Elevated PDGF-BB from Bone Impairs Hippocampal Vasculature by Inducing PDGFRβ Shedding from Pericytes

Abstract

Evidence suggests a unique association between bone aging and neurodegenerative/cerebrovascular disorders. However, the mechanisms underlying bone-brain interplay remain elusive. Here platelet-derived growth factor-BB (PDGF-BB) produced by preosteoclasts in bone is reported to promote age-associated hippocampal vascular impairment. Aberrantly elevated circulating PDGF-BB in aged mice and high-fat diet (HFD)-challenged mice correlates with capillary reduction, pericyte loss, and increased blood-brain barrier (BBB) permeability in their hippocampus. Preosteoclast-specific Pdgfb transgenic mice with markedly high plasma PDGF-BB concentration faithfully recapitulate the age-associated hippocampal BBB impairment and cognitive decline. Conversely, preosteoclast-specific Pdgfb knockout mice have attenuated hippocampal BBB impairment in aged mice or HFD-challenged mice. Persistent exposure of brain pericytes to high concentrations of PDGF-BB upregulates matrix metalloproteinase 14 (MMP14), which promotes ectodomain shedding of PDGF receptor β (PDGFRβ) from pericyte surface. MMP inhibitor treatment alleviates hippocampal pericyte loss and capillary reduction in the conditional Pdgfb transgenic mice and antagonizes BBB leakage in aged mice. The findings establish the role of bone-derived PDGF-BB in mediating hippocampal BBB disruption and identify the ligand-induced PDGFRβ shedding as a feedback mechanism for age-associated PDGFRβ downregulation and the consequent pericyte loss.

Keywords: PDGFRβ; blood-brain barrier permeability; bone; circulating platelet-derived growth factor-BB; receptor shedding.

Figure 1

Elevation of circulating PDGF‐BB correlates with hippocampal pericyte loss and BBB impairment. A,B) ELISA analysis of serum (A) and plasma (B) PDGF‐BB concentrations in C57BL/6 mice at 3, 6, 9, and 22 months of age. C) Representative confocal images of CD31 (green) and CD13 (red) double‐immunofluorescence staining in dentate gyrus (DG) (left) and CA1 (right) region of 3‐month‐ and 22‐month‐old mice. DAPI stains nuclei as blue. Scale bar, 100 µm. D) Quantification of CD13⁺ pericyte coverage of the capillaries in hippocampus. E,F) Quantification of percentage of vessel length (E) and vessel area (F) in hippocampus area. n = 5. G) Spearman correlation of serum PDGF‐BB level and pericyte coverage of capillaries in hippocampus in 3‐ and 22‐month‐old mice. H,I) BBB permeability was quantified from the leaking of 10 kDa dextran‐conjugated fluorophores into the DG and CA1 parenchymal space outside the vessels in 3‐ and 22‐month‐old WT mice. Vessels were identified by the CD31(green), while leaks were identified by leakage (red) of fluorescence outside the vessels (H). Scale bar, 100 µm. DAPI stains nuclei as blue. (I) BBB leakage is quantified by the percentage of Dextran⁺ signal area comparing in hippocampus of 3‐ and 22‐month‐old WT mice. n = 5. J) In vivo Evans blue permeability assay in cortex, hippocampus, and thalamus of 3‐, 12‐, and 22‐month‐old mice. Data are shown as the mean ± SD, **p<0.01, ***p<0.001, as determined by unpaired two‐tailed Student's t test (for two group comparison) or One‐way ANOVA (for multiple group comparison).

