forming of collagen
Within the first few minutes after the injury, platelets (thrombocytes) aggregate (join together) at the injury site to form a fibrin clot, which reduces active bleeding (hemostasis). The speed of wound healing depends on bloodstream levels of platelets, fibrin, and hormones such as oxytocin. When tissue is first wounded, blood touches collagen, causing blood platelets to secrete inflammatory factors.[11] Platelets also express sticky glycoproteins on their cell membranes that allow them to aggregate.
Platelets, the most numerous cells soon after wounding, into the blood release such substances as ECM proteins and cytokines, including growth factors.[11] Growth factors stimulate cells to speed their rate of division. Platelets also release other pro inflammatory factors like serotonin, bradykinin, prostaglandins, prostacyclins, thromboxane, and histamine,[2] which serve such purposes as increasing cell proliferation and migration to the area and causing blood vessels to become dilated and porous
Within an hour of wounding, polymorphonuclear neutrophils (PMNs) arrive at the wound site and become the predominant cells in the wound for the first two days after the injury occurs, with especially high numbers on the second day.[15] Fibronectin, growth factors, and such substances as kinins attract them to the wound. Neutrophils phagocytise debris and bacteria, kill bacteria by releasing free radicals in what is called a 'respiratory burst,' and cleanse the wound by secreting proteases that decompose damaged tissue. Neutrophils usuallyundergo apoptosis after completing their tasks and are engulfed and degraded by macrophages.[18]
Other leukocytes to enter the area include helper T cells, which secrete cytokines to cause more T cells to divide and to increase inflammation and enhance vasodilation and vessel permeability. T cells also increase the activity of macrophages.
Fibrin and fibronectin link together and form a plug that traps proteins and particles and prevents further blood loss.[12] This fibrin-fibronectin plug is also the main structural support for the wound until collagen is deposited. Migratory cells use this plug as a matrix across which to crawl, and platelets adhere to it and secrete factors. The clot is eventually lysed and replaced with granulation tissue and then later with collagen.
.The classic model of wound healing comprises three or four sequential, yet overlapping, phases: (1) Hemostasis , (2) inflammation, (3) Proliferation and (4) Matuaration or remodelling. Upon injury to the skin, a set of complex biochemical events takes place in a closely orchestrated cascade to repair the damage
Just before the inflammatory phase, the clotting cascade occurs in order to achieve hemostasis, or stop blood loss by way of a fibrin clot. Various soluble factors (including chemokines and cytokines) thereafter are released to attract cells that phagocytise debris, bacteria, and damaged tissue and release signaling molecules that initiate the proliferative phase of wound healing.
Tissue damage and the activation of clotting factors during the vascular phase stimulates the release of inflammatory mediators such as prostaglandins and histamine from cells such as mast cells. These mediators cause blood vessels adjacent to the injured area to become more permeable and to vasodilate. This inflammatory response can be detected by the presence of localised heat, swelling, erythema, discomfort and functional disturbance.1
Although the clinical signs are similar, inflammation should not be confused with wound infection. The classic signs of inflammation are due to increased blood flow to the area and the accumulation of fluid in the soft tissues. Wound exudate is produced during
this stage of healing due to the increased permeability of the capillary membranes.
Exudate contains proteins and a variety of nutrients, growth factors and enzymes which facilitate healing. It also has antimicrobial properties .Exudate production, which is most
prolific during the inflammatory phase of healing, bathes the wound with nutrients and actively cleanses the wound surface. It also acts as a growth medium for phagocytic cells. However, excessive exudate production can cause skin sensitivities and tissue maceration.
Neutrophils are the first type of white blood cell to be attracted into the wound, usually arriving within a few hours of injury. These phagocytic cells have a short life span but provide initial protection against micro-organisms as they engulf and digest bacteria and cell debris by phagocytosis . After 2–3 days macrophages become the predominant leucocyte in the wound bed. Their function at this stage is to cleanse the wound. Macrophages are present throughout all stages of the healing process, producing a variety of substances that regulate healing including growth factors, prostaglandins and complement factors (complex proteins) Patients who are immunosuppressed are often unable to produce a typical inflammatory response, so may fail to activate the normal healing process.
Slough formation is common during the inflammatory stage and occurs when a collection of dead cellular debris accumulates on the wound surface. It may be creamy yellow due to the large amounts of leucocytes present. Chronic wounds may develop areas of fibrous tissue covering the wound base. This often combines with slough, making it harder to remove.
Formation of new tissue in the wound bed will not occur until the macrophages have stimulated the proliferative phase by the release of growth factors and the wound bed has been sufficiently cleansed by the inflammatory process. Macrophages are responsible for control-ling the transition between the inflammatory and proliferative phases of healing
During the inflammation phase, bacteria and debris removed from the wound by white blood cells. Blood factors are released into the wound that cause the migration and division of cells during the proliferative phase.
During this phase the wound is filled with new connective tissue. A decrease in wound size is achieved by a combination of the physiological processes of granulation, contraction and epithelialisation.
The proliferation phase is characterized by angiogenesis, In fibroplasia and granulation tissue formation, fibroblasts grow and form a new, extracellular material called ground substance . (ECM) by excreting collagen and fibronectin.[5] These provide the scaffolding into which new capillaries will grow to form connective tissue. The growth of new blood vessels is termed angiogenesis. This is stimulated by macrophage activity and tissue hypoxia resulting from the disruption of blood flow at the time of injury. The role of oxygen in wound healing is a must where hydrogen peroxide is used to cleanse the wound several times /day to remove the cell debris and bacteria toxins cytokines etc from wound surface which interfere with healing of wounds due t o low macrophages , and also supply of oxygen to wounds to stimulate exchange of gases to enhance blood supply to new tissue for supply of nutrients .
Macrophages produce a variety of substances that stimulate angiogenesis.
These include transforming growth factor (TGF), which promotes formation of new tissue and blood vessels, and tumour necrosing factor (TNF), which facilitates the breakdown of necrotic tissue, stimulating proliferation.
