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Distribution of Extracellular Matrix Molecules in Human Hair Follicles A. G. MESSENGERp KATHERINE ELLIOTT: GILLIAN E. WESTGATE,b AND W. T. GIBSONb aDepartment of Dermatology Royal Hallamshire Hospital Shefield SIO U F ,England bUnilever Research Colworth House Sharnbrook Bedford MK44 ILQ, England

INTRODUCTION The dermal papilla and the connective tissue sheath surrounding the lower part of the hair follicle are derived from a condensate of mesenchymal cells that appears at an early stage of embryogenesis directly beneath the developing epithelial hair peg.' Experiments on the rat vibrissa follicle have shown that the dermal papilla is responsible for inducing and maintaining differentiation of the hair matrix epithelium in the adult This is analogous to embryological development, in which follicular morphogenesis is dependent upon interactions between the epithelial and associated mesenchymal component^.^,^ There is also evidence that the dermal papilla is involved in determining other follicular characteristics, such as size and hair fiber and in mediating responses to androgen^.^ The lower part of the connective tissue sheath in the rat vibrissa follicle is able to form a new dermal papilla when the original papilla is surgically removed, suggesting that this structure also participates in regulating hair growth. The nature of the signals that mediate dermal papilla function is uncertain. However, it is known from studies on rodent hair follicles that the dermal papilla, and to a certain extent the connective tissue sheath, elaborate a distinctive extracellular matrix (ECM) that differs from that of nonfollicular dermis. Moreover, the volume and composition of the ECM change during the hair growth cycle.1°-13 In anagen, dermal papilla cells lie in an extensive ECM, rich in basement membrane proteins, fibronectin, and proteoglycans, and show ultrastructural evidence of synthetic activity.I4The volume of the ECM diminishes during catagen, to become almost indiscernible in the telogen follicle, where the papilla appears as a tightly packed ball of cells directly beneath the secondary epithelial germ. Reentry into anagen is associated with a resumption of ECM deposition. The expression of certain ECM components, such as fibronectin and chondroitin sulfate proteog1ycan,l1J3varies during the hair growth cycle, suggesting that these molecules may be involved in epithelial-mesenchymal interactions in the hair follicle. While human and rodent hair follicles have a similar anatomy, their growth characteristics differ. For example, the duration of anagen in human scalp follicles is very much longer than in rodent pelage follicles. To establish whether the findings in animal studies are applicable to man, we have studied the expression of a similar spectrum of ECM molecules during the human hair growth cycle. These studies were performed on tissue sections from normal human scalp or beard skin using immu253



noperoxidase and immunofluorescence techniques. Primary antibodies with the following specificities were used: type I collagen, type 111 collagen, type IV collagen, laminin, bullous pemphigoid antigen, fibronectin, chondroitin-6-sulfate, unsulfated chondroitin, dermatan sulfate/chondroitin-4-sulfate,and heparan sulfate proteoglycan (for further details see Refs. 15 and 29).

INTERSTITIAL COLLAGENS Types I and I11 collagen are major components of most connective tissues, including the dermis. It was somewhat unexpected, therefore, when Couchman found that dermal papillae of rat pelage follicles showed little or no immunoreactivity for interstitial collagens and that cells grown from rat vibrissa papillae did not express type I or type I11 collagen in early primary culture.'* In contrast, we found that human follicles stain strongly for types I and I11 collagen in the dermal papilla and the connective tissue sheath (FIG.la).ls Although changes occur in the volume of ECM in the dermal papilla, staining for interstitial collagens is apparent throughout the hair growth cycle. Also, unlike the rat, cultured human dermal papilla cells also stain immunochemically for interstitial collagens in early primary culture.

FIGURE 1. a. Anagen follicle: type I collagen. There is uniform staining of the dermal papilla and the connective tissue sheath. Pigment in hair cortex is melanin. Immunoperoxidase. Magnification: 100 x. b. Anagen follicle: laminin. There is staining throughout the dermal papilla ECM and linear staining of the outer root sheath basement membrane. Perifollicular capillaries are also positive. Staining of Henle's layer is nonspecific. Immunoperoxidase. Magnification: 100 x. (From Messenger et a1.15 Reprinted by permission from Elsevier Science Publishing Co., Inc.)



