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At least 883 records · Page 49Linked to original sources
PubMed2006

Identification of CD63 as a tissue inhibitor of metalloproteinase-1 interacting cell surface protein.

This study identified CD63, a member of the tetraspanin family, as a TIMP-1 interacting protein by yeast two-hybrid screening. Immunoprecipitation and confocal microscopic analysis confirmed CD63 interactions with TIMP-1, integrin beta1, and their co-localizations on the cell surface of human breast epithelial MCF10A cells. TIMP-1 expression correlated with the level of active integrin beta1 on the cell surface independent of cell adhesion. While MCF10A cells within a three-dimensional (3D) matrigel matrix form polarized acinar-like structures, TIMP-1 overexpression disrupted breast epithelial cell polarization and inhibited caspase-mediated apoptosis in centrally located cells, necessary for the formation and maintenance of the hollow acinar-like structures. Small hairpin RNA (shRNA)-mediated CD63 downregulation effectively reduced TIMP-1 binding to the cell surface, TIMP-1 co-localization with integrin beta1, and consequently reversed TIMP-1-mediated integrin beta1 activation, cell survival signaling and apoptosis inhibition. CD63 downregulation also restored polarization and apoptosis of TIMP-1 overexpressing MCF10A cells within a 3D-matrigel matrix. Taken together, the present study identified CD63 as a cell surface binding partner for TIMP-1, regulating cell survival and polarization via TIMP-1 modulation of tetraspanin/integrin signaling complex.

Antigens, CD↗
PubMed1999

Angiogenesis induced in muscle by a recombinant adenovirus expressing functional isoforms of basic fibroblast growth factor.

The present work studies the effects of a replication-deficient adenovirus (Ad), Ad-RSVbFGF, bearing the human basic fibroblast growth factor (bFGF) cDNA, as a potential vector for therapeutic angiogenesis of ischemic diseases. The different isoforms of the protein were expressed from the viral vector in various cell types and, although the cytoplasmic isoform does not possess a signal peptide, we observed its release from a muscle cell line. The proteins were fully functional when tested in a long-term survival assay of quiescent fibroblasts. After endothelial cell infection with Ad-RSVbFGF, we observed an 80&percnt increase in the mean length of the capillary-like tubes that differentiated in a three-dimensional model of angiogenesis. We evaluated angiogenesis directly in mice 14 days after subcutaneous injection of Matrigel plugs containing Ad-RSVbFGF. A marked neovascularization was observed in the Matrigel plugs and in the surrounding tissues. Finally, the recombinant virus was injected into the hindlimb muscles of mdx mice. A 2.5-fold increase in bFGF content of the muscle was observed 6 days after injection, without any significant variations detected in the animal sera. Immunohistological detection showed an increased number of large-caliber vessels in the treated muscles as compared with control muscles. These results demonstrate that Ad-mediated transfer of the human bFGF gene can induce angiogenesis in muscle, making this tissue a potential target for the treatment of ischemic diseases.

Adenoviridae↗
PubMed2003

A neovascularized organoid derived from retrovirally engineered bone marrow stroma leads to prolonged in vivo systemic delivery of erythropoietin in nonmyeloablated, immunocompetent mice.

Marrow stromal cells (MSCs) are postnatal progenitor cells that can be easily cultured ex vivo to large amounts. This feature is attractive for cell therapy applications where genetically engineered MSCs could serve as an autologous cellular vehicle for the delivery of therapeutic proteins. The usefulness of MSCs in transgenic cell therapy will rely upon their potential to engraft in nonmyeloablated, immunocompetent recipients. Further, the ability to deliver MSCs subcutaneously - as opposed to intravenous or intraperitoneal infusions - would enhance safety by providing an easily accessible, and retrievable, artificial subcutaneous implant in a clinical setting. To test this hypothesis, MSCs were retrovirally engineered to secrete mouse erythropoietin (Epo) and their effect was ascertained in nonmyeloablated syngeneic mice. Epo-secreting MSCs when administered as 'free' cells by subcutaneous or intraperitoneal injection, at the same cell dose, led to a significant - yet temporary - hematocrit increase to over 70% for 55+/-13 days. In contrast, in mice implanted subcutaneously with Matrigel trade mark -embedded MSCs, the hematocrit persisted at levels >80% for over 110 days in four of six mice (P<0.05 logrank). Moreover, Epo-secreting MSCs mixed in Matrigel elicited and directly participated in blood vessel formation de novo reflecting their mesenchymal plasticity. MSCs embedded in human-compatible bovine collagen matrix also led to a hematocrit >70% for 75+/-8.9 days. In conclusion, matrix-embedded MSCs will spontaneously form a neovascularized organoid that supports the release of a soluble plasma protein directly into the bloodstream for a sustained pharmacological effect in nonmyeloablated recipients.

