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Activated protein C mediates a healing phenotype in cultured tenocytes.

Xue M, Smith MM, Little CB, Sambrook P, March L, Jackson CJ - J. Cell. Mol. Med. (2008)

Bottom Line: When tenocytes were pre-treated with RCR252 or EPCR siRNA the effect of APC on proliferation, MMP-2 and type 1 collagen synthesis and MAP kinases was blocked.APC promotes the growth, MMP-2 activity, type I collagen deposition and migration of tenocytes.Furthermore, EPCR is expressed by tenocytes and mediates the actions of APC, at least partly by signalling through selective MAP kinases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bone and Joint Research, Kolling Institute of Medical Research, The University of Sydney at Royal North Shore Hospital, St. Leonards, NSW, Australia.

ABSTRACT
Tendon injuries cause considerable morbidity in the general adult population. The tenocytes within the tendon have the full capacity to heal the tendon intrinsically. Activated protein C (APC) plays an important role in coagulation and inflammation and more recently has been shown to promote cutaneous wound healing. In this study we examined whether APC can induce a wound healing phenotype in tenocytes. Sheep tenocytes were treated with APC, endothelial protein C receptor (EPCR) blocking antibody (RCR252) and/or EPCR small interfering (si)RNA. Cell proliferation and migration were measured by crystal violet assay and a scratch wounding assay, respectively. The expression of EPCR, matrix metalloproteinase (MMP)-2, type I collagen and MAP kinase activity were detected by real time PCR, zymography, immunofluorescence, immunohistochemistry and Western blotting. APC stimulated proliferation, MMP-2 activity and type I collagen deposition in a dose-dependent manner and promoted migration of cultured tenocytes. APC dose-dependently stimulated phosphorylated (P)-ERK2 and inhibited P-p38. Interestingly, tenocytes expressed EPCR protein, which was up-regulated by APC. When tenocytes were pre-treated with RCR252 or EPCR siRNA the effect of APC on proliferation, MMP-2 and type 1 collagen synthesis and MAP kinases was blocked. APC promotes the growth, MMP-2 activity, type I collagen deposition and migration of tenocytes. Furthermore, EPCR is expressed by tenocytes and mediates the actions of APC, at least partly by signalling through selective MAP kinases. These data implicate APC as a potential healing agent for injured tendons.

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Blocking EPCR prevents APC-induced MMP-2 synthesis and proliferation. (A) ECPR protein expression in response to EPCR siRNA treatment at 48 hrs detected by Western blot using whole cell lysates. β-actin was used as the loading control. (B) Cell proliferation was measured after cells were treated with combinations of APC, siRNA and RCR252 for 72 hrs. Cell proliferation is expressed as a percentage of control (mean ± S.D., n= 3 experiments). *P < 0.05 and **P < 0.01 compared to negative control (first bar), ++P < 0.01 compared to APC (fourth bar). Images of gels represent one of three independent experiments. (C) MMP-2 was measured in supernatants of cells trans-fected with either control siRNA (0) or EPCR siRNA for 72 hrs, using zymography. (D) MMP-2 was measured in supernatants of cells treated with APC alone or APC plus RCR252 for 24 hrs using Western blot analysis. β-actin used as the loading control.
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fig05: Blocking EPCR prevents APC-induced MMP-2 synthesis and proliferation. (A) ECPR protein expression in response to EPCR siRNA treatment at 48 hrs detected by Western blot using whole cell lysates. β-actin was used as the loading control. (B) Cell proliferation was measured after cells were treated with combinations of APC, siRNA and RCR252 for 72 hrs. Cell proliferation is expressed as a percentage of control (mean ± S.D., n= 3 experiments). *P < 0.05 and **P < 0.01 compared to negative control (first bar), ++P < 0.01 compared to APC (fourth bar). Images of gels represent one of three independent experiments. (C) MMP-2 was measured in supernatants of cells trans-fected with either control siRNA (0) or EPCR siRNA for 72 hrs, using zymography. (D) MMP-2 was measured in supernatants of cells treated with APC alone or APC plus RCR252 for 24 hrs using Western blot analysis. β-actin used as the loading control.

Mentions: We next investigated whether APC stimulation of cell proliferation and MMP-2 was mediated through EPCR. Specific siRNA was used to inhibit endogenous EPCR expression. The efficacy of EPCR siRNA was detected by Western blot, which showed >90% knockdown of protein expression in cell lysates treated for 24 hrs (Fig. 5B). EPCR siRNA treatment for 72 hrs inhibited cell proliferation by more than 20%, which suggests that either EPCR itself can stimulate proliferation or endogenously derived PC can bind to EPCR and enhance cell growth. This inhibition could not be recovered by addition of the exogenous APC (Fig. 5B), indicating that APC requires EPCR to augment proliferation. This was further confirmed by adding EPCR blocking antibody, RCR252, which abolished the stimulatory effect of APC on cell proliferation (Fig. 5B). Similarly, MMP-2 production was reduced by more than 50% after tenocytes were transfected with EPCR siRNA for 48 hrs as detected by zymography (Fig. 5C). This result was confirmed by pre-treating APC-stimulated cells with RCR 252 for 24 hrs and detected by Western blot (Fig. 5D).


