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VEGF-induced paracellular permeability in cultured endothelial cells involves urokinase and its receptor¹

M. ALI BEHZADIAN^*,,2, L. JACK WINDSOR, NAGLA GHALY^*, GREGORY LIOU^*,, NAI-TSE TSAI^* and RUTH B. CALDWELL^*,,||

^* Vascular Biology Center, Departments of
Pharmacology & Toxicology,
^|| Cellular Biology & Anatomy, and
Ophthalmology, Medical College of Georgia, Augusta, Georgia, USA; and
Department of Oral Biology, Indiana University, Indianapolis, Indiana, USA

²Correspondence: Vascular Biology Center, Department of Pharmacology & Toxicology, Medical College of Georgia, 1120 15th Street, Augusta, Georgia 30912, USA. E-mail: abehzadi{at}mail.mcg.edu

SPECIFIC AIMS

The purpose of this work was to study the functional role of extracellular proteinases as mediators of vascular endothelial growth factor (VEGF)-induced blood/tissue barrier breakdown associated with pathologic angiogensesis. We hypothesized that angiogenic factors induce expression and/or activation of proteolytic enzymes, which modify adhesion proteins of vascular endothelial cells. This will generate leaky vessels, allowing the endothelial cells to migrate and proliferate.

PRINCIPAL FINDINGS

1. The biphasic pattern of VEGF-induced permeability and involvement of urokinase (uPA)
Treatment with VEGF or uPA increases permeability of endothelial cell monolayers (Fig. 1 A). The uPA-induced permeability is rapid and remains unchanged over the first 6 h, with a slight decrease following overnight incubation. The kinetics of the VEGF-induced permeability, conversely, displays a biphasic profile—an early and transient phase followed by a sustained permeability increase starting 4–6 h post-VEGF treatment. In our experimental model, primary cultures of the BRE cells grown on porous membranes (Transwell) separate two chambers: The upper chamber represents the luminal and the lower chamber, the abluminal surface of the endothelium. Using this model, the effects of enzymes, cytokines, and potent pharmaceutical reagents on the permeability of endothelial cells have been previously analyzed by us and by others. For kinetic analysis, sets of four independent Transwells were designated for each time point, where VEGF or other reagents were added to the upper chambers. At the end, the tracer (HRP) was added simultaneously to all upper chambers, and exactly 1 h later, aliquots were withdrawn from the lower chambers for HRP assay. In other words, regardless of the duration of treatment, the amounts of tracer passing across the monolayers were measured simultaneously and for a 1-h period in all Transwells.

View larger version (23K): [in this window] [in a new window]   Figure 1. Kinetics of VEGF-induced permeability and involvement of uPA. A) Bovine retinal microvascular endothelial (BRE) cells grown to confluent monolayers on porous membranes (Transwell) were incubated with VEGF or uPA for designated times, and the flux of tracer molecules [horseradish peroxidase (HRP)] from the upper to the lower chamber was measured during the last hour. B) Designated antibodies were added 30 min before treatment started, and permeability was measured. ON, Overnight; C, control; uPAR, uPA receptor. C) Transendothelial electrical resistance (TER) was measured under the same conditions as in A.

It was only by this method that we were able to reveal, for the first time, the biphasic nature of the VEGF-induced permeability increase. The delayed and sustained phase of VEGF-induced permeability resembles the exogenous uPA effect and is likely to involve uPA/uPAR activity, as it is inhibited by anti-uPA and anti-uPAR antibodies (Fig. 1B ). This delayed phase is accompanied by a decline in TER of the monolayer, whereas the early phase does not involve TER changes (Fig. 1C ). Previous work in our laboratory indicates that the early phase of permeability is transcellular, and it is mediated by plasma membrane-derived caveolae. The late phase of VEGF-induced permeability as well as the acute uPA-induced permeability involves redistribution of junction proteins and decline in electrical resistance; therefore, it is very likely to be paracellular. Data in Figure 1 represent results of quadruplicate assays ± SD, which have been repeated at least two more times.

