Wound Healing
Diseases

Abnormal microenvironment and altered cellular function in hypertrophic scars

WOUND HEALING

Wound healing is the process of tissue repair involving the tissue response to injury. It is divided into four phases: haemostasis, inflammation, repair and remodelling of the scar tissue, each being controlled and regulated by biologically active polypeptides called growth factors.

• Hemostasis: Platelets aggregate at the site of injury activating the thrombin-fibrinogen-fibrin pathway, which finally polymerizes to form a stable clot. The fibrin clot provides a matrix into which fibroblasts, vascular endothelial cells and epidermal cells migrate. The platelets degranulate releasing chemoattractants for inflammatory cells and growth factors such as PDGF, IGF-1, EGF, FGF and FGF-β. These growth factors stimulate the proliferation of keratinocytes and fibroblasts and promote the migration of inflammatory cells.
• Inflammatory Phase: During this phase, platelet degranulation with histamine and growth factors realease promote vasodilation and increase capillary permeability. Vascular permeability enables a massive margination of phagocytic cells, neutrophils, mast cells, complement and antibody.
  The primary role of neutrophils is to kill bacteria (by generating ROS) and to degrade damaged components of the extra cellular matrix (by releasing proteases). They also release important inflammatory mediators such as TNF-α and IL-1.
  
  Monocytes migrate into the wound and differenciate into tissue macrophages. They have a major phagocitic role and produce collagenases and elastases to break down devitalized tissue. They also mediate the transition from inflammatory to proliferative phase by secreting TNF-α, TGF-α, TGF-β, PDGF, IL-1 and -6, IGF-1 and FGF. Macrophages continue to stimulate inward migration of fibroblasts, epithelial cells and vascular endothelial cells into the wound to form granulation tissue.
• Proliferative Phase: The activation of fibroblasts starts: they migrate into the wound becoming the most common cell-type. They utilize matrix metalloproteinases (MMPs) to digest the provisional fibrin matrix and deposit glycosaminoglycans (GAGs), collagen (type I and III) and fibronectin in a disorganized fashion. This initial scar matrix acts like a bridge for a sheet of epidermal cells to migrate, followed by keratinocyte proliferation forming a multilayered stratified epidermis.

• Remodeling: Keratinocytes migrate from the basal membrane to the newly formed surface using specific integrin receptors and release MMPs essential for the controlled degradation, synthesis and reorganization of molecules in the matrix. If a balance is reached between synthesis of ECM components and their degradation by proteases, the migrating epithelium moves over the initial scar matrix and eventually stratifies and differentiates.

  HYPERTROPHIC SCAR

  Hypertrophic scars occur in case of imbalance between the anabolic and catabolic phases of the healing process: more collagen is produced than is degraded, and the scar grows in all directions. The hypertrophic scar is elevated above the skin erythematous and stiff but typically does not expand beyond the boundaries of the initial injury.
  Which cells and biochemical pathways could possibly be involved as major key-roles in this abnormal healing process?
  
  Hereby, considering the skin as one of the widest organs of the human body endowed with fascinating metabolic functions, we provide a brief insight on the “wound healing gone bad” mechanisms.
  
  In the mid-late phase of wound healing cellular interactions become dominated by the interplay of keratinocytes with fibroblasts in a double paracrine manner.
  
  
  Keratinocytes, which represent a constantly renewing cellular compartment in the skin, instruct fibroblasts to synthesize and secrete growth factors and cytokines such as: keratinocyte/fibroblast growth factor 7 (KGF/FGF7), IL-6 and GM-CSF. Expression of these factors is induced by keratinocyte-derived IL-1. The expression of KGF is strongly up regulated in fibroblasts not only by IL-1 but also by TNFα and PDGF.
  

  Database	KGF
  Wikigenes	URL
  GeneCards	URL
  iHOP	URL
  OMIM	URL

  IL-1 and TNFα are powerful proinflammatory cytokines produced at high levels during the first hours of injury. Blocking in the TNFα-pathway results in enhancement of TGFβ and VEGF expression, angiogenesis and collagen production, thus in accelerated healing of the wound. This suggests that inflammatory signals can balance TGFβ expression and its effects.
  
  TGFβ is supplied at early time points by thrombocytes, later by keratinocytes and is responsible for the differentiation of fibroblasts into myofibroblasts: mesenchymal cells that express smooth muscle cell actin isoform (α-SMA). Actin is important for the contractile activity of these highly differentiated fibroblasts that bring the edges of the wound together.
  
  In hypertrophic scars TGFβ is either over expressed, or non-sufficiently counterbalanced by TNFα, thus resulting in increased collagen I, α-SMA, fibronectin and keratin production.
  
  Type I collagen is a fibrillar protein formed by three polypeptide chains resulting in a unique triple-helical structure and is the most abundant type of collagen in repairing scar tissues. The distinctive feature of collagen is the precise amino acid pattern Gly-Pro-Y or Gly-X-Hyp, where proline and hydroxyproline make up to about 1/6 of the total amino acid content. Proline and Hydroxiproline with their geometrically voluminous rings are responsible for the tendency of single strands to form left-handed helices spontaneously, while Glycine, the smallest amino acid with no side chain is required every third position to construct the internal axis of the triple helix structure.
  

