Molecular and Cellular Regulation of Wound Healing. Gregory Schultz, Ph.D.

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1 What Goes Wrong When Wounds Fail to Heal or Heal Too Much? rof. 1 rofessor of Obstetrics/Gynecology Institute for Wound Research University of Florida 2 Overview of topics Consider wound healing as a spectrum of outcomes: normal scars, fibrotic scars, or chronic wounds Review sequential phases of normal wound healing and recognize the beneficialeffects of controlledinflammation and protease activities Understand the detrimentaleffects on healing of chronic inflammation caused by planktonicand biofilmbacteria, which leads to elevated MM activity in wounds that destroy proteins that are essential to healing (ECM, GFs, receptors) Learn about the key roles that TGFβand CTGF play in stimulating excessive scar formation (fibrosis) and how to reduce pathological scar formation Think of wound healing as a spectrum of clinical outcomes Inadequate healing (Chronic) Normal healing (Repair) Excessive healing (Fibrosis) 3 Venous leg ulcer Good skin scar Hypertrophic scar 1

2 Sequence of molecular and cellular events in skin wound healing Four phases of healing 1. Hemostasis 2. Inflammation 3. Repair 4. Remodeling 1. Clotting 2. Vascular response 3. Inflammation 4. Scar formation 5. Epithelial healing 6. Contraction 7. Scar remodeling 4 Hemostasis Fibrin clot & platelets 5 Vascular response, blood clotting, and platelet release of growth factors DGF EGF VEGF Aggregated platelets FGF TGFβ Fibrin net RBC 6 Key points 1. Fibrin clot forms a provisional wound matrix that promotes coagulation and migration of fibroblasts, vascular endothelial cells 2. latelets release growth factors that initiate healing by stimulating chemotaxis, proliferation, and matrix synthesis 2

3 Sequential phases of cytokine and growth factor expression Day 0 to 1 Days 1 to 5 Days 5 to 21 latelets Neutrophils Macrophages Wound tissue cells 7 8 Major families of growth factors Growth factor family Cell source Actions Transforming Growth Factor β TGF-β1 TGF-β2 TGF-β3 latelet Derived Growth Factor DGF-AA, DGF-BB VEGF Fibroblast Growth Factor Acidic FGF, Basic FGF KGF Insulin-like Growth Factor IGF-I, IGF-II Insulin Epidermal Growth Factor EGF, HB-EGF, TGF-α, Amphiregulin, Betacellulin Connective Tissue Growth Factor CTGF latelets Fibroblasts Macrophages latelets Macrophages Keratinocytes Fibroblasts Macrophages Endothelial cells Fibroblasts Liver Skeletal muscle Fibroblasts Macrophages Neutrophils Keratinocytes Macrophages Fibroblasts Endothelial cells Epithelial cells Chemotatic for fibroblast romotes extracellular matrix formation Collagen and TIM synthesis MM synthesis Reduces scarring Collagen Fibronectin Activates immune cells and fibroblasts romotes ECM formation Collagen and TIM synthesis MM synthesis Angiogenesis Angiogenesis Keratinocyte proliferation and migration ECM deposition Keratinocyte & fibroblast proliferation Angiogenesis Collagen synthesis ECM formation Cell metabolism Keratinocyte proliferation and migration ECM formation Collagen synthesis Mediates action of TGF-β on collagen synthesis Inflammation Cytokines, neutrophils, macrophages, proteases, and Reactive Oxygen Species 9 3

