Fibroblast heterogeneity and its implications for engineering organotypic skin models in vitro

AbstractAdvances in cell culture methods, multidisciplinary research, clinical need to replace lost skin tissues and regulatory need to replace animal models with alternative test methods has led to development of three dimensional models of human skin. In general, these in vitro models of skin consist of keratinocytes cultured over fibroblast-populated dermal matrices. Accumulating evidences indicate that mesenchyme-derived signals are essential for epidermal morphogenesis, homeostasis and differentiation. Various studies show that fibroblasts isolated from different tissues in the body are dynamic in nature and are morphologically and functionally heterogeneous subpopulations. Further, these differences seem to be dictated by the local biological and physical microenvironment the fibroblasts reside resulting in “positional identity or memory”. Furthermore, the heterogeneity among the fibroblasts play a critical role in scarless wound healing and complete restoration of native tissue architecture in fetus and oral mucosa; and excessive scar formation in diseased states like keloids and hypertrophic scars. In this review, we summarize current concepts about the heterogeneity among fibroblasts and their role in various wound healing environments. Further, we contemplate how the insights on fibroblast heterogeneity could be applied for the development of next generation organotypic skin models

Opioids and the skin--where do we stand?

International audienceThe common ectodermal origin of the skin and nervous systems can be expected to predict likely interactions in the adult. Over the last couple of decades much progress has been made to elucidate the nature of these interactions, which provide multidirectional controls between the centrally located brain and the peripherally located skin and immune system. The opioid system is an excellent example of such an interaction and there is growing evidence that opioid receptors (OR) and their endogenous opioid agonists are functional in different skin structures, including peripheral nerve fibres, keratinocytes, melanocytes, hair follicles and immune cells. Greater knowledge of these skin-associated opioid interactions will be important for the treatment of chronic and acute pain and pruritus. Topical treatment of the skin with opioid ligands is particularly attractive as they are active with few side effects, especially if they cannot cross the blood-brain barrier. Moreover, cutaneous activation of the opioid system (e.g. by peripheral nerves, cutaneous and immune cells, especially in inflamed and damaged skin) can influence cell differentiation and apoptosis, and thus may be important for the repair of damaged skin. While many of the pieces of this intriguing puzzle remain to be found, we attempt in this review to weave a thread around available data to discuss how the peripheral opioid system may impact on different key players in skin physiology and pathology

The Role of Skin Opioid Receptor System in Itch

Opioid receptors are key players in induction of chronic itch. This could be confirmed using opiate receptor knockout mice experiments and clinical studies on patients with chronic itch. We have induced a dry skin dermatitis as a model for chronic itching on -(MOR) and -(KOR) opioid receptor knockout (KO) mice. MOR KO mice scratched significantly less than wild type (WT). Additionally the epidermal hypertrophy caused by chronic dermatitis and the amount of epidermal nerve endings in MOR KO mice were significantly decreased than in WT mice. KOR KO mice showed similar scratching behavior as MOR KO mice; however the changes were less significant. In addition, we performed a double blind, placebo controlled, cross over study using topically applied opioid receptor antagonist, Naltrexone, on patients with pruritus in atopic dermatitis. The results revealed significant effects of the topical application of Naltrexone in patients with chronic pruritus (45% improvement of pruritus by VAS compared to placebo, n=24), but not in patients with acute pruritus (7%, n=15). These studies establish the clinical relevance of MOR system and the peripheral, epidermal nerve endings in chronic pruritus and warrant further research and therapeutic potential for such research

Specific Targeting of Melanotic Cells with Peptide Ligated Photosensitizers for Photodynamic Therapy

A strategy combining covalent conjugation of photosensitizers to a peptide ligand directed to the melanocortin 1 (MC1) receptor with the application of sequential LED light dosage at near-IR wavelengths was developed to achieve specific cytotoxicity to melanocytes and melanoma (MEL) with minimal collateral damage to surrounding cells such as keratinocytes (KER). The specific killing of melanotic cells by targeted photodynamic therapy (PDT) described in this study holds promise as a potentially effective adjuvant therapeutic method to control benign skin hyperpigmentation or superficial melanotic malignancy such as Lentigo Maligna Melanoma (LMM).ISSN:2045-232

Microbiome in the hair follicle of androgenetic alopecia patients.

Androgenetic alopecia is the most common form of hair loss in males. It is a multifactorial condition involving genetic predisposition and hormonal changes. The role of microflora during hair loss remains to be understood. We therefore analyzed the microbiome of hair follicles from hair loss patients and the healthy. Hair follicles were extracted from occipital and vertex region of hair loss patients and healthy volunteers and further dissected into middle and lower compartments. The microbiome was then characterized by 16S rRNA sequencing. Distinct microbial population were found in the middle and lower compartment of hair follicles. Middle hair compartment was predominated by Burkholderia spp. and less diverse; while higher bacterial diversity was observed in the lower hair portion. Occipital and vertex hair follicles did not show significant differences. In hair loss patients, miniaturized vertex hair houses elevated Propionibacterium acnes in the middle and lower compartments while non-miniaturized hair of other regions were comparable to the healthy. Increased abundance of P. acnes in miniaturized hair follicles could be associated to elevated immune response gene expression in the hair follicle

Investigating endogenous µ-opioid receptors in human keratinocytes as pharmacological targets using novel fluorescent ligand

<div><p>Opioids in skin function during stress response, regeneration, ageing and, particularly in regulating sensation. In chronic pruritus, topical treatment with Naltrexone changes μ-opioid receptor (μ-OR) localization to relieve itch. The molecular mechanisms behind the effects of Naltrexone on μ-OR function in reduction of itching behavior has not been studied. There is an immediate need to understand the endogenous complexity of μ-OR dynamics in normal and pathological skin conditions. Here we evaluate real-time behavior of μ-OR-Endomorphine complexes in the presence of agonist and antagonists. The μ-OR ligand Endomorphine-1 (EM) was conjugated to the fluorescent dye Tetramethylrhodamine (TAMRA) to investigate the effects of agonist and antagonists in N/TERT-1 keratinocytes. The cellular localization of the EM-TAMRA was followed through time resolved confocal microscopy and population analysis was performed by flow cytometry. The <i>in vitro</i> analyses demonstrate fast internalization and trafficking of the endogenous EM-TAMRA-μ-OR interactions in a qualitative manner. Competition with Endomorphine-1, Naltrexone and CTOP show both canonical and non-canonical effects in basal and differentiated keratinocytes. Acute and chronic treatment with Naltrexone and Endomorphine-1 increases EM-TAMRA binding to skin cells. Although Naltrexone is clinically effective in relieving itch, the mechanisms behind re-distribution of μ-ORs during clinical treatments are not known. Our study has given insight into cellular mechanisms of μ-OR ligand-receptor interactions after opioid agonist and antagonist treatments <i>in vitro</i>. These findings potentially offer opportunities in using novel treatment strategies for skin and peripheral sensory disorders.</p></div

HPLC parameters.

<p>HPLC parameters.</p

Competition with μ-OR ligands shows specificity of EM-TAMRA binding and differences between basal and differentiated keratinocyte populations.

<p>(A) For live cell imaging, cells were pre-incubated with 10 μM competitor for five min before addition of EM-TAMRA (200 nM) in the presence of 10 μM competitor at 37°C. As compared to the control, Endomorphine-1 was more effective in competing for membrane binding with EM-TAMRA than CTOP and Naltrexone in basal N/TERT-1 cells. Scale bar represents 10 μm. (B) Flow cytometry analysis after 30 min pre-treatment with ligands on ice show significant reduction in EM-TAMRA positive populations during competition with Endomorphine-1 at low (100 nM) as well as high (10 μM) concentrations in both basal and differentiated keratinocytes. (C) CTOP (100 nM and 1 μM) was able to block EM-TAMRA binding in basal N/TERT-1 but not in differentiated cells. (D) Naltrexone shows competition at higher concentrations of 1 μM to 10 μM but not at low concentration of 100 nM. Data are represented as mean ± SD from four replicate experiments (N = 4) and were subjected to ordinary One-way ANOVA using Dunnett’s multiple comparison <i>post hoc</i> test. * P < 0.05; ** P < 0.01; *** P < 0.001. EM = Endomorphine-1; NTX = Naltrexone.</p

Chemical characterization of Endomorphine-TAMRA conjugates.

<p><b>(A)</b> Chemical structure of EM-TAMRA indicated by modified Endomorphine-1 peptide in blue and TAMRA-Maleimide in magenta. Absorbance and Emission spectra of <b>(B)</b> 200 μM TAMRA-Maleimide and <b>(C)</b> 20 μM EM-TAMRA at pH 7.24. <b>(D)</b> Absorbance of 20 μM EM-TAMRA at different pH 2.15–10. I_Normalized = Normalized intensity; I_abs = Normalized absorbance.</p