Research ArticleNEUROPATHOLOGY

Reversal of endothelial dysfunction reduces white matter vulnerability in cerebral small vessel disease in rats

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Science Translational Medicine  04 Jul 2018:
Vol. 10, Issue 448, eaam9507
DOI: 10.1126/scitranslmed.aam9507
  • Fig. 1 Pathological changes to the BBB and OPCs in brains of young DM rats and presymptomatic humans.

    (A to H) Immunofluorescent images from brain deep white matter and graph (I) showing number of blood vessels (CD34+, red) with claudin-5–positive tight junctions (TJs, green) in DM rats (B and D) compared to controls (A and C), as well as in diseased human brains (F and H) compared to controls (E and G). Arrows indicate blood vessels without claudin-5–positive TJs. (C), (D), (G), and (H) show enlarged images of the white boxes in (A), (B), (E), and (F), respectively. (J to O) Number of OPCs (Olig2+ Nogo-A; indicated by arrows) per square millimeter (J and N) and mature oligodendrocytes (Olig2+ Nogo-A+) (K and O) in DM rats and in diseased human brains compared to controls [Olig2, all oligodendroglia (green); Nogo-A, mature oligodendrocytes (red)]. (P) Number of proliferating OPCs (Olig2+ PCNA+) per square millimeter in DM rats and in the diseased human brains compared to controls [mean ± SEM; *P < 0.05 and **P < 0.01, two-way analysis of variance (ANOVA) with Tukey’s post hoc tests; 3 weeks, n ≥ 4 animals; 4 weeks, n ≥ 3 animals; 5 weeks, (P) n = 3 animals; others, n = 10 animals and n = 5 humans]. Scale bars, 25 μm. PCNA, proliferating cell nuclear antigen.

  • Fig. 2 Endothelial cells are dysfunctional at 3 weeks of age in DM rats and in human brains with SVD.

    (A to F) Slice data demonstrating early changes are not due to BBB leakage. (A and B) Western blot and quantification of claudin-5 in brain slices cultured from DM rats for 5 weeks compared to controls (*P < 0.05, paired t test; n = 3 experiments). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. (C to F) Immunofluorescent images and quantification of brain slices stained for Olig2 (all oligodendroglia, green) and Nogo-A (mature oligodendrocytes, red) in DM rat slices (D) compared to control (C) (*P < 0.05, paired t test; n = 5 experiments). (G and H) Western blot and quantification of eNOS in 3-week-old DM rat brain tissue compared to controls (Ctrl), with GAPDH used as a loading control (*P < 0.05, unpaired t test; n = 3 animals). (I and J) Immunofluorescent images showing proliferating (PCNA+, red) ECs (CD34+, green) (indicated by white arrows) in the deep white matter of brains from 3-week-old control (I) and DM rats (J). Enlargements of boxed areas are shown. (K and L) Colorimetric immunostaining of proliferating (PCNA+, blue) human ECs (CD34+, brown) (indicated by red arrows). (M) Quantification of the number of proliferating ECs per square millimeter in DM rat brains and the diseased human brains compared to their controls (mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, two-way ANOVA with Tukey’s post hoc tests; 3 weeks, n = 5 animals; 4 and 5 weeks, n = 3 animals and n = 5 humans). (N) Assay of nitrates and nitrites (NOx) as a proxy for cell production of NO in the media of BMECs isolated from DM rats compared to controls. Each color represents a different paired repeat (mean ± SEM; *P < 0.05, paired t test; n = 3 BMEC preparations from different litters). (O and P) Immunofluorescent staining showing claudin-5 (mature TJP, green) and ZO-1 (immature TJP, red) in BMEC cultures isolated from control (O) and DM (P) rats (Hoechst, blue), quantified in (Q) and (R) (*P < 0.05, paired t test; n = 3 BMEC preparations from different litters). Scale bars, 25 μm.

  • Fig. 3 HSP90α produced by BMECs reduces OPC maturation.

