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. 2020 Aug 27:8:849.
doi: 10.3389/fcell.2020.00849. eCollection 2020.

TRPV4-Mediated Regulation of the Blood Brain Barrier Is Abolished During Inflammation

Sina C Rosenkranz  1 , Artem Shaposhnykov  1 , Oliver Schnapauff  2 , Lisa Epping  3 , Vanessa Vieira  1 , Karsten Heidermann  1 , Benjamin Schattling  1 , Volodymyr Tsvilovskyy  4 , Wolfgang Liedtke  5 , Sven G Meuth  3 , Marc Freichel  4 , Mathias Gelderblom  2 , Manuel A Friese  1
Affiliations

TRPV4-Mediated Regulation of the Blood Brain Barrier Is Abolished During Inflammation

Sina C Rosenkranz et al. Front Cell Dev Biol. .

Abstract

Blood-brain barrier (BBB) dysfunction is critically involved in determining the extent of several central nervous systems (CNS) pathologies and here in particular neuroinflammatory conditions. Inhibiting BBB breakdown could reduce the level of vasogenic edema and the number of immune cells invading the CNS, thereby counteracting neuronal injury. Transient receptor potential (TRP) channels have an important role as environmental sensors and constitute attractive therapeutic targets that are involved in calcium homeostasis during pathologies of the CNS. Transient receptor potential vanilloid 4 (TRPV4) is a calcium permeable, non-selective cation channel highly expressed in endothelial cells. As it is involved in the regulation of the blood brain barrier permeability and consequently cerebral edema formation, we anticipated a regulatory role of TRPV4 in CNS inflammation and subsequent neuronal damage. Here, we detected an increase in transendothelial resistance in mouse brain microvascular endothelial cells (MbMECs) after treatment with a selective TRPV4 inhibitor. However, this effect was abolished after the addition of IFNγ and TNFα indicating that inflammatory conditions override TRPV4-mediated permeability. Accordingly, we did not observe a protection of Trpv4-deficient mice when compared to wildtype controls in a preclinical model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), and no differences in infarct sizes following transient middle cerebral artery occlusion (tMCAO), the experimental stroke model, which leads to an acute postischemic inflammatory response. Furthermore, Evans Blue injections did not show differences in alterations of the blood brain barrier (BBB) permeability between genotypes in both animal models. Together, TRPV4 does not regulate brain microvascular endothelial permeability under inflammation.

Keywords: TRPV4; blood brain barrier; experimental autoimmune encephalomyelitis; stroke; transendothelial resistance.

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Figures

FIGURE 1
FIGURE 1
TRPV4 inhibition increases transendothelial electrical resistance (TEER) in mouse brain microvascular endothelial cells (MbMECs) under homeostatic conditions. (A) TEER of MbMECs treated with 5 nM of GSK2193874 in comparison to vehicle-treated MbMECs. One representative experiment out of 12 is shown. (B–D) Quantification of TEER of MbMECs treated with 5 nM of GSK2193874 (n = 12) in comparison to vehicle treated MbMECs (n = 12) at 6, 12, and 24 h. TEER was normalized to the time-point of GSK treatment (0 h). Data are presented as box plots. Statistical analysis was performed by two-tailed Student’s t-test. *P < 0.05.
FIGURE 2
FIGURE 2
Inflammatory conditions abolish the effect of TRPV4 inhibition on transendothelial electrical resistance (TEER) in mouse brain microvascular endothelial cells (MbMECs). (A) TEER of MbMECs treated with 5 nM of GSK2193874 in comparison to vehicle-treated MbMECs, both were exposed to 50 U/ml TNFα and INFγ. One representative experiment out of 14 is shown. (B–D) Quantification of TEER of MbMECs treated with 5 nM of GSK2193874 (n = 14) in comparison to vehicle-treated MbMECs (n = 14) at 6, 12, and 24 h, both were exposed to 50 U/ml TNFα and INFγ. TEER was normalized to the time-point of GSK treatment (0 h). (E) Relative Trpv4 mRNA expression assessed by qPCR in MbMECs exposed to 50 U/ml TNFα and INFγ (n = 4) in comparison to homeostatic conditions (n = 8). Data are presented as box plots. Statistical analysis was performed by two-tailed Student’s t-test (A) and (B–D) or two-tailed Mann Whitney test (E). *P < 0.05.
FIGURE 3
FIGURE 3
No difference between Trpv4–/– mice and WT controls on the disease course and BBB permeability in EAE. (A) Day of onset of WT (n = 33) and Trpv4–/– mice (n = 32) during the course of EAE. (B) Clinical scores of WT (n = 27) and Trpv4–/– mice (n = 23) undergoing EAE. (C) Evans Blue (EB) quantification in brain and spinal cord of WT (n = 5) and Trpv4–/– (n = 5) mice at day 13 after EAE immunization. Evans Blue concentration was normalized to the concentration of the right kidney of the corresponding animal. (D) Representative images of brain, spinal cord and kidney of WT- and Trpv4–/–-EAE mice 2 h after Evans Blue injection. Data in (A,C) are presented as box plots, in (B) as mean values ± s.e.m. Statistical analysis was performed by two-tailed Student’s t-test in (A) by two-tailed Mann Whitney test in (B,C).
FIGURE 4
FIGURE 4
No difference between Trpv4–/– mice and WT controls on disease course and BBB permeability in tMCAO. (A) Clinical scores of WT (n = 13) and Trpv4–/– mice (n = 13) after tMCAO; h = hours, d = days. (B) Representative TTC staining of WT and Trpv4–/–brains 3 days after tMCAO. (C) Quantification of infarct volume of WT (n = 8) and Trpv4–/– (n = 9) 3 days after tMCAO. (D) Regional cerebral blood flow (rCBF) of WT (n = 13) and Trpv4–/– mice (n = 13) was measured during occlusion by laser Doppler and normalized to contralateral hemispheres. (E) Evans Blue (EB) quantification in brains of WT (n = 6) and Trpv4–/– (n = 6) mice 24 h after tMCAO. Evans Blue concentration was normalized to the concentration of the right kidney of the corresponding animal. (F) Representative images of brains and kidneys of WT and Trpv4–/– mice 24 h after tMCAO and Evans Blue injection. Data in (A) are presented as mean values ± s.e.m, in (C–E) as box plots. Statistical analysis was performed by two-tailed Mann Whitney test for (A) and two-tailed Student’s t-test for (C–E).
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