Activation of heparanase by ultraviolet B irradiation leads to functional loss of basement membrane at the dermal–epidermal junction in human skin

Recently, we reported that heparanase plays important roles in barrier-disrupted skin, leading to increased interaction of growth factors between epidermis and dermis and facilitating various cutaneous changes, including epidermal hyperplasia and wrinkle formation. However, the role of heparanase in sun-exposed skin remains unknown. Here, we show that heparanase in human keratinocytes is activated by ultraviolet B (UVB) exposure and that heparan sulfate of perlecan is markedly degraded in UVB-irradiated human skin. The degradation of heparan sulfate resulted in a marked reduction of binding activity of the basement membrane for vascular endothelial growth factor, fibroblast growth factor-2 and -7 at the dermal–epidermal junction. Degradation of heparan sulfate was observed not only in acutely UVB-irradiated skin, but also in skin chronically exposed to sun. Interestingly, heparan sulfate was found to be degraded in sun-exposed skin, but not in sun-protected skin. These findings suggest that chronic UVB exposure activates heparanase, leading to degradation of heparan sulfate in the basement membrane and increased growth factor interaction between epidermis and dermis. These changes may facilitate photo-aging.     

Influence of heparan sulfate chains in proteoglycan at the dermal-epidermal junction on epidermal homeostasis

Basement membrane (BM) plays important roles in skin morphogenesis and homeostasis by controlling dermal-epidermal interactions. However, it remains unclear whether heparan sulfate (HS) chains of proteoglycan in epidermal BM contribute to epidermal homeostasis. To explore the function of HS chains at the dermal-epidermal junction (DEJ), we used a skin equivalent (SE) model. This model lacked HS at the DEJ and showed abnormal expression of the differentiation markers filaggrin and loricrin; similar changes were seen in ultraviolet B-irradiated human skin. Perlecan (core-protein of HS proteoglycan) remained localized at the DEJ in both SE and UV-irradiated human skin. Heparanase, which degrades HS, was increased in epidermis of UV-irradiated skin, compared with unirradiated skin. We found that deposition of HS at the DEJ in the SE model was markedly augmented by a synthetic heparanase inhibitor, and release of HS into conditioned medium was suppressed. The inhibitor also increased filaggrin and loricrin expression. Moreover, the recovery of HS was associated with an increase of Ki67-positive basal cells, compared with control SE cultured without inhibitor. Comparative gene expression analysis in epidermis of SE cultured in the presence and absence of heparanase inhibitor, using DNA microarrays, showed that recovery of HS was associated with increased expression of differentiation-related genes and down-regulation of degradation-enzyme-related genes. These results indicate that degradation of HS at the DEJ by heparanase impairs epidermal homeostasis in SE, leading to abnormal differentiation and proliferation behaviour. Thus, HS chains in epidermal BM appear to play an important role in epidermal homeostasis.     

Key role of heparan sulfate chains in assembly of anchoring complex at the dermal-epidermal junction

Epidermal basement membrane forms anchoring complex composed of hemidesmosomes, anchoring filaments, lamina densa and anchoring fibrils to link epidermis to dermis. However, the anchoring complex is rarely formed in skin equivalent models, probably because of degradation of extracellular matrix (ECM) proteins and heparan sulfate chains by matrix metalloproteinases (MMPs) and heparanase, respectively. To explore the roles of ECM proteins and heparan sulfate in anchoring complex assembly, we used specific inhibitors of MMPs and heparanase, and the formation of anchoring complex was analysed in terms of polarized deposition of collagen VII, BP180 and β4 integrin at the dermal-epidermal junction (DEJ) by means of immunohistochemistry and transmission electron microscopy (TEM). The deposition of collagen VII was polarized to the basal side by the addition of MMP inhibitor, and the staining intensity was increased by combined treatment with MMP inhibitor and heparanase inhibitor, which enhanced anchoring fibril formation as observed by TEM. BP180 was polarized to the basal side by heparanase inhibitor, which protects HS chains, but not by MMP inhibitor. MMP inhibitor improved the polarization of β4 integrin. Hemidesmosomes were formed in the presence of each inhibitor, as observed by TEM, and formation was greatly enhanced by the combined treatment. These findings suggest that heparan sulfate chains, in addition to ECM proteins at the DEJ, play an important role in the assembly of anchoring complex, especially hemidesmosomes and anchoring fibrils.     

