ABSTRACT
Prostate cancer (PCa) is the most prevalent non-cutaneous cancer in men. Early PCa detection has been made possible by the adoption of screening methods based on the serum prostate-specific antigen and Gleason score (GS). The aim of this study was to correlate gene expression with the differentiation level of prostate adenocarcinomas, as indicated by GS. We used data from The Cancer Genome Atlas (TCGA) and included 497 prostate cancer patients, 52 of which also had normal tissue sample sequencing data. Gene ontology analysis revealed that higher GSs were associated with greater responses to DNA damage, telomere lengthening, and cell division. Positive correlation was found with transcription factor activator of the adenovirus gene E2 (E2F) and avian myelocytomatosis viral homolog (MYC) targets, G2M checkpoints, DNA repair, and mitotic spindles. Immune cell deconvolution revealed high M0 macrophage counts and an increase in M2 macrophages dependent on the GS. The molecular pathways most correlated with GSs were cell cycle, RNA transport, and calcium signaling (depleted). A combinatorial approach identified a set of eight genes able to differentiate by k-Nearest Neighbors (kNN) between normal tissues, low-Gleason tissues, and high-Gleason tissues with high accuracy. In conclusion, our study could be a step forward to better understanding the link between gene expression and PCa progression and aggressiveness.
Subject(s)
Gene Regulatory Networks , Prostatic Neoplasms , Male , Humans , Prostatic Neoplasms/genetics , Cell Cycle , Cell Division , AdenoviridaeABSTRACT
Despite neutrophil involvement in inflammation and tissue repair, little is understood about their inflammatory status in acute coronary syndrome (ACS) patients with poor outcomes. Hence, we investigated the potential correlation between neutrophil inflammatory markers and the prognosis of ACS patients with/without diabetes and explored whether neutrophils demonstrate a unique inflammatory phenotype in patients experiencing an adverse in-hospital outcome. The study enrolled 229 ACS patients with or without diabetes. Poor evolution was defined as either death, left ventricular ejection fraction (LVEF) <40%, Killip Class 3/4, ventricular arrhythmias, or mechanical complications. Univariate and multivariate analyses were employed to identify clinical and paraclinical factors associated with in-hospital outcomes. Neutrophils isolated from fresh blood were investigated using qPCR, Western blot, enzymatic assay, and immunofluorescence. Poor evolution post-myocardial infarction (MI) was associated with increased number, activity, and inflammatory status of neutrophils, as indicated by significant increase of Erythrocyte Sedimentation Rate (ESR), C-reactive protein (CRP), fibrinogen, interleukin-1ß (IL-1ß), and, interleukin-6 (IL-6). Among the patients with complicated evolution, neutrophil activity had an important prognosis value for diabetics. Neutrophils from patients with unfavorable evolution revealed a pro-inflammatory phenotype with increased expression of CCL3, IL-1ß, interleukin-18 (IL-18), S100A9, intracellular cell adhesion molecule-1 (ICAM-1), matrix metalloprotease (MMP-9), of molecules essential in reactive oxygen species (ROS) production p22phox and Nox2, and increased capacity to form neutrophil extracellular traps. Inflammation is associated with adverse short-term prognosis in acute ACS, and inflammatory biomarkers exhibit greater specificity in predicting short-term outcomes in diabetics. Moreover, neutrophils from patients with unfavorable evolution exhibit distinct inflammatory patterns, suggesting that alterations in the innate immune response in this subgroup may exert detrimental effects on disease progression.
Subject(s)
Acute Coronary Syndrome , Inflammation , Neutrophils , Humans , Neutrophils/metabolism , Neutrophils/immunology , Acute Coronary Syndrome/blood , Acute Coronary Syndrome/complications , Male , Female , Prognosis , Middle Aged , Aged , Inflammation/blood , Inflammation/pathology , Biomarkers/blood , Diabetes Mellitus/blood , Diabetes Mellitus/immunology , Diabetes Mellitus/pathologyABSTRACT
The aim of the study was to use transcriptomics data to identify genes associated with advanced/aggressive breast cancer and their effect on survival outcomes. We used the publicly available The Cancer Genome Atlas (TCGA) database to obtain RNA sequence data from patients with less than five years survival (Poor Prognosis, n = 101), patients with greater than five years survival (Good Prognosis, n = 200), as well as unpaired normal tissue data (normal, n = 105). The data analyses performed included differential expression between groups and selection of subsets of genes, gene ontology, cell enrichment analysis, and survival analyses. Gene ontology results showed significantly reduced enrichment in gene sets related to tumor immune microenvironment in Poor Prognosis and cell enrichment analysis confirmed significantly reduced numbers of macrophages M1, CD8 T cells, plasma cells and dendritic cells in samples in the Poor Prognosis samples compared with Good Prognosis. A subset of 742 genes derived from differential expression analysis as well as genes coding for immune checkpoint molecules was evaluated for their effect on overall survival. In conclusion, this study may contribute to the better understanding of breast cancer transcriptomics and provide possible targets for further research and eventual therapeutic interventions.
