GK, IS, and JHS designed the study. GK, CT, OS, LD, IS, and JHS analyzed the data. GK and JHS enrolled patients. GK, CT, OS, LD, IS, and JHS contributed to writing the paper. GK, RH, DG, and PC collected data or did experiments for the study.
The authors have declared that no competing interests exist.
Skin atrophy is a common manifestation of aging and is frequently accompanied by ulceration and delayed wound healing. With an increasingly aging patient population, management of skin atrophy is becoming a major challenge in the clinic, particularly in light of the fact that there are no effective therapeutic options at present.
Atrophic skin displays a decreased hyaluronate (HA) content and expression of the major cell-surface hyaluronate receptor, CD44. In an effort to develop a therapeutic strategy for skin atrophy, we addressed the effect of topical administration of defined-size HA fragments (HAF) on skin trophicity. Treatment of primary keratinocyte cultures with intermediate-size HAF (HAFi; 50,000–400,000 Da) but not with small-size HAF (HAFs; <50,000 Da) or large-size HAF (HAFl; >400,000 Da) induced wild-type (wt) but not CD44-deficient (CD44−/−) keratinocyte proliferation. Topical application of HAFi caused marked epidermal hyperplasia in wt but not in CD44−/− mice, and significant skin thickening in patients with age- or corticosteroid-related skin atrophy. The effect of HAFi on keratinocyte proliferation was abrogated by antibodies against heparin-binding epidermal growth factor (HB-EGF) and its receptor, erbB1, which form a complex with a particular isoform of CD44 (CD44v3), and by tissue inhibitor of metalloproteinase-3 (TIMP-3).
Our observations provide a novel CD44-dependent mechanism for HA oligosaccharide-induced keratinocyte proliferation and suggest that topical HAFi application may provide an attractive therapeutic option in human skin atrophy.
Mouse and human data suggest that topical application of intermediate-size hyaluronate fragments holds therapeutic potential for skin atrophy.
Time wreaks many changes in the human body but the skin is where one of the first visible signs of aging—wrinkles—occurs. The skin consists of three main layers. The outermost layer is the epidermis. It is the thickness of a sheet of paper and forms a barrier that prevents the body losing water or infectious agents entering it. The cells in the epidermis are mainly keratinocytes. These specialized skin cells are continually produced at the base of the epidermis. From there, they move toward the skin's surface where they are shed. The middle layer is the dermis. It is about ten times thicker than the epidermis and contains the blood vessels that feed the skin, nerves, sebaceous glands, and hair follicles. The final, subcutaneous layer contains sweat glands, some hair follicles, blood vessels and fat. The dermis contains collagen fibers that support the skin and elastin fibers that provide flexibility. Human skin begins to age in early adulthood. By the time a person is 80 years old, their epidermis may be half its original thickness because of decreased keratinocyte proliferation. The dermis also thins, and loss of collagen and elastin fibers means that the skin becomes less elastic. The gradual loss of epidermis and dermis—skin atrophy—is clinically important because aging skin is more fragile and heals slower than young skin and is also prone to ulceration.
No one knows why skin atrophy occurs, but it is becoming more common as people live longer, and there is no effective treatment for it. One characteristic of atrophic skin is that, compared to normal skin, it contains less hyaluronate (also called hyaluronan and hyaluronic acid)—a large carbohydrate component of the extracellular matrix, the material that surrounds cells. It also contains less CD44, a cell-surface protein that interacts with hyaluronate. This interaction can stimulate cell proliferation and migration. Given these observations, in this study the researchers have investigated whether treating atrophic skin with fragments of hyaluronate might counteract atrophy.