Figure 2

Aberrantly elevated circulating PDGF‐BB is sufficient to induce hippocampal microvascular impairment. A,B) ELISA analysis of serum (A) and plasma (B) PDGF‐BB concentration in Pdgfb^cTG mice and WT littermates at 1.5, 3, 6, 9, and 22 months of age. C) Representative confocal images of CD31 (green) immunofluorescence staining in DG region of Pdgfb^cTG mice and WT littermates at 1.5, 3, 6, and 9 months of age. DAPI stains nuclei as blue. Scale bar, 100 µm. D,E) Quantification of vessel length (D) and percentage of vessel area (E). F) Representative confocal images of CD31 (green) and CD13 (red) double‐immunofluorescence staining in DG region of 6‐month‐old Pdgfb^cTG mice and WT littermates. DAPI stains nuclei as blue. Scale bar, 100 µm. G) Quantification of CD13⁺ pericyte coverage of the capillaries in hippocampus. n = 5. Data are shown as the mean ± SD, *p<0.05, **p<0.01 and ***p<0.001, as determined by unpaired two‐tailed Student's t test. H) Spearman correlation of serum PDGF‐BB level and pericyte coverage of capillaries in hippocampus in 3‐, 6‐, and 9‐month‐old Pdgfb^cTG mice and WT littermates. n = 15.

Figure 3

Conditional Pdgfb transgenic mice recapitulate aged BBB phenotype. A–C) MRI measurements of 6‐month‐old Pdgfb^cTG mice and WT littermates. (A) Presents the WEPCAST images, red ellipsoid shows the calculated vein of mouse brain. (B) Water Remain Fraction and (C) brain volume. The great cerebral vein of Galen was marked with an ellipsoid to show the WEPCAST signal difference between WT and Pdgfb^cTG mice. D,E) BBB permeability was quantified from the leaking of 10 kDa dextran‐conjugated fluorophores into the DG and CA1 parenchymal space outside the vessels in 6‐month‐old Pdgfb^cTG mice and WT littermates. Vessels were identified by the CD31(green), while leaks were identified by leakage (red) of fluorescence outside the vessels (D). Scale bar, 100 µm. DAPI stains nuclei as blue. (E) BBB leakage is quantified by percentage of Dextran⁺ signal area comparing in hippocampus of 6‐month‐old Pdgfb^cTG mice and WT littermates. n = 5. F) In vivo Evans blue permeability assay in hippocampus in 6‐month‐old Pdgfb^cTG mice and WT littermates. n = 5. G) Representative immunofluorescence images of DG and CA1 region of hippocampus from Pdgfb^cTG mice and WT littermates using antibody against albumin. DAPI stains nuclei blue. Scale bar, 100 µm. H) Quantification of Albumin⁺ signal covered area using Image J. n = 5. I) Representative confocal images of CD31 (green) and Caveolin‐1 (red) double‐immunofluorescence staining in DG region of 6‐month‐old Pdgfb^cTG mice and WT littermates. DAPI stains nuclei as blue. Scale bar, 100 µm. J) Quantification of Caveolin‐1 expression of the capillaries in hippocampus. K) Representative confocal images of CD31 (green) and ALPL (red) immunofluorescence staining in DG region of 6‐month‐old Pdgfb^cTG mice and WT littermates. DAPI stains nuclei as blue. Scale bar, 100 µm. L) Quantification of ALPL expression of the capillaries in hippocampus. n = 5. M) Representative confocal images of CD31 (green) and TFRC (red) double‐immunofluorescence staining in DG region of 6‐month‐old Pdgfb^cTG mice and WT littermates. DAPI stains nuclei as blue. Scale bar, 100 µm. N) Quantification of TFRC expression of the capillaries in hippocampus. Data are shown as the mean ± SD, **p<0.01, ***p<0.001, as determined by unpaired two‐tailed Student's t test.

Figure 4

Conditional Pdgfb transgenic mice have declined cognitive function. Cognitive mouse behaviors were assessed in 3‐ and 6‐month‐old male Pdgfb^cTG mice and WT littermates. A,B) Mice were tested for open‐field, C) spontaneous alternation and D) spatial recognition in Y‐maze, and E,F) novel object recognition. n = 10–12. Data are shown as the mean ± SD, *p<0.05, as determined by unpaired two‐tailed Student's t test.

