A treadmill exercise reduced cardiac fibrosis, inflammation and vulnerability to ischemia-reperfusion in rat pristane-induced arthritis.

AIMS: To explore cardiac structural and functional parameters and myocardial sensitivity to ischemia in a rat model of chronic arthritis, pristane-induced arthritis (PIA), and to investigate the effects of a running exercise protocol on cardiac disorders related to rheumatoid arthritis (RA). MAIN METHODS: 3 groups of male Dark Agouti rats were formed: Controls, PIA and PIA-Exercise. The PIA-Exercise group was subjected to an individualized treadmill running protocol during the remission phase. At acute and chronic phases of PIA, cardiac structure was analyzed by histology. Cardiac function was explored in isolated hearts to measure left ventricular developed pressure (LVDP), cardiac compliance and infarct size before and after ischemia/reperfusion. Cardiac inflammation was evaluated through VCAM-1 mRNA expression by RT-qPCR. Plasma irisin levels were measured by ELISA. KEY FINDINGS: PIA rats exhibited myocardial hypertrophy fibrosis and inflammation at the 2 inflammatory phases of the model. At chronic phase only, LVDP and cardiac compliance were lower in PIA compared to controls. As compared to sedentary PIA, exercise did not change cardiac function but reduced fibrosis, inflammation, infarct size, and arthritis severity and increased irisin levels. Cardiac inflammation positively correlated with fibrosis, while irisin levels negatively correlated with cardiac inflammation and fibrosis. SIGNIFICANCE: In the PIA model that recapitulated most cardiac disorders of RA, a daily program of treadmill running alleviated cardiac fibrosis and inflammation and improved resistance to ischemia. These data provide arguments to promote the practice of exercise in RA patients for cardiac diseases prevention.

Therapeutic role of FNDC5/irisin in attenuating liver fibrosis via inhibiting release of hepatic stellate cell-derived exosomes.

OBJECTIVE: Cleavage of fibronectin type III domain-containing protein 5 (FNDC5), a membrane-bound precursor protein, would cleave into a myokine, irisin, which is also expressed in the liver. FNDC5/Irisin has been reported to play a critical role in maintaining glucose and lipid homeostasis in the liver and in combating liver fibrosis. Recently, several studies have shown that extracellular vesicles (EVs) derived from hepatic stellate cells (HSCs) could modulate liver fibrosis; however, there is a large gap in understanding whether inhibition of fibrogenic EVs derived from HSCs could alleviate the progression of liver fibrosis. Here, we investigated the role of FNDC5/irisin in liver fibrosis and the mechanism of its inhibitory role in the release of HSC-derived fibrogenic EVs. METHODS: Experiments were performed in wild-type and FNDC5-/- mice, primary mouse HSCs, and human hepatic stellate cell line (LX2). Mice were treated with carbon tetrachloride (CCl4) or bile duct ligation (BDL) to induce liver fibrosis. EVs derived from HSCs were purified and injected intraperitoneally into mice. RESULTS: Our results showed that FNDC5 deficiency exacerbated CCl4-induced liver fibrosis and activation of HSCs in mice. Moreover, fibrogenic EVs derived from PDGF-BB-treated HSCs promoted HSC migration in vitro and liver fibrosis in vivo. However, administration of irisin, a cleavage of FNDC5, inhibited the release of fibrogenic EVs and activation of HSCs by promoting ubiquitylation degradation of Rab27b. In vivo, the promoting role of HSC-derived fibrogenic EVs in liver fibrosis was also reversed by irisin. CONCLUSION: All these results demonstrate that FNDC5/irisin is a novel therapeutic agent for chronic liver fibrosis.

Irisin attenuates fine particulate matter induced acute lung injury by regulating Nod2/NF-κB signaling pathway.

