ABSTRACT
BACKGROUND: MET and AXL dysregulation is reported as a bypass mechanism driving tumour progression in non-small cell lung cancer (NSCLC) with acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI). This non-comparative phase I study investigated the combination of gefitinib with S49076, a MET/AXL inhibitor, in advanced EGFR TKI-resistant NSCLC patients with MET and/or AXL dysregulation. METHODS: Patients received S49076 at escalating doses of 500 or 600 mg with a fixed dose of 250 mg gefitinib orally once daily in continuous 28day cycles. MET and AXL dysregulation and EGFR/T790M mutation status were centrally assessed in tumour biopsies at screening. Tumour response was evaluated using Response Evaluation Criteria in Solid Tumors (RECIST). EGFR TKI resistance mechanisms were analysed by next-generation sequencing. The clonal evolution of tumours was monitored with the analysis of circulating tumour DNA. RESULTS: Of 92 pre-screened patients, 22 met the molecular inclusion criteria and 14 were included. The recommended dose was 600 mg daily S49076. Best overall responses were 2 partial responses (1 patient with MET dysregulation only, 1 MET and AXL co-dysregulation) and 8 patients with stable disease. Other potential concomitant mechanisms of resistance to EGFR TKI were identified in more than half of the included patients. CONCLUSIONS: S49076 plus gefitinib demonstrated a good tolerability with limited anti-tumour activity. Due to the low number of eligible patients, no tendency in term of activity appeared in any specific molecular subset and the data did not allow for identification of AXL overexpression as an oncogenic driver.
Subject(s)
Carcinoma, Non-Small-Cell Lung , Lung Neoplasms , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/genetics , Drug Resistance, Neoplasm/genetics , ErbB Receptors/genetics , Gefitinib/therapeutic use , Humans , Indoles , Lung Neoplasms/drug therapy , Lung Neoplasms/genetics , Mutation , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , ThiazolidinedionesABSTRACT
Trifluridine/tipiracil (FTD/TPI) is a new antimetabolite agent used to treat chemorefractory metastatic colorectal cancer. FTD/TPI induced immunogenic cell death (ICD) in vitro in the microsatellite-stable (MSS) CT26 mouse colon carcinoma cell line, as well as in various human MSS colorectal cancer cell lines (SW620, Caco-2, and Colo-320). The combination of FTD/TPI with oxaliplatin synergized to promote ICD. In vivo, the combination was able to induce ICD, but not the single agents, although all treatment groups showed T-cell dependency. In addition, FTD/TPI and oxaliplatin did not affect regulatory T cells or myeloid-derived suppressor cells but eliminated type-2 tumor-associated macrophages (TAM2), resulting in higher cytotoxic CD8+ T-cell infiltration and activation. This effect was concomitantly associated with PD-L1 expression on tumor cells and PD-1 induction on CD8+ T cells, leading to secondary T-cell exhaustion. Finally, although anti-PD-1 was unable to synergize with FTD/TPI or oxaliplatin monotherapy, concomitant administration of anti-PD-1 to FTD/TPI and oxaliplatin enhanced the antitumor efficacy of the double chemotherapy. Our study showed a novel immunomodulatory role of FTD/TPI and oxaliplatin in depleting TAM2. The combination of oxaliplatin and FTD/TPI induced ICD in vivo, providing a rationale for the use of these drugs to eliminate immunosuppressive cells and boost checkpoint efficacy in patients with metastatic colorectal cancer.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Colorectal Neoplasms/drug therapy , Macrophages/drug effects , Oxaliplatin/therapeutic use , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Pyrrolidines/therapeutic use , Trifluridine/therapeutic use , Uracil/analogs & derivatives , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Line, Tumor , Colorectal Neoplasms/immunology , Drug Combinations , Female , Humans , Immunogenic Cell Death/drug effects , Mice, Inbred BALB C , Mice, Nude , Oxaliplatin/pharmacology , Pyrrolidines/pharmacology , Thymine , Trifluridine/pharmacology , Uracil/pharmacology , Uracil/therapeutic useABSTRACT
Non-small cell lung cancer (NSCLC) tumors harboring mutations in EGFR ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs). Here, we show that resistant cells without the p.T790M or other acquired mutations are sensitive to the Aurora B (AURKB) inhibitors barasertib and S49076. Phospho-histone H3 (pH3), a major product of AURKB, is increased in most resistant cells and treatment with AURKB inhibitors reduces the levels of pH3, triggering G1/S arrest and polyploidy. Senescence is subsequently induced in cells with acquired mutations while, in their absence, polyploidy is followed by cell death. Finally, in NSCLC patients, pH3 levels are increased after progression on EGFR TKIs and high pH3 baseline correlates with shorter survival. Our results reveal that AURKB activation is associated with acquired resistance to EGFR TKIs, and that AURKB constitutes a potential target in NSCLC progressing to anti-EGFR therapy and not carrying resistance mutations.
