In this study, we defined a therapeutic approach for inflammatory CNS disease using inhibition of BBB breakdown to restrict clinical severity and permanent pathology, which we believe to be novel. This approach is based on our identification of a mechanism whereby reactive astrocytes mediate disruption of BBB integrity (Figure ). Our findings showed that the angiogenic factor VEGF-A was an important effector used by reactive astrocytes to drive vascular permeability and CNS damage in acute inflammatory lesions. We identified CLN-5 and OCLN as targets of VEGF-A action in cortical and spinal cord endothelial cells and showed that both proteins, and BBB breakdown, were rescued in inflammatory and demyelinating plaques in the CNS of GfapCre:Vegfafl/fl mice, in which VEGF-A inactivation was targeted to reactive astrocytes. We further identified the downstream effector eNOS as mediating the effects of VEGF-A on CLN-5 and the BBB. Finally, we showed that treatment with the selective eNOS inhibitor cavtratin during the acute clinical phase of EAE protected against paralysis and pathology, similar to the EAE phenotype seen in GfapCre:Vegfafl/fl mice. Collectively, our findings suggested blockade of endothelial VEGF-A signaling as an avenue by which to restrict clinical severity and tissue damage in inflammatory CNS conditions (Figure ).
Proposed mechanism underlying BBB disruption in CNS inflammatory lesions.
Recent work has examined the role of pericytes in BBB maintenance (6
), but earlier studies also define important astrocytic functions at the BBB (9
), and it was on the latter cell type that our work here focused. Astrocytes respond to CNS inflammation or injury with a graded spectrum of molecular expression, hypertrophy, and, in severe cases, scarring (45
). These changes, called reactive astrogliosis, affect surrounding neural and non-neural cells beneficially or detrimentally, depending on the pathways driving the astroglial phenotype (34
). Studies have identified astrocytes as regulators of BBB induction and maintenance (9
) and have implicated astrogliosis, particularly induced by IL-1, as a driver of both BBB breakdown and repair (10
). The mediators producing the effects of reactive astrocytes are incompletely characterized, and our data revealed VEGF-A as an important astrocyte-derived inducer of BBB disruption and pathology in vivo. Although VEGF-A–induced vascular permeability has previously been implicated in pathogenesis of disorders, including myocardial infarction, CNS hypoxia/reperfusion injury, and tumor growth and metastasis (49
), and we and others have previously speculated on its role in BBB breakdown (12
), this study is the first to our knowledge to show the significance of astrocyte-derived VEGF-A in lesion pathogenesis and generation of clinical deficit in models of CNS inflammatory disease.
The BBB uses endothelial tight junctions to generate a physical barrier, allowing control of transcellular molecular trafficking via endothelial transporters and channels (1
). Disruption of the endothelial tight junction transmembrane proteins CLN-3, CLN-5, and OCLN correlates with BBB opening in EAE (4
), and downregulation of CLN-5 is linked to BBB breakdown in glioblastoma multiforme (50
) and models of amyotrophic lateral sclerosis (51
) and viral encephalomyelitis (52
). Redistribution of CLN-5 and OCLN away from the cell membrane has also been implicated in BBB disruption in inflammatory models (53
). Importantly, our data showed that VEGF-A downregulated endothelial expression of both CLN-5 and OCLN, and we observed rescue of both proteins, and BBB integrity, in CNS inflammation in GfapCre:Vegfafl/fl
mice. To quantify BBB breakdown, we measured extravasation of fibrinogen, albumin, and Ig. In the healthy CNS, these proteins are confined to the vasculature, and their leakage into the parenchyma has been widely used as an endogenous marker of BBB disruption (8
). CNS entry of Igs and fibrinogen has also been linked to exacerbation of inflammation and demyelination and to inhibition of repair (17
), suggesting relevance to lesion pathogenesis.
