MSc/MRes Human Tissue Repair PDF

MSc/MRes Human Tissue Repair Tissue Repair and Regeneration (MEDC0064-TRAR) Human skin wound healing - tissue repair and principles of regeneration Prof Krista Rombouts, PhD UCL Institute for Liver and Digestive Health Regenerative Medicine & Fibrosis Group...

MSc/MRes Human Tissue Repair Tissue Repair and Regeneration (MEDC0064-TRAR) Human skin wound healing - tissue repair and principles of regeneration Prof Krista Rombouts, PhD UCL Institute for Liver and Digestive Health Regenerative Medicine & Fibrosis Group Royal Free Hospital, London, UK [email protected] Learning objectives:  Fundamental information to understand injury, cellular and vascular response  Sequence in wound healing  Definitions of Regeneration and Repair  Liver and regeneration Examples of skin damage Sun burn / UV Itchy wounds (bacterial / nutrition….) Burning wounds Itchy wounds due to chronic autoimmune diseases such as Psoriasis cancer In healing wounds, exudate supports healing and provides a moist wound environment. The main role of the exudate is facilitating the diffusion of vital healing factors (eg growth-and immune factors) and the migration of cells across the wound bed. The exudate also promotes cell proliferation, provides nutrients for cell metabolism, and aids autolysis of necrotic or damaged tissue. Steps in repair by scar tissue formation 1. Inflammatory response by polymorphs and macrophages and removal of damaged and dead tissue. 2. Proliferation and migration of parenchymal and connective tissue cells. 3. Formation of new blood vessels (angiogenesis) and granulation tissue. https://en.wikipedia.org/wiki/Wound_healing  Wound Healing Exhibits a Defined Sequence The initial phase of the repair reaction: typically begins with haemorrhage into the tissues. 1. A fibrin clot forms and fills the gap created by the wound. Fibronectin in the extravasated plasma is cross- linked to fibrin, collagen, and other Extracellular Matrix (ECM) components. This cross-linking provides a provisional mechanical stabilization of the wound (0–4 hours). 2. Macrophages recruited to the wound area and process cell remnants and damaged ECM. The binding of fibronectin to cell membranes, collagens, proteoglycans, DNA, and bacteria facilitates phagocytosis by these macrophages and contributes to the removal of debris (1–3 days). 3. Fibronectin, cell debris, and bacterial products are chemoattractants for a variety of cells that are recruited to the wound site (2–4 days). The intermediate phase of the repair reaction. Repair, Regeneration, and Fibrosis. Gregory C. Sephel and Stephen C. Woodward (Moodle)  Wound Healing Exhibits a Defined Sequence 4) As a new ECM is deposited at the wound site, the initial fibrin clot is lysed by a combination of extracellular proteolytic enzymes and phagocytosis (2–4 days). (5) Concurrent with fibrin removal, there is deposition of a temporary matrix formed by proteoglycans, glycoproteins, and type III collagen (2–5 days). (6) Final phase of the repair reaction. Eventually the temporary matrix is removed by a combination of extracellular and intracellular digestion, and the definitive matrix, rich in type I collagen, is deposited (5 days–weeks). The following are characteristic of skin wounds: Sequence of different cell types Cell migration uses the most important mechanism of wound healing, namely the response of cells to chemical signals (cytokines and chemokines) and insoluble substrates of the extracellular matrix. Leukocytes arrive at the wound site early and migrate rapidly by forming small focal adhesions or focal contacts. A family of small peptide chemoattractants (chemokines and cytokines), are capable of restricted or broad recruitment of particular leukocytes. Polymorphonuclear leukocytes are rapidly recruited from the bone marrow and invade the wound site within the first day. They degrade and destroy non-viable tissue by releasing their granular contents. Macrophages arrive shortly after neutrophils but persist for days or longer. They phagocytose debris and orchestrate the developing granulation tissue by the release of cytokines and chemoattractants.  