CpG island was observed in several leukemias and lymphomas. These lymphoid neoplasms also show increased expression of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and a key mediator of angiogenesis. To evaluate a possible relationship between p73 status and VEGF expression, we have studied the effect of ectopically expressed p73 on the regulation of the VEGF gene. Our results demonstrate that p73 can down-regulate endogenous VEGF gene expression on mRNA and protein level. This effect is mediated by transcriptional repression of the VEGF promoter and involves the promoter region −85 to −50 bp, containing a cluster of Sp 1 binding sites. Our results suggest a regulatory role for p73 in tumor angiogenesis.

Increase of c-jun mRNA upon hypo-osmotic cell swelling of rat hepatoma cells.

c‐jun mRNA levels were increased in rat hepatoma cells (H4‐II‐E‐C3) when exposed to hypotonie medium (205 ) with a maximal induction observed after 1 h of hypotonie exposure. At this time point an approximate 5‐fold increase in c‐jun expression could be detected in relation to nonnotonic control incubations (305 ). Hypertonic exposure (405 mosmol/1) had only a slight effect on c‐jun expression. In contrast to the increased c‐jun mRNA levels under hypotonic conditions, expression of the c‐fos proto‐oncogene was unaffected by changes in the osmolarity. The hypotonicity‐induced increase in c‐jun expression was also detectable in the presence of a protein kinase C (PKC) inhibitor. This indicates that PKC is not involved in the signal transduction pathway leading to c‐jun expression upon hypotonic cell swelling in these cells.

Activated Neu/ErbB-2 induces expression of the vascular endothelial growth factor gene by functional activation of the transcription factor Sp 1.

The neu(c-erbB-2or HER2) proto-oncogene which encodes a receptor protein homologous to the epidermal growth factor receptor is overexpressed in 20%–30% of human breast and ovarian cancers. Oncogenic activation of Neu can also occur through multiple molecular mechanisms, including a point mutation in the transmembrane domain, deletion of the extracellular domain and short in-frame deletions of 7–12 amino acids in the extracellular region proximal to the transmembrane domain. Because of the highly vascularized phenotype of breast and ovarian cancers and the contribution of the Neu receptor to the development and progression of these tumors, we investigated the effect of Neu on the expression of the tumor angiogenesis factor VEGF. Expression of various activated Neu receptors but not wild-type Neu in Rat-1 cells, leads to increased VEGF expression on mRNA as well as on protein level. This effect is mediated by transcriptional activation of the VEGF promoter via a cluster of Sp 1 binding sites. Molecular analysis of the activation mechanism of Sp 1 revealed that neither the VEGF promoter binding activity of Sp 1 nor the expression of Sp 1 is affected by Neu transformation of the cells. Instead, functional Neu-induced transactivation of Sp 1 was observed by using a GAL4-based transactivation assay. These results demonstrate that functional changes of the transcription factor Sp 1 mediates a Neu-signaling cascade leading to VEGF promoter activation.

Endothelial progenitor cells from peripheral blood support bone regeneration by provoking an angiogenic response

Neovascularization is crucial for fracture healing and plays an important role in long-time graft survival in tissue engineering applications. Endothelial progenitor cells (EPCs) can be isolated from peripheral blood avoiding donor site morbidity, which makes them attractive for autologous cell-based engineering of neovessels. However, contradictory results are published concerning the vasculogenic potential of this cell type. We could previously show that implanted human endothelial vein cells (HUVECs) gave rise to the formation of a complex functional human neovasculature in a heterotopic (subcutaneous) as well as in an orthotopic (calvarial defect) model of severe combined immunodeficiency (SCID) mice, where vessel formation could even be increased by coimplanting mesenchymal stem cells (MSCs) functioning as perivascular cells. In this study, we investigated whether coimplantation of MSCs which have been predifferentiated in vitro into SMCs (SMC-MSCs) may enable pbEPCs to form blood vessels upon implantation and, if this would be the case, whether the resulting enhanced vascularization may support bone regeneration. For this purpose, pbEPCs and SMC-MSCs were mono- or cocultured in collagen matrices and seeded into scaffolds consisting of decalcified processed bovine cancellous bone (PBCB, Tutobone). Neovascularization and osteogenesis were evaluated using a calvarial bone defect-model in SCID mice. Our experiments could show that the missing vasculogenic potential of pbEPCs is not rescued by coimplantation of SMCs derived from MSCs predifferentiated along the vascular smooth muscle lineage. However, implantation of both cell types alone, or in combination induced an angiogenic response, which correlated in a positive manner with bone formation within the implants.

