Tet-Kin Yeo

Increased Vascular Endothelial Growth Factor Levels in the Vitreous of Eyes With Proliferative Diabetic Retinopathy

The vitreous levels of the angiogenic polypeptide vascular endothelial growth factor (also known as vascular permeability factor) were measured and compared in eyes with and without proliferative diabetic retinopathy. Undiluted vitreous samples from 20 eyes were collected at the time of vitrectomy, and vascular endothelial growth factor levels were determined by using a time-resolved immunofluorometric assay. Vitreous vascular endothelial growth factor levels were significantly higher in eyes with proliferative diabetic retinopathy than in eyes without proliferative diabetic retinopathy (P = .006; Wilcoxon Rank Sum Test). The median vitreous concentration in the eyes with proliferative diabetic retinopathy was 29.1 pM and exceeded the known concentration required for the maximal proliferation of vascular endothelial cells in vitro. These data are consistent with vascular endothelial growth factor serving as a physiologically relevant angiogenic factor in proliferative diabetic retinopathy.

Vascular permeability factor (VPF, VEGF) in tumor biology

SummaryVascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine expressed and secreted at high levels by many tumor cells of animal and human origin. As secreted by tumor cells, VPF/VEGF is a 34–42 kDa heparin-binding, dimeric, disulfide-bonded glycoprotein that acts directly on endothelial cells (EC) by way of specific receptors to activate phospholipase C and induce [Ca2+]i transients. Two high affinity VPF/VEGF receptors, both tyrosine kinases, have thus far been described. VPF/VEGF is likely to have a number of important roles in tumor biology related, but not limited to, the process of tumor angiogenesis. As a potent permeability factor, VPF/VEGF promotes extravasation of plasma fibrinogen, leading to fibrin deposition which alters the tumor extracellular matrix. This matrix promotes the ingrowth of macrophages, fibroblasts, and endothelial cells. Moreover, VPF/VEGF is a selective endothelial cell (EC) growth factorin vitro, and it presumably stimulates EC proliferationin vivo. Furthermore, VPF/VEGF has been found in animal and human tumor effusions by immunoassay and by functional assays and very likely accounts for the induction of malignant ascites. In addition to its role in tumors, VPF/VEGF has recently been found to have a role in wound healing and its expression by activated macrophages suggests that it probably also participates in certain types of chronic inflammation. VPF/VEGF is expressed in normal development and in certain normal adult organs, notably kidney, heart, adrenal gland and lung. Its functions in normal adult tissues are under investigation.

Function of glycoprotein glycans

Abstract Recent results, obtained simultaneously in several laboratories, are reviewed which strengthen earlier notions that the glycan moieties of some glycoproteins play important roles in (1) maintenance of protein conformation and solubility, (2) proteolytic processing and stabilization of the polypeptide against uncontrolled proteolysis, (3) mediation of biological activity, (4) intracellular sorting and externalization of glycoproteins, and (5) embryonic development and differentiation.

Glycosylation is essential for efficient secretion but not for permeability-enhancing activity of vascular permeability factor (vascular endothelial growth factor)

The hyperpermeability of the microvasculature supplying solid tumors is largely attributable to a heterodimeric Mr 34,000-43,000 tumor-secreted protein, vascular permeability factor. Upon reduction, the vascular permeability factor secreted by line 10 tumor cells is resolved by SDS-PAGE into 3 discrete bands of Mr 24,000, 19,500, and 15,000. We demonstrate here that line 10 vascular permeability factor is an N-linked glycoprotein. Nonglycosylated vascular permeability factor migrates on reduced SDS-PAGE as two bands of Mr 20,000 and 15,000. Pulse-chase studies demonstrated that all three chains of native vascular permeability factor were secreted rapidly following synthesis and at equal rates, with a cellular half-retention time of approximately 37 min. When glycosylation was prevented by tunicamycin, individual bands of nonglycosylated vascular permeability factor were also secreted at equivalent rates, but much more slowly (approximately 60 min) than native glycoprotein. Both glycosylated and nonglycosylated forms of vascular permeability factor were equally potent at increasing dermal vessel permeability.