Immunohistological pointers to a possible role for excessive cathelicidin (LL-37) expression by apocrine sweat glands in the pathogenesis of hidradenitis suppurativa/acne inversa

Summary Background The cause of follicular occlusion, a key early event in the pathogenesis of hidradenitis suppurativa (HS), also known as acne inversa, remains unknown. Objectives To identify changes, if any, in the antimicrobial peptide (AMP) and cytokine expression profile of HS affected human skin. Methods Quantitative immunohistomorphometry was used to compare the in situ protein expression of selected AMPs and cytokines in lesional HS skin from 18 patients with that in healthy skin (n = 12). The lesional skin from patients with HS was histologically subclassified based on the predominance of inflammation vs. scarring. Results Compared with healthy controls, significantly increased immunoreactivity for cathelicidin (LL‐37) was noted in the apocrine sweat gland and distal outer root sheath (ORS) of the hair follicle (HF) epithelium in lesional HS skin. Immunoreactivity for LL‐37, psoriasin, human β‐defensin 3 (hBD3), α‐melanocyte stimulating hormone (α‐MSH), macrophage migration inhibitory factor (MIF), tumour necrosis factor (TNF)‐α and interleukin (IL)‐8 was significantly increased in HS epidermis. LL‐37 and TNF‐α immunoreactivity was also increased in the dermis of lesional HS skin. In contrast, lysozyme expression was decreased in the epidermis of lesional HS skin, while that of TNF‐α and IL‐8 was decreased in the proximal ORS of HFs in HS lesions. These differences were most pronounced in HS with predominant inflammation. Conclusions Our observations raise the question as to whether excessive secretion of AMPs by the skin, in particular by the apocrine sweat glands, distal HF epithelium, and epidermis, may attract inflammation and thus facilitate or promote HS development.     

Topical hesperidin prevents glucocorticoid‐induced abnormalities in epidermal barrier function in murine skin

Systemic and topical glucocorticoids (GC) can cause significant adverse effects not only on the dermis, but also on epidermal structure and function. In epidermis, a striking GC‐induced alteration in permeability barrier function occurs that can be attributed to an inhibition of epidermal mitogenesis, differentiation and lipid production. As prior studies in normal hairless mice demonstrated that topical applications of a flavonoid ingredient found in citrus, hesperidin, improve epidermal barrier function by stimulating epidermal proliferation and differentiation, we assessed here whether its topical applications could prevent GC‐induced changes in epidermal function in murine skin and the basis for such effects. When hairless mice were co‐treated topically with GC and 2% hesperidin twice‐daily for 9 days, hesperidin co‐applications prevented the expected GC‐induced impairments of epidermal permeability barrier homoeostasis and stratum corneum (SC) acidification. These preventive effects could be attributed to a significant increase in filaggrin expression, enhanced epidermal β‐glucocerebrosidase activity and accelerated lamellar bilayer maturation, the last two likely attributable to a hesperidin‐induced reduction in stratum corneum pH. Furthermore, co‐applications of hesperidin with GC largely prevented the expected GC‐induced inhibition of epidermal proliferation. Finally, topical hesperidin increased epidermal glutathione reductase mRNA expression, which could counteract multiple functional negative effects of GC on epidermis. Together, these results show that topical hesperidin prevents GC‐induced epidermal side effects by divergent mechanisms.     

