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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.

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Freshly isolated adult rat hepatocytes exhibit a flat, extended morphology when cultured on dried rat tail collagen in the presence of growth factors; they actively synthesize DNA and express high levels of cytoskeletal mRNAs and proteins (actin, tubulin, cytokeratins, vinculin, a-actinin, and desmoplakin), while exhibiting low levels of liver-specific mRNAs (albumin, a,-inhibitor III, and a1-antitrypsin) and limited synthesis and secretion of albumin. Hepatocytes cultured on hydrated gel matrix from the Engelbreth-HolmSwarm (EHS) mouse tumor form small spherical aggregates and exhibit low DNA, cytoskeletal mRNA, and protein synthesis, while at the same time exhibiting elevated liver-specific mRNAs and albumin production; these cells, therefore, more nearly conform to the program of gene expression seen within the normal animal. Hepatocytes on hydrated rat tail collagen resemble those on dry collagen when cultured at low density, but at high density they form compact trabecular aggregates, synthesize negligible amounts of DNA, and maintain a pattern of gene expression resembling that of hepatocytes seeded on the EHS matrix. If cell morphology is compact, as on EHS or on hydrated rat tail collagen when densely populated, DNA synthesis and expression of cytoskeletal genes are low, while liver-specific mRNAs are abundant. When cells are extended the opposite is the case. Without the growth supplement DNA synthesis is low throughout but gene expression is little affected. These studies point to the importance of cell-cell and cell-matrix interactions in determining the differentiated phenotype of hepatocytes, and they reveal an inverse relationship between cytoskeletal and liver-specific protein expression.Studies in a variety of cell culture systems suggest that changes in cell morphology are involved in the regulation of growth and gene expression and in the maintenance of differentiated functions (for reviews, see refs. 1 and 2). Cell morphology reflects the organization of the intracellular cytoskeletal network, which is partially determined by the interaction between the cells and their surrounding extracellular matrix (ECM) (for reviews, see refs. 3-5). This suggests that the ECM influences the expression of differentiated functions through organization of the cytoskeleton (6, 7). Several studies in vitro have demonstrated cytoskeletal involvement in transmitting extracellular signals (mitogenic and differentiation-specific) that regulate various programs of gene expression (for reviews see refs. 1 and 2). It is not known as yet whether mechanisms that regulate the cytoskeleton, both at the level of gene expression and synthesis and at the level of organization of its proteins, are important components of the differentiation programs of highly specialized cells.It has been demonstrated that the expression of certain differentiated functions in cultured hepatocytes is dependent on the interaction of these cells with constituents of the ECM (8-13). Evidence suggests that particular cytoskeletal genes may...

'Vascular endothelial growth factor-A (VEGF-A) blockade has been recently validated as an effective strategy for the inhibition of new blood vessel growth in cancer and ocular pathologies. However, several studies have also shown that anti-VEGF therapy may not be as effective in the treatment of established unwanted blood vessels, suggesting they may become less dependent on VEGF-A for survival. The VEGF-A dependence of vessels may be related to the presence of vascular mural cells (pericytes or smooth muscle cells). Mural cell recruitment to the growing endothelial tube is regulated by platelet-derived growth factor-B (PDGF-B) signaling, and interference with this pathway causes disruption of endothelial cell-mural cell interactions and loss of mural cells. We have investigated the basis of blood vessel dependence on VEGF-A in models of corneal and choroidal neovascularization using a combination of reagents (an anti-VEGF aptamer and an anti-PDGFR-beta antibody) to inhibit both the VEGF-A and PDGF-B signaling pathways. We demonstrate that neovessels become refractory to VEGF-A deprivation over time. We also show that inhibition of both VEGF-A and PDGF-B signaling is more effective than blocking VEGF-A alone at causing vessel regression in multiple models of neovascular growth. These findings provide insight into blood vessel growth factor dependency and validate a combination therapy strategy for enhancing the current treatments for ocular angiogenic disease.

Diseases characterized by retinal neovascularization are among the principal causes of visual loss worldwide. The hypoxia-stimulated expression of vascular endothelial growth factor (VEGF)

Gaucher disease, the most common lysosomal storage disease, can be treated with enzyme replacement therapy (ERT), in which defective acid-β-glucosidase (GlcCerase) is supplemented by a recombinant, active enzyme. The X-ray structures of recombinant GlcCerase produced in Chinese hamster ovary cells (imiglucerase, Cerezyme®) and in transgenic carrot cells (prGCD) have been previously solved. We now describe the structure and characteristics of a novel form of GlcCerase under investigation for the treatment of Gaucher disease, Gene-ActivatedTM human GlcCerase (velaglucerase alfa). In contrast to imiglucerase and prGCD, velaglucerase alfa contains the native human enzyme sequence. All three GlcCerases consist of three domains, with the active site located in domain III. The distances between the carboxylic oxygens of the catalytic residues, E340 and E235, are consistent with distances proposed for acid–base hydrolysis. Kinetic parameters (Km and Vmax) of velaglucerase alfa and imiglucerase, as well as their specific activities, are similar. However, analysis of glycosylation patterns shows that velaglucerase alfa displays distinctly different structures from imiglucerase and prGCD. The predominant glycan on velaglucerase alfa is a high-mannose type, with nine mannose units, while imiglucerase contains a chitobiose tri-mannosyl core glycan with fucosylation. These differences in glycosylation affect cellular internalization; the rate of velaglucerase alfa internalization into human macrophages is at least 2-fold greater than that of imiglucerase.

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