Proteoglycans in Normal and Healing Skin

Abstract

Significance: Proteoglycans have a distinct spatial localization in normal skin and are essential for the correct structural development, organization, hydration, and functional properties of this tissue. The extracellular matrix (ECM) is no longer considered to be just an inert supportive material but is a source of directive, spatial and temporal, contextual information to the cells via components such as the proteoglycans. There is a pressing need to improve our understanding of how these important molecules functionally interact with other matrix structures, cells and cellular mediators in normal skin and during wound healing.

Recent Advances: New antibodies to glycosaminoglycan side chain components of skin proteoglycans have facilitated the elucidation of detailed localization patterns within skin. Other studies have revealed important proliferative activities of proteinase-generated fragments of proteoglycans and other ECM components (matricryptins). Knockout mice have further established the functional importance of skin proteoglycans in the assembly and homeostasis of the normal skin ECM.

Critical Issues: Our comprehension of the molecular and structural complexity of skin as a complex, dynamic, constantly renewing, layered connective tissue is incomplete. The impact of changes in proteoglycans on skin pathology and the wound healing process is recognized as an important area of pathobiology and is an area of intense investigation.

Future Directions: Advanced technology is allowing the development of new artificial skins. Recent knowledge on skin proteoglycans can be used to incorporate these molecules into useful adjunct therapies for wound healing and for maintenance of optimal tissue homeostasis in aging skin.

Margaret Mary Smith, PhD

James Melrose, PhD

Introduction

Scope and significance

The scope of this review is to detail the complexity and localization of proteoglycans in skin. These structurally diverse molecules are now recognized as important in the development, function, metabolism, damage (whether by aging, ultraviolet [UV] irradiation, or injury), and healing of this tissue.

Translational relevance

Proteoglycans support the hydration of the extracellular matrix (ECM) of normal skin, providing resilience, viscoelasticity, and a cushioned environment conducive to cellular function and development. Proteoglycans also act in supportive scaffolding roles as struts and connectors, which aid in the proper alignment of fibrous and elastic components in skin. Many proteoglycans have the ability to sequester and control the bioavailability of growth factors in the ECM surrounding cells. These growth factors stimulate cell populations in skin that orchestrate the normal turnover and repair.

Clinical relevance

There is a critical need to recapitulate the normal intricately organized ECM of healthy young skin after injury. Armed with a greater knowledge of normal skin composition, structural organization, and the functional properties of its constituent proteoglycans, we will better understand deviations in these components that occur in aged and damaged skin, where healing may be slower, incomplete, and/or aberrant (fibrosis/scarring). This will lead to new treatments aimed at altering the content of certain proteoglycan components of the skin ECM to enhance repair and, ultimately, scarless wound healing.

Proteoglycans

Proteoglycans are glycosylated molecules where one or more specific glycosaminoglycan (GAG) and/or O- and N-linked oligosaccharides are attached to a core protein. The GAGs are usually sulfated; chondroitin sulfate/dermatan sulfate (CS/DS), keratan sulfate (KS), and heparan sulfate (HS)/heparin are the most common.¹ GAG chain length, degree, and position of sulfation and degree of epimerization greatly vary, (1) between different proteoglycans, (2) on the same proteoglycan at different sites, and (3) between the same proteoglycans in different tissues. These variations in GAG attachments are of both functional and developmental significance. Chondroitin 4-O-sulfation is required for proper CS localization and modulation of signaling pathways in tissue morphogenesis and emerging biological roles in mammalian development.^2,3 Detailed structural analyses on HS and heparin indicate these molecules are important in information storage and transfer.⁴

The complexity of these sugar-protein structures suggests new facets to an old paradigm in developmental biology, with the emergence of the sugar code and realization that dynamic changes in HS produce a characteristic (nonrandom) heparanome for cells.¹ GAGs can interact with many bioactive binding partners to trigger cell signaling, proliferation, ECM production, and differentiation, underscoring their importance in developmental processes.⁵ Proteoglycans can be classified on the basis of the type of GAG chain they possess and by their tissue location, with a clear distinction between those that reside in the ECM and those that are cell-associated. ECM proteoglycans are usually substituted with CS, DS, and/or KS; cell-associated proteoglycans are more commonly substituted with HS. Most work conducted on growth factor/morphogen interactivity with GAGs has centered on HS and DS.² Heparin is a component of the intracellular proteoglycan of mast cells; serglycin⁶ and similar proteoglycans are synthesized by monocytes/macrophages, T-lymphocytes, and endothelial cells. Often these molecules contain oversulfated chondroitins in addition to HS.

Skin composition

As the largest organ in the body, skin is also one of the most dynamic and complex of organs, with a constant renewal of both ECM and cells throughout life. The varied population of resident cell types throughout the well-defined layers of skin (epidermis and dermis), include epithelial, fibroblasts, keratinocytes, vascular endothelial, lymphatic endothelial, melanocytes, and nerve cells, each of which are capable of producing a unique set of ECM proteoglycans. The main cell types of the epidermis and the dermis are the keratinocyte and the fibroblast respectively. The cellular complexity of skin is further increased during healing responses with cells including mast cells, monocytes/macrophages, polymorphonuclear leucocytes, and cytotoxic T-lymphocytes migrating into the tissue as a response to infection.

The macrostructure of normal skin heavily depends on the three-dimensional (3D) organization of the constituent collagen fibers. The most prevalent ECM structural component in skin is collagen type I, shown localized to the dermis in fetal tissue in Figure 1A–E. Normal healthy collagenous matrix alignment requires certain proteoglycans for both initial formation and maintenance. The proteoglycan composition and structural complexity of skin ECM layers will thus be a product of the metabolism (both synthesis and turnover) of the tissue's current and previous cell populations.

Figure 1.

Immunolocalization of type I collagen (A–E), periodic acid-Schiff-positive glycosaminoglycans (GAGs) (F, G), and perlecan (H, I) in vertical sections of human fetal big toe (14 weeks gestation). The boxed areas in photosegment (A) represent selected areas of the underside and upper-surface of the big toe presented at higher magnification in (B, E, G) and (C, D, F, H, I) respectively. The central cartilage rudiments of the most distal toe joint and the nail bed are labeled for orientation of the specimen. The specimens were fixed in 10% neutral buffered formalin, embedded in paraffin, and then 4 μm microtome sections were adhered to positively charged microscope slides. Type I collagen was immunolocalized using a mouse monoclonal antibody (2 μg/mL; Clone I-8H5 from MP Biomedicals) overnight at 4°C. Primary antibody localization was visualized using a biotinylated-anti-mouse IgG-conjugated secondary antibody using diaminobenzidene as chromogen with 10 min color development.²¹⁶ Type I collagen was prominently immunolocalized in the dermis but not the epidermis and clearly demarcated these tissues.^216,217 Periodic acid-Schiff-positive staining of the upper (F) and underside of skin (G) visualized GAG localized at the epidermal–dermal junction and pericellularly in the epidermis.^218–220 The boxed areas labeled P1 and P2 in (A) represent areas where perlecan immunolocalization is also depicted at higher magnification in (H, I). Monoclonal antibody A7L6 to perlecan domain IV was used for immunolocalization using NovaRED as chromogen.^216,217 Perlecan is a prominent proteoglycan of the epidermis and epidermal–dermal junction and the nail bed but is largely absent from the keratinized tissue overlying the nail bed in (H). Scale bars 1 mm in (A), 200 μm in (B, C) 50 μm in (D, E, H, I), and 100 μm in (F, G). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Proteoglycans are also localized in other structures within skin. Hair follicles consist of a hair shaft sprouting from differentiated keratinocytes within an inner and outer root sheath located in the epidermis and a dermal papilla and connective tissue sheath below, in the dermis. Each of these separately developed tissues is rich in a variety of different ECM proteoglycans.⁷

Extracellular Proteoglycans

Most of the proteoglycans expressed in tissue ECM through the body are also present in skin (Table 1; Fig. 1F, G). These include large, aggregating, space-filling, water-retaining proteoglycans, particularly versican, and the small leucine-rich repeat proteoglycans (SLRPs) decorin, biglycan, lumican, keratocan, and fibromodulin. HS-containing proteoglycans, such as perlecan and the syndecans (SDCs), have also been localized to specific sites in hair follicles and to the dermis during the growth phase.

