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p38 MAP kinase—a molecular switch between VEGF-induced angiogenesis and vascular hyperpermeability ¹

KATJA ISSBRÜCKER^*, HUGO H. MARTI, STEFAN HIPPENSTIEL, GEORG SPRINGMANN, ROBERT VOSWINCKEL^*,, ANDREAS GAUMANN^*, GEORG BREIER^*, HANNES C. A. DREXLER^*, NORBERT SUTTORP and MATTHIAS CLAUSS^*2

^* Department of Molecular Cell Biology, Max-Planck-Institute for Physiological and Clinical Research, 61231 Bad Nauheim, Germany;
Institute of Physiology, University of Zürich, Switzerland;
Department of Internal Medicine/Infectious Disease, Charité, Humboldt-University, Berlin, Germany; and
Department of Internal Medicine, Medical Clinic II, University of Giessen, Germany

²Correspondence: Department of Molecular Cell Biology, Max-Planck-Institute for Physiological and Clinical Research, 61231 Bad Nauheim, Germany. E-mail: M.Clauss{at}kerckhoff.mpg.de

SPECIFIC AIMS

A dissection of molecular pathways between VEGF-induced angiogenesis and vascular permeability would be desirable for the development of angiogenic and anti-angiogenic therapies. Therefore, our specific objective was to investigate the role of the p38 MAPK as the signaling molecule that separates these two processes.

PRINCIPAL FINDINGS

1. p38 MAPK has a negative regulatory role in VEGF-induced angiogenesis
To analyze the involvement of VEGF-induced p38 MAPK activation in angiogenesis, we assessed an in vitro assay for endothelial sprout formation. VEGF strongly induced sprout formation in primary human microvascular lung endothelial cells (HLMEC). Inhibition of the p38 MAPK by two small chemical inhibitors (SB203580 and SB202190) led to a more than additive increase in the number of sprouts, indicating a negative regulatory role of the p38 MAPK in this model of sprouting angiogenesis (Fig. 1 A). This finding was not restricted to microvascular endothelial cells from the lung, since an enhancement of VEGF-induced sprout formation in the presence of p38 MAPK inhibitors was observed in adrenal cortex-derived bovine microvascular endothelial (Fig. 1B ). The inhibitory role of p38 MAPK in this assay is not restricted to stimulation by VEGF because inhibition of p38 MAPK was able to enhance sprout formation in HLMEC induced by bFGF as well (Fig. 1A ). Having demonstrated that inhibition of p38 MAPK enhances angiogenesis in vitro, we tested whether this effect could also be observed in vivo. In the chorioallantoic membrane (CAM) assay for angiogenesis, methylcellulose pellets containing either vehicle, VEGF, SB203580, or SB202190 were added onto the top of the membrane at embryonic day 13. Whereas in control pellets no changes in vessel density were observed, pellets containing VEGF alone showed increased vessel density. Combination of VEGF and the p38 MAPK inhibitors led to a further increase in vessel numbers in the tissue underlying the pellet area.

View larger version (30K): [in this window] [in a new window]   Figure 1. p38 MAPK inhibition increases VEGF-induced sprouting angiogenesis in vitro. A) Human lung-derived microvascular endothelial (HLMEC) cells were seeded on cytodex-3 beads and embedded into a 3-dimensional fibrin gel together with 1 ng/mL VEGF or 10 ng/mL bFGF in combination with 10 µM of the p38 MAPK inhibitors SB203580 (S3) or SB202190 (S2). Control values (medium without growth factors) alone had no sprouting activity in this assay. VEGF and bFGF induced significant sprout formation, which was further enhanced by inhibition of p38 MAPK. Values are mean ± SD (n=3) *P < 0.001, **P < 0.01. B) The same experiments were performed with adrenal cortex-derived bovine microvascular endothelial (ACE) cells. Values are mean ± SD (n=3).

