Previous studies in the canine heart had shown that the growth of collateral arteries occurs via proliferative enlargement of pre-existing arteriolar connections (arteriogenesis). In the present study, we investigated the ultrastructure and molecular histology of growing and remodeling collateral arteries that develop after femoral artery occlusion in rabbits as a function of time from 2 h to 240 days after occlusion. Pre-existent arteriolar collaterals had a diameter of about 50 microm. They consisted of one to two layers of smooth muscle cells (SMCs) and were morphologically indistinguishable from normal arterioles. The stages of arteriogenesis consisted of arteriolar thinning, followed by transformation of SMCs from the contractile- into the proliferative- and synthetic phenotype. Endothelial cells (ECs) and SMCs proliferated, and SMCs migrated and formed a neo-intima. Intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) showed early upregulation in ECs, which was accompanied by accumulation of blood-derived macrophages. Mitosis of ECs and SMCs started about 24 h after occlusion, whereas adhesion molecule expression and monocyte adhesion occurred as early as 12 h after occlusion, suggesting a role of monocytes in vascular cell proliferation. Treatment of rabbits with the pro-inflammatory cytokine MCP-1 increased monocyte adhesion and accelerated vascular remodeling. In vitro shear-stress experiments in cultured ECs revealed an increased phosphorylation of the focal contacts after 30 min and induction of ICAM-1 and VCAM-1 expression between 2 h and 6 h after shear onset, suggesting that shear stress may be the initiating event. We conclude that the process of arteriogenesis, which leads to the positive remodeling of an arteriole into an artery up to 12 times its original size, can be modified by modulators of inflammation.

It is suggested that members of the heat-shock protein (HSP) 70 and 90 families are involved in intracellular antigen processing and the presentation of cell-membrane-anchored antigens. We show that non-lethal heat shock (41.8 degrees C) causes comparable rates of HSP72 (about 20x) and HSP73 (about 3x) synthesis in both tumor (including human Ewing's sarcoma, ES and osteosarcoma cells, HOS58) and normal cells (including EBV-transformed B-LCL, PBL and fibroblasts derived from healthy human volunteers). However, following non-lethal heat stress and a recovery period at 37 degrees C, flow cytometric analysis with a specific MAb showed HSP72 to be expressed only on the cell surface of tumor cells. The cell-surface localization of HSP72 was confirmed by Western-blot analysis of separated membranes and by immunoprecipitation with the HSP72-specific MAb. In addition, co-incubation of untreated tumor cells with supernatants from lethally heat-shocked cells, which contain HSP72, did not lead to HSP72 cell-surface expression. Thus, non-specific association of HSP72 molecules with the outer plasma membrane is unlikely. In conclusion, despite comparable cytoplasmic HSP72 induction, human tumor cells differ from normal cells in their capacity to express HSP72 on their surface. This might imply clinical application as a means to target a stress-inducible, tumor-specific immune response.

In this study, an in vitro model of the blood-brain barrier, consisting of porcine brain-derived microvascular endothelial cells (BMEC), was used to evaluate the mechanism of hypoxia-induced hyperpermeability. We show that hypoxia-induced permeability in BMEC was completely abolished by a neutralizing antibody to vascular endothelial growth factor (VEGF). In contrast, under normoxic conditions, addition of VEGF up to 100 ng/ml did not alter monolayer barrier function. Treatment with either hypoxia or VEGF under normoxic conditions induced a twofold increase in VEGF binding sites and VEGF receptor 1 (Flt-1) mRNA expression in BMEC. Hypoxia-induced permeability also was prevented by the nitric oxide (NO) synthase inhibitor N G-monomethyl-l-arginine, suggesting that NO is involved in hypoxia-induced permeability changes, which was confirmed by measurements of the cGMP level. During normoxia, treatment with VEGF (5 ng/ml) increased permeability as well as cGMP content in the presence of several antioxidants. These results suggest that hypoxia-induced permeability in vitro is mediated by the VEGF/VEGF receptor system in an autocrine manner and is essentially dependent on reducing conditions stabilizing the second messenger NO as the mediator of changes in barrier function of BMEC.

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In vivo, ischemia is known to damage the blood-brain barrier (BBB) leading to the development of vasogenic brain edema. Hypoxia-induced vascular endothelial growth factor (VEGF) has been shown to be a key regulator of these permeability changes. However, the signaling pathways that underlie VEGF-induced hyperpermeability are incompletely understood. In this study, we demonstrate that hypoxia- and VEGF-induced permeability changes depend on activation of phospholipase Cgamma (PLCgamma), phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), and protein kinase G (PKG). Inhibition of mitogen-activated protein kinases (MAPK) and of the protein kinase C (PKC) did not affect permeability at all. Paralleling hypoxia- and VEGF-induced permeability changes, localization of the tight junction proteins occludin, zonula occludens-1 (ZO-1), and ZO-2 along the cell membrane changed from a continuous to a more discontinuous expression pattern during hypoxia. In particular, localization of ZO-1 and ZO-2 expression moved from the cell membrane to the cytoplasm and nucleus whereas occludin expression remained at the cell membrane. Inhibition of PLCgamma, PI3-kinase, and PKG abolished these hypoxia-induced changes. These findings demonstrate that hypoxia and VEGF induce permeability through rearrangement of endothelial junctional proteins which involves activation of the PLCgamma and PI3-K/AKT pathway leading to the activation of PKG.