Table 1.

Skin proteoglycans

Proteoglycan	Core-protein (kDa)	GAG	Tissue/cellular location
Extracellular proteoglycans
Versican Vo isoform	373	CS	ECM, widespread distribution in epidermis, dermis, basal muscle, and vasculature and elastic networks in skina,10
Versican V1 isoform	265	CS
Versican V2 isoform	180	CS
Versican V3 isoform	72	GAG absent
Aggrecan		CS/KS	Absent from normal skin; accumulates in scar tissue
Perlecan	467	HS, varies with cell typeb	Ubiquitous cell associated pericellular and ECM component, basement membrane basal epidermis/dermis40,191,223
Decorin	36	CS/DS	ECM, collagen fibril associated201
Biglycan	38	CS/DS	ECM and cell associated201
Fibromodulin	42	KS	ECM, collagen fibril associated201
Lumican	38	KS	ECM, collagen fibril associated176, 177, 201
Keratocan	37	KS	ECM75
Cell-associated proteoglycans
GPC-1	56	HS	Epidermis, keratinocyte pericellular matrix107
SDC-1	33	HS, CS/DS	Epidermis/dermis, pericellular component of keratinocytes, fibroblasts also expressed by capillary endothelial and glandular cells187
SDC-2	23	HS
SDC-4	22	HS
NG2/CSPG-4	251	CS	Transmembrane81
Intracellular proteoglycans
Serglycin	10–19	HS,C6S,CS-B, CS-E, DS	Intracellular proteoglycan of secretory granules in mast cells, endothelial cells, neutrophils, cytotoxic T-lymphocytes115,192
Part time proteoglycan co-receptors
CD44 (includes epican)	37–81c	CS/DS or HSd	Transmembrane HA receptor, some variants identified in inflammatory and autoimmune skin conditionse,143,224
Betaglycan	110	HS/CS	Transmembrane, type III TGF-β coreceptor225
Endoglin	68 dimer	HS/CS	TGF-β co-receptor, modulates TGF-β signaling136,137,226

^aVersican exists as four isoforms, however, it is not fully known how their localizations vary in skin.

^bOf the perlecans that have been isolated and characterized for specific cell types, endothelial cells produce the archetypal form of perlecan, which only contains HS substitution; smooth muscle cells produce a hybrid for containing C4S and HS while keratinocytes are unique in the proteoglycan field producing a form of perlecan containing HS, CS, and KS.

^cVariable core protein sizes reflects the 19 variant forms identified.

^dHS substitution identified in CD44 variant epiphycan.

^eCD44s, CD44v3, CD44v6, and CD44v7 have been identified in the epidermis/dermis of normal skin and in cutaneous lupus erythematosus, dermatomyositis, scleroderma, and scleromyxedema.

GAG, glycosaminoglycan; GPC, glypican; CS, chondroitin sulfate; DS, dermatan sulfate; ECM, extracellular matrix; HA, hyaluronan; HS, heparan sulfate; KS, keratan sulfate; NG2, nerve-glial antigen-2; CSPG, chondroitin sulfate proteoglycan; SDC, syndecan; TGF-β, transforming growth factor-β.

Versican

Versican is so named from its multiple protein-binding motifs and its versatility of function. The versican gene, originally cloned from fibroblasts, encodes a chondroitin sulfate proteoglycan (CSPG),⁸ which is structurally related to the other large space-filling proteoglycan, aggrecan, possessing terminal domains analogous to the aggrecan G1 and G3 regions (Fig. 2). Versican does not contain an interglobular domain (IGD) or G2 region like aggrecan and its central GAG-attachment regions (GAGα and GAGβ) also differ in amino acid sequence and GAG attachments. The human versican gene (VCAN) is composed of 15 exons, with alternative splicing occurring in exons encoding the GAG-attachment region.⁹ This generates four VCAN mRNAs. The presence of both the GAGα and GAGβ regions gives rise to the V0 form of versican, the presence of only the GAGβ region gives rise to the V1 form, the presence of only the GAGα region gives rise to the V2 form, and the absence of both the GAGα and GAGβ regions gives rise to the V3 form. We can detect both the GAGα and GAGβ regions in young healthy mouse skin (Fig. 3), which agrees with the presence of V0 and V1 isoforms; a recent study was unable to detect V2 and V3 in adult human skin.¹⁰ The V1 isoform of versican is the most common form found in the ECM of many tissues and can be cleaved by aggrecanases (a disintegrin and metalloproteinase with thrombospondin motifs [ADAMTS]-1, -4, -5, and -9)¹¹ and matrix metalloproteinases (MMPs).¹²

Figure 2.

Structural depiction of the major extracellular matrix proteoglycans of skin versican (A), aggrecan (B), perlecan (C). The disulfide stabilized (green) globular domains of versican and aggrecan are circled, and domains I–V of perlecan are in bolded capitals. Core proteins are depicted in blue, chondroitin sulfate (CS) side chains in red, keratan sulfate (KS) in orange, and heparan sulfate (HS) chains in cerise. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Figure 3.

Localization of versican GAGα (A) and GAGβ (B) splice variants in formalin-fixed paraffin-mounted sections of back skin from 12-week-old C57BL/6 wild-type mice. The immunolocalizations were undertaken using rabbit polyclonal antibodies to the GAGα (amino acids 535–598) and GAGβ (amino acids 1,360–1,439) core protein domains of mouse versican^216,221 purchased from Chemicon through Invitrogen and visualized with the Dako Envision rabbit detection system (NovaRED chromogen). An isotype negative control for versican GAGα is given in (C). The surface (S) and underlying muscle (M) are marked. Arrows indicate the pericellular localization of versican. Scale bar=100 μm. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Versican does not have a KS attachment region on the core protein and, compared to aggrecan, contains significantly fewer CS chains that are more sparsely distributed along a 400-kDa core protein. Like aggrecan, the G1 region of versican is a functional hyaluronan (HA)-binding region. Versican, together with HA, a variable molecular weight nonsulfated linear GAG, forms macro-aggregates that are important for skin hydration and viscoelasticity.¹³ Highly hydrated versican-HA aggregates yield looser matrices than aggrecan-HA aggregates and are hence more conducive to cellular migration, an important property for wound healing. Versican is a major component of blood vessels in skin and associated with elastic networks through G3-mediated interactions with fibulin-1,¹⁴ a component of elastic microfibrils. As with aggrecan, the lectin domain within the G3 region of versican also has the ability to interact with tenascins and matrilin-1 and -3, enabling stabilization of extended collagen-HA-versican networks within tissues.¹⁵ These networks are presumed to be important for sensing of the mechanical microenvironment at the local level, allowing these mechanosensory processes to regulate tissue metabolism.¹⁵

Versican not only acts as a structural entity but it also influences cellular function through G3-mediated interactions with epidermal growth factor (EGF) receptors. Versican can promote mammary tumor and melanoma progression^16,17 and is overexpressed in cutaneous malignant melanoma.¹⁸ Aggrecanase-cleaved fragments of versican regulate the regression of ECM after apoptosis in embryonic interdigital webs.¹¹