The following are characteristic of skin wounds: Sequence of different cell types Fibroblasts, myofibroblasts, pericytes, and smooth muscle cells are recruited by growth factors and matrix degradation products, arriving in a skin wound by day 3 or 4. These cells are responsible for fibroplasia, synthesis of connective tissue matrix, tissue remodelling, wound contraction, and wound strength. Endothelial cells form nascent capillaries by responding to growth factors and are visible in a skin wound beyond day 3. The development of capillaries is necessary for the exchange of gases, the delivery of nutrients, and the influx of inflammatory cells. Epithelial cells in the epidermis move across the surface of a skin wound, penetrate the provisional matrix and migrate upon stromal collagen, which is coated with plasma glycoproteins, fibrinogen, and fibronectin. Stem cells from bone marrow, the bulb of the hair follicle, and the basal epidermal layer, provide a renewable source of epidermal and dermal cells capable of differentiation, proliferation, and migration. Under appropriate conditions, these cells form new blood vessels and new epithelium and regenerate skin structures, such as hair follicles and glands.  Pro-inflammatory and anti-inflammatory cells Neutrophils and mast cells: first defense and inflammatory responses. Short-lived neutrophils are the first immune cells to arrive on the scene after injury and are crucial in host defense and wound detection, which is rapidly executed to restore barrier integrity and tissue homeostasis. Mast cells are an additional early participant of the innate immune system’s first defense response, secreting a heterogeneous array of effector molecules, including chemoattractants, to recruit eosinophils and monocytes. Macrophages: paracrine mediators of tissue regeneration. Macrophages are abundant during all stages of tissue repair and have an important influence on the progress and resolution of tissue damage. Macrophages have been categorized conventionally into pro-inflammatory M1 and tissue- repairing M2 phenotypes and exist in a spectrum of states that depend on their environment. Although the full spectrum of macrophages activated by tissue damage has not been fully characterized, ‘classically activated’ macrophages have pivotal roles in fighting pathogens. After the acute phase of tissue injury, there’s a switch in the predominant macrophage phenotype to the ‘non-classical’or wound-healing phenotype. Regulatory T cells: ensuring more than tolerance. Among the many cell types involved in adaptive immune responses in regeneration, including both T and B lymphocytes, regulatory T (Treg) cells have emerged as key players in the response to tissue injury. Preparing the ground for tissue regeneration: from mechanism to therapy. Stuart J Forbes, N Rosenthal. Nature Medicine: 20, 857–869, 2014  Chemokines in early and late phases of wound healing (a) Early wound healing, including clot formation, inflammation, and proliferation. (1) Clot formation occurs to prevent the loss of blood and (2) platelets are activated and release (3) α-granules, which in turn release (4) CXCL4 as an early inhibitor of angiogenesis. Once the clot has fully formed other chemokines such as CXCL8, CXCL1, and CXCL2 are released by α-granules to recruit inflammatory cells, including (5) neutrophils and (6) macrophages. Neutrophils are increased early in the healing process, then macrophages soon take over as the primary inflammatory cell. Neutrophils and macrophages release (7) chemokines such as CCL2, CCL3, and CCL5 into the wound to promote the recruitment of more inflammatory cells that release pro-angiogenic growth factors that (8) increase neovessel formation (neo-angiogenesis) in the wound. (↓: indicates decrease;↑: indicates decrease)  Chemokines in early and late phases of wound healing (b) Late wound healing is the remodeling stage. In this stage, the wound is fully healed and (1) a scar has formed. Type 3 collagen converts to (2) type 1 collagen to promote scar formation and create a more stable wound seal. During the remodeling process (3), angiostatic chemokines (CXCL10, CXCL11) promote the (4) regression of neovessels, as there is no longer a requirement for enhanced blood flow or the recruitment of immunological cells to the wound site. (↓: indicates decrease;↑: indicates decrease) The Role of Chemokines in Wound Healing. Ridiandries A et al., Int. J. Mol. Sci. 2018, 19(10), 3217 Learning objectives:  Fundamental information to understand injury, cellular and vascular response  Sequence in wound healing  