Subject(s)
Breast Neoplasms , Humans , Female , Breast Neoplasms/genetics , Prognosis , Aggression , CD8-Positive T-Lymphocytes , Data Analysis , Tumor Microenvironment/geneticsABSTRACT
Resident macrophages from dorsal root ganglia are important for the development of traumatic-induced neuropathic pain. In the first 5-7 days after a traumatic sciatic nerve injury (i.e., spinal nerve ligation (SNL), spared nerve injury (SNI), sciatic nerve transection or sciatic nerve ligation and transection), Ionized binding adapter protein 1 (Iba1) (+) resident macrophages cluster around dorsal root ganglia neurons, possibly contributing to nerve injury-induced hypersensitivity. Since infiltrating macrophages gradually recruited to the lesion site peak at about 7 days, the first few days post-lesion offer a window of opportunity when the contribution of Iba1 (+) resident macrophages to neuropathic pain pathogenesis could be investigated. Iba1 is an actin cross-linking cytoskeleton protein, specifically located only in macrophages and microglia. In this study, we explored the contribution of rat Iba1 (+) macrophages in SNL-induced neuropathic pain by using intra-ganglionic injections of naked Iba1-siRNA, delivered at the time the lesion occurred. The results show that 5 days after Iba1 silencing, Iba1 (+) resident macrophages are switched from an M1 (pro-inflammatory) phenotype to an M2 (anti-inflammatory) phenotype, which was confirmed by a significant decrease of M1 markers (CD32 and CD86), a significant increase of M2 markers (CD163 and Arginase-1), a reduced secretion of pro-inflammatory cytokines (IL-6, TNF-α and IL-1ß) and an increased release of pro-regenerative factors (BDNF, NGF and NT-3) which initiated the regrowth of adult DRG neurites and reduced SNL-induced neuropathic pain. Our data show for the first time, that it is possible to induce macrophages towards an anti-inflammatory phenotype by interacting with their cytoskeleton.
Subject(s)
Neuralgia , Animals , Rats , Analgesics/pharmacology , Anti-Inflammatory Agents/pharmacology , Ganglia, Spinal/metabolism , Hyperalgesia/metabolism , Macrophages/metabolism , Neuralgia/genetics , Neuralgia/therapy , Spinal Nerves/metabolismABSTRACT
Iba1 (ionized calcium binding adapter protein 1) is a cytoskeleton protein specific only for microglia and macrophages, where it acts as an actin-cross linking protein. Although frequently regarded as a marker of activation, its involvement in cell migration, membrane ruffling, phagocytosis or in microglia remodeling during immunological surveillance of the brain suggest that Iba1 is not a simple cytoskeleton protein, but a signaling molecule involved in specific signaling pathways. In this study we investigated if Iba1 could also represent a drug target, and tested the hypothesis that its specific silencing with customized Iba1-siRNA can modulate microglia functioning. The results showed that Iba1-silenced BV2 microglia migrate less due to reduced proliferation and cell adhesion, while their phagocytic activity and P2x7 functioning was significantly increased. Our data are the proof of concept that Iba1 protein is a new microglia target, which opens a new therapeutic avenue for modulating microglia behavior.
