Fibronectin Content Research Articles

Unraveling the Local Relation Between Tissue Composition and Human Brain Mechanics Through Machine Learning.

The regional mechanical properties of brain tissue are not only key in the context of brain injury and its vulnerability towards mechanical loads, but also affect the behavior and functionality of brain cells. Due to the extremely soft nature of brain tissue, its mechanical characterization is challenging. The response to loading depends on length and time scales and is characterized by nonlinearity, compression-tension asymmetry, conditioning, and stress relaxation. In addition, the regional heterogeneity–both in mechanics and microstructure–complicates the comprehensive understanding of local tissue properties and its relation to the underlying microstructure. Here, we combine large-strain biomechanical tests with enzyme-linked immunosorbent assays (ELISA) and develop an extended type of constitutive artificial neural networks (CANNs) that can account for viscoelastic effects. We show that our viscoelastic constitutive artificial neural network is able to describe the tissue response in different brain regions and quantify the relevance of different cellular and extracellular components for time-independent (nonlinearity, compression-tension-asymmetry) and time-dependent (hysteresis, conditioning, stress relaxation) tissue mechanics, respectively. Our results suggest that the content of the extracellular matrix protein fibronectin is highly relevant for both the quasi-elastic behavior and viscoelastic effects of brain tissue. While the quasi-elastic response seems to be largely controlled by extracellular matrix proteins from the basement membrane, cellular components have a higher relevance for the viscoelastic response. Our findings advance our understanding of microstructure - mechanics relations in human brain tissue and are valuable to further advance predictive material models for finite element simulations or to design biomaterials for tissue engineering and 3D printing applications.

POSSIBILITIES OF ARGININE-CONTAINING THERAPY IN THE TREATMENT OF NEUROLOGICAL MANIFESTATIONS OF OSTEOCHONDROSIS OF THE LUMBAR PART OF THE SPINE

Purpose of the study – to estimate the pathogenetic expediency of L-arginine use inpatients with neurological manifestations of osteochondrosis of the spine lumbar parton the basis of estimation of the clinical picture, cerebral Doppler data and analysis ofthe blood plasma levels of some indices of endothelial dysfunction, such as fibronectin,soluble fms-similar tyrosine kinase 1 (sFlt - 1) and tumor necrotic factor (TNF-α),using Glutargine as an example. Material and methods. 104 male patients with neurological manifestations ofosteochondrosis of the lumbar spine at the age of 25-45 years (mean age 34.05 ± 5.7)without obesity, somatic and vascular pathology and 25 practically healthy persons,who did not differ in age from groups of the patients under study, have been examined.Among the examined patients, there were 50 patients with radiculopathies against abackground of herniated intervertebral discs, verified by means of neuroimaging, and54 patients with reflex manifestations of osteochondrosis of the lumbar part of the spine.The study of the clinical picture of osteochondrosis neurological manifestations of thelumbar spine was carried out by the method of standard neurological examinationusing a visual- analogue pain scale.The state of the autonomic nervous system was assessed using standard tests forautonomic reactivity and autonomic provision of activity, as well as using theQuestionnaire to identify signs of vegetative changes and the Study Scheme to identifysigns of autonomic disorders (O.M. Wein, 1993).The study of cerebral circulation was carried out by the method of extra-intracranialDoppler ultrasonography according to the standard techniques on the Sonomed-350apparatus using the Overshut test for cerebrovascular reactivity.Concentration of fibronectin in the blood plasma, tumor necrosis factor alpha andsoluble fms- similar tyrosine kinase was investigated by enzyme immunoassay accordingto the manufacturer's procedure under laboratory conditions.The paired Student's test and Fisher's angular Phi-transformation are used.Results. Statistically significant decrease of the pain syndrome has been revealedaccording to the VAS scale in the group of patients, who took Glutargin in the complextreatment. Significant changes in autonomic homeostasis have not been revealed whenusing Glutargin. The index of endothelium-dependent vasodilation in the group ofpatients, who took glutargin after treatment, approached the values of the control group(0.3 ± 0.06 at the values in the control group - 0.3 ± 0.096).Analysis of the data of fibronectin investigation, soluble fms-similar tyrosine kinase-1(sFlt - 1) and tumor necrotic factor (TNF-α) in the groups of patients with radicularmanifestations of osteochondrosis of the spine lumbar part before and after treatmenttestifies to that there were positive changes in the studied parameters in both groupsof patients, both with the use of standard treatment, and with the use of Glutargin,however, only in the group of patients, who took Glutargin in addition to the standardtreatment, a decrease in the content of fibronectin and tumor necrotic factor in bloodplasma acquired statistical significance. The increase in the indices of soluble fmslike tyrosine kinase-1 (sFlt - 1) in both groups of patients was insignificant and notstatistically veritable.Conclusions. Glutargin use in the complex treatment of patients with radicularneurological manifestations of lumbar osteochondrosis has a positive effect on theclinical course, reliably reducing the intensity of the pain syndrome and leads to animprovement in the endothelial functional state.

