New N-Substituted-1,2,4-triazole Derivatives of Pyrrolo[3,4-d]pyridazinone with Significant Anti-Inflammatory Activity-Design, Synthesis and Complementary In Vitro, Computational and Spectroscopic Studies.

Regarding that the chronic use of commonly available non-steroidal and anti-inflammatory drugs (NSAIDs) is often restricted by their adverse effects, there is still a current need to search for and develop new, safe and effective anti-inflammatory agents. As a continuation of our previous work, we designed and synthesized a series of 18 novel N-substituted-1,2,4-triazole-based derivatives of pyrrolo[3,4-d]pyridazinone 4a-c-9a-c. The target compounds were afforded via a convenient way of synthesis, with good yields. The executed cell viability assay revealed that molecules 4a-7a, 9a, 4b-7b, 4c-7c do not exert a cytotoxic effect and were qualified for further investigations. According to the performed in vitro test, compounds 4a-7a, 9a, 4b, 7b, 4c show significant cyclooxygenase-2 (COX-2) inhibitory activity and a promising COX-2/COX-1 selectivity ratio. These findings are supported by a molecular docking study which demonstrates that new derivatives take position in the active site of COX-2 very similar to Meloxicam. Moreover, in the carried out in vitro evaluation within cells, the title molecules increase the viability of cells pre-incubated with the pro-inflammatory lipopolysaccharide and reduce the level of reactive oxygen and nitrogen species (RONS) in induced oxidative stress. The spectroscopic and molecular modeling study discloses that new compounds bind favorably to site II(m) of bovine serum albumin. Finally, we have also performed some in silico pharmacokinetic and drug-likeness predictions. Taking all of the results into consideration, the molecules belonging to series a (4a-7a, 9a) show the most promising biological profile.

Response to Inhibition of Receptor-Interacting Protein Kinase 1 (RIPK1) in Active Plaque Psoriasis: A Randomized Placebo-Controlled Study.

Receptor-interacting protein kinase 1 (RIPK1), a regulator of inflammation and cell death, is a potential therapeutic target in immune-mediated inflammatory diseases (IMIDs). The objective of this phase IIa multicenter, randomized, double-blind, placebo-controlled study was to evaluate safety, tolerability pharmacokinetics, pharmacodynamics, and preliminary efficacy of GSK2982772, a RIPK1 inhibitor, in plaque-type psoriasis. Psoriasis patients (N = 65) were randomized to 60 mg twice daily (b.i.d.) or three times daily (t.i.d.), or placebo for 84 days. Most adverse events (AEs) were mild with no severe drug-related AEs reported. Plaque Lesion Severity Sum improved with b.i.d. treatment compared with placebo; interpretation of t.i.d. treatment results was complicated by a high placebo response. Reductions in epidermal thickness and infiltration by CD3+ T cells in the epidermis and dermis were observed compared with placebo. Results support the rationale for additional studies on RIPK1 inhibition in IMIDs.

Photoactivated resveratrol controls intradermal infection by Staphylococcus aureus in mice: a pilot study.

Staphylococcus aureus is one of the main causative agent of infections acquired in both community and hospital environment. In this context, photodynamic therapy (PDT) consists in using a photosensitizer that, activated by light, evokes the formation of reactive oxygen species (ROS), which lead to the death of microorganisms due to oxidative damage; it is useful tool since this action, harmful to pathogens, does not significantly injure human cells. In view of this, this work proposes a more in-depth study on the use of resveratrol (RSV) as a possible photosensitizer. It was observed, in the intradermal infection model in animals' ear dermis, that photoactivated resveratrol promotes an increase in myeloperoxidase expression with reduced bacterial load in the draining lymph node. Besides that, the draining lymph node of the animals treated with photoactivated RSV controls inflammation through IL-10 production. These are pioneers data and this work being a pilot study; then, other works must be conducted with the objective of elucidate the photoactivated resveratrol mechanism of action.

Physical properties of the photodamaged human skin dermis: Rougher collagen surface and stiffer/harder mechanical properties.