To directly demonstrate uPA involvement, BRE cells were treated with VEGF and transforming growth factor-ß (TGF-ß), and conditioned media were analyzed by substrate gel electrophoresis. It was found that BRE cells constitutively express the 50-kDa intact uPA. VEGF increased total uPA activity and generated a low molecular weight (33–35 kDa) uPA (data not shown), which has been previously characterized as an active enzyme with a truncated receptor-binding domain. Treatment with TGF-ß alone or combined with VEGF reduced uPA and blocked the VEGF-induced S-uPA formation.

2. Induction of uPAR gene expression by VEGF
That anti-uPAR antibody blocked VEGF-induced permeability persuaded us to test whether VEGF induces uPAR gene expression. Reverse transcriptase-polymerase chain reaction (PCR) analysis showed that uPAR mRNA was almost undetectable or very low in quiescent BRE cells but was substantially increased by VEGF treatment. uPAR gene expression was quantified by kinetic PCR using a light cycler apparatus. A time-dependent increase in uPAR mRNA was observed starting at 1 h post-VEGF treatment and reached a maximum of 45-fold at 3 h, which remained at that level following overnight incubation (not shown).

3. Effects of VEGF and uPA on the distribution of occludin and ß-catenin in BRE cells
To further establish the functional role of VEGF-induced uPA/uPAR in endothelial permeability, we compared the effects of VEGF and uPA on redistribution of cell-adhesion proteins. The VEGF-induced permeability increase was accompanied by decreased levels of the tight junction protein occludin (not shown). Confocal microscopic analysis showed a reduction in occludin immunoreactivity in membrane-bound and later in cytosolic levels (not shown). Transmission electron microscopy clearly showed openings in tight junctions along the inter-endothelial cell attachments (see full-length article) in endothelial cells treated overnight with VEGF. Tight junctions remained closed in untreated cells or in monolayers treated with VEGF for only a short time. It is interesting that the temporal distinctions between uPA- and VEGF-induced permeability are also reflected in their effects on occludin mobilization and cell morphology. Within 10 min of uPA treatment, membrane-bound occludin is almost totally diminished, and immunoreactive occludin aggregates appeared in the cytoplasm. Within 30 min of uPA treatment, cells appeared larger and thinner with a more hexagonal shape. At the 6-h time point, only small amounts of fragmented membrane-bound occludin could be seen. The VEGF effects, although comparable with the uPA effects, emerged with a 6-h delay.

As uPAR expression by Wnt signaling has been reported to involve the transcriptional activation of ß-catenin, we next examined the VEGF effects on subcellular redistribution of ß-catenin. Confocal microscopy showed that membrane-bound ß-catenin appeared in the cytoplasm within 10 min of VEGF treatment (Fig. 2 A). Western blot analysis and double-labeling confocal microscopy demonstrated that following an increase in cytosolic ß-catenin, nuclear-associated ß-catenin was also increased. Optical sections through the center of nuclei showed the presence of ß-catenin within the nucleus (Fig. 2B , yellow). Taken together, our data indicate that VEGF-induced uPAR expression requires ß-catenin’s signaling pathway, the biological consequence of which corresponds to the paracellular phase of VEGF-induced permeability.

View larger version (85K): [in this window] [in a new window]   Figure 2. Redistribution of ß-catenin in BRE cells treated with VEGF. Confocal microscopy of VEGF-treated BRE cells showing redistribution of immunoreactive ß-catenin. A) Within 10 min of VEGF treatment, ß-catenin aggregates appear in the cytoplasm. B) Digital, 3-dimensional image analysis shows the presence of ß-catenin within the nuclei at 3 h post-VEGF treatment (yellow). Original bar represents 40 µm.

