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The process of repair and regeneration that occurs immediately after tissue injury or surgery represents one of the most essential defence mechanisms of the human organism against its external environments. Wound healing is often described as a series of events or cascade that involuntary takes place after injury. Inflammation is an automatic response of the body following injury. This action of the body is primarily achieved by the increased movement of leukocytes from the blood into the injured site, followed by a cascade of biochemical reactions that proliferate and mature the inflammatory response, involving the local vascular system, the immune system and various cells at the injured sites. A crucial step in the process is initiated by different interactions.
The main cascade that is fundamental in the repair is the formation of new blood vessels termed angiogenesis. Angiogenesis, which is the term used to describe the formation or the growth of new blood vessels from the pre-existing vessels is fundamental in wound healing and tissue repair or re-generation. The healing process of the wound fully depends on the co-ordinated manner of events that occur natural, including angiogenesis. In humans, angiogenesis is achieved via controlled and balanced growth factors. Angiogenesis is important in wound healing as it provides constant supply of oxygen and nutrients to the damaged tissue at a desirable level to help restore the tissue to health. It is particularly helpful in any type of wound, like a surgical wound when the blood vessels are damaged.
Current estimates show that the frequency of none healing or infected wounds remains high despite present treatments widely available. With rates of wound infections being this high, persons with non-healing wounds or infected wounds represent a significant part of the population, particularly people with diabetes. Due to the discomfort of non healing wound, they often have a devastating effect on the lives of the persons suffering. The psychological effects of a wound can be just as major as physiological effects as such patients often have a very negative attitude towards life.
Even though, clinician have a more broad understanding of wound healing and capability to attain effective wound healing has improved extensively over the years, particularly as a result of advances and technologies such as gene therapy, vacuum assisted closure therapy, the use of growth factors, the ability to grow cells in vitro and the development of bioengineered tissue and the various agents used to aid in wound healing that have been tested to determine which is ideal and most effective in promoting the healing of wounds, there still seems to be a challenge for the medical team as these treatments are not always successful and there is still a high number of people suffering. The fact that there still seem to be wound related complication such as infections and amputations due to poor wound healing provides enough evidence that the fundamental problems have not yet been solved and further research is necessary to finding therapeutic for wound that is cost effective with less side effects.
Non healing wound are those that fail to fully heal within a reasonable period of time by use of typical treatments like dressings and anti infection medications such as antibiotics. However, according to Gogia (1995)certain factors contribute to poor wound healing include age, diet, weight, conditions such as poor circulation as the wound healing depends on the constant supply of oxygenated blood to the wound site. Reduced blood supply inhibits the fibroblast migration. Conditions such as atherosclerosis as the blood supply to the site of injury are significantly reduced. Diabetes as it decreases collagen synthesis and phagocytosis.
Delayed wound healing is a major issue particularly in patients with diabetes and cancer due to the defected immune system as the cancer cells continue to grow. Such patients are more prone to experience delayed wound healing or suffer severe infections. Therapeutic angiogenesis modalities represent a broad range of interventions that generate new blood vessel growth to promote wound healing. The therapeutic goal is to stimulate angiogenesis to improve perfusion, deliver oxygen and nutrients to sites of tissue repair, and help restore health and function to the tissue.
Angiogenesis, which is the term used to describe the formation or the growth of new blood vessels from the pre-existing vessels is fundamental in wound healing and tissue repair or re-generation. The healing process of the wound fully depends on the synchronized manner of events that occur naturally, including angiogenesis. In humans, angiogenesis is achieved via controlled and balanced growth factors. Angiogenesis is important in wound healing as it provides constant supply of oxygen and nutrients to the damaged tissue at a desirable level to help restore the tissue to health. It is particularly helpful in any type of wound, like a surgical wound. However, this is not the only time that angiogenesis occurs. For instance women also go through a normal angiogenesis cycle monthly and these new blood vessels are key to forming the uterine lining.
The objective of the experiment aims to identify the activity of heparin sulphate in the regulation and stimulation of angiogenesis in wound healing. It involves the isolation and purification of heparan sulphate, a sugar belonging to glucosaminoglycans (GAG's). Heparin sulphate will be isolated from two different sources of tissue, in this instance from mussel and lobster. Once isolated and purified, using a cell culture the heparin sulphate will be added to it in order to assess its activity in the wound healing and it's potential to be used as therapeutics in the treatments of many conditions, in this case wound healing in cancer after surgery. In particular, heparin sulphate will be discussed the most as it is known heparin sulfate possesses high affinity binding properties for angrogenic growth factors such as FGF.
