PubMed Health⌕ Search

Biomedical subjects

D R Senger

Publications and source records attributed to D R Senger.

At least 19 recordsLinked to original sources
PubMed2001

Low IGF-I suppresses VEGF-survival signaling in retinal endothelial cells: direct correlation with clinical retinopathy of prematurity.

Retinopathy of prematurity is a blinding disease, initiated by lack of retinal vascular growth after premature birth. We show that lack of insulin-like growth factor I (IGF-I) in knockout mice prevents normal retinal vascular growth, despite the presence of vascular endothelial growth factor, important to vessel development. In vitro, low levels of IGF-I prevent vascular endothelial growth factor-induced activation of protein kinase B (Akt), a kinase critical for endothelial cell survival. Our results from studies in premature infants suggest that if the IGF-I level is sufficient after birth, normal vessel development occurs and retinopathy of prematurity does not develop. When IGF-I is persistently low, vessels cease to grow, maturing avascular retina becomes hypoxic and vascular endothelial growth factor accumulates in the vitreous. As IGF-I increases to a critical level, retinal neovascularization is triggered. These data indicate that serum IGF-I levels in premature infants can predict which infants will develop retinopathy of prematurity and further suggests that early restoration of IGF-I in premature infants to normal levels could prevent this disease.

Base Sequence↗
PubMed2001

Angiogenesis as a potential biomarker in prostate cancer chemoprevention trials.

Prostate cancer is a multistep process in which progression rather than initiation may be the rate-limiting step. A strong possibility is that prostatic intraepithelial neoplasia lesions that switch to angiogenic phenotype eventually progress to cancer. However, it is a challenging task to quantitate angiogenesis in preneoplastic lesions. A promising approach to measuring angiogenesis involves real-time TaqMan polymerase chain reaction to quantitate mRNAs encoding a panel of angiogenesis markers. This highly sensitive molecular technique has potential for quantitating angiogenesis in clinical settings and can be used as a high-throughput screening procedure in prostate cancer clinical trials.

Animals↗
PubMed1999

Regulation of vascular endothelial growth factor-dependent retinal neovascularization by insulin-like growth factor-1 receptor.

Although insulin-like growth factor 1 (IGF-1) has been associated with retinopathy, proof of a direct relationship has been lacking. Here we show that an IGF-1 receptor antagonist suppresses retinal neovascularization in vivo, and infer that interactions between IGF-1 and the IGF-1 receptor are necessary for induction of maximal neovascularization by vascular endothelial growth factor (VEGF). IGF-1 receptor regulation of VEGF action is mediated at least in part through control of VEGF activation of p44/42 mitogen-activated protein kinase, establishing a hierarchical relationship between IGF-1 and VEGF receptors. These findings establish an essential role for IGF-1 in angiogenesis and demonstrate a new target for control of retinopathy. They also explain why diabetic retinopathy initially increases with the onset of insulin treatment. IGF-1 levels, low in untreated diabetes, rise with insulin therapy, permitting VEGF-induced retinopathy.

Animals↗
PubMed1997

Angiogenesis promoted by vascular endothelial growth factor: regulation through alpha1beta1 and alpha2beta1 integrins.

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors-the alpha1beta1 and alpha2beta1 integrins-through induction of mRNAs encoding the alpha1 and alpha2 subunits. In contrast, VEGF did not induce increased expression of the alpha3beta1 integrin, which also has been implicated in collagen binding. Integrin alpha1-blocking and alpha2-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and alpha1-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of alpha1beta1 and alpha2beta1 expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of alpha1-blocking and alpha2-blocking Abs. In vivo, a combination of alpha1-blocking and alpha2-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of alpha1beta1 and alpha2beta1 expression by EC is an important mechanism by which VEGF promotes angiogenesis and that alpha1beta1 and alpha2beta1 antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.

Animals↗
PubMed1996

Cell migration promoted by a potent GRGDS-containing thrombin-cleavage fragment of osteopontin.