Air pollution consisting of fine particulate matter (PM2.5) can induce or aggravate pulmonary inflammatory injury. Irisin has been shown to inhibit inflammation and help to protect against acute kidney, lung or brain injury. However, the role of irisin in lung inflammation after exposure to PM2.5 remains unclear. The aim of this study was to investigate the effect and molecular mechanism of irisin supplementation on in vitro and in vivo models of PM2.5-induced acute lung injury（ALI). C57BL/6 mice and alveolar macrophage cell line (MH-S) were treated with PM2.5. Histopathological examination and FNDC5/ irisin immunofluorescence staining was performed on lung tissue sections. MH-S cell viability was determined by CCK-8 assay. The levels of Nod2, NF-κB p65 and NLRP3 were detected by qRT-PCR and western blotting. The levels of cytokines (IL-1ß, IL-18 and TNF-α) were detected by ELISA. PM2.5 exposure induced increased secretion of pro-inflammatory factors and activation of Nod2, NF-κB p65 and NLRP3 as well as endogenous levels of irisin. In vivo and in vitro inflammation was alleviated by irisin supplementation. Irisin significantly decreased IL-1ß, IL-18, and TNF-α production at both mRNA and protein level. Expression levels of Nod2, NF-κB p65, and NLRP3 were all significantly affected by irisin. In vivo the degree of pulmonary injury and inflammatory infiltration was weakened after irisin administration. In vitro, irisin could inhibit the activation of the NLRP3 inflammasome for a sustained period of 24 h, and its inhibitory ability was gradually enhanced. In conclusion, our findings indicate that irisin can modulate the inflammatory injury of lung tissue caused by PM2.5 through the Nod2/NF-κB signaling pathway, suggesting that irisin can be a candidate for the therapeutic or preventive intervention in acute lung inflammation.

Perivascular PDGFRB+ cells accompany lesion formation and clinical evolution differentially in two different EAE models.

BACKGROUND: Multiple Sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS) that may lead to progressive disability. Here, we explored the behavioral pattern and the role of vasculature especially PDGFRB+ pericytes/ perivascular cells, in MS pathogenesis. METHODS: We have evaluated vascular changes in two different experimental allergic encephalomyelitis (EAE) mice models (MOG and PLP-induced). PDGFRB+ cells demonstrated distinct and different behavioral patterns. In both models, fibrosis formation was detected via collagen, fibronectin, and extracellular matrix accumulation. RESULTS: The PLP-induced animal model revealed that fibrosis predominantly occurs in perivascular locations and that PDGFRB+ cells are accumulated around vessels. Also, the expression of fibrotic genes and genes coding extracellular matrix (ECM) proteins are upregulated. Moreover, the perivascular thick wall structures in affected vessels of this model presented primarily increased PDGFRB+ cells but not NG2+ cells in the transgenic NG2-DsRed transgenic animal model. On the other hand, in MOG induced model, PDGFRB+ perivascular cells were accumulated at the lesion sites. PDGFRB+ cells colocalized with ECM proteins (collagen, fibronectin, and lysyl oxidase L3). Nevertheless, both MOG and PLP-immunized mice showed increasing EAE severity, and disability parallel with enhanced perivascular cell accumulation as the disease progressed from earlier (day 15) to later (day 40). CONCLUSION: As a result, we have concluded that PDGFRB+ perivascular cells may be participating in lesion progression and as well as demonstrating different responses in different EAE models.

TRPM7 restrains plasmin activity and promotes transforming growth factor-ß1 signaling in primary human lung fibroblasts.

Sustained exposure of the lung to various environmental or occupational toxins may eventually lead to pulmonary fibrosis, a devastating disease with no cure. Pulmonary fibrosis is characterized by excessive deposition of extracellular matrix (ECM) proteins such as fibronectin and collagens. The peptidase plasmin degrades the ECM, but protein levels of the plasmin activator inhibitor-1 (PAI-1) are increased in fibrotic lung tissue, thereby dampening plasmin activity. Transforming growth factor-ß1 (TGF-ß1)-induced activation of SMAD transcription factors promotes ECM deposition by enhancing collagen, fibronectin and PAI-1 levels in pulmonary fibroblasts. Hence, counteracting TGF-ß1-induced signaling is a promising approach for the therapy of pulmonary fibrosis. Transient receptor potential cation channel subfamily M Member 7 (TRPM7) supports TGF-ß1-promoted SMAD signaling in T-lymphocytes and the progression of fibrosis in kidney and heart. Thus, we investigated possible effects of TRPM7 on plasmin activity, ECM levels and TGF-ß1 signaling in primary human pulmonary fibroblasts (pHPF). We found that two structurally unrelated TRPM7 blockers enhanced plasmin activity and reduced fibronectin or PAI-1 protein levels in pHPF under basal conditions. Further, TRPM7 blockade strongly inhibited fibronectin and collagen deposition induced by sustained TGF-ß1 stimulation. In line with these data, inhibition of TRPM7 activity diminished TGF-ß1-triggered phosphorylation of SMAD-2, SMAD-3/4-dependent reporter activation and PAI-1 mRNA levels. Overall, we uncover TRPM7 as a novel supporter of TGF-ß1 signaling in pHPF and propose TRPM7 blockers as new candidates to control excessive ECM levels under pathophysiological conditions conducive to pulmonary fibrosis.