Aggrecan

Aggrecan is the major proteoglycan in articular cartilage and can form extremely large, link protein-stabilized aggregates with HA. It has a core protein of 250 kDa, three IGDs (G1, G2, and G3) and specific CS and KS-binding domains (Fig. 2). Aggrecan is normally a minor constituent of skin (Table 1), however it can accumulate in this tissue in mice when ADAMTS-5 (aggrecanase) activity is ablated.¹⁹ This accumulation occurs in the pericellular matrix of progenitor fibroblasts, perhaps preventing them from maturing sufficiently to assist in wound healing. Aggrecan gene (ACAN) expression is also elevated during keloid scarring (see Proteoglycans in Wound Healing section). Aggrecan is absent from the dermis of normal adult skin but can be seen in the connective tissue sheath and under the hair matrix in follicles.⁷

Antitumor and antiangiogenic agents based on the G1 domain of aggrecan and versican have been developed and are examples of antagonistic matricryptins. Neovastat (AE-941) has undergone phase I/II clinical trials in plaque psoriasis;²⁰ Metastatin from cartilage aggrecan²¹ and a 42-mer amino acid peptide based on Metastatin's HA-binding motif (BH-P)²² potently inhibit the proliferation and colony-forming capability of B16 melanoma cells by activating caspase-3 and -8 pathways to selectively trigger apoptosis only in these cells.

Perlecan

Perlecan (heparin sulfate proteoglycan 2 [HSPG2]) is a basement membrane proteoglycan named after its “string of pearls” appearance under electron microscopy. The perlecan core protein is large (467 kDa) and contains five domains. Endothelial cells produce perlecan containing three HS chains in domain-I, smooth muscle cell, and chondrocyte; perlecan has some HS chains replaced by CS, while keratinocytes produce a form of perlecan containing KS, CS, and HS.²³ Although perlecan has a ubiquitous pericellular distribution in fetal and adult skin, it is a particularly prominent component of the human fetal epidermis and less abundant in fetal dermis (Fig. 1H, I). Based on the temporal distribution of perlecan in other tissues (meniscus and cartilage),^24,25 we deduce that the amount of perlecan will be diminished in adult compared with fetal skin; however, this has yet to be confirmed.

Each of the five core protein domains of perlecan display homology to growth factors, protein modules involved in lipid metabolism, cell adhesion, and matrix assembly/stabilization.²⁶ The HS chains mediate growth factor and morphogen interactions that are extremely important during organ development. Perlecan interacts with fibroblast growth factors (FGF)-1, -2, -7, -9, and -18, platelet-derived growth factor (PDGF), vascular endothelial cell growth factor (VEGF), hepatocyte growth factor, bone morphogenetic protein (BMP)-1, -2, -4, and -7, hedgehog, Wnt, and Activin A to promote cell proliferation, differentiation, and ECM production²⁷ and with laminin, fibronectin, thrombospondin, α5β1, and α2β1 integrin to promote cell attachment and recruitment in development and tissue remodeling. Perlecan also interacts with proline/arginine-rich and leucine-rich repeat protein (PRELP), von Willebrand factor A domain-related protein, types IV, VI, XIII, and XVIII collagen, fibrillin-1 and -2, nidogen-1 and -2, latent transforming growth factor (TGF) beta-binding protein (LTBP)-1 and -2, fibulin-2, and tropoelastin to promote and stabilize ECM assembly.^26,28,29 Perlecan is a low affinity FGF co-receptor participating in cell signaling to drive cell proliferation and ECM production.³⁰ Recent studies have also shown perlecan-HS interactions are important in fibrillin and elastin assembly^28,29,31 and ECM stabilization when located on dermal elastic fibers in skin.³²

Studies in Hspg2 exon 3 null mice³³ reveal the specific role perlecan-HS chains have in skeletal development. Healing of skin lesions in these knockout mice was significantly delayed with impaired angiogenesis.³⁴ Little is known of how perlecan is processed in skin; this could potentially be through alternative splicing and/or proteolytic processing. Mast cells contain multiple, truncated forms of perlecan,³⁵ some of which have known matrikine functions. Tolloid-like metalloproteinase (BMP-1) cleaves between the LG2 and LG3 domains of domain V, releasing an antiangiogenic peptide termed endorepellin,^36–38 another matricryptin. Endorepellin interacts with α2β1 integrin and disrupts endothelial-basement membrane interactions critical for the stabilization of tube formation. This forms the basis of its antiangiogenic activity, contrasting with native perlecan, which promotes cellular attachment and vasculogenesis.^36–38

Endothelial cell perlecan is also susceptible to cleavage in domains IV and V on digestion with MMP-1, -3, and plasmin.³⁹ A recent study examining the supramolecular organization of the epidermis–dermis basement membrane of normal skin revealed a composite structure composed of independent laminin 332 and type IV collagen networks held together by perlecan aggregates at strategic points in a spot-welding type manner.⁴⁰ Perlecan-deficient keratinocytes assemble a very thin and poorly organized epidermis due to premature apoptosis and a failure to lay down a normal stratified epidermal layer.⁴¹ Perlecan regulates the survival and terminal differentiation of keratinocytes in the epidermis; thus, deficient levels are detrimental to the integrity of the basement membrane.⁴¹

The SLRPs

The SLRPs contain multiple leucine-rich repeat (LRR) motifs⁴² and are categorized into subfamilies on the basis of gene organization, LRR number, GAG type, and structural organization.⁴³ The SLRPs are important not only as components of most ECM structures but are also responsible for development, organization, maintenance, and remodeling of the ECM during injury and repair.^43,44 The comparative content of SLRPs in the ECM of different canine tissues establishes skin (with cartilage and cornea) as one of the highest localizations for these proteoglycans.⁴⁵ SLRPs in skin include decorin, biglycan, lumican, fibromodulin, keratocan, and nonglycanated proline arginine-rich protein (PRELP, prolargin) (Fig. 4). The CS/DS attachment sites in human decorin and biglycan are within the extreme amino terminus of their core.⁴³ Lumican and fibromodulin contain four N-linked oligosaccharide chains within their central LRR regions that may be modified to KS. The mature core proteins of decorin and biglycan have 14 and 21 amino acid pro-regions removed.⁴⁶ Small N-terminal signal peptides are also removed from fibromodulin and lumican to form their mature core proteins. Fibromodulin and lumican also contain sulfated tyrosine residues clustered at their N-termini in the mature protein, contributing to their anionic nature.^47,48 Many of the SLRPs interact with the TGF-β isoforms (TGF-β1, TGF-β2, and TGF-β3), controlling their bioavailability in skin. SLRPs are therefore important in fibrosis, where excessive TGF-β activity is evident. All the SLRPs also interact with collagen VI, XII, and XIV, fibronectin and elastin, EGF, insulin growth factor (IGF), and tumor necrosis factor (TNF)-α.

Figure 4.

