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Trauma Tissue: Ligaments or soft tissues

Molecular Biology of Human ACL Ligament Injury and repairing KL Paul Sung ( 宋国立 ) Department of Orthopaedics (SOM) Department of Bioengineering (SOE) University of California at San Diego (UCSD) College of Bioengineering, Chongqing University CQU Lecture on Nov/10/2016. Trauma Tissue:

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Trauma Tissue: Ligaments or soft tissues

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  1. Molecular Biology of Human ACL Ligament Injury and repairingKL Paul Sung (宋国立)Department of Orthopaedics (SOM) Department of Bioengineering (SOE) University of California at San Diego (UCSD)College of Bioengineering, Chongqing UniversityCQU Lecture on Nov/10/2016

  2. Trauma Tissue: Ligaments or soft tissues *

  3. Tissue Healing (1) (analogous to many soft tissues healing) Inflammatory phase: Release of mediators and growth factors stimulate leukocytes to remove damaged tissue and fibroblasts migrate into injury site. These mediators can recruit potential stem cells and inflammatory cells to produce catabolic cytokines such as matrix metalloproteinases (MMPs) for the tissue remodeling.

  4. Tissue Healing (2) (analogous to many soft tissues healing) • Hyper-cellular (Granulation) phase: Fibroblast synthesis (growth factors) of collagen type III into a disorganized extracellular scar matrix with neovasculature (hypoxia). • Hypo-cellular (Scar tissue remodeling) phase: Collagen III remodeling into organized collagen I matrix by catabolic (MMP) and anabolic (lysyl oxidase) enzymes and increase in vascularity and re-innervations of scar tissue.

  5. Extracellular Events in Tissue remodeling • Normal physical properties of wound are lost due to structural and functional deficiency of scar tissue. • Collagen crosslinking (Lysyl Oxidase), remodeling, and degradation which occur after 2-3 weeks of wound injury are important in developing wound strength. • Scar tissue remodeling involves a balance between collagen synthesis and degradation by tissue collagenases (Matrix Metalloproteinases) in normal tissue healing.

  6. What is MMP?

  7. MATRIX METALLOPROTEINASES (MMPs) • Family of endo-proteinases • Produced by most cell types • Degrade extracellular matrix(ECM) proteins (collagen, fibronectin, laminin etc.)

  8. Generalized domain structure of MMP • MMPs contain the same basic structural features despite the observed variability in substrate specificity. • The two zinc (II) ions are located in the catalytic domain.

  9. MMPs: 5 families with 28 members Collagenases: MMP-1 Gelatinases: MMP-2 Stromelysin: MMP-3 Membrane type MMPs: MT-1-MMP Others: matrilysin

  10. Role of MMPs in Disease • MMPs is correlated with a number of disease states: • Arthritis, cardiovascular disease, stroke, atherosclerosis and tumor genesis, growth, and metastasis • Congestive heart failure has been detected in overproduce MMP-1. • Collagenases level increasing in synovial fluid of arthritis patients. • MMP-7 deficient mice demonstrated a 60% reduction in tumor multiplicity and substantial decrease in tumor size. • Lacking MMP-2 results in a decrease in tumor volume of 39% and 24% implantation of melanoma and Lewis lung carcinoma cells in mice, respectively.

  11. Case of Orthopaedic studies • ACL healing problem: It is a “cancer” like problem in orthopaedic clinic. • ACL: A non-healing tissue for more than 6 decades and thousands scientists have failed to heal raptured ACL. • ACL reconstruction has been introduced.