Angiogenic regulatory influence of extracellular matrix deposited by resting state asthmatic and non-asthmatic airway smooth muscle cells is similar.

The extracellular matrix (ECM) is the tissue microenvironment that regulates the characteristics of stromal and systemic cells to control processes such as inflammation and angiogenesis. Despite ongoing anti‐inflammatory treatment, low levels of inflammation exist in the airways in asthma, which alters ECM deposition by airway smooth muscle (ASM) cells. The altered ECM causes aberrant behaviour of cells, such as endothelial cells, in the airway tissue. We therefore sought to characterize the composition and angiogenic potential of the ECM deposited by asthmatic and non‐asthmatic ASM. After 72 hours under non‐stimulated conditions, the ECM deposited by primary human asthmatic ASM cells was equal in total protein, collagen I, III and fibronectin content to that from non‐asthmatic ASM cells. Further, the matrices of non‐asthmatic and asthmatic ASM cells were equivalent in regulating the growth, activity, attachment and migration of primary human umbilical vein endothelial cells (HUVECs). Under basal conditions, asthmatic and non‐asthmatic ASM cells intrinsically deposit an ECM of equivalent composition and angiogenic potential. Previous findings indicate that dysregulation of the airway ECM is driven even by low levels of inflammatory provocation. This study suggests the need for more effective anti‐inflammatory therapies in asthma to maintain the airway ECM and regulate ECM‐mediated aberrant angiogenesis.

High carbohydrate high fat diet induces the production of connective tissue growth factors, increased blood pressure, and changes in the aortic wall in aged rats

Age and diabetes are a risk factor for arterial hypertension. In the presented work, the morphological basis of an increase in blood pressure under the influence of a high-carbohydrate high-fat diet in old rats was investigated. It was determined the connective tissue growth factor levels and morphological changes in the aortic wall. A study was carried out in male Wistar rats weighing 350–400 g, 140 day old and rats weighing 530-620 g, 540 day old at the end of research. All procedures conformed to the Directive 2010/63/EU of the European Parliament and FASEB Statement of Principles for the use of Animals in Research and Education. Ethical approval was granted by the Ethical Committee of Research Institute of Cardiology, Tomsk National Research Medical Center. The high carbohydrate high fat diet (HCHFD) consisted from proteins 16%, fats 21%, carbohydrates 46%, including 17% fructose, 0.125% cholesterol, 90 days. Control group rats were fed a standard rat diet. It was found that the severity of arterial hypertension (Fig. 1), histological changes in the aortic wall (Fig. 2), and the level of connective tissue growth factors (TGFbeta, CTGF, fibronectin; Fig. 1) were significantly increased in elderly rats kept on a high-carbohydrate high-fat diet than young animals. In young rats kept on a high-carbohydrate, high-fat diet, arterial hypertension has a transient hypervolumic hallmark and does not cause changes in the aortic wall. The rise in blood pressure has a positive correlation with CTGF and fibronectin content (Fig. 1). These findings indicate that the formation of increased blood pressure in old rats when kept on a high carbohydrate high fat diet is associated with changes in the morphological structure of the aorta and is due to increased production of connective tissue growth factors.

KCa3.1 Inhibition Decreases Size and Alters Composition of Atherosclerotic Lesions Induced by Low, Oscillatory Flow