Fragmentation of collagen fibrils and aberrant elastic material (solar elastosis) in the dermal extracellular matrix (ECM) is among the most prominent features of photodamaged human skin. These alterations impair the structural integrity and create a dermal microenvironment prone to skin disorders. The objective of this study was to determine the physical properties (surface roughness, stiffness and hardness) of the dermal ECM in photodamaged and subject-matched sun-protected human skin. Skin samples were sectioned and analysed by histology, atomic force microscopy and nanoindentation. Dermal ECM collagen fibrils were more disorganized (ie, rougher surface), and the dermal ECM was stiffer and harder, in photodamaged forearm, compared to sun-protected underarm skin. Cleavage of collagen fibrils in sun-protected underarm dermis by recombinant human matrix metalloproteinase-1 resulted in rougher collagen fibril surface and reduced dermal stiffness and hardness. Degradation of elastotic material in photodamaged skin by treatment with purified neutrophil elastase reduced stiffness and hardness, without altering collagen fibril surface roughness. Additionally, expression of two members of the lysyl oxidase gene family, which insert cross-links that stiffen and harden collagen fibrils, was elevated in photodamaged forearm dermis. These data elucidate the contributions of fragmented collagen fibrils, solar elastosis and elevated collagen cross-linking to the physical properties of the dermal ECM in photodamaged human skin. This new knowledge extends current understanding of the impact of photodamage on the dermal ECM microenvironment.

ERK activating peptide, AES16-2M promotes wound healing through accelerating migration of keratinocytes.

Wound healing is an important issue that influences quality of life, and the need for products associated with wound healing is growing annually. New materials and therapies for skin wounds are being continuously researched and developed in order to increase treatment efficacy. Here, we show that the peptide AES16-2M comprised of five short amino acid sequences (REGRT) demonstrates efficacy in wound healing. AES16-2M exerted more effective healing than the control in an acute wound model, and tissue regeneration was similar to that of normal tissue in AES16-2M-treated skin. We found that the increase in re-epithelialization by AES16-2M early in wound development was due to migration of keratinocytes; a scratch assay using a human keratinocyte cell line (HaCaT) also demonstrated effective wound closure by AES16-2M. The migration of keratinocytes effected by AES16-2M was promoted through ERK phosphorylation and blocked with U0126, an ERK inhibitor. Moreover, AES16-2M treatment stimulated human dermal fibroblast (HDF) migration as well as keratinocyte. Taken together, these results suggest that AES16-2M can be an effective therapeutic agent for wound healing by promoting migration of keratinocytes and fibroblasts via ERK phosphorylation.

Protein kinase Cε regulates nuclear translocation of extracellular signal-regulated kinase, which contributes to bradykinin-induced cyclooxygenase-2 expression.

The proinflammatory mediator bradykinin stimulated cyclooxygenase-2 (COX-2) expression and subsequently prostaglandin E2 synthesis in dermal fibroblasts. The involvement of B2 receptors and Gαq in the role of bradykinin was suggested by using pharmacological inhibitors. The PKC activator PMA stimulated COX-2 mRNA expression. Bradykinin failed to induce COX-2 mRNA expression in the presence of PKC inhibitors, whereas the effect of bradykinin was observed in the absence of extracellular Ca2+. Bradykinin-induced COX-2 mRNA expression was inhibited in cells transfected with PKCε siRNA. These observations suggest that the novel PKCε is concerned with bradykinin-induced COX-2 expression. Bradykinin-induced PKCε phosphorylation and COX-2 mRNA expression were inhibited by an inhibitor of 3-phosphoinositide-dependent protein kinase-1 (PDK-1), and bradykinin-induced PDK-1 phosphorylation was inhibited by phospholipase D (PLD) inhibitors, suggesting that PLD/PDK-1 pathway contributes to bradykinin-induced PKCε activation. Pharmacological and knockdown studies suggest that the extracellular signal-regulated kinase 1 (ERK1) MAPK signaling is involved in bradykinin-induced COX-2 expression. Bradykinin-induced ERK phosphorylation was attenuated in the cells pretreated with PKC inhibitors or transfected with PKCε siRNA. We observed the interaction between PKCε and ERK by co-immunoprecipitation experiments. These observations suggest that PKCε activation contributes to the regulation of ERK1 activation. Bradykinin stimulated the accumulation of phosphorylated ERK in the nuclear fraction, that was inhibited in the cells treated with PKC inhibitors or transfected with PKCε siRNA. Consequently, we concluded that bradykinin activates PKCε via the PLD/PDK-1 pathway, which subsequently induces activation and translocation of ERK1 into the nucleus, and contributes to COX-2 expression for prostaglandin E2 synthesis in dermal fibroblasts.