Animals↗
PubMed2004

Prevention of angiogenesis by naked DNA IL-12 gene transfer: angioprevention by immunogene therapy.

IL-12 is thought to induce a cytokine cascade with antiangiogenic effects mediated by IFN-gamma and angiostatic CXCR3 chemokine ligands. Naked DNA intramuscular injection of an expression vector plasmid producing IL-12 resulted in significant, well-tolerated elevation of serum IL-12 levels. Injection of the IL-12 plasmid at least 2 days, and up to 20 days, before subcutaneous injection of matrigel with angiogenic factors resulted in strong prevention of angiogenesis in both C57/bl and nude mice. A single injection of the IL-12 plasmid contemporarily with the matrigel or 2 days after resulted in partial, statistically not significant, inhibition. Control plasmid injection did not affect either angiogenesis or angiogenesis inhibition by IL-12 protein in vivo. Angiogenesis inhibition was observed in NK cell-depleted C57/bl and nude mice as well as in IFN-gamma(-/-) and CXCR3(-/-) knockout mice, indicating that NK- and/or T-cell-initiated IFN-gamma-chemokine cascades were not involved in the angiogenesis inhibition observed in vivo. Finally, IL-12 plasmid DNA gene transfer significantly prevented the growth and vascularization of highly angiogenic KS-Imm Kaposi's sarcoma and TS/A murine mammary carcinoma tumors in nude and/or syngeneic mice. These data suggest that a preventive gene therapy approach using antiangiogenic cytokines can effectively inhibit tumor angiogenesis and KS, representing an example of angioimmunoprevention.

Animals↗
PubMed2004

Telomerized presenescent osteoblasts prevent bone mass loss in vivo.

Previously, we showed that human osteoblasts expressing the human telomerase reverse transcriptase (hTERT) gene exhibited specific survival advantages--the result of breaching the replicative senescence barrier and maintaining the phenotypic and functional properties of primary osteoblasts in vitro over the total replicative capacity of primary osteoblasts. We postulated that rejuvenated osteoblasts may have a potential to correct bone loss or osteopenia in age-related osteoporotic diseases. In the present study, we studied whether telomerized presenescent osteoblasts prevent bone mass loss in vivo. After obtaining the informed consent from a patient with osteoarthritis who underwent the arthroplastic knee surgery, osteoblastic cells were isolated from donor bone sample. We transfected the gene encoding hTERT into human osteoblastic cells. Human bone fragments from a donor were incubated with human hTERT-transfected presenescent (in vitro aged) osteoblasts or mock-transfected presenescent osteoblasts in culture medium containing Matrigel. We subcutaneously implanted human bone fragments with telomerized presenescent osteoblasts or primary presenescent osteoblasts as three-dimensional Matrigel xenografts in severe combined immunodeficiency (SCID) mice (each group: six mice) and analyzed the grafts at 6 weeks after implantation. We also determined whether telomerized osteoblasts affect the bone-forming capacity in vivo, using a well-established mouse transplantation model in which ceramic hydroxyapatite/tricalcium phosphate particles are used as carrier vehicle. Telomerized presenescent osteoblasts were rejuvenated, and maintained the functional properties of young osteoblasts in vitro. Bone mineral content (BMC) and bone mineral density (BMD) were measured by ash weight and dual-energy X-ray absorptiometry, respectively. Whereas BMC and BMD of human bone fragments, which were inoculated with aged osteoblasts in SCID mice, decreased with time, telomerized presenescent osteoblasts maintained the BMC and BMD of human bone fragments, indicating that telomerized and rejuvenated osteoblasts may be functional to prevent bone mass loss in vivo. In xenogenic transplants, telomerized osteoblasts generated more bone tissue with lamellar bone structure and cellular components, than did control osteoblasts. These findings suggest that telomerized/rejuvenated presenescent osteoblasts may be used in the development of tissue engineering or cell-based therapy for bone regeneration and repair.

