Neutral endopeptidase and angiotensin-converting enzyme – key enzymes terminating the action of neuroendocrine mediators

Abstract:  Zinc-metalloproteases, such as neutral endopeptidase (NEP) and angiotensin-converting enzyme (ACE), effectively control the bioavailability of peptide mediators released from sensory nerves, immune and skin cells during the cutaneous response to endogenous or exogenous noxious stimuli. Functional inactivation of NEP or ACE by transient inhibition or permanent genomic deletion results in a relative abundance of substance P (SP) and bradykinin (BK); this augments murine allergic contact dermatitis (ACD) by affecting ACD sensitization and elicitation, which involves neurokinin 1 receptors (NK₁), BK receptors (B₂) and an intact cutaneous sensory nervous system. Present evidence suggests that increased SP via NK₁ is capable of boosting important functions of SP- and NK₁-expressing dendritic cells (DCs) and T cells (TCs) in an auto- or paracrine manner, which promotes ACD antigen sensitization. Moreover, skin inflammation or wounding in vivo , as well as treatment of epidermal and dermal cells by UV light and inflammatory mediators in vitro , regulates NEP and ACE expression and activity. Likewise, NEP and ACE are capable of processing neuroendocrine hormones, such as adrenocorticotropin and α-melanocyte-stimulating hormone. Thus, present data indicate that ACE and NEP, via proteolytic cleavage of peptide mediators and growth factors, represent important control factors for the inflammatory response in skin disorders such as psoriasis or allergic inflammation, but may also be capable of affecting pigmentation, cell survival, wound healing and tissue regeneration.     

Putative cancer stem cells in cutaneous malignancies

Recent experimental data offer convincing evidence for the existence of cancer stem cells in leukaemia, brain tumors and breast cancer. These cells are responsible for the maintenance of tumor growth and relapses after cytoreductive treatments. This paper provides a brief overview of current data supporting the idea of cancer stem cells in the pathogenesis of cutaneous malignancies, including skin carcinoma, malignant melanoma and cutaneous T-cell lymphoma. The characterization of putative cancer stem cells is important to develop new therapies selectively targeting these cells.     

Increased expression of an epidermal stem cell marker, cytokeratin 19, in cutaneous squamous cell carcinoma

Background Cytokeratin 19 (CK19) has been considered to be a putative marker for epidermal stem cells in the hair follicle bulge. Cumulative reports have shown that epidermal stem cells play an important role in skin carcinogenesis. However, to date there has been no report on the clinical alteration of the stem cells in squamous cell carcinoma (SCC). Objectives To investigate alteration of the stem cells and proliferating cells and to assess their relationship and potential contribution to SCC. Methods Thirty paraffin‐embedded neoplastic skin lesions, consisting of 10 cases each of actinic keratosis (AK), Bowen disease (BD) and SCC, were examined immunohistologically for CK19 and Ki‐67. Results Positive reactivity for CK19 was seen in 30% of AK, 50% of BD and 80% of SCC lesions. There was significantly higher expression levels of CK19 in SCC than in AK and BD (P < 0·05). In addition, BD lesions harboured a significantly higher number of CK19‐positive cells than did AK lesions (P < 0·05). There were significant differences in Ki‐67 labelling indices between AK and BD and between AK and SCC (P < 0·001), but not between BD and SCC (P > 0·05). Furthermore, a serial section comparison study showed that there was a minor population of cells co‐expressing CK19 and Ki‐67 in a subset of the tumour cells of SCC samples. The percentage of CK19+ cells significantly correlated with that of Ki67+ cells in all examined neoplastic skin lesions. Conclusions These results suggest that CK19 expression may be associated with the retention of stem cell characteristics or a state that is uncommitted to terminal squamous differentiation.     

