INTRODUCTION
The passive and active movement of solutes from the intravascular space to the extracellular compartment is controlled by the endothelial barrier. Central to this process is the cytoskeleton, wherein dynamic reorganization of the actin filaments is crucial for the control of fluid exchange [
1]. Vasodilator-stimulated phosphoprotein (VASP) is a central cytoskeletal protein that holds significant impact on the active reorganization of the cytoskeleton. In endothelial cells, VASP functions in membrane ruffling, aggregation, and tethering of actin filaments during the formation of endothelial cell–substrate and cell–cell contacts. Moreover, VASP expression is increased in endothelial cells during angiogenesis and at most phases involving cell shape change [
2]. At resting conditions, siRNA-mediated downregulation of VASP does not affect transendothelial resistance (TER) but increases permeability to fluorescein isothiocyanate-conjugated dextran (FITC-dextran) [
3,
4]. Similarly, murine microvascular myocardial endothelial (MyEnd) cells derived from
VASP
−/−
mice show no difference in TER when compared to control MyEnd cells but exhibit an increase in permeability to FITC-dextran under resting conditions [
5‐
7].
During periods of hypoxia or inflammation, the endothelial barrier becomes dysfunctional and fluid passes from the intravascular to the extravascular compartment [
8]. This process is associated with the formation of stress fibers within endothelial cells [
9‐
11]. VASP prevents the formation of stress fibers and as such is protective for the maintenance of the endothelial barrier function. Furman
et al. demonstrated that a reduction of the Ena/VASP expression is detrimental during embryologic development. In this study, the authors demonstrated that in the absence of Ena/VASP, the vasculature exhibits patterning defects and lacks structural integrity, leading to edema, hemorrhaging, and, as a result, late stage embryonic lethality [
12]. We have previously demonstrated that VASP is repressed during hypoxia, and this repression results in a reduction of intestinal barrier function during periods of tissue hypoxia
in vivo [
4]. However, the role of endothelial VASP for the maintenance of barrier function during hypoxia
in vivo has not been investigated yet.
In the present study, we pursued the role of VASP for barrier function during hypoxia in vivo. We found a significant repression of endothelial VASP through a hypoxia-inducible factor-1 (HIF-1α)-dependent mechanism which correlated with increased tissue permeability. Studies employing VASP
−/−
animals identified VASP to be of great importance for vascular barrier function during conditions associated with tissue hypoxia.
DISCUSSION
Given the importance of VASP for the endothelial cytoskeleton, the repression of VASP might have functional impact on the endothelial barrier function in vivo. In the presented study, we found a significant repression VASP in endothelial cells during hypoxia which was associated with altered barrier properties during hypoxia in vitro and in vivo as demonstrated through the exposure of VASP
−/−
mice to hypoxia.
VASP mediates actin dynamics within endothelial and epithelial cells and is involved in cell shape change [
2]. In addition, passive cell retraction as a result of cytoskeletal rearrangement plays a key role in mediating cellular contractile response and changes paracellular permeability [
17‐
21]. This rearrangement transposes its force on cell–cell junctions through indirect attachment of actin fibers with tight and adherens junctions. Previous reports have shown that VASP may protect the endothelial barrier during exposure to H
2O
2 or lipopolysaccharide (LPS) through a prevention of stress fiber formation. In endothelial cells, this stress fiber formation or destruction of cytoskeletal structures is associated with an increase in permeability [
22‐
24]. Downregulation of VASP using VASP siRNA techniques in human pulmonary artery endothelium exacerbates the H
2O
2-induced decrease in TER, whereas in human lung microvascular ECs, it potentiated LPS-induced decrease in TER [
3,
25]. Thus, VASP appears to be a common downstream target for oxidants and inflammatory mediators increasing vascular permeability. A redistribution of actin in cells exposed to chemical hypoxia persists longer than 3 h and is also associated with increased paracellular flux [
8,
26]. In addition, ischemia, or as such hypoxia, is a cause for cells to lose their polarity, to open tight junctions, and, as a result, to increase paracellular permeability [
27]. We have demonstrated previously that VASP is repressed in response to inflammatory cytokines in endothelial cells and epithelial cells [
28]. The presented study extends these findings and identifies HIF-1α to be responsible for the observed repression. An involvement of NF-κB, which is also induced during periods of tissue hypoxia, in the observed VASP repression during periods of hypoxia seems rather unlikely given the results of this study [
29]. The possible interaction between hypoxia and inflammation through the IκB kinase complex, a regulatory component of NF-κB, and in the regulation of HIF-1α transcription by NF-κB before and during inflammation, however, has to be kept in mind [
8,
29,
30].
Sites of acute inflammation are characterized by shifts in the supply and demand of metabolites that result in limited oxygen availability (inflammation-associated hypoxia) [
8,
31,
32]. But hypoxia itself represents an inflammatory stimulus [
8,
33‐
35]. Just as hypoxia can induce inflammation, inflamed lesions often become severely hypoxic [
8]. Moreover, exposure of mice to ambient hypoxia (e.g., 8% oxygen over 4–8 h) induces increased leakage through epithelial or endothelial barriers and induces inflammatory cell accumulation in mucosal organs. This plays a critical role in several human clinical conditions including solid organ transplantation (e.g., lung or liver) [
36‐
41]. Although protective pathways are triggered during periods of tissue hypoxia, the effect of these potential pathways on vascular leakage during conditions of VASP repression has to be seen critical [
42]. This highlights the fact that VASP is protective for barrier properties
in vivo. This was also demonstrated by Furman
et al. evaluating the edema formation of mice with gene-targeted deletion of the Ena/VASP complex [
12]. This study demonstrated that during embryonic development, Ena/VASP repression resulted in reduced vascular barrier properties and in tissue edema formation [
12]. We have demonstrated that VASP deficiency does not alter barrier properties at baseline but results in a significant difference during an acute inflammatory response within the lung [
28,
42]. Profirovic
et al. extended these findings demonstrating increased vascular permeability in response to thrombin in
VASP
−/−
deficient lungs [
43]. We now demonstrate that
VASP
−/−
animals do not demonstrate a difference in vascular permeability at baseline but have increased vascular permeability during a hypoxic challenge. Therefore, our findings are in accordance with previous
in vivo reports and further the knowledge about the role of VASP for barrier protection.
In summary, we demonstrate that endothelial VASP has significant impact on vascular barrier function during periods of hypoxia in vivo but does not influence baseline fluid exchange. The results of this and other studies related to the role of VASP in endothelial barrier function during hypoxia may be helpful for development of efficient pharmacological treatment of conditions associated with hypoxia and vascular leak.
ACKNOWLEDGMENTS
We thank Alice Mager, Michaela Hoch-Gutbrot, and Stefanie Laucher for technical assistance. This work was supported by a grant from the Fonds National de la Recherche (FNR) to M.A.S. and a grant from the Deutsche Forschungsgemeinschaft (DFG) DFG-RO 3671/4-1 to P.R.