Glycosaminoglycans in neurofibromas

Glycosaminoglycan (GAG) contents in neurofibromas (NFs) were examined to clarify how well they corresponded to their histological features. Eight cutaneous NFs and three plexiform NFs from five patients with Recklinghausen's (R) disease, two senile NFs and control dermis were subjected to the isolation of GAGs. The GAGs were then fractionated and quantitated with two-dimensional electrophoresis on cellulose acetate membranes. Dry weight/wet weight ratios of the NFs were lower than those of the controls; the plexiform NFs were the lowest. In these plexiform NFs, hyaluronic acid (HA) content was most increased and dermatan sulfate (DS) content most decreased, resulting in a marked decrease in the DS/HA ratio. Both cutaneous and senile NFs showed moderate decreases in DS content, increases in chondroitin sulfate and heparan sulfate contents, and slight increases in HA content, resulting in moderate decreases in the DS/HA ratio. Considering that cutaneous or senile NFs are relatively more fibrotic tumors than plexiform NFs, these differences in the GAGs between plexiform NFs and cutaneous or senile NFs appear to be consistent with the changes in GAGs previously described in physiological and pathological conditions such as fibrotic diseases. Thus these results suggest that studies of hereditary disorders like R disease might be useful for understanding the pathogenesis of so-called acquired diseases with unknown etiology.     

Analysis of increased urinary acid glycosaminoglycans in a patient with relapsing polychondritis

We analysed the composition of glycosaminoglycans (GAGs) found in an increased amount in the urine from a patient with relapsing polychondritis (RP) by means of electrophoresis, since to our knowledge no analytical studies on the composition of GAGs in the urine from the patients with RP have been performed. Unexpectedly, the major GAGs detected in increased amount in the urine were found to be dermatan sulfate (DS) and hyaluronic acid (HA), the excretion of which correlated with the activity of the disease. Both DS and HA are the major components not of the cartilage but of the skin. Therefore, our findings suggest that the increased GAGs in RP are derived from skin or other tissues containing these GAGs rather than from the cartilage, unless there is an extensive cartilage destruction.     

银屑病患者糖胺聚糖代谢的研究

本研究采用改良的咔唑—硫酸法测定了30例银屑病患者和15例健康人24小时尿液中糖胺聚糖(GAG)的含量,并用单向电泳法分析其尿中GAG的种类和相对含量。结果显示:银屑病患者尿中GAG含量较正常对照组明显增高(P<0.05),单向电泳分析表明,银屑病患者尿中排出增多的GAG主要为硫酸乙酰肝素(HS),经直线相关分析得两者呈正相关(r=0.6236,P<0.001),这些结果证明银屑病患者的表皮细胞间质和真皮结缔组织中存在着代谢紊乱。     

Structural characterization of the skin glycosaminoglycans in patients with pseudoxanthoma elasticum

Background Complex polysaccharides, glycosaminoglycans (GAGs), their amount, and fine structure were determined in the skin (epidermis + dermis) of pseudoxanthoma elasticum (PXE)‐affected patients in comparison with healthy subjects. Methods Nonlesional skin GAGs were extracted and specifically determined by enzymatic treatment and high‐performance liquid chromatography separation. Results Dermatan sulfate (DS) and hyaluronic acid (HA) were found to be the major GAG species in normal subjects, with contents of approximately 20% for DS and 58% for HA. The chondroitin sulfate (CS) content (unsaturated six‐sulfated disaccharide) was approximately 21%. Skin from patients with PXE showed similar HA (61%), DS (22%), and CS (16.7%) contents. No change in the total charge density or nonsulfated/sulfated GAG ratio was noted in PXE‐affected subjects, and no modification of the position of the sulfate groups (4s/6s) on the CS/DS backbone. A significant increase (approximately 88%; P < 0.01) in the total amount of GAGs (HA + DS + CS) was found in the PXE group vs. normal subjects, however. Conclusions In the skin of PXE‐affected patients, the altered metabolic processes produce an increase in the total amount of GAGs able to accumulate salts, in particular calcium ions, within the elastic fibers, producing ion precipitates that affect the organization of the matrix fiber.     