Animals↗
PubMed2004

Bone marrow endothelial cells increase the invasiveness of human multiple myeloma cells through upregulation of MMP-9: evidence for a role of hepatocyte growth factor.

The migration of multiple myeloma (MM) cells from the circulation into the bone marrow (BM) implicates that they must have the capacity to cross the BM endothelium including the subendothelial basement membrane. In this study, human CD138+ MM cells were immunomagnetically isolated from BM samples of MM patients and their invasion through Matrigel, that is, a reconstituted basement membrane, was determined. We demonstrated that primary MM cells have the capacity to transmigrate through basement membrane and that this invasiveness was considerably increased when assessed on Matrigel filters coated with BM endothelial cells (EC) (4LHBMEC line) (transendothelial invasion). The isolated MM cells were shown by zymography to secrete matrix metalloproteinase (MMP)-9 and anti-MMP-9 antibodies inhibited transendothelial invasion, indicating that MMP-9 is involved in this process. BM EC were found to increase the MMP-9 secretion in MM cells, indicating that EC enhance MM cell invasion through stimulation of MMP-9 secretion. BM EC were found to produce hepatocyte growth factor (HGF), and this cytokine also stimulated MMP-9 secretion in MM cells, while anti-HGF antibodies significantly inhibited EC-stimulated MM cell invasion. In summary, our findings provide evidence that MM cell-BM EC interactions enhance the invasion of human MM cells through stimulation of MMP-9 secretion.

Bone Marrow Cells↗
PubMed1997

Adenovirus-mediated p16/CDKN2 gene transfer suppresses glioma invasion in vitro.

Malignant gliomas extensively infiltrate the surrounding normal brain, and their diffuse invasion is one of the most important barriers to successful therapy. Recent studies indicate that the progression of gliomas from low-grade to high-grade may depend on the acquisition of a new phenotype and the subsequent addition of genetic defects. One of the most frequent abnormalities in the progression of gliomas is the inactivation of tumor-suppressor gene p16, suggesting that loss of p16 is associated with acquisition of malignant characteristics. Consistent with this hypothesis, our previous studies showed that restoring wild-type p16 activity into p16-null malignant glioma cells modified their phenotype. In order to understand whether the biological consequences of p16 inactivation in high-grade gliomas included facilitating invasiveness, we used a recombinant replication-deficient adenovirus carrying the cDNA of the p16/CDKN2 gene to infect and express high levels of p16 protein in p16-null SNB19 glioma cells. Invasion of SNB19 glioma cells was tested into two models: invasion of glioma cells through Matrigel-coated transwell inserts and invasion of tumor-cell spheroids into fetal rat-brain aggregates in a co-culture system. Matrigel invasion assays showed that the SNB19 cells expressing exogenous p16 exhibited significantly reduced invasion. Similarly, invasion of p16-treated SNB19 cells into fetal rat-brain aggregates was reduced during a 72 h time period compared to invasion of the adenovirus-control and mock-infected cells. Expression of matrix metalloproteinase-2 (MMP-2), an enzyme involved in tumor-cell invasion, in SNB19 cells expressing p16 was significantly reduced compared to that of parental SNB19 and vector-infected cells. Our results show that restoring wild-type p16 activity into p16-null SNB19 glioma cells significantly inhibits tumor-cell invasion, thus suggesting a novel function of the p16 gene.

Adenoviridae↗
PubMed1999

Transformation of intestinal epithelial cells by chronic TGF-beta1 treatment results in downregulation of the type II TGF-beta receptor and induction of cyclooxygenase-2.

