Pathophysiology of wound healing and alterations in venous leg ulcers-review

Introduction

Chronic venous disease (CVD) is a spectrum that clinically includes patients presenting with varicose veins, edema, skin changes and venous leg ulceration (VLU). Chronic venous insufficiency (CVI) is associated with increased ambulatory venous pressure and is usually seen with advanced forms of CVD (edema, skin changes, VLU). CVI affects 10–35% of US adults. Primary (chronic venous disease resulting in venous hypertension from varicose veins with idiopathic valvular insufficiency) CVD is more common than secondary (chronic venous disease resulting in venous hypertension usually from venous thrombosis and valvular insufficiency and/or obstruction) with the latter responsible for about 18% to 28% of limbs with CVD. In the Western population skin changes and ulceration affects 2–5% of Western population. Importantly, VLU wounds occur in up to 5% of the population over 65 years of age.^1,2 Wound healing is a complex process that involves hemostasis with platelet function and coagulation cascade within the first day, this is followed by an inflammatory phase involving neutrophils and macrophages that last several days, a proliferative phase which begins after 3 to 4 days and progresses for months involving growth factors, angiogenesis, fibroblast activity with collagen and extracellular matrix deposition, keratinocytes for epithelialization, and finally a remodeling phase, which begins at several months from the inciting event and progresses for months to years, leading to wound contracture (Table 1).^3,4 A fundamental principle in chronic VLU is that an inflammatory condition exist that persists leading to disease presentation and progression. The important steps in inflammation involves leukocytes, particular macrophages and monocytes as well as T-lymphocytes, changes in shear stress on the endothelium and glycocalyx alterations, inflammatory modulators and chemokines, cytokine expression, growth factors, and matrix metalloproteinase activity. There are genetic and environmental conditions that predispose individuals to CVD likely leading to altered shear stress on the endothelial cells, causing endothelial cells to release vasoactive agents, express E-selectin, inflammatory molecules, chemokines, and prothrombotic precursors.^4–6 Mechanical forces, low shear stress, and endothelial-smooth muscle stretch are sensed by the endothelial cells via intercellular adhesion molecule-1 (ICAM-1, CD54) and the mechano-sensitive transient receptor potential vanilloid channels (TRPVs) that are present in the endothelium.^2,7 It is well known that patient with CVD have increased expression of ICAM-1, which is expressed on endothelial cells, and activate the recruitment of leukocytes and initiate endothelial transmigration, setting up an inflammatory cascade. Initiating events likely involve altered shear stress and mechanical stress forces on the endothelium and its glycocalyx (a glycosaminoglycan on the surface of endothelial cells), with perturbations on nitric oxide production, vasoactive substance release, expression of monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1, CD106), expression of L-selectins and E-selectins, ICAM-1, with recruitment of leukocytes leading to leukocyte transmigration into the vein wall and interstitial tissue space, setting up an inflammatory cascade and production of several cytokines (transforming growth factor-β1 (TGF-β1), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-1 IL-1)) and increased expression of matrix metalloproteinases (MMPs).^7,8 As a result of persistent inflammation the venous wall and valves are affected and venous hypertension ensues, propagating the disturbances on the endothelium, microcirculation, and inflammatory response (Figure 1). OPEN IN VIEWER

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Table 1.

Phases of wound healing. There are four phases to wound healing. It begins with hemostasis involving vasoconstriction and platelet activation and aggregation, followed by an inflammatory phase with neutrophils acting within the first 24 hours and then replaced by macrophages with lymphocytes having a role later in the phase. There are many chemokines, cytokines, growth factors, and proteases that are produced during the inflammatory phase. The proliferative phase is begins the structural repair of the tissue with fibroblasts producing collagen, elastin, and extracellular matrix proteins. Angiogenesis and epithelialization are characteristic of this phase and occur within the first week and continue for months. The final phase involves remodeling which involves myofibroblasts and is the wound continues to gain tensile strength over months to years.

