Mechanical stretch and hypoxia inducible factor-1 alpha affect the vascular endothelial growth factor and the connective tissue growth factor in cultured ACL fibroblasts.
OBJECTIVE
The adult human anterior cruciate ligament (ACL) has poor functional healing response. Hypoxia plays an important role in regulating the microenvironment of the joint cavity after ACL injury, however, its role in mechanical injury is yet to be examined fully in ACL fibroblasts. In this study, we used CoCl2to induce Hypoxia-inducible factor-1α (HIF-1α) in our experimental model to study its affect on matrix metalloproteinase-2 (MMP-2), vascular endothelial growth factor (VEGF), and connective tissue growth factor (CTGF) expression in ACL fibroblasts after mechanical stretch.


METHODS
Cell treatments were performed in the stretch chamber in all experimental groups. Quantitative real-time PCR was used to check mRNA expression levels of MMP-2, CTGF, VEGF, and HIF-1α. Western blot was used to detect the HIF-1α production. Enzyme-Linked immunosorbent assay was performed to check the VEGF and CTGF protein contents in supernatant. MMP-2 activity was assayed by gelatin zymography.


RESULTS
The real-time PCR results show that mechanical stretch or CoCl2treatment increases the expression of MMP-2, VEGF, CTGF, and HIF-1α; however, the combined effects of mechanical stretch and CoCl2-induced HIF-1α increased MMP-2 production but decreased the VEGF and CTGF expression, compared to the CoCl2treatment group alone. Western blot analysis and ELISA also confirmed these results.


CONCLUSIONS
Our results demonstrated that mechanical stretch and CoCl2-induced HIF-1α together increased the level of MMP-2 and decreased the levels of VEGF and CTGF in cultured ACL fibroblasts. The differential expression and production of HIF-1α, VEGF, MMP-2, and CTGF might help to explain the poor healing ability of ACL.
a Institute of Forensic Medicine and Laboratory Medicine , Jining Medical University , Jining , China.|b Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College , Chongqing University , Chongqing , China.|c "111" Biomechanics and Tissue Repair Laboratory, Bioengineering College , Chongqing University , Chongqing , China.
Yequan
Y
Wang
a Institute of Forensic Medicine and Laboratory Medicine , Jining Medical University , Jining , China.
Zhen
Z
Dang
a Institute of Forensic Medicine and Laboratory Medicine , Jining Medical University , Jining , China.
Wen
W
Cui
b Key Laboratory of Biorheological Science and Technology, Ministry of Education, Bioengineering College , Chongqing University , Chongqing , China.|c "111" Biomechanics and Tissue Repair Laboratory, Bioengineering College , Chongqing University , Chongqing , China.
Li
L
Yang
D000328:Adult; D016118:Anterior Cruciate Ligament / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D055513:Connective Tissue Growth Factor / Q000096:biosynthesis*; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000096:biosynthesis*; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis; D008875:Middle Aged; D013314:Stress, Mechanical; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis*
C526317:CCN2 protein, human; C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D055513:Connective Tissue Growth Factor; D003035:Cobalt; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C018021:cobaltous chloride
ACL; CTGF; HIF-1α; VEGF; mechanical stretch