Low-Level Light Therapy Downregulates Scalp Inflammatory Biomarkers in Men With Androgenetic Alopecia and Boosts Minoxidil 2% to Bring a Sustainable Hair Regrowth Activity.

BACKGROUND AND OBJECTIVES: Low-level light therapies using visible to infrared light are known to activate several cellular functions, such as adenosine triphosphate and nitric oxide synthesis. However, few clinical observations report its biological consequences for skin and scalp homeostasis. Since scalp inflammation was recognized as a potential physiological obstacle to the efficacy of the reference hair regrowth drug Minoxidil in vivo and since perifollicular inflammation is the hallmark of about 50%-70% follicular units in androgenetic alopecia, we decided to investigate whether the anti-inflammatory activity of LLLT/GentleWaves® device were assigned to L'Oréal by Light BioScience L.L.C., Virginia Beach, VA (US) could enhance hair regrowth activity of Minoxidil. STUDY DESIGN/MATERIALS AND METHODS: We conducted a first experimental clinical study on 64 men with androgenetic alopecia using LLLT/GentleWaves®, 590-nm predominant wavelength 70 seconds, specifically pulsed once per day, for 3 days, and we performed a whole-genome analysis of treated scalp biopsies. In a second clinical study, including 135 alopecic volunteers, we evaluated the hair regrowth activity in response to the upgraded LLLT/GentleWaves® device and Minoxidil. RESULTS: In the first clinical study, whole-genome analysis of treated scalp biopsies showed downregulation of scalp inflammatory biomarkers, such as AP1/FOSB messenger RNA (mRNA) and mir21, together with the disappearance of CD69 mRNA, specific to scalp-infiltrating T cells of about 50% of the studied volunteers prior to the LLLT/GentleWaves® treatment. In the second clinical study, we observed that LLLT/GentleWaves® was able to boost the hair regrowth activity of a Minoxidil 2% lotion to the extent of the highest concentration (5%) in terms of efficacy, number of responders, and perceived performance. CONCLUSIONS: Altogether, these observations suggest the potential benefit of LLLT/GentleWaves® as a noninvasive adjunctive technology for skin and scalp conditions, where a mild perifollicular inflammation is involved. Lasers Surg. Med. 2021. Copyright © 2021 Wiley Periodicals LLC.

Large scale study of epidermal recovery after stratum corneum removal: dynamics of genomic response.

The stratum corneum (SC) is a superficial skin compartment that protects the body from the outside environment. Any disturbance of this function induces cascading steps of molecular and cellular repair in the whole epidermis. The aim of this study was to investigate epidermal gene expression following SC removal by tape stripping. Twenty-nine healthy male volunteers were included (27 +/- 4 years old). Tape stripping was processed on one inner forearm, the other unstripped forearm served as a control. Epidermis samples were collected at 2, 6, 19, 30 and 72 h after tape stripping. Trans-epidermal water loss measurements were performed at each step to monitor barrier restoration. Total RNA was extracted from collected epidermis samples and analysed by using DermArray cDNA microarrays. Among 4000 genes under investigation, we found that the expression of 370 genes varied significantly at least once during the time following stripping. Using an original clustering method, the modulated genes were gathered into eight groups. A functional characterization of the clusters enabled us to get a dynamic and global view of the main molecular processes taking place during epidermal recovery.

Author Correction: Reconstructed Skin Models Revealed Unexpected Differences in Epidermal African and Caucasian Skin.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Reconstructed Skin Models Revealed Unexpected Differences in Epidermal African and Caucasian Skin.

Clinical observations of both normal and pathological skin have shown that there is a heterogeneity based on the skin origin type. Beside external factors, intrinsic differences in skin cells could be a central element to determine skin types. This study aimed to understand the in vitro behaviour of epidermal cells of African and Caucasian skin types in the context of 3D reconstructed skin. Full-thickness skin models were constructed with site matched human keratinocytes and papillary fibroblasts to investigate potential skin type related differences. We report that reconstructed skin epidermis exhibited remarkable differences regarding stratification and differentiation according to skin types, as demonstrated by histological appearance, gene expression analysed by DNA microarray and quantitative proteomic analysis. Signalling pathways and processes related to terminal differentiation and lipid/ceramide metabolism were up-regulated in epidermis constructed with keratinocytes from Caucasian skin type when compared to that of keratinocytes from African skin type. Specifically, the expression of proteins involved in the processing of filaggrins was found different between skin models. Overall, we show unexpected differences in epidermal morphogenesis and differentiation between keratinocytes of Caucasian and African skin types in in vitro reconstructed skin containing papillary fibroblasts that could explain the differences in ethnic related skin behaviour.

TGF-beta induces connexin43 gene expression in normal murine mammary gland epithelial cells via activation of p38 and PI3K/AKT signaling pathways.