Osteopontin (OPN) is a secreted adhesive glycoprotein with a gly-arg-gly-asp-ser (GRGDS) cell binding domain. Several independent studies have suggested that OPN functions in tumor growth and metastasis, and one likely possibility is that OPN facilitates tumor invasion by promoting tumor cell migration. Consistent with this hypothesis, immobilized OPN promoted concentration-dependent tumor cell migration (i.e., haptotaxis) in modified Boyden chambers. In particular, cleavage of OPN by thrombin, which likely occurs in the tumor microenvironment, resulted in enhancement of OPNs haptotactic activity; and assays performed with purified preparations of the two individual OPN thrombin-cleavage fragments demonstrated that all detectable activity was associated with the GRGDS-containing fragment. In contrast to the activity of both OPN and its GRGDS-containing fragment in promoting haptotaxis, neither of these proteins in solution promoted chemotaxis, indicating that each must be immobilized to promote cell migration. In haptotaxis assays, antibody LM609 to integrin alpha v beta 3 blocked > 80% cell migration towards the GRGDS-containing OPN fragment, implicating alpha v beta 3 as its principal functional receptor. In comparison with equimolar quantities of other adhesive proteins, the GRGDS-containing OPN thrombin-cleavage fragment was not only > 2-fold more effective than intact OPN at promoting haptotaxis, but also > 8-fold and > 6-fold more effective than fibrinogen and vitronectin, respectively, indicating that this OPN fragment is highly active relative to other alpha v beta 3 ligands.

Amino Acid Sequence↗
PubMed1996

Stimulation of endothelial cell migration by vascular permeability factor/vascular endothelial growth factor through cooperative mechanisms involving the alphavbeta3 integrin, osteopontin, and thrombin.

We have identified several mechanisms by which the angiogenic cytokine vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) likely regulates endothelial cells (EC) migration. VPF/VEGF induced dermal microvascular EC expression of mRNAs encoding the alphav and beta3 integrin subunits resulting in increased levels of the alphavbeta3 heterodimer at the cell surface, and VPF/VEGF also induced mRNA encoding osteopontin (OPN), an alphavbeta3 ligand. OPN promoted EC migration in vitro; and VPF/VEGF induction of alphavbeta3 was accompanied by increased EC migration toward OPN. Because thrombin cleavage of OPN results in substantial enhancement of OPN's adhesive properties, and because VPF/VEGF promotes increased microvascular permeability leading to activation of the extrinsic coagulation pathway, we also investigated whether VPF/VEGF facilitates thrombin cleavage of OPN in vivo. Consistent with this hypothesis, co-injection of VPF/VEGF together with OPN resulted in rapid cleavage of OPN by endogenous thrombin. Furthermore, in comparison with native OPN, thrombin-cleaved OPN stimulated a greater rate of EC migration in vitro, which was additive to the increased migration associated with induction of alpha v beta 3. Thus, these data demonstrate cooperative mechanisms for VPF/VEGF regulation of EC migration involving the alphavbeta3 integrin, the alphavbeta3 ligand OPN, and thrombin cleavage of OPN. These findings also illustrate an operational link between VPF/VEGF induction of EC gene expression and VPF/VEGF enhancement of microvascular permeability, suggesting that these distinct biological activities may act accordingly to stimulate EC migration during angiogenesis.

Animals↗
PubMed1995

Structural requirements for dimerization, glycosylation, secretion, and biological function of VPF/VEGF.

Vascular permeability factor (VPF) also known as vascular endothelial growth factor (VEGF), is a dimeric protein that affects endothelial cell (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys-25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactive in both vascular permeability and endothelial cell mitotic assays. VPF Cys-145 mutant protein was efficiently secreted as a glycosylated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full vascular permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.

Animals↗
PubMed1995

Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer.

