VEGF increases paracellular transport without altering the solvent-drag reflection coefficient.

Vascular endothelial growth factor (VEGF) increases microvascular permeability and has been implicated in the development of numerous pathologies including diabetic retinopathy (DR), hypoxia/ischemia, and tumor biology. The transport pathways by which water and solutes cross the endothelium in response to VEGF, however, are not completely understood. We measured, in real time, bovine retinal endothelial cell (BREC) hydraulic conductivity (Lp), 70 kDa dextran permeability (Pe), and the solvent-drag reflection coefficient (sigma) before and after addition of 50 ng/ml VEGF. The diffusional permeability coefficient for dextran (Pd) was measured before pressure gradient application. The sudden application of a 10-cm H2O hydrostatic pressure gradient induced water and solute fluxes that decayed to steady-state values after approximately 2 h. Subsequently, the addition of VEGF significantly increased Lp and Pe by 4.3-fold +/- 0.7-fold and 3.0-fold +/- 0.3-fold, respectively, after 110 min; however, the reflection coefficient remained approximately constant throughout the experiment (approximately 0.8). These observations suggest that water and dextran utilize common paracellular channels across BREC monolayers. Furthermore, the addition of VEGF increases the number or availability of channels but does not alter the selectivity of the monolayer to 70 kDa dextran.

IGF-I prevents glutamate-mediated bax translocation and cytochrome C release in O4+ oligodendrocyte progenitors.

Oligodendroglial death due to overactivation of the AMPA/kainate glutamate receptors is implicated in white matter damage in multiple CNS disorders. We previously demonstrated that glutamate induces caspase-3 activation and death of the late oligodendrocyte progenitor known as the pro-oligodendroblast (pro-OL) via activation of the AMPA/kainate glutamate receptors. We also demonstrated that IGF-I had the unique ability to sustain activation of Akt in the pro-OL and provide long-term protection of these cells from glutamate-mediated apoptosis. The goal of these studies was to investigate the mechanisms of glutamate toxicity and IGF-I-mediated survival in the pro-OL. IGF-I prevented glutamate-induced loss of mitochondrial membrane potential, cytochrome c release, and caspase-9 activation. In contrast to IGF-I mediated survival mechanisms in neurons, IGF-I had no effect on the influx or recovery of intracellular calcium levels or on levels of major pro- and anti-apoptotic molecules including Bax or Bcl-xL. Rather, IGF-I prevented the glutamate-induced translocation of Bax to the mitochondria. Moreover, IGF-I prevented caspase-3 activation in pro-OLs as long as 8 h after exposure of the cells to glutamate, suggesting that delayed activation of IGF-I-mediated survival pathways can block glutamate-mediated apoptosis in pro-OLs. The results of these experiments define the mechanisms by which glutamate kills oligodendrocyte progenitor cells and by which IGF-I blocks glutamate-induced apoptosis in these cells. The data also demonstrate that IGF-I disrupts the glutamate-mediated apoptotic pathway in the pro-OL through mechanisms that are distinct from its survival-promoting actions in neurons.

A transmural pressure gradient induces mechanical and biological adaptive responses in endothelial cells.

