Figure 4. MEKK3 critically regulates the neonatal vascular permeability to small size molecule via suppressing the Rho signals.

(a–d) In vivo leakage experiments show only small-molecular-weight tracers leaked from brain microvessels in Mekk3 iKO and iEC^−/− neonatal mice. (a) Sulfo-NHS-biotin (556.6 dalton) was injected into the hearts of the tamoxifen-treated Mekk3 NCL or Mekk3 iKO neonatal pups. Brain sections were analysed for Sulfo-NHS-biotin leakage by staining. Objective lens power: 2.5 × . (b–d) Fluorescent-labelled tracers with different molecular weights were injected into tamoxifen-treated Mekk3 NCL or Mekk3 iEC^−/− P7 neonatal pups' hearts. After euthanization, brains were fixed and sectioned and tracer leakage to the brain was determined. (b) Hoechst 33342 (616 dalton; blue) plus Dextran–Rhodamine (2 M Dalton; red), N=7, (c) Dextran–FITC (MW: 4K Dalton; green) plus Dextran–Rhodamine (2 M Dalton)(red), N=4, (d) Dextran–FITC (40 K Dalton; green) plus Dextran–Rhodamine (2 M Dalton; red). Bar graphs on the left of each panel show quantification of the relative leakage of the tracers normalized to the intensity of Dextran–Rhodamine, and shows no leakage in either NCL nor Mekk3 iEC^−/− neonatal pups. Error bars indicate s.d. N=4. Objective lens power: 10 × . (e) ROCK inhibitor Y27632 partially rescues survival of Mekk3 iEC^−/− neonatal pups. NCL or Mekk3 iEC^−/− pups were treated with tamoxifen at P1, and continued daily in the absence (open circle and black diamond, respectively) or presence of Y27632 (cross and black triangle, respectively). Survival of pups was monitored daily until P20. (f) Hoechst 33342 (616 Dalton; blue) plus Dextran K–Rhodamine (2 M Dalton; red) were injected into tamoxifen-treated Mekk3 iEC^−/− P7 neonatal pups' hearts fed either with water or Y27632. After euthanization, brains were fixed and sectioned at 30-μm thickness and leakage determined as described above. N=5. Objective lens power: 10 × . (g) Wild-type P7 neonatal pups were treated with cell-permeable wild-type Mekk3-peptide (MEKK3^N-peptide) or A6D/L7D mutated Mekk3-peptide (MEKK3^{mutant-N-peptide}). Brain leakage determined using Hoechst 33342 (blue) plus Dextran K–Rhodamine (red). N=5. Objective lens power: 10 × . (h) Proposed signalling pathway. The interactions of CCM2 and MEKK3 are critical for maintenance of vasculature integrity and permeability by control of Rho/ROCK signalling.