Neuropeptide substance P alters stem cell fate to aid wound healing and promote epidermal stratification through asymmetric stem cell divisions.

Loss of sensory innervation delays wound healing and administration of the neuropeptide substance P improves re-epithelialization. Keratinocyte hyperproliferation post-wounding may result from symmetric stem cell (SC) self-renewal, asymmetric SC self-renewal, committed progenitor divisions, or a combination of these. However, the effects of sensory denervation and of neuropeptides on SC proliferation are not known. Here we show that early after wounding both asymmetric and symmetric SC self-renewal increase, without significant committed progenitor (CP) activation. Decreased sensory innervation is associated with a decrease in both SC and CP proliferation. Based on previous work showing that substance P is decreased in capsaicin-treated mice and improves wound healing in normal skin, we examined the effects of substance P on SC and CP proliferation during wound healing. Substance P restored asymmetric SC proliferation in skin with decreased sensory innervation, both at baseline and following wounding. Epidermis with decreased sensory innervation was severely thinned. Consistent with this, substance P-induced asymmetric SC proliferation resulted in increased stratification in skin with both normal and decreased innervation. Lapatinib prevented the substance P-induced increase in asymmetric SC divisions in murine epidermis, as well as the increase in epidermal stratification, suggesting that asymmetric SC divisions are required for epidermal stratification.

What's new in dermatology: epidermal stem cells.

The study of stem cells in the epidermis is a rapidly emerging field. Great advances have been made in both basic and clinical research. Advances in basic science include the ability to assay stem cells of the epidermis in vivo, new strong evidence for the existence of an independent interfollicular epidermal stem cell, and improved ability to analyze individual stem cell divisions. Advances in the clinic include recognition of the importance of stem cells for wound repair and for gene therapy and promising results have been obtained in a patient with junctional epidermolysis bullosa over a 12 month period of observation. This article discusses these recent advances in cutaneous stem cell biology.

Stem cells and tissue-engineered skin.

Advances in tissue engineering of skin are needed for clinical applications (as in wound healing and gene therapy) for cutaneous and systemic diseases. In this paper we review the use of epidermal stem cells as a source of cells to improve tissue-engineered skin. We discuss the importance and limitations of epidermal stem cell isolation using biomarkers, in quest of a pure stem cell preparation, as well as the culture conditions necessary to maintain this purity as required for a qualitatively superior and long-lasting engineered skin. Finally, we review the advantages of using additional multipotent stem cell sources to functionally and cosmetically optimize the engineered tissue.

In search of the elusive epidermal stem cell.

Recent studies are beginning to reveal that our basic concepts of epidermal stem cell biology may be based on somewhat tenuous ground. For example, it is often assumed that colony-forming cells represent epidermal stem cells, although this has not proved to be the case in hematopoietic cell lineages. In addition, although most stem cells are not cycling, label-retaining cells are used as a primary measure of epidermal stem cells. Moreover, the locations of stem cell niches in epidermis are still being debated. Finally, while putative stem cell markers abound, the most effective isolation procedure for stem cells has not been determined, and the relative efficiency of various methods of stem cell isolation remains unknown. With a functional assay for epidermal stem cells (analogous to the in-vivo competitive assay used for hematopoiesis), we appear to be in a better position to more clearly define the molecular signature of the true long-term repopulating cell/stem cell of the epidermis. Nonetheless, significant progress has been made in regenerative therapy of the epidermis for ulcer and burn treatment, and for corrective gene therapy for inherited skin diseases.

Measuring stem cell frequency in epidermis: a quantitative in vivo functional assay for long-term repopulating cells.