Healthy granulation tissue does not bleed easily and is a pinky red colour. The condition of granulation tissue is often a good indicator as to how the wound is healing. Granulation tissue which is dark in colour may signal that the wound is ischaemic or infected.
.
Molecular Recognition in the Assembly of Collagens: Terminal Noncollagenous Domains Are Key Recognition Modules in the Formation of Triple Helical Protomers*
+ Author Affiliations
Previous SectionNext Section Collagens are modular triple helical proteins that constitute the major structural components of the extracellular matrix of all animals. They occur as diverse suprastructures such as fibrils, microfibrils, and networks, which serve as self-organizing scaffolds for the attachment of other macromolecular complexes including laminin networks, proteoglycans, and cell surface receptors. The suprastructures play functional roles in cell adhesion, cell differentiation, tissue development, and the structural integrity of organs.
Collagen suprastructures are assembled from a large family of gene products called α-chains. To date, 43 unique α-chains that belong to 28 types of collagens (types I–XXVIII) have been discovered in vertebrates. Based on their supramolecular architectures, they are further classified as fibril-forming, fibril-associated containing interrupted triple helices (FACIT),3 beaded filament, anchoring fibril, network-forming, and transmembrane collagens (1–4).
The assembly of all collagen suprastructures begins with the association of three type-specific α-chains (trimerization) that subsequently intertwine to form triple helical protomers, the building blocks of larger assemblies (1–5). Protomers are homotrimers or heterotrimers, composed of up to three different α-chains. They have in common at least one triple helical collagenous domain of varying length and two noncollagenous domains (NC) of variable sequence, size, and shape that are positioned at the N and C termini, designated herein as N-NC and C-NC domains, respectively. The terminal NC domains are excised, modified, or incorporated directly into the final suprastructure, depending on protomer type and function. Subsequently, specific protomers oligomerize into distinct suprastructures involving interactions that form end-to-end connections, lateral associations, and supercoiling of helices. Thus, protomer formation and oligomerization involve pivotal recognitions steps that target specific α-chains to assemble into a particular type of suprastructure.
How the α-chains selectively recognize each other is a fundamental question in matrix biology that remains largely unanswered. Early studies on collagens I and III suggested that the C-NC domains play a critical role in the trimerization step that involves selection, binding, and registration of three α-chains (6–9). In this article, we review recent findings on the trimerization of collagens, with an emphasis on collagens I and IV, which are the best understood.
Previous SectionNext Section Fibril-forming Collagens Fibril-forming collagens include the widely distributed types I, III, and V, types II and XI, prominent in cartilage and eye, and the newly discovered types XXIV and XXVII (10–12). All of the 12 fibril-forming α-chains share a long uninterrupted collagenous domain flanked by N- and C-terminal noncollagenous propeptides. The α-chains assemble into at least 12 type-specific protomers, characterized as homo- and heterotrimers. The terminal propeptides are further cleaved by specific proteases, which promote oligomerization and fibril formation.
Type I collagen is composed of α1- and α2-chains, forming preferably an α1·α1·α2 heterotrimer (Fig. 1). However, if only the triple helical domains are considered, the (α1)3 homotrimer would be the preferred form (13, 14). The pro-α1 homotrimer is formed as the default structure in the absence of pro-α2-chain, whereas the opposite is not true (15). However, if the α2 C-NC domain is replaced with an artificial trimeric NC domain, an α2 homotrimer can form (16, 17). Thus the α2 C-NC domain is the key domain required for heterotrimer assembly, although the α1 C-NC domain contains sufficient information for directing homotrimer formation. Disulfide bonding in the α2 C-NC domain is crucial for heterotrimer formation (18), particularly within the most C-terminal sequence of the α2 C-NC domain (19, 20).
View larger version: FIGURE 1. Protomer assembly of collagen I. Assembly is initiated by specific interactions of the C-NC domains of the α1- and α2-chains, after which folding of the triple helical domain proceeds toward the amino terminus forming a heterotrimeric protomer. Subdomains dock through interactions involving recognition sites. A computer-generated animation is available at www.mc.vanderbilt.edu/cmb/collagen.
The putative conformation and energy-minimized structures of the human α1 C-NC and α2 C-NC reveal (21, 22) that each contains five subdomains (Fig. 1A) (8, 23). Domain I folds into subdomains Ia and Ib without α-helical or β-sheet conformations, inconsistent with suggestions that subdomain Ia participates in trimerization by forming α-helical coiled-coils (23–25). Subdomain Ib contains the interchain disulfide bonds in the assembled C-NC trimer. Domains II and IV fold into globular regions G1 and G2, respectively. These are linked by an antiparallel β-sheet assembled from domains III and V (Fig. 1).
Energy minimization and molecular dynamics simulations have revealed key steps in the folding, docking, and assembly of two α1 C-NC (α11 and α12) and α2 C-NC domains into a heterotrimer (26). Folding is initiated at the C terminus by bimolecular association of the α2 and α12, near the junction of domain III and domain IV-G2, and proceeds toward the N terminus (Fig. 1B). The α2 domain V appears to have a dominant effect on heterotrimer formation (19, 20). Trimerization proceeds via a second interaction between the α2-α12 dimer and the α11 at domain II-G1 after which the interchain disulfide bonds become established in domain Ib. Folding of domain Ia is the last and slowest folding step but eventually drives the folding through the C-telo-Ia junction (26, 27). Overall, the α2 C-NC domain appears to provide the driving force for heterotrimer formation. Computer modeling of collagen I C-propeptide (26) also suggests that the homotrimer could resemble a cruciform as seen in the collagen III C-propeptide (28).
