P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma

Hotspot mutations in the NRAS gene are causative genetic events associated with the development of melanoma. Currently, there are no FDA-approved drugs directly targeting NRAS mutations. Previously, we showed that p38 acts as a tumor suppressor in vitro and in vivo with respect to NRAS-mutant melanoma. We observed that because of p38 activation through treatment with the protein synthesis inhibitor, anisomycin leads to a transient upregulation of several targets of the cAMP pathway, representing a stressed cancer cell state that is often observed by therapeutic doses of MAPK inhibitors in melanoma patients. Meanwhile, genetically induced p38 or its stable transduction leads to a distinct cellular transcriptional state. Contrary to previous work showing an association of invasiveness with high p38 levels in BRAF-mutated melanoma, there was no correlation of p38 expression with NRAS-mutant melanoma invasion, highlighting the difference in BRAF and NRAS-driven melanomas. Although the role of p38 has been reported to be that of both tumor suppressor and oncogene, we show here that p38 specifically plays the role of a tumor suppressor in NRAS-mutant melanoma. Both the transient and stable activation of p38 elicits phosphorylation of mTOR, reported to be a master switch in regulating autophagy. Indeed, we observed a correlation between elevated levels of phosphorylated mTOR and a reduction in LC3 conversion (LCII/LCI), indicative of suppressed autophagy. Furthermore, a reduction in actin intensity in p38–high cells strongly suggests a role of mTOR in regulating actin and a remodeling in the NRAS-mutant melanoma cells. Therefore, p38 plays a tumor suppressive role in NRAS-mutant melanomas at least partially through the mechanism of mTOR upregulation, suppressed autophagy, and reduced actin polymerization. One or more combinations of MEK inhibitors with either anisomycin, rapamycin, chloroquine/bafilomycin, and cytochalasin modulate p38 activation, mTOR phosphorylation, autophagy, and actin polymerization, respectively, and they may provide an alternate route to targeting NRAS-mutant melanoma

Olive Leaf Extract Attenuates Inflammatory Activation and DNA Damage in Human Arterial Endothelial Cells

Olive leaf extract (OLE) is used in traditional medicine as a food supplement and as an over-the-counter drug for a variety of its effects, including anti-inflammatory and anti-atherosclerotic ones. Mechanisms through which OLE could modulate these pathways in human vasculature remain largely unknown. Serum amyloid A (SAA) plays a causal role in atherosclerosis and cardiovascular diseases and induces pro-inflammatory and pro-adhesive responses in human coronary artery endothelial cells (HCAEC). Within this study we explored whether OLE can attenuate SAA-driven responses in HCAEC. HCAEC were treated with SAA (1,000 nM) and/or OLE (0.5 and 1 mg/ml). The expression of adhesion molecules VCAM-1 and E-selectin, matrix metalloproteinases (MMP2 and MMP9) and microRNA 146a, let-7e, and let-7g (involved in the regulation of inflammation) was determined by qPCR. The amount of secreted IL-6, IL-8, MIF, and GRO-α in cell culture supernatants was quantified by ELISA. Phosphorylation of NF-κB was assessed by Western blot and DNA damage was measured using the COMET assay. OLE decreased significantly released protein levels of IL-6 and IL-8, as well as mRNA expression of E-selectin in SAA-stimulated HCAEC and reduced MMP2 levels in unstimulated cells. Phosphorylation of NF-κB (p65) was upregulated in the presence of SAA, with OLE significantly attenuating this SAA-induced effect. OLE stabilized SAA-induced upregulation of microRNA-146a and let-7e in HCAEC, suggesting that OLE could fine-tune the SAA-driven activity of NF-κB by changing the microRNA networks in HCAEC. SAA induced DNA damage and worsened the oxidative DNA damage in HCAEC, whereas OLE protected HCAEC from SAA- and H2O2-driven DNA damage. OLE significantly attenuated certain pro-inflammatory and pro-adhesive responses and decreased DNA damage in HCAEC upon stimulation with SAA. The reversal of SAA-driven endothelial activation by OLE might contribute to its anti-inflammatory and anti-atherogenic effects in HCAEC