Definitions of Regeneration and Repair  Liver and regeneration Regeneration and Repair: definitions Please watch this video: https://www.youtube.com/ watch?v=erMCADOJcHk REGENERATION: The term regeneration implies a well-coordinated restoration of cells, tissues, and organs that have been physically or functionally lost. This reparative process must accomplish the recognition and recapitulation of missing structures, while simultaneously achieving functional integration between recently formed and pre-existing tissues, in order to direct physiological and structural alterations. Furthermore, regeneration involving cellular proliferation requires instructive signals with the capacity to efficiently regulate cell cycle, resulting in a finite number of cells that undergo division and complete repairs. Regeneration: Participating cells must be precisely guided to needed areas, and once regeneration is complete specific cues are required to report regenerative success and signal termination. Otherwise, the initial response would continue indefinitely, causing undesirable consequences for body homeostasis. Independent of magnitude, a regenerative event always seeks to maintain or re-establish both form and function (morphostasis). IMPORTANT: the process is not infallible, as demonstrated by growing evidence showing the associating between regeneration and cancer-related cellular abnormalities. Exploitation of Apoptotic Regulation in Cancer. David S. Ucker D.S. et al Front. Immunol., 27 February 2018. https://doi.org/10.3389/fimmu.2018.00241  Ontogeny and phylogeny of tissue regenerative capacity (a) Regeneration as an evolutionary variable is well illustrated by the differences in cardiac regenerative capacity amongst vertebrate species. In lower vertebrates such as zebrafish and salamanders, lost or damaged cardiac tissue in the adult is restored through a canonical repair process programmed by the immune system. In mammals, regeneration depends on the developmental stage. Preparing the ground for tissue regeneration: from mechanism to therapy. Stuart J Forbes, N Rosenthal. Nature Medicine: 20, 857–869, 2014 (b) The regenerative response to injury in adult mammals depends on age. There is a regenerative response in neonatal mice before day 7 after birth that engages an effective cardiac tissue repair similar to that in fish and amphibians. In contrast, the adult mammalian heart launches an acute myocardial infarction–associated inflammatory response in response to injury that leads to chronic irreversible pathological remodelling of the heart and compromised cardiac function. Although adult mammals can replace a small percentage of damaged cardiomyocytes, this is not sufficient to revitalize the organ after major injury. Preparing the ground for tissue regeneration: from mechanism to therapy. Stuart J Forbes, N Rosenthal. Nature Medicine: 20, 857–869, 2014 Learning objectives:  Fundamental information to understand injury, cellular and vascular response  Sequence in wound healing  Definitions of Regeneration and Repair  Liver and regeneration  General Model of Tissue Repair and Regeneration Healthy tissue and damage Regeneration Scarring (a) In healthy tissue, there is little monocyte trafficking or neutrophil efflux into tissues, an intact basement membrane and no scar tissue. (b) Upon tissue damage, there is loss of epithelia, neutrophil influx, activation of resident macrophages (Mf) and recruitment of inflammatory monocytes, in addition to release of inflammatory factors and activation of pericytes into myofibroblasts. (c) During tissue regeneration, there is epithelial proliferation for reconstitution, macrophage activation into a wound-healing phenotype and matrix remodeling. In addition, Treg cells are recruited to decrease chronic inflammation. (d) During aberrant tissue repair, excessive scarring occurs as a result of continued activation of inflammatory cells and impaired epithelial regeneration. Preparing the ground for tissue regeneration: from mechanism to therapy. Stuart J Forbes, N Rosenthal. Nature Medicine: 20, 857–869, 2014  Liver regeneration process Priming phase: quiescent hepatocytes convert from G0 to G1 of the cell cycle when faced with multiple stimulations Proliferation phase: with the help of mitogens, hepatocytes progress beyond the restriction point to the G1 phase and then the mitosis