L-4-thiazolylalanine (Protinol), a novel non-proteinogenic amino acid, demonstrates epidermal and dermal efficacy with clinically observable benefits.

OBJECTIVE: Facial skin undergoes major structural and functional changes as a result of intrinsic and extrinsic factors. The goal of the current work is to demonstrate L-4-thiazolylalaine (L4, Protinol), a non-proteinogenic amino acid shown to stimulate the production of dermal proteins by fibroblasts, is an alternative efficacious topical ingredient for visible signs of ageing. METHODS: In vitro studies using 3D human skin tissue models were performed to show changes in protein and gene expression of key dermal markers in samples treated with 0.3% L4 compared to vehicle control. In vivo evaluation of skin turnover was measured in volunteers after treatment with L4 compared to retinol. Skin biopsies (n = 30) were taken to investigate epidermal and dermal changes in cases treated with L4 and compared to retinol. Finally, a clinical evaluation (n = 28) was conducted to assess the efficacy of L4 over a base formulation using various ageing parameters within a population of women 46-66 years old with mild-to-moderate wrinkles. RESULTS: In vitro studies on 3D tissues displayed significant changes in the dermal matrix via an increase in HA and pro-collagen I production and a decrease in the expression of inflammatory genes. In vivo biopsy studies demonstrated that L4 and retinol independently increased epidermal thickness and collagen remodelling significantly more compared with the base formula. Clinical evaluation showed firmer and smoother skin at day 28 post-treatment with L4 over the vehicle control without causing side effects such as redness or irritation. CONCLUSION: L4 is a novel, multi-functional ingredient which offers a superior alternative to currently available technologies for improving epidermal and dermal parameters that change during ageing and photodamage.

OBJECTIF: La peau du visage est sujet à des changements majeurs structuraux et fonctionnels dus à des facteurs intrinsèques et extrinsèques. Dans cette étude, nous montrons que l'acide aminé non-protéinogène L-4-thiazolylalanine (L4, Protinol) est une alternative intéressante pour une application topique. MÉTHODES: Des modèles 3D de peaux ont été utilisés pour mesurer les changements d'expressions géniques et protéiques de marqueurs clés du derme à partir d'échantillons traités avec L4 comparés à une condition contrôle. In vivo, après un traitement L4, le renouvellement cutané a été mesuré chez les volontaires et comparé à un traitement au rétinol. Des biopsies de peaux (n = 30) traitées soit à L4 soit au rétinol ont été isolées afin d'évaluer les changements au niveau du derme et de l'épiderme. Pour finir, une étude clinique (n = 28) a été menée pour évaluer l'efficacité de L4 par rapport à une formulation de base en utilisant différents paramètres de vieillissement au sein d'une population de femmes de 46 à 66 ans présentant des rides légères à modérées. RÉSULTATS: Les études in vitro sur tissues 3D ont montré des changements dans la matrice du derme avec une augmentation de la production d'acide hyaluronique et de procollagène I et une diminution d'expression de gènes pro-inflammatoires. Les études menées in vivo sur biopsies ont démontré que L4 et rétinol augmentaient indépendamment tous deux significativement l'épaisseur de l'épiderme et le remodelage du collagène par rapport à leur base seule. Pour finir, une peau plus ferme et plus lisse a été mesurée cliniquement après 28 jours de traitement L4 par rapport au véhicule et cela sans effets indésirables tels que rougeur et irritation. CONCLUSION: L4 est un ingrédient, innovant et multifonctionnel. Il offre une sérieuse alternative aux technologies actuellement disponibles dans les traitements contre le vieillissement de la peau ou le photodommage.

Analysis of the function of ADAM17 in iRhom2 curly-bare and tylosis with esophageal cancer mutant mice.

Tylosis with oesophageal cancer (TOC) is a rare familial disorder caused by cytoplasmic mutations in inactive rhomboid 2 (iRhom2 or iR2, encoded by Rhbdf2). iR2 and the related iRhom1 (or iR1, encoded by Rhbdf1) are key regulators of the membrane-anchored metalloprotease ADAM17, which is required for activating EGFR ligands and for releasing pro-inflammatory cytokines such as TNFα (or TNF). A cytoplasmic deletion in iR2, including the TOC site, leads to curly coat or bare skin (cub) in mice, whereas a knock-in TOC mutation (toc) causes less severe alopecia and wavy fur. The abnormal skin and hair phenotypes of iR2cub/cub and iR2toc/toc mice depend on amphiregulin (Areg) and Adam17, as loss of one allele of either gene rescues the fur phenotypes. Remarkably, we found that iR1-/- iR2cub/cub mice survived, despite a lack of mature ADAM17, whereas iR2cub/cub Adam17-/- mice died perinatally, suggesting that the iR2cub gain-of-function mutation requires the presence of ADAM17, but not its catalytic activity. The iR2toc mutation did not substantially reduce the levels of mature ADAM17, but instead affected its function in a substrate-selective manner. Our findings provide new insights into the role of the cytoplasmic domain of iR2 in vivo, with implications for the treatment of TOC patients.