Loxl2 is dispensable for dermal development, homeostasis and tumour stroma formation.

Lysyl oxidase-like 2 (LOXL2) is a copper-dependent monoamine oxidase that contributes to the remodelling of the extracellular matrix (ECM) by cross linkage of collagen and elastin fibres and has emerged as a potential therapeutic target in cancer and fibrosis. In the skin, LOXL2 is essential for epidermal cell polarity and differentiation. However, its role in the dermis has not been evaluated. We found that Loxl2 is dispensable for mouse dermal development, maturation and homeostasis, yet affects dermal stiffness. Neither loss of Loxl2 nor increased Loxl2 expression affected dermal architecture following treatment with the phorbol ester TPA. Furthermore, Loxl2 expression did not alter the stroma of DMBA-TPA-induced tumours. We conclude that, although Loxl2 is expressed in both dermis and epidermis, its function appears largely confined to the epidermis.

Lipo-PGE1 suppresses collagen production in human dermal fibroblasts via the ERK/Ets-1 signaling pathway.

Dysregulation of collagen production contributes to various pathological processes, including tissue fibrosis as well as impaired wound healing. Lipo-prostaglandin E1 (Lipo-PGE1), a lipid microsphere-incorporated prostaglandin E1, is used as a vasodilator for the treatment of peripheral vascular diseases. Lipo-PGE1 was recently shown to enhance human dermal fibroblast (HDF) migration and in vivo wound healing. No published study has characterized the role of Lipo-PGE1 in collagen regulation in HDFs. Here, we investigated the cellular signaling mechanism by which Lipo-PGE1 regulates collagen in HDFs. Collagen production was evaluated by the Sircol collagen assay, Western blot analysis of type I collagen and real time PCR. Unexpectedly, Lipo-PGE1 decreased mRNA expression of collagen 1A1, 1A2, and 3A1. Lipo-PGE1 markedly inhibited type I collagen and total soluble collagen production. In addition, Lipo-PGE1 inhibited transforming growth factor-ß-induced collagen expression via Smad2 phosphorylation. To further investigate whether extracellular signal-regulated kinase (ERK)/Ets-1 signaling, a crucial pathway in collagen regulation, is involved in Lipo-PGE1-inhibited collagen production, cells were pretreated with an ERK-specific inhibitor, PD98059, prior to the addition of Lipo-PGE1. Lipo-PGE1-inhibited collagen mRNA expression and total soluble collagen production were recovered by pretreatment with PD98059. Moreover, Lipo-PGE1 directly induced the phosphorylation of ERK. Furthermore, silencing of Ets-1 recovered Lipo-PGE1-inhibited collagen production and PD98059 blocked Lipo-PGE1-enhanced Ets-1 expression. The present study reveals an important role for Lipo-PGE1 as a negative regulator of collagen gene expression and production via ERK/Ets-1 signaling. These results suggest that Lipo-PGE1 could potentially be a therapeutic target in diseases with deregulated collagen turnover.

Salmon nasal cartilage proteoglycan enhances growth of normal human dermal fibroblast through Erk1/2 phosphorylation.

Proteoglycan (PG) is a heavily glycosylated protein, localized to cell surface and extracellular matrix, and has various functions. Recently, it has been gradually revealed that PG interacts with various growth factors and morphogens and regulates cellular functions. Although salmon nasal cartilage PG (Salmon-PG) increases proliferation of immortalized cells, its mechanism remains unclear. In this study, we confirmed the effect of Salmon-PG on normal human dermal fibroblast (NHDF) and investigated the mechanism of PG action on NHDF. Salmon-PG dose- and time-dependently increased NHDF proliferation. Receptor tyrosine kinase array revealed that Salmon-PG increased only Erk1/2 signaling. Erk1/2 phosphorylation was significantly increased by Salmon-PG in a time-(10 min) and dose-(400 or 800 µg/mL) dependent manner. MEK inhibitor suppressed the enhancement of NHDF proliferation by Salmon-PG. The overall findings indicate that Salmon-PG plays a role as a growth factor in NHDF via Erk1/2 activation, suggesting that Salmon-PG contributes to the maintenance of skin homeostasis.