Distribution of Biglycan and Its Propeptide Form in Rat and Bovine Aortic Tissue

The matrix proteoglycan biglycan was identified in bovine and rat aortic tissue by Western blot analysis and by immunohistochemistry, using polyclonal antibodies raised against peptides of the propeptide and the hypervariable region of the rat biglycan core protein. Western blot analysis of proteoglycans isolated from bovine and rat aortas by ion exchange chromatography and treated with chondroitin ABC lyase, with antibody against propeptide, demonstrated core proteins with molecular weights ranging from 43,000 to 45,000 daltons. Similar results were obtained with Western blot studies using the peptide antibody to the hypervariable region of biglycan, except the antibody did not recognize the core protein of bovine biglycan. Location of biglycan within bovine and rat aortic tissue sections by immunoperoxidase histochemistry using the antibody raised against the propeptide revealed intense intracellular staining of medial myocytes and endothelial cells but no extracellular staining. In contrast, immunohistochemistry performed with the purified antibody to the hypervariable region revealed significant extracellular staining of the adventitia proximate to the media and of the endothelial lining but no intracellular staining of rat aortic tissue, with no observable staining of bovine aortic tissue. These data demonstrate that, in contrast to cultured smooth muscle cells, biglycan containing the propeptide is not secreted and deposited within the extracellular matrix by smooth muscle cells and endothelial cells from aortic tissue.

Differential transport kinetics of chondroitin sulfate and dermatan sulfate proteoglycan by monkey aorta smooth muscle cells

Pulse-chase studies were performed to study the kinetics of chondroitin sulfate proteoglycan (CSPG) and dermatan sulfate proteoglycan (DSPG) transport in monkey aorta smooth muscle cells. During a short pulse (5 min) with [35S]Na2SO4 (500 microCi/ml), the cells synthesized 59% DSPG, 38% CSPG, and 3% heparan sulfate proteoglycan. Both DSPG and CSPG were transported out of the cell very rapidly after sulfate incorporation. At various chase times, proteoglycans (PGs) were isolated from four cellular compartments: (a) medium, (b) total cell lysate, (c) intracellular pool, and (d) extracellular pool. The PGs from the different pools were analyzed by Sepharose CL-2B column chromatography. The data of intracellular DSPG loss fitted a double exponential decay model: approximately 90% was secreted quickly with a t1/2 of 7 min, and the remaining 10% had a dramatically slower rate of secretion (t1/2 of 130 min). DSPG was rapidly secreted into the medium without prior accumulation in the extracellular matrix. In contrast, the loss of intracellular CSPG fitted a single exponential decay model with a t1/2 of 8 min; however, there was a significant accumulation of CSPG in the extracellular matrix compartment before release into the medium, resulting in a relatively slower secretion of CSPG into the medium (t1/2 of about 31 min). This delay in CSPG secretion into the medium is probably due to aggregation in the extracellular matrix, since addition of short hyaluronan oligomers (8-14 oligosaccharides) to the medium during the chase increased the rate of CSPG being secreted into the medium. We concluded that in aortic smooth muscle cell cultures, CSPG and DSPG are secreted via two distinct pathways through the cellular compartments.

Bromoconduritol treatment delays intracellular transport of secretory glycoproteins in human hepatoma cell cultures

Previous studies in our laboratory have shown that specific glycan structures are required for the normal secretion of some glycoproteins. Bromoconduritol is known to inhibit the removal of the innermost glucose moiety from the Glc3Man9(GlcNAc)2 precursor of N-linked glycoproteins. We have used this inhibitor to investigate the possible role of glycan structure in the intracellular transport of secretory glycoproteins of Hep G2 cultures. Cells were pretreated with 1mM bromoconduritol for 1h, pulsed with [35S]-methionine for 10min and chased for varying intervals. Specific glycoproteins and albumin were immunoprecipitated from the cell lysate and medium. We found that bromoconduritol-treatment inhibited the secretion of alpha 1-protease inhibitor, ceruloplasmin, alpha 2-macroglobulin, transferrin, and alpha-fetoprotein. Apparently, the glucosylated high-mannose intermediate is not secreted, since glycoproteins in the medium are of complex form. We conclude that the removal of the innermost glucose residue from secretory glycoprotein represents an important regulatory step in the intracellular transport pathway.