Epidermal stem cells play a central role in tissue homeostasis, wound repair, tumor initiation, and gene therapy. A major impediment to the purification and molecular characterization of epidermal stem cells is the lack of a quantitative assay for cells capable of long-term repopulation in vivo, such as exists for hematopoietic cells. The tremendous strides made in the characterization and purification of hematopoietic stem cells have been critically dependent on the availability of competitive transplantation assays, because these assays permit the accurate quantitation of long-term repopulating cells in vivo. We have developed an analogous functional assay for epidermal stem cells, and have measured the frequency of functional epidermal stem cells in interfollicular epidermis. These studies indicate that cells capable of long-term reconstitution of a squamous epithelium reside in the interfollicular epidermis. We find that the frequency of these long-term repopulating cells is 1 in 35,000 total epidermal cells, or in the order of 1 in 104 basal epidermal cells, similar to that of hematopoietic stem cells in the bone marrow, and much lower than previously estimated in epidermis. Furthermore, these studies establish a novel functional assay that can be used to validate immunophenotypic markers and enrichment strategies for epidermal stem cells, and to quantify epidermal stem cells in various keratinocyte populations. Thus further studies using this type of assay for epidermis should aid in the progress of cutaneous stem cell-targeted gene therapy, and in more basic studies of epidermal stem cell regulation and differentiation.

Alterations in cytokine regulation in aged epidermis: implications for permeability barrier homeostasis and inflammation. I. IL-1 gene family.

Acute disruption of the cutaneous permeability barrier with either solvents or tape-stripping stimulates a homeostatic metabolic response in the subjacent nucleated layers of the epidermis that results in a rapid restoration of normal permeability barrier function. When the aged epidermal permeability barrier is stressed, it reveals a diminished capacity for recovery, in comparison to young epidermis, analogous to other organs in the aged when stressed. Although the signals that regulate this homeostatic response by the epidermis have not yet been resolved, acute permeability barrier disruption stimulates release of prestored IL-1alpha, and increased production of potentially regulatory cytokines, including IL-1alpha and TNFalpha in the epidermis. In these studies, we addressed the hypothesis that cytokine dysregulation explains the permeability barrier abnormality in aged epidermis, assessing the regulation of IL-1 and TNF signaling in aged vs young mice. To determine whether the IL-1 family of cytokines plays a key role in the permeability barrier abnormality of the aged, permeability barrier recovery rates were compared in transgenic mice lacking the functional IL-1 type 1 receptor vs wild-type mice at various ages. Knockout of the IL-1 type 1 receptor exacerbates the defect in permeability barrier homeostasis that is seen in age-matched, wild-type counterparts. Furthermore, the sluggish permeability barrier recovery in aged epidermis is associated with, and at least in part attributable to, altered expression of the IL-1 family of cytokines and receptors both under basal conditions and after acute barrier perturbations. Whereas modulations in cytokine expression with epidermal permeability barrier perturbation are qualitatively similar in aged epidermis, they greatly differ quantitatively. In contrast, examination of TNFalpha mRNA and protein basally, and following barrier perturbation revealed no alterations in aged epidermis. Together, these results show that selective alterations in the IL-1 family of cytokines occur with aging and that defects in IL-1 signaling may contribute to the epidermal permeability barrier abnormality of aged skin.

Testosterone perturbs epidermal permeability barrier homeostasis.

Although there are no known gender-related differences in permeability barrier function in adults, estrogens accelerate whereas testosterone retards barrier development in fetal skin, and male fetuses demonstrate slower barrier development than female littermates. Moreover, prenatal administration of the androgen receptor antagonist, flutamide, equalizes developmental rates in male and female fetuses. Therefore, we evaluated the effects of changes in testosterone on barrier homeostasis in adult murine and human skin. Hypogonadal mice (whether by castration or by treatment with systemic flutamide) displayed significantly faster barrier recovery at 3, 6, and 12 h than did controls, and testosterone replacement slowed barrier recovery in castrated mice. Moreover, testosterone directly effects the skin, as topical flutamide also accelerated barrier recovery in normal male mice. These findings appear to be of physiologic significance, since prepubertal male mice (age 5 wk) displayed accelerated barrier recovery in comparison with adult postpubertal (11 wk) males. These studies also appear to be relevant for humans, as a hypopituitary human subject demonstrated repeated changes in barrier recovery in parallel with peaks and nadirs in serum testosterone levels during intermittent testosterone replacement. Mechanistic studies showed that differences in epidermal lipid synthesis do not account for the testosterone-induced functional alterations. Instead, epidermal lamellar body (LB) formation and secretion both decrease, resulting in decreased extracellular lamellar bilayers in testosterone-replete animals. These studies demonstrate that fluctuations in testosterone modulate barrier function, and that testosterone repletion can have negative consequences for permeability barrier homeostasis.