Subject(s)
Calcium-Binding Proteins/metabolism , Cytoskeletal Proteins/metabolism , Gene Silencing , Microfilament Proteins/metabolism , Microglia/metabolism , Animals , Cell Adhesion , Cell Count , Cell Line , Cell Movement , Cell Proliferation , Leukocyte Common Antigens/metabolism , Mice , Opsonin Proteins/metabolism , Phagocytosis , RNA, Small Interfering/metabolism , Receptors, Purinergic P2X7/metabolism , Reproducibility of Results , Zymosan/metabolismABSTRACT
Adipose tissue-derived stem cells (ADSCs) are pluripotent mesenchymal stem cells found in relatively high percentages in the adipose tissue and able to self-renew and differentiate into many different types of cells. "Extracellular vesicles (EVs), small membrane vesicular structures released during cell activation, senescence, or apoptosis, act as mediators for long distance communication between cells, transferring their specific bioactive molecules into host target cells". There is a general consensus on how to define and isolate ADSCs, however, multiple separation and characterization protocols are being used in the present which complicate the results' integration in a single theory on ADSCs' and their derived factors' way of action. Metabolic syndrome and type 2 diabetes mellitus (T2DM) are mainly caused by abnormal adipose tissue size, distribution and metabolism and so ADSCs and their secretory factors such as EVs are currently investigated as therapeutics in these diseases. Moreover, due to their relatively easy isolation and propagation in culture and their differentiation ability, ADSCs are being employed in preclinical studies of implantable devices or prosthetics. This review aims to provide a comprehensive summary of the current knowledge on EVs secreted from ADSCs both as diagnostic biomarkers and therapeutics in diabetes and associated cardiovascular disease, the molecular mechanisms involved, as well as on the use of ADSC differentiation potential in cardiovascular tissue repair and prostheses.
Subject(s)
Adipose Tissue/cytology , Cardiovascular Diseases/metabolism , Diabetes Mellitus/metabolism , Extracellular Vesicles/metabolism , Mesenchymal Stem Cells/metabolism , Metabolic Syndrome/metabolism , Adipose Tissue/pathology , Animals , Biomarkers/metabolism , Cardiovascular Diseases/pathology , Cardiovascular Diseases/therapy , Diabetes Mellitus/pathology , Diabetes Mellitus/therapy , Extracellular Vesicles/transplantation , Humans , Metabolic Syndrome/pathology , Metabolic Syndrome/therapyABSTRACT
In this study, we evaluated the effects of epigallocatechin-3-O-gallate (EGCG) in two cancer cell lines, A-431 overexpressing ErbB1 and SK-BR-3, overexpressing ErbB2. EGCG treatment showed dose-dependent collapse of mitochondrial membrane potential (Δψm), increase in reactive oxygen species (ROS) production, changes in nuclear morphology and reduced viability. Flow cytometry data indicated that EGCG partially decreases the phosphorylation of several proteins involved in cell proliferation and survival: pErbB1(Y1173, Y1068), pAkt(S473) and pERK(Y204). EGCG affected the clonogenic growth in both cell lines with an EC50 of 2.5 and 5.4 µM for A-431 and SK-BR-3, respectively. Wound scratch assay demonstrated that EGCG inhibited the healing in dose-dependent manner and the effect was correlated with partial reduction in phosphorylation of pFAK(S910). Our data suggest that EGCG administration might reduce the unfavourable traits, particularly associated with ErbB1/EGFR overexpression.
Subject(s)
Breast Neoplasms/classification , Breast Neoplasms/drug therapy , Catechin/analogs & derivatives , Cell Proliferation/drug effects , Catechin/chemistry , Catechin/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Gene Expression Regulation, Neoplastic , Humans , Membrane Potential, Mitochondrial/drug effects , Molecular Structure , Reactive Oxygen SpeciesABSTRACT
We have investigated the growth-suppressive action of epigallocatechin-3-gallate (EGCG) on human leukemia Jurkat T cells. Results show a strong correlation between the dose-dependent reduction of clonogenic survival following acute EGCG treatments and the EGCG-induced decline of the mitochondrial level of Ca(2+). The cell killing ability of EGCG was synergistically enhanced by menadione. In addition, the cytotoxic effect of EGCG applied alone or in combination with menadione was accompanied by apoptosis induction. We also observed that in acute treatments EGCG displays strong antioxidant properties in the intracellular milieu, but concurrently triggers some oxidative stress generating mechanisms that can fully develop on a longer timescale. In parallel, EGCG dose-dependently induced mitochondrial depolarization during exposure, but this condition was subsequently reversed to a persistent hyperpolarized mitochondrial state that was dependent on the activity of respiratory Complex I. Fluorimetric measurements suggest that EGCG is a mitochondrial Complex III inhibitor and indicate that EGCG evokes a specific cellular fluorescence with emission at 400nm and two main excitation bands (at 330nm and 350nm) that may originate from a mitochondrial supercomplex containing dimeric Complex III and dimeric ATP-synthase, and therefore could provide a valuable means to characterize the functional properties of the respiratory chain.