The researchers isolated keratinocytes from normal mice and from CD44-deficient mice (CD44−/− mice) and treated them with different sized fragments of hyaluronate. Intermediate sized hyaluronate fragments (so-called HAFi) but not large or small fragments increased the proliferation of normal keratinocytes but not CD44−/− keratinocytes. This suggests that proliferation in response to HAFi is CD44-dependent. Similarly, a cream of HAFi applied to the backs of normal mice caused thickening of the epidermal layer but had no effect on CD44−/− mice. Finally, topical application of HAFi for one month caused skin thickening and clinical improvement in six people with skin atrophy but had no effect on normal human skin. The collagen, elastic fiber, and blood vessel content of the dermis also increased in treated patients. By using antibodies to block the function of various proteins, the researchers also discovered that heparin-binding epidermal growth factor (HB-EGF, a protein that stimulates keratinocyte proliferation), erbB1 (a cell-surface protein that binds HB-EGF), and matrix metalloproteinases (proteins that activate HB-EGF) are all required for the stimulation of keratinocyte proliferation by HAFi.
Taken together, these results provide the first indication that application of HAFi to atrophic skin might be useful therapeutically. The absence of any effect on normal human skin is reassuring but puzzling given the thickening seen in normal mouse skin, so this finding needs confirmation before hyaluronate fragments are used clinically. Longer trials in more people are also needed to characterize the clinical effects fully. Finally, the mechanism by which hyaluronate fragments have their effect needs to be studied in more depth. Such studies might reveal other potential therapeutic options for the treatment of skin atrophy.
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Skin atrophy is a frequent and clinically relevant manifestation of aging, often complicated by ulceration and impaired wound healing. Experimental evidence suggests that defective function of the principal cell-surface hyaluronate (HA) receptor CD44 [
Cellular responses to HA are primarily mediated by CD44, the most broadly expressed cell-surface HA receptor whose function is regulated at transcriptional, translational, and post-translational levels. Expression levels, variant isoform selection, and glycosylation all play an important role in the ability of CD44 to bind HA [
HA from rooster comb (IAL) for clinical application was provided by Transbussan (
Groups of five adult (>3 mo-old) SKH1 hairless, DBA/1 (The Jackson Laboratory,
Seven healthy young adults (seven males) between 19 and 32 y (mean age, 25.5 y), ten healthy women in menopause without hormone replacement therapy between 55 and 65 y (mean age, 60 y), three patients with advanced age-related skin atrophy (two females, one male) between 60 and 88 y (mean age, 76 y) and three patients with skin atrophy due to prolonged use of oral corticosteroids for rheumatoid arthritis (three females) between 74 and 86 y (mean age, 81 y) were included in this study after obtaining informed consent. Clinical studies were conducted with the authorization and according to the guidelines of Ethical Commission on Human Research of the University Hospital of Geneva. HAFi (1%) or vehicle cream samples of 0.5 g were applied twice daily for 1 mo on the posterior side of the right or left arm, respectively.
Dorsal skin samples were fixed in 10% phosphate-buffered formaldehyde, embedded in paraffin, and processed for histological analysis. Sections were cut at 5 μm, mounted onto slides, and stained with hematoxylin-eosin, Sirius red, and van Gieson elastin according to standard procedures.
Paraffin-embedded sections (5 μm) were mounted onto slides, dewaxed in xylene, rehydrated in a graded ethanol series, and prepared for immunoperoxidase staining according to standard procedures. Primary antibodies included anti-CD44v3 (1:100; Bender MedSystems,
Hyaluronidase treatment of tissues was performed by incubating the tissue sections with 1.5 μg/ml bovine testicular hyaluronidase (Sigma) in phosphate-buffered saline (PBS) for 5 h at 37 °C. The sections were then stained either with colloidal iron or with HABP as described above. The hyaluronidase digestion experiments also included negative controls incubated under otherwise similar conditions but lacking the enzyme. An HA-secreting mesothelioma section was used as a positive control.
Epidermal thickness of mice was measured by a graded ocular and multiplied by ten to correct the scale. Cutaneous thickness measurements of the healthy participants and patients were performed using a skin ultrasound system (Episcan; Longport,
Epidermal and dermal Ki-67+ cells were counted in ten fields per section at 40× magnification; the average value was calculated.