Low, oscillatory flow/shear patterns are associated with atherosclerotic lesion development. Increased expression of KCa3.1 has been found in Vascular Smooth Muscle (VSM), macrophages and T-cells in lesions from humans and mice. Increased expression of KCa3.1, is also required for VSM cell proliferation and migration. Previously, we showed that the specific KCa3.1 inhibitor, TRAM-34, could inhibit coronary neointimal development following balloon injury in swine. Atherosclerosis develops in regions with a low, oscillatory (i.e. atheroprone) flow pattern. Therefore, we used the Partial Carotid Ligation (PCL) model in high-fat fed, Apoe-/- mice to determine the role of KCa3.1 in atherosclerotic lesion composition and development. PCL was performed on 8–10 week old male Apoe-/- mice and subsequently placed on a Western diet (TD.88137, Teklad) for 4 weeks. Mice received daily s.c. injections of TRAM-34 (120 mg/kg) or equal volumes of vehicle (peanut oil, PO). 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34) treatment reduced lesion size ~50% (p < 0.05). In addition, lesions from TRAM-34 treated mice contained less collagen (6% ± 1% vs. 15% ± 2%; p < 0.05), fibronectin (14% ± 3% vs. 32% ± 3%; p < 0.05) and smooth muscle content (19% ± 2% vs. 29% ± 3%; p < 0.05). Conversely, TRAM-34 had no effect on total cholesterol (1455 vs. 1334 mg/dl, PO and TRAM, resp.) or body weight (29.1 vs. 28.8 g, PO and TRAM, resp.). Medial smooth muscle of atherosclerotic carotids showed diminished RE1-Silencing Transcription Factor (REST)/Neural Restrictive Silencing Factor (NRSF) expression, while REST overexpression in vitro inhibited smooth muscle migration. Together, these data support a downregulation of REST/NRSF and upregulation of KCa3.1 in determining smooth muscle and matrix content of atherosclerotic lesions.

The right ventricular transcriptome signature in Ossabaw swine with cardiometabolic heart failure: implications for the coronary vasculature.

Heart failure (HF) patients with deteriorating right ventricular (RV) structure and function have a nearly twofold increased risk of death compared with those without. Despite the well-established clinical risk, few studies have examined the molecular signature associated with this HF condition. The purpose of this study was to integrate morphological, molecular, and functional data with the transcriptome data set in the RV of a preclinical model of cardiometabolic HF. Ossabaw swine were fed either normal diet without surgery (lean control, n = 5) or Western diet and aortic-banding (WD-AB; n = 4). Postmortem RV weight was increased and positively correlated with lung weight in the WD-AB group compared with CON. Total RNA-seq was performed and gene expression profiles were compared and analyzed using principal component analysis, weighted gene co-expression network analysis, module enrichment analysis, and ingenuity pathway analysis. Gene networks specifically associated with RV hypertrophic remodeling identified a hub gene in MAPK8 (or JNK1) that was associated with the selective induction of the extracellular matrix (ECM) component fibronectin. JNK1 and fibronectin protein were increased in the right coronary artery (RCA) of WD-AB animals and associated with a decrease in matrix metalloproteinase 14 protein, which specifically degrades fibronectin. RCA fibronectin content was correlated with increased vascular stiffness evident as a decreased elastin elastic modulus in WD-AB animals. In conclusion, this study establishes a molecular and transcriptome signature in the RV using Ossabaw swine with cardiometabolic HF. This signature was associated with altered ECM regulation and increased vascular stiffness in the RCA, with selective dysregulation of fibronectin.

Evaluation of Wound-Healing and Antioxidant Effects of Marantodes pumilum (Blume) Kuntze in an Excision Wound Model

Marantodes pumilum (MP) is a great source of herbal medicine used traditionally by both men and women for various purposes. MP may have potential wound-healing effects due to its diverse biological properties. An extensive study was conducted in a normal male rat model for determining the effects of MP var. pumila (MPvp) and var. alata (MPva) on the wound healing process. Here, 126 male Sprague-Dawley rats were divided randomly into seven groups as follows: sham-operated (SH), vehicle dressing (VD), flavine dressing (FD), MPvp leaves (PL), MPvp roots (PR), MPva leaves (AL), and MPva roots (AR). The parameters studied were the percentage of wound contraction, histomorphology study by hematoxylin and eosin (H&E), Masson–Goldner trichrome (MGT), and immunohistochemistry (IHC) staining. In addition, the levels of enzymatic antioxidants and malondialdehyde were also measured in the wound tissue homogenates. Wounds treated with extracts (PL, PR, AL, and AR) showed significantly faster healing (p < 0.05) compared to untreated and control groups (SH, VD, and FD). Histological analysis among MP-treated groups revealed better re-epithelialization, higher collagen deposition, enhanced fibronectin content and fibroblast cells, and higher fiber transformation from collagen-III to collagen-I, accompanied with a significant surge in enzymatic antioxidant activities and a decline in lipid peroxidation. MP has antioxidant effects that may enhance wound healing in the rat model.