The precise role of TGF-beta in colorectal carcinogenesis is not clear. The purpose of this study was to determine the phenotypic alterations caused by chronic exposure to TGF-beta in non-transformed intestinal epithelial (RIE-1) cells. Growth of RIE-1 cells was inhibited by >75% following TGF-beta1 treatment for 7 days, after which the cells resumed a normal growth despite the presence of TGF-beta1. These 'TGF-beta-resistant' cells (RIE-Tr) were continuously exposed to TGF-beta for >50 days. Unlike the parental RIE cells, RIE-Tr cells lost contact inhibition, formed foci in culture, grew in soft agarose. RIE-Tr cells demonstrated TGF-beta-dependent invasive potential in an in vitro assay and were resistant to Matrigel and Na-butyrate-induced apoptosis. The RIE-Tr cells were also tumorigenic in nude mice. The transformed phenotype of RIE-Tr cells was associated with a 95% decrease in the level of the type II TGF-beta receptor (TbetaRII) protein, a 40-fold increase in cyclooxygenase-2 (COX-2) protein, and 5.9-fold increase in the production of prostacyclin. Most RIE-Tr subclones that expressed low levels of TbetaRII and high levels of COX-2 were tumorigenic. Those subclones that express abundant TbetaRII and low levels of COX-2 were not tumorigenic in nude mice. A selective COX-2 inhibitor inhibited RIE-Tr cell growth in culture and tumor growth in nude mice. The reduced expression of TbetaRII, increased expression of COX-2, and the ability to form colonies in Matrigel were all reversible upon withdrawal of exogenous TGF-beta1 for the RIE-Tr cells.

Animals↗
PubMed2000

Demonstration in vivo that stromelysin-3 functions through its proteolytic activity.

Stromelysin-3 (ST3), a matrix metalloproteinase (MMP) expressed in aggressive carcinomas, has been shown to promote tumor development in different in vivo experimental models. However, the inability of its mature form to degrade extracellular matrix components casts doubt on whether ST3 functions in vivo as a protease. In this study, we evaluated whether the ST3 tumor-promoting effect could be ascribed to its proteolytic activity and whether this putative protease could be targeted with MMP inhibitors. Catalytically inactive mutant cDNA of human (h) ST3 or mouse (m) ST3 were generated and transfected into MCF7 cells. When injected into nude mice in the presence of matrigel, the mutant-bearing cells did not exhibit the enhanced tumorigenicity elicited by MCF7 cells transfected with wild-type ST3 cDNA. In a second approach, TIMP2 overproduction in MCF7 cells expressing hST3 was induced by retroviral infection. The co-expression of ST3 and TIMP2 failed to enhance the tumorigenicity of MCF7 cells. Notably, matrigel depleted of low-molecular-weight proteins and growth factors failed to promote the tumorigenicity of ST3-expressing MCF7 cells. These findings provide the first in vivo evidence that ST3 is indeed a protease that can modulate cancer progression by remodeling extracellular matrix and probably by inducing it to release the necessary microenvironmental factors. Thus, ST3 represents an interesting target for specific MMP inhibition.

Adenocarcinoma↗
PubMed2003

Vinblastine inhibits the angiogenic response induced by adrenomedullin in vitro and in vivo.

Adrenomedullin (ADM) is protumorigenic by stimulating tumor cell growth and angiogenesis. In this context, ADM is identified as a novel target for antiangiogenic therapy. In this study, we addressed the possibility that vinblastine (VBL), as demonstrated in other experimental conditions, may act as an angiostatic molecule in the angiogenic response induced by ADM in two assays, such as Matrigel tube formation in vitro and angiogenesis in the chick embryo chorioallantoic membrane (CAM) in vivo. When tested on Matrigel, ADM caused a morphogenetic effect. In fact, endothelial cells spread and aligned with each other to form branching anastomosing tubes with multicentric junctions that gave rise to a meshwork of capillary-like structures. When ADM was administered in the presence of VBL, the capillary-like tubes were interrupted, most cells were spherical, either isolated or aggregated in small clumps. In the CAM assay, ADM induced a strong angiogenic response, which was counteracted by the treatment with VBL. Overall, these observations implicate ADM as a promoter of tumor growth and a possible target for anticancer strategies, such as the use of VBL at very low, nontoxic doses. Nevertheless, the antiangiogenic activity of low-dose VBL deserves further investigation, alone or together with other antiangiogenic agents for the treatment of tumors characterized by enhanced angiogenesis.

