Bone-derived PDGF-BB drives brain vascular calcification in male mice
Jiekang Wang, Ching-Lien Fang, Kathleen Noller, Zhiliang Wei, Guanqiao Liu, Ke Shen, Kangping Song, Xu Cao, Mei Wan
Jiekang Wang, Ching-Lien Fang, Kathleen Noller, Zhiliang Wei, Guanqiao Liu, Ke Shen, Kangping Song, Xu Cao, Mei Wan
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Research Article Bone biology Vascular biology

Bone-derived PDGF-BB drives brain vascular calcification in male mice

Abstract

Brain vascular calcification is a prevalent age-related condition often accompanying neurodegenerative and neuroinflammatory diseases. The pathogenesis of large-vessel calcifications in peripheral tissue is well studied, but microvascular calcification in the brain remains poorly understood. Here, we report that elevated platelet-derived growth factor BB (PDGF-BB) from bone preosteoclasts contributed to cerebrovascular calcification in male mice. Aged male mice had higher serum PDGF-BB levels and a higher incidence of brain calcification compared with young mice, mainly in the thalamus. Transgenic mice with preosteoclast-specific Pdgfb overexpression exhibited elevated serum PDGF-BB levels and recapitulated age-associated thalamic calcification. Conversely, mice with preosteoclast-specific Pdgfb deletion displayed diminished age-associated thalamic calcification. In an ex vivo cerebral microvascular culture system, PDGF-BB dose-dependently promoted vascular calcification. Analysis of osteogenic gene array and single-cell RNA-Seq (scRNA-Seq) revealed that PDGF-BB upregulated multiple osteogenic differentiation genes and the phosphate transporter Slc20a1 in cerebral microvessels. Mechanistically, PDGF-BB stimulated the phosphorylation of its receptor PDGFRβ (p-PDGFRβ) and ERK (p-ERK), leading to the activation of RUNX2. This activation, in turn, induced the transcription of osteoblast differentiation genes in PCs and upregulated Slc20a1 in astrocytes. Thus, bone-derived PDGF-BB induced brain vascular calcification by activating the p-PDGFRβ/p-ERK/RUNX2 signaling cascade in cerebrovascular cells.

Authors

Jiekang Wang, Ching-Lien Fang, Kathleen Noller, Zhiliang Wei, Guanqiao Liu, Ke Shen, Kangping Song, Xu Cao, Mei Wan

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Figure 8

PDGF-BB activates Slc20a1 gene transcription through ERK/RUNX2 signaling.

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PDGF-BB activates Slc20a1 gene transcription through ERK/RUNX2 signaling...
(A) Schematic representation of the Slc20a1 promoter region. The location of the consensus RUNX2 binding site (TGTGGT) and the regions chosen for PCR amplification by the primers in the ChIP-qPCR assays are indicated. (B) Cerebral microvessels isolated from brain were treated with 20 ng/mL PDGF-BB or vehicle. Chromating DNA was immunoprecipitated using a specific antibody against RUNX2 or mouse IgG (negative control). DNA fragments were amplified with primers specific for the Slc20a1 promoter. As a positive control, an antibody against RNA polymerase II (RNA PolII) was used for immunoprecipitation, and primers specific for GAPDH were used for PCR. (C) Cerebral microvessels isolated from brain were treated with 20 ng/mL PDGF-BB or vehicle. Western blot analysis of p-RUNX2 and total RUNX2 (t-RUNX2). n = 3. (D) The relative density of p-RUNX2 to t-RUNX2 was calculated using ImageJ. n = 3. (E) Immunofluorescence staining of frozen brain tissue sections from 3- and 22-month-old male mice using antibodies against RUNX2. n = 3. Scale bar: 100 μm. Magnification ×10. (F) Cerebral microvessels isolated from brain were treated with increasing concentrations of PDGF-BB. Western blot analysis of p-ERK and p-AKT. n = 3. (G) Relative densities of p-ERK and t-ERK were calculated using ImageJ (n = 3). (H) Schematic model showing the molecular mechanisms underlying PDGF-BB–induced brain vascular calcification. All data are shown as the mean ± SD. *P < 0.05 and **P < 0.01, by ordinary 1-way ANOVA for multiple-group comparisons (G). *P < 0.05, by unpaired, 2-tailed Student’s t test for 2-group comparisons (D).