Differentiation potential of human embryonic mesenchymal stem cells for skin-related tissue

BACKGROUND: Mesenchymal stem cells (MSC) have the capacity to differentiate into cells of connective tissue lineages, including bone, fat, cartilage and muscle, but the differentiation of embryonic MSC into epidermal cells by mesenchymal-epithelial transition has not been confirmed. OBJECTIVES: To determine the biological characteristics of human embryonic MSC (hMSC) and their potential for differentiation into epithelial cells. METHODS: hMSC were derived from 4-7-week-old embryos; they were localized and isolated, then primary culture was done. The biological characteristics of hMSC were detected by immunohistochemical methods and flow cytometry. Their differentiation potential was determined by coculture with conditioning medium and in vivo injection. RESULTS: hMSC express the relative specific antigens of MSC, such as SH2, alpha-smooth actin, CD29, CD44, CD90 and S100. After stimulation and in vivo transplantation, hMSC possess the potential to differentiate into epidermal cells with the production of keratin 19 and E-cadherin. CONCLUSIONS: hMSC derived from the early human embryo have the ability to transform into epidermal cells in vivo and in vitro.     

Human skin cancer stem cells: a tale of mice and men

Carcinomas, cancers of epithelial tissues, are the commonest malignancies and cause the greatest cancer mortality worldwide. Among these, the incidence of keratinocyte-derived non-melanoma skin cancers (NMSC), by far the greatest, is increasing rapidly. Yet despite access to tumor tissue, acceptance of human NMSC as a model carcinoma has been hindered by the lack of a reliable xenograft model. Instead, we have relied on the murine two-step carcinogenesis protocol as a reproducible squamous cell carcinoma (SCC) model, but this differs from their human counterpart in cause, site, genetic basis and biological behaviour. By xeno-engraftment of primary human SCC, we were recently successful in demonstrating the presence of primary human SCC cancer stem cells or tumor-initiating cells. These findings once more align the study human SCC as the archetypal carcinoma model. In this review, we describe the evidence for the existence of tumor-initiating cells, with emphasis on skin cancer, limiting our discussions to primary human cancer studies where possible.     

Diaminobenzidine cytochemistry in unfixed human epidermis: a marker for epidermal differentiation and for mitochondria

Intubation of unfixed and unfrozen slices of skin in diaminobenzidine allows visualization of a peroxidatic activity in perinuclear envelope of suprabasal keratinocytes undergoing orthokeratotic differentiation. Basal keratinocytes and melanocytes are always negative. This enzyme is absent in mucous and parakeratotic (psoriatic) differentiation. Mitochondria are also strongly stained by this technique and it was shown that the number of epidermal mitochondria is greatly increased in psoriatic lesions.     

Association between the insertion/deletion polymorphism of the angiotensin I-converting enzyme gene and risk for psoriasis in a Chinese population in Taiwan

BACKGROUND: Genetic factors play an important role in susceptibility for psoriasis. The angiotensin I-converting enzyme (ACE) is expressed by keratinocytes. Administration of ACE inhibitors may induce or exacerbate psoriasis in clinical practice. Thus, ACE gene variants may contribute to the genetic background of psoriasis. OBJECTIVES: To assess the role of the ACE insertion/deletion (I/D) polymorphism in psoriasis among ethnically Chinese Taiwanese subjects. METHODS: In total, 312 patients with psoriasis and 615 control subjects were analysed for the ACE I/D polymorphism by polymerase chain reaction. RESULTS: A marginally significant difference (P=0 x 035) was found in the distribution of ACE I/D genotype frequencies between patients with psoriasis and controls. The frequency of the II genotype in patients with psoriasis was significantly higher than that in the control group (55 x 1% vs. 46 x 7%, respectively, P=0 x 015). Although the I allele frequency in patients with psoriasis (72 x 4%) was higher than that in the control group (68 x 2%), the difference was not significantly different (P=0 x 062). After adjusting for age and gender, carriers of the II genotype were 1 x 45 (95% confidence interval 1 x 09-1 x 92) times more likely than noncarriers to have psoriasis (P=0 x 010). CONCLUSIONS: Our results suggest that the presence of the I allele may confer susceptibility to development of psoriasis among ethnically Chinese Taiwanese individuals.     