Would Phase	Timing	Cellular Events
Hemostasis	0–1 day	Vasoconstriction, platelet aggregation, coagulation cascade
Inflammatory	1–3 days	neutrophils, monocytes, macrophages, lymphocytes, chemokines, cytokines, growth factors
Proliferative	4 days–months	Fibroblasts, angiogenesis, extracellular matrix, epithelialization
Remodelling	Months–years	Remodeling, wound contracture, myofibroblasts

VLU have several areas of activated pathways due to the persistent inflammatory response that include oxidative stress with iron overload, cytokine and chemokine expression, alteration in collagen turnover, and overexpression of MMPs leading to tissues destruction of the dermis with eventual skin changes and ulcer formation. This review will cover some important aspects of general wound healing, and then discuss the important aspect of the pathophysiology of wound healing in VLU, differentiate VLU that heal versus VLU that are classified as non-healing with respect to the biochemical properties of the wound.

Wound Healing

Wound healing has four phases that overlap as one phase regresses and the next phase initiates (Table 1). Following the development of a wound, an immediate event occurs called the hemostasis phase. This phase begins with vasoconstriction by released vasoactive substances (thromboxane A2, serotonin, adrenalin). Platelets are rapidly recruited to the injured area and activation by the injured blood vessel exposing collagen leads to ADP and glycoprotein adhesion molecules (fibrinogen, fibronectin, vitronectin, thrombospondin, and von Willebrand’s factor) release and aggregation. Prothrombin is activated by platelets and forms thrombin, which catalyzes the formation of fibrin from fibrinogen making a thrombus and acting as a provisional matrix. An important protein is fibronectin which contains several integrin receptors and will be important in stimulating the migration and adhesion of fibroblasts, keratinocytes, and endothelial cells. Platelets also have integrin receptors and add further aggregation to thrombus and secrete growth factors (platelet derived growth factor (PDGF), transforming growth factor-α (TGF-α), TGF-β, basic fibroblast growth factor (bFGF), insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF)), which are necessary compounds to recruit inflammatory cells. The second phase begins at 1 to 3 days of wounding involves the inflammatory response which initiates the recruitment of neutrophils and monocytes/macrophages. The cells release a host of inflammatory chemokines and cytokines, as well as growth factors. Neutrophils are recruited by several chemokines including thrombin, leukotrienes, PDGF, TGF-β, and bacteria as well as complement, initiate the defensive mechanisms to infection. Further cytokines are released and after 24 hours neutrophils undergo apoptosis with the insurgence of monocyte/macrophages leading to further inflammation and phagocytosis. Macrophages produce cytokines, MMPs, and growth factors (IL-1, IL-6, IL-8, and TNF-α, TGF-β, PDGF, TGF-α, bFGF, heparin-binding epidermal growth factor-like growth factor (HB-EGF), IL-1, IL-6, and a variety of MMPs 1,2,3,9,10) needed to both clear cellular debris and initiate the process of recruiting fibroblasts for collagen and matrix deposition. Further initiation of angiogenesis is initiated by the direction of VEGF, epidermal growth factor (EGF) and PDGF. Lymphocytes also participate in the later stages of inflammation. The proliferative phase follows and is dominated by fibroblast function with collagen deposition and further extracellular matrix proteins that takes place within the first week and progresses for months. The provisional fibrin matrix is now replaced with collagen, elastin, proteoglycans, glycosaminoglycans, and noncollagenous glycoproteins. Fibroblasts are under the regulation of a number of growth factors (PDGF, epidermal growth factor (EGF), IGF-1, and TGF-β). Collagen deposition (type I and III) increases up to the third week and then begins to decline. Epithelialization comes from the wound edge and skin appendages (sweat glands, hair follicles), and requires the detachment of keratinocytes to migrate, proliferate and cover the collagen rich matrix. This is regulated by growth factors (TGF-α, EGF, keratinocyte growth factor (KGF), and TGF-β) as well as MMP-2, MMP-9 which are important in degrading type IV collagen for cellular detachment and MMP-1, MMP-3, and MMP-10 promoting new epithelial cells to migrate along fibronectin and the extracellular matrix components. Throughout the process of proliferation phase, angiogenesis is taking place to restore new blood vessels in the injured area now enveloped by connective tissues and extracellular matrix, and under the regulation of angiogenic factors (VEGF, PDGF, MMPs), which brings in endothelial cells forming new capillaries for oxygen and carbon dioxide transport and nutrients. The final phase involves remodeling and begins within months and may continue for several years. This phase involves wound contracture and is performed by myofibroblasts differentiated from fibroblast. The characteristics of this phase is the progressive increase in wound tensile strength, increase of total collagen especially type I, increased cross-linking of collagen, where the strength of the tissue reaches 80% of that of unwounded tissue.^3,9,10

