TGF beta-induced cartilage repair is maintained but fibrosis is blocked in the presence of Smad7

Abstract

Cartilage damage in osteoarthritis (OA) is considered an imbalance between catabolic and anabolic factors, favoring the catabolic side. We assessed whether adenoviral overexpression of transforming growth factor-beta (TGFbeta) enhanced cartilage repair and whether TGFbeta-induced fibrosis was blocked by local expression of the intracellular TGFbeta inhibitor Smad7. We inflicted cartilage damage by injection of interleukin-1 (IL-1) into murine knee joints. After 2 days, we injected an adenovirus encoding TGFbeta. On day 4, we measured proteoglycan (PG) synthesis and content. To examine whether we could block TGFbeta-induced fibrosis and stimulate cartilage repair simultaneously, we injected Ad-TGFbeta and Ad-Smad7. This was performed both after IL-1-induced damage and in a model of primary OA. In addition to PG in cartilage, synovial fibrosis was measured by determining the synovial width and the number of procollagen I-expressing cells. Adenoviral overexpression of TGFbeta restored the IL-1-induced reduction in PG content and increased PG synthesis. TGFbeta-induced an elevation in PG content in cartilage of the OA model. TGFbeta-induced synovial fibrosis was strongly diminished by simultaneous synovial overexpression of Smad7 in the synovial lining. Of great interest, overexpression of Smad7 did not reduce the repair-stimulating effect of TGFbeta on cartilage. Adenoviral overexpression of TGFbeta stimulated repair of IL-1- and OA-damaged cartilage. TGFbeta-induced synovial fibrosis was blocked by locally inhibiting TGFbeta signaling in the synovial lining by simultaneously transfecting it with an adenovirus overexpressing Smad7.

Figure 1

Effect of adenoviral transforming growth factor-β (TGF-β) expression on proteoglycan (PG) synthesis and content in cartilage. (a) PG synthesis was measured by ³⁵SO₄^2- incorporation into patellar cartilage 4 days after interleukin-1 (IL-1) injection. (CPM = counts per minute) PG synthesis increased after IL-1 injection (p < 0.005), and this increase was boosted by TGF-β (p < 0.0005 compared with IL-1). (b) PG content of cartilage was measured by Safranin O staining intensity of the cartilage in Safranin O/Fast Green stained sections. The mean PG content of non-treated knee joints was set at 100%. After IL-1 exposure, a clear reduction in PG content was observed (p < 0.05). By adenoviral expression of TGF-β after IL-1-induced damage, the PG content of the cartilage was almost normal. Error bars display standard error.

Figure 2

Synovial fibrosis was assessed in knee joints 2 weeks after intra-articular injection of Ad-transforming growth factor-β (TGF-β) combined with Ad-luciferase or Ad-Smad7. As a measure of fibrosis, the synovial width opposite the growth plates in the femur was measured (a) In addition, the amount of cells staining positive in immunohistochemically stained sections for procollagen type I was calculated with a computerized imaging system (b). The data are represented as an increase of the viral control. Histological representations of the measurements give an indication of actual thickness (c) and procollagen positive cells (d) There were no differences between viral and non-injected controls. TGF-β overexpression resulted in an increase in synovial thickness and number of procollagen type I-positive cells (p < 0.05). By co-expression with Smad7, most of the TGF-β-induced fibrosis was prevented (p < 0.05). Error bars display standard error.

Figure 3

Proteoglycan (PG) synthesis in patellar cartilage after exposure to both transforming growth factor-β (TGF-β) and Smad7. PG synthesis was measured by ³⁵SO₄^2- incorporation into patellar cartilage 4 days after interleukin-1 (IL-1) injection. (CPM = counts per minute) Simultaneous injection of Ad-TGF-β and Ad-Smad7 significantly increased PG synthesis compared with IL-1 + control virus (p < 0.005) and therefore did not block ³⁵SO₄^2- incorporation. Error bars display standard error.

Figure 4

Cartilage damage was introduced by interleukin-1 (IL-1) injection, and 2 days later transforming growth factor-β (TGF-β) was injected intra-articularly combined with Ad-luciferase or Ad-Smad7. Two weeks after adenoviral transfection, knee joints were isolated for assessment of fibrosis. (a) Synovial thickness had increased significantly (p < 0.005) after exposure to TGF-β. The TGF-β-induced increase was reduced by simultaneous overexpression of Smad7 (p < 0.05). (b) The number of procollagen type I-expressing cells had increased after TGF-β exposures accordingly (p < 0.0005). This was also partially blocked by Smad7 (p < 0.005). Error bars display standard error.

Figure 5

Proteoglycan (PG) content of osteoarthritits (OA) cartilage in STR/ort mice. STR/ort mice (4 weeks old) were injected intra-articularly with Ad-transforming growth factor-β (TGF-β) combined with Ad-Smad7 or Ad-luciferase and injected again 2 weeks later. Four weeks after the first injection, knee joints were isolated and the effect of TGF-β overexpression on PG content was assessed. TGF-β exposure resulted in a significantly higher PG content of cartilage than luciferase-injected controls (p < 0.05). Co-expression with Smad7 had no effect on the PG content. Error bars display standard error.

Figure 6

Blocking of transforming growth factor-β (TGF-β)-induced synovial fibrosis in osteoarthritits (OA). STR/ort mice (4 weeks old) were injected intra-articularly with Ad-TGF-β combined with Ad-Smad7 or Ad-luciferase (Ad-luc) and injected again 2 weeks later. Two weeks after the last injection, knee joints were isolated for histology and synovial fibrosis was assessed. Synovial thickness was determined by measuring the width of the synovium opposite the growth plates in the femur. (a) Synovial thickness had increased significantly after adenoviral TGF-β overexpression (p < 0.005). More than 50% of this increase in width was prevented by simultaneous expression of Smad7 (p < 0.005). The number of procollagen type I-expressing cells (b) had increased as well after exposure to TGF-β (p < 0.0005). This was also inhibited by more than 50% by co-expression of Smad7. Histological representations of the measurements give an indication of actual thickness (c) and procollagen positive cells (d) Error bars display standard error.