TYPE IV COLLAGEN, LAMININ In human follicles, these basement membrane molecules are localized throughout the dermal papilla ECM (FIG.lb).l5 Staining is not restricted to epithelial or vascular basement membranes, although it is accentuated in these sites in some follicles. The outer root sheath basement membrane (the glassy membrane) also stains, but the connective tissue sheath does not, except for vascular structures within. Staining of the dermal papilla is maintained during catagen, in which the thickening of the glassy membrane is well demonstrated in some follicles, especially in pathological situations in which the glassy membrane is prominent.16Some intercellular staining for these basement membrane molecules is still apparent in dermal papillae of telogen follicles. The basement membrane is also clearly delineated at this stage of the hair cycle. The presence of basement membrane molecules in the dermal papilla has been well documented in other mammalian species.1°-13 Their origin is not certain, and it possible that they derive from epithelial or endothelial cells. However, interrupted basement membrane-like structures around dermal papilla cells have been observed in tissue sections by electron microscopy (FIG.2).17Cultured dermal papilla cells also synthesize laminin and type IV collagen in v i t r ~ suggesting ,~~ that they may be responsible for contributing basement membrane material to the ECM in viva

BULLOUS PEMPHIGOID ANTIGEN Bullous pemphigoid antigen (BPA) is a component of the epidermal basement membrane. In fetal rat skin, staining for BPA becomes continuous at the dermo-

FIGURE 2. Dennal papilla tissue section from anagen follicle showing electron-dense basement membrane-like structure (arrows) adjacent to papilla cell membrane. Electron micrograph. Magnification: 30,000 x.



epidermal junction by about day 20 of gestational age. However, as the hair follicle develops, there is loss of immunostaining for BPA around the hair bulb, which persists throughout the neonatal growth phase.10 A similar phenomenon has been observed during follicular development in the mouse.18 In adult follicles, in both man and rat, immunostaining for BPA varies during the hair growth ~ycle.~OJ5 In human follicles, there is linear staining of the outer root sheath basement membrane, internal to the glassy membrane, extending to the lower tip of the hair bulb. During late catagen and telogen, BPA staining is continuous along the papilla-epithelial interface. However, in anagen follicles, the basement membrane zone around the dermal papilla does not stain, nor does the papilla itself (FIG.3). The absence of BPA staining of the hair matrix basement membrane could be due to masking or degradation of the antigen or to lack of expression. BPA is synthesized by keratinocytes and is expressed in association with hemidesmosomes.19-*2Hemidesmosomes have been observed at the basal surface of hair matrix epithelial cells in human beard folliclesl7 but not in rat f0llicles.~3Our (unpublished) observations suggest that hemidesmosomes are very sparse in this site in human follicles, a finding consistent with absence of BPA expression. The functional significance of changes in BPA expression during the hair growth cycle is unknown.

FIGURE 3. a. Anagen follicle: bullous pemphigoid antigen. There is linear staining of the outer root sheath basement membrane extending to the lower tip of the hair bulb and around the stalk of the dermal papilla. There is no staining of basement membrane around the body of the dermal papilla (arrows) nor within the papilla ECM. Immunofluorescence. Magnification: 250 x. b. Late catagen follicle: bullous pemphigoid antigen. Linear staining is seen at the interface between follicular epithelium and the dermal papilla (shortarrows).There is a well-developed glassy membrane (long arrows), which is not stained. Immunofluorescence. Magnification: 250 x. (From Messenger et aLL5Reprinted by permission from Elsevier Science Publishing Co., Inc.)