Zgodnji gigantocelični arteritis

Gigantocelični arteritis (GCA) je najpogostejši primarni sistemski vaskulitis pri odraslih po 50. letu starosti v Evropi. Prizadene velike in srednje velike arterije in vnetni proces, ki zožuje ali popolnoma zapre svetlino žile, lahko dovede do hudih/trajnih ishemičnih zapletov kot so oslepitev, možganska kap ali miokardni infarkt. V zadnjem desetletju se je z vključitvijo slikovnih preiskav v diagnostični postopek pomembno skrajšal čas do prepoznave bolezni (t.i. zgodnji GCA). Pospešena obravnava bolnikov (ang. “fast track clinic”) je vodila v zmanjšanje pojavnosti najresnejših ishemičnih zapletov bolezni in znižanje stroškov zdravljenja. Vendar pa bolezen praviloma poteka kronično, s poslabšanji, kar skupaj s kroničnim glukokortikoidnem zdravljenjem vodi v kopičenje okvar organov in tkiv. Prav zato se intenzivno preučuje patogeneza bolezni, z možnostjo implementacije izsledkov kot so sodobne molekularno in celično usmerjene tarčne terapije. Glavni cilji našega preglednega članka so bili: a) analiza raziskav z navedenim časom trajanja od začetka simptomov do postavitve diagnoze, b) raziskava obetavnih molekularnih tarč za zdravljenje GCA in c) prepoznava klinično pomembnih celičnih podtipov. Najbolj obetavne tarčne molekule za tarčno zdravljenje so IL-6, IL-12/IL-23 in citototoksični z limfociti T povezan protein 4, medtem ko terapija z zaviralci TNF-α ni bila uspešna. Kliničnih raziskav z učinkovinami, usmerjenimi proti IL-17, še ni. V prispevku pa smo se dotaknili tudi drugih potencialnih terapevtskih tarč, vključno z molekulami, ki sodelujejujo v signalnih poteh

Uncovering the mechanisms of small molecule therapeutics with anti-inflammatory and metabolic actions in targeting pathogenic fibroblasts in systemic sclerosis and rheumatoid arthritis