Damage at the root of cell renewal--UV sensitivity of human epidermal stem cells

Background

The epidermis harbors adult stem cells that reside in the basal layer and ensure the continuous maintenance of tissue homeostasis. Various studies imply that stem cells generally possess specific defense mechanisms against several forms of exogenous stress factors. As sun exposition is the most prevalent impact on human skin, this feature would be of particular importance in terms of sensitivity to UV-induced DNA damage.

Objective

To investigate whether human epidermal stem cells are susceptible to UV-induced DNA damage and subsequent functional impairment.

Methods

A method to isolate human epidermal stem cells from suction blister epidermis was established and validated. Volunteers were treated with solar-simulated irradiation on test areas of the forearm and stem cells were isolated from suction blister material of this region. DNA damage was analyzed by staining for cyclobutane thymidine dimers. The functional consequences of UV-induced damages were assessed by colony forming efficiency assays and gene expression analyses.

Results

Compared to an unirradiated control, stem cells isolated from areas that were exposed to solar-simulated radiation showed significantly more DNA lesions. Although the number of stem cells was not reduced by this treatment, a functional impairment of stem cells could be shown by reduced colony forming efficiency and altered gene expression of stem cell markers.

Conclusions

Despite their essential role in skin maintenance, epidermal stem cells are sensitive to physiological doses of UV irradiation in vivo.     

Isolating a pure population of epidermal stem cells for use in tissue engineering

Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and non-proliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.     

Onycholemmal variant of keratoacanthoma centrifugum marginatum as an expression of mutated committed stem cells in a conceptual pathway

We describe a 43-year-old woman with a 10-year history of grossly hyperkeratotic nodules which progressively extended over the right ring finger. These involuted leaving pale, atrophic skin in their wake. At presentation, the advancing border had an arciform series of nodules in the pattern of keratoacanthoma centrifugum marginatum. The presence of filiform keratinisation that encased the nail plate, gross onychogryphotic masses of keratin on the ventral finger surface and a flat nail-like plate of keratin on the dorsal finger surface were distinctive features. Skin biopsy showed epidermal acanthosis, gross papillomatous cutaneous horn formation that had onycholemmal features. The pathology differed from keratoacanthoma and was not crateriform or infundibulocystic. Although HPV was not detected on immunohistochemistry, pathogenesis may still represent an HPV-related transfection of onycholemmal keratin committed stem cells producing an onycholemmal variant of keratoacanthoma centrifugum marginatum. A conceptual model linked to advances in follicular stem cell biology is formulated to explore this case.     

Suprabasal expression of human amphiregulin in the epidermis of transgenic mice induces a severe, early-onset, psoriasis-like skin pathology: expression of amphiregulin in the basal epidermis is also associated with synovitis

The expression of amphiregulin (AR) in the basal epidermis of transgenic mice [keratin 14 promoter AR gene (K14-ARGE)] has been previously shown to induce an early-onset and severe skin pathology, with many similarities to psoriasis. In this study, it is demonstrated that involucrin enhancer/promoter-dependent expression of human AR (INV-AR) in the suprabasal epidermis of transgenic mice also produces a cutaneous psoriasis-like phenotype. INV-AR mice possess a limited lifespan and scaling, papillomatous, erythematous skin with partial alopecia. INV-AR mouse histopathology also revealed epidermal hyperkeratosis, parakeratosis, acanthosis, and an exaggerated dermal vasculature. A dermal and epidermal infiltrate was also evident and consisted of both neutrophils and CD3(+) T lymphocytes. The histology of synovial joints in both the INV-AR mice and the K14-ARGE mice of our previous investigation was examined. The histologic examination revealed that 3-week-old INV-AR transgenic mice displayed normal knee joint histology, while 2- to 3-week-old K14-ARGE transgenic mice frequently displayed synovitis, as exemplified by the presence of a mixed leukocytic infiltration, increased vascularization, and enhanced deposition of fibrous matrix in the knee synovium. These results demonstrate that AR overexpression in both the basal and suprabasal epidermis of transgenic mice induces a phenotype that mimics cutaneous psoriasis, while basal AR expression is also associated with synovial inflammation, a precursor to the psoriasis-associated arthropathy, psoriatic arthritis. Collectively, the results implicate epidermal AR expression as a possible mediator of innate cutaneous immunity and epidermal proliferation and also as a potential trigger of both cutaneous psoriasis and psoriatic arthritis.     

