Tissue Ablation: Applications and Perspectives.

Tissue ablation techniques have emerged as a critical component of modern medical practice and biomedical research, offering versatile solutions for treating various diseases and disorders. Percutaneous ablation is minimally invasive and offers numerous advantages over traditional surgery, such as shorter recovery times, reduced hospital stays, and decreased healthcare costs. Intra-procedural imaging during ablation also allows precise visualization of the treated tissue volume while minimizing injury to surrounding normal tissues, reducing the risk of complications. Here, we explore the mechanisms of tissue ablation and innovative energy delivery systems, highlighting recent advancements that have reshaped the landscape of clinical practice. We will also discuss current clinical challenges related to tissue ablation, underlining unmet clinical needs for more advanced material-based approaches to improve the delivery of energy and pharmacology-based therapeutics. This article is protected by copyright. All rights reserved.

Catheter-Directed Ionic Liquid Embolic Agent for Rapid Portal Vein Embolization, Segmentectomy, and Bile Duct Ablation.

Embolic materials currently in use for portal vein embolization (PVE) do not treat the tumor, which poses a risk for tumor progression during the interval between PVE and surgical resection. Here, is developed an ionic-liquid-based embolic material (LEAD) for portal vein embolization, liver ablation, and drug delivery. LEAD is optimized and characterized for diffusivity, X-ray visibility, and cytotoxicity. In the porcine renal embolization model, LEAD delivered from the main renal artery reached vasculature down to 10 microns with uniform tissue ablation and delivery of small and large therapeutics. In non-survival and survival porcine experiments, successful PVE is achieved in minutes, leading to the expected chemical segmentectomy, and delivery of a large protein drug (i.e., Nivolumab) with LEAD. In cholangiocarcinoma mouse tumor models and in ex vivo human tumors, LEAD consistently achieved an effective ablation and wide drug distribution. Furthermore, various strains of drug-resistant patient-derived bacteria showed significant susceptibility to LEAD, suggesting that LEAD may also prevent infectious complications resulting from tissue ablation. With its capabilities to embolize, ablate, and deliver therapeutics, ease of use, and a high safety profile demonstrated in animal studies, LEAD offers a potential alternative to tumor ablation with or without PVE for FLR growth.

Photonic control of ligand nanospacing in self-assembly regulates stem cell fate.

Extracellular matrix (ECM) undergoes dynamic inflation that dynamically changes ligand nanospacing but has not been explored. Here we utilize ECM-mimicking photocontrolled supramolecular ligand-tunable Azo+ self-assembly composed of azobenzene derivatives (Azo+) stacked via cation-π interactions and stabilized with RGD ligand-bearing poly(acrylic acid). Near-infrared-upconverted-ultraviolet light induces cis-Azo+-mediated inflation that suppresses cation-π interactions, thereby inflating liganded self-assembly. This inflation increases nanospacing of "closely nanospaced" ligands from 1.8 nm to 2.6 nm and the surface area of liganded self-assembly that facilitate stem cell adhesion, mechanosensing, and differentiation both in vitro and in vivo, including the release of loaded molecules by destabilizing water bridges and hydrogen bonds between the Azo+ molecules and loaded molecules. Conversely, visible light induces trans-Azo+ formation that facilitates cation-π interactions, thereby deflating self-assembly with "closely nanospaced" ligands that inhibits stem cell adhesion, mechanosensing, and differentiation. In stark contrast, when ligand nanospacing increases from 8.7 nm to 12.2 nm via the inflation of self-assembly, the surface area of "distantly nanospaced" ligands increases, thereby suppressing stem cell adhesion, mechanosensing, and differentiation. Long-term in vivo stability of self-assembly via real-time tracking and upconversion are verified. This tuning of ligand nanospacing can unravel dynamic ligand-cell interactions for stem cell-regulated tissue regeneration.

Effects of C-Peptide on Dexamethasone-Induced In Vitro and In Vivo Models as a Potential Therapeutic Agent for Muscle Atrophy.

