Lorraine H. Kligman

Topical retinoic acid enhances the repair of ultraviolet damaged dermal connective tissue.

Ultraviolet (UV) irradiation induces excessive accumulations of elastic fibers in animal and human skin. Collagen is damaged and glycosaminoglycans are vastly increased. Formerly considered an irreversible change, we recently showed, post-irradiation, that a band of normal connective tissue was laid down subepidermally . Because of its ability to stimulate fibroblasts and enhance healing of wounds, we thought it likely that retinoic acid (RA) would promote the formation of this subepidermal zone of reconstruction. Hairless mice were irradiated for 10 weeks with Westinghouse FS20 sunlamps for a total UV dose of 7 J/cm2. Then, 0.05% RA was applied for 5 and 10 weeks. Observations were made by light and electron microscopy. In contrast to controls treated with vehicle, the reconstruction zone was significantly wider in RA-treated mice. The enhanced repair was dose related. Histochemically and ultrastructurally, collagen was normal, fibroblasts were numerous and in a configuration of high metabolic activity.

Sunscreens prevent ultraviolet photocarcinogenesis.

Sunscreens of low or high sun protection factors (SPF*) were tested for their ability to inhibit ultraviolet (UV) carcinogenesis in two varieties of hairless mice. Low protection (SPF = 2) reduced by 50% the number of albino animals developing tumors. High protection (SPF =15) prevented tumor formation. Tumorigenesis was totally prevented in the lightly pigmented variety with either sunscreen, demonstrating the added protection of melanin. In mice and man, UV-induced cancer is a cumulative process. Reducing the amount of UV light reaching the basal layer will retard that process.

Intensification of ultraviolet-induced dermal damage by infrared radiation

SummaryAbnormal dermal deposition of elastic fibers is the earliest and most striking effect of prolonged sun exposure (solar elastosis). The hyperplastic fibers are usually ascribed to ultraviolet (UV) rays. Nonetheless, other portions of the solar spectrum may play contributing roles. Heat, for example, enhances experimental UV tumorigenesis. Heat induces erythema ab igne in which the structural alterations resemble those of actinically damaged skin, including the development of premaligant and malignant lesions. In regions of high insolation, infrared radiation (IR) is a constant companion of UV. To assess the role of IR in actinic damage to the dermis, albino guinea pigs were irradiated for 45 weeks with UV-B and UV-A, with and without IR. Control animals received IR only or no irradiation at all. Unirradiated dermis contains small amounts of elastic fibers in the upper dermis with greater depositions around follicles and sebaceous glands. After irradiation with UV, the fibers became more numerous, thicker, and more twisted; IR alone produced many fine, feathery fibers. The addition of IR to UV resulted in dense matlike elastic fiber depositions that exceeded what was observed with either irradiation alone. In combination or alone UV and IR radiation produced a large increase in ground substance, a finding also seen in actinically damaged human skin. Infrared radiation, in the physiologic range, though pleasant is not innocuous.ZusammenfassungDer früheste und auffälligste Effekt verstärkter Sonnenexposition ist eine abnormale Ablagerung elastischer Fasern in der Dermis. Die hyperplastischen Fasern werden gewöhnlich der Wirkung ultravioletter (UV) Strahlen zugeschrieben. Jedoch können auch andere Anteile des Sonnenspektrums bei deren Entstehung mitwirken. Hitze steigert z. B. die experimentelle UV-Tumorentstehung. Das Erythema ab igne, dessen strukturelle Veränderungen, einschließlich der Entwicklung prämaligner und maligner Läsionen, denen aktinisch geschädigter Haut ähneln, wird durch Hitze verursacht. Infrarotstrahlen (IR) sind konstante Begleiter des UV-Lichtes in Gebieten mit starker Sonnenbestrahlung. Um die Rolle von IR bei der Entstehung von aktinischer Schädigung zu untersuchen, wurden Meerschweinchen für 45 Wochen mit UV-A und UV-B, mit und ohne IR, bestrahlt. Eine Kontrollgruppe erhielt nur IR oder keine Bestrahlung. Unbestrahlte Dermis enthält in den oberen Anteilen geringe Mengen elastischer Fasern mit stärkeren Ablagerungen um die Follikel und Talgdrüsen. Nach UV-Bestrahlung wurden die Fasern zahlreicher, dicker und gewundener. IR alleine verursachte die Bildung vieler feiner, federnartiger Fasern. Die Hinzufügung von IR zu UV führte zur Bildung dichter, netzartiger, elastischer Fasern, die das übertraf, was nach Bestrahlung mit IR oder UV alleine beobachtet wurde. Sowohl die kombinierte als auch die einzelne Applikation von UV und IR erzeugte eine starke Vermehrung von Grundsubstanz, eine Beobachtung, die auch in aktinisch geschädigter menschlicher Haut gemacht wird. Infrarotstrahlung ist zwar in physiologischen Größenordnungen angenehm, jedoch nicht unschädlich.