Glucosamineglycans (GAG's) are complex, linear, negatively charged, unbranched polymers generally composed of repeating sulfated dissaccharide units or chains. Champe, et al (2005). These GAG's are usually isolated as proteoglycans, consisting of a core protein with one or more covalently attached glycosaminoglycan chains. Glucosaminoglycans are linear polysaccharides, whose disaccharide structure consists of an amino sugar, usually D-glucosamine or d-galactosamine. It is known that the amino sugar is acetylated resulting in the negative charge of the whole molecule. As a result of the variation in the composition of the saccharides in the GAG molecules, GAG's can be classified or divided into groups according to their structure. Glycosaminoglycans (GAGs) include heparin (HP), heparan sulfate (HS), dermatan sulfate (DS), chondroitin sulfate (CS), keratan sulfate (KS), and hyaluronic acid (HA). Chondroitin sulphate, and dermatin sulphate in the other hand are galactosaminoglycans. Heparan sulphate and heparin are believed to be the most structurally diverse GAGs. The disaccharide repeat consists of a glucosamine and uronic acid linked via 1-4 glycosidic bonds. Glycosaminoglycans (GAGs) are responsible for the regulation of the activity of several molecules in the extra cellular surroundings via the binding to many proteins. These proteins can be growth factors, enzymes, enzyme inhibitors, and components of the extra cellular matrix.
Heparan sulfates, have an average molecular weight of approcimentaly 12,000, more or less corresponding to 20 disaccharide units. Heparan sulfate is composed mainly of B-D-glucorunic acid and 2 -acetamido - 2 deoxy-x-d-glucose as major saccharide reperating units. These repeating units are joined via 1-4 glucosidie linkages. Within the proteoglycan, the N-sulphated polysaccharides properties are common components of cell surfaces and the extracellular matrix. The presence of PGs and their associated GAGs present on the cell surface and in the extracellular environment permit them to act as a central device for control of various cell behaviours fundamental to many physiological repair processes, main process being the angiogenesis. According to recent research carried out on the function of heparin sulphate concluded that heparan sulfate is compulsory for the proper function of the other PG's, main one being heparin-binding ligands, such as associates of the fibroblast growth factor (FGF). Heparin sulphate also acts by modifying the action of enzymes such as antithrombin III and matrix metalloproteases. Due to the variety of functions, heparan sulfate has been most widely studied and may in the future be used as therapeutics for the treatment of many diseases.
The heparan sulfate (HS) glycosaminoglycan chains bind tightly to the extra-cellular matrix and cell surface proteins, providing a framework for matrix organization, regulation and cell-cell or cell-matrix interactions. However, HSPGs play more than just a structural role. In cooperation with the basement membrane and cell surface heparin sulfate bind a wide variety of protein ligands that are concerned in wound repair. The heparan sulphate polysaccharide chain has a distinctive molecular structure in which the groups of N- and O-sulphated saccharide residues that are separated by regions of low sulphation specifically determine protein binding properties. Furthermore, the haparan sulphate chains are attached to various protein cores, responsible for the determination of the location for the proteoglycan in the cell membrane as well as the extracellular matrix. The varied functions of heparan sulphate include the control of blood coagulation in relation to the regulation of the cell growth, proliferation, differentiation, migration and adhesion. In addition to this, they are also involved in a variety of important biological processes such as inflammation during injury or wounds which is a fundamental event in effective repair. In contrast to heparin, which occurs only in mast cells, Heparan sulfate proteoglycans are expressed and secreted by most, if not all, mammalian cells. HS proteoglycans are consciously positioned on cell surfaces and in the extracellular matrix. Heparan sulphate proteoglycans not only have fewer but also shorter polysaccharide chains compared to heparin proteoglycan.
Heparan sulphate is believed to have an important role in the wound healing process and has recently been investigated. The process of wound healing is a complex biological involuntary process that occurs in stages following injury. Studies have shown that heparan sulphate interacts with fibroblast growth factor (FGF-2) that are highly involved in the regulation, growth and differentiation of many different cell types. Furthermore, FGF-2 is the main regulator of angiogenesis and is involved in a number of processes. However, to achieve the binding of FGF-2 to its receptors it requires heparan sulfate (HS). Basic fibroblast growth factor (bFGF) is another complex mainly involved in angiogenesis. bFGF gains stability by binding with heparan sulphate.
In addition to its structural role in extracellular matrix assembly and integrity, HS confiscates a number of polypeptides that reside in the extracellular matrix. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and intensely affect cell and tissue function. Heparanase is a mammalian endoglycosidase associated with inflammation. Thus, heparanase accessibility, and activity should be kept tightly regulated. Heparan sulphate is not only a substrate for heparanase, but also its regulator. Heparan sulfate glycosaminoglycans (HS-GAGs) are not only the structural elements of tissue construction but also regulate the bioavailability and transduction pathways of heparan sulfate-bound polypeptides released by cells or the extracellular matrix.