Subject(s)
Catechin/analogs & derivatives , Jurkat Cells/drug effects , Vitamin K 3/pharmacology , Apoptosis/drug effects , Calcium/metabolism , Catechin/pharmacology , Cell Survival/drug effects , Humans , Jurkat Cells/metabolism , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Mitochondria/physiology , Oxidative Stress/drug effects , Reactive Oxygen Species/metabolismABSTRACT
Introduction Acute insulin resistance (IR) and hyperglycemia are frequently observed during acute myocardial infarction (AMI), significantly influencing both immediate and long-term patient outcomes, irrespective of diabetic status. Neutrophilia and increased neutrophil activity, which are common in these scenarios, have been associated with poorer prognoses, as demonstrated in our recent findings. While it is well established that neutrophils and stress-induced hyperglycemia exacerbate inflammation and hinder recovery, the complex interplay between these factors and their combined impact on AMI prognosis remains inadequately understood. This study aims to investigate the effects of stress hyperglycemia and IR on AMI patients at the onset of the event and to elucidate the relationship between these metabolic disturbances and inflammatory markers, particularly neutrophils. Methods We conducted a longitudinal prospective study on 219 AMI patients at Elias Emergency Hospital in Bucharest, Romania, from April 2021 to September 2022. Patients were included within 24 hours of AMI with ST-segment elevation and excluded if they had acute infections or chronic inflammatory diseases. Blood samples were collected to study inflammatory biomarkers, including neutrophil extracellular traps (NETs), S100A8/A9, interleukin (IL)-1ß, IL-18, and IL-6. Diabetic and pre-diabetic statuses were defined using glycated hemoglobin (HbA1c) and medical history (ADA 2019 criteria). To assess glycemic parameters, we employed the glycemia ratio (GR) and the homeostatic model assessment of insulin resistance (HOMA-IR) index, enabling a precise evaluation of stress hyperglycemia, acute IR, and their prognostic implications. Patients were stratified into groups based on GR calculations, categorized as under-average glycemia, normal glycemia, and stress hyperglycemia. Results The majority of patients in the stress hyperglycemia group exhibited an unfavorable prognosis. This group also demonstrated significantly elevated neutrophil counts and neutrophil-to-lymphocyte ratios (NLR). The GR was significantly and positively correlated with inflammation markers, including neutrophil count (Pearson's R = 0.181, P = 0.008) and NLR (Pearson's R = 0.318, P < 0.001), but showed no significant correlation with other evaluated inflammatory markers. Conclusions Our findings suggest that poor outcomes in AMI patients may be associated with stress hyperglycemia, as indicated by GR. AcuteIR, quantified by GR and HOMA-IR, exhibits a strong correlation with neutrophil count and NLR within the first 24 hours of AMI onset. However, no significant correlation was observed with other inflammatory markers, such as IL-1ß, IL-18, and IL-6, underscoring the specific interplay between IR and neutrophil activity in this setting.
ABSTRACT
Lung cancer ranks second worldwide after breast cancer and third in Europe after breast and colorectal cancers when both sexes and all ages are considered. In this context, the aim of this study was to emphasize the power of dual analysis of the molecular profile both in tumor tissue and plasma by NGS assay as a liquid biopsy approach with impact on prognosis and therapy modulation in NSCLC patients. NGS analysis was performed both from tissue biopsies and from cfNAs isolated from peripheral blood samples. Out of all 29 different mutations detectable by both NGS panels (plasma and tumor tissue), seven different variants (24.13%; EGFR L858R in two patients, KRAS G13D and Q61H and TP53 G244D, V197M, R213P, and R273H) were detected only in plasma and not in the tumor itself. These mutations were detected in seven different patients, two of them having known distant organ metastasis. Our data show that NGS analysis of cfDNA could identify actionable mutations in advanced NSCLC and, therefore, this analysis could be used to monitor the disease progression and the treatment response and even to modulate the therapy in real time.
