Calcifying vascular smooth muscle cells and osteoblasts: independent cell types exhibiting extracellular matrix and biomineralization-related mimicries

Abstract

Background: Ectopic vascular calcifications represent a major clinical problem associated with cardiovascular disease and mortality. However, the mechanisms underlying pathological vascular calcifications are largely unknown hampering the development of therapies to tackle this life threatening medical condition.

Results: In order to gain insight into the genes and mechanisms driving this pathological calcification process we analyzed the transcriptional profile of calcifying vascular smooth muscle cells (C-VSMCs). These profiles were compared to differentiating osteoblasts, cells that constitute their physiological calcification counterparts in the body. Overall the transcriptional program of C-VSMC and osteoblasts did not overlap. Several genes, some of them relevant for bone formation, were distinctly modulated by C-VSMCs which did not necessarily lose their smooth muscle cell markers while calcifying. Bioinformatics gene clustering and correlation analysis disclosed limited bone-related mechanisms being shared by two cell types. Extracellular matrix (ECM) and biomineralization genes represented common denominators between pathological vascular and physiological bone calcifications. These genes constitute the strongest link between these cells and represent potential drivers for their shared end-point phenotype.

Conclusions: The analyses support the hypothesis that VSMC trans-differentiate into C-VSMCs keeping their own identity while using mechanisms that osteoblasts use to mineralize. The data provide novel insights into groups of genes and biological processes shared in MSC and VSMC osteogenic differentiation. The distinct gene regulation between C-VSMC and osteoblasts might hold clues to find cell-specific pathway modulations, opening the possibility to tackle undesired vascular calcifications without disturbing physiologic bone formation and vice versa.

Figure 1

Characterization of the C-VSMC development and osteoblast differentiation processes. ALP activity (A) and mineralization (B) corrected for protein during the 3 week cell culture period. ALP + cell signal, measured by FACS until the second week of culture, is shown in panel (C). Detailed scatter plots with the distribution of the ALP + signal between the cell populations are depicted in (D) and (E). Value means ± SD (n = 3, *p < 0.05, **p < 1×10^-4).

Figure 2

Principal Component Analysis of the global gene expression changes occurring during C-VSMC development and osteoblast differentiation. 14733 probes expressed by both VSMC/C-VSMC and MSC/osteoblasts (OB) at day 0, 2, 8, 12 and 25 were considered for analysis. Distance between samples is directly proportional to gene expression differences. Each time point is represented by the average of 3 biological replicates with exception for day 0 where n = 4. Between parenthesis in the x- and y-axis is the percentage of variance captured by the two principal components.

Figure 3

Expression profile of known smooth muscle cell markers during C-VSMC development. Intensity values in arbitrary units are based on data at day 0, 2, 8, 12 and 25. For each time point n = 3 with exception for day 0 where n = 4. Value means ± SD.

Figure 4

Clustering of genes with similar expression patterns in C-VSMCs and osteoblasts and respective functional annotation of the clusters. (A) Clusters of genes with similar expression pattern for C-VSMCs and osteoblasts were obtained using k-means clustering (k = 6). For these analysis only differentially expressed genes were used. Average relative gene expression level (log₂ fold-change relative to day 0) for all probes within each cluster at the different time points analyzed is shown. (B) Functional annotation for each of the 6 clusters in C-VSMCs and osteoblasts. Only significant (Bonferroni p-value < 0.05) biological process, cellular compartment and molecular function annotations were considered for analysis. Numbers within grid represent fold-enrichment levels of GO-terms in the distinct clusters. The number of probes/genes comprised in each cluster is also indicated.

Figure 5

Expression pattern of extracellular region and ECM genes differentially expressed during C-VSMC development and osteoblast differentiation. Temporal expression profile of extracellular region and ECM genes shown in Figure 4 clusters 1 and 2 in (A) both cell types, (B) in C-VSMCs only and (C) in osteoblasts only. Numbers in the Venn diagrams indicate number of probes/genes. (D) Expression profile of ECM probes/genes with identical regulation pattern in C-VSMCs and osteoblasts. A smaller subset of these ECM genes is shown in (E). Expression is plotted as log₂ fold-change relative to d0. Each line plotted represents a probe set. Probe/gene identifiers are provided in Additional file 4: Table S2 and Additional file 5: Table S3.

Figure 6

Pearson correlation plot of genes comprised within Gene Ontology (GO)-terms related to bone biology. VSMC/C-VSMC and MSC/osteoblast are plotted against each other to determine their degree of similarity based on (A) biomineral tissue development, (B) regulation of osteoblast differentiation and (C) regulation of BMP signaling genes. As a reference, VSMC/C-VSMC and MSC/osteoblast are also plotted considering (D) all expressed genes and (E-F) randomly selected genes. Dashed boxes highlight the correlation between C-VSMCs and osteoblasts at day 8-25. Average correlation values (r²) for this group of samples is shown within the dashed boxes; blue, negative correlation; red, positive correlation.