Figure 5

Normalizing the circulating PDGF‐BB ameliorates hippocampal microvascular impairment and cognitive decline. A) ELISA analysis of serum PDGF‐BB concentration in 18‐month‐old Pdgfb^cKO mice and WT littermates. B) Pdgfb^cKO mice and WT littermates were fed HFD or CHD for 4 months, starting from 3 months of age. ELISA analysis of serum PDGF‐BB concentration. C) Representative confocal images of CD31 (green) and CD13 (red) double‐immunofluorescence staining in DG and CA1 region in 18‐month‐old Pdgfb^cKO mice and WT littermates. DAPI stains nuclei blue. Scale bar, 100 µm. D–F) Quantification of the percentage of vessel length (D), vessel area (E), and CD13⁺ pericyte coverage of the capillaries in hippocampus (F), n = 5. G) Representative confocal images of CD31 (green) and CD13 (red) double‐immunofluorescence staining in DG and CA1 region in CHD versus HFD Pdgfb^cKO mice. DAPI stains nuclei blue. Scale bar, 100 µm. H–J) Quantification of the percentage of vessel area (H), vessel length (I), and CD13⁺ pericyte coverage of the capillaries in hippocampus (J), n = 5. K) Representative immunofluorescence images of DG and CA1 region of hippocampus from mice of 18‐month‐old Pdgfb^cKO mice and WT littermates using antibody against albumin. DAPI stains nuclei as blue. Scale bar, 100 µm. L) Quantification of Albumin⁺ signal covered area using Image J. M,N) Cognitive mouse behaviors in CHD versus HFD Pdgfb^cKO mice were assessed. Mice were tested for spatial recognition (M) and spontaneous alternation (N) in Y‐maze. n = 10. *p<0.05, **p<0.01, ***p<0.001 as determined by unpaired two‐tailed Student's t test (for 2 group comparison) or One‐way ANOVA (for multiple group comparison).

Figure 6

Persistent exposure to high concentrations of PDGF‐BB ligand leads to its receptor shedding in pericytes. A) PDGFRβ protein expression in hippocampal tissue collected from 1.5‐ and 6‐month‐old Pdgfb^cTG mice and WT littermates was assessed by Western blot analysis. B) Quantification of the relative intensity of PDGFRβ by Image J. n = 3. C) Representative confocal images of CD13 (green) and PDGFRβ (red) double‐immunofluorescence staining in CA1 region of 6‐month‐old Pdgfb^cTG mice and WT littermates. DAPI stains nuclei as blue. Scale bar, 100 µm. D) Quantification of the percentage of PDGFRβ area on CD13⁺ pericytes in hippocampus. n = 5. E–G) Primary human brain pericytes were treated with different dosages of recombinant human PDGF‐BB (rh‐PDGFBB) for 8 (E), 24 (F), and 72 h (G). PDGFRβ protein expression was detected by Western blot analysis. H) Primary human brain pericytes were treated with rh‐PDGFBB for 24 h, and culture medium (CM) was collected. PDGFRβ expression in cell lysate was detected by Western blot analysis (upper panel), and the cleaved sPDGFRβ in CM was immunoprecipitated by a specific PDGFRβ antibody followed by Western blot analysis of PDGFRβ expression (lower panel). I,J) Primary human brain pericytes were treated with rh‐PDGFBB for 24 (I) and 72 h (J), and intracellular ratio was measured by flow cytometry. K,L) Primary human brain pericytes were treated with rh‐PDGFBB together with Chloroquine (K) or MG‐132 (L) for 72 h. PDGFRβ protein expression was detected by Western blot analysis. n = 3. M) ELISA analysis of CSF sPDGFRβ concentrations in C57BL/6 mice at 3 and 22 months of age. n = 4. Data are shown as the mean ± SD, **p<0.01, ***p<0.001, unpaired two‐tailed Student's t test.

