Altered Caveolin-1 Dynamics Result in Divergent Mineralization Responses in Bone and Vascular Calcification

Abstract

Introduction: Though vascular smooth muscle cells adopt an osteogenic phenotype during pathological vascular calcification, clinical studies note an inverse correlation between bone mineral density and arterial mineral-also known as the calcification paradox. Both processes are mediated by extracellular vesicles (EVs) that sequester calcium and phosphate. Calcifying EV formation in the vasculature requires caveolin-1 (CAV1), a membrane scaffolding protein that resides in membrane invaginations (caveolae). Of note, caveolin-1-deficient mice, however, have increased bone mineral density. We hypothesized that caveolin-1 may play divergent roles in calcifying EV formation from vascular smooth muscle cells (VSMCs) and osteoblasts (HOBs).

Methods: Primary human coronary artery VSMCs and osteoblasts were cultured for up to 28 days in an osteogenic media. CAV1 expression was knocked down using siRNA. Methyl β-cyclodextrin (MβCD) and a calpain inhibitor were used, respectively, to disrupt and stabilize the caveolar domains in VSMCs and HOBs.

Results: CAV1 genetic variation demonstrates significant inverse relationships between bone-mineral density (BMD) and coronary artery calcification (CAC) across two independent epidemiological cohorts. Culture in osteogenic (OS) media increased calcification in HOBs and VSMCs. siRNA knockdown of CAV1 abrogated VSMC calcification with no effect on osteoblast mineralization. MβCD-mediated caveolae disruption led to a 3-fold increase of calcification in VSMCs treated with osteogenic media (p < 0.05) but hindered osteoblast mineralization (p < 0.01). Conversely, stabilizing caveolae by calpain inhibition prevented VSMC calcification (p < 0.05) without affecting osteoblast mineralization. There was no significant difference in CAV1 content between lipid domains from HOBs cultured in OS and control media.

Conclusion: Our data indicate fundamental cellular-level differences in physiological and pathophysiological mineralization mediated by CAV1 dynamics. This is the first study to suggest that divergent mechanisms in calcifying EV formation may play a role in the calcification paradox.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-023-00779-7.

Keywords: (3-10) Vascular smooth muscle cells; Calpain; Cardioinformatics; Caveolae; Extracellular vesicles; Matrix vesicles; Methyl-β-cyclodextrin; Osteoblasts.

Fig. 1

Row labels are p-values for inclusion of SNPs and column labels are distance upstream and downstream of the CAV1 coding region where SNPs are included. Heatmaps illustrate (A) − log(p-values) of observed number of inversely associated SNPs between BMD and CAC for B percentage of included SNPs with inverted directions between BMD and CAC and C total counts of included SNPs. For ease of viewing, the − log p-value corresponding to a statistical significance level of 0.05 is 1.30. D Locations of all SNPs included in at least one of the analyses above. The region of included SNPs spans 1 MB above and below the coding region of CAV1. SNPs indicated in red are within the transcription start and stop site of CAV1. E Workflow of sources for CAC and BMD data, as well as their analytical methods

Fig. 2

Alizarin red staining and quantification of VSMCs (A) and HOBs (B) grown in control and OS media after 28 and 21 days, respectively. Alizarin red staining and quantification in control and OS media with siRNA knockdown of CAV1 of VSMCs (C) and HOBs (D). *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001. Mann-Whitney U test and Kruskal-Wallis ANOVA were used as non-parametric analyses to test for differences between groups

Fig. 3

Alizarin red quantification of VSMCs (A) and HOBs (B) in control, OS, and OS + MβCD media after 28 and 21 days, respectively. Cellular CAV1 in VSMCs (C) and HOBs (D) after 14 days of treatment. Cellular TNAP activity in VSMCs (E) and HOBs (F) after 14 days of culture. VSMC extracellular vesicles’ TNAP activity after 21 days of culture (G). TNAP activity of HOB matrix vesicles released after 14 days of treatments (H). *p < 0.05 and **p ≤ 0.01, Kruskal-Wallis ANOVA was used as a non-parametric analysis to test for differences between groups

Fig. 4

Cultures containing Alizarin red staining and quantification in VSMCS (A) and HOB (B) after 28 and 21 days, respectfully. Cellular CAV1 protein (C) and TNAP protein (D) in VSMCs after 14 days of culture. Cellular TNAP activity in VSMCs after 14 days of treatment (E). TNAP activity of EVs released from VSMCs after 14 days of treatment (F). *p < 0.05 and **p ≤ 0.01, Kruskal-Wallis ANOVA was used as a non-parametric analysis to test for differences between groups

Fig. 5

Relative CAV1 content of HOB cell lysate density fractions from control and OS cultures after 14 days