The ultraviolet-irradiated hairless mouse: A model for photoaging

The hairless mouse is proving to be a relevant model for the systematic study of photoaging. As in humans, with chronic ultraviolet radiation, these mice develop elastic fiber hyperplasia, followed by elastosis and ultrastructural degradation. Collagen is damaged and its metabolism is altered, while the normally low levels of proteoglycans and glycosaminoglycans are greatly increased. With this model we have described the effects on dermal connective tissue of UVB (290 to 320 nm), UVA (320 to 400 nm), and the combination of the two. We have also assessed the protective effects of sunscreens. We found that a significant amount of photodamage was repaired when ultraviolet radiation was stopped. Subepidermally in a former region of elastosis, a band of new normal dermis was laid down. Enhancement of the repair was achieved with topical all-trans-retinoic acid in a time- and dose-dependent manner. Retinoic acid was also found to induce angiogenesis in unirradiated mice.

AN ACTION SPECTRUM FOR ULTRAVIOLET INDUCED ELASTOSIS IN HAIRLESS MICE: QUANTIFICATION OF ELASTOSIS BY IMAGE ANALYSIS

Abstract— To determine an action spectrum for ultaviolet (UV)‐induced elastosis, four groups of 24 albino hairless mice each were exposed to four different spectra emitted by a xenon are solar simulator fitted with cut‐off filters (Schott WG 320, 335, 345, and 360). These filters progressively removed more of the shorter wavelengths until, in the final spectrum, only long wavelength UVA (> 335 nm) remained. Exposures continued up to 62 weeks. A fifth group of mice served as controls. Skin biopsies were taken at pre‐determined dose points and were processed for light microscopy. Elastosis was quantified by computerized image analysis, yielding dose‐response curves for each spectrum. The total energy required for a 50% increase in elastic tissue compared to controls was determined graphically for each spectrum. These were: WG 320, 65 J/cm2; WG 335, 865 J/cm2; WG 345, 1230 J/cm2; and WG 360, 2000 J/cm2. Our results were tested against published action spectra for erythema, photocarcinogenesis and elastosis. The erythema spectrum was the most predictive for elastosis except that the longer UVA wavelengths were less effective for elastosis than for erythema. Solar simulating radiation (WG 320 filter) with its UVB component was the most effective in inducing elastosis. Full spectrum UVA (WG 345) required 20 times more energy while long wavelength UVA (WG 360) required 30 times more energy to induce equivalent elastosis.

Impaired wound healing in mice deficient in a matricellular protein SPARC (osteonectin, BM-40)

BackgroundSPARC is a matricellular protein involved in cell-matrix interactions. From expression patterns at the wound site and in vitro studies, SPARC has been implicated in the control of wound healing. Here we examined the function of SPARC in cutaneous wound healing using SPARC-null mice and dermal fibroblasts derived from them.ResultsIn large (25 mm) wounds, SPARC-null mice showed a significant delay in healing as compared to wild-type mice (31 days versus 24 days). Granulation tissue formation and extracellular matrix protein production were delayed in small 6 mm SPARC-null wounds initially but were resolved by day 6. In in vitro wound-healing assays, while wild-type primary dermal fibroblasts showed essentially complete wound closure at 11 hours, wound closure of SPARC-null cells was incomplete even at 31 hours. Addition of purified SPARC restored the normal time course of wound closure. Treatment of SPARC-null cells with mitomycin C to analyze cell migration without cell proliferation showed that wound repair remained incomplete after 31 hours. Cell proliferation as measured by 3H-thymidine incorporation and collagen gel contraction by SPARC-null cells were not compromised.ConclusionsA significant delay in healing large excisional wounds and setback in granulation tissue formation and extracellular matrix protein production in small wounds establish that SPARC is required for granulation tissue formation during normal repair of skin wounds in mice. A defect in wound closure in vitro indicates that SPARC regulates cell migration. We conclude that SPARC plays a role in wound repair by promoting fibroblast migration and thus granulation tissue formation.