Tradicionalni pogled na fibroblaste, kot strukturne celice človeških tkiv, se je v zadnjih letih spremenil z razkritjem številnih aktivnih vlog fibroblastov pri uravnavanju tkivnega mikrookolja. Nenadzorovana aktivacija fibroblastov prispeva k vztrajanju vnetja, brazgotinjenju tkiv in tkivni poškodbi. Kljub ključni vlogi fibroblastov pri različnih boleznih, so strategije za njihovo terapevtsko zaviranje omejene. V doktorski nalogi preučujemo bolezenske odzive fibroblastov ter odkrivamo nove strategije za zaviranje njihove aktivacije pri kroničnem vnetju in fibrozi. K raziskavam pristopamo iz dveh različnih zornih kotovosredotočimo se na pro-vnetne sinovijske fibroblaste pri revmatoidnem artritisu, tipični vnetni bolezni, ter na pro-fibrotične kožne fibroblaste pri sistemski fibrozi (SSc), tipični fibrotični bolezni. Naše raziskave sinovijskega tkiva in v kulturi gojenih sinovijskih fibroblastov kažejo, da predstavljajo sinovijski fibroblasti pomembno celično terapevtsko tarčo zaviralcev JAK. Zaviralec JAK tofacitinib zmanjšuje vnetne odzive v sinovijskih fibroblastih, vendar zavora vnetja pri terapevtskih koncentracijah zdravila ni popolna. Zaviralci Janus kinaz bi lahko zato učinkovito zavrli sklepno vnetje pri bolnikih z RA s prevladujočimi imunskimi in fibroblastnimi tipi sinovije, kar nakazuje na njihovo široko terapevtsko učinkovitost pri RA. Naši rezultati kažejo, da akutna odtegnitev tofacitiniba vodi do fosforilacije pSTAT1/3 v sinovijskih fibroblastih, kar bi lahko prispevalo k akutnemu poslabšanju RA pri bolnikih, ki začasno prekinejo zdravljenje s tofacitinibom. V drugem delu doktorske naloge pokažemo, da kožni fibroblasti, izpostavljeni pro-fibrotičnemu okolju, pomembno prispevajo k globalnemu metabolnemu neravnovesju v fibrotični koži. Metabolni vmesni produkt dimetil alfa-ketoglutarat (dm-akg) učinkovito zmanjšuje pro-vnetno/fibrotično aktivacijo kožnih fibroblastov v 2D in 3D in vitro celičnih modelih. Antifibrotični učinek dm-akg na kožne fibroblaste potrdimo tudi na ex vivo gojeni koži bolnikov z SSc, ter identificiramo kožne fibroblaste kot glavno celično tarčo dm-akg. Doktorska naloga poglablja razumevanje fibroblastnih odzivov in odkriva nove pristope zdravljenja fibroblastne patologije, kar bi lahko utrlo pot razvoju novih antifibroblastnih zdravil za zdravljenje avtoimunskih in drugih bolezni s prevladujočo fibroblastno patologijo.The traditional view of fibroblasts as the structural only framework of human tissue has changed considerably by discovering their active roles in shaping tissue microenvironment in health and disease. Pathogenic fibroblasts represent key drivers of chronic inflammation, fibrosis and tissue damagefibroblast-targeting strategies, however, remain scarce. In PhD thesis, we investigate novel treatment strategies to suppress pathogenic fibroblast behaviours in inflammation and fibrosis. We specifically focus on pro-inflammatory synovial fibroblasts in rheumatoid arthritis (RA) – a prototypic inflammatory disease – and on pro-fibrotic skin fibroblasts in systemic sclerosis (SSc) - a prototypic fibrotic disease. Our studies of human synovium and cultured synovial fibroblasts infer that synovial fibroblasts represent an important therapeutic cell target of JAK inhibitors. We show that therapeutic concentrations of JAK inhibitor tofacitinib significantly decrease pro-inflammatory activities of cultured synovial fibroblasts, albeit not reaching the complete suppression. Thus, JAK inhibitors might interfere with stromal cell activation providing broad targeting across heterogenous synovial pathotypes. Furthermore, the acute tofacitinib withdrawal increases the pSTAT1/3 phosphorylation in synovial fibroblasts, providing a possible mechanistic explanation for acute arthritis worsening upon temporary interruption of tofacitinib therapy. Our research of skin fibrosis unravels a global metabolic dysregulation in the activated skin fibroblasts. We identify a new metabolism-targeting strategy to suppress pro-fibrotic activation of skin fibroblasts. Specifically, the metabolic intermediate dimethyl alpha-ketoglutarate effectively suppresses the pro-inflammatory/fibrotic activities of skin fibroblast in different 2D and 3D in vitro models. Furthermore, in a proof-of-concept study, we uncover the potent anti-fibrotic actions of dm-akg in the ex vivo explanted SSc skin tissue. These findings deduct that dm-akg-regulated cellular pathways might represent new therapeutic targets to combat skin fibrosis in SSc. Our research deepens the current understanding of fibroblast pathology in inflammation and fibrosis, and paves the way toward closing the gap in fibroblast therapeutic targeting in autoimmune diseases and beyond

Hi-JAKi-ng Synovial Fibroblasts in Inflammatory Arthritis With JAK Inhibitors

The Janus kinase (JAK)-Signal transducer and activator of transcription (STAT) pathway is one of the central signaling hubs in inflammatory, immune and cancer cells. Inhibiting the JAK-STAT pathway with JAK inhibitors (jakinibs) constitutes an important therapeutic strategy in cancer and chronic inflammatory diseases like rheumatoid arthritis (RA). FDA has approved different jakinibs for the treatment of RA, including tofacitinib, baricitinib and upadacitinib, and several jakinibs are being tested in clinical trials. Here, we reviewed published studies of jakinib effects on resolving synovial pathology in inflammatory arthritis. We discussed the results of jakinibs on structural joint damage in clinical trials and explored the effects of jakinibs across different in vitro, ex vivo, and in vivo synovial experimental models. We delved rigorously into experimental designs of in vitro fibroblast studies, deconvoluted jakinib efficacy in synovial fibroblasts across diverse experimental conditions and discussed their translatability in vivo. Synovial fibroblasts can readily activate the JAK-STAT signaling pathway in response to cytokine stimulation. We highlighted rather limited effects of jakinibs on the in vitro cultured synovial fibroblasts and inferred that direct and indirect (immune cell-dependent) actions of jakinibs are required to curb the fibroblast pathology in vivo. These actions have not been mimicked optimally in current in vitro experimental designs, where inflammatory stimuli do not naturally clear out with treatment as they do in vivo. While summarizing the broad knowledge of synovial jakinib effects, our review uniquely challenges future study designs to better mimick the jakinib actions in broader cell communities, as occurring in vivo in the inflamed synovium. This can deepen our understanding of collective synovial activities of jakinibs and their therapeutic limitations, thereby fostering jakinib development in arthritis