Accumulation of unglycosylated liver secretory glycoproteins in the rough endoplasmic reticulum

Previous studies in this laboratory (1) have shown that tunicamycin-treatment inhibits the secretion of three secretory glycoproteins--alpha 2-macroglobulin, ceruloplasmin, and alpha 1-protease inhibitor in human hepatoma (Hep G2) cell cultures. In the present study, we have investigated (i) their site of accumulation within the endoplasmic reticulum/Golgi pathway, and (ii) the solubility characteristics of these unglycosylated proteins. Using percoll density gradient centrifugation, we found that tunicamycin-treatment markedly inhibited the transport of alpha 2-macroglobulin, ceruloplasmin and alpha 1-protease inhibitor from the rough endoplasmic reticulum. However, there was no detectable changes in their solubility properties as both the glycosylated and unglycosylated species were associated with the 100,000 xg supernatant fraction following disruption of the microsomal fraction (i) with 0.2% Triton X-100 and (ii) by repeated freeze-thaw cycles. Also no evidence of protein aggregation was detected by liquid chromatography of the unglycosylated proteins on Bio-Gel A-1.5 column.

Hypoxic Induction of Endothelial Cell Growth Factors in Retinal Cells: Identification and Characterization of Vascular Endothelial Growth Factor

BACKGROUND New vessel growth is often associated with ischemia, and hypoxic tissue has been identified as a potential source of angiogenic factors. In particular, ischemia is associated with the development of neovascularization in a number of ocular pathologies. For this reason, we have studied the induction of endothelial cell mitogens by hypoxia in retinal cells. MATERIALS AND METHODS Human retinal pigment epithelium (hRPE) were grown under normoxic and hypoxic conditions and examined for the production of endothelial mitogens. Northern analysis, biosynthetic labeling and immunoprecipitation, and ELISA were used to assess the levels of vascular endothelial growth factor/vascular permeability factor (VEGF) and basic fibroblast growth factor (bFGF), two endothelial cell mitogens and potent angiogenic factors. Soluble receptors for VEGF were employed as competitive inhibitors to determine the contribution of the growth factor to the hypoxia-stimulated mitogen production. RESULTS Following 6-24 hr of hypoxia, confluent and growing cultures of hRPE increase their levels of VEGF mRNA and protein synthesis. Biosynthetic labeling studies and RT-PCR analysis indicate that the cells secrete VEGF121 and VEGF165, the soluble forms of the angiogenic factor. In contrast, hRPE cultured under hypoxic conditions show reduced steady-state levels of basic fibroblast growth factor (bFGF) mRNA and decreased bFGF protein synthesis. Unlike VEGF, bFGF is not found in conditioned media of hRPE following 24 hr of hypoxia. Using a soluble high-affinity VEGF receptor as a competitive inhibitor of VEGF, we demonstrate that a VEGF-like activity is the sole hypoxia-inducible endothelial mitogen produced by cultured hRPE. CONCLUSIONS From this comparison we conclude that hRPE do not respond to hypoxia with a general, nonspecific increase in the overall levels of growth factors, as is seen during cell wounding responses or serum stimulation. The physiological relevance of data from this in vitro model are affirmed by separate studies in an animal model of retinal ischemia-induced ocular neovascularization (1) in which retina-derived VEGF levels have been shown to correlate spatio-temporally with the onset of angiogenesis. Taken together, these data support the hypothesis that the induction of VEGF by hypoxia mediates the rapid, initial angiogenic response to retinal ischemia.