Aberrant tyrosinase expression in an atypical fibroxanthoma: A case report.

Atypical fibroxanthoma (AFX) is a histologic mimicker of a variety of spindle cell neoplasms, and careful microscopic and immunohistochemical evaluation is critical in establishing the correct diagnosis. Here we report the histologic and immunohistochemical work up of a 1 cm nodule involving the left dorsal hand of a 66-year-old patient. Light microscopy revealed fascicles of spindled and pleomorphic cells within the dermis showing increased mitotic activity occurring in the background of sun-damaged skin. There were numerous multinucleated cells with hyperchromatic nuclei and ample finely vacuolated or foamy cytoplasms. There was strong and diffuse CD10 and patchy CD68 expression among the spindled cells and multinucleated cells. The neoplastic cells did not show immunoreactivity against S100, p75-NGFR, HMB-45 or a panel of keratinocytic, vascular and smooth muscle markers. Tyrosinase and Melan-A were not expressed within the spindle cell component of this neoplasm; however, there was tyrosinase expression among numerous multinucleated giant cells. Melan-A expression was also observed among rare multinucleated giant cells. Tyrosinase expression has not previously been reported in AFX.

[Age-related changes in the content of serine-arginine protein kinase 1 (SRPK1) in human dermis.]

The aim of our work was to examine content of serine-arginine protein kinase 1 (SRPK1) in human dermis at different ages (from 20 weeks of pregnancy to 85 years old). SRPK1, proliferating cells nuclear antigen (PCNA ), endothelial marker CD31 were detected in sections of the skin by indirect immunohistochemistry. Results showed, that content of SRPK1 in dermal fibroblasts was increased form antenatal period to 20 years of life followed by a decrease until 61-85 years period. SRPK1 content in dermal blood vessels is slowly gradually increased from antenatal period to 61-85 age interval. The number of fibroblasts and their proliferative activity, the number of CD31 positive blood vessels in dermis were decreased from antenatal period to 61-85 years period of life. Age-dependent decrease in SRPK1 in dermal fibroblasts from 20 years is associated with a reduction in the number and proliferative activity of fibroblasts. Age-related increase in SRPK1 content in dermal blood vessels is associated with a diminishing of the number of blood vessels. Hence, it can be supposed that SRPK1 has different actions on proliferation of differ components of dermis during aging.

Triolein reduces MMP-1 upregulation in dermal fibroblasts generated by ROS production in UVB-irradiated keratinocytes.

BACKGROUND: Cytokine production and oxidative stress generated by ultraviolet radiation B (UVB) skin exposure are main factors of skin photoaging. Interleukin-6 (IL-6) produced by irradiated keratinocytes is proposed to have a role in metalloproteinases (MMPs) expression activation in dermal fibroblasts. OBJECTIVES: We examined the effect of triolein treatment of UVB-irradiated keratinocytes on MMP1 (interstitial collagenase) expression response of dermal fibroblasts. We assayed UVB-irradiated keratinocytes soluble signals, mainly IL-6 and reactive oxygen species (ROS). METHODS: IL-6 expression and ROS generation were assayed in UVB-irradiated keratinocytes. MMP1 mRNA expression response was assayed in fibroblasts grown in keratinocytes conditioned medium. We evaluated the effect of treating keratinocytes with triolein on IL-6 expression and ROS generation in keratinocytes, and MMP1 expression in fibroblasts. RESULTS: The irradiation of epidermal cells with sublethal UVB doses increased IL-6 expression and ROS generation. Conditioned culture medium collected from keratinocytes was used to culture dermal fibroblasts. MMP1 mRNA expression increase was observed in fibroblasts cultured in medium collected from UVB-irradiated keratinocytes. Triolein treatment reduced the IL-6 expression and ROS generation in keratinocytes and this effect was reflected in downregulation of MMP1 expression in fibroblasts. CONCLUSIONS: Triolein reduces both the expression of IL-6 and ROS generation in irradiated keratinocytes. It seems to exert an anti-inflammatory and anti-oxidative stress effect on irradiated keratinocytes that in turn reduces MMP1 expression in dermal fibroblasts. Collectively, these results indicate that triolein could act as a photoprotective agent.