Subject(s)
Antineoplastic Agents/pharmacology , Aurora Kinase B/metabolism , Carcinoma, Non-Small-Cell Lung/drug therapy , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/pharmacology , Adult , Aged , Aged, 80 and over , Animals , Antineoplastic Agents/therapeutic use , Aurora Kinase B/antagonists & inhibitors , Aurora Kinase B/genetics , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/mortality , Carcinoma, Non-Small-Cell Lung/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Progression , Drug Resistance, Neoplasm/genetics , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/genetics , Female , G1 Phase Cell Cycle Checkpoints/drug effects , Histones/metabolism , Humans , Lung Neoplasms/genetics , Lung Neoplasms/mortality , Lung Neoplasms/pathology , Male , Mice , Middle Aged , Organophosphates/pharmacology , Organophosphates/therapeutic use , Phosphorylation/drug effects , Polyploidy , Protein Kinase Inhibitors/therapeutic use , Quinazolines/pharmacology , Quinazolines/therapeutic use , RNA, Small Interfering/metabolism , Survival Analysis , Treatment Outcome , Xenograft Model Antitumor AssaysABSTRACT
INTRODUCTION: Pixantrone is a first-in-class aza-anthracenedione approved as monotherapy for treatment of relapsed or refractory aggressive diffuse B-cell non-Hodgkin's lymphoma (NHL), a patient group which is notoriously difficult to treat. It has a unique chemical structure and pharmacologic properties distinguishing it from anthracyclines and anthracenediones. Areas covered: The chemical structure and mode of action of pixantrone versus doxorubicin and mitoxantrone; preclinical evidence for pixantrone's therapeutic effect and cardiac tolerability; efficacy and safety of pixantrone in clinical trials; ongoing and completed trials of pixantrone alone or as combination therapy; and the risk of cardiotoxicity of pixantrone versus doxorubicin and mitoxantrone. Expert commentary: Currently, pixantrone is the only approved therapy for multiply relapsed or refractory NHL, an area with few available effective treatment options. Pixantrone is currently being investigated as combination therapy with other drugs including several targeted therapies, with the ultimate goal of improved survival in heavily pretreated patients. In order for pixantrone to be acknowledged in the treatment of aggressive NHL, the perception of pixantrone as an anthracycline-like agent that has anthracycline-like activity and cardiotoxicity needs to be changed. Further data from ongoing clinical trials will help in confirming pixantrone as an effective and safe option.