Putative trimerization control sequences have been located within the C-NC domains (17, 29). Replacing the α2(I) C-NC domain of the α2(I)-chain with the homotrimer-directing α1(III) C-NC permitted α2·α2·α2 homo-trimerization (17, 29)), demonstrating that the α1 C-NC domain is sufficient to direct homotrimer assembly of α2-chains with aligned triple helices. Exchanging specific sequences from the α1 C-NC domain of collagen III with the corresponding sequences of the α2 C-NC of collagen I revealed the location of a putative recognition site, a discontinuous sequence of 15 amino acids, located at the C-terminal of subdomain III (Fig. 1). This α1 sequence, when transferred to the sequence-equivalent region of the α2 C-NC domain, enabled the α2-chains to homotrimerize (17, 29). Thus, this recognition site is both necessary and sufficient to ensure that collagen chains discriminate between each other to form type-specific protomers (Fig. 1, A and C).
Previous SectionNext Section Network-forming Collagens The network-forming collagens include types IV, VIII and X. In contrast to the fibril-forming collagens, their C-NC domains are retained in their suprastructures. Importantly, crystal structures of their C-NC domains are known, which provide insight into the mechanisms of chain selection.
Collagen IV—Type IV collagen is the major constituent of basement membranes. It is composed of a family of six homologous α-chains (α1–α6), each characterized by a long collagenous domain of ∼1400 residues, interrupted by about 20 short noncollagenous sequences, which is flanked by a short N-NC sequence of 25 residues and a globular C-NC domain of 230 residues. The six α-chains assemble into three heterotrimeric protomers (α1·α1·α2, α3·α4·α5, and α5·α5·α6), which further assemble into three distinct networks (30).
Collagen IV has provided a unique opportunity to gain insight into chain recognition mechanisms. First, the six α-chains assemble into 3 specific protomers out of 76 possible combinations, reflecting a remarkable specificity for chain selection in vivo (30, 31). Second, the extraordinary capacity of monomeric C-NC domains to recognize each other and reassemble in vitro into their original trimeric and hexameric compositions (32), together with their critical role in trimerization in vivo (33), indicates a recognition role in assembly and provides the means to characterize specificity and binding parameters (34). Third, the crystal structure of the C-NC hexamer is known (31, 35), which reveals the nature of intermolecular interactions and their role in chain selectivity. Fourth, the known primary structure for all six C-NC domains from many species provides information needed to deduce candidate recognition sites from sequence variation (34). Finally, a study of sequence variation, coupled with structural and kinetic analysis, provides the requisite information needed to decipher the location and three-dimensional features of putative recognition sites (34).
Two putative recognition sites were identified in the C-NC domains as prominent mediators in the mechanism of chain recognition (34). These sites (Fig. 2D) constitute a 13-residue-long β-hairpin motif (site 1) involved in the domain swapping mechanism and a 15-residue-long variable region (site 2, VR3, docking site) with sequence hypervariability across all the chains of collagen IV (34). Accordingly, we have proposed that the combination of sequence variations at these two sites and at the neighboring C-NC domains to which they interact constitutes the code that directs chain recognition in the trimerization of collagen IV (34). The relatively higher affinity of α2 C-NC domain for dimer formation with an α1 C-NC domain is the driving force in directing stoichiometry in the assembly of α1·α1·α2 heterotrimers, as determined by kinetic studies using surface plasmon resonance (34).
View larger version: FIGURE 2. Protomer assembly of collagen IV. Assembly is initiated by specific interactions of the C-NC domains (A), after which folding of the triple helical domain proceeds toward the amino terminus, forming a heterotrimeric protomer. Dimerization of protomers occurs via the equatorial faces (A and B) of the NC trimers, yielding NC hexamers that are stabilized by local and extensive hydrophobic and hydrophilic forces. Within each NC trimer, the monomers recognize one another through a domain-swapping mechanism in which a β-hairpin motif (Site 1) of one monomer is swapped into a docking site (Site 2) of its swapping partner (C and D). Dimers also oligomerize at their N termini forming network suprastructures. Assembly is also illustrated by a computer-generated animation available at www.mc.vanderbilt.edu/cmb/collagen.
Collagen VIII—Type VIII collagen is a prominent component of Descemet membrane and in the subendothelium of vascular walls (36). It is composed of highly conserved α1(VIII)- and α2(VIII)-chains, which contain a short collagenous domain of 454 residues flanked by a N-NC domain of 117 residues and a C-NC domain of 173 residues (37). The chains assemble into two distinct homotrimers that assemble into hexagonal lattices (38, 39). The crystal structure of the α1 C-NC homotrimer is known (40), but the basis for the preferred homotrimer structure remains unclear.
Collagen X—Type X collagen is expressed in the endochondral growth plate. The single α1-chain contains a short collagenous domain of 154 residues, flanked by a N-NC domain of 37 residues and a C-NC domain of 161 residues (41). Its protomer is a homotrimer. The C-NC domains function as nucleation sites for trimer and multimer formation, based on experiments using recombinant C-NC domains (42). A comparison of the x-ray crystal structures of the C-NC trimer of collagens X and VIII revealed them to be highly homologous, but they differ in intermolecular contact residues that govern trimer stability. Such differences may also confer specificity (40, 43).
Previous SectionNext Section FACIT Collagens The FACIT collagens include types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII. Collagen IX is composed of three α-chains and all others of one α-chain, each of which is characterized by short collagenous domains interrupted by several NC domains (1, 2). The protomer of collagen IX is a heterotrimer, and all others are homotrimers, accounting for at least 9 distinct protomers. Unlike the fibril-forming collagens, the FACITs have significantly shorter C-NC domains: 75 residues for collagen XII and fewer than 30 residues for collagen IX, whereas those of fibrillar collagen are about 260 residues. The FACITs share a remarkable sequence homology at their COL1/C-NC1 junctions by having two strictly conserved cysteine residues separated by four residues at the NC domain. Several lines of evidence suggest that the COL1 domain at the COL1/C-NC junction (which contains the first cysteine residue) and the C-NC domain (containing the second conserved cysteine) are involved in the mechanism of chain selection in the assembly of collagens XII and XIV (44–46). Independent studies by Mazzorana et al. (44, 45) and Lesage et al. (46) strongly suggest that both the collagenous portion and the NC domain are involved in formation and stabilization of protomers in collagens XII and XIV (47).