МАРКЕР АКТИВНОСТИ СКЛЕРОМНОГО ПРОЦЕССА

Scleroma is a chronic specific infectious disease of the respiratory tract, which has various forms with different activity of the process. In this work, we studied the concentration of fibronectin as a marker of scleroma activity in 39 patients, including 18 patients with an infiltrative form and 21 patients with a cicatricial form. The fibronectin content in the blood serum of patients with the infiltrative form was 708.1 ±29.4 ug ml. In the group of patients with cicatricial scleroma, the fibronectin concentration was 412.5 ± 20.1 ug ml (the differences between the groups are statistically significant, p &lt; 0.001). In none of the groups of patients with scleroma a relationship was found between the level of fibronectin and the duration of the disease, sex and age, and the prevalence of the pathological process in the respiratory tract. The data obtained make it possible to use the study of fibronectin concentration as a marker of the activity of the scleroma process.

Olive leaf extract attenuates adriamycin-induced focal segmental glomerulosclerosis in spontaneously hypertensive rats via suppression of oxidative stress, hyperlipidemia, and fibrosis.

Olive (Olea europaea L.) leaf extract (OLE) possesses powerful antioxidant, antihyperlipidemic, and anti-inflammatory properties. The aim was to investigated the effects of OLE on the hyperlipidemia, antioxidant defense, heme oxygenase/biliverdin reductase (HO/BVR) pathway, inflammation, and fibrosis in spontaneously hypertensive rats with focal segmental glomerulosclerosis (FSGS, a progressive form of chronic kidney disease) induced by adriamycin (2 mg/kg, i.v., twice in a 21-day period). Daily treatment of OLE (80 mg/kg, p.o.) for 6 weeks suppressed protein oxidation and lipid peroxidation (p < .01 and p < .001, respectively), significantly increased antioxidant enzymes activities and normalized antioxidant capacity, leading to the improvement of antioxidant defense independently of the HO/BVR pathway. Furthermore, the values of triglycerides (p < .01), total, and low-density lipoprotein cholesterol (p < .05, both) were improved by OLE. OLE strongly prevented glomerulosclerosis, interstitial inflammation, and fibrosis (renal injury score, FSGS: 8 ± 0.45 vs. FSGS+OLE: 4.20 ± 1.07; p < .01), as evidenced by normalized fibronectin content (p < .001), suppressed interstitial inflammatory cells infiltration and collagen deposition, without changing cytokines expressions. OLE decreased blood pressure with a tendency to reduce urine albumin loss. These data suggest that OLE may be effective in slowing down the progression of FSGS.

Proliferative Anterior Optic Membranes in Hydrophobic Acrylic Intraocular Lenses.

PurposeTo describe the observed stages of development and management of proliferative anterior optic membranes (AOM) seen on a popularly implanted single-piece hydrophobic intraocular lens (IOL).MethodsThis is an observational series of patients with AOMs managed by three surgeons in different geographical areas in the USA.ResultsAOMs may clinically present in one of the four distinct stages. Most patients were visually asymptomatic, but those with decreased visual acuity (Stage 4) required Nd:YAG laser treatment to remove the AOM. These patients had improved visual acuity without recurrence of the AOM during the one-year follow-up period.ConclusionAOMs can present and progress in four distinct stages with variable visual significance. AOMs may be safely and effectively treated with Nd:Yag laser. Occurrence of the membrane may be related to many factors, including high fibronectin content of this IOL. Additional studies are needed to identify incidence, etiology, and best management strategies.

Dynamics of Matrix Metalloproteinase Activity and Extracellular Matrix Proteins Content in the Process of Replicative Senescence of Human Mesenchymal Stem Cells

A comparative analysis of mesenchymal stem cells (MSCs) of differing origin is important because of their specific interaction with a unique microenvironment (niche) in a particular tissue. Some cellular processes are regulated through the interaction of extracellular matrix (ECM) proteins with matrix metalloproteinases (MMPs). In this work, we compared the dynamics of MMP activity and the level of ECM proteins during replicative senescence of three lines of human MSCs obtained from Wharton’s jelly of human umbilical cord (MSCWJ-1 line), eyelid skin (DF-2 line), and human epicardial adipose tissue isolated during coronary artery bypass grafting (ADH-MSC line). The fractions of cells with β-galactosidase enzyme activity (marker of replicative senescence) and the content of ECM proteins (fibronectin and type I collagen), as well as the activity of MMP-1, MMP-2, and MMP-9, were analyzed during long-term cultivation. It was found that three lines differ in the content of fibronectin, type 1 collagen, and MMP activity during the replicative senescence. Cells of the ADH-MSC line are mostly different from the other two lines in terms of aging rate, ECM protein content, and MMP activity. The reason for this discrepancy is that the cells are obtained from the tissue of a patient having heart disease.

