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Home , Collagen loss, Elastin, Tenascin

See related Commentary on page xii Deficiency of -X Causes Abnormalities in Dermal Morphology

ManonC.Zweers,à Ivonne M. van Vlijmen-Willems,à Toin H. van Kuppevelt,w Robert P. Mecham,zy Peter M. Steijlen,à Jim Bristow,z and Joost Schalkwijkà ÃDepartment of Dermatology and wDepartment of Biochemistry, University Medical Center, St Radboud Nijmegen, Nijmegen, The Netherlands; zDepartment of Cell Biology and Physiology, Washington University School of Medicine, St Louis, MO, USA; yDepartment of Medicine, Washington University School of Medicine,StLouis,MO,USA;zDepartment of Pediatrics, University of California at San Francisco, CA, USA

Deficiency of the tenascin-X (TNX) was recently described as the molecular basis of a new, recessive type of Ehlers–Danlos syndrome. Here we report gross abnormalities of the elastic fibers and microfibrils in the of these patients, and reduced dermal content, as determined by quantitative image analysis. The ascending, fine elastic fibers in the papillary dermis were absent or inconspicuous and had few branches. The coarse elastic fibers of the reticular dermis were fragmented and clumped. At the ultrastructural level, irregular and immature fibers and fibers devoid of microfibrils were observed. In TNX-deficient patients the dermal collagen density was reduced, but no structural abnormalities in the collagen fibrils were found. These findings suggest that both elastic fiber abnormalities and reduced collagen content contribute to the observed phenotype in TNX-deficient patients. Key words: /dermis/Ehlers–Danlos syndrome/immunohistochemistry J Invest Dermatol 122:885 –891, 2004

The Ehlers–Danlos syndrome (EDS) is a clinically and Although the association of TNX-deficiency with EDS genetically heterogeneous connective tissue disorder, char- suggested one of the first functions for any of the tenascin acterized by hyperextensibility of the , hypermobile family members, the exact pathophysiological mechanism joints, and tissue fragility (Beighton et al, 1998). The resulting in this phenotype was hitherto unknown. Previous recognition of ultrastructural abnormalities of the collagen studies of the skin of a TNX-deficient EDS patient showed fibrils and the subsequent identification of mutations in normal secretion of type I and type V , and encoding collagen and collagen-modifying ultrastructural analysis did not reveal gross abnormalities of has led to the concept that EDS is a disorder of fibrillar the collagen fibrils (Burch et al, 1997; Mao and Bristow, collagen (Byers, 1994; Hausser and Anton-Lamprecht, 2001). Histopathological examination of skin biopsies from 1994). This paradigm was recently challenged by the finding five TNX-deficient probands suggested reduced staining of that deficiency for the extracellular matrix protein tenascin- the dermal connective tissue (Schalkwijk et al, 2001), and a X (TNX) causes a clinically distinct, recessive form of EDS recent study in TNX null mice also demonstrated a reduced (Burch et al, 1997; Schalkwijk et al, 2001). collagen content in the skin (Mao et al, 2002). Whereas The are a family of large matricellular glycopro- fibrillar collagens provide the connective tissue with tensile teins, which all share a similar modular structure consisting of strength, the elastic fiber system is another major structural an N-terminal domain possibly responsible for oligomerization, matrix component, providing elasticity and resilience to a series of EGF-like repeats, a variable number of fibronectin connective tissues. The mature elastic fiber is largely type III domains and an C-terminal fibrinogen-like domain. To composed of two distinct components: amorphous elastin date three members have been identified in mammals: and microfibrils. Elastogenesis is thought to begin with the tenascin-C, tenascin-R, and TNX (Erickson, 1993; Chiquet deposition of microfibrillar components, which form a Ehrismann et al, 1994; Minet et al, 1999; Jones and Jones, scaffold for the alignment of tropoelastin (Mecham, 1991; 2000). TNX, the largest member of the tenascin family, is widely Debelle and Tamburro, 1999). Perturbation of the elastic expressed during embryonic development and in several fibers can occur at any step of this multi-phase process connective tissues of the adult, including sheats, (Holbrook and Byers, 1989; Christiano and Uitto, 1994). As muscle, blood vessels, and throughout the entire dermis the biomechanical properties of the tissues that were (Bristow et al, 1993; Burch et al, 1995; Geffrotin et al, 1995). affected in our TNX-deficient patients (skin, joints, small This expression pattern coincides with the connective tissue blood vessels) are not only determined by fibrillar collagen defects found in TNX-deficient patients and indicates a pivotal but also by elastic fibers, TNX could very well be involved in role for TNX in maintaining the integrity of connective tissues. development and maintenance of the elastic fiber system. In this study we describe gross abnormalities of the elastic Abbreviations: EDS, Ehlers–Danlos syndrome; FITC, fluorescein fibers and microfibrils in the skin of TNX-deficient EDS iso-thiocyanate; TNX, tenascin-X patients.

