Peeling agents and irritants,unlike tretinoin,do not stimulate collagen synthesis in the photoaged hairless mouse

Tretinoin has been shown to stimulate the synthesis of collagen in photoaged human and hairless mouse skin. It has been suggested that this partial reversal of photodamage by tretinoin is a consequence of low-grade inflammation. The purpose of this study was to compare the effect of tretinoin with a number of irritants and peeling agents on collagen synthesis. Hairless mice were irradiated thrice weekly for 10 weeks with UVB. In the 10-week postirradiation period, the mice were treated topically five times per week with tretinoin (0.05%), glycolic acid (10%), benzalkonium chloride (1.0%), sodium lauryl sulfate (5%), croton oil (5%) and the water—propylene glycol vehicle. Microscopic measurements showed that the tretinoin-induced zone of new collagen was twice the depth of that induced by irritants or vehicle. The salt-soluble collagen content was determined by HPLC analysis of hydroxyproline levels. Type III procollagen was quantified by radioimmunoassay. Tretinoin-treated skin had increased amounts of collagen and type III procollagen whereas irritant- and peeling agent-treated skins were similar to vehicle-treated controls. Immunofluorescence studies were confirmatory. These results demonstrate that these agents, unlike tretinoin, do not have the capacity to enhance collagen synthesis. Therefore, it is likely that the effect of tretinoin does not depend upon irritation. This study was supported in part by Ortho Pharmaceutical Corp. Dermatology Division, Raritan, N.J., USA     

Alterations in dermal collagen in ultraviolet irradiated hairless mice

Chronic exposure of the skin to sunlight results in severe dermal connective tissue damage that is characterized by the basophilic degeneration of collagen and the accumulation of an elastotic material. The aim of this study was to identify changes in collagen (the major structural protein of the skin) in ultraviolet irradiated mouse skin using immunochemical and biochemical techniques. Specific antibodies directed against the aminopropeptide of type III procollagen were used in immunofluorescence and immunoblotting studies. Immunofluorescent staining of irradiated and nonirradiated mice skin showed that the aminopropeptide of type III procollagen was distributed throughout the dermis in a pattern similar to that observed for type I collagen. Extracts of irradiated (5 and 10 weeks) and nonirradiated skins were then subjected to immunoblotting techniques. Levels of pN alpha 1, type III procollagen (measured by radioimmunoassay) were reduced in the extracts prepared from skins of mice that were irradiated for 5 and 10 weeks. Immunoelectron microscopy verified the loss of pN alpha 1 type III procollagen in irradiated skin. Collagen fibers of nonirradiated skin demonstrated normal labeling with antibody directed against the aminopropeptide of type III procollagen. In contrast, collagen fibers of 10 week irradiated skin failed to label with this antibody. The pN alpha 1 type III collagen is known to coat type I collagen fibers of normal skin. Therefore, its absence from the surface of type I collagen fibers of irradiated skin may play a role in the development of the elastotic material.     

Collagen metabolism in ultraviolet irradiated hairless mouse skin and its correlation to histochemical observations

Early biochemical studies of ultraviolet (UV) irradiated human skin reported a loss of insoluble collagen with a concomitant increase in the soluble fraction. Recent work has described an early increase in type III collagen during chronic irradiation of hairless mice as determined by cyanogen bromide digests of whole skin. In order to understand the correlation of these events and those seen with histochemistry, in the present study we irradiated hairless mice for up to 24 weeks with approximately 4 minimal erythema doses (MEDs) of UVB thrice weekly with Westinghouse FS-40 bulbs. Skin samples were taken at 4-week intervals from irradiated and age-matched control mice. Collagen was isolated from other skin proteins by acid extraction, pepsin digestion, and salt precipitation. Estimates of types I and III collagen were made by interrupted polyacrylamide gel electrophoresis and densitometric scanning. Compared with unirradiated controls, there was a small increase in the ratio of type III to total collagen after 8 weeks of UV. There were no significant increases at later time points until after 24 weeks of radiation. Total collagen in normal mouse skin, determined by hydroxyproline content, remained constant over the 24 weeks, while UV radiation produced significant increases at 4, 8, 12, and 16 weeks, returning to control levels at week 20. There was no change in the degree of hydroxylation at any time point in either group. Thus, chronic UV exposure resulted in increased collagen synthesis until late in the course of irradiation. Because there is a lack of consistent change in the ratio of type III to total collagen, the early increases in collagen content may represent both types I and III, synthesized in relatively unchanging proportions.     