Role of iRhoms 1 and 2 in Endochondral Ossification.

Growth of the axial and appendicular skeleton depends on endochondral ossification, which is controlled by tightly regulated cell-cell interactions in the developing growth plates. Previous studies have uncovered an important role of a disintegrin and metalloprotease 17 (ADAM17) in the normal development of the mineralized zone of hypertrophic chondrocytes during endochondral ossification. ADAM17 regulates EGF-receptor signaling by cleaving EGFR-ligands such as TGFα from their membrane-anchored precursor. The activity of ADAM17 is controlled by two regulatory binding partners, the inactive Rhomboids 1 and 2 (iRhom1, 2), raising questions about their role in endochondral ossification. To address this question, we generated mice lacking iRhom2 (iR2-/-) with floxed alleles of iRhom1 that were specifically deleted in chondrocytes by Col2a1-Cre (iR1∆Ch). The resulting iR2-/-iR1∆Ch mice had retarded bone growth compared to iR2-/- mice, caused by a significantly expanded zone of hypertrophic mineralizing chondrocytes in the growth plate. Primary iR2-/-iR1∆Ch chondrocytes had strongly reduced shedding of TGFα and other ADAM17-dependent EGFR-ligands. The enlarged zone of mineralized hypertrophic chondrocytes in iR2-/-iR1∆Ch mice closely resembled the abnormal growth plate in A17∆Ch mice and was similar to growth plates in Tgfα-/- mice or mice with EGFR mutations. These data support a model in which iRhom1 and 2 regulate bone growth by controlling the ADAM17/TGFα/EGFR signaling axis during endochondral ossification.

Blood-induced bone loss in murine hemophilic arthropathy is prevented by blocking the iRhom2/ADAM17/TNF-α pathway.

Hemophilic arthropathy (HA) is a debilitating degenerative joint disease that is a major manifestation of the bleeding disorder hemophilia A. HA typically begins with hemophilic synovitis that resembles inflammatory arthritides, such as rheumatoid arthritis, and frequently results in bone loss in patients. A major cause of rheumatoid arthritis is inappropriate release of the proinflammatory cytokine tumor necrosis factor-α (TNF-α) by the TNF-α convertase (TACE; also referred to as ADAM17) and its regulator, iRhom2. Therefore, we hypothesized that iRhom2/ADAM17-dependent shedding of TNF-α also has a pivotal role in mediating HA. Here, we show that addition of blood or its components to macrophages activates iRhom2/ADAM17-dependent TNF-α shedding, providing the premise to study the activation of this pathway by blood in the joint in vivo. For this, we turned to hemophilic FVIII-deficient mice (F8-/- mice), which develop a hemarthrosis following needle puncture injury with synovial inflammation and significant osteopenia adjacent to the affected joint. We found that needle puncture-induced bleeding leads to increased TNF-α levels in the affected joint of F8-/- mice. Moreover, inactivation of TNF-α or iRhom2 in F8-/- mice reduced the osteopenia and synovial inflammation that develops in this mouse model for HA. Taken together, our results suggest that blood entering the joint activates the iRhom2/ADAM17/TNF-α pathway, thereby contributing to osteopenia and synovitis in mice. Therefore, this proinflammatory signaling pathway could emerge as an attractive new target to prevent osteoporosis and joint damage in HA patients.

ADAM10-Dependent Signaling Through Notch1 and Notch4 Controls Development of Organ-Specific Vascular Beds.

RATIONALE: Endothelial Notch signaling is critical for early vascular development and survival. Yet, previously described mice lacking endothelial a disintegrin and metalloproteinase 10 (ADAM10), a key regulator of Notch signaling, survived into adulthood with organ-specific vascular defects. These findings raised questions about whether these vascular defects were related to Notch signaling or other functions of ADAM10. OBJECTIVE: The aims of the study are to determine whether compensatory or redundant functions of ADAM17 in Notch signaling can explain the survival of Adam10ΔEC mice, explore the contribution of different Tie2-Cre transgenes to the differences in survival, and establish whether the Adam10ΔEC vascular phenotypes can be recapitulated by inactivation of Notch receptors in endothelial cells. METHODS AND RESULTS: Mice lacking ADAM10 and ADAM17 in endothelial cells (Adam10/Adam17ΔEC), which survived postnatally with organ-specific vascular defects, resembled Adam10ΔEC mice. In contrast, Adam10ΔEC mice generated with the Tie2Cre transgene previously used to inactivate endothelial Notch (Adam10ΔEC(Flv)) died by E10.5. Quantitative polymerase chain reaction analysis demonstrated that Cre-mediated recombination occurs earlier in Adam10ΔEC(Flv) mice than in the previously described Adam10ΔEC mice. Finally, mice lacking endothelial Notch1 (Notch1ΔEC) share some organ-specific vascular defects with Adam10ΔEC mice, whereas Notch4(-/-) mice lacking endothelial Notch1 (Notch1ΔEC/Notch4(-/-)) had defects in all vascular beds affected in Adam10ΔEC mice. CONCLUSIONS: Our results argue against a major role for ADAM17 in endothelial Notch signaling and clarify the difference in phenotypes of previously described mice lacking ADAM10 or Notch in endothelial cells. Most notably, these findings uncover new roles for Notch signaling in the development of organ-specific vascular beds.