This study aimed to investigate the effects of C-peptide on C2C12 myotubes and a mouse model. Both in vitro and in vivo experiments were conducted to elucidate the role of C-peptide in muscle atrophy. Various concentrations (0, 0.01, 0.1, 1, 10, and 100 nM) of C-peptide were used on the differentiated C2C12 myotubes with or without dexamethasone (DEX). C57BL/6J mice were administered with C-peptide and DEX for 8 days, followed by C-peptide treatment for 12 days. Compared to the DEX group, C-peptide increased the fusion and differentiation indices and suppressed atrophic factor expression in C2C12 myotubes. However, 100 nM C-peptide decreased the fusion and differentiation indices and increased atrophic factor expression regardless of DEX treatment. In C57BL/6J mice, DEX + C-peptide co-treatment significantly attenuated the body and muscle weight loss and improved the grip strength and cross-sectional area of the gastrocnemius (Gas) and quadriceps (Quad) muscles. C-peptide downregulated the mRNA and protein levels of muscle degradation-related markers, particularly Atrogin-1, in Gas and Quad muscles. This study underscores the potential of C-peptides in mitigating muscle weight reduction and preserving muscle function during muscle atrophy via molecular regulation. In addition, the work presents basic data for future studies on the effect of C-peptide on diabetic muscular dystrophy.

Gelatin methacryloyl and Laponite bioink for 3D bioprinted organotypic tumor modeling.

Three-dimensional (3D)in vitrotumor models that can capture the pathophysiology of human tumors are essential for cancer biology and drug development. However, simulating the tumor microenvironment is still challenging because it consists of a heterogeneous mixture of various cellular components and biological factors. In this regard, current extracellular matrix (ECM)-mimicking hydrogels used in tumor tissue engineering lack physical interactions that can keep biological factors released by encapsulated cells within the hydrogel and improve paracrine interactions. Here, we developed a nanoengineered ion-covalent cross-linkable bioink to construct 3D bioprinted organotypic tumor models. The bioink was designed to implement the tumor ECM by creating an interpenetrating network composed of gelatin methacryloyl (GelMA), a light cross-linkable polymer, and synthetic nanosilicate (Laponite) that exhibits a unique ionic charge to improve retention of biological factors released by the encapsulated cells and assist in paracrine signals. The physical properties related to printability were evaluated to analyze the effect of Laponite hydrogel on bioink. Low GelMA (5%) with high Laponite (2.5%-3.5%) composite hydrogels and high GelMA (10%) with low Laponite (1.0%-2.0%) composite hydrogels showed acceptable mechanical properties for 3D printing. However, a low GelMA composite hydrogel with a high Laponite content could not provide acceptable cell viability. Fluorescent cell labeling studies showed that as the proportion of Laponite increased, the cells became more aggregated to form larger 3D tumor structures. Reverse transcription-polymerase chain reaction (RT-qPCR) and western blot experiments showed that an increase in the Laponite ratio induces upregulation of growth factor and tissue remodeling-related genes and proteins in tumor cells. In contrast, cell cycle and proliferation-related genes were downregulated. On the other hand, concerning fibroblasts, the increase in the Laponite ratio indicated an overall upregulation of the mesenchymal phenotype-related genes and proteins. Our study may provide a rationale for using Laponite-based hydrogels in 3D cancer modeling.

Ketamine versus etomidate for rapid sequence intubation in patients with trauma: a retrospective study in a level 1 trauma center in Korea.