Disturbed epidermal structure in mice with temporally controlled fatp4 deficiency

So far, little is known about the physiological role of fatty acid transport protein 4 (Fatp4, Slc27a4). Mice with a targeted disruption of the Fatp4 gene display features of a human neonatally lethal restrictive dermopathy with a hyperproliferative hyperkeratosis, a disturbed epidermal barrier, a flat dermal-epidermal junction, a reduced number of pilo-sebaceous structures, and a compact dermis, demonstrating that Fatp4 is necessary for the formation of the epidermal barrier. Because Fatp4 is widely expressed, it is unclear whether intrinsic Fatp4 deficiency in the epidermis alone can cause changes in the epidermal structure or whether the abnormalities observed are secondary to the loss of Fatp4 in other organs. To evaluate the functional role of Fatp4 in the skin, we generated a mouse line with Fatp4 deficiency inducible in the epidermis. Mice with epidermal keratinocyte-specific Fatp4 deficiency developed a hyperproliferative hyperkeratosis with a disturbed epidermal barrier. These changes resemble the histological abnormalities in the epidermis of newborn mice with total Fatp4 deficiency. We conclude that Fatp4 in epidermal keratinocytes is essential for the maintenance of a normal epidermal structure.     

Similar appearance,different mechanisms: xerosis in HIV,atopic dermatitis and ageing

Xerosis is one of the most common dermatologic disorders occurring in the elderly and in patients with atopic dermatitis (AD) and human immunodeficiency virus (HIV) infection. Xerosis has been linked to an impaired skin barrier function of the stratum corneum. Using Raman microspectroscopy, we concentrated on deeper skin layers, viable epidermis and dermis of 47 volunteers and associated molecular alterations to the evolution of xerosis and the skin barrier, for example, lipid, water and antioxidant content. A decrease in lipids within the viable epidermis is found for elderly and HIV‐patients. Lipid and water values of AD patients and their healthy reference group are similar. Decreases in lipids and simultaneous increases in water are found in the dermis for HIV and AD patients in comparison to their healthy reference groups. Excessive levels of epidermal carotenoids, mainly lycopene, in HIV‐patients were found potentially leading to adverse effects such as premature skin ageing.     

Hyperpigmentation in human solar lentigo is promoted by heparanase-induced loss of heparan sulfate chains at the dermal-epidermal junction

Background

Skin pigmentation induced by ultraviolet B radiation is caused in part by inflammation mediated by cytokines secreted from keratinocytes and fibroblasts in the irradiated area. Heparanase is also activated in the irradiated skin, and this leads to loss of heparan sulfate at the dermal-epidermal junction (DEJ), resulting in uncontrolled diffusion of heparan sulfate-binding cytokines through the DEJ. However, it is not clear whether heparanase-induced loss of heparan sulfate at the DEJ is involved in the pigmentation process in sun-exposed skin.

Objective

We examined the role of heparan sulfate in the pigmentation process of human pigmented skin and in pigmented skin-equivalent model.

Methods

Heparan sulfate and blood vessels in human pigmented skin, solar lentigo, and non-pigmented skin were evaluated by means of immunohistochemistry. Pigmented skin equivalent models were cultured with or without heparanase inhibitor and the pigmentation levels were compared.

Results

In solar lentigo, heparan sulfate was hardly observed, presumably due to the increase of heparanase at the DEJ, in spite of the deposition of core protein of perlecan (also known as heparan sulfate proteoglycan). The number of blood vessels was significantly increased in solar lentigo. In the pigmented skin equivalent model, heparanase inhibitor increased the staining intensity of heparan sulfate at the DEJ and markedly reduced melanogenesis in the epidermis.

Conclusions

Our results indicate that heparanase-induced loss of heparan sulfate at the DEJ is involved in the pigmentation process of human skin. Consequently, heparanase inhibitors can be expected to exert a protective effect against ultraviolet exposure-induced skin pigmentation.     

Establishment of a mouse skin model of the lichenification in human chronic eczematous dermatitis

