Background
VEGF was originally identified by its properties as both a permogen and a mitogen, key elements in the function of the alveolar-capillary membrane, leading to interest in its role in many forms of lung disease particularly ARDS [
1‐
3]. We and others found that VEGF levels were compartmentalised between the alveolar space and the vascular bed [
4,
5]. Low levels of intrapulmonary VEGF were found in patients with ARDS with increasing intrapulmonary VEGF levels associated with recovery [
5]. In contrast, plasma levels in patients with ARDS were elevated compared with normal, at-risk, or ventilated control subjects, with falling levels associated with recovery [
6]. These data suggest that VEGF is beneficial in the alveolar space but detrimental in the vascular space. To explore the significance of these observations, it is necessary to understand the mechanisms that regulate VEGF bioactivity. VEGF exerts its biological effect through specific receptors, VEGF-R1 and VEGF-R2 and co-receptors, neuropilin-1 and neuropilin-2 [
7]. In addition, alternative splicing of VEGF transcripts leads to the generation of several functionally different isoforms [
8,
9]. We have previously explored changes in VEGF
xxx-isoforms and receptor expression as mechanisms for regulating VEGF bioactivity and suggested that both these factors may contribute [
10] but do not fully explain the reported contradictory findings. The VEGF
xxxb isoform family consists of peptides of the same length as other forms but with a different C-terminal six amino acids-SLTRKD rather than CDKPRR [
11]. The receptor binding and dimerisation domains are intact, but VEGF
xxxb stimulates a unique pattern of VEGF-R2 tyrosine residue phosphorylation, contrasting with those activated by conventional isoforms [
9]. Two specific isoforms, VEGF
165a and VEGF
165b isoforms were shown to have contrasting effects on the epithelial and endothelial sides of the alveolar-capillary membrane [
12]. These data suggest a pneumotropic effect which could be beneficial within the alveolar space following ARDS. However, the effect of these isoforms on vascular permeability another key element of ARDS is unknown.
We hypothesised that VEGF165a and VEGF165b activate different signalling pathways mediating cell permeability, a potential explanation for the conflicting observations on effects in the vascular space. To explore this theory, we used three methods of assessing vascular barrier function and found contrasting effects with VEGF165a increasing permeability and VEGF165b decreasing permeability. We then explored the relationship of downstream pathways to these functional differences. We compared the effects of specific signalling pathway inhibitors of MEK/p38MAPK/PI3K and eNOS on permeability, cell migration and proliferation to identify a mechanism by which increased permeability could be resolved whilst maintaining beneficial cell proliferation and migration.
Methods
A detailed description of materials and methods is given in the online data supplement.
Primary cell culture
Human Pulmonary microvascular endothelial cell (HPMEC) cryopreserved from passage 2 (PromoCell, Heidelberg, Germany) were cultured in endothelial cell basal medium MV2 (C-22221, PromoCell, Germany) complemented with supplement pack (C-39221, PromoCell, Germany) according to manufacturer’s instructions.
For all experiments cells were grown to 80% confluence, quiesced (MV2 media only) and stimulated with combinations of VEGF
165a and VEGF
165b (20 ng/ml as considered physiologically relevant in circulating plasma) [
4,
6] in the presence or absence of specific signalling pathway inhibitors (U0126, SB203580, LY294002 (Cell Signalling, UK) or L-NAME (Calbiochem, UK).
Measurement of TEER by Endohm
Measurement of trans-endothelial electrical resistance (TEER) of the cell monolayer was performed using an Endohm 12 electrode chamber and an endothelial volt/ohm meter EVOM
2 (World precision Instruments, USA) as previously described by Bevan and al [
13].
ECIS
Cells were plated at 20000 cells/cm2 into 8-well arrays (8W10E+; Wolf laboratories Ltd). Data was automatically and continuously collected every 2 min and recorded by computer. Experiments were performed after cells reached confluence with basal TEER values > 1500 Hz.
FITC-BSA passage
Transendothelial permeability to macromolecules was assessed by the passage of FITC-conjugated BSA (relative molecular mass 66,000) across cell monolayers in tissue culture inserts as previously described [
14].
Scratch assay (Migration and proliferation)
Cells were seeded with 100 μl of cell suspension (5 × 105 cells/ml) in an Ibidi culture-chamber (Ibidi GmbH Munich, Germany). Cells were pre-incubated with or without inhibitor for 1 hour before removal of the chamber. Cells were then incubated in MV2 medium alone or MV2 medium with 20 ng/ml of recombinant protein VEGF165a or VEGF165b. Images were captured and analysed at 0 and 24 h.
Western blotting analysis
Cell lysates were separated on sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted. Blots were blocked with 5% bovine serum albumin (BSA) (Fischer Scientific UK,) and incubated overnight at 4 °C with primary antibodies
Immunocytochemistry
HPMEC were stimulated with 100 ng/ml of VEGF165a, VEGF165b, VEGF165a + b or without any stimulation (control) for 10 min. They were then fixed, permeabilised and immunostained for VE-cadherin (Sigma, UK) and Alexa Fluor® 568 Phalloidin (Invitrogen, UK) for staining actin structures.