Previous SectionNext Section Beaded Filament and Anchoring Fibril Collagens Collagen VI—Type VI collagen is ubiquitously expressed in connective tissues. It is composed of three genetically distinct α-chains, α1, α2, and α3, that form extensive beaded filaments (48–50). Each chain contains a relatively short collagenous domain of ∼335 residues flanked by N-NC and C-NC globular domains The α1- and α2-chains are similar in size and contain one N-NC domain and a C-NC domain with two subdomains, C1 and C2. In contrast, the α3-chain is much longer, and the N-NC domain contains ten subdomains, N1–N10, and five C-NC subdomains, C1–C5 (48). The protomer is an α1·α2·α3 heterotrimer. Studies conducted on cell lines lacking or expressing different combinations of recombinant α-chains have shown that the α3-chain contains sequences necessary for chain association and protomer formation (48, 50). Deletion studies have demonstrated that whereas the C5 subdomain of the α3-chain is required for the extracellular microfibril formation, the C1 subdomain, illustrated in Fig. 3, in all chains is sufficient for chain recognition and protomer assembly (48, 50).
View larger version: FIGURE 3. A summary of structural models of the N and C noncollagenous domains of various classes of collagen suprastructures. The NC domains function as recognition modules in the trimerization of cognate α-chains, a pivotal step in the self-assembly of type-specific protomers and suprastructures. MM/MD, molecular modeling/molecular dynamics.
Collagen VII—Type VII collagen is a major component of anchoring fibrils that maintain the epidermal-dermal adherence of skin. It is composed of three identical α-chains, each consisting of a collagenous domain of 1530 residues with 19 interruptions, which are flanked by N-NC and C-NC domains (51). Protomers form anti-parallel dimers that are stabilized by disulfide bonding via the C-NC domains of adjacent protomers. Following proteolytic modification of the C-NC domain, dimers of protomers oligomerize laterally to form anchoring fibrils with large globular N-NC domains at both ends of the microfibril (52). Recent studies, using recombinant constructs and site-directed mutagenesis, have revealed that the C-NC domain mediates protomer formation and oligomerization of protomers (52).
Previous SectionNext Section Transmembrane Collagens Transmembrane collagens include types XIII, XVII, XXIII, and XXV and other collagen-related proteins, such as the macrophage receptor MARCO (53). They function as cell surface receptors and matrix components (54). The α-chain of each type contains an N-terminal NC domain, which is comprised of (a) three subdomains, an intracellular, a single transmembrane, and an extracellular juxtamembrane linker subdomain and (b) a large extracellular domain, which is composed of multiple collagenous domains interrupted by NC domains. The protomer of each α-chain is a homotrimer. The extracellular linker subdomain contains an α-helical coiled-coil, the conformation of which is thought to prompt trimerization and subsequent zipper-like folding of the triple helical domain. Studies using deletion constructs have shown that the N-NC domains of collagens XIII and XVII are necessary for triple helix formation, suggesting that nucleation of the triple helix occurs at the N-terminal region and proceeds in a N- to C-terminal direction, which is opposite to that of all other classes of collagens (55, 56). For collagen XIII, a short sequence of 21 residues located at the juxtamembrane linker subdomain, has been identified as a putative recognition site for trimerization (Fig. 3).
Previous SectionNext Section Other Collagens Little is known about the assembly of collagen XV (57) and XVIII (58) and the newly discovered XXVI (59) and XXVIII (4). Each type is composed of a single α-chain that contains a collagenous domain, with frequent interruptions, flanked by N-NC and C-NC domains. The C-NC domain of XVIII forms trimers in vitro suggesting a role in protomer assembly (60). The crystal structures of fragments of C-NC domains of XV and XVIII are highly similar but distinct (61, 62), and they differ greatly with the three-dimensional structures of the C-NC domains of collagens VIII, X, and IV (see above); such distinctions are consistent with a special role of C-NC domains in chain selection.
Previous SectionNext Section Conclusions and Future Perspectives Over the last decade, findings have emerged on several collagen types that provide new insights into the chain selection mechanism. Based on the body of evidence reviewed here, we infer that the terminal NC domains function as recognition modules that select, bind, and register three cognate α-chains for self-assembly of triple helical protomers. The C-NC domains govern chain selection for all classes except for the transmembrane collagens, which are governed by the N-NC domains (Fig. 3). As recognition modules, they contain sites that are characterized by positive determinants of shape complementarity, electrostatic charge distribution and magnitude, and hydrophobicity for selection of cognate α-chains and negative determinants for the exclusion of all other unrelated α-chains.
Following trimerization, coupled with triple helix formation, new recognition sites are encoded, a consequence of forming quaternary structures that commit protomers to oligomerize with cognate protomers into a particular type of suprastructure such as a fibril or network. The quaternary recognition sites specify such features as enzymatic cleavage sites, end-to-end connections, lateral associations, supercoiling of helices, and cross-link formation, as well as interactions with other macromolecular complexes such as proteoglycans (63) and integrin receptors (64). Therefore, by directing formation of triple helical protomers with distinct chain compositions and quaternary structures, the NC recognition modules are key determinants of specificity that target a type of α-chain to assemble into distinct suprastructures.
Many important features remain to be elucidated about the NC recognition mechanisms. The C-NC modules of heterotrimers composed of three different chains (collagens IV, V, VI, and IX) are the most promising models for study because their trimeric forms display quaternary structural features that can be differentially analyzed with regard to the identity of recognition motifs and the cyclic order of chains in the triple helix. Such mechanistic information provides opportunities for engineering new suprastructures for biomaterials and development of therapeutic strategies that facilitate or interfere with collagen assembly in various disorders.