Subject(s)
Isoquinolines/therapeutic use , Lymphoma, Large B-Cell, Diffuse/drug therapy , Clinical Trials as Topic , Disease-Free Survival , Doxorubicin/adverse effects , Doxorubicin/therapeutic use , Humans , Isoquinolines/adverse effects , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/mortality , Lymphoma, Large B-Cell, Diffuse/pathology , Mitoxantrone/adverse effects , Mitoxantrone/therapeutic use , Survival RateABSTRACT
BACKGROUND AND OBJECTIVES: S49076 is a novel ATP-competitive tyrosine kinase inhibitor of MET, AXL and FGFR with a unique selectivity profile. A phase I open-label study was undertaken to establish the tolerability profile and determine the recommended dose (RD) and administration schedule. MATERIALS AND METHODS: Patients with advanced solid tumours received S49076 orally once-daily (qd) or twice-daily (bid) in continuous 21-day cycles at escalating doses guided by a 3 + 3 design and followed by an expansion phase at the RD. Pharmacokinetic (PK) parameters were assessed and pharmacodynamic end-points were evaluated in pre- and post-treatment tumour biopsies. Preliminary anti-tumour activity was evaluated as per the Response Evaluation Criteria In Solid Tumours 1.1 criteria. RESULTS: A total of 103 patients were treated: 79 in the dose-escalation and 24 in the expansion. Doses from 15 to 900 mg were evaluated. Dose-limiting toxicities were reported in 9 patients and occurred at 30, 760 and 900 mg in the qd arm and at 180, 225 and 285 mg in the bid arm. The RD was defined at 600 mg qd. Adverse events (AEs) occurred with similar frequency in both regimens at an equivalent total daily dose. Overall, 83 patients (81.4%) had drug-related AEs, the majority (93%) of which were grade I-II (National Cancer Institute Common Terminology Criteria for Adverse Events version 4.0) and only 3% led to drug discontinuation. Intratumoural PK analysis at the RD suggested hitting of MET, AXL and FGFR. CONCLUSION: S49076 demonstrated a tolerable safety profile with limited single-agent activity. PK/pharmacodynamic readouts of S49076 are encouraging for further investigation of S49076 in combination therapies. TRIAL REGISTRATION NUMBER: ISRCTN00759419.
Subject(s)
Indoles/therapeutic use , Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Thiazolidinediones/therapeutic use , Adult , Aged , Aged, 80 and over , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Humans , Indoles/adverse effects , Indoles/pharmacokinetics , Male , Middle Aged , Protein Kinase Inhibitors/adverse effects , Protein Kinase Inhibitors/pharmacokinetics , Protein-Tyrosine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Receptor, Fibroblast Growth Factor, Type 1/antagonists & inhibitors , Thiazolidinediones/adverse effects , Thiazolidinediones/pharmacokineticsABSTRACT
Several therapeutic agents targeting HGF/MET signaling are under clinical development as single agents or in combination, notably with anti-EGFR therapies in non-small cell lung cancer (NSCLC). However, despite increasing data supporting a link between MET, irradiation, and cancer progression, no data regarding the combination of MET-targeting agents and radiotherapy are available from the clinic. S49076 is an oral ATP-competitive inhibitor of MET, AXL, and FGFR1-3 receptors that is currently in phase I/II clinical trials in combination with gefitinib in NSCLC patients whose tumors show resistance to EGFR inhibitors. Here, we studied the impact of S49076 on MET signaling, cell proliferation, and clonogenic survival in MET-dependent (GTL16 and U87-MG) and MET-independent (H441, H460, and A549) cells. Our data show that S49076 exerts its cytotoxic activity at low doses on MET-dependent cells through MET inhibition, whereas it inhibits growth of MET-independent cells at higher but clinically relevant doses by targeting Aurora B. Furthermore, we found that S49076 improves the antitumor efficacy of radiotherapy in both MET-dependent and MET-independent cell lines in vitro and in subcutaneous and orthotopic tumor models in vivo In conclusion, our study demonstrates that S49076 has dual antitumor activity and can be used in combination with radiotherapy for the treatment of both MET-dependent and MET-independent tumors. These results support the evaluation of combined treatment of S49076 with radiation in clinical trials without patient selection based on the tumor MET dependency status. Mol Cancer Ther; 16(10); 2107-19. ©2017 AACR.