Copyright r 2004 by The Society for Investigative Dermatology, Inc. 885 886 ZWEERS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Results gross abnormalities. No deviations in epidermal or dermal thickness were noted. Elastin-van Gieson staining of the Histopathology and immunohistochemistry Histopathol- skin biopsies from healthy individuals revealed the normal ogy and immunolocalization of connective tissue compo- pattern of thin elastic fibers oriented perpendicular to the nents were studied in the papillary and reticular dermis of dermo-epidermal junction, and coarse elastic fibers in the biopsies from five TNX-deficient probands, and compared reticular dermis that were arranged more or less parallel to with biopsies from healthy individuals. H&E staining of the the dermo-epidermal junction (Fig 1A). The dermis of all five skin biopsies from TNX-deficient patients did not reveal TNX-deficient EDS patients showed aberrant elastic fibers.

Figure 1 van Gieson’s staining of elastic fibers in biopsies. In normal non-sun exposed skin, Elastin-van Gieson’s staining shows a typical candelabra-like pattern oriented perpendicular towards the basal lamina (A). In TNX-deficient patients (B, C), the candelabra-like structure is grossly disturbed. Note the short, sparsely branched structures in b and the horizontally oriented, coarse fragments and in c fragmentation of elastic fibers in TNX-deficient skin. Morphometric analysis of five patients and eight controls showed a significant reduction in length of the elastic fibers (po0.01) (D), number of branches per elastic fiber (po0.01) (E) and relative collagen density (po0.05) (F) in TNX-deficient dermis. Asterisks denote values à Ãà significantly different from control ( po0.05, po0.01). Scale bar ¼ 50 mm. 122 : 4 APRIL 2004 ELASTIC FIBER ABNORMALITIES IN EDS 887

In the reticular dermis, normal looking, slender elastic fibers were present together with fragmented and clumped fibers. In the papillary dermis, the number of elastic fibers was low, fibers were not arranged perpendicular to the dermal–epidermal junction and elastic fibers appeared highly fragmented (Fig 1B and C). The elastic fibers appeared normal around blood vessels in the dermis. Morphometrical analysis of the elastic fibers on Elastin- van Gieson stained sections showed a significant decrease in elastic fiber length in TNX-deficient EDS patients as compared with age-matched, healthy individuals (po0.01; Fig 1D). Elastic fiber length was reduced from approxi- mately 50 mm in the normal skin, to 20 mm in the TNX- deficient skin. Moreover, the number of branches per elastic fiber was also considerably decreased (po0.01; Fig 1E). The length and number of branches of the elastic fibers did not correlate with the age of the patients or healthy individuals (not shown). The relative collagen density in the papillary dermis was quantified by image analysis in Elastin-van Gieson stained sections of patients and matched controls. As shown in Fig 1F, the relative collagen density in the papillary dermis was significantly lower in TNX-deficient patients (po0.05;

Figure 3 Immunohistochemical staining for elastin and fibrillin-1. Note the typical structure of thin, ascending elastin-positive fibers in the papillary dermis (A) and the thicker fibers in the reticular dermis of normal skin (C). -1 staining shows a similar pattern (E). In the papillary dermis of the TNX-deficient skin, elastin-positive and fibrillin-positive fibers are sparse and fragmented (B) and (F). In the reticular dermis, clumping and fragmentation of elastin-positive structures is observed in TNX-deficient patients (D). Scale bar ¼ 25 mm.

