19 research outputs found
Compartmentalised metabolic programmes in human anagen hair follicles: New targets to modulate epithelial stem cell behaviour, keratinocyte proliferation and hair follicle immune status?
No full textHuman scalp hair follicles (HF) preferentially engage in glycolysis followed by lactate production in the presence of oxygen (i.e. the Warburg effect). Through the spatiotemporally controlled expression of key metabolic proteins, we hypothesise that the Warburg effect and other HF metabolic programmes are compartmentalised by region in order to regulate regional cell fate and phenotypes, such as epithelial stem cell quiescence in the bulge or keratinocyte proliferation in the hair matrix. We further propose that metabolic conditions in the HF are organised in accordance with the lactate shuttle, hypothesised to occur in other tissue systems and tumours, but never before described in the HF. Specifically, we argue that lactate, produced and exported by glycolytic GLUT1+ lower outer root sheath (ORS) keratinocytes. We further propose that lactate is then utilised symbiotically by neighbouring highly proliferative matrix keratinocytes to fuel oxidative metabolism via MCT1-mediated uptake. Furthermore, as lactate has been described to be immunomodulatory, its production and accumulation could enhance immune tolerance in the HF bulb. Here we delineate how to experimentally probe this hypothesis, define major open questions and present preliminary immunohistological evidence in support of metabolic compartmentalisation and lactate shuttling. Overall, we argue that basic and translational hair research needs to rediscover the importance of lactate in human HF biology, well beyond its recognised role in murine HF epithelial stem cells, and should explore how HF metabolism can be therapeutically targeted to modulate hair growth and the immunological HF microenvironment as a novel strategy for managing hair loss disorders.</p
Visualization of global RNA synthesis in a human (mini-) organ in situ by click chemistry
No full textRNA synthesis can be detected by 5-ethynyl uridine (EU) incorporation and click chemistry. Despite identifying a fundamental functional process, this technique has yet to be widely applied to complex human tissue systems. By incorporating EU into human hair follicle (HF) organs cultured ex vivo, nascent RNA synthesis was detected in situ. EU differentially incorporated across the HF epithelium. Interestingly, RNA synthesis did not correlate with protein synthesis, proliferation or epithelial progenitor cell marker expression. By treating human HFs with the cytotoxic cell cycle inhibitor (R)-CR8, which inhibits transcriptional regulators CDK7 and CDK9, it was further shown that this technique can be used to sensitively detect changes in global RNA synthesis in situ. Together, this work delineates new insights into nascent RNA synthesis within a human (mini)- organ and describes a novel read-out parameter that will enrich future ex vivo human tissue research studies.</p
A Primer for Studying Cell Cycle Dynamics of the Human Hair Follicle
Get PDFThe cell cycle is of major importance to human hair follicle (HF) biology. Not only is continuously active cell cycling required to facilitate healthy hair growth in anagen VI HFs, but perturbations in the cell cycle are likely to be of significance in HF pathology (i.e. in scarring, non-scarring, chemotherapy-induced and androgenic alopecias). However, cell cycle dynamics of the human hair follicle (HF) are poorly understood in contrast to what is known in mouse. The current Methods Review aims at helping to close this gap by presenting a primer that introduces immunohistological/immunofluorescent techniques to study the cell cycle in the human HF. Moreover, this primer encourages the exploitation of the human HF as a powerful and clinically relevant tool to investigate mammalian cell cycle biology in situ. To achieve this, we describe methods to study markers of general ‘proliferation’ (nuclei count, Ki-67 expression), apoptosis (terminal deoxynucleotidyl transferase dUTP nick-end labelling, cleaved caspase 3), mitosis (phospho-histone H3, ‘pS780’), DNA synthesis (5-ethynyl-2′-deoxyuridine) and cell cycle regulation (cyclins) in the human HF. In addition, we provide specific examples of dual immunolabelling for instructive cell cycle analyses and for investigating the cell cycle behaviour of specific HF keratinocyte subpopulations, such as keratin 15+ stem/progenitor cells
XMU-MP-1 induces growth arrest in a model human mini-organ and antagonises cell cycle-dependent paclitaxel cytotoxicity
Get PDFBackground: XMU-MP-1 is an inhibitor of the Hippo pathway kinases MST1/2 and has been shown to promote the downstream activation of the pro-proliferative, pro-regenerative and anti-apoptotic transcriptional regulator YAP1. We tested whether XMU-MP-1 can activate YAP1 in a model human mini-organ, namely the hair follicle, to determine whether it can be pharmacologically exploited to promote regeneration in the hair follicle as a novel strategy to treat pathological hair loss disorders.Results: XMU-MP-1 treatment inhibited MOB1 phosphorylation but did not increase active YAP1 in the hair follicle. Rather than promote proliferation, XMU-MP-1 serendipitously decreased the number of Ki-67+, EdU+ and phospho histone H3+ hair matrix keratinocytes and antagonised the cytotoxic effects of paclitaxel.Conclusions: XMU-MP-1 perturbs epithelial cell cycle progression in a model human mini-organ. This may arise as an off-target effect, especially when XMU-MP-1 has been described to strongly inhibit 21 additional kinases beyond MST1/2. Therefore, whilst these effects may be dependent on tissue context, researchers should exercise caution when interpreting the effects of XMU-MP-1, especially in tissues with actively proliferating cell populations.</p