Solid tumors must induce a vascular stroma to grow beyond a minimal size, and the intensity of the angiogenic response has been correlated with prognosis in breast cancer patients. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a secreted protein that has been implicated in tumor-associated angiogenesis. Vascular permeability factor directly stimulates endothelial cell growth and also increases microvascular permeability, leading to the extravasation of plasma proteins, which alter the extracellular matrix in a manner that promotes angiogenesis. To determine whether VPF has a role in breast cancer, we used in situ hybridization to study VPF mRNA expression in normal breast tissue (13 specimens), comedo-type ductal carcinoma in situ (DCIS) (four specimens), infiltrating ductal carcinoma (12 specimens), infiltrating lobular carcinoma (two specimens), metastatic ductal carcinoma (three specimens) and metastatic lobular carcinoma (one specimen). Vascular permeability factor mRNA was expressed at a low level by normal duct epithelium but was expressed at high levels in tumor cells in all cases of comedo-type DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, VPF mRNA was not expressed at high levels in infiltrating lobular carcinoma. We also used in situ hybridization to study the expression of two recently described endothelial cell surface VPF receptors, flt-1 and kdr. Vascular permeability factor receptor mRNA was strongly expressed in endothelial cells of small vessels adjacent to malignant tumor cells in DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, no definite labeling for receptor mRNA was detected in infiltrating lobular carcinoma or nonmalignant breast tissue. The intense expression of VPF mRNA by breast carcinoma cells and of VPF receptor mRNA by endothelial cells of adjacent small blood vessels provides strong evidence linking VPF expression to the angiogenesis associated with comedo-type DCIS, infiltrating ductal, and metastatic ductal breast carcinoma.

Breast Neoplasms↗
PubMed1995

Ultrastructural localization of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) to the abluminal plasma membrane and vesiculovacuolar organelles of tumor microvascular endothelium.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine secreted by many animal and human tumors, activated macrophages, keratinocytes, rheumatoid synovial cells, embryonic tissues, and by cultured epithelial and mesenchymal cell lines. It acts selectively on vascular endothelial cells to increase their permeability to circulating macromolecules and to stimulate their replication. Although not detectably expressed by vascular cells in the human and animal tumors we have studied, VPF/VEGF accumulates in the microvessels supplying tumors and certain inflammatory reactions in which VPF/VEGF is also overexpressed. Light microscopic immunohistochemistry lacked the resolution necessary to localize VPF/VEGF precisely in such vessels. Therefore, we used a pre-embedding immunocytochemical method to localize VPF/VEGF at the ultrastructural level in the new blood vessels that are elicited in the peritoneal walls of mice bearing a transplantable mouse ascites tumor of ovarian origin. Intense immunostaining for VPF/VEGF was observed on the abluminal plasma membrane of tumor-associated microvascular endothelial cells and in vesiculovacuolar organelles (VVOs) present in these same endothelial cells. (VVOs are recently described cytoplasmic organelles present in tumor vascular endothelium that provide an important pathway for extravasation of circulating macromolecules.) In contrast to labeling of the abluminal plasma membrane and VVO vesicles and vacuoles, endothelial cytoplasmic organelles, such as multivesicular bodies and Weibel-Palade bodies, and the underlying basal lamina, did not stain with antibodies to VPF/VEGF. The distribution of VPF/VEGF here described corresponds to that anticipated for high-affinity VFP/VEGF receptors, although binding of VPF/VEGF to other endothelial cell surface structures, such as plasma membrane proteoglycans, is also a possibility.

Animals↗
PubMed1995

Expression of vascular permeability factor/vascular endothelial growth factor by human granulosa and theca lutein cells. Role in corpus luteum development.

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a cytokine that is overexpressed in many tumors, in healing wounds, and in rheumatoid arthritis. VPF/VEGF is thought to induce angiogenesis and accompanying connective tissue stroma in two ways: 1), by increasing microvascular permeability, thereby modifying the extracellular matrix and 2), as an endothelial cell mitogen. VPF/VEGF has been reported in animal corpora lutea and we investigated the possibility that it might be present in human ovaries and have a role in corpus luteum formation. We here report that VPF/VEGF mRNA and protein are expressed by human ovarian granulosa and theca cells late in follicle development and, subsequent to ovulation, by granulosa and theca lutein cells. Therefore, VPF/VEGF is ideally positioned to provoke the increased permeability of thecal blood vessels that occurs shortly before ovulation. VPF/VEGF likely also contributes to the angiogenesis and connective tissue stroma generation that accompany corpus luteum/corpus albicans formation. Finally, VPF/VEGF was overexpressed in the hyperthecotic ovarian stroma of Stein-Leventhal syndrome in which it may also have a pathophysiological role.