A sudden increase in the transmural pressure gradient across endothelial monolayers reduces hydraulic conductivity (L(p)), a phenomenon known as the sealing effect. To further characterize this endothelial adaptive response, we measured bovine aortic endothelial cell (BAEC) permeability to albumin and 70-kDa dextran, L(p), and the solvent-drag reflection coefficients (sigma) during the sealing process. The diffusional permeability coefficients for albumin (1.33 +/- 0.18 x 10(-6) cm/s) and dextran (0.60 +/- 0.16 x 10(-6) cm/s) were measured before pressure application. The effective permeabilities (measured when solvent drag contributes to solute transport) of albumin and dextran (P(ealb) and P(edex)) were measured after the application of a 10 cmH(2)O pressure gradient; during the first 2 h of pressure application, P(ealb), P(edex), and L(p) were significantly reduced by 2.0 +/- 0.3-, 2.1 +/- 0.3-, and 3.7 +/- 0.3-fold, respectively. Immunostaining of the tight junction (TJ) protein zonula occludens-1 (ZO-1) was significantly increased at cell-cell contacts after the application of transmural pressure. Cytochalasin D treatment significantly elevated transport but did not inhibit the adaptive response, whereas colchicine treatment had no effect on diffusive permeability but inhibited the adaptive response. Neither cytoskeletal inhibitor altered sigma despite significantly elevating both L(p) and effective permeability. Our data suggest that BAECs actively adapt to elevated transmural pressure by mobilizing ZO-1 to intercellular junctions via microtubules. A mechanical (passive) component of the sealing effect appears to reduce the size of a small pore system that allows the transport of water but not dextran or albumin. Furthermore, the structures of the TJ determine transport rates but do not define the selectivity of the monolayer to solutes (sigma).

Hydrolysis of Ca2+-sensitive fluorescent probes by perfused rat heart.

Rat hearts were loaded with the fluorescent calcium indicators fura 2, indo 1, rhod 2, or fluo 3 to determine cytosolic calcium levels in the perfused rat heart. With fura 2, however, basal tissue fluorescence increased above anticipated levels, suggesting accumulation of intermediates of fura 2-AM deesterification. To examine this process, we separated the intermediates of the deesterification process using HPLC after incubation of fura 2-AM with tissue homogenates and after loading in the rat heart. Loading of hearts with fura 2-AM resulted in tissue levels of fura 2 free acid that were only 5% of the total heart dye content of all fura 2 species. The parent fura 2-AM form accumulated without accumulation of intermediate products. Similar results were obtained with indo 1-AM. Fluo 3 loaded very poorly in perfused hearts. Unlike other indictors, rhod 2 rapidly loaded in perfused hearts and was completely converted to the free acid form. To determine the subcellular localization of the free acid form of these indictors, mitochondria from indicator-loaded hearts were assayed for the free acid form. Approximately 75% of the total amount of rhod 2 in hearts could be recovered in isolated mitochondria. Subcellular localization of indo 1 and fura 2 was more evenly distributed between mitochondria and nonmitochondrial compartments. We conclude that measurement of calcium in the perfused rat heart using surface fluorescence with either indo 1 or fura 2 is complicated by an inconsistent accumulation of the parent ester and that the resulting signal cannot be easily calibrated using "in situ" methods using the free acid form. Rhod 2 does not display this shortcoming, but like other indicators, it also loads into the mitochondrial matrix.

Hydrocortisone decreases retinal endothelial cell water and solute flux coincident with increased content and decreased phosphorylation of occludin.

Corticosteroids provide an effective treatment to reduce edema for conditions in which the blood-brain or blood-retinal barrier is compromised. However, little is known about the mechanism by which these hormones affect endothelial cell function. We hypothesized that hydrocortisone would reduce transport of water and solutes across bovine retinal endothelial cell (BREC) monolayers coincident with changes to the tight junction protein occludin. Treatment of BREC with 103 nm hydrocortisone for two days significantly decreased water and solute transport across cell monolayers. Immunoblot analysis of occludin extracted in SDS or urea based buffers revealed a 1.65- or 2.57-fold increase in content, respectively. A similar two-fold increase in occludin mRNA was observed by real-time PCR. Immunocytochemistry revealed hydrocortisone dramatically increased both occludin and ZO-1 staining at the cell border. Additionally, 4 h of hydrocortisone treatment significantly reduced occludin phosphorylation. To our knowledge, this is the first example of a regulated decrease in occludin phosphorylation associated with increased barrier properties. In conclusion, hydrocortisone directly affects retinal endothelial cell barrier properties coincident with changes in occludin content, phosphorylation and tight junction assembly. Localized hydrocortisone therapy may be developed as a treatment option for patients suffering from retinal edema due to diabetes.