In vivo mechanisms of vascular endothelial growth factor-mediated increased hydraulic conductivity of Rana capillaries

Abstract

1. Vascular endothelial growth factor (VEGF) increases hydraulic conductivity (L(p)) in vivo. To determine the signal transduction cascade through which this is mediated, we measured the effect of inhibition of various signalling pathways on VEGF-mediated acute increases in L(p) in individually perfused frog mesenteric microvessels. 2. VEGF receptors have previously been shown to activate phospholipase C-gamma (PLCgamma), protein kinase C (PKC) and MEK, the mitogen-activated and extracellular signal-related kinase (ERK) kinase. To determine the role of these signalling pathways we measured the effects of inhibitors of each on the VEGF-mediated increase in L(p). 3. VEGF-mediated increases in L(p) were attenuated by pre-treatment with the PLC inhibitor U73122, but not affected by treatment with the inactive enantiomer U73343. The PLC inhibitor was also able to attenuate the increase in L(p) mediated by the inflammatory mediator ATP. 4. Inhibition of either PKC or MEK activation using the selective inhibitors bisindolylmaleimide (BIM, 1 microM) and PD98059 (30 microM), respectively, did not change the VEGF-mediated increase in L(p). However, PD98059, BIM and U73122 all reduced phosphorylation of ERK1/2 determined by Western blot analysis with anti-phospho-ERK1/2 antibodies. 5. Furthermore, inhibition of the conversion of diacyl glycerol (DAG) to arachidonic acid, by perfusion with the DAG lipase inhibitor RHC80267 (50 microM), did not attenuate the increase in L(p) brought about by VEGF. 6. These data suggest that VEGF acutely increases microvascular permeability in vivo through a mechanism that is dependent on PLC stimulation, but is independent of PKC or MEK activation or production of arachidonic acid from DAG. We therefore propose that VEGF acutely acts to increase L(p) through the direct actions of DAG, independently of PKC or arachidonic acid.

Figure 1. Diagram to show potential signalling pathways for increased vascular permeability

We have previously shown that VEGF increases intracellular calcium and vascular permeability through a store-independent calcium influx (Pocock et al. 2000; dashed line indicates that this pathway is known not to be the mechanism). In this set of experiments we determined whether inhibiting the action of PLC (1), DAG lipase (2), PKC (3) or MEK (4) would prevent the VEGF-mediated increase in vascular permeability. AA, arachidonic acid.

Figure 2. Representative Western blots of frog lung tissue treated with saline or inhibitor, and with VEGF or VEGF and inhibitor

A, blots were probed with mouse anti-phospho-ERK1/2 primary antibody, and HRP-conjugated goat anti-mouse secondary antibody. B, mean ±s.e.m. relative densities, calculated from four independent experiments.

Figure 3. The effect of MEK inhibition on the VEGF-mediated increase in permeability

Time-averaged L_p measurements during 1 nm VEGF perfusion on 10 vessels with (•) and 18 vessels without (□) 20 min pre-treatment with 30 μm PD98059. Values are means ±s.e.m.

Figure 4. The effect of BIM on the VEGF-mediated increase in L_p

A, time-averaged L_p measurements on 10 vessels during 1 nm VEGF perfusion with (•) and without (□) 20 min pre-treatment with 1 μm BIM. Values are means ±s.e.m. B, median baseline (□) and peak (▪) L_p before, during and after perfusion with 1 μm BIM. * Significantly increased compared to baseline (P < 0.01). The peak increases are not significantly different from each other.

Figure 5. The effect of PLC inhibitors on the VEGF-mediated increase in L_p

A, time-averaged L_p measurements on 10 vessels during 1 nm VEGF perfusion with (□) and without (•) 20 min pre-treatment with 10 μm U73122. B, time-averaged L_p measurements on seven vessels during 1 nm VEGF perfusion with (□) and without (•) 20 min pre-treatment with 10 μm of the inactive enantiomer of U73122, U73343. Values are means ±s.e.m.

Figure 6. Comparison of the effects of the PLC inhibitors on store-independent (VEGF) and store-mediated (ATP) increases in L_p

Median ± IQR for baseline (□) and peak (▪) measurements before, during and after perfusion with the inhibitors. A, effect of U73122 on the VEGF-mediated increase in L_p in 10 vessels. B, effect of U73343 on the VEGF-mediated increase in L_p in seven vessels. C, effect of U73122 on the ATP-mediated increase in L_p in seven vessels. D, effect of U73343 on the ATP-mediated increase in L_p in five vessels. *P < 0.05 compared to baseline; † significantly less than without inhibitor (P < 0.05).

Figure 7. The effect of a DAG lipase inhibitor on the VEGF-mediated increase in L_p

A, time-averaged L_p measurements on 10 vessels during 1 nm VEGF perfusion with (□) and without (•) 20 min pre-treatment with 50 μm RHC80267. Values are means ±s.e.m. B, median baseline (□) and peak (▪) L_p before, during and after perfusion with 50 μm RHC80267. * Significantly increased compared to baseline (P < 0.01). The peak increases are not significantly different from each other.