Oroxylin A reversed Fibronectin-induced glioma insensitivity to Temozolomide by suppressing IP3R1/AKT/ß-catenin pathway.

AIMS: Cell adhesion mediated-drug resistance (CAM-DR) is one of main reasons for. the limitation to chemotherapy, but the underlying mechanism remains unclear in glioma. In this study, we investigated the mechanism of CAM-DR induced by Fibronectin (Fn). Besides, we studied the reversal effect of Oroxylin A, a natural flavonoid extracted from Scutellaria radix, on Temozolomide (TMZ) insensitivity of glioma cells. MAIN METHODS: Human Fn protein was used to mimic cell adhesion model and investigate its effect on the insensitivity of glioma cells to TMZ. Moreover, Oroxylin A was studied regarding its reversal effect on TMZ insensitivity of glioma via multiple molecular biological methods such as MTT, cell apoptosis assay, siRNA transfection, western blot, immunofluorescence assay. KEY FINDINGS: Fn could decrease the apoptosis-inducing effect of TMZ and led to the CAM-DR in glioma cells. Further studies showed that up-regulations of IP3R1 and intracellular Ca2+ level induced the activation of AKT kinase which increased the phosphorylation of GSK-3ß and subsequently caused the entry of ß-catenin into the nucleus. Knocking down IP3R1 significantly improved the sensitivity of glioma cells to TMZ. Meanwhile, after treatment with low-toxic concentration of Oroxylin A, the apoptosis induced by TMZ under Fn condition increased dramatically. Furthermore, our results revealed that Oroxylin A markedly inhibited the expression of IP3R1 and the activation of AKT/ß-catenin pathway. SIGNIFICANCE: Oroxylin A could reverse the insensitivity of TMZ via suppressing IP3R1/AKT/ß-catenin pathway and it might be helpful for enhancing the anti-cancer effect of TMZ in glioma.

Cellular Fibronectin Containing Extra Domain A Causes Insulin Resistance via Toll-like Receptor 4.

We determined the role of cellular fibronectin (CFN) containing the alternatively spliced extra domain A (FN-EDA) in causing insulin resistance (IR) through toll-like receptor 4 (TLR4). Circulating FN-EDA level was evaluated in mouse and rat IR models. Specific anti-FN-EDA antibody and TLR4 inhibitor were used to study its role in IR in mice. CFN protein was injected to evaluate TLR4 dependent effect of FN-EDA in IR. Furthermore, FN-EDA was estimated in blood plasma and correlated with demographic and clinical characteristics in healthy human participants (n = 38). High-fat diet feeding significantly increased circulating FN-EDA in both mouse (P = 0.03) and rat (P = 0.02) IR models. Antibody against FN-EDA protected mice from IR by increasing glucose disposal rate following glucose (P = 0.02) and insulin (P = 0.01) tolerance tests. CFN protein injection caused IR, however, TLR4 inhibitor protected the mice from CFN induced IR. Multivariate regression analysis predicted an independent positive correlation between circulating FN-EDA and fasting plasma glucose (P = 0.003) in healthy human participants. In conclusion, FN-EDA may cause IR through TLR4 by decreasing glucose disposal rate following glucose and insulin load. Targeting FN-EDA thus can be considered as a possible therapeutic strategy to delay prediabetes progression to diabetes.