Interstitial changes after reperfused myocardial infarction in swine: morphometric and genetic analysis

BackgroundFollowing myocardial infarction (MI), we aimed to characterize morphometric and genetic changes in extracellular matrix (ECM) components from ischemia onset until late phases after coronary reperfusion in necrotic and salvaged myocardium.ResultsSwine were divided into one control (n = 5) and three MI groups: 90-min of ischemia without reperfusion, or followed by 1-week or 1-month reperfusion (n = 5 per group). In samples from the necrotic and salvaged areas, ECM components were morphometrically quantified and mRNA levels of factors involved in ECM remodeling were evaluated. After 90-min of ischemia, fibronectin, laminin, and elastic fibers content as well as upregulated mRNA expression of tissue inhibitors of metalloproteinases (TIMP)1, TIMP2, TIMP3 and connective tissue growth factor increased in the necrotic and salvaged myocardium. In both reperfused MI groups, collagen-I, collagen-III, elastic fibers, glycosaminoglycans, laminin, and fibronectin levels heightened in the necrotic but not the salvaged myocardium. Moreover, mRNA expression of TIMP1, TIMP2 and TIMP3, as well as metalloproteinase-2 and metalloproteinase-9 heightened in the necrotic but not in the salvaged myocardium.ConclusionsMatrix remodeling starts after ischemia onset in both necrotic and salvaged myocardium. Even if ECM composition from the salvaged myocardium was altered after severe ischemia, ECM makes a full recovery to normal composition after reperfusion. Therefore, rapid coronary reperfusion is essential not only to save cardiomyocytes but also to preserve matrix, thus avoiding impaired left ventricular remodeling.

Effects of Insulin on Fibronectin Alterations in Sciatic Nerve of Diabetic Rats-A Brief Report

Objective: Alteration in the basement membrane proteins maybe associated with diabetic neuropathy. Fibronectin is one of the most important components of peripheral nerves basement membrane. In this study we investigated the effects of insulin administration on prevention of alteration in fibronectin contents of sciatic nerve in diabetic rats. Materials and Methods: Twenty-four wistar rats were divided into control, diabetic and diabetic with insulin treatment groups. Three months after diabetes induction, we measured blood glucose level, body weight and then expression of fibronectin in sciatic nerves of rats were evaluated by real time polymerase chain reaction (PCR) and immunohistochemical study. Results: Intensity of fibronectin immunoreactivity in the perineurium and endoneurium of sciatic nerves significantly increased in diabetic without treatment group compared to control group (P-value&lt; 0.001). Conclusion: This finding suggested that diabetic neuropathy resulted in increased of fibronectin contents in sciatic nerves of rats.

Adhesion to fibronectin via α5β1 integrin supports expansion of the megakaryocyte lineage in primary myelofibrosis

AbstractExcessive accumulation of extracellular matrix (ECM) is a hallmark of bone marrow (BM) milieu in primary myelofibrosis (PMF). Because cells have the ability to adhere to the surrounding ECM through integrin receptors, we examined the hypothesis that an abnormal ECM-integrin receptor axis contributes to BM megakaryocytosis in JAK2V617F+ PMF. Secretion of ECM protein fibronectin (FN) by BM stromal cells from PMF patients correlates with fibrosis and disease severity. Here, we show that Vav1-hJAK2V617F transgenic mice (JAK2V617F+) have high BM FN content associated with megakaryocytosis and fibrosis. Further, megakaryocytes from JAK2V617F+ mice have increased cell surface expression of the α5 subunit of the α5β1 integrin, the major FN receptor in megakaryocytes, and augmented adhesion to FN compared with wild-type controls. Reducing adhesion to FN by an inhibitory antibody to the α5 subunit effectively reduces the percentage of CD41+ JAK2V617F+ megakaryocytes in vitro and in vivo. Corroborating our findings in mice, JAK2V617F+ megakaryocytes from patients showed elevated expression of α5 subunit, and a neutralizing antibody to α5 subunit reduced adhesion to FN and megakaryocyte number derived from CD34+ cells. Our findings reveal a previously unappreciated contribution of FN-α5β1 integrin to megakaryocytosis in JAK2V617F+ PMF.

Protective effects of curcumin on bleomycin-induced changes in lung glycoproteins.