starts Termination phase: cells terminate proliferation under the control of negative factors Liver Regeneration: Analysis of the Main Relevant Signaling Molecules. Yachao Tao et al., Volume 2017, Article ID 4256352. https://doi.org/10.1155/2017/4256352  Liver Regeneration and cells Molecule-mediated liver regeneration through interactions between mature hepatocytes and non-parenchymal liver cells: Kupffer cells (KC), liver sinusoidal endothelial cells (LSEC), biliary endothelial cells, hepatic stellate cells (HSC). In liver tissue regeneration; matrix metalloproteinases (MMPs) and their specific inhibitors (tissue inhibitors of metalloproteinases, TIMPs) play a pivotal role in both fibrogenesis and fibrolysis. !!!!! Experimental and clinical data indicate that the regeneration signals facilitate also the growth of both primary and secondary liver tumours and can alter their malignant potential. This is an important mechanism underlying tumour recurrence after liver surgery. New therapeutic strategies based on better insight into the relationship between liver regeneration and tumour biology are needed. Exploitation of Apoptotic Regulation in Cancer. David S. Ucker D.S. et al Front. Immunol., 27 February 2018. https://doi.org/10.3389/fimmu.2018.00241 Effect of liver regeneration on malignant hepatic tumors. Shi JH, Line PD. World J Gastroenterol 2014; 20(43): 16167-16177. DOI: https://dx.doi.org/10.3748/wjg.v20.i43.16167  Tissue Repair. ECM homeostasis : MMPs vs TIMPs In healthy liver, homeostasis of extracellular matrix (ECM) is sustained by a precisely regulated permanent turn- over directed by a group of enzymes called matrix metalloproteinases (MMPs) and their specific inhibitors i.e. tissue inhibitors of metalloproteinases (TIMPs). Upon chronic damage of liver tissue, hepatic stellate cells (HSCs) become activated and differentiate into a fibroblast-like phenotype, key in producing ECM. In activated HSCs especially the expression of TIMP-1 is upregulated leading to the inhibition of MMP activity and subsequent accumulation of matrix proteins in the extracellular space. In chronic liver diseases: A substantial change in ECM composition is the deposition of collagens, mainly fibril-forming types I, III, and IV which increase in fibrotic ECM up to tenfold.  Normal Regeneration versus Aberrant regeneration A. In the normal adult liver, hepatocytes are mitotically quiescent (G0 of the cell cycle). Endothelial cells (LSEC) secrete TGF-β, which acts as a proliferation brake on hepatocytes. B. Following hepatectomy or acute injury, LSEC downregulate TGF-β during the early phase of regeneration. Endothelial cells, hepatic stellate cells and Kuppfer cells secrete growth factors and cytokines. These factors act with circulating factors to stimulate hepatocyte and biliary epithelial proliferation. This is followed by proliferation of endothelial cells, hepatic macrophages and stellate cells. Normal Regeneration versus Aberrant regeneration (C) the chronically damaged liver, there is increased expression of TIMPs, which oppose the scar-resolving function of MMPs and thus an excessive scar is deposited, which inhibits the proliferation of mature epithelial cells. During hepatocyte damage, macrophages phagocytose hepatocyte debris and stimulate hepatic regeneration.  Liver Regeneration : Experimental Partial hepatectomy (PH) Figure 4: Murine liver regeneration following partial hepatectomy (PH). This series of photos is a time-course of liver regeneration after a two-thirds PH. The volume of the remaining liver increases gradually and the restored organ rapidly begins to store lipids used in the regeneration process. The peak of steatosis occurs on the first day. As steatosis diminishes with time, the remaining liver enlarges to its pre-resection size in about 1 week. The rat partial hepatectomy model (75% reduction in liver size) is the classic model of liver regeneration and has been studied for decades. Following a 75%, or so-called two-thirds partial hepatectomy, the remaining liver remnant undergoes a series of rapid vascular endothelial, inflammatory and epithelial changes. The peak of liver regeneration, as measured by the number of hepatocytes in DNA synthetic phase, termed S phase, occurs ~24 h following resection. By 7–10 days after hepatectomy, the rat has largely regrown a normal-sized liver (93%) by hyperplasia.  