4 Controlled wound inflammation is beneficial Neutrophil Macrophage O 2 - H 2 O 2 HOCl MMs Elastase 10 Inflammatory cells kill microorganisms and release proteases (MMs, elastase) that remove denatured ECM components and permit wound healing to proceed. Wounds that are contaminated by bacteria and fungus must not be closed Respiratory burst in neutrophils & macrophages produces Reactive Oxygen Species (ROS) that kill bacterial & fungi In the membranes of neutrophils, NADH oxidase generates superoxide (O 2- ), which spontaneously dismutates to H 2O 2, and is converted to hypochlorous acid (HOCl) by myeloperoxidase (MO). These reactive oxygen species (ROS), especially HOCl, participate in the killing of bacteria. The right panels show a bacteria being phagocytized and production of ROS (red color) surrounding the yeast cell 11 Major cytokines involved in wound healing Cytokine Cell source Biological activity ro-inflammatory cytokines Tumor Necrosis Factor (TNF-α) Interleukin-1 (IL-1) Interleukin-6 (IL-6) Interleukin-8 (IL-8) Interferon-γ(INF-γ) Anti-inflammatory cytokines Interleukin-4 (IL-4) 12 Interleukin-10 (IL-10) macrophages macrophages, keratinocytes macrophages, keratinocytes, MNs macrophages, fibroblasts macrophages, T- lymphocytes T-lymphocytes, basophils, mast cells T-lymphocytes, macrophages, keratinocytes MN margination and cytotoxicity MM synthesis fibroblast and keratinocyte chemotaxis, MM synthesis fibroblast proliferation macrophage and MN chemotaxis collagen synthesis macrophage and MN activation collagen synthesis MM synthesis TNF-α, IL-1, IL-6 synthesis fibroblast proliferation, collagen synthesis TNF-α, IL-1, IL-6 synthesis macrophage and MN activation 4

5 Repair phase Conversion of the provisional wound matrix into initial scar tissue 13 rovisional wound matrix is replaced by initial scar tissue rovisional wound matrix (fibrin clot) is replaced by new collagen, elastin, proteoglycans and glycoproteins synthesized by fibroblasts that migrate into the wound. TGFβand CTGFare dominant growth factors that stimulate scar formation. 14 Angiogenesis is stimulated by hypoxia-induced VEGF and controlled MM activity A B C D E F Tissue injury (A) causes hypoxia -induces Hypoxia Inducible Factor (HIF)-stimulates release of angiogenic growth factors like VEGF -induces MMs -erode holes in the basement membrane (B) surrounding capillaries. Vascular endothelial cells proliferate & migrate to ischemic area (C)- creating new capillary arcs (D, E, F). 15 5

6 Contraction of wounds by myofibroblasts (A) Full thickness excision for melanoma on thigh cannot be closed by surgical means because of its extent and depth. It heals by secondary intention and closes mainly by the formation of new tissue and contraction (skin grafting was not performed at patient s request). (B) After 10 days, granulation tissue can be seen in wound bed. (C) On the 21 st day, the size of the wound bed has markedly decreased through contraction. (D) After 2 years the wound is closed and epithelialized. Tension lines caused by contraction are running towards the center of the wound. MMs secreted by myofibroblasts are required for matrix contraction 16 D Epithelial healing of deep skin wounds Moist healing Dry healing 17 Epithelialization of deep partial thickness or full thickness wounds occurs predominately from the edge of the wound by proliferation and migration of epithelial cells. Epithelialization is more rapid under moist conditions (left) than dry conditions (right) because migrating epithelial cells must penetrate under scab and desiccated matrix Remodeling phase Slow removal of initial irregular scar tissue by proteases and replacement with extracellular matrix that is has more normal structure and composition 18 6

7 Remodeling phase (2) 19 Controlled MMS are necessary for wound healing Debridement, angiogenesis, contraction, epithelial migration, remodeling MMS are necessary for several key process in wound healing 1. Removing denatured matrix 2. Degrading capillary basement membrane for angiogenesis 3. Contraction of ECM by myofibroblasts 4. Migration of epidermal cells 5. Remodeling of scar Is there a common molecular pathology of chronic wounds?? Diabetic foot ulcer Arterial ulcer 21 ressure ulcer Venous ulcer 7

