We have shown previously that vascular endothelial growth factor (VEGF) synthesized by the cellular constituents of small vessels per se, viz. endothelial cells and pericytes, participates in the hypoxia-driven proliferation of both cell types (Nomura, M., Yamagishi, S.,
Vascular endothelial cells (EC) exhibit organ‐to‐organ heterogeneity in their functions and morphologies. In particular, brain capillary EC have unique characteristics exemplified by the blood‐brain barrier (BBB). The formation and the maintenance of BBB have been ascribed to EC responses to inductive signal(s) or factor(s) from astrocytes that encircle microvessels in the central nervous system. These EC responses were demonstrated in numerous in vivo studies, exemplified by those of Janzer and Raff (Nature 325:253, 1987) and Tout et al. (Neuroscience 55:291, 1993) showing that transplanted astrocytes induced BBB properties in non‐neural vascular EC. In this study, we constructed a heterologous co‐culture system, in which rat fetal brain astrocytes were cultivated on one surface of a porous membrane and human umbilical vein EC on the opposite surface. Electron microscopic examination revealed that astrocytes passed their endfeet through the pores, making contact with EC. In this system, γ‐glutamyltranspeptidase (γ‐GTP) activity in EC was found to be significantly increased by contacting astrocytes in a density‐and time‐dependent manner, but not when the astrocyte feeder layer was apart from EC or replaced by COS cells; astrocyte‐derived extracellular matrix partially activated γ‐GTP. mRNAs for some of the representative BBB markers, including transferrin receptor, P‐glycoprotein, brain‐type glucose transporter(GLUT‐1), and γ‐GTP were also demonstrated by reverse transcription‐polymerase chain reaction to be upregulated in EC co‐cultured with astrocytes. Astrocyte inductions of close membrane apposition resembling a zonula occludens and of an increase in the content of mitochondria in EC were also noted in electron micrographs. Furthermore, an increased barrier activity against inulin was conferred on EC when they were lined with astrocytes. The results obtained with this heterologous co‐culture system thus indicate that through contact with their feet, astrocytes are capable of transdifferentiating non‐neural EC into the brain type, endowing them with the BBB properties. Glia 19:13–26, 1997 © 1997 Wiley‐Liss, Inc.
Vascular endothelial cells (EC) exhibit organ-to-organ heterogeneity in their functions and morphologies. In particular, brain capillary EC have unique characteristics exemplified by the blood-brain barrier (BBB). The formation and the maintenance of BBB have been ascribed to EC responses to inductive signal(s) or factor(s) from astrocytes that encircle microvessels in the central nervous system. These EC responses were demonstrated in numerous in vivo studies, exemplified by those of Janzer and Raff (Nature 325:253, 1987) and Tout et al. (Neuroscience 55:291, 1993) showing that transplanted astrocytes induced BBB properties in non-neural vascular EC. In this study, we constructed a heterologous co-culture system, in which rat fetal brain astrocytes were cultivated on one surface of a porous membrane and human umbilical vein EC on the opposite surface. Electron microscopic examination revealed that astrocytes passed their endfeet through the pores, making contact with EC. In this system, gamma-glutamyltranspeptidase (gamma-GTP) activity in EC was found to be significantly increased by contacting astrocytes in a density- and time-dependent manner, but not when the astrocyte feeder layer was apart from EC or replaced by COS cells; astrocyte-derived extracellular matrix partially activated gamma-GTP. mRNAs for some of the representative BBB markers, including transferrin receptor, P-glycoprotein, brain-type glucose transporter (GLUT-1), and gamma-GTP were also demonstrated by reverse transcription-polymerase chain reaction to be upregulated in EC co-cultured with astrocytes. Astrocyte inductions of close membrane apposition resembling a zonula occludens and of an increase in the content of mitochondria in EC were also noted in electron micrographs. Furthermore, an increased barrier activity against inulin was conferred on EC when they were lined with astrocytes. The results obtained with this heterologous co-culture system thus indicate that through contact with their feet, astrocytes are capable of transdifferentiating non-neural EC into the brain type, endowing them with the BBB properties.