BACKGROUND: Repeated mechanical stresses, such as scratching and rubbing, on a lesional skin area induce a rough skin condition known as lichenification in patients with chronic eczematous dermatitis. For ethical reasons, the pathomechanisms involved are difficult to study, so an animal model is required. OBJECTIVES: To study the pathomechanisms of the unique rough skin changes seen in chronic eczematous dermatitis, we established a mouse skin model by repeated tape stripping to inflict stratum corneum (SC) barrier disruption. The skin characteristics of the model were investigated biologically, histologically and pharmacologically. METHODS: Tape stripping was done on mouse back skin three times a week for 4 weeks. The skin changes were studied by obtaining negative replicas, haematoxylin and eosin staining, immunostaining for CD31 and BrdU, and measuring epidermal and cutaneous thickness and skin capacitance. Activities of matrix metalloproteinase (MMP)-2, 9 and urokinase-type plasminogen activator (uPA) in the skin tissues were analysed by zymography. The effects of MMP inhibitor and glycine were assessed. RESULTS: The repeated tape stripping produced crusting and desquamation at 48 h, followed 1 week later by the formation of shallow furrows, which became much deeper after 4 weeks, appearing as fine and regular wrinkles. The resultant wrinkled skin resembled lichenified skin seen in patients with chronic eczematous dermatitis. Histopathologically, we found acanthosis, hypergranulosis and hyperkeratosis even at 48 h, and the skin was 2.5 times thicker than untreated control skin at 4 weeks. We observed angiogenesis in the upper dermis at 1 and 4 weeks. Skin capacitance, a parameter of SC hydration, displayed consistently low levels throughout the experimental period. Although the dermis was also thickened, the activity of MMP-9 was sharply increased only at 24 and 48 h after tape stripping, declining thereafter to the control level. Topical applications of CGS-27023A (CGS), an MMP inhibitor, failed to suppress this tape-stripping-induced wrinkle formation. In contrast, topical applications of a barrier recovery accelerator, glycine, effectively inhibited the wrinkle formation induced by repeated tape stripping. CONCLUSIONS: The induction of fine and regular wrinkles by inflicting chronic SC barrier disruption in this model involves mainly epidermal changes, which is in sharp contrast to the mainly dermal changes induced by chronic ultraviolet B irradiation.     

1‐(2‐Hydroxyethyl)‐2‐imidazolidinone,a heparanase and matrix metalloproteinase inhibitor,improves epidermal basement membrane structure and epidermal barrier function

Daily exposure to sunlight is known to affect the structure and function of the epidermal basement membrane (BM), as well as epidermal differentiation and epidermal barrier function. The aim of this study is to clarify whether the inhibition of BM‐degrading enzymes such as heparanase and matrix metalloproteinase 9 (MMP‐9) can improve the epidermal barrier function of facial skin, which is exposed to the sun on a daily basis. 1‐(2‐hydroxyethyl)‐2‐imidazolidinone (HEI) was synthesized as an inhibitor of both heparanase and MMP‐9. HEI inhibited not only the BM damage at the DEJ but also epidermal proliferation, differentiation, water contents and transepidermal water loss abnormalities resulting from ultraviolet B (UVB). This was determined in this study by the use of UVB‐induced human cultured skins as compared with the control without HEI. Moreover, topical application of HEI improved epidermal barrier function by increasing water content and decreasing transepidermal water loss in daily sun‐exposed facial skin as compared with non‐treated skins. These results suggest that the inhibition of both heparanase and MMP‐9 is an effective way to care for regularly sun‐exposed facial skin by protecting the BM from damage.     

S100A7 (psoriasin) inhibits human epidermal differentiation by enhanced IL‐6 secretion through IκB/NF‐κB signalling

Psoriasin (S100A7), a member of the S100 protein family, is a well‐known antimicrobial peptide and a signalling molecule which regulates cellular function and is highly expressed in hyperproliferative skin conditions such as atopic dermatitis (AD) and psoriasis with disrupted skin barrier function. However, its role in epidermal differentiation remains unknown. We examined the effect of S100A7 on epidermal differentiation in normal human keratinocytes (NHKs) and on a reconstituted human epidermis model. When NHKs were exposed to disruptive stimuli such as Staphylococcus aureus, ultraviolet irradiation and retinoic acid, the secretion of S100A7 into the culture medium increased and the expression of epidermal differentiation markers decreased. Treatment of NHKs with S100A7 significantly inhibited epidermal differentiation by reducing the expression of keratin 1, keratin 10, involucrin and loricrin and by increasing the expression of abnormal differentiation markers (keratin 6 and keratin 16). We verified that the MyD88‐IκB/NF‐κB signal cascade was activated via RAGE after S100A7 treatment, resulting in the upregulation of interleukin‐6. Finally, we confirmed that S100A7 is a negative regulator of epidermal differentiation using a reconstituted human epidermis model. This study suggests that S100A7‐related signalling molecules could be potent targets for recovering skin barrier function in AD and psoriasis where S100A7 is accumulated excessively.     