BACKGROUND: Ketamine and etomidate are commonly used as sedatives in rapid sequence intubation (RSI). However, there is no consensus on which agent should be favored when treating patients with trauma. This study aimed to compare the effects of ketamine and etomidate on first-pass success and outcomes of patients with trauma after RSI-facilitated emergency intubation. METHODS: We retrospectively reviewed 944 patients who underwent endotracheal intubation in a trauma bay at a Korean level 1 trauma center between January 2019 and December 2021. Outcomes were compared between the ketamine and etomidate groups after propensity score matching to balance the overall distribution between the two groups. RESULTS: In total, 620 patients were included in the analysis, of which 118 (19.9%) were administered ketamine and the remaining 502 (80.1%) were treated with etomidate. Patients in the ketamine group showed a significantly faster initial heart rate (105.0 ± 25.7 vs. 97.7 ± 23.6, p = 0.003), were more hypotensive (114.2 ± 32.8 mmHg vs. 139.3 ± 34.4 mmHg, p < 0.001), and had higher Glasgow Coma Scale (9.1 ± 4.0 vs. 8.2 ± 4.0, p = 0.031) and Injury Severity Score (32.5 ± 16.3 vs. 27.0 ± 13.3, p < 0.001) than those in the etomidate group. There were no significant differences in the first-pass success rate (90.7% vs. 90.1%, p > 0.999), final mortality (16.1% vs. 20.6, p = 0.348), length of stay in the intensive care unit (days) (8 [4, 15] (Interquartile range)), vs. 10 [4, 21], p = 0.998), ventilator days (4 [2, 10] vs. 5 [2, 13], p = 0.735), and hospital stay (days) (24.5 [10.25, 38.5] vs. 22 [8, 40], p = 0.322) in the 1:3 propensity score matching analysis. CONCLUSION: In this retrospective study of trauma resuscitation, those receiving intubation with ketamine had greater hemodynamic instability than those receiving etomidate. However, there was no significant difference in clinical outcomes between patients sedated with ketamine and those treated with etomidate.

Skin-interfaced electronics: A promising and intelligent paradigm for personalized healthcare.

Skin-interfaced electronics (skintronics) have received considerable attention due to their thinness, skin-like mechanical softness, excellent conformability, and multifunctional integration. Current advancements in skintronics have enabled health monitoring and digital medicine. Particularly, skintronics offer a personalized platform for early-stage disease diagnosis and treatment. In this comprehensive review, we discuss (1) the state-of-the-art skintronic devices, (2) material selections and platform considerations of future skintronics toward intelligent healthcare, (3) device fabrication and system integrations of skintronics, (4) an overview of the skintronic platform for personalized healthcare applications, including biosensing as well as wound healing, sleep monitoring, the assessment of SARS-CoV-2, and the augmented reality-/virtual reality-enhanced human-machine interfaces, and (5) current challenges and future opportunities of skintronics and their potentials in clinical translation and commercialization. The field of skintronics will not only minimize physical and physiological mismatches with the skin but also shift the paradigm in intelligent and personalized healthcare and offer unprecedented promise to revolutionize conventional medical practices.

Photoswitchable Microgels for Dynamic Macrophage Modulation.

Dynamic manipulation of supramolecular self-assembled structures is achieved irreversibly or under non-physiological conditions, thereby limiting their biomedical, environmental, and catalysis applicability. In this study, microgels composed of azobenzene derivatives stacked via π-cation and π-π interactions are developed that are electrostatically stabilized with Arg-Gly-Asp (RGD)-bearing anionic polymers. Lateral swelling of RGD-bearing microgels occurs via cis-azobenzene formation mediated by near-infrared-light-upconverted ultraviolet light, which disrupts intermolecular interactions on the visible-light-absorbing upconversion-nanoparticle-coated materials. Real-time imaging and molecular dynamics simulations demonstrate the deswelling of RGD-bearing microgels via visible-light-mediated trans-azobenzene formation. Near-infrared light can induce in situ swelling of RGD-bearing microgels to increase RGD availability and trigger release of loaded interleukin-4, which facilitates the adhesion structure assembly linked with pro-regenerative polarization of host macrophages. In contrast, visible light can induce deswelling of RGD-bearing microgels to decrease RGD availability that suppresses macrophage adhesion that yields pro-inflammatory polarization. These microgels exhibit high stability and non-toxicity. Versatile use of ligands and protein delivery can offer cytocompatible and photoswitchable manipulability of diverse host cells.

Therapeutic Potential of Targeting Periostin in the Treatment of Graves' Orbitopathy.