Our studies in vivo also showed that reduced BBB disruption in GfapCre:Vegfafl/fl
mice was accompanied by mitigation of lymphocyte infiltration and implicated BBB integrity as an important mechanism underlying this effect. Whereas BBB breakdown was restricted in AdIL-1–induced lesions in GfapCre:Vegfafl/fl
mice, we found that endothelial adhesion molecule and chemokine expression and CNS induction of MMPs were the same as in other genotypes. VCAM-1 and ICAM-1 (54
), as well as CC and CXC chemokines (55
), are important in driving inflammatory cell transmigration, whereas MMPs facilitate entry through the glia limitans and infiltration into the CNS parenchyma (56
). Using the focal, directly induced AdIL-1 model in GfapCre:Vegfafl/fl
mice allowed us to selectively examine the effect of VEGF-A–induced BBB breakdown on lymphocyte entry while retaining these other important variables. Critically, we also found in these experiments and in EAE studies that inhibition of BBB breakdown and leukocyte infiltration in GfapCre:Vegfafl/fl
mice was associated with restriction of gray and white matter pathology and with reduced clinical paralysis. Thus, our data implicate inhibition of VEGF-A signaling as a protective avenue in patients with CNS inflammation.
The transcription factor HIF-1α induces VEGF-A during development (36
) and is induced transcriptionally by IL-1β in astrocytes, suggesting relevance to hypoxic/ischemic and inflammatory disease (23
). However, although these data suggest HIF-1α as a potential route to inactivation of astrocytic VEGF-A, conditional GfapCre:Hif1afl/fl
mice displayed little effect on VEGF-A expression or BBB disruption in AdIL-1 lesions. These findings indicate that mechanisms regulating VEGF-A in embryogenesis and inflammation are likely distinct. However, our data do not invalidate roles in inflammatory lesion pathogenesis for other members of the family, for example, HIF-2α, which has previously been implicated as a regulator of astrocytic VEGF-A production in VHL-deficient mice and in oxygen-induced retinopathy (57
). In addition, our findings focused on inflammation and did not address astrocytic responses to hypoxic/ischemic injury.
The results of our analyses of endothelial VEGF-A signaling were more directly translational, identifying a therapeutic avenue by which to inhibit BBB breakdown. In CNS MVEC cultures, we found that VEGF-A downregulation of CLN-5 and OCLN occurred via VEGFR2 signaling through PLCγ and its effector, eNOS. Notably, targeting events within the endothelial cell allows for systemic delivery and avoids issues with CNS access, and our studies examining eNOS blockade in vivo validated this approach in a therapeutic setting. Tonic eNOS activity controls vascular tone and leukocyte trafficking in the resting state, whereas the activated form drives permeability and extravasation in disease (58
). The phenotype of eNOS mutants combines inactivation of the tonic and activated forms and produces complex findings in EAE and carrageenan-induced inflammation (59
). Understanding the translational potential of eNOS has been facilitated by development of selective inhibitors, notably cavtratin, a cell-permeable recombinant consisting of the eNOS-binding domain of caveolin-1 coupled to the homeodomain of Drosophila
). Cavtratin binds to eNOS via the caveolin-1 scaffolding domain, specifically inhibiting its enzymatic activity via protein-protein interaction (40
). Previous work has also shown that cavtratin does not block activity of other forms of NOS (40
), and our experiments in vitro support these results (Figure F and Supplemental Figure 6E). Cavtratin reduces tumor microvascular permeability and progression (41
) and restricts edema in inflammatory models without influencing systemic hemodynamics (40
). In CNS MVECs, our results showed that cavtratin matched the effect of eNOS siRNA in abrogating VEGF-A–induced downregulation of CLN-5 and OCLN, and its administration in vivo produced a phenotype similar to that seen in GfapCre:Vegfafl/fl
mice in terms of blockade of BBB breakdown, lymphocyte infiltration, and tissue damage in inflammatory lesions. Importantly, treatment in the acute stages of EAE was also protective, suggestive of relevance to human disease.
These data suggest further studies to investigate roles of VEGF-A and eNOS in other inflammatory CNS conditions. For example, blocking the pathway might be effective in preventing CNS entry of extrathecally produced autoantibodies in diseases such as neuromyelitis optica. These approaches may also represent a means by which to examine the role of BBB permeability in disorders characterized by slowly accumulating tissue damage accompanied by low-grade inflammation and BBB leakage, such as primary or secondary progressive MS (16
). It might also be interesting to combine inhibition of inducers of BBB disruption, such as VEGF-A–eNOS and MMPs, with activation of pathways promoting barrier formation and/or repair, such as Shh signaling (11
), for potentially additive or synergistic outcomes.