8 Hypothesis of chronic wound pathophysiology Repeated injury, ischemia and planktonic and biofilm bacteria TNF-α IL-1β, IL-6 rolonged, elevated inflammation neutrophils macrophages mast cells Imbalanced proteases & inhibitors roteases (MMs, elastase, plasmin) and inhibitors (TIMs, α1i) Destruction of essential proteins growth factors / receptors, ECM degradation cell migration, cell proliferation 22 Chronic non-healing wound B.A. Mast and G.S. Schultz. Wound Rep Reg 4: , 1996 Imbalanced molecular environments of healing and chronic wounds Healing wounds: Low levels of planktonic bacteria Low inflammatory cytokines Low proteases, ROS, RNS Intact functional matrix High mitogenic activity Mitotically competent cells Chronic wounds: High levels of bacteria biofilm, MRSA High inflammatory cytokines High proteases, altered nitric oxide Degraded nonfunctional matrix Low mitogenic activity Senescent cells 23 B.A. Mast and G.S. Schultz. Wound Rep Reg4: , 1996 High levels of MM activity in chronic wounds decrease as wounds heal 24 Trengove, Stacey, Macauley, Bennett, Gibson, Burslem, Murphy, Schultz. Wound Rep Reg 7: ,

9 25 Low protease activity in chronic wound fluids of pressure ulcers predicts the rate and extent of healing Ratio of MM-9 (pro+active):tim-1 (ng/ml) Good healing >95% area healed; n=12 Intermediate healing <95% & >65% area healed; n=36 oor healing < 65% area healed; n=8 Day-0 Day-10 Day-36 Time course Ladwig, Robson, Liu, Kuhn, Muir, Schultz. Ratios of activated matrix metalloproteinase-9 to tissue inhibitor of matrix metalloproteinase-1 in wound fluids are inversely correlated with healing of pressure ulcers, Wound Repair Reg 10: 26-37, 2002 MM levels in healthy skin versusvenous ulcer tissues before and after compression therapy pg/µg total protein Healthy skin VU Before Tx VU After Tx N= S.K. Beidler, et al., Multiplexed analysis of matrix metalloproteinases in leg ulcer tissue of patients with chronic venous insufficiency before and after compression therapy, Wound Rep Regen 2008 Inverse correlation between percent wound closure and elevated MM-1 levels in venous ulcers after compression therapy % wound closure at 4 wks MM1 level after 4 weeks of compression (pg/µg) S.K. Beidler, et al.,multiplexed analysis of matrix metalloproteinases in leg ulcer tissue of patients with chronic venous insufficiency before and after compression therapy, Wound Rep Regen

10 Fibronectin is rapidly degraded by chronic wound fluids Fibronectin is absent in the base of chronic venous ulcers Fibronectin reappears in ulcer base during healing lasma Venous ulcer 1 Venous ulcer 2 Diabetic ulcer 1 Diabetic ulcer 2 Mastectomy fluid 1 Mastectomy fluid 2 lasma + CWF 1 lasma + CWF 2 Fibronectin is degraded in non-healing ulcer Fibronectin reappears (stable) as ulcer heals 28 Fibronectinprofile in plasma shows a single intact band at 250 kda. In contrast, fibronectinis degraded to lower molecular weight fragments in venous stasis ulcers and in diabetic ulcers Wysockiand Grinnell, Lab Invest. 63: 825, 1990 Herrick, Sloan, McGurk, Freak, McCollum and Ferguson. Am J athol. 141, 1992 Fibronectin promotes cell attachment, spreading, and migration No fibronectin With fibronectin A B 29 (A) Fibroblasts cultured on glass do not spread. C (B) In contrast, the fibroblasts cultured on fibronectin coated slides attach and spread quickly (within 1 hour) (C) Addition of fibronectin(fn) promotes migration of vascular endothelial cells following scrape wound in vitro DGF-AA immunostaining Low in normal skin High in acute healing wound Low in chronic wound, and higher in healing chronic wound 30 ierce et al., J ClinInvest 96: ,