Studies on human scar tissue proteoglycans

The proteoglycans (PGs) in pooled normal scars and pooled hypertrophic scars were extracted with 4 M guanidinium chloride and isolated by DEAE-cellulose chromatography. The PG samples were then fractionated further by dissociative CsCl density gradient sedimentation. Following cleavage of the density gradient PG fractions with alkaline NaB3H4, the glycosaminoglycan (GAG) constituents were isolated and analyzed by quantitative cellulose acetate electrophoresis. In addition, single samples of normal skin and a keloid scar were also analyzed. The results showed that the hypertrophic scars had a higher average content of extractable and also residual PGs than did the normal scars but a wide range of values was obtained for each type of scar. Some differences were noted in the amounts and distribution of the GAGs in CsCl gradient fractions, in the different types of scar tissue. The PGs in tissues were distributed in low-, medium-, and high density fractions and are, therefore, heterogeneous. Dermatan sulfate (DS) was the major GAG in each tissue and smaller quantities of chondroitin sulfate (CS), heparan sulfate (HS), and heparin (HP) were also present. In addition, 2 other GAG constituents were also detected. Based on the susceptibility of these GAGs to enzymes and nitrous acid treatments, one was a HS or HP while the second was a DS. The major differences in the PG composition of the scar tissues were the higher proportions of low-density CS-PGs in the keloid scar and of low density DS-PGs in hypertrophic and keloid scars.     

Extrinsic ageing in the human skin is associated with alterations in the expression of hyaluronic acid and its metabolizing enzymes

Abstract: Extrinsic skin ageing or ‘photoageing’, as opposed to intrinsic skin ageing, is the result of exposure to external factors, mainly ultraviolet irradiation. Glycosaminoglycans (GAG) and particularly hyaluronic acid (HA) are major components of the cutaneous extracellular matrix involved in tissue repair. However, their involvement in extrinsic skin ageing remains elusive. In this study, we investigated the expression of HA and its metabolizing enzymes in photoexposed and photoprotected human skin tissue specimens, obtained from the same patient. Total GAG were isolated, characterized using specific GAG‐degrading enzymes and separated by electrophoresis on cellulose acetate membranes and polyacrylamide gels. Quantitation of HA in total GAG was performed using ELISA. Gene expression of hyaluronan synthases (HAS), hyaluronidases (HYAL) and HA receptors CD44 and receptor for HA‐mediated motility (RHAMM) was assessed by RT‐PCR. We detected a significant increase in the expression of HA, of lower molecular mass, in photoexposed skin as compared with photoprotected skin. This increase was associated with a significant decrease in the expression of HAS1 and an increase in the expression of HYAL1‐3. Furthermore, the expression of HA receptors CD44 and RHAMM was significantly downregulated in photoexposed as compared with photoprotected skin. These findings indicate that extrinsic skin ageing is characterized by distinct homoeostasis of HA. The elucidation of the role of HA homoeostasis in extrinsic skin ageing may offer an additional approach in handling cutaneous ageing.     

Comparative biochemistry of human skin: glycosaminoglycans from different body sites in normal subjects and in patients with localized scleroderma

OBJECTIVES: The aim of this investigation is to compare the relative proportions of disaccharides of chondroitinase-digestible glycosaminoglycans (GAGs) among the different body sites in control human skin and in the skin lesions of patients with localized scleroderma. METHODS: The disaccharide relative proportions were determined using high-performance liquid chromatography (HPLC). RESULTS: DeltaDi-4S, the main disaccharide unit of dermatan sulphate (DS), was the major skin GAG disaccharide (approximately 70% of the total) in control skin among all different body sites studied here. In scleroderma there was an increase in the relative proportion of both deltaDi-HA, the main disaccharide unit of hyaluronic acid (HA), and deltaDi-diS(B) (alpha-deltaUA(2SO4)-1-->3-GalNAc(4SO4)), derived from DS, and a decrease in deltaDi-4S, as compared with the uninvolved skin or the site-matched control skin. CONCLUSION: DS is the major GAG species in normal skin from different body sites. In addition, our results suggest a decrease and also a structural change in DS and an increase in the proportion of HA in scleroderma skin.     