2. p38 MAPK inhibition enhances survival, plasminogen activation, and Erk-1/2 MAPK phosphorylation
Based on our finding that the p38 MAPK plays a negative regulatory role in VEGF-induced angiogenesis, we analyzed its effect on plasminogen activation, proliferation, survival, and migration, major components of angiogenesis. First we investigated the influence of p38 MAPK on survival in HLMEC. Under conditions of serum deprivation, we found that SB203580 enhanced VEGF-induced survival of HLMEC. Next we investigated the role of the p38 MAPK on plasminogen activation. When confluent HLMEC were stimulated in serum-free medium with or without VEGF alone or in combination with SB203580, SB203580 further enhanced VEGF-induced plasminogen activation as measured by the conversion of a chromogenic substrate. However, proliferation and migration were not enhanced when cells were treated with VEGF in the presence of p38 MAPK inhibitors.

Because endothelial survival and plasminogen activation have both been described to be dependent on phosphorylation of the Erk1/2 MAPK, we asked whether the effect of p38 MAPK inhibition on increased endothelial survival and plasminogen activation could be explained by an increased phosphorylation state of the Erk1/2 MAPK. Indeed, inhibition of the p38 MAPK with SB202190 or SB203580 caused a significant increase in phosphorylation of Erk1/2 MAPK. Intriguingly, addition of SB202190 or SB203580 enhanced VEGF-dependent phosphorylation of Erk1/2 MAPK for as long as 24 h.

3. Role of p38 MAPK in VEGF-induced vascular endothelial permeability
We wanted to examine the function of p38 MAPK in VEGF-induced vascular hyperpermeability, so we performed in vivo and in vitro permeability assays. First, we injected intravenous (i.v.) FITC-dextran in the CAM of the developing egg at E13 and measured the amount of fluorescence in the surrounding tissue. In line with the fact that VEGF is up-regulated in normal embryogenesis and is related to hyperpermeability in chick gestation, prestimulation with VEGF showed no significant influence on permeability (data not shown). However, prestimulation with SB203580 dramatically decreased hyperpermeability in the CAM (Fig. 2 A). Together, these data suggest that the p38 MAPK is essential for VEGF-mediated increase in vascular permeability and are in line with our previous report that the p38 MAPK is essential for VEGF-induced vascular permeability in the dermis.

View larger version (24K): [in this window] [in a new window]   Figure 2. p38 MAPK inhibition abrogates vascular permeability in vitro and in vivo. A) Fertilized eggs were i.v. injected with 100 µg SB203580 or vehicle and incubated for 2 h. To quantify vascular permeability of the CAM vessels, 25 µg FITC-dextran was i.v. injected and fluorescence was determined. Values are mean ± SE (n5). **P < 0.03. B) Adult mice were intraperitoneally injected with 20 µg SB203580 or vehicle and exposed to normobaric hypoxia at 8% oxygen for 24 h or kept at room air. To quantify vascular permeability of brain vessels, 200 µL sodium-fluorescein (6 mg/mL in PBS) was injected through the tail vein and the fluorescence of the brain parenchyma determined. p38 MAPK inhibition completely blocked the hypoxia-induced increase in vascular permeability but had no effect under normoxic conditions. Values are mean ± SE (n6). *P < 0.05. C) To HUVEC grown on polycarbonate filter membranes, 1 ng/mL VEGF, and/or 10 µM SB202190, respectively, or solvent was applied to the upper chamber, and hydraulic conductivity was determined in the presence of 10 cm hydrostatic pressure. 0.2 pM TNF was added 3 h before all experiments. VEGF-induced permeability was abolished by SB202190.

Next we wanted to know whether the p38 MAPK is also involved in increased vascular hyperpermeability in the brain. This is of clinical relevance because edema formation in the brain is a major complication in ischemic conditions. In animals submitted to systemic hypoxia, VEGF production is continuously increased in the brain when oxygen availability is progressively decreased, leading to brain edema formation. Pretreatment of animals with p38 MAPK inhibitor completely inhibited the increase in vascular permeability under hypoxic conditions but had no influence under normoxic settings (Fig. 2B ).