pH in nature,humans and skin

The pH plays an important physiological role in nature and humans. pH varies from 1 to 8 in human organs with tight regulation in blood and epithelia of barrier organs. The physiological pH of the stratum corneum is 4.1–5.8 and several mechanisms contribute to its formation: filaggrin degradation, fatty acid content, sodium‐hydrogen exchanger (NHE1) activation and melanosome release. First, the acidic pH of the stratum corneum was considered to present an antimicrobial barrier preventing colonization (e.g. by Staphylococcus aureus and Malassezia). Later on, it was found that the pH influences skin barrier function, lipid synthesis and aggregation, epidermal differentiation and desquamation. Enzymes of ceramide metabolism (e.g. β‐glucocerebrosidase or acid sphingomyelinase) as well as proteases (e.g. chymotryptic enzyme or cathepsin D linked to epidermal differentiation and desquamation) are regulated by the pH. Experimental disruption of the physical barrier leads to an increase of pH, returning to normal levels only after many hours. Inflammatory skin diseases and diseases with an involvement of the epidermis exhibit a disturbed skin barrier and an increased pH. This is known for atopic dermatitis, irritant contact dermatitis, ichthyosis, rosacea and acne, but also for aged and dry skin. Normalizing the pH by acidification through topical treatment helps to establish a physiological microbiota, to repair skin barrier, to induce epidermal differentiation and to reduce inflammation.     

Modulations in epidermal calcium regulate the expression of differentiation-specific markers

Mammalian epidermis normally displays a distinctive calcium gradient, with low levels in the basal/spinous layers and high levels in the stratum granulosum. Although changes in stratum granulosum calcium regulate the lamellar body secretory response to permeability barrier alterations, whether modulations in calcium also regulate the expression of differentiation-specific proteins in vivo remains unknown. As acute barrier perturbations reduce calcium levels in stratum granulosum, we studied the regulation of murine epidermal differentiation after loss of calcium accompanying acute barrier disruption and by exposure of such acutely perturbed skin sites to either low (0.03 M) or high (1.8 M) calcium. Three hours after acute barrier disruption, coincident with reduced calcium and ultrastructural evidence of accelerated lamellar body secretion, both northern analyses and in situ hybridization revealed decreased mRNA levels for loricrin, profilaggrin, and involucrin in the outer epidermis, but protein levels did not change significantly. Moreover, exposure of acutely disrupted skin sites to low calcium solutions sustained the reduction in mRNA levels, whereas exposure to high calcium solutions restored normal mRNA levels (blocked by the L-type calcium channel inhibitor, nifedipine). Finally, with prolonged exposure to a low (<10% relative humidity) or high (>80% relative humidity) humidity, calcium levels increased and declined, respectively. Accordingly, mRNA and protein levels of the differentiation-specific markers increased and decreased at low and high relative humidity, respectively. These results provide direct evidence that acute and sustained fluctuations in epidermal calcium regulate expression of differentiation-specific proteins in vivo, and demonstrate that modulations in epidermal calcium coordinately regulate events late in epidermal differentiation that together form the barrier.     

Staphylococcus aureus skin colonization is promoted by barrier disruption and leads to local inflammation

Experimental mouse models of bacterial skin infections that have been described show that pathogenic microorganisms can readily invade the epidermis and dermis to produce localized infections. We used an epicutaneous mouse skin infection model to determine how the level of barrier disruption by tape‐stripping correlates with persistence of Staphylococcus aureus skin colonization, concomitant induction of cutaneous inflammation and infection. Furthermore, we investigated how murine skin responds to S. aureus colonization in a physiologic setting by analysing proinflammatory cytokines and antimicrobial peptides in mouse skin. We show that previous cutaneous damage allows skin inflammation to develop and favours S. aureus persistence leading to cutaneous colonization, suggesting an interdependence of cutaneous bacteria and skin. Our study suggests that skin barrier defects favour S. aureus skin colonization, which is associated with profound cutaneous inflammation.     