Periostin is a matricellular protein that is ubiquitously expressed in normal human tissues and is involved in pathologic mechanism of chronic inflammatory and fibrotic disease. In this study we investigate periostin in the pathogenesis of Graves' orbitopathy (GO) using human orbital adipose tissue obtained from surgery and primary cultured orbital fibroblasts in vitro. POSTN (gene encoding periostin) expression in Graves' orbital tissues and healthy control tissues was studied, and the role of periostin in GO pathologic mechanism was examined through small-interfering RNA (siRNA)-mediated silencing. POSTN gene expression was significantly higher in Graves' orbital tissues than healthy control tissues in real-time PCR results, and immunohistochemical staining revealed higher expression of periostin in Graves' orbital tissues than normal tissues. Silencing periostin using siRNA transfection significantly attenuated TGF-ß-induced profibrotic protein production and phosphorylated p38 and SMAD protein production. Knockdown of periostin inhibited interleukin-1 ß -induced proinflammatory cytokines production as well as phosphorylation of NF-κB and Ak signaling protein. Adipocyte differentiation was also suppressed in periostin-targeting siRNA transfected GO cells. We hypothesize that periostin contributes to the pathogenic process of inflammation, fibrosis and adipogenesis of GO. Our study provides in vitro evidence that periostin may be a novel potential therapeutic target for the treatment of GO.

Systematic Screening and Therapeutic Evaluation of Glyconanoparticles with Differential Cancer Affinities for Targeted Cancer Therapy.

Cancer-targeting ligands used for nanomedicines have been limited mostly to antibodies, peptides, aptamers, and small molecules thus far. Here, a library of glycocalyx-mimicking nanoparticles as a platform to enable screening and identification of cancer-targeting nanomedicines is reported. Specifically, a library of 31 artificial glycopolymers composed of either homogeneous or heterogeneous display of five different sugar moieties (ß-glucose, ß-galactose, α-mannose, ß-N-acetyl glucosamine, and ß-N-acetyl galactosamine) is converted to a library of glyconanoparticles (GlyNPs). GlyNPs optimal for targeting CT26, DU145, A549, and PC3 tumors are systematically screened and identified. The cypate-conjugated GlyNP displaying α-mannose and ß-N-acetyl glucosamine show selective targeting and potent photothermal therapeutic efficacy against A549 human lung tumors. The docetaxel-contained GlyNP displaying ß-glucose, ß-galactose, and α-mannose demonstrate targeted chemotherapy against DU145 human prostate tumors. The results presented herein collectively demonstrate that the GlyNP library is a versatile platform enabling the identification of cancer-targeting glyconanoparticles and suggest its potential applicability for targeting various diseased cells beyond cancer.

A bilirubin-derived nanomedicine attenuates the pathological cascade of pulmonary fibrosis.

Pulmonary fibrosis is an irreparable and life-threatening disease with only limited therapeutic options. The recent outbreak of COVID-19 has caused a sharp rise in the incidence of pulmonary fibrosis owing to SARS-CoV-2 infection-mediated acute respiratory distress syndrome (ARDS). The considerable oxidative damage caused by locally infiltrated immune cells plays a crucial role in ARDS, suggesting the potential use of antioxidative therapeutics. Here, we report the therapeutic potential of nanoparticles derived from the endogenous antioxidant and anti-inflammatory bile acid, bilirubin (BRNPs), in treating pulmonary fibrosis in a bleomycin-induced mouse model of the disease. Our results demonstrate that BRNPs can effectively reduce clinical signs in mice, as shown by histological, disease index evaluations, and detection of biomarkers. Our findings suggest that BRNPs, with their potent antioxidant and anti-inflammatory effects, long blood circulation half-life, and preferential accumulation at the inflamed site, are potentially a viable clinical option for preventing Covid-19 infection-associated pulmonary fibrosis.

Biomimetic lipid Nanocomplexes incorporating STAT3-inhibiting peptides effectively infiltrate the lung barrier and ameliorate pulmonary fibrosis.