Pathophysiology in Venous Leg Ulcer Wound Healing

Chronic VLU are characterized by perpetual inflammatory phase, with provisional matrix but inability to continue to the proliferative and remodeling phases of wound healing. The appearance of wound ulcer that have an inflammatory state is characterized by open wounds which have drainage, have necrotic tissue overlying the wound bed, have pain as well as induration around the surrounding irregular borders (Figure 2). The characteristics leading to VLU are multifactorial, and the biologic environment is a complex interplay of imbalances in a number of biomolecules and processes which include: inflammatory cells, chemokines and cytokines, inflammatory modulators, extracellular matrix proteins, collagen turnover, oxidative stress, and protease activity especially MMP2- and MMP-9. ¹¹ It is important to discuss cellular and biochemical properties of the wound which differentiate VLU that heal versus VLU that are non-healing, that could provide insight into the mechanisms and potential treatments of VLU (Table 2). OPEN IN VIEWER

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Table 2.

Important pathophysiologic events in venous leg ulcers. There are a number of cellular and biochemical events involved in the pathophysiology of venous leg ulcer. These events have a pathology that is associated with the ulcer, and how the wound environment changes the condition of the cellular components or biomolecules can predict if healing of the venous leg ulcer will occur.

Cellular/Biochemical Event	Pathology	Predictive of VLU Healing
Inflammatory Cells	Macrophages, Mast Cells, Lymphocytes persist	Reduction in leukocytes seen in CVD without skin changes
Chemokines Cytokines	Overexpressed in VLU	Reduction after compression increased VLU healing, IL-1ra increased in VLU healing
Growth Factors	Present in VLU, may be inactive due to proteolytic activity	PDGF and TGF-β are increased in VLU healing
Inflammatory Modulators	Overexpression of lactotransferrin, annexin A1, S100A9	Reduction in healing VLU Function in VLU remains to be determined
Collagen Deposition	Collagen dysregulation of type I and III	Healed VLU have increased collagen deposition/turnover
Oxidative stress	Increased ferritin and total oxidative state, free radicals	Lower ferritin and total antioxidant state healed VLU
Matrix Metalloproteinase	Increased proteinase activity	Decreased MMP-2, 3, 9 in healing VLU

Conclusions

VLU have a complex pathophysiology, and have reduced wound healing due to a persistent inflammatory state. There are many inflammatory pathways that are activated within the VLU environment. Genetic and environmental factors predispose patients to CVD and changes on the endothelial surface and protective glycocalyx are likely early initiating events. The inflammatory cells are activated by activation of endothelial cells and adhesion molecules, which results in the expression of many cytokines, inflammatory modulators, MMPs, and oxidative stressors. There is clinical and scientific evidence in the literature that modulation of cytokines by compression leads to VLU healing. In addition, favorable changes in the proteolytic, collagen turnover, and oxidative environment of the VLU, leads to increased healing. This would suggest that these pathways, when dysregulated and unopposed, are important in development of VLU, and that further research in the inflammatory and proteolytic mechanism of VLU should be pursued to determine appropriate biomolecules that are involved in VLU development, but also as potential markers for prognosis and targeted therapy.