Effect of intercritical annealing on the microstructure and mechanical properties of 0.1C-13Cr-3Ni martensitic stainless steel

Standard heat treatment of martensitic stainless steel consists of quenching and tempering. However, this results in high strength and hardness, while Charpy impact toughness shows lower values and a large deviation in its values. Therefore, a modified heat treatment of 0.1C-13Cr-3Ni martensitic stainless steel (PK993/1CH13N3) with intercritical annealing between Ac1 and Ac3 was introduced before tempering to study its effect on the microstructure and mechanical properties (yield strength, tensile strength, hardness and Charpy impact toughness). The temperatures of intercritical annealing were 740, 760, 780 and 800 °C. ThermoCalc was used for thermodynamic calculations. Microstructure characterization was performed on an optical and scanning electron microscope, while XRD was used for the determination of retained austenite. Results show that intercritical annealing improves impact toughness and lowers deviation of its values. This can be attributed to the dissolution of the thin carbide film along prior austenite grain boundaries and prevention of its re-occurrence during tempering. On the other hand, lower carbon concentration in martensite that was quenching from the intercritical region resulted in lower strength and hardness. Intercritical annealing refines the martensitic microstructure creating a lamellar morphology

Increased L-selectin on monocytes is linked to the autoantibody profile in systemic sclerosis

Monocytes are known to be implicated in the pathogenesis of systemic sclerosis (SSc), as they exert prominent migratory, adhesive, and chemotactic properties. The aim of our study was to characterize the surface expression of adhesion/chemotactic molecules (CD62L, CD11b, CCR2, CCR5) on the SSc monocytes and determine correlations with the clinical presentation of SSc. We included 38 SSc patients and 36 healthy age-and sex-matched controls. Isolated monocytes, as well as in vitro serum-treated monocytes, were analyzed by flow cytometryadditionally, soluble CD62L was measured in serum. We found increased soluble CD62L in the SSc serum samples and increased CD62L on the surface of the SSc monocytes in the in the same set of patients. Among samples with determined SSc-specific autoantibodies, the surface CD62L was the lowest in patients positive for anti-PM/Scl autoantibodies and the highest in patients with anti-topoisomerase I autoantibodies (ATA). The treatment of isolated healthy monocytes with ATA-positive SSc serum resulted in increased surface CD62L expression. Moreover, surface CCR5 was reduced on the monocytes from SSc patients with interstitial lung disease but also, along with CCR2, negatively correlated with the use of analgesics/anti-inflammatory drugs and immunosuppressants. In conclusion, increased CD62L on SSc monocytes, particularly in ATA-positive patients, provides new insights into the pathogenesis of SSc and suggests CD62L as a potential therapeutic target

Gene and miRNA expression in giant cell arteritis-a concise systematic review of significantly modified studies

Giant cell arteritis (GCA) is a systemic vasculitis in individuals older than 50 years, characterized by headaches, visual disturbances, painful scalp, jaw claudication, impairment of limb arteries, and systemic inflammation, among other symptoms. GCA diagnosis is confirmed by a positive temporal artery biopsy (TAB) or by imaging modalities. A prominent acute phase response with inflammation is the hallmark of the disease, predominantly targeting large- and medium-sized arteries leading to stenosis or occlusion of arterial lumen. To date, there are no reliable tissue markers specific for GCA. Scarce reports have indicated the importance of epigenetics in GCA. The current systematic review reports significantly changed candidate biomarkers in TABs of GCA patients compared to non-GCA patients using qPCR