Early changes in dermal collagen of mice exposed to chronic UVB irradiation and the effects of a UVB sunscreen

Our previous studies have shown that hairless albino mice receiving chronic UVB irradiation show an increase in type III collagen, in the irradiated skin, after 12-36 weeks treatment. In this study we wished to find the earliest time at which such collagen changes were detectable and also to ascertain whether a topically applied UV-B sunscreen could prevent such changes. Groups of 10 hairless albino mice were irradiated, dorsally, with a daily dose of 62 mJcm-2 UVB for 3, 6, 9, and 12 weeks. Three parallel groups were used. They received a) no topical treatment, b) sunscreen vehicle alone, and c) sunscreen (containing 2-ethylhexyl 4'-methoxy-cinnamate) applied dorsally at 2 microliter cm-2. Dorsal and ventral (non-irradiated) skin samples were taken and the types I and III collagen quantified densitometrically after cyanogen bromide digestion and polyacrylamide gel electrophoresis. The ratio of dorsal/ventral type III collagen (D/V III) was determined for each mouse. The topical treatments caused no change in D/V III in the non-irradiated animals. In the non-topically treated group the irradiated animals showed a considerable increase in D/V III, compared with controls, at 12 weeks (P less than 0.001). The group treated with sunscreen vehicle alone also showed a significant increase in D/V III at week 12 (P less than 0.01). Both these groups showed a slight elevation in D/V III at week 6. The group treated with sunscreen showed no significant difference in D/V III between irradiated and control animals at any time.     

Generalized morphea with blisters. A case report

A patient suffering from generalized morphea developed blisters in the morpheic plaques on her buttocks. The plaques had an increased concentration of serum aminoterminal propertice of type III procollagen, an echo response and thickened skin on ultrasound scanning, and compact bundles of collagen fibrils with bimodal distribution of the diameters. The blisters appeared as an echo-free band in the subepidermal zone by ultrasound scanning. Electron microscopy revealed blisters in the upper papillary dermis, surrounded by degraded collagen fibrils.     

High-concentration all-trans retinoic acid induces dermal inflammation and reduces the accumulation of type I procollagen in human skin in vivo

Background Because inflammation is a factor promoting ageing, all‐trans retinoic acid (RA)‐induced irritation may have a negative influence on collagen accumulation in human skin despite its stimulation of collagen production. Objectives To determine whether RA‐induced irritation detrimentally affects RA efficacy as represented by new collagen synthesis. Methods Retinoic acid (0·01%, 0·025% or 0·05%) or vehicle was applied to the buttock skin of elderly male volunteers three times a week for 8 weeks under continuous occlusion. Every 2 weeks, biopsy specimens were obtained and immunohistochemical analysis was performed to determine levels of type I procollagen expression and inflammatory cell infiltration. Results Topical RA regardless of concentration increased type I procollagen expression in human skin in vivo after 2 weeks. However, only 0·01% RA continuously increased type I procollagen expression up to 8 weeks. After 4 weeks, significant infiltrations of macrophages and neutrophils were observed in 0·025% and 0·05% RA‐treated skin, and procollagen expression had returned to baseline. Conclusions Excessive RA‐induced inflammation might prevent collagen accumulation in aged skin despite the positive effect of RA on collagen production.     

Identification of collagen fibrils in scleroderma skin

Skin from early and late stages of scleroderma has been shown to contain large amounts of thin (30-40 nm diameter) collagen fibrils that may be present in bundles or intermingled with large diameter fibrils (90-120 nm). The nature of these fibrils is unknown. Skin biopsies were obtained from involved areas of nine patients with progressive systemic sclerosis (PSS), one case of generalized morphea, one case of morphea, and six normal controls. Intact skin was analyzed by immunoelectron microscopy (IEM), while extracts were subjected to sodium dodecyl polyacrylamide gel electrophoresis (SDS-PAGE), Western immunoblotting, radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA). Fine fibrils 20-40 nm in diameter in the mid to lower dermis of scleroderma skin were labeled with antibodies directed against the aminopropeptide (AP) of type III procollagen. Antibodies directed against the AP of type I procollagen labelled fine fibrils in the lower dermis. Larger fibrils (80-120 nm) did not label. pN alpha 1 (III) was found to be present in both normal and scleroderma skin. Extracts of scleroderma skin contained 2.5 times the amount of pN (III) collagen and 3.0 times the amount of fibronectin as did extracts of normal skin. The data indicate that the increase in thin fibrils in scleroderma skin is most likely due to an increase in type III collagen, which retains the AP at its surface.     