Activation of signal transducer and activator of transcription 3 (STAT3) under conditions of inflammation plays a crucial role in the pathogenesis of life-threatening pulmonary fibrosis (PF), initiating pro-fibrotic signaling following its phosphorylation. While there have been attempts to interfere with STAT3 activation and associated signaling as a strategy for ameliorating PF, potent inhibitors with minimal systemic toxicity have yet to be developed. Here, we assessed the in vitro and in vivo therapeutic effectiveness of a cell-permeable peptide inhibitor of STAT3 phosphorylation, designated APTstat3-9R, for ameliorating the indications of pulmonary fibrosis. Our results demonstrate that APTstat3-9R formulated with biomimetic disc-shaped lipid nanoparticles (DLNPs) markedly enhanced the penetration of pulmonary surfactant barrier and alleviated clinical symptoms of PF while causing negligible systemic cytotoxicity. Taken together, our findings suggest that biomimetic lipid nanoparticle-assisted pulmonary delivery of APTstat3-9R may be a feasible therapeutic option for PF in the clinic, and could be applied to treat other fibrotic diseases.

Bilirubin nanomedicine alleviates psoriatic skin inflammation by reducing oxidative stress and suppressing pathogenic signaling.

Psoriasis is a prevalent chronic inflammatory skin disease characterized by thickening of the epidermis accompanied by lesional erythema, scaling, and induration as a result of abnormal proliferation of keratinocytes. During the development of psoriasis, levels of intracellular reactive oxygen species (ROS) within psoriatic lesions are elevated, activating a pro-inflammatory signaling cascade. Here, we evaluated the therapeutic efficacy and mode of action of bilirubin nanoparticles (BRNPs), based on the potent, endogenous antioxidant bilirubin, in a preclinical psoriasis model. We found that topical treatment of psoriatic lesions with BRNPs effectively attenuated upregulation of intracellular ROS levels within keratinocytes and ameliorated the symptoms of psoriasis. A subsequent mechanistic study showed that preventing oxidative stress in activated keratinocytes suppressed the secretion of inflammatory mediators and recruitment of immune cells. Subsequent expression of the antigen-presenting cell (APC) maturation markers, class II major histocompatibility complex (MHC class II), cluster of differentiation (CD) 80 and CD86, was significantly decreased, resulting in a reduction in the differentiation of naïve CD4+ T cells into interleukin (IL)-17-producing T-helper (Th) 17 cells. Unlike the commercial corticosteroid drug, clobetasol propionate (CLQ), BRNPs, composed of the endogenous antioxidant bilirubin and the approved polymer polyethylene glycol (PEG), did not exert systemic cytotoxicity. Collectively, these findings highlight the potential of BRNPs as a novel nanomedicine for ameliorating psoriasis-like skin inflammation through topical treatment and suggest that their use could be further expanded to treat other chronic skin inflammation diseases, including atopic dermatitis.

Tubulin-Based Nanotubes as Delivery Platform for Microtubule-Targeting Agents.

Tubulin-based nanotubes (TNTs) to deliver microtubule-targeting agents (MTAs) for clinical oncology are reported. Three MTAs, docetaxel (DTX), laulimalide (LMD), and monomethyl auristatin E (MMAE), which attach to different binding sites in a tubulin, are loaded onto TNTs and cause structural changes in them, including shape anisotropy and tubulin layering. This drug-driven carrier transformation leads to changes in the drug-loading efficiency and stability characteristics of the carrier. TNTs coloaded with DTX and LMD efficiently deliver dual drug cargoes to cellular tubulins by the endolysosomal pathway, and results in synergistic anticancer and antiangiogenic action of the drugs in vitro. In in vivo tests, TNTs loaded with a microtubule-destabilizing agent MMAE suppress the growth of tumors with much higher efficacy than free MMAE did. This work suggests a new concept of using a drug's target protein as a carrier. The findings demonstrate that the TNTs developed here can be used universally as a delivery platform for many MTAs.

Probiotic treatment induced change of inflammation related metabolites in IBS-D patients/double-blind, randomized, placebo-controlled trial.