The present study investigated the therapeutic effect of curcumin on bleomycin (BLM)-induced alterations in glycoprotein components in the fibrotic lungs. Analysis of the bronchoalveolar lavage fluid (BALF) demonstrated increased fibronectin content at 3, 5, 7, and 14days after BLM administration. Similarly, lung tissue fibronectin content revealed a progressive increase at various times (days 3, 5, 7, 14, and 28) during the development of lung fibrosis. In addition, alveolar macrophage release of fibronectin was also elevated in BLM-treated rats. Analysis of carbohydrate moieties of glycoproteins revealed an increase in total hexose, fucose, sialic acid and hexosamine levels at 7, 14, and 28days after BLM treatment. Furthermore, the activities of lung glycosidases such as N-acetyl-β-D-glucosaminidase, β-glucosidase, β-galactosidase, and β-fucosidase in the fibrotic rats were elevated. Importantly, curcumin significantly inhibited the BLM-induced increases in BALF and lung fibronectin levels. Treatment of BLM rats with curcumin dramatically suppressed alveolar macrophage release of fibronectin. Curcumin also inhibited the increases in complex carbohydrates and glycosidases in the fibrotic lungs. These findings suggest that BLM-induced lung fibrosis is associated with accumulation of glycoproteins, and curcumin has the ability to suppress the enhanced deposition of glycoproteins in the fibrotic lung.

Influence of high-fructose diet on fibronectin level in rat blood serum

In recent years, the consumption of diets containing a large amount of fructose has increased sharply, which is associated with the observed increase in the incidence of obesity, diabetes mellitus, metabolic syndrome, and cardiovascular diseases. Changes in lipid and carbohydrate metabolism due to increased consumption of fructose are one of the causes of destructive processes in the tissues and cells of the organism. Fibronectin, which is an adhesive and regulatory protein of the intercellular matrix, can serve as one of the markers of these processes. The aim of the research was to study the effect of a high-fructose diet on rat serum fibronectin level. Material and methods. The experiment was performed on 50 outbred male rats aged 5-6 months. Rats from the control group (n=25) received a balanced diet of vivarium. Animals of the experimental group (n=25) for 35 days were fed a diet containing 60% fructose, 20% protein, 14% fiber, 6% fat. From the 36th to the 60th day, all rats were fed usual vivarium diet. On the 60th day, the formation of metabolic changes in the long-term period was evaluated. The animals of each group were weighed before and when removed from the experiment on the 21st, 35th and 60th day. Serum was obtained from blood samples collected during decapitation on an empty stomach under short-term ether anesthesia. The concentration of cholesterol, high and low density lipoproteins, triglycerides, glucose, insulin and fibronectin were determined in serum. The atherogenic index and insulin resistance indices were calculated using the corresponding formulas. Results and discussion. The experiment revealed a significant increase in glucose concentration from 6.52±0.35 (in control) to 7.74±0.73, 7.97±1.91 and 7.87±0.65 mmol/l and in insulin level from 1.6±0.6 to 2.3±0.4, 2.6±0.6 and 3.1±0.9 μU/ml on the 21nd, 35th and 60th days of the experiment, respectively, in animals of the experimental group, which is possibly associated with a sharp rise in the concentration of free fatty acids due to the special metabolism of fructose and the occurring oxidative stress, which causes cell damage and the formation of insulin resistance. This is reflected by insulin resistance indices that are different from the control: HOMA by 42.3, 49.2 and 81.3% higher, CARO by 13.3, 16.0 and 28.8% less than the control in the dynamics of the experiment. There was a tendency to an increase in cholesterol level with a significant rise on the 35th day of the experiment by 58.9% relative to the control. A statistically significant increase in the content of low-density lipoproteins was also observed by 158, 141 and 229% compared with the control. Such a rise can be associated with both increased formation of free fatty acids and lipogenetic activity of insulin. In addition, we revealed a significant increase in the content of fibronectin on days 21, 35 and 60 of the experiment, respectively, from 56.5±2.8 (in control) to 74±8.1, 79±3.8 and 98±2.1 μg/ml. Fibronectin plays a significant role in tissue repair processes, providing cell migration and adhesion. Its blood level can correlate with the degree of destructive and reparative processes in the cells of liver and vascular endothelium. Conclusion. A high-fructose diet in rats causes an increase in the studied biochemical parameters of carbohydrate and lipid metabolism, as well as an increase in blood level of the main regulatory protein of the intercellular matrix - fibronectin.

Downregulation of SEMA4C Inhibit Epithelial-Mesenchymal Transition (EMT) and the Invasion and Metastasis of Cervical Cancer Cells via Inhibiting Transforming Growth Factor-beta 1 (TGF-β1)-Induced Hela cells p38 Mitogen-Activated Protein Kinase (MAPK) Activation.