Figure 7

MMP14 mediates PDGF‐BB‐induced PDGFRβ shedding. A) Gene‐array analysis on rh‐PDGFBB‐treated or control human brain pericytes for 3 days. Heatmaps showed the magnitude of differential expression with fold change (on the right) of each gene. B) Quantitative real‐time PCR analysis of MMP14 mRNA expression in human brain pericyte with or without rh‐PDGFBB treatment for 24 h. n = 3. C) Western blot analysis of MMP14 protein expression in primary human brain pericyte with or without rh‐PDGFBB treatment for 24 h. D) Quantification of the relative intensity of MMP14 using Image J. n = 3. E) Primary human brain pericytes were treated with rh‐PDGFBB for 1 or 2 h. Double‐immunofluorescence staining of the cells was performed using antibodies against MMP14 (red) and PDGFRβ (green). F,H) Representative confocal images of PDGFRβ (red) and MMP14 (green) double‐immunofluorescence staining in DG region of 3‐ and 22‐month‐old WT mice (F) and 6‐month‐old Pdgfb^cTG mice and WT littermates (H). DAPI stains nuclei as blue. Scale bar, 100 µm. G,I) Quantification of the percentage of MMP14⁺ cells in PDGFRβ ⁺ pericytes in hippocampus. n = 5. J) Representative images of gel zymography showing the activity of MMP in the hippocampus of 3‐ and 20‐month‐old WT mice. K) Primary human brain pericytes were treated with rh‐PDGFBB in the presence or absence of MMP inhibitor GM6001 for 24 h, and culture medium (CM) was collected. PDGFRβ expression in cell lysate was detected by Western blot analysis (upper panel), and the cleaved sPDGFRβ in CM was immunoprecipitated by a specific PDGFRβ antibody followed by Western blot analysis of PDGFRβ expression (lower panel). L) Two different MMP14 siRNAs or scrambled control siRNA were individually transfected into human brain pericytes. MMP14 (upper panel) and GAPDH (lower panel) expression levels were detected by Western blot analysis. M) Primary human brain pericytes were transfected by MMP14 siRNA or scrambled control siRNA followed by rh‐PDGFBB treatment for 24 h. PDGFRβ expression in cell lysate was detected by Western blot analysis (1st Row), and the cleaved sPDGFRβ in CM was immunoprecipitated by a specific PDGFRβ antibody followed by Western blot analysis of PDGFRβ expression (2nd Row). MMP14 (3rd Row) and GAPDH (4th Row) expression levels in cell lysate were also detected by Western blot analysis. Data are shown as the mean ± SD, ***p<0.001, unpaired two‐tailed Student's t test.

Figure 8

GM6001 rescue MMP14‐induced PDGFRβ shedding and aged‐associated BBB leakage. A–I) Pdgfb^cTG mice were treated with GM6001 or vehicle every other day by i.p. injection for 2 weeks. Representative confocal images of PDGFRβ (red) and CD13 (green) double‐immunofluorescence staining in DG (A), CA1 (D), and cortex (G). DAPI stains nuclei as blue. Scale bar, 100 µm. Quantification of the percentage of PDGFRβ expression area on CD13⁺ cells (B, E, H) and CD13⁺ pericyte area (C, F, I) in the three regions. J–M) 18‐month‐old mice were treated with GM6001 or vehicle every other day by i.p. injection for 4 weeks. Representative confocal images of CD31 (green) and Dextran (red) double‐immunofluorescence staining in DG (J) and CA1 (L). DAPI stains nuclei as blue. Scale bar, 100 µm. Quantification of the percentage of Dextran⁺ signal area in these two areas (K and M). n = 5. Data are shown as the mean ± SD, *p<0.05, **p<0.01, ***p<0.001, as determined by unpaired two‐tailed Student's t test (for two group comparison) or One‐way ANOVA (for multiple group comparison).