Elaine Schwartz

COLLAGEN ALTERATIONS IN CHRONICALLY SUN-DAMAGED HUMAN SKIN

Abstract The major histological characteristic of sun‐damaged skin is the accumulation of an elastotic material that appears to replace collagen. This elastotic material consists primarily of elastin and histological studies suggest a large loss of collagen in the dermis of chronically sun‐damaged skin. In this study, we examine the content and distribution of collagen and procollagen in sun‐damaged human skin. The total collagen content of sun‐damaged skin was 20% less than nonsolar‐exposed skin (524 μg collagen per mg total protein in sun‐damaged skin and 667 μg collagen per mg total protein in nonsolar‐exposed skin). In addition, there was a 40% decrease in the content of intact amino propeptide moiety of type III procollagen in sun‐damaged skin (0.68 U per 50 mg wet weight) as compared to nonsolar‐exposed skin (1.12 U per 50 mg wet weight). The data suggest that this change in collagen content is due to increased degradation. The distribution of collagen in sun‐damaged skin was examined by indirect immunofluo‐rescence. Mild digestion of sun‐damaged skin with elastase removed the elastin and revealed the presence of collagen in the elastotic material. Therefore, the elastin appears to mask the presence of collagen fibers in the dermis of sun‐damaged skin.

Abnormalities of connective tissue components in lesional and non-lesional tissue of patients with pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a disorder of connective tissue in which abnormalities of elastic tissue and collagen are found. The purpose of this study was to examine the ultrastructure and distribution of connective tissue components in lesional and non-lesional skin of patients by means of indirect immunofluorescence, electron microscopy and indirect immunoelectron microscopy. Prominent abnormalities of elastic tissue were seen on electron microscopy and confirmed by immunoelectron microscopy. Abnormal elastic fibers containing electron-dense bodies and holes were seen even in non-lesional skin. In addition, the normal pattern of collagen bundles was disrupted in lesional skin, but not in non-lesional skin of patients with PXE. The majority of individual collagen fibrils appeared normal by electron microscopy. The distribution of type IV collagen and laminin was normal in small blood vessels. Finally, abnormalities in the distribution of fibronectin were seen. The finding of atypical elastic fibers in non-lesional skin supports an early role for elastic tissue components in the pathogenesis of PXE. Interactions between elastin, collagen and other matrix substances may explain some of the abnormalities seen.

Association of elastin with oxytalan fibers of the dermis and with extracellular microfibrils of cultured skin fibroblasts.

The formation of a mature elastic fiber is thought to proceed by the deposition of elastin on pre-existing microfibrils (10-12 nm in diameter). Immunohistochemical evidence has suggested that in developing tissues such as aorta and ligamentum nuchae, small amounts of elastin are associated with microfibrils but are not detected at the light microscopic and ultrastructural levels. Dermal tissue contains a complex elastic fiber system consisting of three types of fibers--oxytalan, elaunin, and elastic--which are believed to differ in their relative contents of microfibrils and elastin. According to ultrastructural analysis, oxytalan fibers contain only microfibrils, elaunin fibers contain small quantities of amorphous elastin, and elastic fibers are predominantly elastin. Using indirect immunofluorescence techniques, we demonstrate in this study that nonamorphous elastin is associated with the oxytalan fibers. Frozen sections of normal skin were incubated with antibodies directed against human aortic alpha elastin and against microfibrillar proteins isolated from cultured calf aortic smooth muscle cells. The antibodies to the microfibrillar proteins and elastin reacted strongly with the oxytalan fibers of the upper dermis. Oxytalan fibers therefore are composed of both microfibrils and small amounts of elastin. Elastin was demonstrated extracellularly in human skin fibroblasts in vitro by indirect immunofluorescence. The extracellular association of nonamorphous elastin and microfibrils on similar fibrils was visualized by immunoelectron microscopy. Treatment of these cultures with sodium dodecyl sulfate/mercaptoethanol (SDS/ME) solubilized tropoelastin and other proteins that reacted with the antibodies to the microfibrillar proteins. It was concluded that the association of the microfibrils with nonamorphous elastin in intact dermis and cultured human skin fibroblasts may represent the initial step in elastogenesis.