BackgroundEpithelial-mesenchymal transition (EMT) plays a key role in promoting invasion and metastasis of tumor cells. SEMA4C can regulate the generation of transforming growth factor-beta 1 (TGF-β1)-induced EMT in cervical cancer. This study investigated the relationship between the regulation of SEMA4C on TGF-β1-induced p38 mitogen-activated protein kinase (MAPK) activation and invasion and metastasis of cervical cancer.Material/MethodsHela-shSEMA4C cell line was established and the success of transfection was confirmed with fluorescence intensity. Cell experiments were divided into 2 groups. Group 1 was Hela, Hela-shNC, and Hela-shSEMA4C; and Group 2 was Hela, Hela-shNC, Hela-shSEMA4C, Hela+TGF-β1, Hela-shNC+TGF-β1, and Hela-shSEMA4C+TGF-β1. Group 1 was detected for SEMA4C mRNA expression by real-time polymerase chain reaction (RT-PCR), cell viability by Cell Counting Kit-8 (CCK-8), F-actin fluorescence intensity by immunofluorescence, cell migration by scratch test, and cell invasion by invasion test. Group 2 was analyzed for E-cadherin fluorescence intensity by immunofluorescence, human fibronectin (FN) content by enzyme-linked immunosorbent assay (ELISA), and SEMA4C, E-cadherin and p-p38 expressions by Western blot.ResultsFor Group 1, compared with Hela and Hela-shNC subgroups, the SEMA4C mRNA expression, cell viability, F-actin fluorescence intensity, cell migration and invasion ability in the Hela-shSEMA4C subgroup were significantly decreased (P<0.05). For Group 2, compared with Hela and Hela-shNC subgroups, the E-cadherin expression and fluorescence intensity in the Hela-shSEMA4C subgroup were significantly increased (P<0.01), while the FN content, SEMA4C, and p-p38 MAPK expressions were significantly decreased (P<0.01). Compared with Hela-shNC+TGF-β1 and Hela+TGF-β1 subgroups, the E-cadherin expression and fluorescence intensity in the Hela-shSEMA4C+TGF-β1 subgroup were significantly increased (P<0.01), while the FN content, SEMA4C and p-p38 expressions were significantly decreased (P<0.01).ConclusionsDownregulation of SEMA4C can inhibit EMT and the invasion and metastasis of cervical cancer cells via inhibiting TGF-β1-induced Hela cells p38 MAPK activation.

Chanzyme TRPM7 protects against cardiovascular inflammation and fibrosis.

AimsTransient Receptor Potential Melastatin 7 (TRPM7) cation channel is a chanzyme (channel + kinase) that influences cellular Mg2+ homeostasis and vascular signalling. However, the pathophysiological significance of TRPM7 in the cardiovascular system is unclear. The aim of this study was to investigate the role of this chanzyme in the cardiovascular system focusing on inflammation and fibrosis.Methods and resultsTRPM7-deficient mice with deletion of the kinase domain (TRPM7+/Δkinase) were studied and molecular mechanisms investigated in TRPM7+/Δkinase bone marrow-derived macrophages (BMDM) and co-culture systems with cardiac fibroblasts. TRPM7-deficient mice had significant cardiac hypertrophy, fibrosis, and inflammation. Cardiac collagen and fibronectin content, expression of pro-inflammatory mediators (SMAD3, TGFβ) and cytokines [interleukin (IL)-6, IL-10, IL-12, tumour necrosis factor-α] and phosphorylation of the pro-inflammatory signalling molecule Stat1, were increased in TRPM7+/Δkinase mice. These processes were associated with infiltration of inflammatory cells (F4/80+CD206+ cardiac macrophages) and increased galectin-3 expression. Cardiac [Mg2+]i, but not [Ca2+]i, was reduced in TRPM7+/Δkinase mice. Calpain, a downstream TRPM7 target, was upregulated (increased expression and activation) in TRPM7+/Δkinase hearts. Vascular functional and inflammatory responses, assessed in vivo by intra-vital microscopy, demonstrated impaired neutrophil rolling, increased neutrophil: endothelial attachment and transmigration of leucocytes in TRPM7+/Δkinase mice. TRPM7+/Δkinase BMDMs had increased levels of galectin-3, IL-10, and IL-6. In co-culture systems, TRPM7+/Δkinase macrophages increased expression of fibronectin, proliferating cell nuclear antigen, and TGFβ in cardiac fibroblasts from wild-type mice, effects ameliorated by MgCl2 treatment.ConclusionsWe identify a novel anti-inflammatory and anti-fibrotic role for TRPM7 and suggest that its protective effects are mediated, in part, through Mg2+-sensitive processes.