Divergent proliferation patterns of distinct human hair follicle epithelial progenitor niches in situ and their differential responsiveness to prostaglandin D2
No full textHuman scalp hair follicles (hHF) harbour several epithelial stem (eHFSC) and progenitor cell sub-populations organised into spatially distinct niches. However, the constitutive cell cycle activity of these niches remains to be characterized in situ. Therefore, the current study has studied these characteristics of keratin 15+ (K15), CD200+ or CD34+ cells within anagen VI hHFs by immunohistomorphometry, using Ki-67 and 5-ethynyl-2'-deoxyuridine (EdU). We quantitatively demonstrate in situ the relative cell cycle inactivity of the CD200+/K15+ bulge compared to other non-bulge CD34+ and K15+ progenitor compartments and found that in each recognized eHFSC/progenitor niche, proliferation associates negatively with eHFSC-marker expression. Furthermore, we also show how prostaglandin D2 (PGD2), upregulated in balding scalp, differentially impacts on the proliferation of distinct eHFSC populations. Namely, 24h organ-cultured hHFs treated with PGD2 resulted in reduced Ki-67 expression and EdU incorporation in bulge resident K15+ cells, but not in supra/proximal bulb outer root sheath K15+ progenitors. This study emphasises clear differences between the cell cycle behaviours of spatially distinct stem/progenitor cell niches in the hHF, and demonstrates a possible link between PGD2 and perturbed proliferation dynamics in epithelial stem cells
Human epithelial stem cell survival within their niche requires "tonic" cannabinoid receptor 1-signalling-Lessons from the hair follicle
Get PDFThe endocannabinoid system (ECS) regulates multiple aspects of human epithelial physiology, including inhibition/stimulation of keratinocyte proliferation/apoptosis, respectively. Yet, how the ECS impacts on human adult epithelial stem cell (eSC) functions remains unknown. Scalp hair follicles (HFs) offer a clinically relevant, prototypic model system for studying this directly within the native human stem cell niche. Here, we show in organ-cultured human HFs that, unexpectedly, selective activation of cannabinoid receptor-1 (CB1)-mediated signalling via the MAPK (MEK/Erk 1/2) and Akt pathways significantly increases the number and proliferation of cytokeratin CK15+ or CK19+ human HF bulge eSCs in situ, and enhances CK15 promoter activity in situ. In striking contrast, CB1-stimulation promotes apoptosis in the differentiated progeny of these eSCs (CK6+ HF keratinocytes). Instead, intrafollicular CB1 gene knockdown or CB1 antagonist treatment significantly reduces human HF eSCs numbers and stimulates their apoptosis, while CB1 knockout mice exhibit a reduced bulge eSCs pool in vivo. This identifies "tonic" CB1 signalling as a required survival stimulus for adult human HF eSCs within their niche. This novel concept must be taken into account whenever the human ECS is targeted therapeutically.</p
Activation of the integrated stress response in human hair follicles.
No full textUnravelling how energy metabolism and stress responses are regulated in human scalp hair follicles could reveal novel insights into the controls of hair growth and provide new targets to manage hair loss disorders. The Mitochondrial Pyruvate Carrier (MPC) imports pyruvate, produced via glycolysis, into the mitochondria, fuelling the TCA cycle. Previous work has shown that MPC inhibition promotes lactate generation, which activates murine epithelial hair follicle stem cells (eHFSCs). However, by pharmacologically targeting the MPC in short-term human hair follicle ex vivo organ culture experiments using UK-5099, we induced metabolic stress-responsive proliferative arrest throughout the human hair follicle epithelium, including within Keratin 15+ eHFSCs. Through transcriptomics, MPC inhibition was shown to promote a gene expression signature indicative of disrupted FGF, IGF, TGFβ and WNT signalling, mitochondrial dysfunction, and activation of the integrated stress response (ISR), which can arrest cell cycle progression. The ISR, mediated by the transcription factor ATF4, is activated by stressors including amino acid deprivation and ER stress, consistent with MPC inhibition within our model. Using RNAScope, we confirmed the upregulation of both ATF4 and the highly upregulated ATF4-target gene ADM2 on human hair follicle tissue sections in situ. Moreover, treatment with the ISR inhibitor ISRIB attenuated both the upregulation of ADM2 and the proliferative block imposed via MPC inhibition. Together, this work reveals how the human hair follicle, as a complex and metabolically active human tissue system, can dynamically adapt to metabolic stress
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Does dysfunctional autophagy contribute to immune privilege collapse and alopecia areata pathogenesis?