Adolescent↗
PubMed1994

Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue.

Vascular permeability factor (VPF, also known as vascular endothelial growth factor or VEGF), is a potent microvascular permeability enhancing cytokine and a selective mitogen for endothelial cells. It has been implicated in tumor angiogenesis and ascites fluid accumulation. Since development of the destructive synovial pannus in rheumatoid arthritis (RA) is associated with changes in vascular permeability (synovial fluid accumulation), synovial cell hyperplasia, and angiogenesis, we examined synovial fluids (SFs) and joint tissue for the expression and local accumulation of VPF/VEGF. VPF/VEGF was detected in all of 21 synovial fluids examined and when measured by an immunofluorimetric assay, ranged from 6.9 to 180.5 pM. These levels are biologically significant, since < 1 pM VPF/VEGF can elicit responses from its target cells, endothelial cells. Levels of VPF/VEGF were highest in rheumatoid arthritis fluids (n = 10), with a mean value (+/- SEM) of 59.1 +/- 18.0 pM, vs. 21.4 +/- 2.3 pM for 11 SFs from patients with other forms of arthritis (p = 0.042). In situ hybridization studies that were performed on joint tissues from patients with active RA revealed that synovial lining macrophages strongly expressed VPF/VEGF mRNA, and that microvascular endothelial cells of nearby blood vessels strongly expressed mRNA for the VPF/VEGF receptors, flt-1 and KDR. Immunohistochemistry performed on inflamed rheumatoid synovial tissue revealed that the VPF/VEGF peptide was localized to macrophages within inflamed synovium, as well as to microvascular endothelium, its putative target in the tissue. Together, these findings indicate that VPF/VEGF may have an important role in the pathogenesis of RA.

Adult↗
PubMed1994

Adhesive properties of osteopontin: regulation by a naturally occurring thrombin-cleavage in close proximity to the GRGDS cell-binding domain.

Osteopontin (OPN) is a secreted adhesive glycoprotein with a functional glycine-arginine-glycine-aspartate-serine (GRGDS) cell-binding domain. An interesting feature of OPN structure is the presence of a thrombin-cleavage site in close proximity to the GRGDS region. Cleavage of OPN by thrombin is likely to be of physiological importance, because cleavage of blood plasma OPN occurs naturally after activation of the blood coagulation pathway. To investigate functional consequences of OPN cleavage by thrombin, cell attachment and spreading assays were performed with uncleaved and cleaved forms of OPN. For all cell lines examined, thrombin-cleaved OPN promoted markedly greater cell attachment and spreading than uncleaved OPN. Cell attachment and spreading on thrombin-cleaved OPN was inhibited both by the soluble GRGDS peptides and an OPN-specific antibody raised to the GRGDS domain of OPN, thus implicating the GRGDS region in mediating the increased cell attachment and spreading observed on thrombin-cleaved OPN. Because the GRGDS sequence in OPN is only six residues from the thrombin-cleavage site, the data suggest that possibility that thrombin cleavage allows greater accessibility of the GRGDS domain to cell surface receptors. To investigate receptors that recognize uncleaved and thrombin-cleaved OPN, affinity chromatography was performed on placental extracts; the cell surface integrin alpha v beta 3 bound to columns constructed either with native or thrombin-cleaved OPN and was selectively eluted from each with soluble GRGDS peptide and EDTA. Moreover, adhesion assays performed in the presence of alpha v beta 3 blocking monoclonal antibody LM609 identified alpha v beta 3 as a major functional receptor for thrombin-cleaved OPN. Several lines of evidence suggest that cleavage of OPN by thrombin occurs in vivo, such as in tumors and at sites of tissue injury, and adhesion assay data presented here indicate that such cleavage is important in the regulation of OPN function.