Frozen mouse epidermis or dermis and human biopsy samples were incubated in extraction buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 10 mM EDTA, 1% SDS, 10% glycerol, and protease inhibitor cocktail (complete; Boehringer,
Samples were loaded in nonreducing SDS sample buffer, subjected to electrophoresis, and transblotted onto 0.45 μm pore–size nitrocellulose membrane. Antibodies used for Western blot analysis were anti-CD44 standard (Bender MedSystems); anti-CD44v3 (Bender MedSystems); anti–MMP-7 (G-20; Santa Cruz Biotechnology); anti–pro-HB-EGF (M-18; Santa Cruz Biotechnology); anti–HB-EGF neutralizing antibody (R&D Systems,
Lysates were incubated overnight at 4 °C with anti-CD44 antibodies and protein A+G agarose beads (Pierce Biotechnology) in the presence of BSA and rat IgG. Elution of antigen–antibody complex was performed in nonreducing SDS sample buffer by heating 5 min at 95 °C. After centrifugation for 3 min at 15,000
Quantification of CD44, HA, and erbB1 in the skin samples by an enzyme-linked binding protein assay was performed using the sCD44 ELISA Kit (Bender MedSystems), the Corgenix Hyaluronic Acid Quantitative Test Kit (Endotell,
Epidermal keratinocytes were isolated and cultured in 96-well plates (Becton Dickinson,
Cultured keratinocytes from the dorsal skin of wt and CD44−/− DBA/1 mice were incubated with HAF generated from high-molecular-weight HA by sonication, enzymatic digestion, and size exclusion gel filtration (
(A) Elution profile of HAF. HAFs, HAFi, or HAFl were run on a Sephacryl S-400 column after sonification and enzymatic digestion.
(B) HAFi, but not HAFs or HAFl, induces in vitro mouse keratinocyte proliferation that is inhibited by anti-erbB1 antibody and TIMP-3. Keratinocytes from SKH1 and DBA/1 wt− mice were cultured in 96-well plates. On day 5 of culture, HAFs, HAFi, or HAFl (100 μg/ml), monoclonal anti-human AR neutralizing antibody (100 ng/ml), monoclonal anti-human erbB1 neutralizing antibody (isotype IgG1; 100 ng/ml), or mouse recombinant TIMP-3 (100 ng/ml) was added to the cultures. Mouse IgG1 was used as a control for anti-erbB1. Later (48 h), 0.037 MBq of [3H]-thymidine was added to each well. All experiments were done in triplicate and repeated five times. The results are presented as the mean incorporated counts per min ± standard error of the mean (SEM) of three wells per group. ***
(C) In vitro proliferative response of mouse keratinocytes to HAFi is CD44 and HB-EGF dependent. Keratinocyte proliferation in response to HAFi in vitro. Keratinocytes from SKH1, DBA/l wt, and DBA/l CD44−/− mice were cultured in 96-well plates. On day 5 of culture, HAFi (100 μg/ml), human HB-EGF (50 ng/ml), or mouse anti-human HB-EGF–neutralizing antibody (100 ng/ml) was added to the cultures. Later (48 h), 0.037 MBq of [3H]-thymidine was added to each well. All experiments were done in triplicate and repeated five times. The results are presented as the mean incorporated counts per min ± SEM of three wells per group. **
(D) Keratinocyte proliferation in response to TPA and EGF in vitro. Keratinocytes from DBA/l wt and DBA/l CD44−/− mice were cultured in 96-well plates. On day 5 of culture, TPA (1 ng/ml), EGF (50 ng/ml), or HAFi (100 μg/ml) was added to the cultures. Later (48 h), 0.037 MBq of [3H]-thymidine was added to each well. All experiments were done in triplicate and repeated five times. The results are presented as the mean incorporated counts per min ± SEM of three wells per group. **
(E) Fibroblast proliferation in response to bFGF in vitro. Human fibroblasts were cultured in 96-well plates. On day 2 of culture, bFGF (1 ng/ml) or HAFi (100 μg/ml) was added to the cultures. Later (48 h), 0.037 MBq of [3H]-thymidine was added to each well. All experiments were done in triplicate and repeated five times. The results are presented as the mean incorporated counts per minute ± SEM of three wells per group. ***
