In the present study, we found a rapid, specific, and concentration-dependent reduction of P-glycoprotein transport activity in isolated brain capillaries exposed to VEGF. Tight junctional permeability was not altered. The VEGF effect on transport was fully reversible and did not involve reduced protein expression of P-glycoprotein. VEGF acted through the flk-1 receptor and Src kinase, but not through PI3K/Akt or PKC. Direct activation of Src kinase by YEEIP mimicked the effects of VEGF on P-glycoprotein activity in brain capillaries. Finally, ICV injection of VEGF increased brain distribution of the P-glycoprotein substrates morphine and verapamil without increasing BBB permeability to sucrose. This effect was blocked by peripheral administration of the Src kinase inhibitor PP2. Thus, inhibition of P-glycoprotein transport activity by VEGF occurred both in isolated brain capillaries in vitro and the intact BBB in vivo.
Recent experiments with a rat brain endothelial cell line showed increased interaction of caveolin-1 with P-glycoprotein is associated with decreased transporter activity, and that Tyr-14 phosphorylation of caveolin-1 by transfected Src kinase promotes this interaction (Barakat et al., 2007
). We show here that exposing intact brain capillaries to either VEGF or the Src kinase activating peptide YEEIP increased specific Tyr-14 phosphorylation of caveolin-1; VEGF-induced caveolin phosphorylation was blocked by the Src kinase inhibitor, PP2. However, in co-immunprecipitation experiments, we did not see increased association of caveolin-1 with P-glycoprotein following VEGF treatment. Several possibilities could explain this discrepancy. First, immortalization and culture of brain capillary endothelial cells causes changes in protein expression and function; indeed, expression and function P-glycoprotein is especially sensitive to culture conditions (Gaillard et al., 2000
). Second, induced association of these proteins may be more dynamic and/or less strong than their constitutive association (note that P-glycoprotein was pulled down with caveolin-1 in all conditions). The induced association may not survive tissue lysis and protein extraction. Finally, phosphorylation of caveolin-1 may occur independently of P-glycoprotein inhibition and not be necessary for VEGF signaling to P-glycoprotein. Further, the effects of PP2 could be explained by direct inhibition of flk-1 tyrosine kinase activity by PP2. Though we are not aware of any interaction between PP2 and flk-1 that does not involve inhibition of Src activity, we cannot rule out this possibility. However, the inhibitory effect of PP2 together with mimicry of VEGF effects by the Src activator YEEIP strongly suggest that VEGF acts on P-glycoprotein via Src, and implies that other factors associated with increased Src signaling may acutely regulate P-glycoprotein.
Loss of P-glycoprotein transport activity could reflect decreased expression of the transporter protein, removal of the protein from the cell surface, a shift of the protein to a different plasma membrane microdomain, or allosteric modulation, possibly via protein-protein interactions. For VEGF-induced loss of P-glycoprotein activity, we found no reduction in transporter protein expression nor any effect of a proteosome inhibitor, ruling out reduced expression as an underlying mechanism. At present, we cannot distinguish with certainty among the remaining possibilities. Nevertheless, loss of activity was fully reversible when VEGF was removed from the medium and was blocked when capillaries were pretreated with nocodazole, a microtubule polymerization inhibitor, in the present study. These findings suggest involvement of cytoskeletal-dependent removal of the protein from the cell surface or protein movement to a different plasma membrane microdomain where it is inactive. In preliminary experiments, VEGF exposure decreased proteolysis of P-glycoprotein in brain capillaries following brain perfusion with a protease solution (Hawkins et al., unpublished data), suggesting that VEGF stimulates internalization of P-glycoprotein. Confirmation of this finding will begin to illuminate the mechanism by which VEGF regulates P-glycoprotein.
