Faculty Bibliography

Recent molecular genetics studies in humans and mice showed that transforming growth factor-beta (TGF-beta) is involved in vasculogenesis and maintenance of blood vessel integrity. These results confirm earlier in vitro studies demonstrating generation of active TGF-beta when endothelial cells are cocultured with smooth muscle cells or pericytes. TGF-beta is secreted as a latent, inactive complex and becomes active only when released. Latent TGF-beta binds covalently to proteins (LTBP) that target it to the extracellular matrix. Thus, the latency of TGF-beta is essential to the regulation of the bioavailability and activity of this cytokine. The development of methods for measuring activation of latent TGF-beta in cell cultures and identification of the proteins contained in the latent TGF-beta complex have shed new light on the mechanism of activation of latent TGF-beta possibly involved in vasculogenesis, angiogenesis, and other processes

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin

Most growth factors are released from cells in a form that does not permit immediate interaction with their high affinity receptors. An important mechanism for presentation of these released latent growth factors is activation by the plasminogen activator-plasmin system. The involvement of this system in the biology of Transforming Growth Factor-beta (TGF-beta) is reviewed

PURPOSE: To assess the effect of diltiazem, a calcium channel blocking agent, on oxygen induced retinopathy (OIR) in a mouse model using neovascular nuclei quantitation and a quantitative scoring system based on examining fluorescein perfused retinal whole mount preparations. METHODS: The mouse model of oxygen induced retinopathy consisting of a 5 day exposure to 75% oxygen from postnatal day 7 to 12 was used to produce retinal neovascularization. Fluorescein conjugated dextran angiography of retinal vasculature was performed and retinal whole mounts were prepared to score features of retinopathy. The parameters that were scored in a masked fashion included blood vessel growth, blood vessel tuft formation, extra retinal neovascularization, degree of central vasoconstriction, retinal hemorrhage, and tortuosity of vessels. Diltiazem (0.05-0.5 mg/kg/day subcutaneously for five days) was administered to mice pups during exposure to oxygen to determine if calcium channel blockade altered retinopathy. In addition, quantification of retinal neovascular nuclei was performed in a masked fashion with periodic acid Schiff (PAS) staining of frozen eye sections. RESULTS: Animals that were exposed to hyperoxia for five days had a median (25th, 75th quartile) retinopathy score of 9 (8,11) versus control animals that had a retinopathy score of 1 (0,1) with p<0.001. Subscores for blood vessel growth, blood vessel tufts, extra-retinal neovascularization, central vasoconstriction, hemorrhage, and blood vessel tortuosity were all significantly different between control and treated animals. In addition, quantification of neovascular nuclei showed a significant increase in the number of nuclei extending beyond the inner limiting membrane into the vitreous in hyperoxic treated animals. Diltiazem at doses of 0.2 and 0.5 mg/kg/day improved the retinopathy as measured by the total retinopathy score [5 (4,6) and 4 (3.75,5.25), respectively]. The average number of extraretinal neovascular nuclei per retinal section (mean +/-standard deviation) was significantly decreased by diltiazem at doses of 0.2 and 0.5 mg/kg/day (31.4+/-18.8 and 20.9+/-6.9, respectively) when compared to hyperoxic treated animals (56.1+/-21.5). CONCLUSIONS: Diltiazem reduces oxygen induced retinopathy in the mouse as measured by a scoring system based on a retinal whole mount method of retinal neovascularization and by quantification of extra retinal neovascular nuclei

By using the yeast two-hybrid system, we identified the ribosomal protein L6/TAXREB107 as an intracellular partner for FGF-2. L6/TAXREB107 also mediates the DNA binding of the HTLV-1 transactivator Tax. In vitro binding experiments indicated that both the high-molecular-weight forms (HMW) and the 18-kDa form of FGF-2 bind to L6/TAXREB107. Deletion analysis suggested that L6/TAXREB107 has two binding sites for HMW FGF-2 and one binding site for 18-kDa FGF-2, implying that the unique N-terminal extension of the HMW FGF-2 is one of the binding domains for L6/TAXREB107. Transfection assays showed that high expression of either HMW or 18 kDa FGF-2 stimulates Tax-mediated transactivation in NIH 3T3 cells. This result suggests a possible role of FGF-2 in Tax-mediated HTLV-1 transformation as well as FGF-2 binding to ribosomes and/or their precursors.

BACKGROUND: Many cytokines regulate processes involved in the pathogenesis of proliferative vitreoretinopathy. Transforming growth factor-beta (TGF-beta) is an example of a pluripotent growth factor that regulates cell proliferation, extracellular matrix (ECM) deposition, cell migration, and differentiation--all biological activities involved in the formation and progression of proliferative vitreoretinopathies. METHODS: A review of experimental results that demonstrate how vascular cells generate biologically active TGF-beta is presented. Most cell types--including endothelial cells and pericytes, which form the retinal microvasculature--express TGF-beta as a large latent TGF-beta complex. Mature TGF-beta, the biologically active form, must be generated from the large latent complex before it can signal by binding to its high affinity cell surface receptors. RESULTS: A critical step in regulating TGF-beta effects may be the activation of the large latent TGF-beta complex. Activation of the complex can be achieved by chemical and enzymatic treatments, or by various cell systems. We have identified that co-culturing bovine smooth muscle cells or pericytes and endothelial cells generates active TGF-beta. CONCLUSION: The mechanism of latent TGF-beta activation self-regulates through effectors of plasmin generation. Studying TGF-beta generation by co-cultures of pericytes and endothelial cells can provide us with insights into how disruption of latent TGF-beta activation may lead to unregulated endothelial proliferation, ECM deposition, and cellular infiltration, as observed clinically in neovascular- and fibrotic-related pathologies