MECHANISM(S) OF LIVER REGENERATION: HYPERPLASIA The progression of liver regeneration is segmented roughly in three phases: priming (G0 to G1 phase), proliferation (G1 to M phase) and termination. Liver regeneration progression is highly coordinated by the signal communication between hepatocytes and non-parenchymal cells. The process of restoration of liver volume is initiated by the replication of various types of intrahepatic cells. Non- parenchymal cells, such as liver sinusoidal endothelial cells, Kupffer cells, and biliary duct cells replicate in a delayed fashion, but demonstrate a similar synchronicity in DNA synthesis and mitosis as seen in hepatocytes. In parallel with the cellular replication, there is also a breakdown and remodelling of the extracellular matrix (ECM) by MMPs and TIMPs, promoted by cell-ECM interactions, which is also of vital importance in regulating liver regeneration. This hyperplasia associated with standard partial hepatectomy (PH) is due to the synchronized entry of approximately 95% of hepatocytes into the S phase of the cell cycle, followed by mitosis. Within minutes of liver resection, hepatocytes undergo a coordinated cellular activation termed the ‘acute phase response after PH’. This highly regulated process is mediated by multiple growth factors and cytokines that coordinate to transduce the response signal into kinase and transcription factor activation. As a result of the acute response, a surviving hepatocyte initiates the transcription of more than 100 early genes and accumulates triacylglycerol and cholesterol esters in intracellular lipid droplets. These droplets supply the energy and building materials needed to support rapid cell division and tissue regrowth and so are essential for normal liver regeneration. Effect of liver regeneration on malignant hepatic tumors. Shi JH, Line PD. World J Gastroenterol 2014; 20(43): 16167-16177. DOI: https://dx.doi.org/10.3748/wjg.v20.i43.16167  Regeneration in the ‘abnormal’ liver The original size and weight of the liver is re-established within one week after hepatectomy in rodents and about 3 months in humans. The growth of the remnant liver or graft is a restoration of function by induced hyperplasia. Thereafter, the hepatocytes enter their non-replicative and quiescent, functional state. The lobes are slowly reorganized and hepatic histology is completely restored after 2-3 weeks in rodents and 3-6 months in humans. Clinical data shows that liver regeneration is significantly impaired in damaged livers due to other chronic liver diseases, such as viral hepatitis, alcohol abuse and fatty liver. In the clinic, the regeneration of normal liver is relevant; for example, when a healthy relative donates part of their liver to a recipient with liver disease, so called living donor liver transplantation. Here, the donor will have been specifically screened to exclude clinically significant liver disease. Maria Agnese Della Fazia M.A. et al., Foie gras and liver regeneration: a fat dilemma. Cell Stress, Vol. 2, No. 7, pp. 162 - 175; doi: 10.15698/cst2018.07.144  Background information The Role of Chemokines in Wound Healing. Ridiandries A et al., Int. J. Mol. Sci. 2018, 19(10), 3217 Preparing the ground for tissue regeneration: from mechanism to therapy. Stuart J Forbes, N Rosenthal. Nature Medicine: 20, 857–869, 2014 Preparing the ground for tissue regeneration: from mechanism to therapy. Stuart J Forbes, N Rosenthal. Nature Medicine: 20, 857–869, 2014 Repair, Regeneration, and Fibrosis. Gregory C. Sephel and Stephen C. Woodward (Moodle) Exploitation of Apoptotic Regulation in Cancer. David S. Ucker D.S. et al Front. Immunol., 27 February 2018. https://doi.org/10.3389/fimmu.2018.00241 Liver Regeneration: Analysis of the Main Relevant Signaling Molecules. Yachao Tao et al., Volume 2017, Article ID 4256352. https://doi.org/10.1155/2017/4256352 Effect of liver regeneration on malignant hepatic tumors. Shi JH, Line PD. World J Gastroenterol 2014; 20(43): 16167-16177. DOI: https://dx.doi.org/10.3748/wjg.v20.i43.16167 Maria Agnese Della Fazia M.A. et al., Foie gras and liver regeneration: a fat dilemma. Cell Stress, Vol. 2, No. 7, pp. 162 - 175; doi: 10.15698/cst2018.07.144 Exploitation of Apoptotic Regulation in Cancer. David S. Ucker D.S. et al Front. Immunol., 27 February 2018. https://doi.org/10.3389/fimmu.2018.00241