  12. PCL ACL 目前的 ACL 重建

  13. Summary (US and European) • Overall complication rate is 21.6% in pain/unstability • 11% need re-surgery for restricted range of motion • 65% have much higher rate unpredictable re-injury episodes • 60% of developing arthritis Knee surgery is the #1 surgery in the US Are patients’ or/and doctors’ choice? Here is my motivation

  14. Last 8 years: Focusing on injured ACL (9000 samples, 1000 gels: healing Cocktail development) (Human: C, 2, 10, 30, 90 days; Rats: 1500) Last 4 years: Focusing on MMP & LOX (3rd generation healing cocktail development)

  15. MMP-2 amount in patient knee fluids Blood Day 1 Day 2 Day 3 Control MMP-2 Pro- Active 4 3 2 Ratio to Control 1 0 Blood Normal Day 1 Day 2 Day 3 (10 times diluted)

  16. M IK IK C C 213 120 Pro - MMP-9 Pro - MMP-9 76 Active Pro - MMP-2 Pro - MMP-2 Active 47 10 X Hurdle factors of ACL healing (Zymographic analysis) MMP-2/-9 Activity in Injured ACL Knee Fluids: Human (left) and Rat Model*(right) *Lee and Sung et at., ORS 2004

  17. Methodology Zymograph Studies of Injured ACL (cellular & molecular) • Inhibitors: MMPpi/MMPi • Inflammatory Factors • Growth Factors • Mechanical Injury

  18. Cellular injury: Mechanical stretch • homogeneous strain Homogeneous strain Quantitative strain homogeneous strain

  19. In Vitro Equi-biaxial Stretch Apparatus Allows homogeneous strain distribution over membrane F F Indenter Culture Well Medium Fibroblasts Silicone Membrane Rubber O-ring X-Sectional View

  20. Zymography: Up-regulated MMP-2 in ACL/MCL After Injurious Stretching ( 6, 12 & 14%) ACL % Stretch MW 6 12 14 6 12 14 6 12 14 MCL 16 20 24 Collection Time (hr)

  21. Inflammatory Factor: IL-1a Restores JNK-inhibited MMP-2 Activity MMP Inhibition Responses: ACL Mechanical Stretch Injury Injury can generate multi-signals Inflammation restored MMP-2 level

  22. Rat Fractured ACL repaired under different conditions ( 2nd Generation Cocktails) ACL Rupture Suture (10-0 or 9-0) Inhibitor RxA,RxT AP-1 † Fracture Strength (N) Mean ± SD EGF † + + – – 6.3 ± 2.1 + + + – 15.8 ± 9.1* + + + + 27.1 ± 2.8 + – – – ACL disappeared * 5 and 6 weeks grouped together; Control: 35 ± 4.7 N

  23. MMPi, MMPPi, & Growth Factor Cocktails (3rd generation)

  24. Third generation healing cocktails

  25. The 3rd generation ACL healing cocktail • In in vivo (rat): reaches to 37 N grapping force in healed injured-leg • In ex vivo (Bone-ACL-Bone): 92% resumed the ACL strength (27 N) which still lacks of 8% Satisfy? yes or no

  26. Photo of Experiment With only Suture (Too weak, easily ruptured 6N) Suture + Inhibitors (ACL thin and weak 15N) Suture + Inhibitors + Growth Factors (Similar to original ACL 27N) No Treatment (ACL disappeared)

  27. The 4th generation of cocktail • Cocktail array to get cocktails of cocktail (double cocktail) which allow us to search primary signaling instead secondary, tertiary or quaternary. • Cocktail array will include anabolic enzymes (lysyl oxidase). • To rebuild ACL tensile strength to 100 percent or more.

  28. Lysyl Oxidase

  29. Introduction • Lysyl oxidase (LOX) is the copper and quinone-containing enzyme amine oxidase that plays a critical role in the biogenesis of connective tissue matrices by cross linking the extracellular matrix proteins, e.g., collagen and elastin. • The formation of covalent cross-links in collagen and elastin is essential to normal embryonic development, connective tissue function, and wound healing.

  30. LOX Catalytic Mechanism LOX deaminates the ε-amino group of lysines of collagen or elastin. The resulting aldehydes (AAS) condense to form cross-linkages between monomers of collagen or elastin.