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Dandruff lesional scalp skin exhibits epidermal T cell infiltration and a weakened hair follicle immune privilege
No full textDandruff is characterised by the presence of perivascular leukocytes and mild inflammation; however, the immune microenvironment of dandruff-affected scalp skin and the potential changes to the hair follicle's (HF) physiological immune privilege (HF IP) remain unknown. Here, we characterised the HF immune microenvironment and immune privilege status in dandruff-affected scalp skin.
We assessed relevant key parameters in healthy versus dandruff-affected human scalp biopsies using quantitative immunohistomorphometry, laser capture microdissection, and RNA sequencing.
The number of epidermal CD4
and CD8
T cells was increased in lesional dandruff scalp skin, while the number of MHC class II
/CD1a
Langerhans cells was decreased in the infundibulum. The number of intrafollicular and perifollicular CD4
T cells and CD8
T cells, perifollicular CD68
macrophages, and tryptase
mast cells remained unchanged. Interestingly, MHC class Ia and ß2-microglobulin protein expression were significantly increased specifically in the suprabulbar outer root sheath (ORS) compartment of dandruff-associated HFs. RNAseq analysis of laser capture micro-dissected suprabulbar ORS compartment revealed antigen presentation pathway as the top regulated canonical pathway, along with the upregulation of HF-IP genes such as HLA-C, HLA-DP, and TAP1, which are normally down-regulated in healthy HFs. Intrafollicular protein expression of known HF IP guardians (CD200 and α-MSH) and 'danger signals' (MICA and CXCL10) remained unaltered at the IP sites of dandruff lesional HFs compared to non-lesional and healthy HFs. Instead, the expression of macrophage migration inhibiting factor (MIF), another HF IP guardian, was reduced.
Together, this work shows that dandruff is associated with epidermal T-cell infiltration and a weakened HF IP in the suprabulbar ORS of HFs in dandruff lesional scalp
Vascular endothelial growth factor blockade induces dermal endothelial cell apoptosis in a clinically relevant skin organ culture model
Get PDFBackground/Aims: Vascular endothelial growth factor (VEGF), a key mediator of angiogenesis, plays a key role in physiological processes and is a major contributor to several diseases including cancer and psoriasis. Anti-VEGF therapies are widely used as cancer and ophthalmological treatments. There is some evidence that VEGF blockade may have utility in the management of psoriasis, although their potential has been largely unexplored. We hypothesised that a human skin organ culture could provide a stable ex vivo model in which the cutaneous microvascular network could be studied and experimentally manipulated. Methods: Punch biopsies (3 mm) of skin, donated by healthy individuals (39-72 years old, n=5) were incubated with monoclonal antibody (mAb) to human VEGF (bevacizumab) at doses based on data from animal and clinical studies. After three day culture, cell death and proliferation as well as vascular endothelial cell changes were assessed using quantitative immunohistomorphometry. Results: Anti-VEGF mAb at 0.8 mg/mL induced a significant increase in cleaved caspase-3 expression in CD31+ cells (p<0.05). None of the doses tested increased TUNEL or decreased Ki-67 expression in the basal layer of the epidermis, confirming the model’s viability. In addition, the lactate dehydrogenase (LDH) assay showed no increase in LDH activity in treated samples compared to untreated control. The highest anti-VEGF mAb dose (0.8 mg/mL) induced an increase in TUNEL expression in the upper epidermis, which did not correlate with caspase-3 immunoreactivity. Further investigation revealed that anti-VEGF mAb did not change the expression of markers of terminal differentiation such as keratin 10, filaggrin and involucrin; suggesting that VEGF depletion does not affect keratinocyte terminal differentiation. In contrast to the control group, levels of VEGF protein were undetectable in the culture supernatant of samples treated with 0.8 mg/mL of anti-VEGF mAb, suggesting sufficient dose. Conclusion: Our pilot study, provides the first evidence that anti-VEGF therapy promotes endothelial cell apoptosis in human skin ex vivo. Our pragmatic human skin organ culture assay offers a valuable tool for future preclinical endothelial cell and translational microvascular network / anti-angiogenesis research in human skin