Platelets, the most numerous cells soon after wounding, into the blood release such substances as ECM proteins and cytokines, including growth factors.[11] Growth factors stimulate cells to speed their rate of division. Platelets also release other pro inflammatory factors like serotonin, bradykinin, prostaglandins, prostacyclins, thromboxane, and histamine,[2] which serve such purposes as increasing cell proliferation and migration to the area and causing blood vessels to become dilated and porous
Within an hour of wounding, polymorphonuclear neutrophils (PMNs) arrive at the wound site and become the predominant cells in the wound for the first two days after the injury occurs, with especially high numbers on the second day.[15] Fibronectin, growth factors, and such substances as kinins attract them to the wound. Neutrophils phagocytise debris and bacteria, kill bacteria by releasing free radicals in what is called a 'respiratory burst,' and cleanse the wound by secreting proteases that decompose damaged tissue. Neutrophils usuallyundergo apoptosis after completing their tasks and are engulfed and degraded by macrophages.[18]
Other leukocytes to enter the area include helper T cells, which secrete cytokines to cause more T cells to divide and to increase inflammation and enhance vasodilation and vessel permeability. T cells also increase the activity of macrophages.
Fibrin and fibronectin link together and form a plug that traps proteins and particles and prevents further blood loss.[12] This fibrin-fibronectin plug is also the main structural support for the wound until collagen is deposited. Migratory cells use this plug as a matrix across which to crawl, and platelets adhere to it and secrete factors. The clot is eventually lysed and replaced with granulation tissue and then later with collagen.
.The classic model of wound healing comprises three or four sequential, yet overlapping, phases: (1) Hemostasis , (2) inflammation, (3) Proliferation and (4) Matuaration or remodelling. Upon injury to the skin, a set of complex biochemical events takes place in a closely orchestrated cascade to repair the damage
Just before the inflammatory phase, the clotting cascade occurs in order to achieve hemostasis, or stop blood loss by way of a fibrin clot. Various soluble factors (including chemokines and cytokines) thereafter are released to attract cells that phagocytise debris, bacteria, and damaged tissue and release signaling molecules that initiate the proliferative phase of wound healing.
Tissue damage and the activation of clotting factors during the vascular phase stimulates the release of inflammatory mediators such as prostaglandins and histamine from cells such as mast cells. These mediators cause blood vessels adjacent to the injured area to become more permeable and to vasodilate. This inflammatory response can be detected by the presence of localised heat, swelling, erythema, discomfort and functional disturbance.1
Although the clinical signs are similar, inflammation should not be confused with wound infection. The classic signs of inflammation are due to increased blood flow to the area and the accumulation of fluid in the soft tissues. Wound exudate is produced during
this stage of healing due to the increased permeability of the capillary membranes.
Exudate contains proteins and a variety of nutrients, growth factors and enzymes which facilitate healing. It also has antimicrobial properties .Exudate production, which is most
prolific during the inflammatory phase of healing, bathes the wound with nutrients and actively cleanses the wound surface. It also acts as a growth medium for phagocytic cells. However, excessive exudate production can cause skin sensitivities and tissue maceration.
Neutrophils are the first type of white blood cell to be attracted into the wound, usually arriving within a few hours of injury. These phagocytic cells have a short life span but provide initial protection against micro-organisms as they engulf and digest bacteria and cell debris by phagocytosis . After 2–3 days macrophages become the predominant leucocyte in the wound bed. Their function at this stage is to cleanse the wound. Macrophages are present throughout all stages of the healing process, producing a variety of substances that regulate healing including growth factors, prostaglandins and complement factors (complex proteins) Patients who are immunosuppressed are often unable to produce a typical inflammatory response, so may fail to activate the normal healing process.
Slough formation is common during the inflammatory stage and occurs when a collection of dead cellular debris accumulates on the wound surface. It may be creamy yellow due to the large amounts of leucocytes present. Chronic wounds may develop areas of fibrous tissue covering the wound base. This often combines with slough, making it harder to remove.
Formation of new tissue in the wound bed will not occur until the macrophages have stimulated the proliferative phase by the release of growth factors and the wound bed has been sufficiently cleansed by the inflammatory process. Macrophages are responsible for control-ling the transition between the inflammatory and proliferative phases of healing
During the inflammation phase, bacteria and debris removed from the wound by white blood cells. Blood factors are released into the wound that cause the migration and division of cells during the proliferative phase.
During this phase the wound is filled with new connective tissue. A decrease in wound size is achieved by a combination of the physiological processes of granulation, contraction and epithelialisation.
The proliferation phase is characterized by angiogenesis, In fibroplasia and granulation tissue formation, fibroblasts grow and form a new, extracellular material called ground substance . (ECM) by excreting collagen and fibronectin.[5] These provide the scaffolding into which new capillaries will grow to form connective tissue. The growth of new blood vessels is termed angiogenesis. This is stimulated by macrophage activity and tissue hypoxia resulting from the disruption of blood flow at the time of injury. The role of oxygen in wound healing is a must where hydrogen peroxide is used to cleanse the wound several times /day to remove the cell debris and bacteria toxins cytokines etc from wound surface which interfere with healing of wounds due t o low macrophages , and also supply of oxygen to wounds to stimulate exchange of gases to enhance blood supply to new tissue for supply of nutrients .
Macrophages produce a variety of substances that stimulate angiogenesis.
These include transforming growth factor (TGF), which promotes formation of new tissue and blood vessels, and tumour necrosing factor (TNF), which facilitates the breakdown of necrotic tissue, stimulating proliferation.
Healthy granulation tissue does not bleed easily and is a pinky red colour. The condition of granulation tissue is often a good indicator as to how the wound is healing. Granulation tissue which is dark in colour may signal that the wound is ischaemic or infected.
.