  31. The AAS can condense with a neighboring ε-amino group of an unmodified lysine to form the anhydro-lysinor-leucine (deLNL) crosslink. Similarly, two of these AAS residues can react to form the aldol condensation product (ACP).

  32. LOX Family Members on chromosome 5q23 [Hamalainen, Genomics,1991] LOX LOXL1 on chromosome 15q23 [Kim, Bio-Chemisty,1995] Gene mapping LOXL2 on chromosome 8p21 [Jourdan-Le, Bio-Chemisty1996] LOXLs LOXL3 on chromosome 2p13 [Jourdan-Le, Genomics ,2001] on chromosome 10q24 [L. Asuncion, MatrixBiology,2001] LOXL4

  33. Following intracellular signal peptide cleavage, LOX and LOXL1 are secreted as proproteins which are proteolytically cleaved to release the C-terminal free catalysts and the N-terminal propeptide regions. • The regions immediately following the signal peptide sequences of LOXL2, LOXL3 and LOXL4 each contain four scavenger receptor cysteine-rich domains (SRCR domain). In contrast, the propeptides of LOX and LOXL1 do not contain cysteine residues.

  34. LOX and disease Abnormal expression of LOX can induce numerous disease, such as: • Scleroderma • Alzheime‘s disease • Amyotrophic lateral sclerosis • Menkes disease • Wilson disease • Cancer

  35. Comparisons of LOX and LOXL-1 gene expressions after 6% and 12% stretch in ACL and MCL (6%) (12%)

  36. Comparisons of LOX-2 and -3 gene expressions after 6% and 12% stretch in ACL and MCL (6%) (12%)

  37. Comparisons of LOX-4 gene expressions after 6% and 12% stretch in ACL and MCL (6%) (12%)

  38. The LOXs proteins showed the time-dependent increase after injury Control 12h 24h 48h 72h Figure 2. Protein expressions of LOX family in ACL. After 16 hours starvation in 2% FBS, the medium were switched to 1% FBS and the cells in chambers were stretched to 12%. Medium samples were collected at 12, 24, 48, 72h. Samples were subjected to western blot. Our results showed the time-dependent increase of LOXs in ACL and MCL, furthermore LOXs in MCL had higher expressions than in ACL.

  39. The 4th generation of cocktail • Cocktail array to get cocktails of cocktail (double cocktail) which allow us to search primary signaling instead secondary, tertiary or quaternary. • Cocktail array will include anabolic enzymes (lysyl oxidase). • To rebuild ACL tensile strength to 100 percent or more.

  40. Micro-environomse Bio-informatics Systems Biology

  41. Activation of the receptor (R (GPCR)) • Activation of G-protein (free G ) L: ligand • G binds to PLC • Generation of IP3 • Inactivation • GRK binds to G  • active receptor is phosphorylated and internalized T: GTP, D: GDP, G: G-protein Mechanism of Ligand-Induced IP3 Generation • Activation

  42. There is tremendous need for computational models and tools able to distill specific pathways of interest from large molecular interaction databases. • We are screening the current network of 20,000 known yeast interactions in several ways, including: • Gene expression • Single deletion phenotypes (e.g., sensitivity to a condition) • Comparison of two networks

  43. Begley et al., Mol Cancer Res, (2002)

  44. Ligament Homeostasis Micro-environomse in injured ligament Anabolic Enzymes: LOXs Catabolic Enzymes: MMPs Inflammatory agents Mechanical Stimuli - ? + + + ? Growth Factors Tissue Synthesis Tissue Remodeling + + - Hypoxia

  45. ACL Tissue Engineering in UCSD: 5 years (1997-2002) 10 people

  46. Regenerative Medicine in XiangShan Sciences Meeting (2005) Theme of XiangShan meeting Challenge: Non-healing ACL Opposite Idea: Tissue Engineering Unusual Concept: Healing Cocktail

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