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Role of vasodilator-stimulated phosphoprotein in PKA-induced changes in endothelial junctional permeability¹

Center for Experimental Therapeutics and Reperfusion Injury, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA

²Correspondence: Center for Experimental Therapeutics & Reperfusion Injury, Brigham and Women’s Hospital, Thorn Bldg. 704, 75 Francis St., Boston, MA 02115, USA. E-mail: colgan{at}zeus.bwh.harvard.edu

SPECIFIC AIMS

At sites of ongoing inflammation, polymorphonuclear leukocytes (PMN, neutrophils) migrate across vascular endothelia; such transmigration has the potential to disturb barrier properties and can result in intravascular fluid loss and edema. We sought to define molecular mechanisms of PMN-regulated endothelial barrier function.

PRINCIPAL FINDINGS

1. Role of PKA in regulation of endothelial barrier function
It was earlier reported that PMN interactions with endothelia promote barrier function and may represent an endogenous pathway for resealing vascular endothelia after PMN transmigration. An important component of this response included PMN-derived compounds, such as adenosine and AMP, via activation of surface adenosine A_2b receptors. As an extension of these data, incubation of human microvascular endothelial (HMVEC) monolayers with increasing numbers of physiologically localized PMN (i.e., apical surface) resulted in a concentration-dependent decrease in endothelial permeability to 70 kDa FITC-dextran. Consistent with previous findings, addition of the adenosine A_2b receptor antagonist alloxazine (10 µM) significantly hampered such changes in permeability elicited by PMN. Next, we examined the direct role of PKA in this response. Vascular endothelial cells were pre-exposed to the PKA inhibitor H89 (30 µM, 60 min; conditions that maximally inhibit PKA activation), followed by exposure to the stable adenosine analog NECA and examination of paracellular permeability. Pre-exposure to H89 significantly blocked the concentration-dependent increase in barrier function (reflected as a decrease in paracellular flux) elicited by NECA in both HMVEC and bovine aortic endothelia (BAE). Such data indicate a dominant role for PKA in adenosine-elicited changes in endothelial permeability.

2. Role of VASP in regulation of endothelial barrier
After demonstrating that PKA may significantly contribute to regulation of endothelial barrier, we examined PKA-mediated phosphorylation of a tight junction complex-associated protein(s). Since PKA phosphorylates only serine/threonine residues, zonula occludens (ZO-1) immunoprecipitates from NECA-treated HMVEC were probed for the presence of ZO-1-associated phospho-proteins (Fig. 1 A). This analysis revealed the presence of a dominant band at 50 kDa. Microsequence analysis of tryptic peptides derived from this protein revealed three peptides (sequences MQPDQQVVINCAIVR, WLPAGTGPQAFSR, VQIYHNPTANSFR) with direct sequence homology to vasodilator-stimulated phosphoprotein (VASP). It was recently shown that VASP may localize to epithelial cell-cell junctions, and since VASP is an established substrate for PKA, we investigated whether conditions that promote endothelial barrier function result in VASP phosphorylation. Western blot analysis was used to examine phosphorylation of VASP (phosphorylation of serine at position 157 leads to a marked shift in apparent molecular mass of VASP by SDS-PAGE from 46 to 50 kDa). As can be seen in Fig. 1B-D , HMVEC express VASP and, under basal conditions, the majority of VASP (>95% by densitometry) is in the unphosphorylated (46 kDa) form. In marked contrast, agents that elevate intracellular cAMP [adenosine (0–300 µM), NECA (0–50 µM), or forskolin (10 µM, positive control for PKA activation in each instance)] result in a concentration-dependent increase in VASP phosphorylation (50 kDa form), with EC₅₀s of 0.3 and 0.1 µM for adenosine and NECA, respectively. Such phosphorylation elicited by NECA (0.05–50 µM) was inhibited by pre-exposure to H89 (Fig. 1D ). Similar results were found using BAE as a source of endothelia. These data define VASP as a potential tight junction target for PKA.

View larger version (60K): [in this window] [in a new window]   Figure 1. Association of VASP with ZO-1 and phosphorylation by adenosine and adenosine analogs. A) HMVECs were stimulated with NECA (3 µM, +) or vehicle (-) for 15 min, lysed, and ZO-1 immunoprecipitated. Shown is a representative phospho-serine/threonine Western blot derived from a ZO-1 immunoprecipitation; a nonspecific antibody control is also shown (IP Ctl). The arrow signifies the 50 kDa protein identified as VASP by microsequence analysis. HMVECs were stimulated with various concentrations of 5'AMP (0–300 µM), B) adenosine (0–300 µM), C) NECA (0–50 µM) or forskolin (10 µM). Samples were separated on SDS-PAGE and immunoblotted with an anti-VASP antibody. Phosphorylated VASP runs as a 50 kDa protein and the unphosphorylated protein runs at 46 kDa. D) HMVECs were pretreated ± H89 (30 µM) before stimulation with NECA (0–50 µM).

3. Phospho-VASP localizes to endothelial cell-cell junctions
To begin defining the potential role of phospho-VASP in endothelial permeability, we generated a phospho-specific VASP anti-peptide Ab using a phospho-peptide spanning the PKA binding site (Ser157). This phospho-VASP Ab recognizes only the 50 kDa form of VASP, corresponding to Ser157 phosphorylation and the associated shift in relative molecular mass.