Association study between keratinocyte-derived growth factor gene polymorphisms and susceptibility to vitiligo vulgaris in a Taiwanese population: potential involvement of stem cell factor

Background  Vitiligo vulgaris is a depigmentary disorder resulting from the disappearance of functional melanocytes. Currently, the pathogenesis of this disorder remains obscure.
Objectives  Genetic analysis of patients with vitilgo may provide important clues for elucidating the complex pathomechanisms involved in the disease process. Because dysfunctional keratinocytes have recently been implicated in the pathogenesis of vitiligo vulgaris, we conducted a case–control association study to investigate this phenomenon.
Patients and methods  Fifty-one patients with vitiligo vulgaris and 118 healthy controls from Taiwan were recruited to investigate the association between relevant keratinocyte-related genes and the occurrence of vitiligo vulgaris. This study genotyped 11 single-nucleotide polymorphisms (SNPs) in five genes including stem cell factor ( SCF , also known as KITLG ), basic fibroblast growth factor ( bFGF , also known as NuDT6 ), endothelin-1 ( EDN1 ), hepatocyte growth factor ( HGF ) and stem cell growth factor ( SCGF , also known as CLEC11A ).
Results  Our results revealed that the A allele for SNP rs11104947 in the SCF gene and the T allele for SNP rs13866 in the SCGF gene were, respectively, associated with a 1·95- and a 2·14-fold risk of developing vitiligo vulgaris. A higher risk was also detected among subjects who carried the SCF rs995029/rs11104947 C/A haplotype (odds ratio = 2·45). Furthermore, the at-risk alleles for SCF rs11104947 (A allele) and for SCGF SNP rs13866 (T allele) were found to display a 7·92-fold increased gene–gene combined risk. No significant relationship between polymorphic frequency for genes bFGF , EDN1 as well as HGF and occurrence of vitiligo vulgaris was observed.
Conclusions  These novel genetic findings provide new insights in relation to the mechanisms that might be involved in the development of vitiligo vulgaris.     

Characterization of the expression and activation of the epidermal growth factor receptor in squamous cell carcinoma of the skin

BACKGROUND: Locally advanced skin cancers including squamous cell carcinoma (SCC) of the skin are increasing in incidence. Patients are often elderly with significant comorbidities and therapy can be difficult. New targeted therapies, such as treatment directed at the epidermal growth factor receptor (EGFR), may be effective and less toxic in these patients. However, before designing appropriate clinical trials it is necessary to characterize the expression and activation of targets such as the EGFR to evaluate the rationale of using EGFR inhibitors (EGFRIs) in the treatment of this type of cancer. OBJECTIVES: To characterize the expression and activation by phosphorylation of EGFR in SCC of the skin by quantitative Western blotting using the LiCor immunofluorescence detection system with validation by immunohistochemistry. Secondary objectives were to evaluate downstream targets of EGFR expression and activation in SCC of the skin and to examine the associations between EGFR, pathological features and clinical behaviour of these tumours. METHODS: Twenty-one mainly locally advanced skin SCCs collected in our institution and stored in our tissue bank over a 4-year period were used for the study. RESULTS: Nine of 21 (43%) tumours expressed EGFR above background. Of those nine, five expressed phosphorylated EGFR. There was no correlation with downstream activation of canonical signalling pathways, pathological features or clinical behaviour. CONCLUSIONS: EGFR is expressed in a minority of tumours and then is not always activated. These results show that, before designing a trial with a targeted agent such as an EGFRI in SCC of the skin, it is important to verify the presence of the appropriate target to maximize the best outcome.     

