Exploration Of Relationship Between Fibronectin And A Disintegrin And Metalloproteinase-8(ADAM-8) In Intervertebral Disc Degeneration

BackgroundIntervertebral disc (IVD) degeneration and related back pain are common clinical problems in developed countries.Current treatments are limited to surgical removal of degenerative IVDs and other palliative measures aimed toward alleviation of pain. Biologic treatments directed toward restoring IVD structure and function are currently unavailable. The hallmark of the degenerative discs is loss of extracellular matrix (ECM) organization, leading to loss of water-binding capacity and diminished ability to dissipate loads within the spine. Further understanding of the molecular biology and immunology and pathophysiology and biomechanics of IVD degeneration is necessary to develop such treatments.Fibronectin (FN), a multifunctional glycoprotein found in the ECM and plasma, the molecular shape like "V", is a key factor in organizing the ECM. It is expressed by multiple cell types and plays important roles in cell adhesion and migration, cell differentiation, and cell function.FN is encoded by a single gene.However, alternative splicing produces multiple FN per-mRNAs, and as a result, up to20different isoforms and variants exist.ADAMs (A disintegrin and metalloproteinase) belong to the metzincin family of enzymes, which are dependent onzinc for catalytic function. Of20encoded human genes, approximately half of these proteins are predicted to contain the histidine triad motif known toco-ordinate zinc that is necessary for proteolytic activity. ADAM play vital roles in regulating the hydrolysis of extracellular matrix and interaction of cell-cell and cell-matrix. The ADAM family relate to membrane fusion (cell adhesion and cell fusion), shedding of cytokines and growth factors, the control of cell migration, some process of muscle development, and fertilization and cell fate decision. Correlational research of pathology suggestion the inflammation and cancer is also affected by this family.The roles of FN and its fragments in cartilage have been studied extensively. Homand berg et al first described presence and effects of FN fragments (FN-f) in articular cartilage. In contrast, the only published description suggesting a relationship between FN and disc degeneration is by Oegema et al. Recently, The VRAA neoepitope antibody recognizes the C-terminus of the N-terminal fibronectin fragment resulting from cleavage by ADAM-8,result in FN loss the biological role and function, then cartilage degeneration process is exacerbated. Because IVD tissues share similarities in terms of cellular and ECM properties with cartilage, Therefore, the goals of the present study were to provide firm evidence from related experiment of human intervertebral disc and ADAM-8transgenic mice for relationship between the ADAM-8and FN in intervertebral disc degeneration of the occurrence and development. Above all, providing a new theory and method for the intervertebral disc degeneration treatment. Chapter one:The effect of ADAM-8play the role of FN in the human intervertebral disc tissues.ObjectTo determine the effects of ADAM-8overexpression and gene silencing on fibronectin fragmentation in Samples of intervertebral disc with surgery patients and organ donors in vitro.Methods1.Patient selection and sample collection. IVD tissues were collected from patients undergoing spinal surgery and from deceased organ donors. The disc and articular cartilage (AC) tissues were rinsed in1XPBS containing protease inhibitors (Roche) as described before9, and stored at-70℃.2.Quantitative Western blots.Identified of full length FN, FN-fs and the existence and content in human intervertebral disc degeneration of different grade.3.Quanlitative Western blots and Immunostaining for identified of ADAM-8existence and distribution in the human intervertebral disc.Results1.The fibronectin (FN) antibody (to the neoepitope VRAA)and the N-terminal antibody (MAB1936) specifically recognize full-length FN and its fragments (FN-fs) in human disc tissues.2.The content of fibronectin N-terminal fragments increases as human disc degeneration progresses.3.ADAM-8is present in the both the human adult NP and AF tissues by Western blot and immunostaining. We also find that ADAM-8generated the VRAA neoepitope in IVD tissues.4.The content of mature ADAM-8increases with higher degrees of human intervertebral disc degeneration.Conclusion1.This is the first report demonstrating that a fragment containing the VRAA neoepitope, previously described in degenerative articular cartilage, is present in both the NP and AF tissues.2.The content of fibronectin N-terminal fragments increases as human disc degeneration progresses.3.We have further identified ADAM-8in both the degenerative and normal appearing human cadaveric NP and AF tissues, and it has been shown to generate the VRAA neoepitope in degenerative and normal discs.The presence of ADAM-8has not been previously described in the human IVD tissues.4.The content of mature ADAM-8increases with higher degrees of human intervertebral disc degeneration,Similar to the content of fibronectin N-terminal fragments increases as human disc degeneration progresses.Suggestion that ADAM-8and FN-fs played the critical role of intervertebral disc degeneration. Chapter two:Observation of the ADAM-8play the role of influence of fibronectin about two type transgenic mice in vivoObjectTo determine the effects of ADAM-8gene inactivation on fibronectin N-terminal fragmentation in transgenic mice in vivo.Methods1.Histology for ADAM-8two type transgenic mice, At a fixed point in time collect KO and WT two genotypes of lumbar samples,which in effect to relationship between FN-f and ADAM-8in intervertebral disc degeneration.2.Qualitative Western blot test for ADAM-8two type transgenic mice, which in effect to relationship between FN-f and ADAM-8in intervertebral disc degeneration.Result1.Compare to histological observation for two type ADAM-8transgenic mice, the overexpression type(WT) intervertebral disc degeneration degree higher than silencing type(KO).2.Western blot test has not been shown to generate the VRAA neoepitope in ADAM-8KO type transgenic mice, but WT type represent the neoepitope.Conclusion Compare to histological observation and Western blot test for two type ADAM-8transgenic mice,indication that ADAM-8can be generated by a specific site cleaved fibronectin FN-s, then ADAM-8was the important reason result in the intervertebral disc degeneration.