The effect of platelet-derived growth factor on cell division and glycosaminoglycan synthesis by human skin and scar fibroblasts

The effect of platelet-derived growth factor (PDGF) on cell division and glycosaminoglycan (GAG) synthesis by fibroblasts isolated from skin and scar was measured. We found that PDGF stimulates cell division more efficiently in normal skin fibroblasts than in scar fibroblasts and decreases GAG synthesis in skin and scar fibroblasts. Using a 4-h pulse label with [3H]thymidine ([3H]Thd) following a 20-h incubation of confluent monolayer cultures with 0-5 units PDGF/ml Dulbecco's modified Eagle's medium, we found a concentration-dependent increase in [3H]Thd incorporation. After incubation of fibroblasts with [3H]glucosamine and 35SO4 in the presence or absence of PDGF, labeled constituents were isolated from the extracellular, pericellular, and cellular fractions by pronase digestion and column chromatography on Sepharose CL4B or DEAE-cellulose and analyzed by cellulose acetate electrophoresis. The presence of PDGF decreased the total amount of 35S incorporated into macromolecules by skin and scar fibroblasts and resulted in an altered distribution of labeled GAGs. Dermal fibroblasts exposed to PDGF for 24 h incorporated a greater percentage of radiolabeled 35S into dermatan sulfate prime (DS') and less into dermatan sulfate (DS) in the extracellular fractions and a greater percentage of 35S into heparan sulfate (HS) in the pericellular fractions than did parallel cultures grown in the absence of PDGF. It is thought than PDGF may have an effect on scar formation by increasing the fibroblast population in the wound tissue and by affecting the total amount and types of matrix components synthesized.     

THE ENZYMATIC DETERMINATION OF ACIDIC GLYCOSAMINOGLYCANS IN SCAR AND KELOID WITH CHONDROITINASE

Acidic glycosaminoglycans (GAG) prepared from scar and keloid, was assayed for separation and quantitative measurement with the paper chromatographic characteristic of the unsaturated disaccharide unit after chondroitinase digestion. The following results were obtained:

• 1) It was found that the total amount of GAG in scars was greatly increased, especially in earlier phases, in comparison with that of normal tissue, and it gradually decreased with time. Even in different phases of scar, a decrease of hyaluronic acid (HA) was commonly observed in contrast to an increase in sulfated GAG, i.e. dermatan sulfate (DS) in particular amoung constituents.
• 2) A similar behavior of GAG to their mentioned above was observed in keloid specimens.
• 3) Any striking difference of GAG constituents between the scar in the hypertrophic phase and the keloid remained indistinguishable.

     

Proteoglycans synthesized by adult human epidermis in whole skin organ culture.

Adult human epidermis was cultured in whole skin organ culture under serum-free conditions in the presence of 35SO4. Proteoglycans (PG) comprised about 25% of the total (35SO4)-labeled material produced by epidermis. The rest of the incorporated activity displayed solubility characteristics typical of lipids. The molecular mass and the composition of the 35SO4-labeled epidermal PG and glycosaminoglycans (GAG) were studied using gel filtrations and agarose gel electrophoresis. The 35SO4-label of the epidermal PG was located in heparan sulfate (HS, approximately 75%) and chondroitin/dermatan sulfate (CS/DS, 25%), but not in keratan sulfate as determined by nitrous acid, chondroitinase AC II, chondroitinase ABC, and keratanase digestions, respectively. The molecular mass of the GAG chains was 10-40 kDa. The 35SO4-labeled PG were distributed between 60 and 600 kDa in agarose gel electrophoresis, with the highest activity at 350 kDa. Smaller activity peaks occurred at 150 and 60 kDa. Digestion of the PG with heparitinase removed most of the activity at 350 and 150 kDa, whereas chondroitinase ABC removed that at 60 kDa. A small amount of activity migrating between 600 and 1000 kDa was not affected by any of the GAG-degrading enzymes. Pulse chase experiments showed that the epidermal PG had an average half life of 24 h. The results thus demonstrate that human epidermis produces at least three different, rapidly metabolized PG. The PGs from 150 to 350 kDa contained heparan sulfate chains, whereas those at 60 kDa were chondroitin/dermatan sulfate PG.     