    (A and B) Immunofluorescent images showing OPCs (NG2+, green) and MBP+ mature oligodendrocytes (red) in cultures grown in DM (B) compared to control (A) BMEC–CM (Hoechst, blue) [*P < 0.05, paired t test; n = 3 experiments, quantified in (C) and (D)]. (E and F) Proliferating Ki67-expressing cells (red) in cultures of OPCs grown in DM (F) compared to control (E) BMEC CM (Hoechst, blue) [*P < 0.05, paired t test; n = 3 experiments, quantified in (G)]. (H to J) OPCs [Olig2+ (all oligodendroglia, green) Nogo-A (mature oligodendrocytes, red), arrows indicating Olig2+ NogoA OPCs] in wild-type brain slices grown in culture for 5 weeks in DM BMEC CM (J) compared to control CM (I) [quantified in (H) normalized to slices grown in unconditioned media; *P < 0.05, paired t test; n = 3 experiments]. (K) Western blot for HSP90α in CM from DM BMECs compared to controls (*P < 0.05, paired t test; n = 3 experiments, quantified in (L)], including Ponceau stain of the membrane showing total protein used as a loading control, with prominent band of bovine serum albumin. (M) ELISA for HSP90α in CM from DM BMECs compared to controls (P = 0.05, t test; n = 4 experiments). (N to Q) Effect of addition of recombinant (r)HSP90α on maturation of wild-type OPCs in culture (immature NG2+ OPCs, green; mature MBP+ oligodendrocytes, red; Hoechst, blue) [quantified in (P) and (Q) with each color representing a different paired repeat; *P < 0.05, paired t test; n = 4 experiments]. (R to V) Effect of addition of HSP90α blocking antibody (BA) on OPC cultures grown in DM CM (immature NG2+ OPCs, green; mature MBP+ oligodendrocytes, red; Hoechst, blue). (U and V) Quantification of the percentage of NG2+ cells is normalized to control CM (such that 1 = an average of 23% NG2+ cells) and quantification of the percentage of MBP+ cells is normalized to control CM (such that 1 = an average of 15% MBP+ cells) (mean ± SEM; *P < 0.05 and **P < 0.01, one-way repeated measures ANOVA with Bonferroni post hoc tests; n = 4 separate experiments with cells from different litters). (W) Western blot showing full-length and cleaved, secreted forms of HSP90α (with GAPDH used as a loading control) in rat and human control and diseased brains [*P < 0.05, unpaired t test; n = 3 rats, each group quantified in (X); P = 0.28, unpaired t test; n = 5 humans, each group quantified in (Y)]. Scale bars, 25 μm.

  • Fig. 4 Drugs that reduce EC dysfunction also reduce white matter vulnerability.

    (A) Timeline of drug trial showing the ages at which pathologies appear, the timing of blood pressure measurements, and the beginning and end of treatment. (B) Table of treatment groups and effects. All drugs were administered daily through an oral route at the following concentrations: simvastatin, 2 mg/kg; perindopril, 2 mg/kg; cilostazol, 60 mg/kg; and H+H, 16 mg/kg. (C) Blood pressure readings over the experiment. (D to K) Immunofluorescence images showing the effect of simvastatin on EC proliferation, mature TJs, OPC and oligodendrocyte numbers, and OPC proliferation, with quantification in graphs (L to P). (Q) Blinded ranking of myelin rarefaction on Luxol blue/cresyl violet–stained brain sections [mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001, (L to P) one-way ANOVA with Bonferroni post hoc tests, and (Q) Kruskal-Wallis test with Dunn’s post hoc tests; cilostazol, n = 4; others, n = 5 animals per treatment group]. Scale bars, 25 μm.

  • Fig. 5 Atp11b is mutated in DM rats and leads to dysfunctional ECs.

