Multidirectional activity of bakuchiol against cellular mechanisms of facial ageing - Experimental evidence for a holistic treatment approach.

OBJECTIVE: Skin ageing is a multifactorial process involving formation of reactive oxygen species, consecutive inflammation with reduced epidermal and dermal cell viability and resulting damage to the extracellular matrix. Effective dermocosmetic treatment modalities should ideally address these hallmarks in a holistic approach. Here, we determined the corresponding activity profile of bakuchiol, a plant-derived meroterpene, in an array of in vitro, ex vivo and in vivo studies and compared it to retinol, currently considered as gold standard in topical antiageing cosmetics. METHODS: The antioxidative capacity and power of bakuchiol and retinol were analysed by measuring 2,2'-diphenyl-1-picrylhydrazyl (DPPH) reduction via its absorption decay and electron spin resonance spectroscopy, respectively. Effects on prostaglandin E2 (PGE2), macrophage migration inhibitory factor (MIF), fibroblast growth factor 7 (FGF7), collagen type I and VII (COL1A1, COL7A1), fibronectin (FN) levels as well as the metabolization of water-soluble tetrazolium 1 (WST-1) were determined in human dermal fibroblasts. Epidermal regeneration was assessed utilizing an in vitro wound healing model. FN protein levels were analysed ex vivo after treatment with a formulation containing bakuchiol, retinol or vehicle using suction blister fluid. Skin condition improvement was determined in vivo in a split-face comparison study after application of bakuchiol or vehicle. RESULTS: In contrast to retinol, bakuchiol demonstrated high antioxidative efficacy. Levels of PGE2 and MIF were significantly decreased by both bakuchiol and retinol. Bakuchiol but not retinol significantly increased FGF7 protein levels. WST-1 metabolization levels were significantly augmented by bakuchiol and retinol. Bakuchiol and retinol application led to a significant augmentation of COL1A1, COL7A1 and FN protein levels. Wounds supplemented with bakuchiol but not retinol displayed a significant increase in epidermis regeneration. Clinically, areas treated with a bakuchiol-containing formulation showed a statistically significant increase in FN protein values after a 4-week application compared to untreated areas and areas treated with vehicle. CONCLUSION: These data provide evidence for the multidirectional efficacy of bakuchiol against cellular hallmarks of skin ageing. Its activity profile shares some common features with retinol but demonstrates several hitherto unknown biopositive effects in our studies, namely stimulation of the critical extracellular matrix component FN, and accelerated epidermal regeneration and wound healing.

OBJECTIF: le vieillissement de la peau est un processus multifactoriel impliquant la formation de dérivés réactifs de l'oxygène, une inflammation consécutive qui entraîne une viabilité réduite des cellules du derme et de l'épiderme, et endommage la matrice extracellulaire. Pour être efficaces, les traitements dermocosmétiques devraient dans l'idéal traiter ces caractéristiques selon une approche holistique. Ici, nous avons déterminé le profil d'activité correspondant du bakuchiol, un méroterpène d'origine végétale, dans une série d'études in vitro, ex vivo et in vivo, et l'avons comparé au rétinol, qui est aujourd'hui considéré comme la référence parmi les cosmétiques anti-âge topiques. MÉTHODES: la capacité antioxydante et la puissance du bakuchiol et du rétinol ont été analysées en mesurant la réduction du 2,2-diphényl-1-picrylhydrazyl (DPPH) selon sa décroissance par absorption et à l'aide d'une spectroscopie par résonance magnétique électronique, respectivement. Les effets sur la prostaglandine E2 (PGE2), le facteur inhibiteur de la migration (FIM) des macrophages, le facteur de croissance des fibroblastes 7 (FGF7), le collagène de type I et VII (COL1A1, COL7A1), les taux de fibronectine (FN), ainsi que la métabolisation du tétrazolium 1 soluble dans l'eau (WST-1) ont été déterminés dans des fibroblastes dermiques humains. La régénération épidermique a été évaluée à l'aide d'un modèle de cicatrisation des plaies in vitro. Les taux de fibronectine ont été analysés ex vivo après un traitement avec une formulation contenant du bakuchiol, du rétinol ou un excipient à l'aide d'un liquide d'aspiration sous forme de vésicules. L'amélioration de l'état de la peau a été déterminée in vivo dans une étude de comparaison en hémiface après l'application de bakuchiol ou d'un excipient. RÉSULTATS: Contrairement au rétinol, le bakuchiol s'est avéré présenter une efficacité antioxydante élevée. Les taux de PGE2 et de FIM ont significativement diminué avec le bakuchiol et le rétinol. L'application de bakuchiol s'est accompagnée d'une augmentation significative des taux de protéine FGF7, mais pas celle de rétinol. Les taux de métabolisation du WST-1 ont augmenté de façon significative avec le bakuchiol et le rétinol. L'application de bakuchiol et de rétinol a entraîné une augmentation significative des taux de protéines COL1A1, COL7A1 et fibronectine. Les plaies supplémentées en bakuchiol, mais pas en rétinol, ont montré une augmentation significative de la régénération épidermique. Sur le plan clinique, les zones traitées avec une formulation contenant du bakuchiol ont montré une augmentation statistiquement significative des taux de fibronectine après une application de 4 semaines par rapport aux zones non traitées et aux zones traitées avec un excipient. CONCLUSION: ces données fournissent des preuves de l'efficacité multidirectionnelle du bakuchiol contre les caractéristiques cellulaires du vieillissement de la peau. Son profil d'activité partage certaines caractéristiques communes avec le rétinol, mais démontre plusieurs effets biopositifs jusqu'alors inconnus dans nos études : la stimulation de la fibronectine, composante essentielle de la matrice extracellulaire, et une régénération épidermique et une cicatrisation accélérée des plaies.