One of the shared physiological roles between TGF-beta and connexin family members is to inhibit epithelial cell cycle progression and consequently, to provide protection against malignant transformation. Herein, we demonstrated that TGF-beta1 induces the expression of connexin43 (Cx43) in normal murine mammary gland (NMuMG) cell lines at the protein and mRNA levels, and transcriptionally. Using overexpression of a truncated dominant-negative form of Cx43, we determined that the modulation of gap junctional communication by TGF-beta1 plays a key role in the control of NMuMG cells proliferation by TGF-beta1. In addition, using overexpression of truncated dominant-negative forms of either Smad2 or Smad3, and MDA-MB-468 human breast carcinoma cells deficient for Smad4, we determined that the Smad cascade is not implicated in TGF-beta1 effect on Cx43 expression. Using specific pharmacologic inhibitors for JNK, ERK, p38, and PI3K/AKT signaling pathways, we demonstrated the cooperative role of p38 and PI3K/AKT signaling in TGF-beta1-induced Cx43 expression and gap junctional communication. Furthermore, transfection of a c-jun antisense expression vector significantly prevented TGF-beta1-induced Cx43 gene expression demonstrating the involvement of c-Jun/AP-1 pathway together with p38 and PI3K/AKT pathways in mediating TGF-beta1-induced Cx43 gene expression.

Involvement of ERK signaling in halofuginone-driven inhibition of fibroblast ability to contract collagen lattices.

Halofuginone, an alkaloid isolated from the plant Dichroa febrifuga, has been shown to be a potent inhibitor of tissue fibrosis. We herein demonstrate that, at concentrations below 10(-7) M, halofuginone does not affect the cell cycle but efficiently induces extracellular signal-regulated kinases(1,2) (ERK(1,2)), p38 and Jun NH2-terminal kinases(1,2) (JNK(1,2)) phosphorylation. In addition, at these non cytotoxic concentrations, halofuginone diminishes the capacity of fibroblasts to contract mechanically unloaded collagen lattices, an effect that is specifically blocked by the ERK inhibitors PD98059 and U0126, not by inhibitors of the JNK or p38 pathways. These data thus indicate that the inhibitory effect of halofuginone on fibroblast contractile activity, a key function for wound healing implicated in the development of tissue fibrosis, is an ERK-mediated mechanism.

The steroid receptor co-activator-1 (SRC-1) potentiates TGF-beta/Smad signaling: role of p300/CBP.

The three related 160-kDa proteins, SRC-1, TIF-2 and RAC-3, were initially identified as factors interacting with nuclear receptors. They have also been reported to potentiate the activity of other transcription factors such as AP-1 or NF-kappaB. The aim of this work was to identify whether SRC-1 interferes with the TGF-beta/Smad signaling pathway, and if so, to identify its underlying mechanisms of action. Using transient cell transfection experiments performed in human dermal fibroblasts with the Smad3/4-specific (SBE)4-lux reporter construct, as well as the human PAI-1 promoter, we determined that SRC-1 enhances TGF-beta-induced, Smad-mediated, transcription. Likewise, SRC-1 overexpression potentiated TGF-beta-induced upregulation of PAI-1 steady-state mRNA levels. Using a mammalian two-hybrid system, we demonstrated that SRC-1 interacts with the transcriptional co-activators p300/CBP, but not with Smad3. Overexpression of the adenovirus E1A oncoprotein, an inhibitor of CBP/p300 activity, prevented the enhancing effect of SRC-1 on Smad3/4-mediated transcription, indicating that p300/CBP may be required for SRC-1 effect. Such hypothesis was validated, as expression of a mutant form of SRC-1 lacking the CBP/p300-binding site failed to upregulate Smad3/4-dependent transcription, while full-length SRC-1 potentiated p300.Smad3 interactions. These results identify SRC-1 as a novel Smad3/4 transcriptional partner, facilitating the functional link between Smad3 and p300/CBP.

Analysis of gene expression dynamics revealed delayed and abnormal epidermal repair process in aged compared to young skin.

With aging, epidermal homeostasis and barrier function are disrupted. In a previous study, we analyzed the transcriptomic response of young skin epidermis after stratum corneum removal, and obtained a global kinetic view of the molecular processes involved in barrier function recovery. In the present study, the same analysis was performed in aged skin in order to better understand the defects which occur with aging. Thirty healthy male volunteers (67 ± 4 years old) were involved. Tape-strippings were carried out on the inner face of one forearm, the other unstripped forearm serving as control. At 2, 6, 18, 30 and 72 h after stripping, TEWL measurements were taken, and epidermis samples were collected. Total RNA was extracted and analyzed using DermArray(®) cDNA microarrays. The results highlighted that barrier function recovery and overall kinetics of gene expression were delayed following stripping in aged skin. Indeed, the TEWL measurements showed that barrier recovery in the young group appeared to be dramatically significant during the overall kinetics, while there were no significant evolution in the aged group until 30 h. Moreover, gene expression analysis revealed that the number of modulated genes following tape stripping increased as a function of time and reached a peak at 6 h after tape stripping in young skin, while it was at 30 h in aged skin, showing that cellular activity linked to the repair process may be engaged earlier in young epidermis than in aged epidermis. A total of 370 genes were modulated in the young group. In the aged group, 382 genes were modulated, whose 184 were also modulated in the young group. Only eight genes that were modulated in both groups were significantly differently modulated. The characterization of these genes into 15 functional families helped to draw a scenario for the aging process affecting epidermal repair capacity.