Structural depiction of members of the small leucine-rich repeat proteoglycan family that have been identified in skin: decorin (DCN), biglycan (BGN), fibromodulin (FMOD), lumican (LUM), keratocan (KERA), and prolargin (PRELP). Core proteins are depicted in blue, CS side chains in red, KS in orange. Amino terminal arginine, proline, and sulfated tyrosine clusters are also indicated. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

Decorin

Decorin is so-named as it “decorates” the surface of collagen fibrils. The decorin gene (DCN) encodes a 36-kDa core protein, containing a single CS or DS chain.⁴⁹ Decorin interacts with the “d” and “e” bands of collagen type I fibrils, fibronectin, C1q, EGF receptor, TGF-β, and thrombospondin.⁵⁰ Decorin has important regulatory functions in collagen fibrillogenesis,⁵¹ fibrosis in vivo, and bioavailability of TGF-β.⁵⁰ The regions of decorin that interact with collagen are located within LRR regions at dissimilar sites to those involved in its interaction with TGF-β. Molecular modeling shows decorin accommodates a single collagen molecule within its concave face^52,53; this surface localization of decorin may protect the collagen molecules from proteolysis by collagenases.⁵⁴ The decorin core protein is cleaved by MMPs in vitro,^55,56 by three isoforms of BMP-1⁵⁷ and by membrane type-1 matrix metalloproteinase (MT1-MMP).⁵⁸ Proteinase-cleaved fragments of decorin, particularly from LRR regions, can activate EGF receptor-mediated receptor phosphorylation and result in anti-proliferative (quiescence) effects.⁵⁹

Decorin is expressed by cultured dermal keratinocytes and detected in the suprabasal layers of the epidermis. It modulates the formation of ECM in skin, where the protein and its mRNA are present in high concentrations in the reticular but not papillary dermis.⁶⁰ Postnatal skin also contains decorin with shorter GAG chains and catabolic fragments of decorin.¹⁰ DCN knockout mice have fragile skin and abnormally large and irregularly shaped collagen fibrils, reminiscent of those observed in Ehlers–Danlos Syndrome.⁵¹ DS epimerase 1 (Dse) knockout mice have shorter DS changes on decorin and consequently larger diameter skin collagen fibrils.⁶¹ Decorin is thus important for the formation of uniform correctly sized collagen fibrils during development, healing, and chronic inflammation, although the exact mechanisms of its interactions with collagen molecules are unknown.

Biglycan

The biglycan gene (BGN) encodes a 38-kDa core protein, with two CS/DS chains, although nonglycanated forms of biglycan have also been detected. Pro-biglycan is cleaved by BMP-1⁴⁶ and mature biglycan by MMPs.⁵⁶ Biglycan interacts with the lattice-forming type VI collagen, which form chondron basket-like structures around cells and interact with BMPs and TGF-β in the initiation of the inflammatory response during tissue stress. In skin, biglycan is localized to the ECM of the epidermis and the basement membranes and connective tissue sheath of the hair follicle.⁷ Biglycan has emerging roles as a signaling molecule with an extensive repertoire of molecular interactions with growth factors and receptors that regulate cell growth, morphogenesis, and immunity.⁴⁴

Fibromodulin

The fibromodulin gene (FMOD) encodes a 42-kDa core protein, is KS-substituted and shares significant sequence homology with decorin and biglycan.⁶² It contains four N-linked oligosaccharide sites within the LRR domains that can be substituted with KS. Fibromodulin also contains a number of N-terminal sulfated tyrosine residues that facilitate interaction with clusters of basic residues in a variety of bioactive, heparin-binding proteins.^47,48 Nonglycanated forms of fibromodulin and lumican can accumulate in tissues due to an age-dependant decline in KS synthesis.⁶³ Fibromodulin interacts with types I and II collagen fibrils, inhibiting fibrillogenesis in vitro.⁶⁴ It also interacts with TGF-β and C1q, thus having roles in TGF-β sequestration and bioregulation.^44,50 FMOD is expressed in human epidermal keratinocytes in vitro and in human epidermis in vivo.⁶⁵ The fragile skin of FMOD-knockout mice demonstrate the importance of fibromodulin to collagen assembly and stability in the ECM of this tissue,⁶⁶ although detailed mechanistics are unknown. When bound to collagen, fibromodulin is susceptible to cleavage by MMP-13, which removes the N-terminal sulfated tyrosine cluster. However, soluble fibromodulin is not susceptible to cleavage by MMP-13, neither is it degraded by MMP-2, -8, and -9.⁶⁷

Lumican

The lumican gene (LUM) encodes a 38-kDa core protein,⁶⁸ that has four N-linked sites within the LRR domain that can be substituted with KS. Lumican interacts with similar partners as fibromodulin, and has similar roles in collagen fibril formation. Both SLRPs have homologous sequences in the five to seven LRRs that compete for collagen binding⁶⁹ and they bind to the same regions of type I collagen fibrils.⁷⁰ Conversely, despite significant similarities between lumican and decorin, they have dissimilar binding sites on type I collagen⁷¹ and independently modulate collagen fibrillogenesis.⁷² Like other SLRP members, lumican is also susceptible to degradation by MMPs and it can also be cleaved by MT1-MMP.⁷³ Lumican is expressed by melanoma cells but not by normal melanocytes and was thus suggested as a marker of malignant melanoma.⁷⁴

Keratocan

Keratocan is a 60–70-kDa KS substituted member of the SLRP proteoglycan family with a widespread distribution in cornea, cartilage, tendon, sclera, aorta, lung, skeletal muscle, and skin.⁷⁵ Corneal keratocan has a 37-kDa core protein that contains three to four KS chains with attachment points in the LRR region; skin keratocan is less sulfated.⁷⁵ Keratocan shares 35% amino acid identity with fibromodulin and lumican but only 10–12% homology with decorin or biglycan. Its expression is downregulated by FGF-2- and TGF-β1-activation of JNK signaling pathway in activated corneal keratocytes during corneal stromal healing⁷⁶ so we can speculate it has a similar role in skin.

Proline/arginine-rich and leucine-rich repeat protein

PRELP (prolargin) is a 55-kDa nonglycanated SLRP so classified on the basis of its sequence and general structural organization.⁷⁷ It contains N terminal clusters of arginine and proline with anchoring roles in basement membranes.^78,79 The N-terminus of PRELP binds to the heparin and the HS of perlecan and acts as a basement membrane anchor and/or linking module to other collagenous networks in skin.⁸⁰

Cell-Associated Proteoglycans

Nerve-glial antigen-2/CSPG-4

CSPG-4, also known as high molecular weight melanoma associated antigen in humans and nerve-glial antigen-2 (NG2) in rodents, is a transmembrane CSPG that is expressed by immature progenitor cells including oligodendrocyte, chondroblasts/osteoblasts, myofibroblasts, smooth muscle cells, pericytes, interfollicular epidermal, and hair follicle cells.^81,82 CSPG-4 occurs as a 250-kDa glycoprotein, a 450-kDa C4S-proteoglycan, or as a nonglycanated form (Fig. 5C).^83,84 The CS side chain of CSPG-4 facilitate interactions with α4β1 integrin and fibronectin, influences its cell surface distribution, and has roles in the activation of pro-MMP2 by transmembrane MMPs. This ability to influence integrin and MMP activation associates CSPG-4 with the regulation of cellular migration and invasion in skin and implicates this proteoglycan as a molecular target in the prevention of melanoma spread.^85,86 CSPG-4 may participate in cell signaling both directly, as a co-receptor with another receptor tyrosine kinase, or by association with cytoplasmic kinases such as focal adhesion kinase or extracellular regulated kinase (ERK)-1, -2. The core protein of CSPG-4 binds FGF-1 and PDGF and presents these mediators to their cognate receptors in situ.⁸⁷ The central nonglobular domain of CSPG-4 binds to collagen V and VI^88,89 and other ECM molecules, facilitating cellular attachment and ECM stabilization. Binding of CSPG-4 to ECM components also induces cytoskeletal reorganization conducive to cell spreading and migration.^90,91

Figure 5.