FIBRONECTIN The fibronectins are a family of glycoproteins, encoded by a single gene, that are widely distributed in extracellular matrices and body fluidsdZ4 The fibronectin molecule possesses a number of domains that mediate binding to other ECM components, such as collagen and heparin, and to cellular integrins. Thus, fibronectin appears to be involved in organization of extracellular matrices and in promoting adhesion of cells to the ECM. Fibronectin is also thought to be important in regulating cell migration, notably during embryonic development and wound healing in which it is associated with the epithelial basement membrane zone.24~~ In rat hair follicles, the dermal papilla stains strongly for fibronectin during anagen but shows little staining during catagen and telogen. * I However, reexpression occurs in very early anagen in a distinctive basement membrane pattern at the papilla-epithelial interface. This occurs after the onset of proliferative activity in the secondary epithelial germ and may relate to the cell migration occurring at this stage of follicular development. The distribution of fibronectin in human follicles is very similar (FIG. 4). Most anagen follicles show strong staining throughout the dermal papilla ECM and in the connective tissue sheath. However, in some anagen follicles staining of the papilla and connective tissue sheath is less prominent and is distributed mainly around blood vessels. This latter pattern may indicate that follicles are in a late stage of anagen, as very little staining for fibronectin is seen in catagen or telogen follicles. In early anagen, fibronectin is reexpressed in the dermal papilla, with accentuation of staining in the basement membrane region at the papilla-epithelial interface in a distribution similar to that seen in the rat. At the same time there is a diffuse increase of fibronectin staining in the connective tissue sheath. Staining of the papilla ECM increases during anagen development.

GLYCOSAMINOGLYCANS Glycosaminoglycans (GAG) consist of linear polysaccharide chains characterized by a repeating sequence of hexosamine and uronic acid residues, the different types being distinguished from each other by the nature of the sugars and their degree of sulfation.26With the exception of hyaluranon, GAGS are covalently bound to a core protein to form proteoglycans. It has long been known that the dermal papilla is rich in GAGS by virtue of its histochemical staining properties.27.B Recently, chondroitin-6-sulfate (C6S), chondroitin sulfate proteoglycan (CSPG), and heparan sulfate proteoglycan (HSPG) have been immunolocalized in rat dermal papillae.12J3Staining for CSPG, but not HSPG, diminished during catagen and was absent in telogen follicles. In human follicles, the dermal papilla and connective tissue sheath stain with antibodies to C6S, unsulfated chondroitin (COS), and dermatan sulfate (DS) during anagen.29 Staining for C6S (FIG.5) and COS in the dermal papilla and the connective tissue sheath around the regressing epithelial stalk diminishes during catagen and is absent in telogen follicles. Both of these GAGS are reexpressed in early anagen. DS, but not C6S or COS, is found throughout the dermis in a distribution similar to that of interstitial collagens. C6S, but not COS, is also found at the epidermal basement membrane zone. However, in the lower part of the hair follicle, C6S is deposited in association with collagen fibrils and cells in the connective tissue sheath external to the outer root sheath basement membrane and does not appear to be an integral basement membrane component in this site. In contrast, the distribution of basement membrane-associated HSPG is identical to that of type



FIGURE 4. a. Anagen follicle: fibronectin. There is strong staining throughout the dermal papilla ECM and in the connective tissue sheath. Immunoperoxidase. Magnification: 100 x. b. Catagen follicle: fibronectin. There is only faint staining in the dermal papilla and connective tissue sheath. Immunoperoxidase. Magnification: 100 x. c. Anagen I: fibronectin. Staining of basement membrane zone at papilla-epithelial interface with diffuse staining in perifollicular connective tissue. Immunoperoxidase. Magnification: 250 x. d. Anagen 111: fibronectin. Staining of basement membrane is maintained, and there is increasing staining within the dermal papilla ECM. Immunoperoxidase. Magnification: 250 x.



FIGURE 5. a. Anagen follicle: chondroitin-6-sulfate. There is diffuse staining of the dermal papilla and in the inner layer of the connective tissue sheath. Immunoperoxidase. Magnification: 100 x. b. Late catagen/telogen: chondroitin-6-sulfate. The dermal papilla (arrow) is unstained, but faint staining persists in the connective tissue sheath external to the glassy membrane. Immunoperoxidase. Magnification: 100 x.