Molecular pathology of the basement membrane zone in heritable blistering diseases:: The paradigm of epidermolysis bullosa.

Epidermolysis bullosa (EB), a phenotypically heterogeneous group of skin fragility disorders, is characterized by blistering and erosions with considerable morbidity and mortality. Mutations in as many as 18 distinct genes expressed at the cutaneous basement membrane zone have been shown to be associated with the blistering phenotype, attesting to the role of the corresponding proteins in providing stable association of the epidermis to the dermis through adhesion at the dermo-epidermal basement membrane zone. Thus, different forms of EB have been highly instructive in providing information on the physiological functions of these proteins as integral components of the supramolecular adhesion complexes. In addition, precise information of the underlying genes and distinct mutations in families with EB has been helpful in subclassification of the disease with prognostic implications, as well as for prenatal testing and preimplantation genetic diagnosis. Furthermore, knowledge of the types of mutations is a prerequisite for application of allele-specific treatment approaches that have been recently developed, including read-through of premature termination codon mutations and chaperone-facilitated intracellular transport of conformationally altered proteins to proper physiologic subcellular location. Collectively, EB serves as a paradigm of heritable skin diseases in which significant progress has been made in identifying the underlying genetic bases and associated aberrant pathways leading from mutations to the phenotype, thus allowing application of precision medicine for this, currently intractable group of diseases.

Intense pulsed light photorejuvenation: a histological and immunohistochemical evaluation.

BACKGROUND: The use of intense pulsed light (IPL) for facial rejuvenation had been the topic of many studies. However, few of them discussed quantitative changes in extracellular matrix proteins after IPL therapy. OBJECTIVE: To objectively quantify the histological changes in extracellular matrix proteins after IPL treatment for facial wrinkles. METHODS: Biopsy specimens were obtained from the periocular area of six volunteers of Fitzpatrick skin type III-IV and Glogau's class I-III wrinkles. They were subjected to three months of IPL treatment (six sessions at two-week intervals). Using histological and immunostaining analysis coupled with computerized morphometric analysis, quantitative evaluation of collagen types I, III and VII, newly synthesized collagen, total elastin and tropoelastin was performed for skin biopsies at baseline, end of treatment, and three months post-treatment. RESULTS: Clinical assessment of volunteers did not show clinically noticeable improvement in facial wrinkles after IPL treatment. Furthermore, quantitative evaluation of extracellular matrix proteins showed no statistically significant changes (P>0.05) in response to IPL treatment CONCLUSION: Although 50 percent of volunteers showed mild improvement in skin texture at the end of IPL treatment, none of them reported improvement in skin tightening or wrinkles. No statistically significant histological changes were observed three months post IPL treatment.

Early onset collagen VI myopathies: Genetic and clinical correlations.

OBJECTIVE: Mutations in the genes encoding the extracellular matrix protein collagen VI (ColVI) cause a spectrum of disorders with variable inheritance including Ullrich congenital muscular dystrophy, Bethlem myopathy, and intermediate phenotypes. We extensively characterized, at the clinical, cellular, and molecular levels, 49 patients with onset in the first 2 years of life to investigate genotype-phenotype correlations. METHODS: Patients were classified into 3 groups: early-severe (18%), moderate-progressive (53%), and mild (29%). ColVI secretion was analyzed in patient-derived skin fibroblasts. Chain-specific transcript levels were quantified by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and mutation identification was performed by sequencing of complementary DNA. RESULTS: ColVI secretion was altered in all fibroblast cultures studied. We identified 56 mutations, mostly novel and private. Dominant de novo mutations were detected in 61% of the cases. Importantly, mutations causing premature termination codons (PTCs) or in-frame insertions strikingly destabilized the corresponding transcripts. Homozygous PTC-causing mutations in the triple helix domains led to the most severe phenotypes (ambulation never achieved), whereas dominant de novo in-frame exon skipping and glycine missense mutations were identified in patients of the moderate-progressive group (loss of ambulation). INTERPRETATION: This work emphasizes that the diagnosis of early onset ColVI myopathies is arduous and time-consuming, and demonstrates that quantitative RT-PCR is a helpful tool for the identification of some mutation-bearing genes. Moreover, the clinical classification proposed allowed genotype-phenotype relationships to be explored, and may be useful in the design of future clinical trials.

The protease domain of procollagen C-proteinase (BMP1) lacks substrate selectivity, which is conferred by non-proteolytic domains.

Procollagen C-proteinase (PCP) removes the C-terminal pro-peptides of procollagens and also processes other matrix proteins. The major splice form of the PCP is termed BMP1 (bone morphogenetic protein 1). Active BMP1 is composed of an astacin-like protease domain, three CUB (complement, sea urchin Uegf, BMP1) domains and one EGF-like domain. Here we compare the recombinant human full-length BMP1 with its isolated proteolytic domain to further unravel the functional influence of the CUB and EGF domains. We show that the protease domain alone cleaves truncated procollagen VII within the short telopeptide region into fragments of similar size as the full-length enzyme does. However, unlike full-length BMP1, the protease domain does not stop at this point, but degrades its substrate completely. Moreover, the protease domain cleaves other matrix proteins such as fibronectin, collagen I and collagen IV, which are left intact by the full-length enzyme. In addition, we show for the first time that thrombospondin-1 is differently cleaved by both BMP1 and its catalytic domain. In summary, our data support the concept that the C-terminal domains of BMP1 are important for substrate recognition and for controlling and restricting its proteolytic activity via exosite binding.