Structural organization of cell surface proteoglycans identified in skin, glypican (GPC) (A), syndecan (SDC) (B), and nerve-glial antigen-2 (NG2)/chondroitin sulfate proteoglycan (CSPG)-4 (C). Core proteins are depicted in blue, with extracellular (ED), transmembrane (TMD), and cytoplasmic (CD) domains indicated. CS side chains are depicted in NG2 in red and HS chains in SDC and GPC in cerise; the lipid bilayer of the plasma membrane is depicted in yellow. Organization of the extracellular sub domains D1, D2, and D3 of NG2 proteoglycan are also indicated; D1 contains two laminin G type domains, D2 contains a single CS side chain. Although not all forms of NG2 are glycanated, this region of NG2 interacts with type V, VI collagen, growth factors, integrins, and matrix metalloproteinases (MMPs). Two MMP cleavage sites have been demonstrated in the D3 domain, which result in shedding of the extracellular portion of NG2. Protein kinase C alpha (PKCα) and extracellular regulated kinase (ERK)-1, -2 interactive regions in the CD where tyrosine phosphorylation occurs during cell signaling are indicated. GPC contains multiple HS chains in the ED. Detail of the GPC core protein attachment region to the phospholipid bilayer of the plasma membrane by a triphospho-mannose-glucosamine-inositol-glycerophosphate linkage are indicated in the exploded region in (C). Modified from O'Connell and Weeraratna²²² and Price et al.⁸⁴ with permission. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

In early postnatal mouse skin, NG2 is expressed in the dermis, outer root sheath of hair follicles, and basal keratinocytes of the epidermis. 81 In adult skin, NG2 proteoglycan is most prominently expressed by stem cells in the hair follicles and regulated by TGF-β. In NG2 knockout mice, the epidermis is very thin compared with wild-type mice due to the reduced proliferation of the basal keratinocytes in the epidermis, providing clues as to the likely role of this proteoglycan in skin development, homeostasis, and its possible role in melanoma progression.^81,84

The glypican family

The glypicans (GPCs) are a family of HS proteoglycans, which are anchored to the cell-surface via a covalent linkage to glycosylphosphatidylinositol (GPI) (Fig. 5A). Six GPC family members that share 17–67% identity have so far been identified. The HS chains on GPC are attached to a C-terminal region near the GPI anchor point at the cell membrane. These HS chains bind growth factors and morphogens and modify cell signaling pathways, cellular proliferation, and matrix production.^104–106 GPC-1 has been identified in skeletal and smooth muscle epidermis and hair folllicles in rodents but not in the dermis.¹⁰⁷ Neither GPC-2,¹⁰⁸ a major neural tissue proteoglycan, nor GPC-3, -4, or -5¹⁰⁹ are expressed in skin. GPC-1 has been immunolocalized to the pericellular matrix of keratinocytes in normal skin and shown to be an intracellular proteoglycan of epidermal cells at the edge of chronic ulcers.⁹⁵

The syndican family

The SDCs are transmembrane HS-proteoglycans that carry three to five HS and CS/DS chains. They facilitate interactions with a large variety of ligands, including FGF, VEGF, and TGF-β family members. They are thus involved in wound healing, inflammation, cell adhesion, and vascular biology.^92–94 All SDCs have an N-terminal signal peptide, an ectodomain, a membrane-anchoring hydrophobic transmembrane region, and a C-terminal cytoplasmic domain (CD) (Fig. 5B). The SDCs are differentially expressed in skin; SDC-1 is highly expressed by keratinocytes, fibroblasts, and epithelial cells.⁹⁵ SDC-1 mediates cell interactions with the ECM, cell attachment and migration, and responses to growth factors. The interaction of SDC-1 with laminin 322 is essential for keratinocyte migration⁹⁶ and is consistent with reduced migration of keratinocytes in SDC1 knockout mice and enhanced TGF-β signaling.⁹⁷ Changes in SDC-1 and SDC-4 expression correlate with the progression of skin carcinogenesis and the metastatic potential of melanoma.^98,99 SDC1 expression is reduced in basal cell, squamous and metastatic human skin cancers and has been linked to malignant conversion in skin carcinogenesis.⁹⁹ SDC1 expression is elevated in psoriatic epidermis.¹⁰⁰

SDC-2 is also expressed by keratinocytes and endothelial cells and by dermal fibroblasts and glandular cells. In normal skin, SDC-2 is confined to the keratinocytes of the superficial epidermis where it is an intracellular proteoglycan. SDC-2 influences the migratory potential of melanoma cells¹⁰¹ and SDC-4 affects the migration of adult dermal fibroblasts.¹⁰² SDC-4 is pericellularly expressed by keratinocytes in normal skin, weakly by fibroblasts, and appears in capillaries. SDC-4 has been associated with leg ulcers.¹⁰³

Intracellular Proteoglycans

Serglycin

Serglycin is an intracellular proteoglycan produced by endothelial cells, mast cells, and lymphocytes. Serglycin is so named from the high proportion of repeating serine and glycine residues in its core protein. It is localized to the α-secretory granules of platelets and mast cells, where it binds and controls the activity of various mediators including factor-4 in platelets, serotonin, dopamine, or histamine in mast cells or proteases like tryptase and chymase.^110,111 Serglycin also localizes neutrophil elastase to the azurophilic granules of neutrophils,¹¹² and granzyme-B in cytotoxic T-lymphocytes.^113,114 Mast cells show significant heterogeneity depending on tissue location. In rodents, mast cells are classified as mucosal or connective tissue types, with the former primarily residing in the intestinal mucosa and the latter residing in skin, peritoneum and elsewhere. The GAG substitution on serglycin isolated from mast cell subclasses is quite variable. Serglycin is decorated with CS chains in the secretory granules of circulating basophils, whereas in resident mast cells it is decorated with heparin.¹¹⁰ Endothelial cell serglycin in blood vessels of dermatomyositic and discoid lupus erthythamotosus skin are highly substituted with C6S.¹¹⁵ Mast cell numbers dramatically increase in response to infection where proteases are released from the secretory granules at specific tissue sites to inactivate invading organisms. Serglycin has important roles to play in inflammatory, allergic, and immune reactions in skin.^116–119 Extracellular release of serglycin may result in interactions with chemokines produced by inflammatory cells; therefore, mast cell heparin may regulate eosinophil recruitment by binding to the chemokine CCL11 thus protecting it from proteolytic degradation in situ.¹¹⁹

Cell-Surface Co-Receptors and “Part Time” HS Proteoglycans

Some cell-surface co-receptors are also termed “part time” CS–HS proteoglycans, since the proteins do not always have a GAG chain attached, thus nonglycanated forms of these proteoglycans have also been identified. These include endoglin, betaglycan, and CD44 variants 3–10, and 8–10. Keratinocytes also produce a variant HS-substituted CD44 (epican).^120–122

Endoglin

Endoglin is a glycoprotein/proteoglycan co-receptor expressed on endothelial cells, macrophages, keratinocytes, and stromal cells in the epidermis/dermis and bulb region of hair follicles and sweat glands.¹²² It is upregulated under inflammatory conditions and in skin lesions where endothelial cell proliferation is prevalent. Endoglin is a 68-kDa, dimeric, type 1 integral membrane protein containing extracellular, transmembrane, and CDs.^123–125 Mouse and porcine endoglin share 71% and 96% sequence identity respectively with human endoglin and almost identical transmembrane and CDs.^126,127 Human, porcine, and mouse endoglin also display similarities in their extracellular domains to the type III TGF-β receptor (TβRIII) betaglycan, but mouse and porcine endoglin do not contain an Asp-Gly-Arg cell receptor interactive motif.¹²⁶ Endoglin, like betaglycan, is a TGF-β co-receptor that modulates TGF-β signaling in endothelial cells and macrophages.^128,129 Unlike betaglycan, which binds all TGF-β isoforms with high affinity, endoglin binds TGF-β1 and -β3 in the presence of TβRII but does not bind TGF-β2.^128,129 Although endoglin is not a signaling receptor, it has been shown to associate with TGF-β signaling receptors (TβRII, activin receptor-like kinase [ALK]1 and ALK5) and to modulate their phosphorylation status and downstream Smad-dependent and independent signaling in endothelial cells.¹³⁰