Fig 1F). In addition to collagen, we studied the presence of glycosaminoglycans, which represent another major constituent of the dermal extracellular matrix. Safranin-O staining, which detects negatively charged polysacchar- ides, did not reveal differences between the TNX-deficient and normal skin (Fig 2A and B). Using immunohistochem- Figure 2 istry, we analyzed the distribution of , an abundant Safranin-O histochemistry and decorin immunostaining. Safranin-O dermal that colocalizes and binds to TNX staining shows a similar glycosaminoglycan distribution in control (A) (Elefteriou et al, 2001), and interacts with elastic fiber and TNX-deficient skin (B). Decorin is equally present in control (C) and components (Reinboth et al, 2002) and collagen (Brown and TNX-deficient skin (D). TNX is distributed throughout the entire dermis of control skin (E) and is absent from TNX-deficient skin (F). Scale Vogel, 1989). Again, no differences were noted between bar ¼ 25 mm for A–D,50mm for E and F. the normal and TNX-deficient skin (Fig 2C and D). For 888 ZWEERS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY comparison, TNX was present throughout the entire dermis Discussion of control skin, whereas it was absent from the TNX- deficient skin (Fig 2E and F). We observed disturbed elastic fiber morphology and Immunohistochemical staining of the elastic fiber com- reduced collagen density in skin biopsies of TNX-deficient ponent elastin showed a distribution similar to the Elastin- EDS patients, demonstrating an essential role of TNX in the van Gieson staining (Fig 3A–D). In addition, the skin formation and/or maintenance of dermal connective tissue sections were stained for fibrillin-1, which is a component architecture. An obvious interpretation of these findings is of both the elastic fibers and the microfibrillar network that that TNX is involved in regulation of structural integrity exists independent of the elastic fibers. In healthy indivi- of elastic fibers and microfibrils, and that the absence of duals, the staining for fibrillin-1 revealed a dense network of TNX precludes proper biomechanical functioning of these fine branching fibers in the papillary dermis arranged structures. Since TNX is present early in embryonic perpendicular to the dermal-epidermal junction (Fig 3E). In development in different connective tissues (Matsumoto the deeper reticular dermis microfibrils colocalize with the et al, 1994; Burch et al, 1995), TNX could be involved in the elastic fibers and fibrillin-1 staining showed a distribution development of the elastic fibers through direct interaction accordingly. Skin biopsies of all five TNX-deficient EDS with elastic fiber components. TNX contains a number of patients showed a strongly decreased staining for fibrillin-1. different domains, including fibronectin type III domains and Few fibrillin-1 positive fibers ascending from the papillary EGF-repeats that might be involved in intermolecular dermis to the basal membrane of the were interactions (Bristow et al, 1993; Erickson, 1993; Jones present (Fig 3F). The reticular dermis contained many small and Jones, 2000). TNX could also provide anchorage fragments of fibrillin-positive material as compared with of elastic fibers to cells mediated by interaction with the control biopsies (not shown). cell-surface integrin avb3 (Elefteriou et al, 1999), thereby stabilizing and organizing the elastic fibers, as has been shown for the microfibrillar component fibrillin-1 (Pfaff et al, Ultrastructure of elastic fibers TNX-deficient EDS patients 1996; Sakamoto et al, 1996). Deposition and production showed a number of elastic fiber abnormalities as compared of fibrillin-1 by TNX-deficient fibroblasts in vitro was not with healthy individuals (Fig 4). Irregularly shaped elastic altered, therefore it is unlikely that TNX regulates elastic fibers were present in all TNX-deficient EDS