Sunscreens promote repair of ultraviolet radiation-induced dermal damage

Chronic UV irradiation profoundly damages the dermis of human and animal skin. These alterations were thought to be irreversible. Recently, we showed that substantial repair occurred in hairless mice after stopping UV exposure. A band of new connective tissue was laid down subepidermally. The present study focussed on whether repair would occur if animals were protected by sunscreens after dermal damage was induced and irradiation was continued. Albino hairless mice were exposed to Westinghouse FS20 sunlamps thrice weekly for 30 weeks. The daily dose of UV (UVB + UVA) was 0.17 J/cm2. Sunscreens of sun protection factors (SPF) 6 and 15 were applied after 10 and 20 weeks of irradiation. Biopsies were taken at 10, 20, 30, and 45 weeks of the experiment. With both sunscreens, especially SPF-15, previously damaged dermis was repaired during continued irradiation. Repair occurred in situ and, in severely damaged skin, in the novel form of subepidermal reconstruction zones of new connective tissue with parallel collagen bundles and a network of fine elastic fibers.     

Dermal collagen production following irradiation by dye laser and broadband light source

BACKGROUND : Improvement in the appearance of wrinkles has been observed following exposure to short-pulsed 585 nm laser light. The assumed effect is a specific absorption of light in the blood vessels of the superficial dermis, resulting in release of inflammatory mediators into the interstitium followed by stimulated fibroblast activity. The fibroblasts effectively initiate tissue repair mechanisms, which include enhanced new collagen production. METHODS : Quantitative measures of collagen synthesis rate in the skin can be obtained from determinations of the aminoterminal propeptide of type III procollagen level in suction blister fluid using a radioimmunoassay. RESULTS : A single laser treatment at subpurpura energy level showed that the 585 nm laser source induced an increase of 84% ( p < 0.05) in the type III procollagen production rate compared with a non-treated control site. A broadband, pulsed, white light source at 4 J/cm 2 showed no measurable increase, whilst the skin area treated with 7 J/cm 2 increased the procollagen production rate by 17% (NS, p > 0.05). A second treatment 2 weeks later further improved the laser-induced increase in procollagen production rate to 148% ( p < 0.05) compared with the control site. The broadband, pulsed, white light-irradiated skin sites showed that at 4 J/cm 2 the procollagen production rate was increased by 21.4% and at 7 J/cm 2 by 32.1% compared with the corresponding non-treated control site (NS, p > 0.05). CONCLUSIONS : Irradiation by the haemoglobin-specific short-pulsed 585 nm laser induced a fivefold increase in procollagen production rate compared with a biologically comparable fluence delivered in a broadband spectrum. An additional treatment after 2 weeks further increased the effect of the short-pulsed 585 nm laser to 148% of the control. Vascular-specific light/tissue interactions seem to play a key role in stimulating skin collagen production.     

Fermentable metabolite of Zymomonas mobilis controls collagen reduction in photoaging skin by improving TGF-beta/Smad signaling suppression