(F) Vascular endothelial cell proliferation in response to VEGF in vitro. HUVECs were cultured in 96-well plates. On day 2 of culture, VEGF (20 ng/ml) or HAFi (100 μg/ml) was added to the cultures. Later (48 h), 0.037 MBq of [3H]-thymidine was added to each well. All experiments were done in triplicate and repeated five times. The results are presented as the mean incorporated counts per min ± SEM of three wells per group. ***
(G) Keratinocyte proliferation in response to HAFi-treated fibroblast or HUVEC supernatants in vitro. Keratinocytes from DBA/l wt mice, fibroblasts, or HUVECs were cultured in 96-well plates. On day 2 of culture, HAFi (100 μg/ml) was added to the fibroblast or HUVEC cultures. On day 5 of culture, supernatants alone or supplemented with HAFi (100 μg/ml) were added to the keratinocyte cultures (1:4 ratio). Later (48 h), 0.037 MBq of [3H]-thymidine was added to each well. All experiments were done in triplicate and repeated five times. The results are presented as the mean incorporated counts per min ± SEM of three wells per group. ***
To verify that the inability of HAFi to induce proliferation of CD44−/− keratinocytes was indeed due to the lack of CD44 and not to a more general proliferation defect of these cells, we assessed the response of CD44−/− keratinocytes to phorbol ester and EGF stimulation. CD44−/− keratinocytes were observed to mount a proliferative response to both mitogens, albeit to a lesser degree than wt cells (
The stimulatory effect of HAFi was not limited to cultured keratinocytes. Incubation of primary human fibroblasts from a healthy donor and HUVECs with HAFi at concentrations that induced keratinocyte proliferation resulted in increased proliferation of both cell types (
To determine whether HA fragments may induce keratinocyte proliferation in vivo, the effect of repeated local administration of HAFi to the dorsal skin of wt and CD44−/− DBA/l mice was assessed. To control for possible strain specific effects, SKH1 hairless mice were also subjected to local HAFi treatment. Daily topical application of a solution of 0.2% HAFi for 3 d resulted in significant epidermal hyperplasia (
(A) HAFi-induced hyperplasia of mouse skin is CD44 dependent. Histological sections of vehicle-treated (a and c) or HAFi-treated (b and d) DBA/1 (a and b) or CD44−/− (c and d) dorsal mouse skin. Note the epidermal hyperplasia in DBA/1 but not in CD44−/− mice.
(B) Epidermal thickness in vehicle- or HAFi-treated SKH1, DBA/1, and CD44−/− mouse back skin measured with an ocular micrometer. Ten measurements were performed per mouse, and the average value was calculated. The results are presented as the mean epidermal thickness ± SEM of six animals per group. ***
(C) In vivo proliferative response of mouse epidermis to HAFi is CD44 dependent. Ki67 staining of vehicle-treated (a and c) or HAFi-treated (b and d) DBA/1 (a and b) or CD44−/− (c and d) dorsal mouse skin.
(D) Dermal cellularity in vehicle- or HAFi-treated SKH1, DBA/1, and CD44−/− mouse back skin. Samples were counted at 40× magnification. Ten counts were made per mouse, and the average value was calculated. The results are presented as the mean number of cells ± SEM of six animals per group. ***
(E) HAFi corrects age- and corticosteroid-related atrophy in human skin. Atrophic human forearm skin 1 mo after topical treatment with vehicle (a) or 1% HAFi (b). Note the decrease of wrinkles, hemorrhage (yellow arrow), and pseudoscars (red arrows), the visibility of superficial vessels (blue arrow), and the smoothening of the skin with after HAFi treatment.
(F) HAFi corrects age- and corticosteroid-related atrophy in human skin. Histology of atrophic human forearm skin 1 mo after topical treatment with vehicle (a) or 1% HAFi (b). Note the significant epidermal hyperplasia after HAFi treatment.