Efficacy of RetroNectin-activated cytokine-induced killer cell therapy in metastatic brain tumor patients.

BACKGROUND: The aim of this study was to investigate the clinical efficacy of RetroNectin-activated cytokine-induced killer cell (R-CIK) therapy following conventional therapies in patients with metastatic brain tumors. METHODS: This study included 20 patients with metastatic brain tumors. Patients received R-CIK therapy following conventional therapies (including chemotherapy and target therapy). Progression-free survival (PFS), overall survival (OS), and prognostic factors were evaluated. RESULTS: Of the 4 breast cancer patients in our cohort, 2 remained alive and 2 died. Of the 14 non-small cell lung cancer (all adenocarcinoma) patients, 3 had a partial response, 8 had stable disease, and 3 had progressive disease after receiving R-CIKs. The overall response rate was 21.4% (3/14), and the disease control rate was 78.6% (11/14). The median PFS and OS were 7.7 months (95% confidence interval (CI) 3-16.5 months) and 12.6 months (95% CI 6-21 months), respectively. CONCLUSION: R-CIKs combined with conventional therapies could improve the prognosis of metastatic brain tumor patients, especially of those with adenocarcinoma of the lung.

Phase 2 study of CT-322, a targeted biologic inhibitor of VEGFR-2 based on a domain of human fibronectin, in recurrent glioblastoma.

VEGF signaling through VEGFR-2 is the major factor in glioblastoma angiogenesis. CT-322, a pegylated protein engineered from the 10th type III human fibronectin domain, binds the VEGFR-2 extracellular domain with high specificity and affinity to block VEGF-induced VEGFR-2 signaling. This study evaluated CT-322 in an open-label run-in/phase 2 setting to assess its efficacy and safety in recurrent glioblastoma. Eligible patients had 1st, 2nd or 3rd recurrence of glioblastoma with measurable tumor on MRI and no prior anti-angiogenic therapy. The initial CT-322 dose was 1 mg/kg IV weekly, with plans to escalate subsequent patients to 2 mg/kg weekly if tolerated; within each CT-322 dose cohort, patients were randomized to ±irinotecan IV semiweekly. The primary endpoint was 6-month progression-free survival (PFS-6). Sixty-three patients with a median age of 56 were treated, the majority at first recurrence. One-third experienced serious adverse events, of which four were at least possibly related to study treatment (two intracranial hemorrhages and two infusion reactions). Twenty-nine percent of subjects developed treatment-emergent hypertension. The PFS-6 rate in the CT-322 monotherapy groups was 18.6 and 0.0 % in the 1 and 2 mg/kg treatment groups, respectively; results from the 2 mg/kg group indicated that the null hypothesis that PFS-6 ≤12 % could not be rejected. The study was terminated prior to reaching the planned enrollment for all treatment groups because data from the completed CT-322 2 mg/kg monotherapy treatment arm revealed insufficient efficacy. Despite biological activity and a tolerable side effect profile, CT-322 failed to meet the prespecified threshold for efficacy in recurrent glioblastoma.

When do we transfuse cryoprecipitate?

BACKGROUND: The 2001 National Health and Medical Research Council/Australasian Society of Blood Transfusion Clinical Practice Guidelines for cryoprecipitate are being updated, and cryoprecipitate has been incorporated into new Patient Blood Management modules. AIMS: This clinical audit sought to clarify current cryoprecipitate use in Victoria, Tasmania and the Australian Capital Territory; assess adherence to guidelines; and gain insights into deviations from recommended practice. This information can be utilised in updating guidelines to make them more relevant, to identify areas for clinician education and to form a baseline of practice prior to release of the 2011 guidelines. METHODS: Participating institutions were invited to audit up to 30 consecutive episodes of cryoprecipitate transfusion over an 11-month period in 2008. The audits were conducted using a standardised pro forma and involved review of patient records. These were collated electronically using algorithms to determine alignment versus non-alignment with guidelines. RESULTS: Cryoprecipitate is used in a variety of situations with surgery accounting for the highest volume. Twenty-six per cent (26%) of transfusions were aligned with 2001 guidelines rising to 61% with a modified fibrinogen trigger. Fibrinogen levels did not appear to dictate all clinical decisions regarding cryoprecipitate use perhaps owing to the acuity of many cases. Additional bleeding risk together with low fibrinogen levels (e.g. thrombocytopenic patients) may contribute to empiric cryoprecipitate use. CONCLUSIONS: These results highlight discrepancies between guidelines and practice, providing rationale for the update of the guidelines that is currently underway. Cryoprecipitate has attendant risks, and it is appropriate that transfusion be restricted to situations with good evidence or sound principles to underpin use.