There have been many studies suggesting that probiotics are effective in patients with diarrhea-predominant irritable bowel syndrome (IBS-D). However, its mechanism of action as well as prediction of response is still to be elucidated. In the present study, to find out metabolomic characteristics of probiotic effect in IBS-D, we compared IBS symptom changes and metabolomic characteristics in the subjects' urine samples between multi-strain probiotics (one strain of Lactobacillus sp. and four strains of Bifidobacterium sp.) group (n = 32) and placebo group (n = 31). After 8 weeks' administration (3 times/day), dissatisfaction in bowel habits and stool frequencies were significantly improved. Also, probiotics group had significantly changed seven metabolites including palmitic acid methyl ester (PAME) and 4,6-dihydroxyquinoline, 4-(2-aminophenyl)-2,4-dioxobutanoic acid (DOBA). According to IBS-SSS and IBS-QoL questionnaires, IBS-SSS responders showed higher PAME levels and IBS-QoL responders showed lower DOBA levels. This suggests potential role of these metabolites as a biomarker to predict probiotics effect in IBS-D patients.

Sequential and Timely Combination of a Cancer Nanovaccine with Immune Checkpoint Blockade Effectively Inhibits Tumor Growth and Relapse.

We describe a small lipid nanoparticle (SLNP)-based nanovaccine platform and a new combination treatment regimen. Tumor antigen-displaying, CpG adjuvant-embedded SLNPs (OVAPEP -SLNP@CpG) were prepared from biocompatible phospholipids and a cationic cholesterol derivative. The resulting nanovaccine showed highly potent antitumor efficacy in both prophylactic and therapeutic E.G7 tumor models. However, this vaccine induced T cell exhaustion by elevating PD-L1 expression, leading to tumor recurrence. Thus, the nanovaccine was combined with simultaneous anti-PD-1 antibody treatment, but the therapeutic efficacy of this regimen was comparable to that of the nanovaccine alone. Finally, mice that showed a good therapeutic response after the first cycle of immunization with the nanovaccine underwent a second cycle together with anti-PD-1 therapy, resulting in suppression of tumor relapse. This suggests that the antitumor efficacy of combinations of nanovaccines with immune checkpoint blockade therapy is dependent on treatment sequence and the timing of each modality.

Therapeutic Effect of Guggulsterone in Primary Cultured Orbital Fibroblasts Obtained From Patients with Graves' Orbitopathy.

Purpose: Inflammation, hyaluronan production, and adipogenesis are the main pathological events leading to Graves' orbitopathy (GO). Guggulsterone (GS), a phytosterol found in the resin of the guggul plant, is a well-known treatment for several inflammatory disorders, such as arthritis, obesity, and hyperlipidemia. Here we investigated the effects of GS treatment on GO pathology. Methods: Using primary cultures of orbital fibroblasts from GO patients and non-GO controls, we examined the effects of GS on hyaluronan production and the production of proinflammatory cytokines induced by interleukin (IL)-1ß, using real-time reverse transcription-polymerase chain reaction analysis, western blots, and enzyme-linked immunosorbent assays. Further, adipogenic differentiation was evaluated by quantification of Oil Red O staining and assessment of protein levels of peroxisome proliferator activator gamma (PPARγ), CCAAT-enhancer-binding proteins (C/EBP) α and ß, and sterol regulatory element-binding protein-1 (SREBP-1). Results: Treatment with noncytotoxic concentrations of GS resulted in the dose-dependent inhibition of IL-1ß-induced inflammatory cytokines, including IL-6, IL-8, MCP-1, and COX-2, at both mRNA and protein levels. The hyaluronan level was also significantly suppressed by GS. Moreover, GS significantly decreased the formation of lipid droplets and expression of PPARγ, C/EBP α/ß, and SREBP-1 in a dose-dependent manner. GS pretreatment attenuated the phosphorylation of nuclear factor-kappa B induced by IL-1ß. Conclusions: Our data show significant inhibitory effects of GS on inflammation, production of hyaluronan, and adipogenesis in orbital fibroblasts. To our knowledge, this is the first in vitro preclinical evidence of the therapeutic effect of GS in GO.

Gold nanorods with an ultrathin anti-biofouling siloxane layer for combinatorial anticancer therapy.