Endoglin is expressed in normal and psoriatic skin; its levels are significantly elevated in psoriatic skin where it appears to have a role in the extravasation of peripheral blood mononuclear cells via the endothelium.¹³¹ Endoglin is also significantly upregulated on dermal blood vessels in scleroderma¹³² and primary malignant melanomas.¹³³ Endoglin controls the expression of ALK1 and ALK5 by endothelial cells and regulates endothelial cells by upregulating TGF-β/ALK1 cell signaling.¹³⁴ Endoglin counteracts TGF-β-mediated suppression of CD4(+) T cell proliferation by induction of Smad-independent signaling via ERK.¹³⁵ Endoglin has a profibrotic role in scleroderma fibroblasts promoting TGF-β1/ALK1/Smad1 cell signaling and collagen production,¹³⁶ however, results have been variable with reports of both positive and negative enhancement of TGF-β effects in fibrosis.¹³⁷ Endoglin is essential for angiogenesis with its expression upregulated in inflammation and may have regulatory roles in transendothelial leucocyte trafficking.¹³⁸

Betaglycan

Betaglycan/TβRIII is a 300-kDa membrane anchored cell-surface CS/HS proteoglycan that binds to several members of the TGF-β superfamily via its core protein, and FGF-2 via its HS chains. Betaglycan presents TGF-β to the type II TGF-β signaling receptor serine threonine kinase to effect cell signaling.¹³⁹ Betaglycan is not directly involved in TGF-β signal transduction, but by binding all TGF-β isoforms at the cell surface¹⁴⁰ with relatively high affinity, it acts as a positive regulator of TGF-β access to signaling receptors. Soluble betaglycan can also bind TGF-β in free solution, which anatagonizes TGF-β signaling through TβRI/TβRII, since the betaglycan-TGF-β complex cannot now bind to the membrane-bound betaglycan TGF-β type III co-receptor.¹³⁹ This may regulate tissue fibrosis through excessive TGF-β activity. TGF-β is localized to the hair follicle bulb, the putative stem cell niche, in young skin.¹⁴¹

CD44/epican

CD44 (Pgp-1, ECMRIII) has a widespread distribution, with most cells expressing the CD44s standard form of 363 amino acids and a predicted molecular weight of 37 kDa. The apparent molecular mass of CD44s protein estimated by gel electrophoresis is ∼80 kDa. The higher molecular weight value than predicted from the number of amino acids encoded for CD44 can be accounted for by post-translational modifications including N- and O-linked glycosylation, CS, HS, and sialic acid substitution. Epican, the largest variant form of CD44 containing peptides from all variant exons, is substituted with HS and has a molecular weight in excess of 200 kDa. A schematic of the structural organization of CD44 is provided in Figure 6. Some epithelial cells also express a larger isoform (CD44E)¹⁴² with insertion of v8–v10 exons at the alternative splice site. CD44 is the major HA receptor in skin,¹⁴³ however, in the alternatively spliced CD44v3, HS is substituted for CS and this form of CD44 can additionally bind FGF-2 and EGF. In normal skin, CD44 is expressed by eccrine (sweat) gland cells,¹⁴³ where its distribution is asymmetric, with intense dermal but little luminal staining. In skin undergoing inflammation or neoplastic attack, CD44 expression can be widespread on keratinocytes and infiltrating lymphocytes.^144,145

Figure 6.

Schematic depiction of the structural organization of CD44. The extracellular region of the core protein is depicted in blue with the amino terminal disulphide stabilized A, B, and B′ loops that form the hyaluronan (HA)-binding region indicated. Areas of putative CS side chain, N- and O- linked oligosaccharide substitution are also indicated. The standard CD44s is encoded by exons s1-s10 and alternative splicing of CD variants by exons v1–v10 inserted between s5 and s6. The transmembrane and CDs are also shown and cytoskeletal interactive proteins that have roles in cell signaling. Modified from Goodison et al.¹⁴⁷ with permission. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

CD44 mediates cell interaction with HA, cell aggregation, migration, proliferation, and activation; cell–cell and cell–substrate adhesion; endocytosis of HA; and assembly of pericellular matrices, for example, between HA and versican. The importance of CD44 to skin homeostasis is immediately apparent in CD44 antisense transgenic mice where defective accumulation of HA accompanies severe disturbances in the basal keratinocytes, which display aberrations in their normal mitogen and growth factor-mediated proliferative responses, decrease in skin elasticity and hydration, and impaired responses to local inflammation and tissue repair.¹⁴⁵ This strongly supports an important role for HA and CD44 in skin physiology and tissue repair. CD44 is also a direct and functional target of the micro RNA miR-34a, which targets CD44 in prostate cancer stem cells; knockdown of CD44 inhibits tumor invasion.¹⁴⁶

CD44 is a “part time” proteoglycan, as not all variants of CD44 are glycosylated. CD44 is a highly variable cell surface receptor, and different cell types synthesize CD44 of considerable structural diversity. Alternative splicing of variant exons and post-translational modifications in the glycosylation pattern of CD44 generates a diverse collection of CD44 isoforms (Fig. 6). Three broad size categories of CD44 molecules have been identified, (1) a predominant category synthesized by most cells of hemopoietic origin in a 80–90-kDa size range, (2) epithelial CD44 of a size range of 110–160 kDa, and (3) a proteoglycan form of CD44 substituted with CS chains of relatively large size range>200 kDa.¹⁴⁷ Keratinocytes also synthesize a 250-kDa form of CD44 substituted with HS chains.^120,121

Patients with malignant melanomas have significantly lower serum levels of soluble CD44.¹⁴⁸ As soluble recombinant CD44v6 disrupted the interaction of cellular CD44 with HA, inhibited tumor formation, and halted metastasis;¹⁴⁹ it may have a positive clinical outcome for melanoma patients.

Summary of Proteoglycan Localization in Normal Skin

Figure 7 is a summary schematic of the diverse and discrete localizations of the various proteoglycans identified in skin where information is available in the medical literature. None of the proteoglycans studied are ubiquitous throughout the ECM and cells of skin tissue, with the possible exception of HA. This summary is by no means complete; further study is required to more fully define the complex distribution of proteoglycans in skin tissues.

Figure 7.

Summary schematic of known proteoglycan localizations in normal human skin. Variations in staining density are relative within the section for each individual proteoglycan and not between proteoglycans. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/wound

The Effect of Age, Injury, and Disease on Proteoglycans in Skin

Proteoglycans in wound healing

Wound healing is a dynamic, highly ordered, and tightly regulated process comprised of inflammatory, proliferative, remodeling, and maturation phases.^150–152 These healing processes are driven by cellular responses to ECM changes and are optimal in fetal skin and impaired in both aged and diseased skin. Wound healing becomes much slower with age but, after menopause, scarring is also reduced, most likely due to loss of estrogen-induced TGF-β release from fibroblasts.¹⁵³ There is no documented correlation between the speed of a wound repair and the amount of scarring that occurs. TGF-β plays a major role in normal skin in both the differentiation of progenitor cell populations in the follicle bulb niche and in skin homeostasis. Recruitment of inflammatory cells is essential for normal wound healing to induce EGF, TGF-β1, and FGF release at the wound site, where granulation tissue fills the defect site as a temporary containment measure. These growth factors induce focal recruitment, maturation, and proliferation of fibroblasts, fibrocytes, keratinocytes, and myofibroblasts that lay down collagen, proteoglycans, and other ECM components at the damage site during the proliferative phase of wound repair with the original fibrin clot being replaced with a more mechanically stable matrix containing collagen, fibronectin, and HA.