IV collagen and laminin-that is, it is found in the dermal papilla and the outer root sheath basement membrane-and staining for HSPG is maintained in the papilla during catagen.

DISCUSSION Interactions between cells are central to many biological and pathological processes, such as embryological development, regeneration, wound healing, and neopla~ia.~.31 Many such interactions involve both mesenchymal and epithelial tissue, a phenomenon exemplified in the hair follicle. Cells may communicate by humoral factors (hormones, growth factors) or by cell-cell contacts, either via cell membrane receptors or by direct coupling through permeable membrane junctions. It is also thought that tissue morphogenesis is highly dependent on dynamic compositional and spatial changes in the extracellular and cell surface mat~ix.32.~~ Basement membranes, for example, are believed to have a variety of biologic properties, including structural organization and compartmentalization, the filtering of macromolecules, Fibronectin may also and the modulation of cell metabolism and differentiati~n.~~ influence cell behavior directly through binding to cell membrane integrins that are linked to the cytoskeleton. The expression of ECM molecules in hair follicle mesenchyme-derived tissue is distinctive and differs from that of the interfollicular dermis. Furthermore, the ex-



pression and spatial distribution of basement membrane proteins, BPA, fibronectin, and GAGs/proteoglycans are similar in rodent and human hair follicles, suggesting a fundamental role in follicular physiology. This conclusion is supported by the observations of Link and colleagues, who found that treatment of vibrissa follicle organ cultures with dispase (which degrades type IV collagen and fibronectin) caused condensation of the dermal papilla and inhibited thymidine labeling in the epithelial matrix.35The major difference between rat and human hair follicle ECM is in immunostaining for interstitial collagens.lzJs The significance of this observation is not yet clear, although it may possibly relate to the different growth characteristics of these follicle types. It should be noted that the antibodies used in these two studies were not the same, and their specificities may be different. However, ultrastructural studies have demonstrated collagen fibers in human dermal papillae (although they are more sparse and not arranged into bundles as in the dermis).17 For the most part, the functional properties of ECM molecules in hair growth have to be inferred from studies in other tissues. The unusual distribution of type IV collagen, laminin, and HSPG in the dermal papilla suggests that basement membranes have an important physiological role, but the maintenance of expression in catagen argues against a direct effect on epithelial growth and differentiation. It is possible that these molecules are necessary for maintaining the structural integrity of the dermal papilla. The expression of other ECM components, notably fibronectin and chondroitin proteoglycans, varies in concert with the hair growth cycle. The basement membrane deposition of fibronectin in early anagen is particularly interesting, as this phenomenon appears to be common to situations in which cell migration is taking place. Perhaps the best evidence for a direct role in modulating hair growth is available for GAGs. This comes from a number of experimental and clinical observations: (i) Hypertrichosis may be a feature of disorders in which there is accumulation of

GAGs in the skin, such as pretibial myxoedema36 and the mucopolysaccharidoses.37 (ii) Injection of GAGs into the skin has been reported to stimulate hair growth in rabbits.38 (iii) Inhibition of GAG metabolism in chick embryo skin disrupts development of feather follicles (which may be regarded as the avian analogue of the mammalian hair f ~ l I i c l e ) . ~ ~ GAGsIproteoglycans are highly heterogeneous molecules that have many different biological properties. Their function in hair growth is not yet understood, although it has been suggested that they may confer a state of immunological privilege on the follicle, providing a permissive or protective benefit for growth.40 In conclusion, the unusual and distinctive array of ECM molecules that are expressed in hair follicle mesenchyme provides further evidence of the specialized nature of this tissue. The changes that occur in the dermal papilla ECM during the hair growth cycle suggest an important functional role for these molecules in mediating the epithelial-mesenchymal interactions that are known to occur at this site. However, many questions remain. For example, we need to know more about the control of turnover of ECM molecules during the hair growth cycle and whether the changes observed are causes or effects. The precise functional role of different ECM components in hair growth is also, at present, largely conjectural. The application of new techniques for studying human hair growth, such as the use of in vitro models, may help to provide some of the answers in future years.



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Distribution of extracellular matrix molecules in human hair follicles.

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