A new Vitreoscilla filiformis extract grown on spa water-enriched medium activates endogenous cutaneous antioxidant and antimicrobial defenses through a potential Toll-like receptor 2/protein kinase C, zeta transduction pathway.

Vitreoscilla filiformis (VF) biomass (VFB) has been widely used in cosmetic preparations and shown to modulate the major inducible free-radical scavenger mitochondrial superoxide dismutase in skin cells. By adding La Roche-Posay (LRP) thermal spring water to the VF culture medium, we obtained a biomass (LRP-VFB) with a similar mitochondrial superoxide dismutase activation capacity to VF. Also, the new biomass more powerfully stimulated mRNA expression and antimicrobial peptides in reconstructed epidermis. Interestingly, a predictive computer model that analyzed transducing events within skin epidermal cells suggested that this protective activity may involve the Toll-like receptor 2/protein kinase C, zeta transduction pathway. Protein kinase C, zeta inhibition was effectively shown to abolish VFB-induced gene stimulation and confirmed this hypothesis. This thus opens new avenues for investigation into the improvement of skin homeostatic defense in relation to the control of its physiological microbiota and innate immunity.

Modulation of collagen and MMP-1 gene expression in fibroblasts by the immunosuppressive drug rapamycin. A direct role as an antifibrotic agent?

We have examined whether rapamycin, an immunosuppressive drug, may exert part of its antifibrotic activity by directly targeting fibroblast extracellular matrix deposition. Incubation of human lung fibroblast (WI-26) cultures with rapamycin led to dose- and time-dependent reduction in the expression of types I and III collagens, both at the protein and mRNA levels. Rapamycin had no effect on collagen promoter activity but accelerated mRNA decay, indicating post-transcriptional control of collagen gene expression. In contrast, rapamycin significantly enhanced the expression of interstitial collagenase (MMP-1) at the protein and mRNA levels and transcriptionally. We determined that rapamycin efficiently activates AP-1-driven transcription by rapidly inducing c-jun/AP-1 phosphorylation with activation of the c-Jun N-terminal kinase (JNK) cascade, resulting in enhanced binding of AP-1.DNA complex formation and AP-1-dependent gene transactivation. Conversely, the JNK inhibitor SP600125 inhibited rapamycin-induced MMP-1 gene transactivation and AP-1/DNA interactions. A c-jun antisense expression vector efficiently prevented rapamycin-induced MMP-1 gene transcription. Pharmacological inhibition of either ERK or p38 MAPK pathways was without effect on rapamycin-induced MMP-1 gene expression. It thus appears that rapamycin may exert direct antifibrotic activities independent from its immunosuppressive action.

A central role for the JNK pathway in mediating the antagonistic activity of pro-inflammatory cytokines against transforming growth factor-beta-driven SMAD3/4-specific gene expression.

We have focused our attention on the molecular events underlying the antagonistic activities of pro-inflammatory cytokines against transforming growth factor-beta (TGF-beta)/SMAD signaling. Using jnk1/2-knockout (jnk(-/-)) and I kappa B kinase-gamma/nemo(-/-) fibroblasts, we have determined the specific roles played by the JNK/AP-1 and NF-kappa B/Rel pathways in this phenomenon. We demonstrate that, in a cellular context devoid of JNK activity (i.e. jnk(-/-) fibroblasts), interleukin-1 and tumor necrosis factor-alpha (TNF-alpha) did not inhibit the formation of SMAD-DNA complexes and the resulting SMAD-driven transcription in response to TGF-beta. On the other hand, lack of NF-kappa B activity in nemo(-/-) fibroblasts did not affect the antagonistic effect of pro-inflammatory cytokines against TGF-beta. In the latter cell type, overexpression of antisense c-jun mRNA or of a dominant-negative form of MKK4 blocked the inhibitory activity of TNF-alpha, similar to what was observed in normal human dermal fibroblasts. Among JNK substrates, c-Jun and JunB (but not activating transcription factor-2) antagonized TGF-beta/SMAD signaling in a JNK-dependent manner. Overexpression of JNK1 in jnk(-/-) fibroblasts restored the ability of cytokines and Jun proteins to interfere with SMAD signaling. In junAA mouse embryo fibroblasts, in which c-Jun can no longer be phosphorylated by JNK, JunB substituted for c-Jun in mediating the cytokine effect against SMAD-driven transcription in a JNK-dependent manner. These results suggest a critical role for JNK-mediated c-Jun and JunB phosphorylation in transmitting the inhibitory effect of pro-inflammatory cytokines against TGF-beta-induced SMAD signaling. In addition, we demonstrate that such a JNK-dependent regulatory mechanism underlies the antagonistic activity of TNF-alpha against TGF-beta-induced up-regulation of type I and III collagens in fibroblasts.