Recessive x-linked ichthyosis: role of cholesterol-sulfate accumulation in the barrier abnormality.

Cholesterol sulfate is a multifunctional sterol metabolite, produced in large amounts in squamous keratinizing epithelia. Because patients with recessive x-linked ichthyosis display not only a 10-fold increase in cholesterol sulfate, but also a 50% reduction in cholesterol, we assessed here whether cholesterol sulfate accumulation and/or cholesterol deficiency produce abnormal barrier function in recessive x-linked ichthyosis. Patients with recessive x-linked ichthyosis display both an abnormal barrier under basal conditions, and a delay in barrier recovery after acute perturbation, which correlate with minor abnormalities in membrane structure and extensive lamellar-phase separation. Moreover, both the functional and the structural abnormalities were corrected by topical cholesterol. Yet, topical cholesterol sulfate produced both a barrier abnormality in intact skin and extracellular abnormalities in isolated stratum corneum, effects largely reversed by coapplications of cholesterol. Together, these results suggest that cholesterol sulfate accumulation rather than cholesterol deficiency is responsible for the barrier abnormality. Despite the apparent importance of cholesterol sulfate-to-cholesterol processing for normal barrier homeostasis, neither steroid sulfatase activity nor mRNA levels are upregulated following acute perturbations. These results demonstrate both a potential role for cholesterol sulfate-to-cholesterol processing in normal permeability barrier homeostasis, and that basal levels of steroid sulfatase are sufficient to accommodate acute insults to the permeability barrier.

Aging and the epidermal permeability barrier: implications for contact dermatitis.

OBJECTIVES: This report examines our present knowledge of the epidermal permeability barrier as it pertains to irritant contact dermatitis and discusses how altered barrier function may affect the clinical manifestations of irritant contact dermatitis in the aged. CONCLUSIONS: Altered barrier function affects the ability of an irritant to penetrate the stratum corneum and produce deleterious effects on both stratum corneum and epidermis. Moreover, a damaged epidermis can be expected to produce a defective stratum corneum, thus resulting in a vicious cycle. Furthermore, although basal transepidermal water loss has traditionally been used as an assessment of barrier function, evidence suggests that dynamic tests of barrier function are more reliable indicators of barrier function in the clinical setting of additive, repetitive, or chronic insults. Finally, barrier disruption per se has now been shown to produce both a cytokine response and an increase in epidermal Langerhans' cell density. Thus, barrier disruption not only alters penetrance of contactants, but also may prime the inflammatory response. Studies of aged epidermal permeability barrier function provide a greater understanding of the modified response to irritants in the aged.

Optimal ratios of topical stratum corneum lipids improve barrier recovery in chronologically aged skin.