Discussion
The pulmonary endothelium is crucial to the regulation of the passage of solutes and molecules between the blood and the interstitial space of the lung, enabling close proximity of the vascular bed to the alveolar space for gaseous exchange to occur. Despite this, there is a very limited understanding of the mechanisms involved in the regulation of pulmonary endothelial cell (EC) barrier function integrity, which is so essential for maintaining this critical function of the lung.
Vascular endothelial growth factor (VEGF) was originally described as both an angiogenic and a permeability factor [
39] and its effects have previously been studied using the human umbilical vein endothelial cell (HUVEC) as the archetypal EC. Large organ functional differences are reflected in the variability of endothelial cell junction structure and composition particularly relevant in the functional differences between the pulmonary and systemic circulation [
40,
41]. To explore our hypothesis and its relationship to previous clinical studies (5, 6) it was important to study the response of human pulmonary microvascular endothelial cell (HPMEC) to VEGF isoforms.
Among all the pathological processes involved in. ARDS increases in lung vessel permeability are critical and non-redundant [
42]. The measurement of permeability in this study has been undertaken only in-vitro models with self-evident limitations [
43]. Transport of plasma proteins, cell and solutes across monolayers occurs paracellularly via specialised endothelial cell-cell junctions, or transcellularly by special transport mechanism including transcytosis, via transcellular channels or cell membrane transporter proteins.
Two types of inter-endothelial junction are present in the endothelium, adherens and tight junctions, the former being dominant in most vascular beds. The integrity of the adherens junction is particularly critical for regulating paracellular permeability via homophilic adhesions between VE-cadherin molecules [
19,
20]. Disruptions of these domains lead to downstream events that result in organisational changes in the actin cytoskeleton [
44]. The transcellular pathway is responsible for the transport of larger molecules such as albumin across endothelial cell monolayers, classically via transcellular pores associated with caveolae and lipid rafts [
45]. Traditionally these pathways have been considered independent but there is now a body of evidence showing interdependence [
46]. Following on from this, the in-vitro methods of measuring permeability that we have used, TEER (thought to reflect only paracellular permeability) and FITC-BSA (thought to only reflect transcellular mechanism) are recognised to have influences from crosstalk between both pathways [
15].
We have demonstrated for the first time that VEGF
165a and VEGF
165b induce differing effects on the permeability of pulmonary microvascular endothelial cells. Specifically, VEGF
165a induced an increase and VEGF
165b a decrease in permeability. The receptor binding and dimerisation domains are intact in the VEGF
xxxb family of VEGF isoforms. However, in porcine aortic endothelial cells, VEGF
165b has been shown to stimulate a unique pattern of VEGF-R2 tyrosine residue phosphorylation, contrasting with those activated by conventional isoforms suggesting activation of differing downstream signalling pathways in addition to partial agonist activity and changes in neuroplin-1 binding [
22,
47,
48]. In this study, differing phosphorylation kinetics were clearly observed following stimulation by VEGF
165a and VEGF
165b using what we considered to be physiologically relevant concentrations of VEGF.
The differential effects of VEGF
165a and VEGF
165b on the vascular permeability in addition to those we have previously shown and published on proliferation in HPMEC (also repeated in the scratch experiments) and human alveolar epithelial cells offer a potential paradigm to explain the apparent compartmentalisation of VEGF between the alveolar and vascular space and the apparent disparity of data relating to the role of VEGF in ARDS [
5,
6,
12].
We identified that VEGF165a and VEGF165b lead to differential functional outcomes with VEGF165a increasing cell permeability in methods suggested to reflect both para and transcellular permeability and VEGF165b reducing paracellular permeability only. This suggested that the differences were due to divergence of signalling pathways and therefore potential targets for amelioration of outcome e.g. reducing permeability whilst preserving a pneumotropic effect. To verify this hypothesis, different protein inhibitors have been used to look at their effect on the change in resistance reflecting the paracellular permeability pathway of pulmonary microvascular ECs.
The VEGF
165a signalling pathways have been studied extensively in HUVEC although studies in HPMEC are limited [
41]. We chose to use inhibitors of pMEK, P38 MAPK, PI3 kinase and eNOS proteins as these proteins have been suggested to be involved in VEGF signalling pathways and look at both permeability and proliferation/migration in an attempt to identify a divergence in functionality and thus an opportunity for selective inhibition.
The use of L-NAME (eNOS inhibitor) and LY294002 (PI3K inhibitor) on HPMEC inhibited the effect of both VEGF
165a and VEGF
165b. Being part of the same signalling pathway these results suggest that VEGF cell paracellular permeability involves the phosphoinositide 3-kinase–AKT pathway, which then further phosphorylates and activates endothelial nitric oxide synthase (eNOS) [
49]. Also, the inhibition of pMEK and p38 MAPK did not affect VEGF isoforms activity on the proliferation and paracellular permeability pathway in HPMEC. In summary, the inhibitors chosen did not allow for the identification of specific differential pathways between VEGF
165a and VEGF
165b. Further studies of other pathways are required in order to unravel the molecules responsible for the differential permeability effects of VEGF isoforms such as Src kinase pathway and its role in the regulation of endothelial-barrier integrity as demonstrated recently by Gao et al. [
50].
Acknowledgements
Not applicable.