(G) HAFi results in skin hyperplasia in atrophic but not normal human skin. Skin thickness in HAFi-treated young (a), nonlesional aged (b), or atrophic aged (c) human skin measured by echography. The results are presented as boxplots with median values (triangles). Young untreated versus nonlesional aged untreated,
The potential effect of HAFi on keratinocyte differentiation in vivo was assessed by staining of HAFi- and vehicle-treated skin sections with antibodies against differentiation markers, including keratin-14, filaggrin, and loricrin. All three differentiation markers were found to display increased expression in hyperplastic HAFi-treated skin that was proportional to the degree of hyperplasia (
The effect of HAFi on the composition of the dermis was assessed by addressing changes in collagen expression and vascularization. Staining with Sirius red revealed an increase in collagen content of the superficial dermis (
Finally, to determine whether HAFi-mediated stimulation of fibroblasts and endothelial cells might indirectly participate in keratinocyte proliferation, keratinocytes derived from wt mice were incubated with 1:4 diluted conditioned culture media from 72-h HAFi- and vehicle-treated fibroblasts and HUVECs. Neither HAFi-treated fibroblasts nor HUVEC culture supernatants displayed any significant effect on keratinocyte proliferation in vitro (
To assess the effect of HAFi administration to human skin, six patients with atrophic skin lesions and 17 control participants, including seven healthy men (age, 29–32 y; mean age, 25.5 y) and ten healthy postmenopausal women who had not received hormone replacement therapy (age, 55–65 y; mean age, 60 y) were subjected to daily topical application to the forearm of a 1% preparation of HAFi for 1 mo. Following termination of the treatment, none of the control participants revealed a measurable increase in skin thickness, signs of inflammation, or scaling (
To begin to address the putative mechanism of HAFi-induced skin hyperplasia, we assessed changes in CD44 expression and HA synthesis in response to local HA application. Topical HAFi application, but not HAFl or HAFs (unpublished data) application, resulted in increased CD44 expression at the RNA and protein levels throughout the epidermis (
(A) HAFi increases CD44 expression in mouse skin. Immunostaining of sections of vehicle-treated (a and c) or HAFi-treated (b and d) DBA/1 mouse dorsal skin with anti-CD44 (a and b) or anti-CD44v3 (c and d) antibodies. Note the hyperplasia and increase in both CD44 and CD44v3 expression in the epidermis.
(B) HAFi increases the epidermal and dermal HA content in mouse skin. HABP staining of sections of vehicle-treated (a) or HAFi-treated (b) DBA/1 mouse dorsal skin showing elevated amounts of HA in the dermis. Epidermal (c) and dermal (d) HA content of HAFi-treated skin of SKH1 hairless mice were quantified by an enzyme-linked binding protein assay. The results are presented as the mean HA concentration ± SEM of six animals per group. **
(C) HAFs and HAFi penetrate mouse skin. Streptavidin-FITC staining of sections of vehicle (a), biotinylated HAFs-treated (b), HAFi-treated (c), or HAFl-treated (d) SKH1 hairless mouse back skin. Note the presence of biotin in HAFs-treated, and to a lesser extent, in HAFi-treated skin (arrows).
(D) Topically applied HAFi is located both intra- and extracellularly. Sections of HAFi-treated dorsal SKH1 hairless mouse skin were stained with streptavidin-FITC (a) or anti-vimentin antibody, biotinylated secondary antibody, and streptavidin-rhodamine (b). Topically applied HAFi (a; green fluorescence, top arrows) shows colocalization (c; yellow fluorescence, arrows) with vimentin (b; red fluorescence, arrows) and outside the cells in the extracellular matrix (a; lower arrows).