Abundance of interstitial heparan sulfate in granuloma annulare but not in other mucinous skin diseases

BACKGROUND: Heparan sulfate (HS), unlike other glycosaminoglycans, is mainly located on cell surfaces but can be shed into the interstitium by a regulated process. It has been found in interstitial fluid drained from cutaneous wounds, but otherwise the conditions under which the release of HS from the cell surface occurs are unknown. To better characterize this process, we have investigated the presence of interstitial HS in various skin diseases with glycosaminoglycan accumulation. METHODS: Histologic routine material was stained immunohistochemically using an antibody recognizing HS. RESULTS: Heparan sulfate immunoreactivity is present in the interstitium of young cutaneous scars and in the interstitium of the inflammatory infiltrate of granuloma annulare. No reactivity was found in a number of non-inflammatory skin diseases with mucin deposition. CONCLUSIONS: The selective presence of interstitial HS in only two of the investigated skin conditions supports the existence of a regulated mechanism to release HS from the surface of cells into the interstitium. It is suggested that HS modulates the biologic actions of growth factors and cytokines not only during wound repair but possibly also in inflammatory skin diseases such as granuloma annulare.     

Effect of platelet homogenate on in vitro glycosaminoglycans production by dermal fibroblasts from systemic sclerosis patients and normal controls

Confluent cultures of dermal fibroblasts from the involved skin of systemic sclerosis patients (SF) and a matched skin site of normal controls (NF) were investigated for the synthesis of glycosaminoglycans (GAG) in response to various concentrations of human platelet homogenate (PH). Experiments were carried out in the presence of 1% and 15% human serum (HS). In the absence of PH, GAG synthesis was higher in SF than NF. An increase in GAG synthesis was demonstrated in both SF and NF as the concentration of PH was increased to 200 micrograms/ml of growth medium. The PH-stimulated GAG synthesis occurred in 15% HS treated SF and NF, but there was no GAG synthesis increase in 1% HS-treated NF. The absolute count of GAG synthesis was always greater in SF than NF. The addition of PH in concentrations higher than 200 micrograms/ml led to cell death of both SF and NF. These findings are the first to indicate a difference between SF and NF response to PH.     

Proteoglycan expression patterns in human hair follicle

BACKGROUND: Proteoglycans (PGs) are known to play key roles in many cellular signalling pathways involved in hair follicle biology. Although some PG core proteins have previously been described in adult human hair follicles, their glycosaminoglycan (GAG) moieties have been less studied. OBJECTIVES: To add knowledge about PG core protein and GAG distributions in human anagen hair follicle and, for selected follicles, during catagen. METHODS: We used immunohistochemistry and immunohistofluorescence to revisit the expression pattern of GAG chains and core proteins in human hair follicle. The studied epitopes included CD44v3, syndecan-1, perlecan, versican, aggrecan, biglycan, heparan sulphate (HS), chondroitin sulphate (CS), dermatan sulphate (DS) and keratan sulphate (KS). RESULTS: The membrane PGs syndecan-1 and CD44v3 were respectively detected in the epithelial part of whole hair and in the outer root sheath basal layer. The dermal part of the hair follicle contained high amounts of extracellular PGs such as perlecan, versican, aggrecan, biglycan and their saccharidic moieties, namely HS, CS, DS and KS. We also observed a variable distribution of these components along the hair follicle. Especially, we noted a PG impoverishment at the very bottom of the anagen bulb. Moreover, while type D chondroitin expression remained unaffected, 4C3-CS and PG4-CS/DS epitopes respectively decreased in the dermal papilla and the connective tissue sheath, at the onset of catagen. CONCLUSIONS: GAG and PG expression along the human anagen hair follicle was characterized by (i) discontinuities mainly affecting the basement membrane and (ii) disappearance of some epitopes at catagen onset. These results are discussed in term of functionalities in nutrient diffusion, cell proliferation and differentiation, and hair protection.     