This study was undertaken to determine whether and how advanced glycation end products (AGE), senescent macroproteins accumulated in various tissues under hyperglycemic states, cause angiogenesis, the principal vascular derangement in diabetic microangiopathy. We first prepared AGE-bovine serum albumin (BSA) and anti-AGE antiserum using AGE-RNase A. Then AGE-BSA was administered to human skin microvascular endothelial cells in culture, and their growth was examined. The AGE-BSA, but not nonglycated BSA, was found to induce a statistically significant increase in the number of viable endothelial cells as well as their synthesis of DNA. The increase in DNA synthesis by AGE-BSA was abolished by anti-AGE antibodies. AGE-BSA also stimulated the tube formation of endothelial cells on Matrigel. We obtained the following evidence that it is vascular endothelial growth factor (VEGF) that mainly mediates the angiogenic activities of AGE. Glucose and other reducing sugars can react nonenzymatically with the amino groups of proteins to form reversible Schiff bases and, then, Amadori products. These early glycation products undergo further complex reactions such as rearrangement, dehydration, and condensation to become irreversibly crosslinked, heterogeneous fluorescent derivatives termed advanced glycation end products (AGE) 1 (1). The formation and accumulation of AGE in various tissues have been known to progress during normal aging and at an extremely accelerated rate in diabetes mellitus. This has been implicated in the development of diabetic micro-and macro-vascular complications (1), which may account for the disabilities and high mortality rate in patients with this disease (2).Microvessels are composed of only two types of cells, endothelial cells and pericytes, and have been known to show both functional and structural abnormalities during prolonged diabetic exposure, resulting in the deleterious effects on the organs that they supply (3-5). Using pericyte-endothelial cell co-culture systems, we have shown previously that pericytes can not only regulate the growth but also preserve the prostacyclin-producing ability and protect against lipid peroxide-induced injury of endothelial cells (6). This has provided a basis for understanding how diabetic retinopathy develops consequent to "pericyte loss," the earliest histopathological hallmark in diabetic retinopathy (5, 7).Recently, we have found that AGE exert a growth inhibitory effect and a cell type-specific immediate toxicity on pericytes through interactions with their receptor for AGE (RAGE), a cell surface receptor belonging to the immunoglobulin superfamily (8), and have proposed a novel mechanism for pericyte loss (9). The AGE-induced, RAGE-mediated decrease in pericyte number would then indirectly cause angiogenesis (6,9).In the present study, we investigated the effects of AGE on the growth and tube formation of human skin microvascular endothelial cells, the key steps of angiogenesis. We demonstrate that AGE exert angiogenic activities directly on microvasc...
Focal adhesion kinase (FAK) and hypoxia-inducible factor (HIF-1alpha) are both up-regulated in glioblastoma multiforme (GBMs), particularly in invasive zones. Because FAK may play an important role in the invasion of glioma cells into the surrounding brain, we sought an agent that causes down-regulation of FAK phosphorylation as a potential inhibitor of brain tumor invasion and growth. Geldanamycin (GA), a benzoquinone ansamycin antibiotic, binds to heat shock protein 90 (Hsp90) and interferes with its function. GA inhibits the proliferation of various non-glial cells and has anti-tumor activity. Moreover, GA blocks HIF-regulated transcription of VEGF and inhibits the VEGF-induced phosphorylation of FAK and migration of endothelial cells. Here, we tested the effect of GA on glioma cell migration in vitro and its potential to down-regulate HIF-1alpha induction. Our results demonstrate that GA (i) decreases U87MG, LN229, and U251MG glioma cell migration; (ii) reduces cell migration independent of p53 and PTEN status; (iii) prevents migration at non-toxic concentrations; (iv) reduces phosphorylation of FAK; and (v) inhibits cobalt chloride (CoCl(2))-mediated induction of HIF-1alpha in glioma cells. To the best of our knowledge, this is the first report showing that GA can inhibit phosphorylation of FAK concomitant with a decrease in cellular migration. One of the most clinically relevant aspects of this study is that GA interferes with the induction of HIF-1alpha that has been linked with glioma cell migration and angiogenesis. Given the fact that GA is a small lipophilic molecule capable of penetrating the blood brain barrier together with the data presented here provide a strong rationale for its use or its analogues in the treatment of highly invasive GBMs.
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