Despite the wide utility of gold nanorods (GNRs) in biomedical fields, only a few methods for modifying or coating the surface of GNRs suitable for biomedical applications are available. In this study, we report a new facile method that enables formation of an ultra-thin (nanometre-thickness) siloxane layer on GNRs with anti-biofouling properties and ligand functionalisation ability. A triblock random copolymer, poly(TMSMA-r-PEGMA-r-NAS), was used to coat GNRs. An ultrathin polymeric shell was formed surrounding GNRs through acid-catalysed crosslinking of silicates of TMSMA. The polymer-coated GNRs (p-GNRs) exhibited high colloidal stability in biological solutions of high ionic strength and long-term stability superior to that of PEG2k-S-GNRs. The functionalities of NAS were demonstrated using two methods for conjugating targeting ligands and loading doxorubicin via electrostatic interactions. The ligand-specific cancer-targeting ability and combinatorial chemo-photothermal anticancer effects were validated in vitro and in vivo, suggesting their potential utility in various fields.

Role of Proprotein Convertase Subtilisin/Kexin Type 9 in the Pathogenesis of Graves' Orbitopathy in Orbital Fibroblasts.

Background: The proprotein convertase subtilisin/kexin type 9 (PCSK9) has been implicated in the pathogenesis of inflammatory diseases. We sought to investigate the role of PCSK9 in the pathogenesis of Graves' orbitopathy (GO) and whether it may be a legitimate target for treatment. Methods: The PCSK9 was compared between GO (n=11) and normal subjects (n=7) in orbital tissue explants using quantitative real-time PCR, and in cultured interleukin-1ß (IL-1ß)-treated fibroblasts using western blot. Western blot was used to identify the effects of PCSK9 inhibition on IL-1ß-induced pro-inflammatory cytokines production and signaling molecules expression as well as levels of adipogenic markers and oxidative stress-related proteins. Adipogenic differentiation was identified using Oil Red O staining. The plasma PCSK9 concentrations were compared between patients with GO (n=44) and healthy subjects (n=26) by ELISA. Results: The PCSK9 transcript level was higher in GO tissues. The depletion of PCSK9 blunted IL-1ß-induced expression of intercellular adhesion molecule 1 (ICAM-1), IL-6, IL-8, and cyclooxygenase-2 (COX-2) in GO and non-GO fibroblasts. The levels of activated nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and phosphorylated forms of Akt and p38 were diminished when PCSK9 was suppressed in GO fibroblasts. Decreases in lipid droplets and attenuated levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer-binding protein ß (C/EBPß), and leptin as well as hypoxia-inducible factor 1α (HIF-1α), manganese superoxide dismutase (MnSOD), thioredoxin (Trx), and heme oxygenase-1 (HO-1) were noted when PCSK9 was suppressed during adipocyte differentiation. The plasma PCSK9 level was significantly higher in GO patients and correlated with level of thyrotropin binding inhibitory immunoglobulin (TBII) and the clinical activity score (CAS). Conclusions: PCSK9 plays a significant role in GO. The PCSK9 inhibition attenuated the pro-inflammatory cytokines production, oxidative stress, and fibroblast differentiation into adipocytes. PCSK9 may serve as a therapeutic target and biomarker for GO.

Proinflammatory Effects of Calprotectin in Graves' Orbitopathy.

Purpose: Early detection and control of inflammation are important to manage Graves' orbitopathy (GO). We investigated the effects of calprotectin (S100A8/A9) on orbital fibroblast inflammation and GO pathogenesis.Methods: We measured serum calprotectin, S100A8 and S100A9 mRNA expression in orbital fat/connective tissue from GO patients and healthy controls, and proinflammatory cytokines in primary cultured orbital fibroblasts.Results: The serum levels of S100A8/A9 and the expression of S100A8/A9 mRNA in orbital tissue were higher in the GO patients than in the healthy controls. The serum calprotectin levels positively correlated with the clinical activity score and serum thyroid-stimulating immunoglobulin levels. In cultured GO orbital fibroblasts, S100A8/A9 increased the expression of interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1, as well as the phosphorylation of extracellular signal-regulated kinase and nuclear factor-κB.Conclusion: We demonstrated the potential of calprotectin as a biomarker of GO severity and proinflammatory responses to S100A8/A9 in GO orbital fibroblasts.