Disaccharide analysis of skin glycosaminoglycans in atrophoderma of Pasini and Pierini

There are divergent opinions as to whether atrophoderma of Pasini and Pierini (APP) is a nosologic entity or a primary atrophic morphoea. In this study, we used high performance liquid chromatography to analyse the skin disaccharide contents of glycosaminoglycan (GAG) in two patients with APP and compared the results with those from a typical atrophic morphoea patient. Perilesional uninvolved skin was used as a control in each patient. In the atrophic phase morphoea, both the total amount of disaccharide per skin punch-biopsy and the amount of DeltaDi-4S(DS) - the main disaccharide unit of dermatan sulphate - per mg dry weight were increased. These changes were consistent with sclerotic phase morphoea. In contrast, the total amount of disaccharide per skin punch-biopsy was decreased and the amount of DeltaDi-4S(DS) per mg dry weight was decreased or unchanged in APP. Our results suggest that GAG metabolism in APP may be unique and quite different from that in morphoea.     

Modulation of cell shedding and glycosaminoglycan synthesis of human malignant keratinocytes by all-trans-retinoic acid and hydrocortisone in vitro

Physiologic concentrations (5 X 10(-8) M) of all-trans-retinoic acid (RA) caused a 2- to 3-fold increase in the rate of cell desquamation of a malignant keratinocyte line (SqCC/Y1) grown in serum-free medium. Measurement of the incorporation of [35S]sulfate and [3H]glucosamine into cetylpyridinium chloride-precipitable glycosaminoglycans (GAGS) demonstrated that RA treatment did not alter total GAG production. In addition, compartmental distribution was not affected by RA, with 50-70% of GAGS being recovered from the medium, 25% from the pericellular matrix, and the remainder from the cells. Relatively small amounts of GAGS were associated with shed cells in RA-treated cultures, presumably reflecting a relatively short association of these cells with the monolayer before desquamation. Chondroitin sulfate (Ch-S), heparin/heparan sulfate (Hep-S), and hyaluronic acid (HA) were the GAG species identified in SqCC/Y1 cultures by gel-exclusion chromatography. RA reduced the relative amount of HA in the trypsin-sensitive pericellular compartment by 50%. Since the proportions of Ch-S and Hep-S were not affected by RA, the findings suggest that the altered ratio of HA to sulfated GAGS in this fraction may contribute to the increased cell desquamation. Hydrocortisone (10(-6) M) reversed the effect of RA on cell shedding, and increased the proportion of pericellular HA relative to that found in cultures exposed to RA alone. These findings support the concept that the relative proportion of HA to sulfated GAGS may be important in the intercellular cohesion of keratinocytes. In addition, the relative decrease in HA and the predominance of Ch-S over Hep-S in SqCC/Y1 cultures differed from results reported with normal keratinocytes, indicating that this property may be associated with the malignant phenotype.     

CUTANEOUS ACID MUCOPOLYSACCHARIDES IN SOME DERMATOSES

Summary.— Quantitative chemical analysis for hyaluronic acid (HA) and derMatan sulphate (DS) was performed on biopsies from 9 healthy volunteers and from the lesions of 30 patients with skin disease. The following observations were made:
(1) Values for both HA and DS in the skin of healthy volunteers were higher than those reported previously for post-mortem material.
(2) A striking decrease was found in the HA, together with a less marked reduction in the DS level, in lesions of discoid L.E. and scleroderma.
(3) A very low DS level was found in a single case of dystrophic epidermolysis bullosa.     

Primary localized cutaneous nodular amyloidosis: case report and biochemical analysis of amyloid.