miR-126 regulates platelet-derived growth factor receptor-α expression and migration of primary human osteoblasts

Abstract Adequate vascularization is an essential requirement for bone development, fracture healing and bone tissue engineering. We have previously described the coculture of primary human osteoblasts (hOBs) and human endothelial cells (HUVECs), designed to investigate the interactions between these cells. In this system, we showed that cocultivation of these two cell types leads to a downregulation of platelet-derived growth factor receptor-α (PDGFR-α) in hOBs, which was a consequence of reduced mRNA stability. In the current study we investigated the possible involvement of microRNAs in this process. Firstly, we performed a microarray analysis of osteoblastic miRNAs following cocultivation with HUVECs, revealing an upregulation of miR-126. This result was confirmed by RT-qPCR, and we observed that the increase is dependent on direct cell-to-cell contacts. Gain-of-function and loss-of-function experiments showed that miR-126 is a negative regulator of PDGFR-α mRNA. Additionally, migration of hOBs was inhibited by miR-126 overexpression and stimulated by miR-126 inhibition. Addition of PDGFR-α blocking antibody to hOB culture also inhibited hOB migration. There was no effect of miR-126 modulation on osteoblast proliferation, apoptosis rate or differentiation. In conclusion, we report that the miR-126/PDGFR-α system regulates the migratory behavior of human osteoblasts, without exerting effects on cell survival and differentiation.

Over-expression of protein kinase C-α enhances platelet-derived growth factor- and phorbol ester- but not calcium ionophore-induced formation of prostaglandins in NIH 3T3 fibroblasts

Over‐expression of human protein kinase C‐α in murine NIH 3T3 fibroblasts is associated with an increased platelet‐derived growth factor‐ and phorbol ester‐mediated formation of prostaglandins, whereas the calcium ionophore‐induced release of arachidonic acid metabolites is unaffected; however, the differences of arachidonic acid and prostaglandin formation are much more pronounced with platelet‐derived growth factor than with phorbol ester. Platelet‐derived growth factor induces an identical elevation of intracellular free calcium in control and protein kinase C‐α over‐expressing cells; the phorbol ester has no effect on intracellular free calcium in both cell lines. These results demonstrate that protein kinase C‐α may couple to arachidonic acid cascade in NIH 3T3 fibroblasts.

Overexpression of hypoxia-inducible factor-1 alpha improves vasculogenesis-related functions of endothelial progenitor cells

Postnatal vasculogenesis is mediated by mobilization of endothelial progenitor cells (EPCs) from bone marrow and homing to ischemic tissues. This feature emphasizes this cell type for cell-based therapies aiming at the improvement of neovascularization in tissue engineering applications and regenerative medicine. In animal models, it was demonstrated that implantation of EPCs from cord blood (cbEPCs) led to the formation of a complex functional neovasculature, whereas EPCs isolated from adult peripheral blood (pbEPCs) showed a limited vasculogenic potential, which may be attributed to age-related dysfunction. Recently, it was demonstrated that activation of hypoxia-inducible factor-1α (Hif-1α) improves cell functions of progenitor cells of mesenchymal and endothelial origin. Thus, we hypothesized that overexpression of Hif-1α may improve the vasculogenesis-related phenotype of pbEPCs. In the present study, we overexpressed Hif-1α in pbEPCs and cbEPCs by using recombinant adenoviruses and investigated effects on stem cell- and vasculogenesis-related cell parameters. Overexpression of Hif-1α enhanced proliferation, invasion, cell survival and in vitro capillary sprout formation of both EPC populations. Migration was increased in cbEPCs upon Hif-1α overexpression, but not in pbEPCs. Cellular senescence was decreased in pbEPCs, while remained in cbEPCs, which showed, as expected, intrinsically a dramatically lower senescent phenotype in relation to pbEPCs. Similarly, the colony-formation capacity was much higher in cbEPCs in comparison to pbEPCs and was further increased by Hif-1α overexpression, whereas Hif-1α transduction exerted no significant influence on colony formation of pbEPCs. In summary, our experiments illustrated multifarious effects of Hif-1α overexpression on stem cell and vasculogenic parameters. Therefore, Hif-1α overexpression may represent a therapeutic option to improve cellular functions of adult as well as postnatal EPCs.