Adrenomedullin↗
PubMed2003

Sequence-specific silencing of MT1-MMP expression suppresses tumor cell migration and invasion: importance of MT1-MMP as a therapeutic target for invasive tumors.

Membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14) has been believed a key enzyme in tumor invasion, because it is expressed in a variety of malignant human tumors, and overexpression of the enzyme enhances the ability of cellular invasiveness. However, it has not necessarily been clarified whether the endogenously expressed MT1-MMP in human tumors plays a critical role in their invasiveness. We used RNA silencing technology to downregulate the endogenous MT1-MMP expression in human tumor cells (fibrosarcoma HT1080 and gastric carcinoma MKN-28 cell lines), and evaluated the effect on the invasion of a reconstituted basement membrane (Matrigel). Transfection of a double-stranded RNA targeted to the MT1-MMP gene decreased the level of the enzyme to less than 10-20% without affecting production of other MMPs. According to the degree of silencing, activation of proMMP-2 was inhibited. CD44 shedding was also inhibited, but only in part. Decreased MT1-MMP levels were also reflected in reduced cell motility on hyaluronan (HA) and invasion in Matrigel. Thus, specific downregulation of MT1-MMP expression was sufficient to cause significant inhibition of the migration and invasion of tumor cells, even though other MMPs continued to be expressed.

Cell Line, Tumor↗
PubMed2004

Expression of activated M-Ras in a murine mammary epithelial cell line induces epithelial-mesenchymal transition and tumorigenesis.

The expression of activated mutants of M-Ras (G22V or Q71L), but not wild-type M-Ras, in a murine mammary epithelial cell line, scp2, resulted in epithelial-mesenchymal transition (EMT) and oncogenic transformation. Cells expressing constitutively active M-Ras continued to grow in the absence of serum and exhibited a loss of the epithelial markers cytokeratin, E-cadherin and beta-catenin, together with a gain of the mesenchymal marker vimentin, a loss of contact inhibition in monolayer growth and a gain of the capacity for anchorage-independent growth. Moreover, unlike the parental cells, they failed to form differentiated mammospheres on Matrigel and instead formed branched networks of cells that grew and invaded the Matrigel. The expression of activated p21 Ras (G12V H-Ras or Q61K N-Ras) also resulted in EMT and tumorigenesis, although there was evidence that expression of higher levels was toxic. Tumors derived from scp2 cells expressing activated M-Ras exhibited activation of Akt and of ERK. The levels of expression of Q71L M-Ras and G12V H-Ras required for tumorigenesis were comparable, although higher levels of the weaker G22V M-Ras mutant were selected for in vivo. These data indicate that the expression of activated mutants of M-Ras was sufficient for oncogenic transformation of a murine mammary epithelial cell line.

Amino Acid Substitution↗
PubMed2004

Angiomotin expression promotes hemangioendothelioma invasion.

Angiomotin was identified by its ability to bind angiostatin and has been shown to mediate its activity in vitro. The family of angiomotin-like protein consists of three members that have coiled-coil domains and conserved c-terminal PDZ-binding motifs. We show here that expression of angiomotin in mouse aortic endothelial (MAE) cells results in stabilization of tubes in the Matrigel assay. Control tubes start to regress after 72 h, whereas MAE-angiomotin (MAE Amot) tubes were stable for over 30 days. In contrast, cells expressing a functional mutant lacking the PDZ protein interaction motif did not migrate and form tubes. Cells from the established tubes invaded into the solidified matrigel. We therefore tested whether angiomotin promotes endothelial invasion. In microcarrier-based invasion in vitro assay, angiomotin-expressing cells invaded collagen matrix and formed tube-like branches. This was confirmed in vivo as injection of MAE-Amot cells promoted tumor growth and invasion into surrounding muscle tissue. Injection of cells transfected with the functional mutant resulted in establishment of noninvasive tumors surrounded by a capsule of fibrous tissue. These tumors remained in constant size or dormant over 3 weeks. Zymogel analysis of the transfected cells did not reveal any differences in proteolytic activity. However, time-lapse photography showed a significant increase in random motility in MAE-Amot cells. We conclude that angiomotin may promote angiogenesis by both stimulating invasion as well as stabilizing established tubes.

