Expressions of local renin-angiotensin system components in chondrocytes

Abstract

In 2013, we reported that local renin-angiotensin system (local RAS) components express during the hypertrophic differentiation of chondrocytes and can modulate it, using ATDC5 cell line that involves differentiation from mesenchymal stem cells to calcified hypertrophic chondrocytes. However, the expressions of local RAS components in normal chondrocytes have not been revealed yet. The purpose of this study is to examine the expression of the local RAS components in chondrocytes in vivo and the conditions allowing the expression. We stained five major regions of 8-week-old C57BL/6 adult mice in which chondrocytes exist, including epiphyseal plates and hyaline cartilages, with antibodies to local RAS components. We also examined the expression of local RAS components in the cultured bovine's articular cartilage chondrocytes using quantitative reverse transcription polymerase chain reaction and western blot analysis. In result, hypertrophic chondrocytes of epiphyseal plates included in the tibia and the lamina terminals expressed local RAS components. However, hyaline chondrocytes, including the knee articular cartilages, the parenchyma of nasal septums and of the tracheal walls, did not express local RAS components. Cultured bovine's articular cartilage chondrocytes also did not express local RAS components. However, inducing hypertrophy by administering interleukin-1β or tumor necrosis factor-α, the cultured articular chondrocytes also expressed angiotensin II type 1 receptor and angiotensin II type 2 receptor. In conclusion, local RAS components express particularly in chondrocytes which occur hypertrophy and do not in hyaline chondrocytes. The results are in accord with our previous in vitro study. We think this novel knowledge is important to investigate cartilage hypertrophy and diseases induced by hypertrophic changes like osteoarthritis.

Figure 1.

Localization of chondrocytes in knee joints, nasal septums, tracheal tubes and spines of 8-week-old C57BL/6 adult female mice: double-staining with alizarin red and Alcian blue. A) Meniscus and articular cartilage of knee joints and epiphyseal plate of the tibia were stained with Alcian blue; †, meniscus; ‡, articular cartilage; *, epiphyseal plate. B,G) Parenchyma of nasal septum was stained with Alcian blue. C,H) Tracheal walls were stained with Alcian blue; †, esophagus; *, intratracheal. D,I) Epiphyseal plates of spine, chondrocytes of lamina terminalis, annulus fibrosus and nucleus pulposus were stained with Alcian blue; *, epiphyseal plate; α, proliferating zone; (β, hypertrophic zone; **, lamina terminalis; †, annulus fibrosus; ‡, nucleus pulposus. E) Epiphyseal plate of the tibia was stained with Alcian blue; *, epiphyseal plate; α, proliferating zone; β, hypertrophic zone. F) Articular cartilage and meniscus of knee joints were stained with Alcian blue. Scale bars: A-D) 100 µm; E-I) 50 µm. Magnifications: A-C) 4×; D-I) 40×.

Figure 2.

Immunohistochemical micrographs of local renin-angiotensin system components in epiphyseal plate of the tibia and the spine. A,B), ACE1. C,D), ANG. E,F), AT1R. G,H), AT2R. A,C,E,G), Tibia. B,D,F,H), Spine. The hypertrophic chondrocytes in epiphyseal plates of each section were stained with each local renin-angiotensin system components; cells stained with antibodies were indicated with arrows; α, proliferating zone; β, hypertrophic zone; ACE1, angiotensin-converting enzyme 1; ANG, angiotensinogen; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2 receptor. Scale bars: 50 µm. Magnification: 100×.

Figure 3.

Immunohistochemical micrographs of local renin-angiotensin system components in articular cartilage and meniscus of knee joint, parenchyma of nasal septum, tracheal wall, lamina terminalis, annulus fibrosus and nucleus pulposus. A-D), ACE1. E-H), ANG. I-L), AT1R. M-P), AT2R. A,E,I,M), articular cartilage and meniscus; †, meniscus; *, articular cartilage. B,F,J,N), parenchyma of nasal septum. C,G,K,O) tracheal wall. D,H,L,P), lamina terminalis, annulus fibrosus and nucleus pulposus; *, lamina terminalis; †, annulus fibrosus; ‡, nucleus pulposus. Each chondrocytes of each section were not stained with local renin-angiotensin system components. ACE1, angiotensin-converting enzyme 1; ANG, angiotensinogen; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2 receptor. Scale bars: A,C-E,G-I,K-M,O,P) 50 µm; B,F,J,N) 100 µm. Magnification: 40×.

Figure 4.

Micrographs of immunohistchemical control stains. A) Knee joint; †, meniscus; ‡, articular cartilage; *, epiphyseal plate. B) Nose. C) Trachea. D) Spine; *, epiphyseal plate; **, lamina terminalis; †, annulus fibrosus; ‡, nucleus pulposus. Unspecific immunohistochemical reactions were not detected. Scale bars: 100 µm. Magnification: 40×.

Figure 5.

Expressions of AT1R and AT2R in cultured bovine’s articular cartilage chondrocytes. A) Both mRNA expressions of AT1R and AT2R were not detected without administering any agents; the expressions of AT1R and AT2R were induced administering IL-1β in a concentration-dependent manner. B) Both mRNA expressions of AT1R and ATR are not detected without administering any agents; the expressions of AT1R and AT2R were induced administering TNF-α in a concentration-dependent manner. C) Both protein synthesis of AT1R and ATR were not detected without administering any agents; the protein synthesis of AT1R and AT2R were induced administering IL-1β. D) Both protein synthesis of AT1R and ATR were not detected without administering any agents; the protein synthesis of AT1R and AT2R were induced administering TNF-α. *, P<0.05 between treatments; AT1R, angiotensin II type 1 receptor, AT2R, angiotensin II type 2 receptor, IL-1β, interleukin-1β; TNF-α, tumor necrosis factor-alpha.