patients fiber assembly and stability directly through interaction with examined. Large amounts of electron-dense elastin were fibrillin-1. No significant correlation was found between the present, indicative of ‘‘immature’’ elastin, that is, many length and number of branches of the elastic fibers and the microfibrils with relatively little cross-linked elastin. Small age of the individuals included in this study (age range 32– elastic fibers were observed in both the papillary and reticular 56), which makes it less likely that TNX-deficiency results dermis in three of four examined TNX-deficient EDS patients. in increased breakdown of elastic fibers. Moreover, the In addition, we observed debris-like inclusions inside phenotype of TNX-deficient patients is already present in the elastic fibers. We also observed abnormalities in the early childhood. Taken together, we speculate that TNX is organization of microfibrils. Excessive amounts of microfibrils essential for collagen and elastin assembly and stability. were occasionally present with a tangled rather than a linear Whether TNX is involved in directly guiding extracellular orientation relative to the elastic fiber. Furthermore, in many matrix assembly, or in maintaining tissue fibers elastin appeared to be deposited independent of the remains unclear. TNX knockout mice, which have recently microfibrillar scaffold, as hardly any microfibrils were present been generated, will provide a suitable model to study the in and around the elastic fibers. Beside atypical elastic fibers, elastic fiber pathology in more detail. These mice are fertile normal elastic fibers were present as well in each patient. and viable and they recapitulate the skin manifestations of The morphology of fibroblasts was not altered; fibroblasts the TNX-deficient EDS patients (Mao et al, 2002). showed a well-developed , indicative The clinical symptoms of TNX-deficiency also include of active protein synthesis. vascular fragility of the small blood vessels and joint (Schalkwijk et al, 2001). Both blood vessels and joint structures such as and contain In vitro production of microfibrils The fragmented mor- TNX, collagen, and elastic fibers. It is not known to what phological appearance of the elastic fibers in adult skin extent collagen and elastic fibers are affected in these could be the result from breakdown of pre-existing intact tissues. It is, however, very likely that similar connective fibers, or alternatively, it could be the result of reduced tissue alterations will be found there, and that they contribute synthesis or a faulty assembly process during development. to disturbance of functional properties. It is not very likely We therefore used an in vitro model for microfibril assembly that TNX-deficiency results in a generalized elastinopathy, as to address this issue. Both fibroblasts of control and TNX- has been implied for fibulin-5 (Loeys et al, 2002; Nakamura deficient individuals formed a network of long, branching et al, 2002; Yanagisawa et al, 2002; Markova et al, 2003). fibrillin-1 positive fibers (Fig 5C and D). The control Although one of the TNX-deficient patients has a chronic fibroblasts and fibroblasts of TNX-deficient patients se- obstructive disease (Schalkwijk et al, 2001), which is creted comparable amounts of intact 350 kDa fibrillin-1 in consistent with loss of elastic fiber structure, no obvious the culture supernatant (Fig 5B). In addition, no proteolytic abnormalities were found in the elastic fibers in the mitral fragments of fibrillin-1 were observed. TNX was secreted by valve of one of the TNX-deficient patients (not shown). control fibroblasts in low amounts (Fig 5A), and localization A previous study reported that the ultrastructure of of TNX was mainly intracellular (Fig 5E). collagen fibrils in the TNX-deficient index patient was 122 : 4 APRIL 2004 ELASTIC FIBER ABNORMALITIES IN EDS 889