Molecular Recognition in the Assembly of Collagens: Terminal Noncollagenous Domains Are Key Recognition Modules in the Formation of Triple Helical Protomers*
- Jamshid Khoshnoodi‡,1,
- Jean-Philippe Cartailler§,1,
- Keith Alvares¶,
- Arthur Veis¶ and
- Billy G. Hudson‡,2
+ Author Affiliations
- ‡Department of Medicine, Vanderbilt University Medical Center, Nashville, Tennessee 37232-2372, §Symmation LLC, Spring Hill, Tennessee 37174, and ¶Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611
- ↵2 To whom correspondence should be addressed: Dept. of Medicine, Vanderbilt University Medical Center, S-3223 Medical Center North, 1161, 21st Ave. South, Nashville, TN 37232-2372. Tel.: 615-322-7299; Fax: 615-322-7381; E-mail: [email protected].
Previous SectionNext Section Collagens are modular triple helical proteins that constitute the major structural components of the extracellular matrix of all animals. They occur as diverse suprastructures such as fibrils, microfibrils, and networks, which serve as self-organizing scaffolds for the attachment of other macromolecular complexes including laminin networks, proteoglycans, and cell surface receptors. The suprastructures play functional roles in cell adhesion, cell differentiation, tissue development, and the structural integrity of organs.
Collagen suprastructures are assembled from a large family of gene products called α-chains. To date, 43 unique α-chains that belong to 28 types of collagens (types I–XXVIII) have been discovered in vertebrates. Based on their supramolecular architectures, they are further classified as fibril-forming, fibril-associated containing interrupted triple helices (FACIT),3 beaded filament, anchoring fibril, network-forming, and transmembrane collagens (1–4).
The assembly of all collagen suprastructures begins with the association of three type-specific α-chains (trimerization) that subsequently intertwine to form triple helical protomers, the building blocks of larger assemblies (1–5). Protomers are homotrimers or heterotrimers, composed of up to three different α-chains. They have in common at least one triple helical collagenous domain of varying length and two noncollagenous domains (NC) of variable sequence, size, and shape that are positioned at the N and C termini, designated herein as N-NC and C-NC domains, respectively. The terminal NC domains are excised, modified, or incorporated directly into the final suprastructure, depending on protomer type and function. Subsequently, specific protomers oligomerize into distinct suprastructures involving interactions that form end-to-end connections, lateral associations, and supercoiling of helices. Thus, protomer formation and oligomerization involve pivotal recognitions steps that target specific α-chains to assemble into a particular type of suprastructure.
How the α-chains selectively recognize each other is a fundamental question in matrix biology that remains largely unanswered. Early studies on collagens I and III suggested that the C-NC domains play a critical role in the trimerization step that involves selection, binding, and registration of three α-chains (6–9). In this article, we review recent findings on the trimerization of collagens, with an emphasis on collagens I and IV, which are the best understood.
Previous SectionNext Section Fibril-forming Collagens Fibril-forming collagens include the widely distributed types I, III, and V, types II and XI, prominent in cartilage and eye, and the newly discovered types XXIV and XXVII (10–12). All of the 12 fibril-forming α-chains share a long uninterrupted collagenous domain flanked by N- and C-terminal noncollagenous propeptides. The α-chains assemble into at least 12 type-specific protomers, characterized as homo- and heterotrimers. The terminal propeptides are further cleaved by specific proteases, which promote oligomerization and fibril formation.
Type I collagen is composed of α1- and α2-chains, forming preferably an α1·α1·α2 heterotrimer (Fig. 1). However, if only the triple helical domains are considered, the (α1)3 homotrimer would be the preferred form (13, 14). The pro-α1 homotrimer is formed as the default structure in the absence of pro-α2-chain, whereas the opposite is not true (15). However, if the α2 C-NC domain is replaced with an artificial trimeric NC domain, an α2 homotrimer can form (16, 17). Thus the α2 C-NC domain is the key domain required for heterotrimer assembly, although the α1 C-NC domain contains sufficient information for directing homotrimer formation. Disulfide bonding in the α2 C-NC domain is crucial for heterotrimer formation (18), particularly within the most C-terminal sequence of the α2 C-NC domain (19, 20).
View larger version: FIGURE 1. Protomer assembly of collagen I. Assembly is initiated by specific interactions of the C-NC domains of the α1- and α2-chains, after which folding of the triple helical domain proceeds toward the amino terminus forming a heterotrimeric protomer. Subdomains dock through interactions involving recognition sites. A computer-generated animation is available at www.mc.vanderbilt.edu/cmb/collagen.
The putative conformation and energy-minimized structures of the human α1 C-NC and α2 C-NC reveal (21, 22) that each contains five subdomains (Fig. 1A) (8, 23). Domain I folds into subdomains Ia and Ib without α-helical or β-sheet conformations, inconsistent with suggestions that subdomain Ia participates in trimerization by forming α-helical coiled-coils (23–25). Subdomain Ib contains the interchain disulfide bonds in the assembled C-NC trimer. Domains II and IV fold into globular regions G1 and G2, respectively. These are linked by an antiparallel β-sheet assembled from domains III and V (Fig. 1).
Energy minimization and molecular dynamics simulations have revealed key steps in the folding, docking, and assembly of two α1 C-NC (α11 and α12) and α2 C-NC domains into a heterotrimer (26). Folding is initiated at the C terminus by bimolecular association of the α2 and α12, near the junction of domain III and domain IV-G2, and proceeds toward the N terminus (Fig. 1B). The α2 domain V appears to have a dominant effect on heterotrimer formation (19, 20). Trimerization proceeds via a second interaction between the α2-α12 dimer and the α11 at domain II-G1 after which the interchain disulfide bonds become established in domain Ib. Folding of domain Ia is the last and slowest folding step but eventually drives the folding through the C-telo-Ia junction (26, 27). Overall, the α2 C-NC domain appears to provide the driving force for heterotrimer formation. Computer modeling of collagen I C-propeptide (26) also suggests that the homotrimer could resemble a cruciform as seen in the collagen III C-propeptide (28).