ABSTRACT
Cardiac pathological hypertrophy is the major risk factor that usually progresses to heart failure. We hypothesized that extracellular vesicles (EVs), known to act as important mediators in regulating physiological and pathological functions, could have the potential to reduce the cardiac hypertrophy and the ensuing cardiovascular diseases. Herein, the effects of mesenchymal stem cell-derived extracellular vesicles (EV-MSCs) on cardiac hypertrophy were investigated. EVs were isolated from the secretome of human adipose tissue-derived stem cells (EV-ADSCs) or bone marrow-derived stem cells (EV-BMMSCs). Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were stimulated with AngII and TGF-ß1, in absence or presence of EVs. The results showed that exposure of hiPSC-CMs to AngII and TGF-ß1 generated in vitro model of hypertrophic cardiomyocytes characterized by increases in surface area, reactive oxygen species production, protein expression of cardiac-specific biomarkers atrial natriuretic factor, migration inhibitory factor, cTnI, COL1A1, Cx43, α-SMA and signalling molecules SMAD2 and NF-kBp50. The presence of EV-ADSCs or EV-BMMSCs in the hiPSC-CM culture along with hypertrophic stimuli reduced the protein expressions of hypertrophic specific markers (ANF, MIF, cTnI, COL1A1) and the gene expressions of IL-6 molecule involved in inflammatory process associated with cardiac hypertrophy and transcription factors SMAD2, SMAD3, cJUN, cFOS with role in cardiomyocyte hypertrophic response induced by AngII and TGF-ß1. The EV-ADSCs were more effective in reducing the protein expressions of hypertrophic and inflammatory markers, while EV-BMMSCs in reducing the gene expressions of transcription factors. Notably, neither EV-ADSCs nor EV-BMMSCs induced significant changes in cardiac biomarkers Cx43, α-SMA and fibronectin. These different effects of stem cell-derived EVs could be attributed to their miRNA content: some miRNAs (miR-126-3p, miR-222-3p, miR-30e-5p, miR-181b-5p, miR-124-3p, miR-155-5p, miR-210-3p hsa-miR-221-3p) were expressed in both types of EVs and others only in EV-ADSCs (miR-181a-5p, miR-185-5p, miR-21-5p) or in EV-BMMSCs (miR-143-3p, miR-146a-5p, miR-93-5p), some of these attenuating the cardiac hypertrophy while others enhance it. In conclusion, in hiPSC-CMs the stem cell-derived EVs through their cargo reduced the expression of hypertrophic specific markers and molecules involved in inflammatory process associated with cardiac hypertrophy. The data suggest the EV potential to act as therapeutic mediators to reduce cardiac hypertrophy and possibly the subsequent cardiovascular events.
ABSTRACT
The number and function of endothelial progenitor cells (EPCs) are reduced in diabetes, contributing to deteriorated vascular repair and the occurrence of cardiovascular complications. Here, we present the results of treating early diabetic dyslipidemic mice or dyslipidemic with disease-matched EPCs modified to overexpress VLA4 (VLA4-EPCs) as compared with the treatment of EPCs transfected with GFP (GFP-EPCs) as well as EPCs from healthy animals. Organ imaging of injected PKH26-stained cells showed little pulmonary first-pass effects and distribution in highly vascularized organs, with splenic removal from circulation, mostly in non-diabetic animals. Plasma measurements showed pronounced dyslipidemia in all animals and glycaemia indicative of diabetes in streptozotocin-injected animals. Echocardiographic measurements performed 3 days after the treatment showed significantly improved aortic valve function in animals treated with VLA4-overexpressing EPCs compared with GFP-EPCs, and similar results in the groups treated with healthy EPCs and VLA4-EPCs. Immunohistochemical analyses revealed active inflammation and remodelling in all groups but different profiles, with higher MMP9 and lower P-selectin levels in GFP-EPCs, treated animals. In conclusion, our experiments show that genetically modified allogeneic EPCs might be a safe treatment option, with bioavailability in the desired target compartments and the ability to preserve aortic valve function in dyslipidemia and diabetes.
ABSTRACT
Atherosclerosis is a progressive, chronic inflammatory disease of the large arteries caused by the constant accumulation of cholesterol, followed by endothelial dysfunction and vascular inflammation. We hypothesized that delivery of extracellular vesicles (EVs), recognized for their potential as therapeutic targets and tools, could restore vascular function in atherosclerosis. We explored by comparison the potential beneficial effects of EVs from subcutaneous adipose tissue stem cells (EVs (ADSCs)) or bone marrow mesenchymal stem cells (EVs (MSCs)) on the consequences of atherogenic diet on vascular health. Also, the influences of siRNA-targeting Smad2/3 (Smad2/3siRNA) on endothelial dysfunction and its key molecular players were analyzed. For this study, an animal model of atherosclerosis (HH) was transplanted with EVs (ADSCs) or EVs (MSCs) transfected or not with Smad2/3siRNA. For controls, healthy or HH animals were used. The results indicated that by comparison with the HH group, the treatment with EVs(ADSCs) or EVs(MSCs) alone or in combination with Smad2/3siRNA of HH animals induced a significant decrease in the main plasma parameters and a noticeable improvement in the structure and function of the thoracic aorta and carotid artery along with a decrease in the selected molecular and cellular targets mediating their changes in atherosclerosis: 1) a decrease in expression of structural and inflammatory markers COL1A1, α-SMA, Cx43, VCAM-1, and MMP-2; 2) a slight infiltration of total/M1 macrophages and T-cells; 3) a reduced level of cytosolic ROS production; 4) a significant diminution in plasma concentrations of TGF-ß1 and Ang II proteins; 5) significant structural and functional improvements (thinning of the arterial wall, increase of the inner diameter, enhanced distensibility, diminished VTI and Vel, and augmented contractile and relaxation responses); 6) a reduced protein expression profile of Smad2/3, ATF-2, and NF-kBp50/p65 and a significant decrease in the expression levels of miR-21, miR-29a, miR-192, miR-200b, miR-210, and miR-146a. We can conclude that 1) stem cell-derived EV therapies, especially the EVs (ADSCs) led to regression of structural and functional changes in the vascular wall and of key orchestrator expression in the atherosclerosis-induced endothelial dysfunction; 2) transfection of EVs with Smad2/3siRNA amplified the ability of EVs(ADSCs) or EVs(MSCs) to regress the inflammation-mediated atherosclerotic process.