VEGF has long been associated with increased vascular permeability, and thought to contribute to increased BBB permeability in hypoxia and some brain tumors (Vogel et al., 2007
). As such, it is critical that we distinguish any apparent decrease in P-glycoprotein transport activity from nonspecific opening of the BBB. In the present study, we found no effect of VEGF on paracellular (tight junction-restricted) permeability to TR in vitro nor to sucrose in vivo at VEGF concentrations and exposure times that significantly reduced P-glycoprotein transport activity. This is in agreement with previous studies that reported VEGF to have no effect on BBB permeability when injected directly into a non-diseased brain, a finding that lead investigators to conclude that “…tumor-derived angiogenic vessels and normal cutaneous vessels respond to permeability mediators in a way that normal brain vessels do not
” (Criscuolo et al., 1990
). It is also important to use caution in comparing data from in vivo models and freshly isolated brain capillaries to data from cell culture studies, as critical changes in brain endothelial cell phenotype do occur in culture. For example, mRNA for the flt-1 receptor is decreased 21-fold in primary brain endothelial cell culture compared to freshly isolated capillaries (Calabria and Shusta, 2008
). Flt-1 is thought to act as a “scavenger” for VEGF, binding it with higher affinity than the flk-1 receptor and thus negatively regulating the biological effects of VEGF mediated by flk-1 (Hiratsuka et al., 1998
). This may explain why developmentally mature, non-diseased brain capillaries (freshly isolated or in situ) are relatively unresponsive to the permeabilizing effect of VEGF compared to endothelial cell cultures, as suggested by our results and others (Criscuolo et al., 1990
; Harrigan et al., 2002
Given the association of increased brain VEGF with CNS disease (Greenberg and Jin, 2005
), the present results suggest that BBB P-glycoprotein activity may be altered by VEGF in certain disease states. Indeed, there is clinical evidence for P-glycoprotein activity being depressed in injured portions of the brain. Ederoth and colleagues used microdialysis to measure brain concentrations of morphine, a P-glycoprotein substrate, in traumatic brain injury (TBI) patients. They found a higher concentration in injured versus non-injured portions of the brain and attributed this finding to impaired drug efflux (Ederoth et al., 2004
). The implications of diminished P-glycoprotein transport activity are twofold. First, loss of P-glycoprotein function could render TBI patients vulnerable to central toxicity and or/ side effects of peripherally-acting drugs, such as ion channel blockers (i.e., verapamil) and immunosuppressants (i.e., CSA). Indeed, the altered brain pharmacokinetics for morphine, a weak P-glycoprotein substrate, argue that this is the case. Thus, the potential for adverse drug reactions may be increased. Conversely, delivery of therapeutics to treat brain injury may be enhanced at the site of injury. Note that agents showing considerable promise as neuroprotective therapeutics in TBI based on animal studies include FK-506 (Singleton et al., 2001
) and CSA (Hatton et al., 2008
), both of which are P-glycoprotein substrates. The neuroprotective efficacy of these agents in TBI may benefit in part from improved distribution to the site of injury as a result of VEGF-induced acute downregulation of P-glycoprotein activity.
It is becoming increasingly clear that functional changes in brain microvascular physiology play a significant role in the pathogenesis and progression of neurodegenerative disease, including Alzhiemer’s disease. Both aberrant angiogenic signaling (including increased levels of VEGF) (Kalaria et al., 1998
; Tarkowski et al., 2002
; Thirumangalakudi et al., 2006
; Desai et al., 2009
) and diminished expression/activity of P-glycoprotein (Vogelgesang et al., 2002
; Vogelgesang et al., 2004
) have been observed in the brains of Alzheimer’s disease patients. Low levels of the homeobox gene MEOX2 (encoding the protein GAX) have been reported in brain endothelial cells from Alzheimer’s patients (Wu et al., 2005
). GAX is a regulator of vascular differentiation, and in transgenics with one copy of the MEOX2 gene deleted, clearance of Aβ by low-density lipoprotein receptor−related protein 1 (LRP) are impaired. Interestingly, angiogenesis in response to hypoxia was also impaired in these animals, despite expected elevations of VEGF in response to hypoxia (Wu et al., 2005
). In other words, the microvasculature in Alzheimer’s disease may be exposed to high levels of VEGF, but cannot respond normally to it. In another study, VEGF was found to co-localize with β-amyloid plaques (Yang et al., 2004
). Endothelial deposition of β-amyloid is inversely correlated with P-glycoprotein expression (Vogelgesang et al., 2002
), and there is evidence that P-glycoprotein also contributes to the brain clearance of Aβ (Cirrito et al., 2005
; Kuhnke et al., 2007
), likely in coordination with LRP and the receptor for advanced glycosylation end products (RAGE) (Zlokovic et al., 2000
). At this point, it is not known whether VEGF is related to the diminished function of BBB P-glycoprotein in Alzheimer’s, though the data presented herein suggest that anti-VEGF therapy could present a novel strategy for improving brain clearance of Aβ.
In summary, our data show that VEGF acutely and reversibly depresses the transport activity of P-glycoprotein, a key BBB drug efflux transporter. VEGF signaled through the flk-1 receptor and Src, and depression of P-glycoprotein activity by VEGF is associated with Src-dependent phosphorylation of caveolin-1, a signal known to trigger caveolin-dependent endocytosis. To our knowledge, this is the first demonstration of VEGF regulating P-glycoprotein in any tissue. Our findings disclose a novel role for VEGF in brain. These findings also imply that brain pharmacokinetics of xenobiotics may be altered via modulation of P-glycoprotein in conditions associated with increased levels of VEGF in the brain including TBI and Alzheimer’s disease.