BACKGROUND: Chronologically aged skin exhibits delayed recovery rates after defined barrier insults, with decreased epidermal lipid synthesis, and particularly a reduction in cholesterol synthesis. Prior studies in young mice (< 10 weeks) and humans (20 to 30 years of age) have shown that application of a mixture of cholesterol, ceramides, and essential/nonessential free fatty acids (FFAs) in an equimolar ratio allows normal barrier recovery, whereas any 3:1:1:1 ratio of these four ingredients accelerates barrier recovery. OBJECTIVE AND METHODS: Our purpose was to compare the ability of equimolar and cholesterol- and FFA-dominant molar lipid mixtures (2% in propylene glycol/n-propanol, 7:3) versus vehicle alone on barrier recovery rates at 0, 3, 6, 24, 48 hours, and 1 week after tape stripping of aged hairless mouse (> 18 months) and chronologically aged human skin (80 +/- 5 years). RESULTS: Whereas a single topical application of the equimolar mixture only allows normal recovery in young mice, it appeared to improve barrier recovery in chronologically aged mice (p < 0.06). Moreover, a 3:1:1:1 mixture with cholesterol as the dominant lipid further accelerated barrier recovery at 3 and 6 hours (p < 0.01 and p < 0.03, respectively, vs 1:1:1:1). Likewise, the cholesterol-dominant, optimal molar ratio mixture significantly accelerated barrier recovery in chronologically aged human skin at 6 hours (p < 0.005; n = 6). In contrast, in aged mice, an FFA-dominant mixture significantly delayed barrier recovery at 3, 6, and 24 hours (p < 0.005, 0.05, and 0.001, respectively), Finally, ultrastructural studies showed that lipid-induced, accelerated recovery in chronologically aged mice is associated with the accelerated replenishment of the stratum corneum interstices with lamellar unit structures. CONCLUSION: These findings show that barrier recovery is accelerated in chronologically aged murine epidermis with optimized ratios of physiologic lipids, provided that cholesterol is the dominant lipid and that the same mixture also accelerates barrier recovery in chronologically aged human skin.

Morphology of lipid alterations in the epidermis: a review.

An overview of the morphological correlates of mammalian epidermal lipids and the cutaneous permeability barrier is provided. The following features are discussed: 1) ultrastructural characterization of the progressive lipid alterations accompanying normal stratum corneum (SC) formation; 2) effects of drugs/inhibitors that interfere with specific aspects of epidermal lipid metabolism; 3) lipid alterations in certain human skin disorders; 4) effects of selected topical emollients on SC lipid organization; and 5) potential pitfalls in interpretation of ultrastructural data in terms of cutaneous function and dysfunction.

Stratum corneum structure and function correlates with phenotype in psoriasis.

Psoriatic epidermis demonstrates a defective program of growth and differentiation, including an abnormal permeability barrier. Despite the fact that damage to the epidermis often initiates the disease, psoriasis is commonly viewed as triggered by aberrant immune phenomena in deeper skin layers. Permeability barrier homeostasis requires the formation and secretion of lamellar body contents, as well as the extracellular processing of lamellar body contents into lamellar bilayers. To address the hypothesis that psoriasis is triggered by exogenous rather than internal factors, we assessed permeability barrier function, lamellar body structure, and extracellular lamellar bilayer formation in untreated patients with different psoriatic phenotypes. Subjects with erythroderma and active plaque phenotypes displayed elevated transepidermal water loss levels, increased numbers of epidermal lamellar bodies (many of which failed to be secreted); i.e., corneocytes displayed retained cytosolic lamellar bodies, and extracellular domains largely devoid of lamellae. In contrast, patients with chronic plaque psoriasis and sebopsoriasis displayed a lesser increase in transepidermal water loss, normal numbers of lamellar bodies with only a few retained organelles, and abundant extracellular lamellar material (although a normal unit bilayer pattern did not form). Thus, both functionally and structurally, permeability barrier homeostasis was more disrupted in erythrodermic and active plaque psoriasis than in chronic plaque psoriasis and sebopsoriasis; i.e., the extent of defective barrier function correlated with abnormalities in the known mechanisms of barrier repair, including lamellar body production and extracellular bilayer formation. These findings are consistent with the hypothesis that both the initial appearance of psoriasis (Koebner phenomenon) and changes in disease phenotype are driven by alterations in barrier function.

Decreased epidermal lipid synthesis accounts for altered barrier function in aged mice.