Although not strictly a proteoglycan, HA is nevertheless an abundant GAG component of skin and of considerable functional importance. The cell regulatory properties of HA in skin are attributable to its participation in signal transduction events through cell surface and intracellular HA receptors such as CD44, receptor for HA mediated motility, lymphatic vessel endothelial cell HA receptor (LYVE)-1, Cdc-37, and P-32.¹⁵⁴ Such interactions can inhibit or stimulate cellular proliferation, cellular migration, and angiogenesis (and consequently tumorogenesis). Many of these cellular effects are dependent on the size of the HA. Large HA (>500 kDa) is anti-inflammatory and antiangiogenic, whereas small molecular weight HA (≤500 kDa) or HA oligosaccharides act in an antagonistic manner to block or counteract effects induced by high molecular weight HA.^154,155 For example, HA oligosaccharides induce angiogenesis and promote wound repair in skin.¹⁵⁶ In the early inflammatory phase of wound repair, abundant HA acts as a promoter of inflammation, which is crucial to the wound-healing process.^157,158 In wound granulation tissue, HA promotes both cell migration into the provisional wound matrix and cell proliferation.¹⁴⁴ Granulation tissue is highly inflammatory but is stabilized by the moderating action of large molecular weight HA on inflammation.¹⁴⁴ HA is also a free-radical scavenger and protects cells from free-radical damage, an important attribute in the context of tissue repair.¹⁵⁹ HA regulates keratinocyte proliferation in response to extracellular stimuli in skin.¹⁴⁴ In normal skin, HA is found in relative high concentrations in the basal layer of the epidermis where proliferating keratinocytes are found.¹⁵⁹ Its suggested role here is in the re-epithelization process: it serves as an integral part of the ECM of basal keratinocytes and has roles in maintenance of an accessible epidermal extracellular space. This allows a hydrated matrix conducive to migration of keratinocytes and nutrients.¹⁴⁴ In wound healing, HA and CD44 are expressed at the wound margins, correlating with the influx of migrating keratinocytes that actively promote tissue repair.^144,145 This HA-containing matrix is slowly resorbed and replaced by a stronger ECM during tissue remodeling at later stages of wound repair. HA oligosaccharides also stimulate a number of cell types to produce elevated levels of active MMPs, which contribute to the metastatic spread of tumors in skin.^160,161

Fibrosis occurs due to a dysregulation of the wound-healing process either at the proliferative or remodeling phase, with in some cases, an irritant persisting in the tissue that drives these processes.¹⁶² Fibroblasts (myofibroblasts) are the major biosynthetic cell type that lay down ECM in wound repair; fibrosis can occur in any tissue where this process occurs. There is now a greater appreciation of the contributions of matrix components and soluble mediators in skin remodeling.^150,163,164 The controlled release of growth factors and cellular mediators (chemokines and cytokines) sequestered by proteoglycans at the wound site actively recruit inflammatory cells that begin the process of controlling the injury and healing the tissue damage. Tight regulation of chemokine bioavailability is critical to control the magnitude of the inflammatory response to tissue injury and an atypical chemokine receptor (D6) recently identified has critical regulatory roles to play in this process.¹⁶⁵

Major differences have long been noted in the healing response of fetal and adult skin.^{153,166–170} It is not known why some fetal wounds heal without scarring while wound repair in mature skin usually leaves a percentage of abnormal tissue, although TGF-β3, or the ratio of TGF-β3 to TGF-β1, may be important.¹⁷¹ These growth factors stimulate ECM production and inhibit ECM catabolism but also attract macrophages, neutrophils, and fibroblasts. The stimulation of HA synthesis by TGF-β3 and the resulting prolonged abundance of this GAG has been linked to scarless fetal wound healing.¹⁷² In addition, high levels of HA are present in scarlessly healed wounds of immunodeficient mice.¹⁷³ Fibromodulin is upregulated and decorin is downregulated in fetal wounds.¹⁵³ Hypertrophic scars have less decorin¹⁷⁴ and more versican and biglycan than normal scars.¹⁷⁵ As mentioned earlier, versican-HA complexes may help hydrate the tissue during the repair processes,¹³ aggrecan may hinder cell migration to the wound,¹⁹ and SDCs are important for growth factor signaling during this process.⁹² Reduced decorin, fibromodulin, and TGF-β3 in the deep dermis leads to postburn hypertrophic scarring.^176,177 Delayed wound closure occurred both in FMOD knockout mice associated with an increase in TGF-β3¹⁷⁸ and in LUM knockout mice with a delay in TGF-β1 expression.¹⁷⁹ Modulation of SLRP binding of TGF-β isoforms may be a key area for future accelerated and/or scarless wound-healing therapies. SDC-1 expression is transiently upregulated in the epidermis upon tissue injury.¹⁸⁰ In keratinocytes, loss of SDC-1 delays wound healing, reduces cell migration, and increases TGF-β expression.

Keloid scarring is a benign, recurring, skin tumor with excessive fibroblastic tissue resulting from abnormal wound healing. Scars can be disfiguring and can limit mobility and growth by restricting skin movement and there is currently no treatment for prevention in susceptible individuals. The ECM is implicated in both hypertrophic and keloid scarring.¹⁸¹ Keloid fibroblasts synthesize less decorin with longer GAG chains than normal skin cells.¹⁸² The expression of the aggrecan gene (ACAN) increased an average of 175-fold in skin biopsies from the margins of keloid scars from four patients compared with corresponding areas of normal skin.¹⁸³ This suggests a change of cell phenotype leading to a dysregulation of proteoglycan metabolism in this scar tissue.

Proteoglycans in aging skin

Aging is an important factor to consider when studying the proteoglycan content of skin and its ability to heal. The molecular aspects of skin aging has been recently reviewed.¹⁸⁴ Changes in aging skin can occur both internally due to chronological age (intrinsic) and from environmental factors such as climatic, particularly sun, exposure (extrinsic). While HA is decreased in intrinsically aged skin,¹⁸⁵ proteoglycans are increased and abnormally distributed in photo-aged skin.¹⁸⁶

Intrinsic aging of skin can be studied in body regions usually clothed and hence rarely exposed to the elements. Sections of aged buttock skin biopsies had reduced Alcian blue, HA, HS, decorin, and SDC-1 staining compared with young skin in both genders.¹⁸⁷ CS GAGs and perlecan staining were also reduced but only in female aged skin. Versican staining increased only in male aged skin.¹⁸⁷ Decreases were observed in the size of the GAG chains attached to decorin with skin aging; the smaller GAG chains would result in changes in collagen fibril packing and skin mechanical properties.¹⁸⁸

Skin damaged by the sun has an increased content of GAGs that appears to be mostly CS on the versican core protein more superficially localized in the dermis compared with versican's localization in normal skin.¹⁸⁹ Versican increases in skin exposed to UVB radiation when reactive oxygen species are generated in an inflammatory response.¹⁹⁰ However, pathological and phenotypical changes in skin following solar elastosis can lead to a loss of the HA-binding ability of versican with attendant effects on the hydration and viscoelastic properties of the dermis.¹³ Increased UV irradiation increases protein levels for HS-proteoglycans in human skin (perlecan, SDC-1, -4) and CD44 v3.¹⁹¹