Angiomotins↗
PubMed2005

Silibinin strongly inhibits growth and survival of human endothelial cells via cell cycle arrest and downregulation of survivin, Akt and NF-kappaB: implications for angioprevention and antiangiogenic therapy.

Recently, we observed that suppression of tumor xenograft growth by silibinin was associated with reduction in tumor vasculature and an increased apoptosis. Here, we provide evidence for molecular events associated with antiangiogenic efficacy of pharmacologically achievable doses of silibinin in endothelial cell culture system. Our data show that silibinin almost completely (P<0.001) inhibits growth of human umbilical vein endothelial cells (HUVEC) and human microvascular endothelial cells (HMVEC-dermal origin) together with induction of cell death in a dose- and time-dependent manner. Growth inhibition was associated with a strong induction of G1 arrest accompanied by an increase in Kip1/p27, Cip1/p21 and p53. Apoptosis induction (up to 14- to 17-fold in both cell lines, P<0.001) was an underlying mechanism in silibinin-induced death of endothelial cells. In the studies elucidating the molecular events involved in apoptosis, silibinin caused loss of mitochondrial membrane potential and an increase in cytochrome c release from mitochondria. An increase in Bax and a decrease in Mcl-1 proteins were also observed. Silibinin-induced apoptosis involved both caspase-dependent and -independent mechanisms. Silibinin also decreased survivin level and inhibited Akt and NF-kappaB signaling. Two different PI-3K inhibitors, wortmannin and LY294002, showed Akt-independent activation of NF-kappaB. Further, silibinin showed a concentration-dependent strong inhibition of capillary tube formation on matrigel, retraction and disintegration of preformed capillary network, inhibition of matrigel invasion and migration, and a decrease in matrix metalloproteinase-2 secretion by HUVEC. Together, these findings identify pleiotropic mechanisms for antiangiogenic efficacy of silibinin, and suggest its usefulness in angioprevention and antiangiogenic therapy.

Angiogenesis Inhibitors↗
PubMed2005

Runx1 promotes angiogenesis by downregulation of insulin-like growth factor-binding protein-3.

Mouse embryos lacking the Runx1 transcription factor exhibit an angiogenic defect accompanied by the absence of hematopoietic stem cells (HSCs). To ask whether Runx1 plays a direct role in angiogenesis, we established a novel endothelial progenitor cell line, designated AEL-DeltaR1, from the aorta-gonad-mesonephros (AGM) region of Runx1-null mouse. We introduced Runx1 cDNA into AEL-DeltaR1 cells under the doxycycline-inducible promoter. The ability of AEL-DeltaR1 cells to form vascular networks on matrigel was highly enhanced by the restored expression of Runx1. By molecular comparison of mRNAs in AEL-DeltaR1 cells before and after the induction of Runx1, we found that mRNA expression of insulin-like growth factor-binding protein 3 (IGFBP-3) is downregulated by Runx1. Gel retardation and reporter assays revealed that Runx1 binds to the promoter region of mouse IGFBP-3 gene and represses its transcription. When IGFBP-3 was exogenously added in the matrigel assay, the angiogenesis-enhancing activity of Runx1 was suppressed in a dose-dependent manner. These results demonstrate that Runx1 is directly involved in angiogenesis by repression of IGFBP-3 mRNA expression.

Animals↗
PubMed2005

FRA-1 expression level regulates proliferation and invasiveness of breast cancer cells.