The epidermis of aged mice displays decreased stratum corneum (SC) lipid content and decreased extracellular bilayers, which result in impaired barrier recovery following the solvent treatment or tape stripping. We assessed the role of altered lipid synthesis as the cause of the abnormal barrier and lipid content in aged epidermis, both under basal conditions and in response to acute barrier perturbations. In aged epidermis ( > or = 18 months), synthesis of one of the three key lipid classes (cholesterol) is decreased under basal conditions, and sterologenesis fails to attain the levels reached in young epidermis following comparable acute perturbations. In contrast, fatty acid and sphingolipid synthesis in aged epidermis increase sufficiently to approach the levels attained in stimulated young epidermis. The abnormalities in sterologenesis in aged epidermis are paralleled by a decrease in activity of its rate-limiting enzyme, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, under basal conditions, and enzyme activity also fails to increase as much as in young epidermis after barrier disruption. That defective lipid generation contributes to the barrier defect is shown directly by the ability of either a cholesterol-containing mixture of SC lipids or cholesterol alone to enhance barrier recovery. Finally, lipid-induced acceleration of barrier recovery in aged epidermis correlates with repletion of the extracellular spaces with normal lamellar structures. Thus, a deficiency in lipid synthesis, particularly in cholesterologenesis, accounts for the barrier abnormality in aged epidermis.

Skin type, but neither race nor gender, influence epidermal permeability barrier function.

BACKGROUND AND DESIGN: Previous studies that compared transepidermal water loss in subjects of different race and sex showed minimal differences in basal permeability barrier function. These studies often did not assess the ability of the stratum corneum to withstand or recover from insults to the epidermal permeability barrier. We compared epidermal permeability barrier function in the following human subjects (age range, 22 to 38 years): white (n = 8) vs Asian (n = 6); male (n = 7) vs female (n = 7); and skin type II/III (n = 14) vs skin type V/VI (n = 7) (scale, I to VI). Basal transepidermal water loss was measured by evaporimetry (three sites) on the volar aspect of the forearm. Barrier integrity then was assessed by determining the number of tape strippings required to reach a transepidermal water loss greater than or equal to 20 g/m2 per hour. The rates of barrier recovery then were compared at 6, 24, and 48 hours and 1 week after abrogation. RESULTS: Neither the number of tape strippings required to perturb the barrier nor the rates of barrier recovery were significantly different in white vs Asian subjects or in female vs male subjects. However, patients with skin types II/III required only 29.6 +/- 2.4 tape strippings to perturb the barrier, while the skin type V/VI group required 66.7 +/- 6.9 tape strippings. Furthermore, while barrier function in skin type II/III recovered by approximately 20% by 6 hours and 55% by 48 hours, barrier function in skin type V/VI, independent of race, recovered more quickly, 43% and 72% at 6 and 48 hours, respectively. Finally, neither the differences in barrier integrity nor in rates of recovery could be attributed to variations in cohesiveness, since stripping appears to remove the same weight of stratum corneum in both groups. CONCLUSIONS: Darkly pigmented skin displays both a more resistant barrier and one that recovers more quickly after perturbation by tape stripping than does the skin of individuals with lighter pigmentation. These findings have potential implications for transdermal delivery of topical or systemic therapeutic agents, the ability of individuals with different skin types to withstand environmental or occupational insults, and the influence of acquired hyperpigmentation or pigment loss to influence permeability.

The aged epidermal permeability barrier. Structural, functional, and lipid biochemical abnormalities in humans and a senescent murine model.