Increases in GAGs due to UV irradiation have been studied using the hairless SKH-1 mouse, however, the results have been mixed.¹⁹² Increases in CS,¹⁹³ DS,¹⁹⁴ and HS¹⁹⁵ in UV-exposed skin have been variously reported, with HA either increasing,^193,194 not changing¹⁹⁵ or decreasing^196,197 after UV exposure. If cultured human dermal fibroblasts are UV-irradiated, gene expression of all the SLRPs, versican and perlecan decrease, with only SDC-1 increasing.¹⁹⁸ Expression of type I and III collagen changes with aging and, following UV irradiation (photo-aging), there is a decrease in the normal collagen to decorin ratio, resulting in the appearance of smaller collagen fiber bundles and consequential changes in the biomechanical properties of skin.¹⁹⁹ A decrease in skin DCN expression was found following short-term UV exposure.²⁰⁰ The free-radical scavenging properties of HA provide protection against solar radiation that can lead to damaging levels of free radical generation,¹⁴⁴ however, chronic UV irradiation of mice downregulated HA synthases, resulting in lower levels of this GAG in mouse dermis.¹⁹⁷

Future Directions

Members of the SLRPs have well-known capability to control TGF-β and have been examined as a means of inhibiting excessive TGF-β activity in tissues that lead to fibrosis and impaired organ function.⁵⁰ Decorin and biglycan in particular would appear prime candidates for further examination of their therapeutic potential in the prevention of skin fibrosis. For example, decorin GAG chains sequester the growth factors, EGF, IGF, and TGFβ in the ECM surrounding cells and may regulate their bioavailability. Decorin can be extensively processed by MMPs, MT1-MMP, and isoforms of BMP-1 to produce a fragment named decorunt.^10,57,201 This contains the LRR region of decorin with a TGFβ-binding site and a strong interactive site for collagen. Decorunt is thus capable of sequestering TGFβ and influencing collagen fibril assembly processes but not processes mediated by the other growth factors and cytokines. Most recently, a heptapeptide (LQVVYLH) TGF-β binding segment of the biglycan core protein, Peniel-2000 (P2K) has been used to repair experimental defects in the intervertebral disc.²⁰² Reattainment of disc height was achieved, with P2K significantly upregulating anabolic gene expression (aggrecan, type II collagen). Significantly, the mode of P2K action was by inhibiting Smad-1/5/8 cell signaling with TGF-β, while Smad-2, -3 signaling through ALK1 was still detected. Thus, P2K selectively switched off the anti-anabolic effects of TGF-β but not its beneficial anabolic gene effects. Similar approaches using bio-active peptides, which directly target functional aspects of components in skin such as TGF-β to promote or inhibit a specific functional property, may also be exploitable for potential skin repair therapies. For such approaches to succeed, it is crucial that we obtain a firm understanding of the biology of key functional components and how they must to be manipulated to provide a positive outcome.

A number of synthetic skin substitutes have been developed including Matriderm^®, Integra^® bilayer artificial dermis, JACE^® cultured epidermal autograft, MyDerm^® bilayered human skin equivalent, Suprathel^® synthetic skin substitute, Renoskin^®, and Hyalomatrix^®.^203,204 These have been seeded with autologous stem cells harvested from debrided human burn skin²⁰⁵ or from adipose tissue²⁰⁶ or human amniotic fluid²⁰⁷ and used in a number of skin repair strategies. Advances in nano-fabrication technology for the preparation of artificial matrices such as those that can be assembled by the Nanospider NS200 using collagen-based electrospinning of nanofibrous textile meshes to produce matrices of controlled 3D morphology are also now possible.²⁰⁸ These matrices are also suitable for seeding with stem cells to effect repair of large skin defects.²⁰⁹ Artificial skin substitutes with a microvascular supply have also been developed and applied to melanoma research.²¹⁰ Epidermal electronics offers particularly exciting possibilities in the area of advanced materials design and in the remote sensing of skin repair processes.²¹¹ Transparent, optical, pressure sensitive artificial skin with large area stretchable electronics have been developed.²¹² These ultrathin (600 μm) devices are laminated on to the skin in a similar manner to a temporary transfer tattoo and are fully contained with their own power supply and communication components. The intrinsic sensing capability of such advanced materials have already found application in smart dressings with the capability of sensing bacterial infection in wounds²¹³ and in development of Theragauze^®, a smart dressing with the ability to regulate antibiotic and moisture delivery across diabetic wounds of the foot,²¹⁴ to promote skin repair processes, and prevent infection in pediatric burns patients.²¹⁵ The use of proteoglycans to sequester cell mediators and refine ECM structure will augment this skin repair technology and has the potential for exciting possibilities in the future.

Take Home Messages

• • Proteoglycans are a multifunctional diverse group of matrix and cell-associated proteins each with a distinct localization in skin.

• • Proteoglycans can interact with other matrix molecules such as collagen to form and stabilize the ECM, thus producing the hydrated integrated structures required for responsive cells in a healthy intact skin.

• • Proteoglycans can bind and sequester growth factors in the skin ECM and either inhibit their activity or present them to cognitive receptors to initiate cell signaling, proliferation, and matrix production.

• • Manipulation of proteoglycan metabolism and homeostasis in skin may lead to effective therapies to accelerate scarless wound healing.

Abbreviations and Acronyms

ADAMTS

a disintegrin and metalloproteinase with thrombospondin motifs

ALK

activin receptor-like kinase

BGN

biglycan

BMP

bone morphogenetic protein

CD

cytoplasmic domain

CS

chondroitin sulfate

CSPG

chondroitin sulfate proteoglycan

DCN

decorin

DS

dermatan sulfate

ECM

extracellular matrix

EGF

epidermal growth factor

ED

extracellular domain

ERK

extracellular regulated kinase

FGF

fibroblast growth factor

FMOD

fibromodulin

GAG

glycosaminoglycan

GPC

glypican

GPI

glycosylphosphatidylinositol

HA

hyaluronan

HS

heparan sulfate

HSPG2

heparin sulfate proteoglycan 2; perlecan

IGD

interglobular domain

IGF

insulin growth factor

KERA

keratocan

KS

keratan sulfate

LRR

leucine-rich repeat

LTBP

latent transforming growth factor beta-binding protein

MMP

matrix metalloproteinase

MT1-MMP

membrane type-1 matrix metalloproteinase

NG2

nerve-glial antigen-2

PDGF

platelet-derived growth factor

PKCα

protein kinase C alpha

PRELP

proline/arginine-rich and leucine-rich repeat protein; prolargin

SDC

syndecan

SLRP

small leucine-rich repeat proteoglycan

TβRI/TβRII/TβRIII

transforming growth factor-β receptor I, II, III

TGF

transforming growth factor

TMD

transmembrane domain

TNF

tumor necrosis factor

UV

ultraviolet

VEGF

vascular endothelial growth factor

WARP

von Willebrand factor A domain-related protein

Acknowledgments and Funding Sources

We gratefully acknowledge the contribution of Susan Smith for technical assistance with the immunohistology. The National Health and Medical Research Council of Australia provided funding for a portion of this work.

Author Disclosure and Ghostwriting

No competing financial interests exist. The content of this article was expressly written by the authors listed. No ghostwriters were used in the creation of this article.

About the Authors

Dr. Margaret Mary Smith and Dr. James Melrose are senior research fellows in the Raymond Purves Research Laboratories at the Kolling Institute (University of Sydney) at Royal North Shore Hospital in Sydney. Both authors study the role of proteoglycans in extracellular matrix structure, function and disease pathology.