Open clinical study of eye drops containing the fibronectin-derived peptide PHSRN for treatment of persistent corneal epithelial defects.

PURPOSE: We have previously shown that the fibronectin-derived peptide PHSRN (Pro-His-Ser-Arg-Asn) promotes corneal epithelial wound healing in vivo. We have now examined the clinical efficacy of eye drops containing the PHSRN peptide for treatment of persistent epithelial defects (PEDs) of the cornea. METHODS: Seven patients (5 men and 2 women; mean age±SD, 78.3±9.4 years) with PEDs were treated by administration of eye drops containing PHSRN. The duration of the PEDs before treatment was 7.4±5.5 weeks. The eye drops were administered as 1 drop per eye 4 times a day. Epithelial defects were observed with a slit-lamp microscope and photographed during the treatment course. RESULTS: Epithelial defects in 5 of the 7 affected eyes (71%) responded to PHSRN treatment as manifested by complete epithelial resurfacing within the 4-week period after treatment initiation. The mean±SD time required for complete epithelial resurfacing in the 5 responding subjects was 15.8±3.4 days. No adverse effects of treatment were observed in any of the subjects. CONCLUSIONS: Eye drops containing the fibronectin-derived PHSRN peptide are clinically efficacious for the treatment of PEDs.

Fibronectin III 13-14 domains induce joint damage via Toll-like receptor 4 activation and synergize with interleukin-1 and tumour necrosis factor.

Cartilage loss is a feature of chronic arthritis. It results from degradation of the extracellular matrix which is composed predominantly of aggrecan and type II collagen. Extracellular matrix degradation is mediated by aggrecanases and matrix metalloproteinases (MMPs). Recently, a number of endogenous matrix molecules, including fibronectin (FN), have been implicated in mediating cartilage degradation. We were interested in studying the C-terminal heparin-binding region of FN since it mediates aggrecan and type II collagen breakdown in cartilage, but the specific FN domains responsible for proteolytic enzyme activity and their receptors in cartilage are unknown. In this study, the ability of recombinant FN domains to induce cartilage breakdown was tested. We found that the FN III 13-14 domains in the C-terminal heparin-binding region of FN are potent inducers of aggrecanase activity in articular cartilage. In murine studies, the FN III 13-14-induced aggrecanase activity was inhibited in Toll-like receptor 4 (TLR4) knockout mice but not wild-type mice. FN III 13-14 domains also synergized with the known catabolic cytokines interleukin-1α and tumour necrosis factor and induced secretion of MMP-1, MMP-3, gp38 and serum amyloid-like protein A in chondrocytes. Our studies provide a mechanistic link between the innate immune receptor TLR4 and sterile arthritis induced by the FN III 13-14 domains of the endogenous matrix molecule FN.

Phase I and pharmacokinetic study of CT-322 (BMS-844203), a targeted Adnectin inhibitor of VEGFR-2 based on a domain of human fibronectin.