Solar ultraviolet (UV) irradiation causes damages on human skin and premature skin aging (photoaging). UV-induced reduction of type I collagen in dermis is widely considered primarily induction of wrinkled appearance of photoaging skin. Type I procollagen synthesis is reduced under UV irradiation by blocking transforming growth factor-beta (TGF-beta)/Smad signaling; more specifically, it is down-regulation of TGF-beta type II receptor (T beta RII). Therefore, preventing UV-induced loss of T beta RII results decreased type I collagen reduction in photoaging skin. Zymomonas mobilis is an alcohol fermentable, gram-negative facultative anaerobic bacterium whose effect on skin tissue is scarcely studied. We investigated the protective effects of fermentable metabolite of Z. mobilis (FM of Z. mobilis) against reduction of type I procollagen synthesis of UV-induced down-regulation of T beta RII in human dermal fibroblasts FM of Z. mobilis was obtained from lyophilization of bacterium culture supernatant. The levels of T beta RII and type I procollagen mRNA in human dermal fibroblasts were measured by quantitative real-time RT-PCR, and T beta RII protein levels were assayed by western blotting. T beta RII, type I procollagen, and type I collagen proteins in human dermal fibroblasts or hairless mouse skin were detected by immunostaining. FM of Z. mobilis inhibited down regulation of T beta RII mRNA, and protein levels in UVB irradiated human dermal fibroblasts consequently recover reduced type I procollagen synthesis. These results indicate UVB irradiation inhibits type I procollagen synthesis by suppression of TGF-beta/Smad signaling pathway, and FM of Z. mobilis has inhibitory effect on UVB-induced reduction of type I procollagen synthesis. While short period UVB irradiation decreased both T beta RII and type I procollagen protein levels in hairless mouse skin, topical application of FM of Z. mobilis prevented this decrease. Wrinkle formation in hairless mouse skin surface was accelerated by continuous 5 month UVB irradiation along with a reduction of type I collagen in the dermis, but this change was prevented by topical application of FM of Z. mobilis. From this experimental data, it is suggested that FM of Z. mobilis is effective for suppression of wrinkle formation in photoaging skin by inhibition of type I procollagen synthesis reduction.     

The carboxylpropeptide of type I procollagen in skin fibrillogenesis

Previous studies suggested that the aminopropeptide of type I procollagen may initiate fibril formation. The purpose of this investigation was to study the location of the carboxylpropeptide of type I procollagen during collagen fibrillogenesis. Chick embryonic and posthatching skin specimens were studied by immunofluorescence and immunoelectron microscopy and by immunoblotting with antibodies against the amino and carboxylpropeptide of type I procollagen. The carboxylpropeptide was demonstrated at the surface of collagen fibrils, 20-40 nm in diameter (10-day embryos) and in fibrils, 40-65 nm (21-day embryos). In addition, the carboxylpropeptide was found at the cell surface and free in the ground substance. The aminopropeptide was only seen in fibrils, 20-30 nm in diameter, as previously reported. Ratios of pN-collagen/pC-collagen increased from 16 days embryonic to 3 and 9 days postembryonic skins. This study suggests that both pN-collagen (aminopropeptide plus collagen) and pC-collagen (carboxylpropeptide plus collagen) participate in fibrillogenesis.     

Lifetime topical application of tretinoin to hairless mice.

The discovery that topical tretinoin can reverse some of the effects of photodamage may lead to its chronic application. Examination of long-term effects was of interest. Three groups of hairless mice (age 6-8 weeks) were treated dorsally with 1) tretinoin (0.025%), 2) cream vehicle, 3) sham treatment. Applications were 3 times weekly and continued for up to 2 years until all mice were sacrificed or had died. Biweekly examinations showed no sign of retinoid toxicity, with growth and longevity similar in all groups. Tretinoin-treated skin was smooth and pink, resembling that of younger mice. Controls had yellowed, irregularly thickened skin. Histologically, tretinoin-treated skin had a hyperplastic epidermis consisting of plump, cytologically normal cells. Control skin had 3-4 compressed cell layers. Foci of new normally staining collagen were present in the subepidermal dermis of tretinoin-treated skin; fibroblasts were large and abundant in these areas. These foci were absent in controls. Mice treated with tretinoin also appeared to have increased amounts of elastic fibers and glycosaminoglycans.     

Sunscreens with low sun protection factor inhibit ultraviolet B and A photoaging in the skin of the hairless albino mouse.