Role of PKC-delta as a signal mediator in epidermal barrier homeostasis

The skin shows an important "epidermal permeability barrier homeostasis" in response to barrier disruption. Calcium ion (Ca(2+)), a major regulator in keratinocyte differentiation and proliferation, plays a crucial role in skin barrier homeostasis. Acute barrier disruption induces an immediate depletion of both extra- and intracellular calcium ions in the epidermis, especially in the upper granular layers, and results in the loss of normal epidermal calcium gradient. Currently, we hypothesize that the change in the intracellular calcium ion concentration triggers the barrier repair responses, such as lamellar body (LB) secretion and increased lipid synthesis in the epidermis. In this article, we suggest that PKC-delta is a signaling mediator for the changes in extracellular and intracellular calcium ion concentration.     

Calcium and potassium inhibit barrier recovery after disruption, independent of the type of insult in hairless mice

Abstract Disruption of the cutaneous permeability barrier induces metabolic responses in the epidermis which result in barrier recovery. Barrier disruption by either solvent treatment or tape stripping results in the loss of the epidermal calcium gradient. Previous studies in acetone treated hairless mice have shown that maintaining this calcium gradient inhibits barrier repair, suggesting that alterations in the epidermal calcium concentration may be an important signal for barrier homeostasis. In the present study, we show that in hairless mice disruption of the barrier by treatment with the detergent. SDS, also results in the loss of the calcium gradient, as demonstrated both semi-quantitatively with ultrastructural cytochemical localization and quantitatively using proton induced X-ray emission (PIXE). Additionally, immersion in calcium containing solutions delays barrier repair after either detergent (SDS treatment) or mechanical (tape stripping) disruption of the barrier, as reported previously for acetone treated skin. These results indicate that barrier disruption, regardless of the insult, induces changes in the epidermal calcium gradient which may play an important role in signaling the metabolic changes required for barrier homeostasis.     

Wie die Sonne unsere Haut altern l?sst

Extrinsic factors (= environmentally induced skin aging) lead to both epidermal and dermal changes. Recent investigations have shown that the dermis plays the decisive role, at least for skin aging caused by ultraviolet (UV) radiation. Exposure to UV radiation results in an accumulation of damage to the mitochondrial DNA of dermal fibroblasts and thus an altered gene expression of the affected cells, which chronically drives both the dermal (e.g., wrinkle formation) and epidermal (atrophy, barrier dysfunction) aging process. This knowledge is currently being used to develop highly effective cosmetic strategies to reverse the skin aging process.     

Skin lipid content during early fetal development

Although little is known about changes in the lipid composition of the skin during fetal development, information regarding the developmental sequence of fetal skin lipid content could be important for understanding the emergence of epidermal barrier function, as well as providing baseline criteria for prenatal diagnosis of certain inherited disorders of cornification. In these studies, epidermis was separated from dermis in fetal skin samples ranging from 50 to 140 d, estimated gestational ages (EGA), and its lipid composition was analyzed by quartz rod microchromatography/flame-ionization and thin layer chromatography. Lipid biochemical data were correlated with developmental milestones observed by electron microscopy (morphologic studies). The lipid composition of epidermal and dermal fractions from skin samples between 50 and 110 d EGA was similar, with both tissues exhibiting a predominance of free sterols and phospholipids. After 110 d EGA dermis became enriched in triglycerides, corresponding to the progressive development of adipocytes after this time. EGA epidermis after 110 d was enriched not only in triglycerides, but also sterol esters. Moreover, ceramides and glycosphingolipids also became increasingly prominent, changes that were greatest in epidermis from older fetuses and from cephalad regions. These changes in epidermal lipid composition corresponded morphologically to the progressive emergence of both folliculocentric epidermal cornification and sebaceous gland development.