(E) HAFi increases the expression of HASs and Hyal2 in mouse skin. Northern blot analysis of HAS1, HAS2, HAS3, and Hyal2 RNA expression in vehicle- or HAFi-treated DBA/1 wt or CD44−/− mouse dorsal skin. The hybridization signals were quantitated by scanning the autoradiograms with a laser densitometer. The results are presented as the mean optical density ± SEM of three animals per group. *
The superficial dermis of HAFi-treated DBA/1 mouse dorsal skin sections displayed strong reactivity with biotinylated HABP, an established probe of tissue HA (
To address the fate of locally administered HAF to the skin, size-fractionated HA was biotinylated, and its localization was traced with fluorescein-labeled streptavidin. Biotinylated HAFs-treated SKHI hairless mice and, to a lesser extent, HAFi- but not HAFl-treated SKH1 hairless mice displayed biotin deposits in the superficial dermis after 3 d of topical application (
To determine whether the absorbed HAF might affect local HA synthesis and degradation, we assessed the expression of HA-polymerizing enzymes HAS1, HAS2, and HAS3 and the major tissue HA-degrading enzyme, hyaluronidase 2 (Hyal2), in HAFi-treated skin of DBA/1 and CD44−/− mice (
Because HAFi-induced CD44v3 expression (
(A) HAFi induces expression of CD44v3, pro-HB-EGF, and active HB-EGF in mouse skin. Western blot analysis on the protein extracts of vehicle- or HAFi-treated SKH1 hairless mice for CD44v3 (∼200 kDa [a]), active HB-EGF (∼15 kDa [b]), pro-HB-EGF (∼25 kDa [c]), and loading control α-tubulin (50 kDa [d]).
(B) CD44 associates with erbB1 in keratinocytes in vitro and in vivo. Western blot analysis of anti-CD44 antibody immunoprecipitates of protein extracts of cultured keratinocytes (a) or epidermis of DBA/1 mouse skin immunoblotted with anti-erbB1. Mock, isotype-matched rat IgG; beads, protein A+G agarose beads treated with anti-CD44 antibody.
(C) CD44, HA and erbB1 levels are diminished in atrophic human skin. CD44 (a), HA (b), and erbB1 (c) expression in forearm skin biopsy specimens of young adults (control) and elderly patients with skin atrophy. The results are presented as the mean CD44, HA, or erbB1 concentration ± SEM of three subjects per group. *
Based on the observations that HAFi treatment enhanced CD44v3, erbB1, and HB-EGF expression and induced HB-EGF activation, we addressed the possibility that these molecules may form part of the functional machinery implicated in the regenerative response of keratinocytes to HAFi. The proliferative response of wt DBA/1 mouse keratinocytes to HAFi was abrogated by anti-human erbB1 neutralizing antibody (
Our observations provide evidence for the first time to our knowledge that topically applied HAF ranging from 50,000 to 400,000 Da penetrate the epidermis and induce keratinocyte proliferation that translates into the thickening of mouse and human skin. Penetration of topically applied HA of 250,000–400,000 Da into mouse and human dermis was recently demonstrated [
At least two types of effects occurred as a result of topical HAFi application: CD44-independent penetration of skin and induction of HAS and Hyal2, and CD44-dependent keratinocyte proliferation. Both fibroblasts and endothelial cells also proliferated in response to HAFi in vitro, providing a possible explanation for the increased dermal collagen deposition and angiogenesis, respectively, observed in vivo. However, the contribution of fibroblast activity and angiogenesis to epidermal hyperplasia was most likely of minor importance, given that conditioned culture media of HAFi-stimulated fibroblasts and endothelial cells failed to augment keratinocyte proliferation. Importantly, an increase in human skin thickness in response to HAFi was observed only in patients with skin atrophy. Although the reasons of the absence of such a response in healthy participants can only be speculative at present, it is possible that physiological HA production saturates tissue CD44 binding capacity in the steady state, such that additional exogenous HA fragments fail to induce a significant CD44-dependent response. Whatever the precise mechanism, the absence of skin hyperplasia in healthy participants in response to HAFi suggests that topical HAFi administration does not present the risk of inducing undesirable local side effects.