We examined the chronic effect of long daily suberythemal, fluorescent solar-stimulated radiation (FSSR; ultraviolet B (UVB)+A(UVA)) and UVA alone on female Skh-1 hairless albino mouse skin. Mice were dorsally irradiated 8 h every weekday for 16 weeks with FSSR or UVA, or 32 weeks with UVA alone. Various topical, low concentration, UVB and/or UVA sunscreens were applied before irradiation. Damage was assessed by skin-fold thickness, histology and biochemically by changes in the proportion of type III collagen. All FSSR-exposed mice showed increased skin thickening, elastic fibre hyperplasia, collagen damage and an increased proportion of type III collagen. Application of the UVB sunscreen (2.00%) resulted in marked protection for all nonbiochemical endpoints. There was no obvious advantage of adding 0.75% UVA sunscreen to the UVB sunscreen, but adding 2.00% UVA sunscreen reduced biochemical changes and connective tissue damage. Sixteen weeks of UVA irradiation caused skin thickening and laxity but the histology and biochemistry were indistinguishable from the controls. The mice irradiated with UVA for 32 weeks showed slight elastic fibre hyperplasia and collagen damage histologically, and increased skin thickening and laxity; these changes were unmodified by the 0.75% UVA sunscreen. These mice showed a significant increase in the proportion of type III collagen against which the UVA sunscreen offered protection. Our data suggest that UVA may be important in photoaging and that the use of low sun protection factor UVB+ UVA sunscreens on a day-to-day basis may offer some protection from solar photoaging.     

Effects of long-term retinoic acid treatment on epidermal differentiation in vivo: specific modifications in the programme of terminal differentiation

Summary To investigate the effects of long-term all-trans-retinoic acid (RA) treatment on epidermal differentiation in vivo , rhino mice were treated topically with 0.005% RA, and their epidermis was analysed histologically and biochemically after 5, 13 and 26 weeks of treatment. Effects of RA were observed first in the living layers of the epidermis, and then in the non-viable stratum corneum. Five weeks of topical RA led to thickening of the spinous and granular compartments, induction of keratins K6, K16 and K17, and suppression of filaggrin expression. After 13 and 26 weeks of RA treatment, the number of anucleate cornified cell layers remained similar to controls, but additional changes in histology and protein expression were observed. The results showed that prolonged administration of topical RA induced epidermal hyperproliferation, but did not suppress differentiation, in contrast to results observed in keratinocyte cultures. However, the distinct histological and biochemical changes observed in the spinous, granular and cornified layers of RA-treated skin after 26 weeks of treatment, suggested that the progeny of RA-treated basal cells undergo a modified programme of terminal differentiation. Considering the present data together with results of previous in vivo studies, we propose that long-term topical RA treatment retards, or specifically modulates, the later stages in epidermal differentiation, or programmed cell death.     

A close relationship between type 1 diabetes and vitamin A-deficiency and matrix metalloproteinase and hyaluronidase activities in skin tissues

Numerous wrinkles are observed in the skin of streptozotocin (STZ)-induced type 1 diabetic rats, which are similar to those seen in vitamin A-deficient (VAD) rats. Retinoic acid (RA), the active form of vitamin A, promotes the production of collagen in dermis and induces cell growth and inhibition of epidermal differentiation in skin tissues. Normal skin function is maintained by the extracellular matrix (ECM)-degrading enzymes, matrix metalloproteinase (MMP) and hyaluronidase (HAase). This study is the first comparison of MMP and HAase activities in skin tissues of type 1 diabetic, VAD and RA-treated animal models. In skin tissues of type 1 diabetic and VAD rats or VAD mice, both MMP-2 and HAase activities increased as compared with controls. In contrast, MMP and HAase activities were reduced in the skin tissues of RA-treated mice. Blood retinol levels in type 1 diabetic rats were lower than controls. These results indicate a close relationship between type 1 diabetes and vitamin A-deficiency on MMP and HAase in skin tissues, suggesting that type 1 diabetic rats could be vitamin A-deficient. Vitamin A-derived RA might be a significant regulator of ECM-degrading enzyme expression and diabetic symptoms.     

Collagen mRNA expression detected by in situ hybridization in keloid tissue

The keloid fibroblasts exhibited increased extracellular matrix gene expression, and prominent elevated type I procollagen mRNA when compared to control fibroblasts cultured from the uninvolved skin of normal people. It also showed markedly elevated type I/III procollagen mRNA ratios, but no synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. By in situ hybridization in keloid tissue, high levels of type I and type III procollagen mRNAs were detected in most of the fibroblasts, suggesting the presence of a subpopulation responsible for the increased collagen production. The levels of type I and type III procollagen mRNAs in these fibroblasts were clearly elevated compared to control skin specimens. And concentration of type I procollagen mRNA was found more predominantly than was type III. These results suggest that deposition of collagen in keloid could result from activation of certain fibroblasts responsible for type I procollagen production.