  

  The key step in the biosynthetic pathway of Proline (and Ornithine, a precursor of Arginine) is the production of P5C’ (D’-pirroline-5 Carboxylic Acid). Proline synthesis requires glutamate, ATP, NADPH and NADH. A first enzyme catalyzes the phosphorylation of glutamate by ATP to form γ-glutamyl phosphate, which is reduced by a second enzyme requiring NADPH to glutamyl-γsemialdehyde. The glutamyl-γsemialdehyde is in balance with its enamin P5C’ which is finally reduced with NADH to form Proline.
  

  The fibroblast proliferation and collagen synthesis is regulated through the mTOR pathway, a serine/threonine protein kinase stimulated by insulin, growth factors and extra cellular amino acids.
  The mammalian target of rapamycin ( mTOR ) functions as a nutrient/energy/redox sensor controlling protein synthesis and is inhibited by low nutrient levels, growth factor deprivation, reductive stress and drugs such as rapamycin.
  
  One of the main targets of mTOR is p70-S6 Kinase1, activated through phosphorilation of a threonine residue leading to initiation of protein synthesis and other components of the translational machinery. Amongst other signals, mTOR upregulates the expression of Hypoxia Inducible Factor (HIF) leading to an increased synthesis of Vascular Endothelial Growth Factor (VEGF), both playing a crucial role in wound healing.
  
  HIFs are proline-rich trascription factors composed of an alpha and a beta subunit, that respond to decreases of oxygen in the cellular environment .
  
  The alpha subunit of HIF is a target for hydroxylation by HIF prolil-hydroxilase. Once hydroxilated HIFα becomes a target for degradation by an ubiquitin ligase, leading to quick degradation by the proteasome.

  Prolyl-hydroxylases are nonheme Iron-containing enzymes: Iron has to be in Fe (II) form to function correctly and Ascorbic Acid is essential to maintain iron reduced. These enzymes belong to Fe (II)-dependent oxygenase super family and utilize molecular oxygen and 2-oxoglutarate (2OG) as substrates. [It is interesting to point out that this family also includes the prolyl 4-hydroxylase, the enzyme that catalyzes the hydroxylation of L-Proline in collagen.] Iron plays a key role in this reaction; indeed divalent ions resembling Iron (i.e. Ni or Co) that could replace Iron in the catalytic site of the enzymes or interfere with its uptake or depletions of Ascorbic Acid can impair the activity of prolyl-hydroxylases.
  In hypoxic or NADPH depleted cells, HIF prolyl-hydroxylase is inhibited, allowing the stabilized HIF alpha to bind to the constitutively expressed beta subunit. HIF-1α and HIF-1β heterodimers can bind to the hypoxia response element (HRE), increasing transcription rates for tyrosine hydroxylase, erythropoietin (Epo), transferrin, transferrin receptor, glycolytic enzymes and VEGF.
  

  

  Another significant alteration in fibroblast function is the reduced expression of Nitric Oxide Syntheses (NOS) and the impaired production of Nitric Oxide (NO). NO is a product of the arginine-citrulline metabolic pathway with several physiologic activities such as: smooth muscle relaxation or vasodilatation, anti-proliferative activity and immune regulation. In hypertrophic scar fibroblasts both constitutively expressed NOS and inducible NOS levels are reduced when compared with normal fibroblasts. Markedly inhibited NO levels could cause over-proliferation of fibroblasts, resulting in hypertrophic scar formation.
  
  NOS is a calmodulin-dependent heme-containing enzyme similar to cytochrome p450 with two catalytic domains (reductase and oxygenase) bearing both Flavin Adenine Dinucleotide (FAD) and Flavin Mononucleotide (FMN). In the presence of NADPH and dioxygen (O2), NOS can synthetize nitric oxide (NO) from the terminal nitrogen atom of L-arginine. NO can activate Guanylate Cyclase increasing intracellular cGMP synthesis, which finally inhibits calcium entry into the cell inducing smooth muscle relaxation. Iron seems to play an important role in this pathway as well, working as an electron acceptor during NO production from L-arginine protecting NOS from oxydative burden.
  

  

  As mentioned above, not only fibroblasts but also keratinocytes play an important role in the mid-late phase of wound healing. Keratinocytes, among many types of high rate proliferating cells, have high/increased needs of Iron supply. Indeed hypertrophic scar keratinocytes have shown increased levels of Transferrine receptor (CD71), the Iron carrier protein in blood stream, suggesting the high proliferation rate of these cells.

  Drawing a conclusion, hypertrophic scars could be considered on one hand the result of hyper nutrient states leading to the crucial activation of mTOR. Indeed high amino acid levels (especially proline), high insulin levels (typically occurring in insulin-resistant obese or overweight individuals) are some of the most powerful ligands leading to mTOR pathway hyper-activation. On the other hand, this abnormal healing can result as a consequence of impaired levels of crucial co-factors and micronutrients that represent the molecular basis of inhibitory/counter-regulatory enzymes such as prolyl-hydroxylase or NOS, with Iron playing a key role among these factors.