PURPOSE: To determine the maximum tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics, immunogenicity, and preliminary antitumor activity of CT-322 (BMS-844203), a VEGFR-2 inhibitor and the first human fibronectin domain-based targeted biologic (Adnectin) to enter clinical studies. EXPERIMENTAL DESIGN: Patients with advanced solid malignancies were treated with escalating doses of CT-322 intravenously (i.v.) weekly (qw), or biweekly (q2w). Plasma samples were assayed for CT-322 concentrations, plasma VEGF-A concentrations, and antidrug antibodies. RESULTS: Thirty-nine patients completed 105 cycles of 0.1 to 3.0 mg/kg CT-322 i.v. either qw or q2w. The most common treatment-emergent grade 1/2 toxicities were fatigue, nausea, proteinuria, vomiting, anorexia, and hypertension. Grade 3/4 toxicities were rare. Reversible proteinuria, retinal artery, and vein thrombosis, left ventricular dysfunction, and reversible posterior leukoencephalopathy syndrome were dose limiting at 3.0 mg/kg. The MTD was 2 mg/kg qw or q2w. CT-322 plasma concentrations increased dose proportionally. Plasma VEGF-A levels increased with dose and plateaued at 2 mg/kg qw. Anti-CT-322 antibodies developed without effects on pharmacokinetics, VEGF-A levels, or safety. Minor decreases in tumor measurements occurred in 4 of 34 evaluable patients and 24 patients had stable disease. CONCLUSIONS: CT-322 can be safely administered at 2 mg/kg i.v. qw or q2w and exhibits promising antitumor activity in patients with advanced solid tumors. The absence of severe toxicities at the MTD, demonstration of plasma drug concentrations active in preclinical models, and clinical pharmacodynamic evidence of VEGFR-2 inhibition warrant further development of CT-322 and suggest strong potential for Adnectin-based targeted biologics.

Diagnóstico de la amenaza de parto prematuro / Diagnosis of preterm labour

La prematuridad está aumentando en todoel mundo y es actualmente uno de los principalesproblemas obstétricos. En la mayoría de loscasos de parto prematuro se produce previamenteun cuadro clínico de trabajo de partopretérmino. El diagnóstico clásico de esta entidadmediante la presencia de contraccionesuterinas y modificaciones cervicales clínicamentemuestra una baja capacidad diagnósticacon una alta tasa de falsos positivos. La valoraciónecográfica transvaginal y la determinaciónde fibronectina en las secreciones cervico-vaginales presentan una buena especificidady valor predictivo negativo y su incorporacióna un algoritmo diagnóstico podría ayudara diferenciar la verdadera de la falsa amenazade parto prematuro

Preterm birth have soared all over the world and is one of the top obstetric problems nowadays. Most cases of preterm delivery present clinical signs of preterm labor. Classic diagnosis of this disease by the presence of uterine contractions and clinical cervix effacement and dilatation show a low diagnostic accuracy with a high false-positive rate. Cervical ultrasound measurement and determination of fibronectin in cervico-vaginal secretions present a high especificity and negative predictive value and its inclusion in a diagnostic algorithm may help to differenciate the true from the false preterm labor (AU)

Fibronectin matrix deposition and cell contractility: implications for airway remodeling in asthma.

The adhesion of cells to the extracellular matrix (ECM) protein, fibronectin, is important in the regulation and coordination of such complex processes as cell growth, migration, differentiation, and ECM organization. The deposition of fibronectin into the ECM is a cell-dependent process that is normally tightly regulated to ensure controlled matrix deposition. Increased deposition of fibronectin and collagen into the subepithelial space of the airways is observed in all forms of asthma and occurs early in the progression of the disease. Experimental evidence suggests a model in which fibronectin matrix accumulation contributes to the progression of asthma by altering both the structural properties of the airways and the functional properties of cells of the airway wall.

Recurrent spontaneous intracerebral hemorrhage in a congenitally afibrinogenemic patient: diagnostic pitfalls and therapeutic options.

BACKGROUND: Coagulation disorders can cause intracerebral bleeding that may be difficult to detect since subsequent aberrant clot formation may mask early detection. This is an important pitfall because, when diagnosed early, bleeding in these patients is treatable. CASE DESCRIPTION: A patient with congenital afibrinogenemia presented with recurrent hemiparesis. Spontaneous intracerebral hemorrhage was diagnosed, despite an initial negative CT scan. Diagnosis, therapy, and complications of therapy are discussed. CONCLUSIONS: Intracerebral hemorrhage must be strongly suspected in any patient with a coagulation disorder presenting with matching clinical symptoms. Therapy must be installed immediately, before additional investigations, and should be continued even when initial neuroimaging is negative.