P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma

Hotspot mutations in the NRAS gene are causative genetic events associated with the development of melanoma. Currently, there are no FDA-approved drugs directly targeting NRAS mutations. Previously, we showed that p38 acts as a tumor suppressor in vitro and in vivo with respect to NRAS-mutant melanoma. We observed that because of p38 activation through treatment with the protein synthesis inhibitor, anisomycin leads to a transient upregulation of several targets of the cAMP pathway, representing a stressed cancer cell state that is often observed by therapeutic doses of MAPK inhibitors in melanoma patients. Meanwhile, genetically induced p38 or its stable transduction leads to a distinct cellular transcriptional state. Contrary to previous work showing an association of invasiveness with high p38 levels in BRAF-mutated melanoma, there was no correlation of p38 expression with NRAS-mutant melanoma invasion, highlighting the difference in BRAF and NRAS-driven melanomas. Although the role of p38 has been reported to be that of both tumor suppressor and oncogene, we show here that p38 specifically plays the role of a tumor suppressor in NRAS-mutant melanoma. Both the transient and stable activation of p38 elicits phosphorylation of mTOR, reported to be a master switch in regulating autophagy. Indeed, we observed a correlation between elevated levels of phosphorylated mTOR and a reduction in LC3 conversion (LCII/LCI), indicative of suppressed autophagy. Furthermore, a reduction in actin intensity in p38–high cells strongly suggests a role of mTOR in regulating actin and a remodeling in the NRAS-mutant melanoma cells. Therefore, p38 plays a tumor suppressive role in NRAS-mutant melanomas at least partially through the mechanism of mTOR upregulation, suppressed autophagy, and reduced actin polymerization. One or more combinations of MEK inhibitors with either anisomycin, rapamycin, chloroquine/bafilomycin, and cytochalasin modulate p38 activation, mTOR phosphorylation, autophagy, and actin polymerization, respectively, and they may provide an alternate route to targeting NRAS-mutant melanoma.ISSN:2072-669

P38 Mediates Tumor Suppression through Reduced Autophagy and Actin Cytoskeleton Changes in NRAS-Mutant Melanoma

Hotspot mutations in the NRAS gene are causative genetic events associated with the development of melanoma. Currently, there are no FDA-approved drugs directly targeting NRAS mutations. Previously, we showed that p38 acts as a tumor suppressor in vitro and in vivo with respect to NRAS-mutant melanoma. We observed that because of p38 activation through treatment with the protein synthesis inhibitor, anisomycin leads to a transient upregulation of several targets of the cAMP pathway, representing a stressed cancer cell state that is often observed by therapeutic doses of MAPK inhibitors in melanoma patients. Meanwhile, genetically induced p38 or its stable transduction leads to a distinct cellular transcriptional state. Contrary to previous work showing an association of invasiveness with high p38 levels in BRAF-mutated melanoma, there was no correlation of p38 expression with NRAS-mutant melanoma invasion, highlighting the difference in BRAF and NRAS-driven melanomas. Although the role of p38 has been reported to be that of both tumor suppressor and oncogene, we show here that p38 specifically plays the role of a tumor suppressor in NRAS-mutant melanoma. Both the transient and stable activation of p38 elicits phosphorylation of mTOR, reported to be a master switch in regulating autophagy. Indeed, we observed a correlation between elevated levels of phosphorylated mTOR and a reduction in LC3 conversion (LCII/LCI), indicative of suppressed autophagy. Furthermore, a reduction in actin intensity in p38–high cells strongly suggests a role of mTOR in regulating actin and a remodeling in the NRAS-mutant melanoma cells. Therefore, p38 plays a tumor suppressive role in NRAS-mutant melanomas at least partially through the mechanism of mTOR upregulation, suppressed autophagy, and reduced actin polymerization. One or more combinations of MEK inhibitors with either anisomycin, rapamycin, chloroquine/bafilomycin, and cytochalasin modulate p38 activation, mTOR phosphorylation, autophagy, and actin polymerization, respectively, and they may provide an alternate route to targeting NRAS-mutant melanoma