BMP1-like proteinases are essential to the structure and wound healing of skin.

Closely related extracellular metalloproteinases bone morphogenetic protein 1 (BMP1) and mammalian Tolloid-like 1 (mTLL1) are co-expressed in various tissues and have been suggested to have overlapping roles in the biosynthetic processing of extracellular matrix components. Early lethality of mice null for the BMP1 gene Bmp1 or the mTLL1 gene Tll1 has impaired in vivo studies of these proteinases. To overcome issues of early lethality and functional redundancy we developed the novel BTKO mouse strain, with floxed Bmp1 and Tll1 alleles, for induction of postnatal, simultaneous ablation of the two genes. We previously showed these mice to have a skeletal phenotype that includes elements of osteogenesis imperfecta (OI), osteomalacia, and deficient osteocyte maturation, observations validated by the finding of BMP1 mutations in a subset of human patients with OI-like phenotypes. However, the roles of BMP1-like proteinase in non-skeletal tissues have yet to be explored, despite the supposed importance of putative substrates of these proteinases in such tissues. Here, we employ BTKO mice to investigate potential roles for these proteinases in skin. Loss of BMP1-like proteinase activity is shown to result in markedly thinned and fragile skin with unusually densely packed collagen fibrils and delayed wound healing. We demonstrate deficits in the processing of collagens I and III, decorin, biglycan, and laminin 332 in skin, which indicate mechanisms whereby BMP1-like proteinases affect the biology of this tissue. In contrast, lack of effects on collagen VII processing or deposition indicates this putative substrate to be biosynthetically processed by non-BMP1-like proteinases.

Novel Protein Arginine Methyltransferase 8 Isoform Is Essential for Cell Proliferation.

Identification of molecular mechanisms that regulate cellular replicative lifespan is needed to better understand the transition between a normal and a neoplastic cell phenotype. We have previously reported that low oxygen-mediated activity of FGF2 leads to an increase in cellular lifespan and acquisition of regeneration competence in human dermal fibroblasts (iRC cells). Though cells display a more plastic developmental phenotype, they remain non-tumorigenic when injected into SCID mice (Page et al. [2009] Cloning Stem Cells 11:417-426; Page et al. [2011] Eng Part A 17:2629-2640) allowing for investigation of mechanisms that regulate increased cellular lifespan in a non-tumorigenic system. Analysis of chromatin modification enzymes by qRT-PCR revealed a 13.3-fold upregulation of the arginine methyltransferase PRMT8 in iRC cells. Increased protein expression was confirmed in both iRC and human embryonic stem cells-the first demonstration of endogenous human PRMT8 expression outside the brain. Furthermore, iRC cells express a novel PRMT8 mRNA variant. Using siRNA-mediated knockdown we demonstrated that this novel variant was required for proliferation of human dermal fibroblasts (hDFs) and grade IV glioblastomas. PRMT8 upregulation in a non-tumorigenic system may offer a potential diagnostic biomarker and a therapeutic target for cells in pre-cancerous and cancerous states. J. Cell. Biochem. 117: 2056-2066, 2016. © 2016 Wiley Periodicals, Inc.

Human placental extract exerts hair growth-promoting effects through the GSK-3ß signaling pathway in human dermal papilla cells.

Human placental extract (HPE) is widely used in Korea to relieve fatigue. However, its effects on human dermal papilla cells (hDPCs) remain unknown. In the present study, in an effort to develop novel therapies to promote hair growth, we screened HPE. We demonstrate that HPE has hair growth­promoting activities and induces ß­catenin expression through the inhibition of glycogen synthase kinase­3ß (GSK­3ß) by phosphorylation in hDPCs. Treatment with HPE significantly increased the viability of the hDPCs in a concentration­dependent manner, as shown by bromodeoxyuridine (BrdU) assay. HPE also significantly increased the alkaline phosphatase (ALP) expression levels. The increased ß­catenin levels and the inhibition of GSK­3ß (Ser9) by phosphorylation suggested that HPE promoted the hair-inductive capacity of hDPCs. We compared the effects of treatment with HPE alone and treatment with HPE in conjunction with minoxidil (MXD). We found that HPE plus MXD effectively inhibited GSK­3ß by phosphorylation (Ser9) in the hDPCs. Moreover, we demonstrated that HPE was effective in inducing root hair elongation in rat vibrissa hair follicles, and that treatment with HPE led to a delay in catagen progression. Overall, our findings suggest that HPE promotes hair growth and may thus provide the basis of a novel therapeutic strategy for the clinical treatment of hair loss.