To confirm that the effects of p38 MAPK in controlling vascular permeability are indeed caused by a direct action of VEGF on endothelial cells, we examined the effect of p38 MAPK on VEGF-induced hyperpermeability in endothelial monolayers grown on polycarbonate filter membranes. In fact, VEGF-induced vascular permeability was completely blocked by addition of p38 MAPK inhibitor (Fig. 2C ), suggesting an involvement of p38 MAPK in the control of endothelial monolayer permeability.

CONCLUSION

In this study, we are the first to provide evidence that p38 MAPK signaling can act as a "molecular switch" between angiogenesis and hyperpermeability. We demonstrate that inhibition of p38 MAPK enhances in vitro and in vivo angiogenesis whereas permeability of endothelial cells is blocked. The proangiogenic activity of p38 MAPK inhibitors could be demonstrated by using two different endothelial growth factors (VEGF and bFGF), suggesting that p38 MAPK signaling is a more general anti-angiogenic mechanism. p38 inhibition was recently shown to accelerate bFGF-mediated angiogenesis in vitro and in in vivo.

A characteristic feature of the main angiogenic growth factor VEGF is that it not only induces angiogenesis but also vascular permeability. We had previously demonstrated that the ability of VEGF to act as vascular permeability factor in the skin is dependent on the endothelial expression of transmembrane TNF and that this effect is based on p38 MAPK activation. Here we confirmed the essential role of the p38 MAPK in VEGF-induced vascular hyperpermeability by in vitro permeability studies using confluent endothelial cell monolayers. We went one step further and demonstrated that p38 MAPK inhibition also ablates hypoxia and VEGF-mediated edema formation in the brain and blocks hyperpermeabiliy during embryogenesis in the CAMs of developing eggs.

Our demonstration that p38 MAPK is an essential signaling molecule for dissection of vascular permeability and angiogenesis is of potential clinical significance, including therapy of ischemic diseases, therapeutic angiogenesis, and antitumor therapy. 1) In stroke patients, edema formation contributes to progression of ischemic injury and is a major cause of death. A regimen leading to reduction edema formation and accelerating angiogenesis may be beneficial for therapeutic intervention in ischemic diseases. Indeed, treatment with p38 MAPK inhibitors was demonstrated to attenuate early neural injury and neuroprotection after ischemia. Whether reduced edema formation is involved in this protection was not analyzed. 2) Our finding of a dual role for p38 MAPK is of relevance for therapeutic angiogenesis. VEGF is used to accelerate angiogenesis and improve blood flow in ischemic tissue including the heart, a strategy accompanied by the side effect of increased vascular permeability and edema formation. Again, it would be desirable to ablate these side effects and to increase the efficacy of the angiogenic therapy. 3) Increased angiogenesis and hyperpermeability due to VEGF production are typical features displayed by many tumors. In this case, however, it may be advantageous to increase vascular permeability in order to facilitate drug delivery. Increasing p38 MAPK activity should therefore enhance vascular permeability but concomitantly reduce angiogenesis in tumors in which VEGF is up-regulated. Inhibition of angiogenesis (but not vascular permeability) is a promising therapeutic intervention in treatment of tumors. These examples suggest that it is important to identify a mechanism that can dissect vascular permeability from angiogenesis induction.

In conclusion, in ischemic disease, p38 MAPK inhibition is expected to reduce edema formation and improve restoration of blood flow. p38 MAPK inhibition can reduce the side effect of hyperpermeability in therapeutic angiogenesis with VEGF and enhance its angiogenic capacity. Such therapeutic approaches are promising because SB203580 and other small chemical inhibitors of the p38 MAPK have already been applied in pulmonary, neuronal, and inflammatory diseases in the absence of clinical side effects.

View larger version (37K): [in this window] [in a new window]   Figure 3. Scheme of the proposed effects of p38 MAPK in permeability and angiogenesis. VEGF has been shown to activate p38 MAPK, Erk1/2 MAPK, and PI3-kinase signaling pathways. p38 MAPK negatively regulates angiogenesis by reducing phosphorylation of Erk1/2 MAPK and increases permeability by yet unidentified mechanisms.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0329fje; to cite this article, use FASEB J. (December 18, 2002) 10.1096/fj.02-0329fje

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