Breast cancer progression is likely a multistep process involving the activation and inactivation of a number of genes. Previously, we showed that the mRNA coding for Fra-1, a FOS family member and an AP-1 transcription factor component, was highly expressed in the more invasive estrogen receptor negative (ER-) breast cancer cell lines. We used a tet-off system to stably overexpress Fra-1 in MCF7 ER+ cells and evaluate the impact of Fra-1 on this aggressive phenotype. Conversely, Fra-1 was silenced in highly invasive ER-MDA-MB231 cells using RNA interference. We report that in both systems the Fra-1 expression level was positively associated with cell proliferation, cell motility and invasiveness assessed in vitro. In addition, Fra-1 inhibition in fibroblastoid ER- cells, which formed colonies with large stellate projections in Matrigel, resulted in morphological changes. Cells acquired an epithelioid shape and had a spherical appearance in Matrigel. Fra-1 regulated several genes, implicated in invasion, angiogenesis and cell proliferation independently of beta1-integrin activation, and directly induced MMP-1 and MMP-9 promoter activity. These overall results show that high Fra-1 expression is associated with a more malignant cell phenotype and suggest that Fra-1 could have a pivotal role in breast cancer progression.

Breast Neoplasms↗
PubMed2005

The human VE-cadherin promoter is subjected to organ-specific regulation and is activated in tumour angiogenesis.

Vascular endothelial (VE)-cadherin is exclusively expressed at interendothelial junctions of normal and tumour vessels. In this report, we characterized the transcriptional activity of the human VE-cadherin promoter. Transient transfection assays revealed that sequences at positions --1135/-744 and -166/-5 base pairs are critical for promoter activity in endothelial cells. We show that specific sequences in the proximal region interact with Ets and Sp1 family members. Transgenic mice were created and the human VE-cadherin promoter was able to confer correct temporal and spatial expression on the LacZ gene in embryos. In adults, the transgene was specifically and strongly expressed in the lung, heart, ovary, spleen and kidney glomeruli, whereas expression was weak or absent in the vasculature of other organs, including the brain, thymus, liver and skeletal muscle. Neovessels in tumour grafts and Matrigel implants harboured strong stainings, indicating that promoter activity is enhanced in angiogenic situations. Furthermore, Matrigel and transfection assays showed that VE-cadherin promoter is subjected to bFGF induction. Transgene expression was also noticed in extravascular sites of the central nervous system, suggesting that silencer elements may be located elsewhere in the gene. These results are a first step towards addressing the organ- and tumour-specific regulation of the VE-cadherin gene.

Animals↗
PubMed2006

Insulin-like growth factor-binding protein-3 inhibition of prostate cancer growth involves suppression of angiogenesis.

Insulin-like growth factor-binding protein-3 (IGFBP-3) is a multifunctional protein that induces apoptosis utilizing both insulin-like growth factor receptor (IGF)-dependent and -independent mechanisms. We investigated the effects of IGFBP-3 on tumor growth and angiogenesis utilizing a human CaP xenograft model in severe-combined immunodeficiency mice. A 16-day course of IGFBP-3 injections reduced tumor size and increased apoptosis and also led to a reduction in the number of vessels stained with CD31. In vitro, IGFBP-3 inhibited both vascular endothelial growth factor- and IGF-stimulated human umbilical vein endothelial cells vascular network formation in a matrigel assay. This action is primarily IGF independent as shown by studies utilizing the non-IGFBP-binding IGF-1 analog Long-R3. Additionally, we used a fibroblast growth factor-enriched matrigel-plug assay and chick allantoic membrane assays to show that IGFBP-3 has potent antiangiogenic actions in vivo. Finally, overexpression of IGFBP-3 or the non-IGF-binding GGG-IGFBP-3 mutant in Zebrafish embryos confirmed that both IGFBP-3 and the non-IGF-binding mutant inhibited vessel formation in vivo, indicating that the antiangiogenic effect of IGFBP-3 is an IGF-independent phenomenon. Together, these studies provide the first evidence that IGFBP-3 has direct, IGF-independent inhibitory effects on angiogenesis providing an additional mechanism by which it exerts its tumor suppressive effects and further supporting its development for clinical use in the therapy of patients with prostate cancer.

Animals↗