ABSTRACT
Diabetes reduces the number and induces dysfunction in circulating endothelial progenitor cells (EPCs) by mechanisms that are still uncovered. This study aims to evaluate the number, viability, phenotype, and function of EPCs in dyslipidemic mice with early diabetes mellitus and EPC infiltration in the aortic valve in order to identify possible therapeutic targets in diabetes-associated cardiovascular disease. A streptozotocin-induced diabetic apolipoprotein E knock-out (ApoE-/-) mouse model was used to identify the early and progressive changes, at 4 or 7 days on atherogenic diet after the last streptozotocin or citrate buffer injection. Blood and aortic valves from diabetic or nondiabetic ApoE-/- animals were collected.EPCs were identified as CD34 and vascular endothelial growth factor receptor 2 positive monocytes, and the expression levels of α4ß1, αVß3, αVß5, ß1, αLß2, α5 integrins, and C-X-C chemokine receptor type 4 chemokine receptor on EPC surface were assessed by flow cytometry. The number of CD34 positive cells in the aortic valve, previously found to be recruited progenitor cells, was measured by fluorescence microscopy. Our results show that aortic valves from mice fed 7 days with atherogenic diet presented a significantly higher number of CD34 positive cells compared with mice fed only 4 days with the same diet, and diabetes reversed this finding. We also show a reduction of circulatory EPC numbers in diabetic mice caused by cell senescence and lower mobilization. Dyslipidemia induced EPC death through apoptosis regardless of the presence of diabetes, as shown by the higher percent of propidium iodide positive cells and higher cleaved caspase-3 levels. EPCs from diabetic mice expressed α4ß1 and αVß3 integrins at a lower level, while the rest of the integrins tested were unaffected by diabetes or diet. In conclusion, reduced EPC number and expression of α4ß1 and αVß3 integrins on EPCs at 4 and 7 days after diabetes induction in atherosclerosis-prone mice have resulted in lower recruitment of EPCs in the aortic valve.
Subject(s)
Diabetes Mellitus, Experimental/metabolism , Dyslipidemias/physiopathology , Endothelial Progenitor Cells/metabolism , Integrin alpha4beta1/metabolism , Integrin alphaVbeta3/metabolism , Stem Cells/metabolism , Streptozocin/therapeutic use , Animals , Aortic Valve Disease , Cells, Cultured , Male , Mice , Mice, KnockoutABSTRACT
The purpose was to evaluate the effect of platelets on functional properties of late endothelial progenitor cells (EPCs), in the direct co-culture conditions, and to investigate the involved mediators, in experimental induced atherosclerosis. The late EPCs obtained from two animal groups, hypertensive-hyperlipidemic (HH) and control (C) hamsters, named late EPCs-HH and late EPCs-C, were co-incubated with or without platelets isolated from both groups. Our results have showed that exposure to platelets from control animals: (i) promoted the late EPCs-C capacity to form colonies and capillary-like structures, and also to proliferate and migrate; (ii) improved the functional properties of late EPCs-HH; (iii) strengthened the direct binding EPCs-platelets; (iv) increased SDF-1α,VEGF, PDGF, and reduced CD40L, IL-1ß,-6,-8 levels; and (v) enhanced miR-223 and IGF-1R expressions. Platelets from HH group diminished functional abilities for both EPC types and had opposite effects on these pro-angiogenic and pro-inflammatory molecules. Furthermore, testing the direct effect of miR-223 and IGF-1R on late EPCs disclosed that these molecular factors improve late EPC functional properties in atherosclerosis in terms of stimulation of the proliferation and migration abilities. In conclusion, in vitro exposure to platelets of healthy origins had a positive effect on functional properties of atherosclerotic late EPCs. The most likely candidates mediating EPC-platelet interaction can be SDF-1α, VEGF, CD40L, PDGF, IL-1ß,-6,-8, miR-223, and IGF-1R. The current study brings evidences that the presence of healthy origin platelets is of utmost importance on functional improvement of EPCs in atherosclerosis.