Elastase-like protease and elastolytic activities expressed in cultured dermal fibroblasts derived from lesional skin of patients with pseudoxanthoma elasticum, actinic elastosis, and cutis laxa.

Pseudoxanthoma elasticum (PXE), actinic elastosis, and cutis ‘laxa are three cutaneous disorders in which alterations in the structure of dermal elastic fibers are seen [1,2]. Elastic fibers in PXE are increased in number, appear clumped and become calcified. The acquired skin disorder, actinic elastosis, is described as a proliferation (elastosis) of abnormal elastic fibers that become thickened and tangled [3]. Cutis rhomboidalis nuchae represents an extensive form of actinic elastosis on the posterior aspect of the neck of individuals 60 years of age or older who have had chronic exposure to the sun. Cutis laxa is considered to be a more generalized elastolysis; the skin is abnormally lax and hangs in folds; histopathological changes include a reduction in elastic fibers. In addition, the remaining elastic fibers appear thin and fragmented. Therefore, these three distinct disorders are all characterized by abnormalities in elastic fibers. It has been suggested that proteolytic degradation by elastase(s) plays a role in the formation of the elastin abnormalities seen in disease states [4-61. These proteases have been found in extracts prepared from dermal fibroblasts [7-lo] and in sera from a patient with cutis laxa [ll]. In this investigation, fibroblast cultures were established from affected areas of patients with pseudoxanthoma elasticum, actinic elastosis (cutis rhomboidalis nuchae), and cutis laxa. Elastase-like protease and elastolytic activities were assayed using succinyl trialanine paranitroanilide (SANA) and radiolabeled insoluble skin elastin in extracts of these cells.

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.

Determination of desmosines in elastin-related skin disorders by isocratic high-performance liquid chromatography☆

Abnormalities in the amount of skin elastin occur in several cutaneous disorders. The number of elastic fibers is increased in elastotic disorders such as pseudoxanthoma elasticum (PXE) and cutis rhomboidalis nuchae (actinic elastosis, AE) and is decreased in elastolytic disorders such as cutis laxa (CL). We describe a procedure to quantify desmosines and elastin in small amounts of skin using high-performance liquid chromatography (HPLC). Biopsies were obtained from normal, nonsolar exposed skin and from the lesional skin of patients with PXE, cutis rhomboidalis nuchae, and CL. Specimens were subjected to hot alkali treatment and the desmosines were released by acid hydrolysis and quantified by HPLC. The mean value for normal skin was 252 +/- 28 ng desmosines per milligram wet weight (SD, n = 5). The disorders of elastosis (PXE and AE) demonstrated a two- to fivefold increased content of desmosines. In contrast, the elastolytic disorder (CL) had only 20% of the normal content of desmosines. Furthermore, PXE and normal skin elastins had the same amount of desmosines per milligram purified elastin. This method could be used to evaluate the extent of elastosis or elastolysis in a particular lesion.

Collagen loss in photoaged human skin is overestimated by histochemistry

It is well known that photoaged skin is characterized by increases in dermal matrix components that include glycosaminoglycans, proteoglycans and masses of abnormal elastic fibers accompanied by substantial collagen loss. Histochemical staining of such tissue gives the impression of “massive” loss of collagen and its replacement by these other matrix components. Early biochemical studies have lent support to this notion with a reported decrease in total collagen of ∼45% compared to protected skin. More recent studies report considerably less, but varying, amounts of collagen loss. Rarely have the two approaches, histochemistry and biochemical analysis, been used in the same study to examine the same tissue. In this study, collagen loss was quantified biochemically in paired biopsies from sun‐protected and sun‐exposed arm skin of moderately photoaged female subjects (age 51–77 years). The values obtained were compared with histochemical and immunochemical findings. Quantitatively, collagen loss on a per mg protein basis was small compared to the histochemical appearance.

