Osteogenic responses in fibroblasts activated by elastin degradation products and transforming growth factor-beta1: role of myofibroblasts in vascular calcification - PubMed (original) (raw)

Osteogenic responses in fibroblasts activated by elastin degradation products and transforming growth factor-beta1: role of myofibroblasts in vascular calcification

Agneta Simionescu et al. Am J Pathol. 2007 Jul.

Abstract

Our objective was to establish the role of fibroblasts in medial vascular calcification, a pathological process known to be associated with elastin degradation and remodeling. Rat dermal fibroblasts were treated in vitro with elastin degradation products and transforming growth factor (TGF)-beta1, factors usually present in deteriorated matrix environments. Cellular changes were monitored at the gene and protein level by reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and von Kossa staining for calcium deposits. By 21 days, multicellular calcified nodules were formed in the presence of elastin degradation products and TGF-beta1 separately and to a significantly greater extent when used together. Before mineralization, cells expressed alpha-smooth muscle actin and large amounts of collagen type I and matrix metalloproteinase-2, characteristic features of myofibroblasts, key elements in tissue remodeling and repair. Stimulated cells expressed increased levels of core-binding factor alpha1, osteocalcin, alkaline phosphatase, and osteoprotegerin, representative bone-regulating proteins. For most proteins analyzed, TGF-beta1 synergistically amplified responses of fibroblasts to elastin degradation products. In conclusion, elastin degradation products and TGF-beta1 promote myofibroblastic and osteogenic differentiation in fibroblasts. These results support the idea that elastin-related calcification involves dynamic remodeling events and suggest the possibility of a defective tissue repair process.

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Figures

Figure 1

Figure 1

Expression of α-SMA in fibroblasts exposed to EDPs and TGF-βl. A: Immunofluorescent cell staining for α-SMA. B: α-SMA gene expression measured by RT-PCR.

Figure 2

Figure 2

SM22 in fibroblasts exposed to EDPs and TGF-βl. Immunofluorescent cell staining for SM22.

Figure 3

Figure 3

Modulation of collagen type I and MMP-2 expression in fibroblasts by EDPs and TGF-βl. A: Immunocytochemical staining of collagen type I (red fluorescence). B: Collagen type I gene expression measured by RT-PCR. MMP-2 gene expression measured by RT-PCR (C) and MMP-2 enzyme activity measured by gelatin zymography (D) (inset) followed by densitometry and expressed as relative density units (RDU).

Figure 4

Figure 4

Osteogenic responses in fibroblasts exposed to EDPs and TGF-βl. A: Cbfa-1 gene expression measured by RT-PCR. B: Levels of Cbfa-1 protein measured in cell extracts by ELISA. C: Osteocalcin (Oc) gene expression measured by RT-PCR. D: Protein levels in culture media assayed with an osteocalcin ELISA kit.

Figure 5

Figure 5

Effects of EDPs and TGF-βl on alkaline phosphatase expression. A: Gene expression. B: Enzyme activity measured with _p_-nitrophenyl phosphate as a substrate. C: Histochemical staining of enzyme activity (dark deposits, arrows). D: Histochemical staining for calcium deposits with the von Kossa stain (arrows).

Figure 6

Figure 6

Expression of OPG in fibroblasts exposed to EDPs and TGF-βl. A: Immunofluorescent cell staining for OPG (red fluorescence). B: OPG gene expression measured by RT-PCR. C: OPG protein expression measured by Western blotting (inset) followed by densitometry and expressed as relative density units (RDU).

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