Bone-derived PDGF-BB drives brain vascular calcification in male mice
Jiekang Wang, … , Xu Cao, Mei Wan
Jiekang Wang, … , Xu Cao, Mei Wan
Published October 10, 2023
Citation Information: J Clin Invest. 2023;133(23):e168447. https://doi.org/10.1172/JCI168447.
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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 6

PDGF-BB stimulates p-PDGFRβ/p-ERK/RUNX2 signaling in PCs to activate multiple key osteoblast differentiation genes.

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PDGF-BB stimulates p-PDGFRβ/p-ERK/RUNX2 signaling in PCs to activate mul...
(A–D) Primary mouse brain PCs were exposed to recombinant mouse PDGF-BB at a concentration of 30 ng/mL for shorter time periods (A and B) and longer time periods (C and D). Expression of the indicated proteins was detected by Western blot analysis (A and C). The relative intensities of the proteins were quantified using ImageJ (B and D). (E) Primary mouse brain PCs were exposed to recombinant mouse PDGF-BB at a concentration of 30 ng/mL for 4 and 8 hours. Western blot analysis of RUNX2 and OPN expression. (F) Double-immunofluorescence staining of frozen brain tissue sections from 3- and 22-month-old male mice using antibodies against CD31 and p-PDGFRβ. n = 3. (G) Primary mouse brain PCs were subjected to an 8-hour treatment with recombinant mouse PDGF-BB. Double immunocytochemical staining was performed using antibodies against PDGFRβ and OPN. Boxed areas are shown at a higher magnification in the corresponding panels to the right. n = 3. (H) Primary mouse brain PCs were treated with 30 ng/mL PDGF-BB for 4 and 8 hours. qRT-PCR analysis was conducted to assess the expression levels of osteogenic marker genes, including Spp1, Runx2, and Sp7. n = 6. (I) Primary brain astrocytes were treated with 30 ng/mL PDGF-BB for 4, 8, and 16 hours. qRT-PCR was conducted to assess the expression levels of osteogenic markers, including Sp7 and Bglap. n = 3. Scale bars: 100 μm (F and G). Relative fold-change results are shown as the mean ± SD. *P < 0.05, **P < 0.01, and ****P < 0.0001, by ordinary 1-way ANOVA for multiple-group comparisons (H and I).