Figure 5 Fibrillin-1 and TNX deposition and secretion. Normal fibroblasts secrete TNX into the medium in contrast to TNX-deficient fibroblasts, as shown by western blotting (A). No difference is seen in fibrillin-1 secretion into the medium (B), or in deposition of fibrillin-1 in the extracellular matrix by control fibroblasts (C) and TNX-deficient fibroblasts (D). Staining of TNX shows intracellular localization in normal fibroblasts (E) and absence of staining in TNX-deficient fibroblasts (F). Scale bar ¼ 25 mm.

patients feels velvety to touch. Clearly, the effect of TNX- deficiency on collagen metabolism and its contribution to Figure 4 connective tissue pathology requires further study. Ultrastructural analysis of elastic fibers in dermis. TNX-deficient Although we cannot exclude the possibility that TNX skin (A–E) shows many abnormalities in elastic fibers. (A) Fragmented affects the stability of the elastic fibers and collagen, appearing elastic fiber. (B) The elastin matrix appears to be deposited independent of the microfibrillar scaffold. Note the absence of thereby promoting degradation, we favor the interpretation microfibrils within the amorphous elastin and the holes containing that TNX acts as an organizer of connective tissue microfibrillar material inside the elastic fiber. (C) Note the blebs of assembly. The absence of TNX could cause quantitative amorphous elastin without microfibrils as indicated by arrows. (D) and qualitative alterations of the extracellular matrix. Over Excessive amounts of elastin containing electron-dense debris. (E) Microfibrils are excessive and appear to be tangled in TNX-deficient the past decade, several causes of heritable diseases of the patients. (F) Elastic fiber in skin from age-matched control. Arrows: elastic fibers have been elucidated, and it has become abnormalities of elastic fibers; arrowheads: debris of microfibrils; apparent that mutations in genes that encode asterisk: normal appearing elastic fiber. Scale bar; A–D, F ¼ 0.4 mm, E ¼ 0.17 mm. involved in elastic fiber biology, will also result in elastic fiber pathology (for review see Handford et al, 2000; Milewicz et al, 2000). This study establishes a role for TNX in elastic fiber biology, which could explain at least part of the normal (Burch et al, 1997), which is in agreement with our phenotype of the TNX-deficient patients. observations. It should be noted that in most ligaments and tendons, elastin content is fairly low compared with collagen. In view of the reduced collagen content found in Materials and Methods the dermis of our patients, and the reduced collagen Patients Four-mm full-thickness punch biopsies were taken from deposition reported in TNX-deficient mice (Mao et al, a non-sun exposed area on the buttock from five TNX-deficient 2002), it is possible that symptoms of joint hypermobility individuals (age range 32–56 y, average 45 8 y), and eight age- are, besides elastic fiber abnormalities, mostly a conse- matched healthy volunteers (age range 25–63 y, average 48 12 quence of quantitative or qualitative collagen fibril altera- y), as described previously (Schalkwijk et al, 2001). Informed tions. in the reticular dermis does not seem to consent was obtained, and the study protocol was approved by be compensated for by an increased content of glycosa- the local medical ethical committee. All clinical investigations were conducted according to Declaration of Helsinki principles. minoglycans. The discrepancy of reduced collagen content and normal skin thickness could possibly be explained by culture Human fibroblasts from skin biopsies of healthy edema, which might also explain why skin of TNX-deficient donors and TNX-deficient patients were grown in Dulbecco’s 890 ZWEERS ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Modified Eagle Medium (DMEM) (Biowhittaker Europe, Verviers, analysis. Length of elastic fibers was analyzed semi-automatically Belgium) supplemented with 10% fetal calf serum (Seralab, using IPLab software (Scananalytics, Fairfax) and number of Nistelrode, The Netherlands) and penicillin/streptomycin (Gibco- branches was counted manually. Relative density of collagen BRL, Life Technologies, Breda, The Netherlands). were was determined on Elastin-van Gieson stained sections using allowed to attach and spread for at least 3 d and cultures were ImagePro Plus software (Media Cybernetics, Silver Spring, Mary- maintained at 371Cinan8%CO2 atmosphere. When the land). This was accomplished by determining the percentage of fibroblasts were grown to confluency, they were allowed to grow unstained area in the papillary dermis in the region of interest. The for an additional 2 wk to induce formation of microfibrillar area of interest was consistently set just below the epidermis structures. across the entire distance of the biopsy, with a depth of 0.3 mm. All measurements were performed at 200 magnification. Immunohistochemistry Biopsies were fixed for 4 h in buffered 4% formalin and embedded in paraffin. Six-micrometer sections Statistical procedures Student’s t test was used for comparison were mounted on 3-aminopropyltriethoxy-silane (Sigma, St Louis, of means between groups. A value of po0.05 was considered Missouri)-coated slides and subsequently deparaffinized and significant. rehydrated. For general histopathology, the sections were stained with haematoxylin and eosin (H&E). Sections to be stained for elastin were pre-treated with 2% trypsin for 15 min at 371C. For We thank Dr Paul Jap for fruitful discussions and valuable advice. We immunohistochemical detection of fibrillin-1 no pre-treatment was thank Dr Anneke Gabree¨ ls-Festen and Lilian Eshuis for their helpful required. Sections were blocked with 20% normal goat serum or contribution. 20% normal swine serum (Vector Laboratories Inc., Burlingham, California) before application of primary antibodies. Sections were DOI: 10.1111/j.0022-202X.2004.22401.x subsequently labeled with primary antibodies: monoclonal anti- Manuscript received February 27, 2003; revised October 17, 2003; elastin (BA-4, Sigma), monoclonal anti-fibrillin-1 (MAB2502; Che- accepted for publication November 10, 2003 micon, Temecula, California), or anti-decorin (Steijlen et al, 1994). Then, sections were washed and incubated with biotinylated Address correspondence to: Manon Zweers, Department of Dermatol- secondary antibody followed by a complex of avidine and ogy, University Medical Center, St Radboud Nijmegen, PO Box 9101, biotinylated horseradish peroxidase according to the manufac- 6500 HB Nijmegen, The Netherlands. Email: [email protected] turer’s instructions (Vector Labs). Aminoethylcarbazol was used as the chromogenic substrate (Vector Labs) and, if necessary, sections were counterstained with Mayer’s haematoxylin (Sigma). References Cryo-sections of skin-biopsy specimens were immunostained for TNX as described before (Schalkwijk et al, 2001). For - Beighton P, De Paepe A, Steinmann B, Tsipouras P, Wenstrup RJ: Ehlers–Danlos ral staining of elastic fibers, Elastin-van Gieson staining was syndromes: Revised nosology, Villefranche, 1997. 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