CONCLUSIONS AND SIGNIFICANCE

Studies of endothelial hyperpermeability and blood/tissue barrier dysfunction have implications for a number of disease entities. Inflammatory disorders, rheumatoid arthritis, and disorders involving pathologic angiogenesis, such as diabetic retinopathy, tumor growth, and metastasis, all begin with leaky vessels. Although VEGF has been initially characterized as a vascular permeability factor (VEGF was found to be 50,000 times more potent than histamine), research has been focused on VEGF’s mitogenic activity, which has implication in tumor angiogenesis and wound healing. But, the mechanism of VEGF-induced endothelial permeability has not been fully understood.

Using primary cultures of endothelial (BRE) cells, we show that VEGF induces uPAR expression and uPA activation in these cells. BRE cells constitutively release the 50-kDa intact uPA into the culture medium, and VEGF treatment increases the total uPA activity only by 50%. Moreover, VEGF treatment generates the 33-kDa short double-chain uPA (S-uPA) that has been characterized as an active enzyme with a truncated receptor-binding domain. It is further demonstrated here that uPA/uPAR expression in BRE cells correlates with a delayed and sustained permeability increase, as measured by two different parameters: the TER and the flux of the tracer HRP across the BRE monolayers. uPAR focuses activation of uPA, plasmin, and metalloproteinase on the cell surface, initiating a cascade of pericellular proteolytic activities leading to tissue remodeling or endothelial cell migration and proliferation. Hence, expression of uPAR provides a crucial link between VEGF’s action in promoting vascular permeability and angiogenesis.

Previously, we presented data indicating that glial cell-derived TGF-ß increases endothelial cell permeability by inducing expression of the matrix metalloproteinase (MMP)-9 and that TGF-ß, when activated under hypoxia or coculture conditions, induces VEGF expression. As latent TGF-ß and pro-MMP-9 are thought to be activated at the cell surface by plasmin, we speculated that uPA/uPAR activity plays a role in TGF-ß-induced permeability as well. Conversely, it has been reported that TGF-ß up-regulates uPA inhibitor PAI-1, which in turn, facilitates the internalization of the uPA–uPAR complex and degradation of uPA. Although TGF-ß and VEGF are implicated in the angiogenesis process, they seem to function in a temporally distinct and sometimes contradictory manner. VEGF promotes endothelial cell proliferation and migration at the onset of the angiogenesis process, and TGF-ß blocks endothelial cell proliferation and promotes their differentiation, steps that are required for the emergence of patent capillaries. Therefore, the mechanism of VEGF and TGF-ß interaction in regulating pericellular proteolytic activities deserves further investigation, as it has significant implications in coordinating vascular stability.

To establish the functional role of VEGF-induced uPA/uPAR activation in endothelial-cell permeability, we compared the effect of VEGF with that of the exogenous uPA on cellular redistribution of occludin, a component of tight junctions that forms diffusion barrier and regulates the flux of ions and hydrophilic molecules through the paracellular pathway. We demonstrate here that the VEGF effect on occludin mimics the uPA effect but with a considerable delay, which probably represents the time required for uPAR expression.

In summary, our data demonstrate that in the delayed phase of VEGF-induced permeability, the tight junction barrier breaks down, allowing paracellular passage of the tracer HRP. This is indicated by a simultaneous drop in TER (Fig. 1C ) and redistribution of occludin. It is interesting that when exogenous uPA is directly added to the culture, the barrier breakdown is instant. This is evident in the permeability assay, TER measurements, and occludin redistribution. Moreover, we are showing that VEGF-induced u-PAR expression and down-regulation of occludin in BRE cells are regulated by a mechanism involving the ß-catenin signaling pathway. This model allows for testing reagents that may specifically block VEGF-induced signals leading to the activation of a uPA/uPAR system and increased permeability in vascular endothelial cells.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0484fje; to cite this article, use FASEB J. (February 19, 2003) 10.1096/fj.02-0484fje

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This Article Full Text (PDF) All Versions of this Article: 17/6/752 02-0484fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by BEHZADIAN, M. A. Articles by CALDWELL, R. B. Search for Related Content PubMed PubMed Citation Articles by BEHZADIAN, M. A. Articles by CALDWELL, R. B.