Runx2 Controls Bone Resorption through the Down-Regulation of the Wnt Pathway in Osteoblasts.

The transcription factor Runx2 and the Wnt/ß-catenin pathway are major regulators of bone formation. Our aim was to assess the interactions between the Wnt/ß-catenin pathway and Runx2 that contribute to bone resorption. Our results indicate that the activity of the canonical Wnt/ß-catenin pathway depends on Runx2. Runx2 overexpression inhibited ß-catenin levels and activity in vitro and in vivo. Inhibition of Gsk3b using lithium chloride in Runx2-overexpressing osteoporotic female mice rescued the Wnt/ß-catenin signaling in vivo and completely restored trabecular bone volume by increasing bone formation and decreasing bone resorption. The activation of Wnt/ß-catenin signaling by lithium chloride treatment reduced the number and activity of bone marrow-derived osteoclast-like cells in vitro, suggesting that the restoration of trabecular bone in vivo was due to decreased bone resorption, consistent with the reduced receptor activator of NF-κB ligand/osteoprotegerin ratio in Runx2-overexpressing osteoblasts. Lithium chloride also increased osteoblast differentiation and activity in vitro in agreement with the increase in mineral apposition rate and osteocalcin expression detected in vivo. Our results indicate that the activity of the canonical Wnt/ß-catenin pathway in osteoblast is modulated by Runx2. To conclude, our in vivo and in vitro results highlight the role of Runx2 as a negative regulator of Wnt/ß-catenin pathway activity in osteoblasts and indicate that the abnormal Wnt/ß-catenin activity seen in Runx2 transgenic mice affects both osteoblast and osteoclast differentiation and activity.

Blood-induced arthropathy in hemophilia: mechanisms and heterogeneity.

Hemophilia A is an X-linked bleeding disorder that can be largely controlled by treatment with recombinant factor VIII. However, this treatment is only partially effective in preventing hemophilic arthropathy (HA), a debilitating degenerative joint disease that is caused by intra-articular bleeding events. The disease progression of HA has several distinct steps, beginning with hemophilic synovitis (HS), a hyperplasia of the synovial lining coupled with a neovascular response, followed by joint erosion with cartilage destruction and erosion of the underlying bone. The early stages of HA have certain features in common with arthritides such as rheumatoid arthritis (RA), whereas the later degenerative stages of HA have some similarities with osteoarthritis (OA). The main purpose of this review is to explore the similarities between HA with RA and OA and discuss how this information could potentially help understand the pathogenesis of HA and uncover new treatment opportunities.

Reversal of age-related oxidative stress prevents hippocampal synaptic plasticity deficits by protecting D-serine-dependent NMDA receptor activation.

Oxidative stress (OS) resulting from an imbalance between antioxidant defenses and the intracellular accumulation of reactive oxygen species (ROS) contributes to age-related memory deficits. While impaired synaptic plasticity in neuronal networks is thought to underlie cognitive deficits during aging, whether this process is targeted by OS and what the mechanisms involved are still remain open questions. In this study, we investigated the age-related effects of the reducing agent N-acetyl-L-cysteine (L-NAC) on the activation of the N-methyl-D-aspartate receptor (NMDA-R) by its co-agonist D-serine, because alterations in this mechanism contribute greatly to synaptic plasticity deficits in aged animals. Long-term dietary supplementation with L-NAC prevented oxidative damage in the hippocampus of aged rats. Electrophysiological recordings in the CA1 of hippocampal slices indicated that NMDA-R-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) were depressed in aged animals, deficits that could be reversed by exogenous D-serine. Chronic treatment with L-NAC, but not acute application of the reducing agent, restored potent D-serine-dependent NMDA-R activation and LTP induction in aged rats. In addition, it is also revealed that the age-related decrease in D-serine levels and in the expression of the synthesizing enzyme serine racemase, which underlies the decrease in NMDA-R activation by the amino acid, was rescued by long-term dietary treatment with L-NAC. Our results indicate that protecting redox status in aged animals could prevent injury to the cellular mechanisms underlying cognitive aging, in part by maintaining potent NMDA-R activation through the D-serine-dependent pathway.