Putative trimerization control sequences have been located within the C-NC domains (17, 29). Replacing the α2(I) C-NC domain of the α2(I)-chain with the homotrimer-directing α1(III) C-NC permitted α2·α2·α2 homo-trimerization (17, 29)), demonstrating that the α1 C-NC domain is sufficient to direct homotrimer assembly of α2-chains with aligned triple helices. Exchanging specific sequences from the α1 C-NC domain of collagen III with the corresponding sequences of the α2 C-NC of collagen I revealed the location of a putative recognition site, a discontinuous sequence of 15 amino acids, located at the C-terminal of subdomain III (Fig. 1). This α1 sequence, when transferred to the sequence-equivalent region of the α2 C-NC domain, enabled the α2-chains to homotrimerize (17, 29). Thus, this recognition site is both necessary and sufficient to ensure that collagen chains discriminate between each other to form type-specific protomers (Fig. 1, A and C).
Previous SectionNext Section Network-forming Collagens The network-forming collagens include types IV, VIII and X. In contrast to the fibril-forming collagens, their C-NC domains are retained in their suprastructures. Importantly, crystal structures of their C-NC domains are known, which provide insight into the mechanisms of chain selection.
Collagen IV—Type IV collagen is the major constituent of basement membranes. It is composed of a family of six homologous α-chains (α1–α6), each characterized by a long collagenous domain of ∼1400 residues, interrupted by about 20 short noncollagenous sequences, which is flanked by a short N-NC sequence of 25 residues and a globular C-NC domain of 230 residues. The six α-chains assemble into three heterotrimeric protomers (α1·α1·α2, α3·α4·α5, and α5·α5·α6), which further assemble into three distinct networks (30).
Collagen IV has provided a unique opportunity to gain insight into chain recognition mechanisms. First, the six α-chains assemble into 3 specific protomers out of 76 possible combinations, reflecting a remarkable specificity for chain selection in vivo (30, 31). Second, the extraordinary capacity of monomeric C-NC domains to recognize each other and reassemble in vitro into their original trimeric and hexameric compositions (32), together with their critical role in trimerization in vivo (33), indicates a recognition role in assembly and provides the means to characterize specificity and binding parameters (34). Third, the crystal structure of the C-NC hexamer is known (31, 35), which reveals the nature of intermolecular interactions and their role in chain selectivity. Fourth, the known primary structure for all six C-NC domains from many species provides information needed to deduce candidate recognition sites from sequence variation (34). Finally, a study of sequence variation, coupled with structural and kinetic analysis, provides the requisite information needed to decipher the location and three-dimensional features of putative recognition sites (34).
Two putative recognition sites were identified in the C-NC domains as prominent mediators in the mechanism of chain recognition (34). These sites (Fig. 2D) constitute a 13-residue-long β-hairpin motif (site 1) involved in the domain swapping mechanism and a 15-residue-long variable region (site 2, VR3, docking site) with sequence hypervariability across all the chains of collagen IV (34). Accordingly, we have proposed that the combination of sequence variations at these two sites and at the neighboring C-NC domains to which they interact constitutes the code that directs chain recognition in the trimerization of collagen IV (34). The relatively higher affinity of α2 C-NC domain for dimer formation with an α1 C-NC domain is the driving force in directing stoichiometry in the assembly of α1·α1·α2 heterotrimers, as determined by kinetic studies using surface plasmon resonance (34).
View larger version: FIGURE 2. Protomer assembly of collagen IV. Assembly is initiated by specific interactions of the C-NC domains (A), after which folding of the triple helical domain proceeds toward the amino terminus, forming a heterotrimeric protomer. Dimerization of protomers occurs via the equatorial faces (A and B) of the NC trimers, yielding NC hexamers that are stabilized by local and extensive hydrophobic and hydrophilic forces. Within each NC trimer, the monomers recognize one another through a domain-swapping mechanism in which a β-hairpin motif (Site 1) of one monomer is swapped into a docking site (Site 2) of its swapping partner (C and D). Dimers also oligomerize at their N termini forming network suprastructures. Assembly is also illustrated by a computer-generated animation available at www.mc.vanderbilt.edu/cmb/collagen.
Collagen VIII—Type VIII collagen is a prominent component of Descemet membrane and in the subendothelium of vascular walls (36). It is composed of highly conserved α1(VIII)- and α2(VIII)-chains, which contain a short collagenous domain of 454 residues flanked by a N-NC domain of 117 residues and a C-NC domain of 173 residues (37). The chains assemble into two distinct homotrimers that assemble into hexagonal lattices (38, 39). The crystal structure of the α1 C-NC homotrimer is known (40), but the basis for the preferred homotrimer structure remains unclear.
Collagen X—Type X collagen is expressed in the endochondral growth plate. The single α1-chain contains a short collagenous domain of 154 residues, flanked by a N-NC domain of 37 residues and a C-NC domain of 161 residues (41). Its protomer is a homotrimer. The C-NC domains function as nucleation sites for trimer and multimer formation, based on experiments using recombinant C-NC domains (42). A comparison of the x-ray crystal structures of the C-NC trimer of collagens X and VIII revealed them to be highly homologous, but they differ in intermolecular contact residues that govern trimer stability. Such differences may also confer specificity (40, 43).
Previous SectionNext Section FACIT Collagens The FACIT collagens include types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII. Collagen IX is composed of three α-chains and all others of one α-chain, each of which is characterized by short collagenous domains interrupted by several NC domains (1, 2). The protomer of collagen IX is a heterotrimer, and all others are homotrimers, accounting for at least 9 distinct protomers. Unlike the fibril-forming collagens, the FACITs have significantly shorter C-NC domains: 75 residues for collagen XII and fewer than 30 residues for collagen IX, whereas those of fibrillar collagen are about 260 residues. The FACITs share a remarkable sequence homology at their COL1/C-NC1 junctions by having two strictly conserved cysteine residues separated by four residues at the NC domain. Several lines of evidence suggest that the COL1 domain at the COL1/C-NC junction (which contains the first cysteine residue) and the C-NC domain (containing the second conserved cysteine) are involved in the mechanism of chain selection in the assembly of collagens XII and XIV (44–46). Independent studies by Mazzorana et al. (44, 45) and Lesage et al. (46) strongly suggest that both the collagenous portion and the NC domain are involved in formation and stabilization of protomers in collagens XII and XIV (47).