We report a patient with scalp lesions of primary localized cutaneous nodular amyloidosis. The extensive examination revealed no systemic involvement. Analysis of glycosaminoglycans (GAGs) in amyloid deposits showed a twofold increase as compared with normal skin, which was due to the increase in dermatan sulfate. Local disorders of GAG metabolism may be related to the amyloid fibril formation. Amyloid fibrils were purified and identified electron-microscopically, which consisted of two major 12,000- and 13,000-dalton and minor 29,000- and 48,000-dalton peptides. Western blotting analysis showed a minor 29,000-dalton peptide reactive with antibodies against both kappa and lambda light chains of immunoglobulin. There is a possibility that some components of amyloid in some cases of primary localized cutaneous nodular amyloidosis may consist of both kappa and lambda immunoglobulin light chains.     

Heparanase activation induces epidermal hyperplasia,angiogenesis, lymphangiogenesis and wrinkles

Please cite this paper as: Heparanase activation induces epidermal hyperplasia, angiogenesis, lymphangiogenesis and wrinkles. Experimental Dermatology 2010; 19 : 965–972. Abstract: To clarify the difference between cutaneous responses to single and repeated barrier disruption, changes of epidermal gene expression were examined by using RT‐PCR. In repeatedly barrier‐disrupted skin, heparanase was specifically up‐regulated in epidermis. In addition, there was a marked decrease in heparan sulfate (HS) chains of perlecan in basement membrane at the dermal–epidermal junction (DEJ) compared with singly disrupted skin. HS chains form a reservoir for heparan sulfate‐binding growth factors. In repeatedly barrier‐disrupted skin, expression of vascular endothelial growth factor‐A (VEGF‐A), an angiogenic factor, was induced in epidermis, whereas thrombospondin‐1 (TSP‐1), an angiogenesis inhibitor, was down‐regulated, and concomitantly blood vessels were elongated and enlarged in dermis. Expression of VEGF‐C, a lymphangiogenesis factor, was augmented in epidermis of repeatedly barrier‐disrupted skin, concomitantly with an increase in the number and size of lymphatic vessels. Topical application of a synthetic heparanase inhibitor, 1‐[4‐(1H‐benzoimidazol‐2‐yl)phenyl]‐3‐[4‐(1H‐benzoimidazol‐2‐yl)phenyl]urea, to skin after barrier disruption significantly suppressed wrinkle formation, degradation of HS chains in the basement membrane, epidermal hyperplasia and the changes of blood and lymphatic vessels. These results suggest that chronic barrier disruption activates heparanase and induces gene expression changes, leading to increased growth factor interaction between epidermis and dermis, and facilitating various cutaneous changes, including wrinkle formation.     

Susceptibility to cumulative and acute irritant dermatitis An experimental approach in human volunteers

Reactivity to repeated daily sodium lauryl sulfate (SLS) applications and patch test reactivity to SLS was studied in 23 females. Skin changes were quantified by transepidermal water loss (TEWL), dielectric water content (DEWC), laser Doppler velocimetry (LDV) and visual scoring (VS). Dermatologic histories (HS) and susceptibility to sunburn (ST) were obtained and clinical skin dryness evaluated (DS). Great interindividual variation occurred in the degrees of changes in the biophysical parameters measured: the variation was most apparent in TEWL. The subjects with HS 1 or more developed greatest TEWL increase after open SLS applications (p less than 0.05). DS showed poor correlation with SLS reactivity and only minor DEWC alterations were seen. ST showed some non-significant correlation with erythema reactivity in the patch test.     

Glycosaminoglycan content in the media of cultured dermal fibroblasts derived from burn scar and normal skin

The glycosaminoglycan (GAG) content of the extracellular matrix of burn scar in humans has been reported to differ from that of normal skin. In order to investigate whether the GAG content altered as a result of functional changes in fibroblasts, the GAG content was determined in culture media of fibroblasts derived from growing burn scar, mature scar, and normal skin tissue. No statistical differences were observed in the population doubling-times of scar and normal skin. Mature scar showed significantly higher values for all the concentrations of uronic acid, hexosamine, and sulfate measured in the glycosaminoglycan, as compared with normal skin values, and the concentrations from growing scar were slightly higher than those for normal skin. The above results may suggest an increase in glycosaminoglycan sulfate synthesis following the hyperplasia of the matrix in burn scar tissue.