Activated protein C mediates a healing phenotype in cultured tenocytes.

Xue M, Smith MM, Little CB, Sambrook P, March L, Jackson CJ - J. Cell. Mol. Med. (2008)

Blocking EPCR prevents APC-induced MMP-2 synthesis and proliferation. (A) ECPR protein expression in response to EPCR siRNA treatment at 48 hrs detected by Western blot using whole cell lysates. β-actin was used as the loading control. (B) Cell proliferation was measured after cells were treated with combinations of APC, siRNA and RCR252 for 72 hrs. Cell proliferation is expressed as a percentage of control (mean ± S.D., n= 3 experiments). *P < 0.05 and **P < 0.01 compared to negative control (first bar), ++P < 0.01 compared to APC (fourth bar). Images of gels represent one of three independent experiments. (C) MMP-2 was measured in supernatants of cells trans-fected with either control siRNA (0) or EPCR siRNA for 72 hrs, using zymography. (D) MMP-2 was measured in supernatants of cells treated with APC alone or APC plus RCR252 for 24 hrs using Western blot analysis. β-actin used as the loading control.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3822881&req=5

fig05: Blocking EPCR prevents APC-induced MMP-2 synthesis and proliferation. (A) ECPR protein expression in response to EPCR siRNA treatment at 48 hrs detected by Western blot using whole cell lysates. β-actin was used as the loading control. (B) Cell proliferation was measured after cells were treated with combinations of APC, siRNA and RCR252 for 72 hrs. Cell proliferation is expressed as a percentage of control (mean ± S.D., n= 3 experiments). *P < 0.05 and **P < 0.01 compared to negative control (first bar), ++P < 0.01 compared to APC (fourth bar). Images of gels represent one of three independent experiments. (C) MMP-2 was measured in supernatants of cells trans-fected with either control siRNA (0) or EPCR siRNA for 72 hrs, using zymography. (D) MMP-2 was measured in supernatants of cells treated with APC alone or APC plus RCR252 for 24 hrs using Western blot analysis. β-actin used as the loading control.
Mentions: We next investigated whether APC stimulation of cell proliferation and MMP-2 was mediated through EPCR. Specific siRNA was used to inhibit endogenous EPCR expression. The efficacy of EPCR siRNA was detected by Western blot, which showed >90% knockdown of protein expression in cell lysates treated for 24 hrs (Fig. 5B). EPCR siRNA treatment for 72 hrs inhibited cell proliferation by more than 20%, which suggests that either EPCR itself can stimulate proliferation or endogenously derived PC can bind to EPCR and enhance cell growth. This inhibition could not be recovered by addition of the exogenous APC (Fig. 5B), indicating that APC requires EPCR to augment proliferation. This was further confirmed by adding EPCR blocking antibody, RCR252, which abolished the stimulatory effect of APC on cell proliferation (Fig. 5B). Similarly, MMP-2 production was reduced by more than 50% after tenocytes were transfected with EPCR siRNA for 48 hrs as detected by zymography (Fig. 5C). This result was confirmed by pre-treating APC-stimulated cells with RCR 252 for 24 hrs and detected by Western blot (Fig. 5D).

Bottom Line: When tenocytes were pre-treated with RCR252 or EPCR siRNA the effect of APC on proliferation, MMP-2 and type 1 collagen synthesis and MAP kinases was blocked.APC promotes the growth, MMP-2 activity, type I collagen deposition and migration of tenocytes.Furthermore, EPCR is expressed by tenocytes and mediates the actions of APC, at least partly by signalling through selective MAP kinases.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bone and Joint Research, Kolling Institute of Medical Research, The University of Sydney at Royal North Shore Hospital, St. Leonards, NSW, Australia.

ABSTRACT
Tendon injuries cause considerable morbidity in the general adult population. The tenocytes within the tendon have the full capacity to heal the tendon intrinsically. Activated protein C (APC) plays an important role in coagulation and inflammation and more recently has been shown to promote cutaneous wound healing. In this study we examined whether APC can induce a wound healing phenotype in tenocytes. Sheep tenocytes were treated with APC, endothelial protein C receptor (EPCR) blocking antibody (RCR252) and/or EPCR small interfering (si)RNA. Cell proliferation and migration were measured by crystal violet assay and a scratch wounding assay, respectively. The expression of EPCR, matrix metalloproteinase (MMP)-2, type I collagen and MAP kinase activity were detected by real time PCR, zymography, immunofluorescence, immunohistochemistry and Western blotting. APC stimulated proliferation, MMP-2 activity and type I collagen deposition in a dose-dependent manner and promoted migration of cultured tenocytes. APC dose-dependently stimulated phosphorylated (P)-ERK2 and inhibited P-p38. Interestingly, tenocytes expressed EPCR protein, which was up-regulated by APC. When tenocytes were pre-treated with RCR252 or EPCR siRNA the effect of APC on proliferation, MMP-2 and type 1 collagen synthesis and MAP kinases was blocked. APC promotes the growth, MMP-2 activity, type I collagen deposition and migration of tenocytes. Furthermore, EPCR is expressed by tenocytes and mediates the actions of APC, at least partly by signalling through selective MAP kinases. These data implicate APC as a potential healing agent for injured tendons.

Show MeSH
Related in: MedlinePlus