ABSTRACT
Diabetes contributes directly to the development of cardiovascular aortic valve disease. There is currently no drug therapy available for a dysfunctional valve and this urges the need for additional research to identify distinctive mechanisms of cardiovascular aortic valve disease evolution. The aim of this study was to evaluate changes of valvular aortic lesions induced in a hyperlipemic ApoE-/- mouse model by early type 1 diabetes onset (at 4 and 7 days after streptozotocin induction). The haemodynamic valve parameters were evaluated by echography and blood samples and aortic valves were collected. Plasma parameters were measured, and inflammatory, remodelling and osteogenic markers were evaluated in the aortic valves. Next, correlations between all parameters were determined. The results showed early aortic valve dysfunction detected by echography after 1 week of diabetes; lesions were found in the aortic root. Moreover, increased expression of cell adhesion molecules, extracellular matrix remodelling and osteogenic markers were detected in hyperlipemic ApoE-/- diabetic mice. Significant correlations were found between tissue valve biomarkers and plasmatic and haemodynamic parameters. Our study may help to understand the mechanisms of aortic valve disease in the diabetic milieu in order to discover and validate new biomarkers of cardiovascular aortic valve disease in diabetes and reveal new possible targets for nanobiotherapies.
Subject(s)
Aortic Valve , Atherosclerosis/complications , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 1/complications , Heart Valve Diseases/etiology , Animals , Aortic Valve/metabolism , Aortic Valve/pathology , Aortic Valve/physiopathology , Atherosclerosis/metabolism , Atherosclerosis/pathology , Blood Glucose/metabolism , Cell Adhesion Molecules/metabolism , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetes Mellitus, Type 1/metabolism , Diabetes Mellitus, Type 1/pathology , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Glycated Hemoglobin/metabolism , Heart Valve Diseases/metabolism , Heart Valve Diseases/pathology , Heart Valve Diseases/physiopathology , Hemodynamics , Inflammation Mediators/metabolism , Lipids/blood , Male , Mice, Knockout, ApoE , Osteogenesis , Time FactorsABSTRACT
AIMS: CXCL1 is a chemokine with pleiotropic effects, including pain and itch. Itch, an unpleasant sensation that elicits the desire or reflex to scratch, it is evoked mainly from the skin and implicates activation of a specific subset of IB4+, C-type primary afferents. In previous studies we showed that acute application of CXCL1 induced a Ca2+ influx of low amplitude and slow kinetics in a subpopulation of transient receptor potential vanilloid type 1 (TRPV1)+/isolectin B4 (IB4)+dorsal root ganglia neurons which also responded to other itch-inducing agents. In this study we explored the mechanism behind the Ca2+ influx to better understand how CXCL1 acts on primary sensitive neurons to induce itch. MATERIALS AND METHODS: Intracellular Ca2+ imaging and patch-clamp recordings on dorsal root ganglia neurons primary cultures and HEK293T cell transiently transfected with TRPV1 and CXCR2 plasmids were used to investigate the acute effect (12min application) of 4nM CXCL1. In primary cultures, the focus was on TRPV1+/IB4+ cells to which the itch-sensitive neurons belong. KEY FINDINGS: The results showed that the Ca2+ influx induced by the acute application of CXCL1 is mediated mainly by TRPV1 receptors and depends on extracellular Ca2+ not on intracellular stores. TRPV1 was activated, not sensitized by CXCL1, in a CXCR2 receptors- and actin filaments-dependent manner, since specific blockers and actin depolymerizing agents disrupted the CXCL1 effect. SIGNIFICANCE: This study brings additional data about the itch inducing mechanism of CXCL1 chemokine and about a new mechanism of TRPV1 activation via actin filaments.