Aged epidermis displays altered drug permeability, increased susceptibility to irritant contact dermatitis, and often severe xerosis, suggesting compromise of the aged epidermal barrier. To delineate the functional, structural, and lipid biochemical basis of epidermal aging, we compared barrier function in young (20-30 yr) vs aged (> 80 yr) human subjects, and in a murine model. Baseline transepidermal water loss in both aged humans and senescent mice was subnormal. However, the aged barrier was perturbed more readily with either acetone or tape stripping (18 +/- 2 strippings vs 31 +/- 5 strippings in aged vs young human subjects, respectively). Moreover, after either acetone treatment or tape stripping, the barrier recovered more slowly in aged than in young human subjects (50 and 80% recovery at 24 and 72 h, respectively, in young subjects vs 15% recovery at 24 h in aged subjects), followed by a further delay over the next 6 d. Similar differences in barrier recovery were seen in senescent vs young mice. Although the total lipid content was decreased in the stratum corneum of aged mice (approximately 30%), the distribution of ceramides (including ceramide 1), cholesterol, and free fatty acids was unchanged. Moreover, a normal complement of esterified, very long-chain fatty acids was present. Finally, stratum corneum lamellar bilayers displayed normal substructure and dimensions, but were focally decreased in number, with decreased secretion of lamellar body contents. Thus, assessment of barrier function in aged epidermis under basal conditions is misleading, since both barrier integrity and barrier repair are markedly abnormal. These functional changes can be attributed to a global deficiency in all key stratum corneum lipids, resulting in decreased lamellar bilayers in the stratum corneum interstices. This constellation of findings may explain the increased susceptibility of intrinsically aged skin to exogenous and environmental insults.

Membrane structural abnormalities in the stratum corneum of the autosomal recessive ichthyoses.

Congenital ichthyosiform erythroderma (CIE) and classic lamellar ichthyosis (LI) are autosomal recessive disorders of cornification (DOC), distinguished previously by clinical, histologic, ultrastructural, and cell kinetic criteria. Whether there is further heterogeneity within the CIE group is uncertain. To address the issue of genetic heterogeneity, and to study the pathogenesis of these DOC, skin biopsies from eight CIE, three LI, and six normal subjects were assessed by electron microscopy, including ruthenium tetroxide postfixation with optical diffraction, to visualize and quantitate intercellular membrane domains. We found abnormal lamellar bodies in CIE and distinctive alterations in intercellular lamellar bilayer architecture among patients with CIE and three patients with LI. Two biopsies from two patients at different sites demonstrated the consistency of these findings. Moreover, in both CIE and the three LI patients, desmosomes persisted throughout the outer layers of the SC, indicative of impaired degradation. Our ultrastructural observations support the previously reported phenotypic distinction between CIE and LI, and the further likelihood of genetic heterogeneity within CIE. However, these studies do not support the division of the autosomal recessive ichthyoses into three subgroups based upon cytosolic structural abnormalities. Finally, these studies provide new insights into the pathogenesis of the autosomal recessive DOC.

Exogenous origin of n-alkanes in pathologic scale.

BACKGROUND: Although n-alkanes accumulate in some disorders of cornification, recent studies using radioactive carbon 14 content by accelerator mass spectrometry point to an exogenous origin for alkanes in normal stratum corneum, and their derivation in congenital ichthyosiform erythroderma remains controversial. DESIGN AND RESULTS: Using 14C content to measure sample age, the n-alkane fractions from two patients with congenital ichthyosiform erythroderma contained no detectable contemporary materials. By electron microscopy, alkane-enriched emollients (petrolatum [Vaseline]) permeated to all levels of stratum corneum of hairless mice, expanding the intercellular domains and distorting membrane bilayers. Similar ultrastructural changes were also observed in the stratum corneum of patients with congenital ichthyosiform erythroderma. When alkanes were excluded, no differences in lipid content were evident between two forms of autosomal recessive ichthyosis. CONCLUSIONS: These data demonstrate that scale n-alkanes in disorders of cornification derive from environmental sources and indicate the pervasiveness of petroleum-based emollients in skin. Therefore, epidermal lipid analyses must be interpreted with caution. However, these studies do not rule out an important therapeutic and/or pathogenic role for exogenous n-alkanes in skin.

Ichthyoses and hyperkeratotic disorders.

Many of the ichthyotic disorders have characteristic ocular findings. These disorders include X-linked ichthyosis, lamellar ichthyosis, Sjögren-Larsson syndrome, KID syndrome, Refsum's disease, neutral lipid storage disease, chondrodysplasia punctata, and Richner-Hanhart syndrome. A knowledge of the ocular manifestations may provide a valuable aid to diagnosis in difficult cases. In some cases, knowledge of the ocular complications results in early referral for optimal ophthalmologic care.