Abnormalities of fibrillin in acquired cutis laxa

BACKGROUND Degeneration of elastic tissue in acquired cutis laxa has been previously described, but microfibrils have not been adequately studied. OBJECTIVE We determined whether the microfibrillar component of elastic tissue is affected in skin of a patient with acquired cutis laxa. METHODS Lesional skin was examined with indirect immunofluorescence and immunoelectron microscopy with antibodies to fibrillin. RESULTS Indirect immunofluorescence showed a reduction in the distribution of fibrillin in the papillary dermis, where there was loss of the usual pattern of microfibrils perpendicular to the epidermis. Immunoelectron microscopy showed a typical distribution of elastic microfibrils around elastin of normal skin. In skin affected by cutis laxa microfibrils appeared morphologically normal but appeared less frequently in selected sites. CONCLUSION The microfibrillar component of elastic fibers was reduced in the papillary dermis of this patient with acquired cutis laxa.

Steady‐state mRNA levels of interleukin‐1, integrins, cJun, and cFos in hairless mouse skin during short‐term chronic UV exposure and the effect of topical tretinoin

We have proposed that UV activation of cytokine and integrin signaling pathways may initiate the photoaging process and that one of the effects of tretinoin treatment may be to alter the cytokine and integrin patterns. In previous results, steady‐state mRNA levels of interleukin‐1α, tumor necrosis factor α, transforming growth factor β, collagenase, stromelysin, collagen, and integrins (α1 and α2) were increased in the skin of hairless mice that were either UV treated or concurrently treated with UV followed by topical tretinoin for 5 weeks. The aim of this study was to focus on the expression of α1, α2 and α5 integrins, IL‐1α, IL‐1β, cJun, and cFos at an earlier time point (3 weeks). Animals were UV irradiated thrice weekly for 3 weeks and were treated topically with either 0.05% tretinoin or the vehicle immediately after each exposure. Total RNA was prepared and used in RT‐PCR with radiolabeled dCTP and specific primers. UV slightly increased steady‐state mRNA levels for α1, α2 and α5 integrins whereas UV+tretinoin increased their expression (3‐, 2‐ and 7‐fold respectively). Steady‐state mRNA levels for IL‐1α, IL‐1β and cJun were increased with UV (3‐, 12‐ and 6‐fold respectively) and with UV+tretinoin (6‐, 7‐ and 9‐fold respectively). In contrast, cFos expression was unchanged. In situ staining for IL‐1α mRNA was slightly more abundant in mice treated for 3 weeks with UV and UV+ tretinoin than in controls whereas 5 weeks of UV+tretinoin treatment gave strongly positive staining. Results are consistent with cytokines and integrins mediating the effects of UV on the skin, with modulation of these effects by tretinoin.

Elastase digestion of normal and pseudoxanthoma elasticum lesional skin elastins.

Pseudoxanthoma elasticum (PXE) is a heritable disorder of connective tissue that is characterized by redundant folds of skin in flexural areas. There is considerable evidence that suggests that the elastic fiber is the main site of the abnormality although the primary molecular defect has not been identified. The aim of this study was to identify differences between PXE and normal skin elastins. Elastins from normal, nonsolar-exposed skin, and pseudoxanthoma elasticum lesional skin were purified and their solubilization by pancreatic elastase was compared. Results demonstrated that elastin derived from normal skin was more susceptible to proteolytic cleavage than elastin purified from either pseudoxanthoma elasticum lesional skin or ligamentum nuchae. Pretreatment of the lesional elastin with testicular hyaluronidase increased its solubilization two-fold and generated a unique 15,000 Da molecular weight fragment. Elastin prepared from PXE skin may contain bound glycosaminoglycans which interfere with elastase activity. The susceptibility of normal skin elastin to proteolytic degradation may have implications in the study of aging skin.