Subject(s)
Chemokine CXCL1/metabolism , TRPV Cation Channels/metabolism , Actin Cytoskeleton/metabolism , Animals , Calcium/metabolism , Capsaicin/pharmacology , Chemokine CXCL1/physiology , Ganglia, Spinal/cytology , HEK293 Cells , Humans , Male , Neurons/drug effects , Pain/metabolism , Patch-Clamp Techniques , Primary Cell Culture , Pruritus/metabolism , Rats , Rats, Wistar , Sensation/drug effectsABSTRACT
In this study, we aimed to identify the mechanisms underlying the different effects of palmitic acid and oleic acid on human pancreatic beta cell function. To address this problem, the oxidative stress, endoplasmic reticulum stress, inflammation, apoptosis and their mediator molecules have been investigated in the insulin releasing beta cells exposed to palmitic and/or oleic acid. Herein, we have demonstrated that in cultured 1.1B4 beta cells oleic acid promotes neutral lipid accumulation and insulin secretion, whereas palmitic acid is poorly incorporated into triglyceride and it does not stimulate insulin secretion from human pancreatic islets at physiologically glucose concentrations. In addition, palmitic acid caused: (1) oxidative stress through a mechanism involving increases in ROS production and MMP-2 protein expression/gelatinolytic activity associated with down-regulation of SOD2 protein; (2) endoplasmic reticulum stress by up-regulation of chaperone BiP protein and unfolded protein response (UPR) transcription factors (eIF2α, ATF6, XBP1u proteins) and by PTP-1B down-regulation in both mRNA and protein levels; (3) inflammation through enhanced synthesis of proinflammatory cytokines (IL6, IL8 proteins); and (4) apoptosis by enforced proteic expression of CHOP multifunctional transcription factor. Oleic acid alone had opposite effects due to its different capacity of controlling these metabolic pathways, in particular by reduction of the ROS levels and MMP-2 activity, down-regulation of BiP, eIF2α, ATF6, XBP1u, CHOP, IL6, IL8 and by SOD2 and PTP-1B overexpression. The supplementation of saturated palmitic acid with the monounsaturated oleic acid reversed the negative effects of palmitic acid alone regulating insulin secretion from pancreatic beta cells through ROS, MMP-2, ATF6, XBP1u, IL8 reduction and SOD2, PTP-1B activation. Our findings have shown the protective action of oleic acid against palmitic acid on beta cell lipotoxicity through promotion of triglyceride accumulation and insulin secretion and regulation of some effector molecules involved in oxidative stress, endoplasmic reticulum stress, inflammation and apoptosis.
ABSTRACT
ErbB proteins overexpression, in both normal and mutated forms, is associated with invasive forms of cancer prone to metastasis and with stronger antiapoptotic mechanisms and therefore more challenging to treat. Downstream effectors of ErbB receptors mediating these phenotypic traits include MAPK, STAT, and PI3K/AKT/mTOR pathways. Various phytochemical compounds were studied for their large number of biological effects including anticancer activity. Among these compounds, epigallocatechin-3-gallate (EGCG), the main catechin from green tea leaves, and curcumin, component of the curry powder, constituted the object of numerous studies. Both compounds were shown to act directly either on ErbB expression, or on their downstream signaling molecules. In this paper we aim to review the involvement of ErbB proteins in cancer as well as the biologic activity of EGCG and curcumin in ErbB expressing and overexpressing malignancies. The problems arising in the administration of the two compounds due to their reduced bioavailability when orally administered, as well as the progress made in this field, from using novel formulations to improved dosing regimens or improved synthetic analogs, are also discussed.
ABSTRACT
The polymorphism of amyloid fibrils is potentially crucial as it may underlie the natural variability of amyloid diseases and could be important in developing a fuller understanding of the molecular basis of protein deposition disorders. This study examines morphological differences in lysozyme fibrils and the implications of these differences in terms of cytotoxicity. The structural characteristics of amyloid fibrils formed under two different experimental conditions (acidic and neutral) were evaluated using spectroscopic methods, atomic force microscopy and image analysis. Growth curves and apoptotic/necrotic assays were used to determine the cytotoxic effect of fibrils on the LLC-PK1 renal cells. The results reveal that both types of mature lysozyme amyloid fibrils are actively involved in the cytotoxic process, however each exhibit different levels of cytotoxicity. Fibrils formed at acidic pH affect cell growth in a dose-dependent manner, but a threshold-dependent inhibition of cell growth was observed in the case of lysozyme fibrils prepared at neutral pH. Experiments examining the mechanism of the cell death suggest that both types of mature lysozyme fibrils trigger late apoptosis/necrosis at different fibril concentrations. Our findings clearly indicate that the intrinsic differences between amyloid fibrils due to their polymorphism result in different degrees of cytotoxicity.