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. 2009 Dec;175(6):2288-98.
doi: 10.2353/ajpath.2009.090474. Epub 2009 Nov 5.

Gla-rich Protein Is a Novel Vitamin K-dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications

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Gla-rich Protein Is a Novel Vitamin K-dependent Protein Present in Serum That Accumulates at Sites of Pathological Calcifications

Carla S B Viegas et al. Am J Pathol. .
Free PMC article

Abstract

Mineralization of soft tissues is an abnormal process that occurs in any body tissue and can greatly increase morbidity and mortality. Vitamin K-dependent (VKD) proteins play a crucial role in these processes; matrix Gla protein is considered one of the most relevant physiological inhibitors of soft tissue calcification know to date. Several studies have suggested that other, still unknown, VKD proteins might also be involved in soft tissue calcification pathologies. We have recently identified in sturgeon a new VKD protein, Gla-rich protein (GRP), which contains the highest ratio between number of Gla residues and size of the mature protein so far identified. Although mainly expressed in cartilaginous tissues of sturgeon, in rat GRP is present in both cartilage and bone. We now show that GRP is a circulating protein that is also expressed and accumulated in soft tissues of rats and humans, including the skin and vascular system in which, when affected by pathological calcifications, GRP accumulates at high levels at sites of mineral deposition, indicating an association with calcification processes. The high number of Gla residues and consequent mineral binding affinity properties strongly suggest that GRP may directly influence mineral formation, thereby playing a role in processes involving connective tissue mineralization.

Figures

Figure 1
Figure 1
Rat GRP gene is highly expressed in nonskeletal tissues. Sites of gene expression were determined by in situ hybridization with digoxigenin-labeled antisense probes in outer ear paraffin sections, and signal was revealed with alkaline phosphatase-coupled antidigoxigenin-AP antibody and nitro blue tetrazolium /5-bromo-4-chloro-3-indolyl phosphate substrate solution, yielding a characteristic blue color. GRP is highly expressed in the skin and its appendages in both epidermis (Ep, A and D) and dermis (De, A and D). Within the dermis GRP is detected in the fibroblasts (Fb, B and D), hair follicles (HF, C), and sebaceous (SG, B and C) and sweat glands (SwG, A). In blood vessels, GRP is detected mainly in vascular smooth muscle cells (VSMC) of the tunica intima (TI, F). In the elastic cartilage (EC) composing the outer ear, GRP is detected in chondrocyte (Cd) cells (G). Negative control performed through in situ hybridization with GRP sense probe is presented in E. Magnifications: A, D, E, ×10; B, C, F, G, ×20.
Figure 2
Figure 2
Rat GRP is highly accumulated in cartilage (A, B, F), bone (C), skin and its appendages (D and E), and in the vascular system (G–J) as determined by immunohistochemistry using the CTerm-GRP primary antibody and peroxidase-conjugated goat anti-rabbit IgG as secondary antibody. Peroxidase activity was visualized using 3,3-diaminobenzidine substrate yielding a brown color. A and B: Sites of GRP accumulation in sections of rib cartilage showing protein detection inside chondrocytes in all stages of maturation: columnar chondrocytes (CC), mature chondrocytes (MC), hypertrophic chondrocytes (HC), and immature chondrocytes (IC). C: GRP detection in bone sections showing protein inside osteocytes (Oc). D–F: GRP detection in outer ear sections showing protein accumulation in skin epidermis (Ep) and dermis (De, D and E) and its appendages: hair follicles (HF, D), sebaceous glands (SG, D), and dermal fibroblast (Fb, E). In addition, GRP was detected in chondrocytes (Cd) of the elastic cartilage (EC, F). G–J: GRP accumulation in the vascular system in sections of outer ear (G and H), heart (I), and kidney (J), showing GRP highly accumulated in the walls of blood vessels (Bv) and capillaries (Cp). TM, tunica media; TI, tunica intima; EL, elastic lamina. Magnifications: A–E and J, ×10; F–H and I, ×20.
Figure 3
Figure 3
Gla proteins detection co-localize in rat skin and cartilage with GRP accumulation. Immunolocalization of Gla proteins was performed using the Gla-specific monoclonal antibody M3B, and peroxidase-conjugated goat anti-mouse IgG as secondary antibody, in sections of rat outer ear (A–C) and rib (D), showing a positive co-localization (brown) with GRP, presented in Figure 2. De, dermis; Ep, epidermis; Fb, fibroblasts (arrowheads); SG, sebaceous gland; and HF, hair follicle. CHC, cartilage of the hypertrophic zone. Black star is located within the calcified cartilage. Magnification, ×10.
Figure 4
Figure 4
GRP is present in human skin (A–F) and vascular system (G–I). A–C: Sites of GRP expression determined by in situ hybridization with digoxigenin-labeled antisense probes (blue) in healthy human skin. D–F: Sites of GRP accumulation determined by immunohistochemistry with the CTerm-GRP primary antibody and peroxidase-conjugated goat anti-rabbit IgG as secondary antibody (brown), in healthy human skin. D–F show consecutive sections of A–C. Human GRP is detected in skin at the levels of epidermis (Ep) and dermis (De, A and D), small capillaries (Cp, A), and skin appendages (B, C, E, F), similarly to what is observed for rat GRP (Figures 1 and 2). Fb, fibroblast; HF, hair follicle; SwG, sweat gland. G–I: GRP accumulation in noncalcified human carotids showing GRP in vascular smooth muscle cells (VSMC) located in the tunica media (TM, G and H), and in small blood vessels (Bv) of the tunica adventitia (TA, G and I). Magnifications: A–G, ×10; H and I, ×20.
Figure 5
Figure 5
GRP is highly accumulated at sites of pathological calcification in human skin diagnosed with dermatomyositis with calcinosis (DC) (A–C) and PXE (D–F). Sites of GRP accumulation were determined by immunohistochemistry using the CTerm-GRP primary antibody and peroxidase-conjugated goat anti-rabbit IgG as secondary antibody (brown pointed by arrows, A and B, D and E). Mineral detection was achieved by staining consecutive sections of DC and serial sections of PXE with silver nitrate by the von Kossa staining method (open arrows, C and F, respectively). In both pathological situations, GRP is co-localized with sites of mineral deposition, either in DC samples when massive mineral deposits are present (A and B) or in PXE samples where disperse small mineral spots are detected (D and E). A–C were counterstained with toluidine blue and F with hematoxylin and eosin. Negative controls were performed by omitting the CTerm-GRP antibody in consecutive sections of DC (NC, inset in A) and PXE (NC, inset in D) samples. Magnification, ×10.
Figure 6
Figure 6
GRP is highly accumulated in the human vascular system at sites of pathological calcification of human arteries from both CDK patients (A–D) and postmortem samples (pM) (E–H). Sites of GRP accumulation were determined by immunohistochemistry using the CTerm-GRP primary antibody and peroxidase-conjugated goat anti-rabbit as secondary antibody (brown) (B, D, F, H), and mineral detection was achieved by staining consecutive sections of both CDK and pM with silver nitrate by the von Kossa staining method (A and C and E and G, respectively). In CDK samples GRP is highly accumulated at sites showing mineral deposition (A and B), when compared with non-calcified areas (C and D). In pM samples GRP is co-localized with sites of disperse (E and F) and massive (G and H) calcification. E–H were counterstained with toluidine blue. Magnification, A–G, ×10.

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