Effects of all trans-retinoic acid on the dermis of hairless mice

The marked degradative changes induced in human and animal dermis by chronic ultraviolet radiation are thought to be irreversible. We have shown in studies with hairless mice that a new normal dermis formed subepidermally after irradiation ceased. Because retinoids stimulate wound repair, we evaluated the ability of all-trans-retinoic acid (RA) to enhance the repair of ultraviolet damage. To produce mild dermal damage, hairless mice were irradiated for 10 weeks with FS20 sunlamps. The mice were then treated topically with various concentrations of RA for either 5 or 10 weeks. The skin was examined by light and electron microscopic techniques. The subepidermal repair zone in RA-treated mice was significantly wider than that in the untreated control group. The greater repair appeared to be retinoid specific and was dose dependent. The collagen was both histochemically and ultrastructurally normal; fibroblasts were numerous and morphologically hyperactive. In addition, topical RA increased dermal vascularity.

BIOCHEMICAL CHANGES IN HAIRLESS MOUSE SKIN COLLAGEN AFTER CHRONIC EXPOSURE TO ULTRAVIOLET‐A RADIATION*

Evidence is mounting that UV‐B and UV‐A radiation affect skin differently in responses as diverse as erythema and elastosis. We found in this study that collagen metabolism was also differentially affected. Albino hairless mice were irradiated with two UV‐A sources: (1) UVASUN 3000 (340–400nm) for cumulative exposures of 4000 and 8000 J/cm2; (2) a xenon solar simulator filtered to provide full spectrum UV‐A (320–400nm) and long wavelength UV‐A (335–400nm) for cumulative exposures of 3000 and 4000 J/cm2 respectively. Collagen was isolated from other skin proteins by acid extraction, pepsin digestion and salt precipitation. Collagen types I and III were separated by interrupted gel electrophoresis. Ultraviolet‐A rendered the collagen highly resistant to pepsin digestion. In age‐matched controls only16–18% of the total collagen remained insoluble, whereas in long wavelength UV‐A‐irradiated skins the insoluble fraction was as high as 87%. A dose response was noted at 4000 and 8000 J/cm2 as delivered by the UVASUN. Recovery of collagen from the pepsin soluble fraction was low in all UV‐A groups and the amount of type III so small that determination of ratios of type III to I collagen was unreliable. These results suggest that chronic UV‐A radiation may increase cross‐linking of dermal collagen.

Ultraviolet B Radiation Increases Hairless Mouse Mast Cells in A Dose‐Dependent Manner and Alters Distribution of UV‐lnduced Mast Cell Growth Factor

Abstract— In studies of the effects of chronic UVB irradiation on dermal connective tissue in the hairless mouse, we observed that the number and size of mast cells was increased. Because mast cells are known to be associated with connective tissue remodeling, we examined and quantified the effect of increasing UVB (290‐320 nm) doses on this cell. Groups of mice were exposed to filtered FS‐40 Westinghouse lamps (290‐400 nm: peak irradiance 313 nm) for 1‐5 minimal erythema doses (MED) thrice weekly for 10 weeks. Appropriate controls were included. Biopsies, processed for light microscopy, were stained with toluidine blue. Mast cells were counted in 15 high‐magnification fields per specimen with upper and lower dermis scored separately. Significant increases in large densely granular mast cells occurred at 2 MED in the lower dermis, in association with a UVB‐exacerbated granulomatous reaction. In the upper dermis, mast cells were significantly increased with 3 MED. These findings suggest that mast cells may play a dual role in UV‐irradiated skin with those in the lower dermis related to inflammation processes and those in the upper dermis involved in connective tissue modeling. To gain understanding of the mechanism of mast cell recruitment and maturation, we examined the effect of UVB on mast cell growth factor expression. This was enhanced in the epidermis by UVB, with a shift from cytoplasmic staining to membrane‐associated or intercellular staining at 2 MED and higher. Dermal dendritic and mononuclear cells also showed increased reactivity.

Topical all-trans-retinoic acid prevents corticosteroid-induced skin atrophy without abrogating the anti-inflammatory effect.

We tested the ability of all-trans-retinoic acid to prevent corticosteroid-induced skin atrophy without lessening the anti-inflammatory effect of the steroids. Histologic study and skin-fold thickness in hairless mice treated topically with various steroids, followed by topical all-trans-retinoic acid, were used to measure prevention of atrophy. By both assessments, all-trans-retinoic acid prevented atrophy. Noninterference with the anti-inflammatory property of steroids was tested in a phorbol ester-induced mouse ear edema model and by histologic assessment of croton oil-induced inflammation of mouse dermis. We found that all-trans-retinoic acid did not interfere with steroid suppression of either edema or dermal inflammation. Thus all-trans-retinoic acid was effective in preventing steroid-induced atrophy without affecting the steroids anti-inflammatory property.