Subject(s)
Aurora Kinase B/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/radiotherapy , Proto-Oncogene Proteins c-met/genetics , Aurora Kinase B/antagonists & inhibitors , Carcinoma, Non-Small-Cell Lung/genetics , Carcinoma, Non-Small-Cell Lung/pathology , Cell Proliferation/drug effects , Drug Resistance, Neoplasm/genetics , ErbB Receptors/antagonists & inhibitors , Gefitinib , Gene Expression Regulation, Neoplastic , Humans , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Quinazolines/administration & dosage , Radiation Tolerance , Receptor Protein-Tyrosine Kinases/genetics , Receptor, Fibroblast Growth Factor, Type 1/genetics , Signal Transduction/drug effects , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine KinaseABSTRACT
Aberrant activity of the receptor tyrosine kinases MET, AXL, and FGFR1/2/3 has been associated with tumor progression in a wide variety of human malignancies, notably in instances of primary or acquired resistance to existing or emerging anticancer therapies. This study describes the preclinical characterization of S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3. S49076 potently blocked cellular phosphorylation of MET, AXL, and FGFRs and inhibited downstream signaling in vitro and in vivo. In cell models, S49076 inhibited the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocked MET-driven migration of lung carcinoma cells, and inhibited colony formation of hepatocarcinoma cells expressing FGFR1/2 and AXL. In tumor xenograft models, a good pharmacokinetic/pharmacodynamic relationship for MET and FGFR2 inhibition following oral administration of S49076 was established and correlated well with impact on tumor growth. MET, AXL, and the FGFRs have all been implicated in resistance to VEGF/VEGFR inhibitors such as bevacizumab. Accordingly, combination of S49076 with bevacizumab in colon carcinoma xenograft models led to near total inhibition of tumor growth. Moreover, S49076 alone caused tumor growth arrest in bevacizumab-resistant tumors. On the basis of these preclinical studies showing a favorable and novel pharmacologic profile of S49076, a phase I study is currently underway in patients with advanced solid tumors. Mol Cancer Ther; 12(9); 1749-62. ©2013 AACR.
Subject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Indoles/pharmacology , Neoplasms/drug therapy , Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Thiazolidinediones/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/metabolism , Bevacizumab , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Proliferation/drug effects , Drug Screening Assays, Antitumor , Female , Humans , Indoles/chemistry , Mice , Mice, Inbred BALB C , Protein Kinase Inhibitors/chemistry , Proto-Oncogene Proteins/antagonists & inhibitors , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Thiazolidinediones/chemistry , Xenograft Model Antitumor Assays , Axl Receptor Tyrosine KinaseABSTRACT
We aimed to characterize circulating microparticles in association with arterial stiffness, inflammation and endothelial dysfunction in aldosterone-salt-induced hypertension in rats and to investigate the preventive effects of red wine polyphenols. Uninephrectomized male Sprague-Dawley rats were treated with aldosterone-salt (1 µg.h(-1)), with or without administration of either red wine polyphenols, Provinols™ (20 mg.kg(-1).day(-1)), or spironolactone (30 mg.kg(-1).day(-1)) for 4 weeks. Microparticles, arterial stiffness, nitric oxide (NO) spin trapping, and mesenteric arterial function were measured. Aldosterone-salt rats showed increased microparticle levels, including those originating from platelets, endothelium and erythrocytes. Hypertension resulted in enhanced aortic stiffness accompanied by increased circulating and aortic NO levels and an upregulation of aortic inducible NO-synthase, NFκB, superoxide anions and nitrotyrosine. Flow-induced dilatation was reduced in mesenteric arteries. These effects were prevented by spironolactone. Provinols™ did not reduce arterial stiffness or systolic hypertension but had effects similar to those of spironolactone on endothelial function assessed by flow-mediated vasodilatation, microparticle generation, aortic NO levels and oxidative stress and apoptosis in the vessel wall. Neither the contractile response nor endothelium-dependent relaxation in mesenteric arteries differed between groups. The in vivo effects of Provinols™ were not mediated by mineralocorticoid receptors or changes in shear stress. In conclusion, vascular remodelling and endothelial dysfunction in aldosterone-salt-mediated hypertension are associated with increased circulating microparticles. Polyphenols prevent the enhanced release of microparticles, macrovascular inflammation and oxidative stress, and microvascular endothelial dysfunction independently of blood pressure, shear stress and mineralocorticoid receptor activation in a model of hyperaldosteronism.