Amino Acid Sequence↗
PubMed1994

Osteopontin: its transglutaminase-catalyzed posttranslational modifications and cross-linking to fibronectin.

Osteopontin (OP) is a component of extracellular, bone, and urinary stone matrices, but the mechanism by which it is stably incorporated into such matrices remains unknown. By SDS-PAGE analysis of [125I]OP, treated with a catalytic amount of TG, we first demonstrate both intra- and intermolecular covalent cross-linking of OP. Most importantly, the analysis of the products generated from reactions containing OP, Fn, and TG by SDS-PAGE, autoradiography, and Western blotting using either OP or Fn antibody, and quantitation of TG-catalyzed epsilon-(gamma-glutamyl)lysine isopeptide formation between OP and Fn demonstrate, for the first time, covalent cross-linking between these two proteins. Similar reactions in the presence of polyamine substrates of TG show OP-Fn intermolecular cross-linking via N,N-bis-(gamma-glutamyl)polyamine formation. Finally, immunoprecipitation of 125I-labeled NRK cell surface proteins with anti-OP and anti-Fn antibodies, SDS-PAGE analysis, and autoradiography provides critical evidence for nonreducible OP-Fn cross-linking in vivo. These results clearly suggest that TG-mediated cross-linking between OP and Fn represents one of the most likely mechanisms by which OP becomes covalently linked to bone matrix, urinary stone matrix, and to ECM.

Amino Acid Sequence↗
PubMed1994

Ultrastructural immunogold localization of osteopontin in human gallbladder epithelial cells.

We used a post-embedding ultrastructural immunogold method to localize osteopontin in human gallbladder epithelial cells. This glycoprotein, originally described in bone but recently found to have a much wider distribution in many epithelia and in some mesenchymal cells, was present in the filamentous glycocalyx, small apical cytoplasmic smooth membrane-bound vesicles, large membrane-bound cytoplasmic granules, and in portions of the Golgi complex in gallbladder columnar epithelial cells. These findings suggest that newly synthesized osteopontin is packaged in Golgi-derived granules that release their contents by classical exocytosis from the cell surface. At least a portion of secreted osteopontin remains on the cell surface, where it becomes integrated into the filamentous glycocalyx coating the luminal surface of gallbladder epithelial cells.

Amino Acid Sequence↗
PubMed1994

Specific reduction in osteopontin synthesis by antisense RNA inhibits the tumorigenicity of transformed Rat1 fibroblasts.

Osteopontin (OPN) is a secreted phosphoglycoprotein abundant in secretory luminal epithelia (Brown et al., 1992) and in bone (Reinholt et al., 1990). It contains a functional gly-arg-gly-asp-ser (GRGDS) integrin binding domain (Oldberg et al., 1986), promotes the adhesion of a variety of cell types (Somerman et al., 1989; Brown et al., 1992) and is a ligand for the vitronectin binding integrin alpha v beta 3 (Miyauchi et al., 1991). Elevated expression of OPN correlates with tumorigenic transformation in a great variety of stromal and epithelial cell lines (Senger et al., 1980, 1983, 1989; Craig et al., 1988; Chambers et al., 1992; Chang & Prince, 1993). The protein is also present in excess in the blood of patients with metastatic disease (Senger et al., 1988). To find whether OPN contributes significantly to the tumorigenic phenotype, we expressed antisense mRNA to OPN in high OPN producing malignant B77-Rat1 fibroblasts. This caused a reduction in their OPN secretion and reduced their ability to form both lung tumors in nude mice after intravenous injection, and colonies in soft agar. Antisense transfectants also showed increased spreading on vitronectin. These observations suggest that OPN overproduction is advantageous to the metastatic phenotype, possibly by altering adhesion via, or signal transduction from, vitronectin receptors.

Animals↗