Penetration of HA fragments into the dermis may occur via hair follicles, which provide a well-recognized route for macromolecular skin penetration [
Proliferation in response to HAFi is a CD44-dependent event. Our present observations provide evidence that in addition to CD44, HB-EGF, erbB1, and MMPs/ADAMs are required for HA-dependent in vitro keratinocyte proliferation. The absence of a proliferative response of CD44−/− keratinocytes to HB-EGF is consistent with the notion that HB-EGF interaction with its receptors requires presentation by heparan sulfate side chains of CD44v3-containing isoforms [
The observation that anti-erbB1– and anti–HB-EGF–blocking antibodies had the same abrogating effect as the absence of CD44 on in vitro keratinocyte proliferation in response to HAFi supports the notion that erbB1 signaling, triggered by HB-EGF, may play a key role in HAFi-induced proliferation. Similar to its role in uterine and mammary epithelia, HA-induced CD44v3 aggregates may recruit a functional cell-surface complex in keratinocytes composed of pro-HB-EGF, erbB1, which replaces erbB4, and an MMP or ADAM [
(A) CD44, HA, and HB-EGF in normal skin (a), atrophic skin (b), and following treatment of atrophic skin treated with HAFi (c).
(B) Schematic representation of the hypothetical assembly of the putative complex: HAFi mediates CD44v3 and heparan sulfate–bound pro-HB-EGF aggregation, resulting in the recruitment and activation of an MMP/ADAM. Pro-HB-EGF is cleaved by the MMP/ADAM, and the resulting active moiety binds and activates erbB1, generating proliferation signals.
Our observations have defined 50,000–400,000 Da HAF as reagents capable of inducing a proliferative response in mouse and human skin. Clinically, topical HAFi application resulted in epidermal hyperplasia with restoration to normal thickness of atrophic human skin as early as 1 mo after initiation of treatment. This effect was accompanied by significant clinical improvement, suggesting that HAFi may provide the basis for the development of novel therapeutic strategies for skin diseases characterized by atrophy.
(A) TPA induces a reduced skin hyperplasia in CD44−/− mice. Histological sections of acetone-treated (parts a and c) or TPA-treated (parts b and d) DBA/1 (parts a and b) or CD44−/− (parts c and d) dorsal mouse skin. Note the decreased epidermal hyperplasia in CD44−/− mice.
(B) Epidermal thickness in acetone- or TPA-treated DBA/1 and CD44−/− mouse back skin measured with an ocular micrometer. Ten measurements were performed per mouse, and the average value was calculated. The results are presented as the mean epidermal thickness ± SEM of six animals per group. ***
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(A) HAFi has no effect on epidermal differentiation in mouse skin. Immunostaining of sections of vehicle-treated (parts a, c, and e) or HAFi-treated (parts b, d, and f) DBA/1 mouse dorsal skin with anti-K14 (parts a and b), anti-filaggrin (parts c and d), or anti-loricrin (parts e and f) antibody. Note the lack of increase in staining intensity despite the increased number of stained cells in HAFi-treated skin.
(B) HAFi increases the collagen, elastin and vascular content in atrophic human skin. Histology of atrophic human forearm skin 1 month after topical treatment with vehicle (part a) or 1% HAFi (part b), stained with Sirius red (parts a and b), van Gieson elastin (parts c and d), or anti-CD31 antibody (parts e and f). Note the increase in dermal collagen, elastic fibers, and vessels after HAFi treatment.
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Western blot analysis on the protein extracts of vehicle- or HAFi-treated SKH1 hairless mice for CD44v3.
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IS and JHS share senior authorship of this article. We thank Eric Augsburger, Marie-Jo Cartier, and Evelyne Leemans at the Dermatopathology Laboratory of the University Hospital of Geneva for their excellent technical help.
a disintegrin and metalloproteinase
amphiregulin
epidermal-like growth factor
hyaluronate
HA-binding protein
HA fragment(s)
intermediate-size HAF
large-size HAF
small-size HAF
HA synthase
heparin-binding EGF
human umbilical vein endothelial cell
matrix metalloproteinase 7
HB-EGF precursor
standard error of the mean
tissue inhibitor of metalloproteinase-3
wild-type