Subject(s)
Antioxidants/therapeutic use , Cell-Derived Microparticles/drug effects , Endothelium, Vascular/drug effects , Hypertension/drug therapy , Mesenteric Arteries/drug effects , Polyphenols/therapeutic use , Spironolactone/therapeutic use , Aldosterone , Animals , Antioxidants/pharmacology , Aorta/drug effects , Aorta/metabolism , Aorta/pathology , Arterial Pressure/drug effects , Cell-Derived Microparticles/metabolism , Cell-Derived Microparticles/pathology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Hypertension/chemically induced , Hypertension/metabolism , Hypertension/pathology , Male , Mesenteric Arteries/metabolism , Mesenteric Arteries/pathology , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/metabolism , Oxidative Stress/drug effects , Polyphenols/pharmacology , Rats , Rats, Sprague-Dawley , Receptors, Mineralocorticoid/metabolism , Sodium Chloride , Spironolactone/pharmacology , Superoxides/metabolism , Vascular Stiffness/drug effects , Vasodilation/drug effectsABSTRACT
BACKGROUND: A challenge in urologic tissue engineering is to obtain well-differentiated urothelium to overcome the complications related to other sources of tissues used in ureteral and urethral substitution. OBJECTIVE: We investigated the effects of in vitro mechanical stimuli on functional and morphologic properties of a human tissue-engineered tubular genitourinary graft (TTGG). DESIGN, SETTING, AND PARTICIPANTS: Using the self-assembly technique, we developed a TTGG composed of human dermal fibroblasts and human urothelial cells without exogenous scaffolding. Eight substitutes were subjected to dynamic flow and hydrostatic pressure for up to 2 wk compared to static conditions (n=8). MEASUREMENTS: Stratification and cell differentiation were assessed by histology, electron microscopy, immunostaining, and uroplakin gene expression. Barrier function was determined by permeation studies with carbon 14-urea. RESULTS AND LIMITATIONS: Dynamic conditions showed well-established stratified urothelium and basement membrane formation, whereas no stratification was observed in static culture. The first signs of cell differentiation were perceived after 7 d of perfusion and were fully expressed at day 14. Superficial cells under perfusion displayed discoidal and fusiform vesicles and positive staining for uroplakin 2, cytokeratine 20, and tight junction protein ZO-1, similar to native urothelium. Mechanical stimuli induced expression of the major uroplakin transcripts, whereas expression was low or undetectable in static culture. Permeation studies showed that mechanical constraints significantly improved the barrier function compared to static conditions (p<0.01 at 14 d, p<0.05 at 7 d) and were comparable to native urothelium. CONCLUSIONS: Mechanical stimuli induced in vitro terminal urothelium differentiation in a human genitourinary substitute displaying morphologic and functional properties equivalent to a native urologic conduit.