GP37 expression in normal and diseased human epidermis: a marker for keratinocyte differentiation

An antiserum prepared against a glycoprotein (GP37) extracted from the upper epidermal layers, was used to stain frozen sections of human oral mucosa, normal and abnormal skin by an indirect immunofluorescence technique. On normal human epidermis, this antiserum mainly reacted with the cytoplasm of granular cells, whereas on buccal mucosa the recognized antigen was observed as scattered dots limited to the upper epithelial layers. In epidermal diseases, alterations in the staining pattern were observed. In psoriasis, the labelling was markedly diminished; in contrast, in lichen planus it was intense and present on the 3-6 uppermost cellular layers. Basal cell epitheliomas were almost negative, except around horn cysts. In Bowen's disease dyskeratotic cells were strongly labelled. In squamous cell carcinomas a clear-cut staining was observed in squamous nests. On cultures, GP37 expression could be induced by growing epidermal cells in vitamin A-depleted medium. The biological significance of the observed staining patterns remains to be precised. Nevertheless, GP37 represents a sensitive marker of epidermal differentiation and may be useful in skin pathology and in in vitro studies.     

Characterization of neurons from adult human skin-derived precursors in serum-free medium : a PCR array and immunocytological analysis

Abstract: Adult stem cells could be small sources of neurons or other cellular types for regenerative medicine and tissue engineering. Recently, pluripotent stem cells have been extracted from skin tissue, which opened a new accessible source for research. To routinely obtain a high yield of functional neurons from adult human skin stem cells with defined serum‐free medium, stem cells from abdominal skin were cultured in serum‐free medium. To differentiate them, we used a defined medium containing growth factors. Differentiated cells were identified using the following methods: (i) Oil‐red‐O staining for adipocytes, immunocytochemistry with antibodies recognising (ii) neurofilaments and PGP9.5 for neural differentiation, (iii) glial fibrillary acidic protein (GFAP) for glial differentiation, (iv) Ki‐67 for proliferative cells, (v) FM1‐43 staining to analyse vesicle trafficking in neuronal cells and (vi) a PCR array was used. Stem cells were floating in spheres and were maintained in culture for 4 months or more. They expressed nestin and Oct 4 and were proliferative. We induced specific differentiation into adipocytes, glial and neuronal cells. The yields of differentiated neurons were high and reproducible. They were maintained for long time (1 month) in the culture medium. Furthermore, these neurons incorporated FM1‐43 dye, which indicates a potent acquisition of synaptic features in neurons. Stem cells from adult human skin could be valuable and reproducible tools/source to obtain high numbers of functional specific cellular types, such as neurons, for tissue engineering. In this work, the possibility to obtain a high yield of differentiated neurons, with the ability of endocytosis and vesicle cell trafficking, was shown.     

皮肤间充质干细胞在促进皮肤愈合中的作用

间充质干细胞(mesenchymal stem cells,MSCs)是当前干细胞研究的热点之一。目前,皮肤愈合正逐渐受到重视。现有的研究认为骨髓间充质干细胞(BM-MSCs)能从多个方面促进皮肤愈合,如促进表皮生长、促进真皮成纤维细胞的增生等。皮肤间充质干细胞(SMSCs)和BM-MSCs均为MSCs,具有很多的相似性,且SMSCs较BM-MSCs更容易得到。所以可从目前对BM-MSCs的研究预测到SMSCs在皮肤创伤愈合中的研究前景,且将来很可能会替代BM-MSCs。