Secretion of fibronectin by human pancreatic stellate cells promotes chemoresistance to gemcitabine in pancreatic cancer cells

BackgroundGemcitabine remains a cornerstone in chemotherapy of pancreatic ductal adenocarcinoma (PDAC) despite suboptimal clinical effects that are partly due to the development of chemoresistance. Pancreatic stellate cells (PSCs) of the tumor stroma are known to interact with pancreatic cancer cells (PCCs) and influence the progression of PDAC through a complex network of signaling molecules that involve extracellular matrix (ECM) proteins. To understand tumor-stroma interactions regulating chemosensitivity, the role of PSC-secreted fibronectin (FN) in the development of gemcitabine resistance in PDAC was examined.MethodsPSC cultures obtained from ten different human PDAC tumors were co-cultured with PCC lines (AsPC-1, BxPC-3, Capan-2, HPAF-II, MIA PaCa-2, PANC-1 and SW-1990) either directly, or indirectly via incubation with PSC-conditioned medium (PSC-CM). Gemcitabine dose response cytotoxicity was determined using MTT based cell viability assays. Protein expression was assessed by western blotting and immunofluorescence. PSC-CM secretome analysis was performed by proteomics-based LC-MS/MS, and FN content in PSC-CM was determined with ELISA. Radiolabeled gemcitabine was used to determine the capacity of PCCs to uptake the drug.ResultsIn both direct and indirect co-culture, PSCs induced varying degrees of resistance to the cytotoxic effects of gemcitabine among all cancer cell lines examined. A variable degree of increased phosphorylation of ERK1/2 was observed across all PCC lines upon incubation with PSC-CM, while activation of AKT was not detected. Secretome analysis of PSC-CM identified 796 different proteins, including several ECM-related proteins such as FN and collagens. Soluble FN content in PSC-CM was detected in the range 175–350 ng/ml. Neither FN nor PSC-CM showed any effect on PCC uptake capacity of gemcitabine. PCCs grown on FN-coated surface displayed higher resistance to gemcitabine compared to cells grown on non-coated surface. Furthermore, a FN inhibitor, synthetic Arg-Gly-Asp-Ser (RGDS) peptide significantly inhibited PSC-CM-induced chemoresistance in PCCs via downregulation of ERK1/2 phosphorylation.ConclusionsThe findings of this study suggest that FN secreted by PSCs in the ECM plays a key role in the development of resistance to gemcitabine via activation of ERK1/2. FN-blocking agents added to gemcitabine-based chemotherapy might counteract chemoresistance in PDAC and provide better clinical outcomes.

Dahuang Zhechong Pill suppresses colorectal cancer liver metastasis via ameliorating exosomal CCL2 primed pre-metastatic niche

Ethnopharmacological relevanceDahuang Zhechong Pill (DZP) is a classical formula from “Synopsis of Prescriptions of the Golden Chamber”. It has been used for treatment of abdominal masses (including tumorous diseases) for thousands of years. Aim of the studyOur previous work showed that DZP suppresses CCl-4 induced hepatic fibrosis by downregulating the expression of interleukin-13. We aimed to test if DZP suppresses the metastasis of colorectal cancer (CRC) by ameliorating the fibrosis status of the future metastatic organ. Materials and methodsLiver metastasis was observed by injection of MC38-EGFP cells with stably expressing enhanced green fuorescence protein beneath the splenic capsule of C57BL/6J mice. MC38-EGFP-derived exosomes were analyzed by Label-free comparative proteomics. mRNA expression was determined by Quantitative PCR. Protein expression was determined by immunohistochemistry, immunofuorescence and Western blot. Collagen deposition was determined by Masson staining. All data were statistically analyzed using SPSS. ResultsDZP drastically reduced the metastatic tumor number and fluorescence intensity in a splenic liver metastasis model. It also lowered the expression of mature TGF-β1 and decreased the fibronectin contents & collagen deposition. Exosome proteomics showed that the upregualted CC chemokine ligand-2 (CCL2) was repressed by DZP treatment. Importantly, DZP markedly lowered the expression of CCL2 and its receptor CCR2 in the liver. Exosomal CCL2 activated macrophage recruitment and shifted the M1/M2 paradigm to a M2 phenotype. DZP reduced the macrophage infiltration and attenuated the M2 polarizaion in tumor-bearing mice liver. It decreased the F4/80 positive areas and specifically reduced the ratio of CCR2+ positive macrophage. Anti-fibrosis and inhibition of CCR2 suppress the growth and metastasis of CRC. ConclusionsDZP inhibits the liver metastasis of CRC by suppressing CCL2 mediated M2-skewing paradigm and ameliorating the pro-fibrotic microenvironment.