Subject(s)
Cell Differentiation , Fibroblasts/physiology , Mechanotransduction, Cellular , Tissue Engineering , Urogenital System/physiology , Urothelium/physiology , Basement Membrane/metabolism , Biomarkers/metabolism , Cells, Cultured , Coculture Techniques , Fibroblasts/metabolism , Fibroblasts/ultrastructure , Fluorescent Antibody Technique , Humans , Hydrostatic Pressure , Keratin-20/metabolism , Membrane Proteins/metabolism , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Permeability , Phosphoproteins/metabolism , RNA, Messenger/metabolism , Stress, Mechanical , Time Factors , Tissue Engineering/methods , Transcription, Genetic , Up-Regulation , Urogenital System/metabolism , Urogenital System/ultrastructure , Uroplakin II/genetics , Uroplakin II/metabolism , Urothelium/metabolism , Urothelium/ultrastructure , Zonula Occludens-1 ProteinABSTRACT
BACKGROUND: Markers of collagen turnover have not been well studied in the context of cardiovascular disease in patients with chronic kidney disease (CKD). We investigated the associations between serum markers of collagen turnover [N-terminal procollagen type 3 propeptide (PIIINP) and carboxy-terminal telopeptide (C1TP)] and both pulse wave velocity (PWV) and left ventricular mass index (LVMI) in a CKD cohort. METHODS: The study included 242 patients (mean age 60 ± 15 years, 53% males, 80% Caucasian, CKD Stages 3-5) from the Renal Research Institute (RRI)-CKD Study. Serum PIIINP and C1TP levels were analyzed by radioimmunoassay. PWV was obtained by applanation tonometry from carotid and femoral pressure wave contours. LVMI was measured by echocardiography. Statistical analyses included Pearson's correlations and multiple linear regression. RESULTS: Both PIIINP and C1TP values were significantly higher in more advanced stages of CKD (P < 0.05). A positive correlation was demonstrated between PWV and LVMI (r = 0.25, P = 0.0018), persisting after adjustment for potential confounders (partial r = 0.27, P = 0.0009). PIIINP correlated with PWV (r = 0.16, p = 0.029) and LVMI (r = 0.16, P = 0.0018), while C1TP correlated with LVMI (r = 0.18, P = 0.013) but not with PWV (r = 0.12, P = 0.09). In multivariable analysis adjusting for race, diabetes, estimated glomerular filtration rate (eGFR) and renin-angiotensin-aldosterone system inhibitors, only PWV (ß = 0.45, P = 0.0017) but not LVMI (P = 0.09) remained significantly associated with serum PIIINP. CONCLUSIONS: Our results demonstrate the association of PIIINP (but not C1TP), a circulating biomarker of collagen synthesis with arterial stiffness (but not with LVMI) in a CKD cohort, independent of eGFR. This suggests that altered collagen turnover may play a role in the pathogenesis of arterial stiffness in CKD.
Subject(s)
Arteries/physiopathology , Biomarkers/metabolism , Collagen/metabolism , Hypertrophy, Left Ventricular/diagnosis , Hypertrophy, Left Ventricular/etiology , Kidney Failure, Chronic/complications , Vascular Stiffness , Academies and Institutes , Cohort Studies , Female , Follow-Up Studies , Glomerular Filtration Rate , Humans , Hypertrophy, Left Ventricular/metabolism , Kidney Failure, Chronic/metabolism , Male , Middle Aged , Peptide Fragments/metabolism , Procollagen/metabolism , Prognosis , Prospective StudiesABSTRACT
PURPOSE: Currently, bladder repair is performed using gastrointestinal segments; however, this technique has a high morbidity rate, and new alternatives are thus needed. The lack of native or synthetic tissue with similar properties of the bladder led us to develop autologous vesical substitutes entirely made by tissue engineering and without exogenous matrices. Watertight function and mechanical resistance are fundamental for the model. The aim of this study was to determine the structural and functional characteristics of our vesical equivalent (VE). MATERIALS AND METHODS: Porcine VEs are produced in 55 days. The cellular types that make up the vesical wall are extracted and purified simultaneously from a small porcine bladder biopsy. Dermal fibroblasts are extracted and cultured in vitro to form cellular sheets. Endothelial cells were seeded on the fibroblast sheets before their superimposition. Urothelial cells are then seeded onto this cellular construction. VEs are characterized by histology, immunostaining, electron microscopy, and cell viability. Mechanical properties of the reconstructed substitutes are evaluated by uniaxial tensile tests, and tissue absorption is verified with (14)C-urea, which quantifies the degree of impermeability. RESULTS: This process allowed us to obtain a highly structured tissue with a total fusion of the fibroblast layers. As expected, histological observations showed a pseudostratification of the urothelium developing on an organized self-secreted extracellular matrix. Positive markers for cytokeratin 8/18 in immunostaining confirmed the presence of a urinary epithelium. Electron microscopy confirmed the normal aspect of urothelial cells. Our VE's permeability to (14)C-urea was significantly similar to porcine bladder, and characterization of the mechanical properties indicated that our tissue could be suitable for grafting since its ultimate tensile strength compares favorably with a native porcine bladder. CONCLUSION: The construction of a VE using this method seems very promising in meeting the needs in the urological field. Our substitute has proven its efficiency as a barrier to urea and has a sufficient mechanical resistance to support suturing. Additionally, this model is completely autologous, and its possible endothelialization could promote the early vascularization process after grafting and thus significantly reducing inflammation and possible rejection.