    (A) Filtering strategy: Venn diagram with overlap of genes with frameshift or stop-gain mutations in the SHRSP and the SHR. Mutations unique to SHRSP were filtered first using Variant Effect Predictor (Ve!P) tool from Ensembl (37) to select those predicted to lead to truncated proteins, then with the Allen Brain Atlas (38) to select genes expressed in the brain, and finally with the Barres Brain RNA-seq database (39) to select genes expressed in brain ECs. (B) DNA sequence showing the 28–base pair deletion in the Atp11b gene and corresponding complementary DNA (cDNA). (C) Western blot with complete loss of ATP11B in DM rat brains compared to controls using an N-terminal antibody. (D) Western blot of knockdown of ATP11B in bEND. 3 cells treated with Atp11b siRNA (Atp11b-KD) compared with nontargeting siRNA (control) [*P < 0.05, paired t test; n = 3 experiments, quantified in (E) with GAPDH used as a loading control]. (F and G) Immunofluorescent images showing proliferation (Hoechst, blue; Ki67+ nuclei, magenta-white arrows) in Atp11b-KD bEND.3 cells (G) compared to controls (F) [quantified in (H); *P < 0.05, paired t test; n = 3 experiments] (Hoechst, blue). (I to L) Immunofluorescent images staining for claudin-5 (mature TJP, green) compared to ZO-1 (immature TJP, red) in Atp11b-KD bEND. 3 cells (J) compared to controls (I) [quantified in (K) and (L); *P < 0.05, paired t test; n = 3 experiments] (Hoechst, blue). (M to P) Differentiation of OPCs (NG2+ OPCs, green; mature MBP+ oligodendrocytes, red; Hoechst, blue) cultured in either CM from Atp11b-KD bEND.3 cells (N) or control cells (M) [quantified in (O) and (P); *P < 0.05, paired t test; n = 4 experiments]. (Q to S) Proliferation (Ki67+, red) of OPCs cultured in CM from Atp11b-KD bEND.3 cells (R) compared to controls (Q), quantified in (S) (Hoechst, blue) (*P < 0.05, paired t test; n = 3 experiments). (T) Western blot of HSP90α in CM from Atp11b-KD bEND.3 cells compared to controls [quantified in (U); *P < 0.05, paired t test; n = 3 experiments], including Ponceau stain of the membrane showing the total protein (prominent band is bovine serum albumin) used as a loading control. Scale bar, 25 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/448/eaam9507/DC1

    Materials and Methods

    Fig. S1. Lower claudin-5 protein in brains of DM rats.

    Fig. S2. No leakage of a large dextran tracer indicates normal vascular integrity in 5-week-old DM rats.

    Fig. S3. No difference in astrocytes or pericytes.

    Fig. S4. Increased number of OPCs due to increased proliferation in DM rats and diseased human brains.

    Fig. S5. Brains of young, prehypertensive DM rats and presymptomatic humans show increased numbers of microglia/macrophages.

    Fig. S6. More HSP90α is secreted by BMECs isolated from DM rats.

    Fig. S7. Addition of rHSP90α or use of blocking antibodies to HSP90α has no effect on OPC proliferation.

    Fig. S8. CM from human ECs treated with ATP11B siRNA reduces OPC maturation.

    Fig. S9. Human cerebral ECs from the HBEC5i and hCMEC/d3 lines do not express endothelial markers.

    Fig. S10. SNPs within ATP11B are associated with WMH in the CHARGE consortium data.

    Fig. S11. Summary of findings illustrating the central role of endothelial dysfunction in SVD pathology.

    Table S1. Summary of human SVD and control postmortem data and pathological characteristics.

    Table S2. Summary of treatment groups in drug study.

    References (7177)

    • This PDF file includes:
    • Materials and Methods
    • Fig. S1. Lower claudin-5 protein in brains of DM rats.
    • Fig. S2. No leakage of a large dextran tracer indicates normal vascular integrity in 5-week-old DM rats.
    • Fig. S3. No difference in astrocytes or pericytes.
    • Fig. S4. Increased number of OPCs due to increased proliferation in DM rats and diseased human brains.
    • Fig. S5. Brains of young, prehypertensive DM rats and presymptomatic humans show increased numbers of microglia/macrophages.
    • Fig. S6. More HSP90α is secreted by BMECs isolated from DM rats.
    • Fig. S7. Addition of rHSP90α or use of blocking antibodies to HSP90α has no effect on OPC proliferation.
    • Fig. S8. CM from human ECs treated with ATP11B siRNA reduces OPC maturation.
    • Fig. S9. Human cerebral ECs from the HBEC5i and hCMEC/d3 lines do not express endothelial markers.
    • Fig. S10. SNPs within ATP11B are associated with WMH in the CHARGE consortium data.
    • Fig. S11. Summary of findings illustrating the central role of endothelial dysfunction in SVD pathology.
    • Table S1. Summary of human SVD and control postmortem data and pathological characteristics.
    • Table S2. Summary of treatment groups in drug study.
    • References (7177)

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