Previous SectionNext Section Beaded Filament and Anchoring Fibril Collagens Collagen VI—Type VI collagen is ubiquitously expressed in connective tissues. It is composed of three genetically distinct α-chains, α1, α2, and α3, that form extensive beaded filaments (48–50). Each chain contains a relatively short collagenous domain of ∼335 residues flanked by N-NC and C-NC globular domains The α1- and α2-chains are similar in size and contain one N-NC domain and a C-NC domain with two subdomains, C1 and C2. In contrast, the α3-chain is much longer, and the N-NC domain contains ten subdomains, N1–N10, and five C-NC subdomains, C1–C5 (48). The protomer is an α1·α2·α3 heterotrimer. Studies conducted on cell lines lacking or expressing different combinations of recombinant α-chains have shown that the α3-chain contains sequences necessary for chain association and protomer formation (48, 50). Deletion studies have demonstrated that whereas the C5 subdomain of the α3-chain is required for the extracellular microfibril formation, the C1 subdomain, illustrated in Fig. 3, in all chains is sufficient for chain recognition and protomer assembly (48, 50).
View larger version: FIGURE 3. A summary of structural models of the N and C noncollagenous domains of various classes of collagen suprastructures. The NC domains function as recognition modules in the trimerization of cognate α-chains, a pivotal step in the self-assembly of type-specific protomers and suprastructures. MM/MD, molecular modeling/molecular dynamics.
Collagen VII—Type VII collagen is a major component of anchoring fibrils that maintain the epidermal-dermal adherence of skin. It is composed of three identical α-chains, each consisting of a collagenous domain of 1530 residues with 19 interruptions, which are flanked by N-NC and C-NC domains (51). Protomers form anti-parallel dimers that are stabilized by disulfide bonding via the C-NC domains of adjacent protomers. Following proteolytic modification of the C-NC domain, dimers of protomers oligomerize laterally to form anchoring fibrils with large globular N-NC domains at both ends of the microfibril (52). Recent studies, using recombinant constructs and site-directed mutagenesis, have revealed that the C-NC domain mediates protomer formation and oligomerization of protomers (52).
Previous SectionNext Section Transmembrane Collagens Transmembrane collagens include types XIII, XVII, XXIII, and XXV and other collagen-related proteins, such as the macrophage receptor MARCO (53). They function as cell surface receptors and matrix components (54). The α-chain of each type contains an N-terminal NC domain, which is comprised of (a) three subdomains, an intracellular, a single transmembrane, and an extracellular juxtamembrane linker subdomain and (b) a large extracellular domain, which is composed of multiple collagenous domains interrupted by NC domains. The protomer of each α-chain is a homotrimer. The extracellular linker subdomain contains an α-helical coiled-coil, the conformation of which is thought to prompt trimerization and subsequent zipper-like folding of the triple helical domain. Studies using deletion constructs have shown that the N-NC domains of collagens XIII and XVII are necessary for triple helix formation, suggesting that nucleation of the triple helix occurs at the N-terminal region and proceeds in a N- to C-terminal direction, which is opposite to that of all other classes of collagens (55, 56). For collagen XIII, a short sequence of 21 residues located at the juxtamembrane linker subdomain, has been identified as a putative recognition site for trimerization (Fig. 3).
Previous SectionNext Section Other Collagens Little is known about the assembly of collagen XV (57) and XVIII (58) and the newly discovered XXVI (59) and XXVIII (4). Each type is composed of a single α-chain that contains a collagenous domain, with frequent interruptions, flanked by N-NC and C-NC domains. The C-NC domain of XVIII forms trimers in vitro suggesting a role in protomer assembly (60). The crystal structures of fragments of C-NC domains of XV and XVIII are highly similar but distinct (61, 62), and they differ greatly with the three-dimensional structures of the C-NC domains of collagens VIII, X, and IV (see above); such distinctions are consistent with a special role of C-NC domains in chain selection.
Previous SectionNext Section Conclusions and Future Perspectives Over the last decade, findings have emerged on several collagen types that provide new insights into the chain selection mechanism. Based on the body of evidence reviewed here, we infer that the terminal NC domains function as recognition modules that select, bind, and register three cognate α-chains for self-assembly of triple helical protomers. The C-NC domains govern chain selection for all classes except for the transmembrane collagens, which are governed by the N-NC domains (Fig. 3). As recognition modules, they contain sites that are characterized by positive determinants of shape complementarity, electrostatic charge distribution and magnitude, and hydrophobicity for selection of cognate α-chains and negative determinants for the exclusion of all other unrelated α-chains.
Following trimerization, coupled with triple helix formation, new recognition sites are encoded, a consequence of forming quaternary structures that commit protomers to oligomerize with cognate protomers into a particular type of suprastructure such as a fibril or network. The quaternary recognition sites specify such features as enzymatic cleavage sites, end-to-end connections, lateral associations, supercoiling of helices, and cross-link formation, as well as interactions with other macromolecular complexes such as proteoglycans (63) and integrin receptors (64). Therefore, by directing formation of triple helical protomers with distinct chain compositions and quaternary structures, the NC recognition modules are key determinants of specificity that target a type of α-chain to assemble into distinct suprastructures.
Many important features remain to be elucidated about the NC recognition mechanisms. The C-NC modules of heterotrimers composed of three different chains (collagens IV, V, VI, and IX) are the most promising models for study because their trimeric forms display quaternary structural features that can be differentially analyzed with regard to the identity of recognition motifs and the cyclic order of chains in the triple helix. Such mechanistic information provides opportunities for engineering new suprastructures for biomaterials and development of therapeutic strategies that facilitate or interfere with collagen assembly in various disorders.