11 TGFβtype-II receptor is decreased in chronic venous ulcers but increases with healing TGFβ1 e u u M N O e u u TGFβ-IIR Q R S Normal skin Ulcer edge Non-healing ulcer base Healing ulcer base A.J. Cowin, N. Hatzirodos, C.A. Holding, V. Dunaiski, R.H. Harries, T.E. Rayner, R. Fitridge, R.D. Cooter, G.S. Schultz and D.A. Belford. Effect of Healing on the Expression of Transforming Growth Factor-βs 31 and Their Receptors in Chronic Venous Leg Ulcers. J Invest Dermatol 117: , 2001 T If chronic wounds fail to heal because of hostile molecular environment, can it be corrected? Yes, by applying the principles of wound bed preparation 32 Wound bed preparation and T I M E Tissue, Inflammation/Infection, Moisture, Edge Tissue debridement Epithelial edge Surgical Infection/Inflammation Biofilms Moisture balance 33 G. Schultz et al.,wound Rep Reg11: S1-S28,

12 New debridement techniques 34 Medical honey Medical larvae Anionic polymers Ultrasonic debridement Better understanding of infection/inflammation Roles of biofilms DNA-based identification of bacteria Diagnostics for proteases rotease inhibiting dressings Better moisture control NWT + Instillation with microbicidal solutions - impacted I and M Advanced super absorbent polymers Better agents to stimulate epithelial cell proliferation and migration Amniotic membranes Dermal matrix dressings Major developments in last 10 years athological scarring - fibrosis 35 Kidney Glomerulosclerosis Signaling pathway in scar formation Tissue injury TGF-β1 Auto-induction TGF-β1 sirna, ASO Binds TGF-β receptor 2 TGF-βR2 sirna Induces CTGF CTGF sirna, ASO 36 Irregular extracellular matrix (collagen synthesis) Neovascularization Scar tissue Differentiation of fibroblast into myofibroblast (α-sma contraction) 12

13 Second generation antisense oligonucleotides Highly evolved 2 MOE Antisense Oligos (ASO) drugs RNase H mrna fragments 37 Antisense oligonucleotide targeting CTGF reduces scarring in rabbit ear wound model Control untreated CTGF ASO treated Conclusions: The CTGF ASO demonstrated a significant reduction in skin scarring compared to saline control M. Sisco, Z.B. Kryger, K.D. O Shaughnessy,.S. Kim, G.S. Schultz, X.Z. Ding, N.K. Roy, N.M. Dean, T.A. Mustoe. Antisense inhibition of connective tissue growth factor (CTGF/CCN2) mrna limits hypertrophic scarring without affecting wound healing in vivo, Wound Rep Reg, 16: , Hypertrophic scar in skin 39 13

14 hase I/IIa/Ilb/llc clinical trials in skin Abdominoplasty hypertrophic scar revision Average skin excision during abdominoplasty: 40 X 12 cm ellipse 32 patients received injection of ASO to one end of incision and scrambled ASO to other end separated by 2 cm in all directions atient returns 12 and 24 8 weeks later for assessment of scar Revision of hypertrophic scar following breast surgery implants or reduction one end (side) injected with ASO the other injected with scrambled ASO evaluated at 24 & 24 weeks post surgery 40 Analysis of incision: Histological Molecular Wound strength ASO or vehicle treatment of incision Untreated Treated 41 Excaliard harmaceuticals, Inc. hysician responses on scar appearance Overall Surface area liability Relief Thickness EXC 001 No difference lacebo igmentation Vascularity 42 Excaliard harmaceuticals, Inc # of physician scores favoring 14

15 43 Summary Healing of acute healing wounds proceeds through four major sequential phases (hemostasis, inflammation, repair, and remodeling) Chronic wounds frequently have bacterial biofilms that cause elevated levels of pro-inflammatory cytokines, leading to chronic inflammation Elevated proteases from inflammatory cells destroy essential growth factors, receptors, and ECM proteins, which impairs healing Fibrosis(excessive healing) involves elevated levels of TGFβ and CTGFthat increase synthesis of ECM molecules (collagen) and decreases proteases Reduction of CTGF gene by ASO decreases scarring 44 15