Vascular endothelial growth factor (VEGF) has the ability to intervene angiogenesis Stringer (2006) reported that a number of in vivo and in vitro studies examined the roles of HS proteoglycans in VEGF activity concluded that Heparan sulphate to be essential to spatially restrict VEGF to create the appropriate gradient to allow blood vessel branching to occur. HS proteoglycans can also re-activate oxidation-damaged forms of VEGF, a function that may be essential in hypoxic sites where new vessels are required. The switch from normal quiescent vasculature to angiogenesis is induced by a change in the balance of many pro- and antiangiogenic factors that are released predominantly by surrounding pericytes and lymphocytes. A large number of these factors have been found to bind to HS (heparan sulphate) proteoglycans or the structurally related heparin, including key angiogenic growth factors such as the FGFs (fibroblast growth factors) and VEGFs (vascular endothelial growth factors) . However, the only growth factor observed almost ubiquitously at sites of angiogenesis and whose levels correlate most closely with the spatial and temporal events of blood vessel growth is VEGF.
Studies by Bernfield, (1992), has shown that syndecans that are of transmembrane core proteins able of carrying heparan sulfate (HS) and chondroitin sulfate (CS) chains enables their interactions with many proteins, these being heparin-binding growth factors such as fibroblast growth factors (FGFs), vascular endothelial growth factors (VEGFs), transforming growth factor-ß (TGF-ß), and platelet-derived growth factors. In another aspect, Heparan sulfate aids the interactions with a range of extracellular matrix proteins such as laminin. These studies have revealed that HS critically regulates angiogenesis by playing a proangiogenic role, and this regulatory function critically depends on HS fine structure. The latter is responsible for enabling cell-surface binding of various pro-angiogenic growth factors that in turn mediate endothelial growth signalling. Studies carried out in vitro in mice have concluded that signaling by multiple growth factors as well as matrix storage of growth factors may be regulated by HS.
During the wound healing process, Woods, (2001) reported that syndecan particularly sundecan 1 and 4 are increased in expression and release in soluble state. In addition, syndecan expression is increased in dermal and mucosal repair, and during response to arterial injury. Syndecan deficient mice models used in vitro according to Woods, (2001), experienced delayed wound healing.
Syndecans are transmembrane heparan sulfate proteoglycans(mucopolysaccharides bound to protein chains occurring in the extracellular matrix of connective tissue. (HSPGs) that are present on most cell types have been known for some time to regulate a variety of biological processes, ranging from coagulation cascades, growth factor signaling, lipase binding and activity, cell adhesion to ECM and subsequent cytoskeletal organization, to infection of cells with microorganisms. They are complex molecules, with specific core protein to which a variable number of glycosaminoglycan (GAG) chains are attached.
This paper has reviewed the scientific literature available as to the importance of glucosaminoglycans, mainly heparin sulphate of wound injuries of any type i.e. surgical wounds. All of the literature studied points out the importance continued research in this area. Although research on wound healing has been ongoing for many years, there is still much to be learned about glucosaminoglycsns in effective wound healing. In addition, all of the benefits of the saccharides are still not fully understood, but the benefits which have been documented may well be a new lead in therapeutics.
The former study, conducted in collaboration with the Dental Faculty, looked into the role of chondroitin sulphate in wound healing after surgical treatment of congenital cleft palate, one of the most common birth defects. The researchers investigated into the function of chondroitin sulphate in cell proliferation, adhesion and migration during palatal wound healing. Their study shows that chondroitin-6-sulphate is indeed involved in regulating cell proliferation. Through an in vitro model, the team also found that wound closure was dramatically slower when chondroitin sulphate formation was inhibited, with only 39.9 per cent reduction in wound gap distance 18 hours after wounding occurred as compared to 92.7 per cent decrease in the control group.
The studies of GAGs in wound healing may be a target for future. Dermatan sulfate (DS) GAGs have also been found to bind heparin-binding growth factors, including FGF-2 (4), hepatocyte growth factor/scatter factor (11), heparin cofactor-II (12, 13), platelet factor-4 (14), fibronectin (15), and protein C inhibitor (16). In human wounds, DS contributes the majority of FGF-2-dependent cell responsiveness, with heparan sulfate having lesser activity
Recently, the potential roles of GAGs in specific biological processes including
angiogenesis 84, tumor growth 85, and bone metabolism 86 have been reported.
ECM components can alter the activity or availability of angiogenic cytokines in interactions such as that between heparan sulphates and fibroblast growth factors (Folkman et al., 1988) or between transforming growth factor fll and collagen IV
(Paralkar et al., 1991) or glycosaminoglycans.
Difference between heparin and heparin
Considerable confusion exists over the definition of heparin and HS. The major differences are summarized in Table 11.7. Heparin is produced by mast cells and sold by pharmaceutical companies as an anticoagulant. In contrast, HS is made by virtually all cells. It also can contain anticoagulant activity, but the crude preparations have much less activity than heparin. During biosynthesis, heparin undergoes more extensive sulfation and uronic acid epimerization, such that more than 85% of the GlcNAc residues are N-deacetylated and N-sulfated and more than 70% of the uronic acid is converted to IdoA.