The role of polyphosphates in the sequestration of matrix metalloproteinases.

This study outlines the potential of a novel therapeutic dressing for the management of chronic wounds. The dressing incorporates polyphosphate, a non toxic compound with a number of beneficial characteristics in terms of wound healing, in a foam matrix. The aim of this study was to identify the potential of polyphosphate incorporated in the foam dressing to sequester the activity of matrix metalloproteinases (MMPs) and proteases derived from Pseudomonas aeruginosa. Methods used included gelatin zymography and milk-casein agar plate analysis. Results have shown that this dressing is effectively capable of reducing the levels of MMP-2 and MMP-9 in both their active and latent forms using an in vitro model. The dressing also demonstrated the compound's potential in the regulation of P. aeruginosa derived proteases.

Proteomic revelations.

The power of proteomics in cultured skin fibroblasts from individuals with either systemic sclerosis or recessive dystrophic epidermolysis bullosa has led to the common finding of senescence and deficiencies in autophagy. Both of these disorders exert high demand on fibroblast activity, and without the protective action of autophagy cellular stress could have many adverse effects that are further amplified by the senescent phenotype.

Identification of inhibitors of inositol 5-phosphatases through multiple screening strategies.

Phosphoinositides are low abundance membrane phospholipids that have key roles in signaling, membrane trafficking, and cytoskeletal dynamics in all cells. Until recently, strategies for robust and quantitative development of pharmacological tools for manipulating phosphoinositide levels have focused selectively on PI(3,4,5)P3 due to the importance of this lipid in growth factor signaling and cell proliferation. However, drugs that affect levels of other phosphoinositides have potential therapeutic applications and will be powerful research tools. Here, we describe methodology for the high-throughput screening of small molecule modulators of the inositol 5-phosphatases, which dephosphorylate PI(4,5)P2 (the precursor for PI(3,4,5)P3) and PI(3,4,5)P3). We developed three complementary in vitro activity assays, tested hit compounds on a panel of 5-phosphatases, and monitored efficacy toward various substrates. Two prominent chemical scaffolds were identified with high nanomolar/low micromolar activity, with one class showing inhibitory activity toward all 5-phosphatases tested and the other selective activity toward OCRL and INPP5B, which are closely related to each other. One highly soluble OCRL/INPP5B-specific inhibitor shows a direct interaction with the catalytic domain of INPP5B. The efficacy of this compound in living cells was validated through its property to enhance actin nucleation at the cell cortex, a PI(4,5)P2 dependent process, and to inhibit PI(4,5)P2 dephosphorylation by OCRL (both overexpressed and endogenous enzyme). The assays and screening strategies described here are applicable to other phosphoinositide-metabolizing enzymes, at least several of which have major clinical relevance. Most importantly, this study identifies the first OCRL/INPP5B specific inhibitor and provides a platform for the design of more potent inhibitors of this family of enzymes.

Loss of collagen VII is associated with reduced transglutaminase 2 abundance and activity.

Absence of collagen VII leads to widespread cellular and tissue phenotypes. However, the underlying molecular mechanisms are not well understood. To gain insights into cellular responses to loss of collagen VII, we undertook a quantitative disease proteomics approach. By using recessive dystrophic epidermolysis bullosa (RDEB), a skin blistering disease caused by collagen VII deficiency, as a genetic model, collagen VII-dependent differences in cellular protein abundances and protein-protein interactions were analyzed. Absence of collagen VII led to alterations of intracellular protein compositions and to perturbations in cell adhesion, protein trafficking, and the turnover pathway autophagy. A potential linker of the different cellular phenotypes is transglutaminase 2 (TGM2), a multifunctional enzyme important for protein cross-linking. TGM2 was identified as a stable interaction partner of collagen VII. In RDEB, both abundance and activity of TGM2 were reduced, accounting not only for diminished adhesion and perturbed autophagy but also for reduced cross-linking of the extracellular matrix and for decreased epidermal-dermal integrity in RDEB.