We next determined the cellular localization of phospho-VASP in resting endothelial cells and cells exposed to conditions that promote barrier function (3 µM NECA). Phospho-VASP staining was diffuse and unorganized in a perinuclear locale in unstimulated, resting endothelia (Fig. 2 A). Increasing periods of PKA activation (Fig. 2) resulted in the dominant localization of phospho-VASP at endothelial cell-cell junctions. Within 7.5 min of activation, phospho-VASP outlined the lengthwise orientation of the actin cytoskeleton; by 15 min, a predominant staining pattern along cell-cell borders was evident. Prominent staining at endothelial cell-cell junctions was evident as late as 60 min postactivation (data not shown). Also shown in Fig. 2 are merged localization of phospho-VASP and F-actin revealing the dominant colocalization of VASP and actin at the periphery of individual cells.

View larger version (67K): [in this window] [in a new window]   Figure 2. Phosphorylated VASP localizes to the junctions. HMVECs were stimulated with NECA (3 µM; 0–15 min), fixed, and permeabilized, followed by confocal localization of phospho-VASP (A, left series) or a combination of f-actin and phospho-VASP (A, right series). Arrows indicate junctional localization of phospho-VASP with PKA activation. A) Bottom series demonstrates background labeling with addition of only 2° antibody (left) and no 2° antibody/no phalloidin (right). B) Transient transfection of GFP-VASP is localized in the presence (right) and absence (left) of NECA (3 µM, 10 min) and demonstrates localization of VASP to the junction (arrows) with NECA activation.

As additional evidence for localization of VASP at cell junctions, BAE were transiently transfected with a plasmid encoding GFP-VASP. As shown in Fig. 2B , under basal conditions, VASP localized to the actin cytoskeleton and to focal adhesions within the endothelial membrane. Upon addition of NECA, GFP-VASP was dominantly localized to cell-cell junctions and diffusely associated with the actin network within the cytoplasm. Such data confirm our hypothesis that phospho-VASP exists in a compartment consistent with junctional regulation.

4. Role of phospho-VASP in endothelial permeability
To determine the role of phospho-VASP in endothelial permeability, truncated forms of VASP were transiently expressed in BAE and PKA-elicited changes in permeability were examined. Plasmids encoding truncated VASP encode proteins missing the EVH1 and proline-rich central domain and consist of truncations within the EVH2 domain, regions within the EVH2 domain that are both necessary and sufficient for VASP oligomerization and F-actin bundling. In the absence of PKA activation, transient transfection of BAEs with tetramerization deletions or F-actin bundling deletions resulted in 60% decreased permeability. Activation of PKA further enhanced the decrease in permeability (presumably through phosphorylation of endogenous VASP protein) in endothelial cells expressing truncated VASP protein. These results were from cells expressing a significant amount of wild-type VASP protein. Such observations indicate that full-length VASP might function in maintaining an open paracellular pathway in vascular endothelia. Taken together, these data identify VASP and structurally modified phospho-VASP as key components in the dynamic regulation of barrier function.

DISCUSSION

Approximately 70 million PMN exit the vasculature per minute. These cells move into underlying tissue by initially passing between endothelial cells that line the inner surface of blood vessels. Successful transendothelial migration (TEM) is accomplished by temporary PMN self-deformation with localized widening of the inter-endothelial junction. After TEM, adjacent endothelial cells appear to ‘reseal’, leaving no residual inter-endothelial gaps. In the present study, we sought to better define the molecular links between coupling of surface receptors and increased endothelial barrier function.

Analysis of ZO-1-associated proteins identified VASP as a dominant tight junction-associated phospho-protein. A significant target for PKA activation in many actin-based responses is VASP. Further investigation in endothelia revealed that VASP is structurally localized at endothelial junctions and is rapidly phosphorylated by agents that activate PKA. Thus, these initial studies placed VASP in a structurally relevant locale for PKA-mediated regulation of permeability. A clearer understanding of VASP phosphorylation in the process of regulated endothelial permeability was aided by using reagents that selectively probe the structure-function aspects of phospho-VASP. First, a phospho-specific antibody directed against the PKA binding site of VASP (Ser157) allowed us to immunolocalize phospho-VASP. These studies determined that basal expression of phospho-VASP was low and morphologically diffuse. However, upon PKA activation, phospho-VASP rapidly outlined actin filaments; within 7.5 min, dominant junctional staining was evident. These kinetics of phospho-VASP appearance closely parallel increases in endothelial barrier function. Moreover, expression of truncated VASP protein lacking the EVH1 domain and the proline-rich region including the preferred PKA binding site resulted in enhanced barrier function. These observations suggest that the full-length VASP protein functions as a negative regulator of actin-based barrier function.

Several lines of evidence implicate phospho-VASP in structural relaxation of the actin cytoskeleton after PKA activation, resulting in enhanced endothelial barrier function (see model in Fig. 3 ). First, inhibitors of PKA result in parallel decreases in VASP phosphorylation and endothelial permeability. Second, conditions of enhanced barrier function (e.g., PKA activation) result in a time-dependent accumulation of phospho-VASP at endothelial cell-cell junctions. Third, expression of VASP fragments lacking the PKA consensus domain enhance barrier function, suggesting that structural changes associated with VASP phosphorylation relax cytoskeletal tension and diminish paracellular permeability.

View larger version (46K): [in this window] [in a new window]   Figure 3. Model of VASP-regulation of endothelial barrier function in response to PMN. Increased levels of cAMP in endothelial cells activate protein kinase A (PKA) and subsequent phosphorylation of VASP. We propose that phospho-VASP interactions with actin and tight junction proteins (e.g., ZO-1) contribute to diminishing paracellular permeability through relaxation of actin cytoskeletal tension.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0739fje; to cite this article, use FASEB J. (February 12, 2002) 10.1096/fj.01-0739fje

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