Subject(s)
Artificial Organs , Plastic Surgery Procedures/methods , Tissue Engineering/methods , Urinary Bladder/surgery , Animals , Cell Survival , Fluorescent Antibody Technique , Humans , Immunoblotting , Materials Testing , Mechanical Phenomena , Permeability , Sus scrofa , Urinary Bladder/pathology , Urinary Bladder/ultrastructureABSTRACT
BACKGROUND: Recombinant human activated Protein C (APC) is used as an adjunctive therapeutic treatment in septic shock. APC seemingly acts on coagulation-inflammation interaction but also by decreasing proinflammatory gene activity, thus inhibiting subsequent production of proinflammatory cytokines, NO and NO-induced mediators, reactive oxygen species production and leukocyte-endothelium interaction. The hemodynamic effects of APC on arterial pressure and cardiac function are now well established in animal models. However, the specific effects of APC on heart and vessels have never been studied. OBJECTIVES: To investigate the potential protective properties of therapeutic ranges of APC on a rat endotoxic shock model in terms of anti-inflammatory and cytoprotective pathways. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. INTERVENTIONS: Rats were exposed to lipopolysaccharide (LPS) (10 mg/Kg intravenous). Endotoxic shock was treated with infusion of saline with or without APC (33 microg/kg/h) during 4 hrs. Hemodynamic parameters were continuously assessed and measurements of muscle oxygen partial pressures, NO and superoxide anion (O2(-)) by spin trapping, of NF-kappaB, metalloproteinase-9 (MMP-9) and inducible NO synthase (iNOS) by Western blotting, as well as leukocyte infiltration and MMP-9 activity were performed at both the heart and aorta level (tissue). MAIN RESULTS: APC partially prevented the reduction of blood pressure induced by LPS and improved both vascular hyporeactivity and myocardial performance. This was associated with a decreased up-regulation of NF-kappaB, iNOS and MMP-9. LPS-induced tissue increases in NO and O2(-) production were decreased by APC. Furthermore, APC decreased tissue leukocyte infiltration/activation as assessed by a decrease in myeloperoxidase and matrix metalloproteinase 9 activity. CONCLUSIONS: These data suggest that APC improves cardiovascular function: 1) by modulating the endotoxin induced-proinflammatory/prooxidant state, 2) by decreasing endothelial/leukocyte interaction and 3) by favoring stabilization of the extracellular matrix.
Subject(s)
Anti-Infective Agents/therapeutic use , Blood Vessels/metabolism , Inflammation/prevention & control , Oxidative Stress/drug effects , Protein C/therapeutic use , Shock, Septic/drug therapy , Animals , Blood Vessels/drug effects , Endotoxemia/complications , Heart/drug effects , Lipopolysaccharides/administration & dosage , Male , Myocardium/metabolism , Rats , Rats, Wistar , