laser-capture-microdissection-on-surgical-tissues-to-identify

Laser Capture Microdissection on Surgical Tissues to Identify Aberrant Gene Expression in Impaired Wound Healing in Type 2 Diabetes

The global prevalence Type 2 diabetes mellitus (T2DM) is escalating at a rapid rate. Patients with T2DM suffer from a multitude of complications and one ...

University of Bradford

Pomiferin is a unique, prenylated isoflavonoid that can be isolated and purified from the fruits of Maclura pomifera (Osage Orange). The molecule typically is isolated with a small amount of a molecule called Osajin which is structurally similar to Pomiferin but lacks an aromatic hydroxyl group. As a consequence, Osajin has been shown to be a less effective antioxidant than Pomiferin. In vitro studies on Normal Human Dermal Fibroblasts demonstrate that Pomiferin is a potent extracellular matrix protein stimulant, showing increases in collagen, elastin and fibrillin expression comparable or superior to equivalent concentrations of retinol. Ex vivo hair follicle assays demonstrate comparable effects on expression of collagen and elastin at Pomiferin concentrations in the range of 0.05-5 ppm. Taken together, the results from the two assays conducted on different models indicate that Pomiferin may be a very interesting ingredient for topical skin and scalp treatments where modulation of the expression of extracellular matrix proteins is important.

In vitro and ex vivo examination of topical Pomiferin treatments.

Skin pigmentation is directed by epidermal melanin units, characterized by long-lived and dendritic epidermal melanocytes (MC) that interact with viable keratinocytes (KC) to contribute melanin to the epidermis. Previously, we reported that MC:KC contact is required for melanosome transfer that can be enhanced by filopodi, and by UVR/UVA irradiation, which can upregulate melanosome transfer via Myosin X-mediated control of MC filopodia. Both MC and KC express Ca -dependent E-cadherins. These homophilic adhesion contacts induce transient increases in intra-KC Ca , while ultraviolet radiation (UVR) raises intra-MC Ca via calcium-selective ORAI1 ion channels; both are associated with regulating melanogenesis. However, how Ca triggers melanin transfer remains unclear. Here we evaluated the role of E-cadherin in UVR-mediated melanin transfer in human skin cells. MC and KC in human epidermis variably express filopodia-associated E-cadherin, Cdc42, VASP and β-catenin, all of which were upregulated by UVR in human MC in vitro. Knockdown of E-cadherin revealed that this cadherin is essential for UVR-induced MC filopodia formation and melanin transfer. Moreover, Ca induced a dose-dependent increase in filopodia formation and melanin transfer, as well as increased β-catenin, Cdc42, Myosin X and E-cadherin expression in these skin cells. Together, these data suggest that filopodial proteins and E-cadherin, which are upregulated by intracellular (UVR-stimulated) and extracellular Ca availability, are required for filopodia formation and melanin transfer. This may open new avenues to explore how Ca signalling influences human pigmentation.

E-cadherin mediates ultraviolet radiation- and calcium-induced melanin transfer in human skin cells.

Canities (or hair greying) is an age-linked loss of the natural pigment called melanin from hair. While the specific cause(s) underlying the loss of melanogenically-active melanocytes from the anagen hair bulbs of affected human scalp remains unclear, oxidative stress sensing appears to be a key factor involved. In this study, we examined the follicular melanin unit in variably pigmented follicles from the aging human scalp of healthy individuals (22-70 years). Over 20 markers were selected within the following categories: melanocyte-specific, apoptosis, cell cycle, DNA repair/damage, senescence and oxidative stress. As expected, a reduction in melanocyte-specific markers in proportion to the extent of canities was observed. A major finding of our study was the intense and highly specific nuclear expression of Ataxia Telangiectasia Mutated (ATM) protein within melanocytes in anagen hair follicle bulbs. ATM is a serine/threonine protein kinase that is recruited and activated by DNA double-strand breaks and functions as an important sensor of reactive oxygen species (ROS) in human cells. The incidence and expression level of ATM correlated with pigmentary status in canities-affected hair follicles. Moreover, increased staining of the redox-associated markers 8-OHdG, GADD45 and GP-1 were also detected within isolated bulbar melanocytes, although this change was not clearly associated with donor age or canities extent. Surprisingly, we were unable to detect any specific change in the expression of other markers of oxidative stress, senescence or DNA damage/repair in the canities-affected melanocytes compared to surrounding bulbar keratinocytes. By contrast, several markers showed distinct expression of markers for oxidative stress and apoptosis/differentiation in the inner root sheath (IRS) as well as other parts of the hair follicle. Using our in vitro model of primary human scalp hair follicle melanocytes, we showed that ATM expression increased after incubation with the pro-oxidant hydrogen peroxide (HO). In addition, this ATM increase was prevented by pre-incubation of cells with antioxidants. The relationship between ATM and redox stress sensing was further evidenced as we observed that the inhibition of ATM expression by chemical inhibition promoted the loss of melanocyte viability induced by oxidative stress. Taken together these new findings illustrate the key role of ATM in the protection of human hair follicle melanocytes from oxidative stress/damage within the human scalp hair bulb. In conclusion, these results highlight the remarkable complexity and role of redox sensing in the status of human hair follicle growth, differentiation and pigmentation.

Stress-sensing in the human greying hair follicle: Ataxia Telangiectasia Mutated (ATM) depletion in hair bulb melanocytes in canities-prone scalp.

In women, ageing leads to reduced hair density and thinner fibers and can result in female pattern hair loss (FPHL). However, the impact of the ageing dermal environment on female scalp hair follicles (HFs) remains unclear. Here we document in situ changes in 22 women (19-81yrs), and in primary cultures of dermal fibroblast (DF) and dermal sheath (DS) cells. In situ, the papillary/reticular boundary was indistinguishable in young scalp, but prominent over 40yrs, accompanied by reduced podoplanin (PDPN) expression, increased versican (VCAN) expression, and changes in collagen organisation. HFs were shorter, not reaching the adipose layer. Hyaluronic acid synthase 2 (HAS2) was highly expressed, while matrix metalloproteinase-1 (MMP1) was elevated in the DP and DS in situ. Primary DF cultures confirmed MMP1 increased with age, while in DS cells, HAS2 and PDPN increased and α-smooth muscle actin (αSMA) decreased. Both exhibited increased cartilage oligomeric protein (COMP) expression. Proteomics revealed an increase in DS proteins in the DF secretome with age. In summary, ageing female scalp shows striking structural and biological changes in the HF environment that may impact hair growth.

Article	Year
In vitro and ex vivo examination of topical Pomiferin treatments. Fitoterapia\| PubMed ID: 24513505	2014
E-cadherin mediates ultraviolet radiation- and calcium-induced melanin transfer in human skin cells. Experimental dermatology\| PubMed ID: 28636748	2017
Stress-sensing in the human greying hair follicle: Ataxia Telangiectasia Mutated (ATM) depletion in hair bulb melanocytes in canities-prone scalp. Scientific reports\| PubMed ID: 33128003	2020
Age-related changes in female scalp dermal sheath and dermal fibroblasts: how the hair follicle environment impacts hair ageing. The Journal of investigative dermatology\| PubMed ID: 33326808	2020

Stephen K. Sikkink

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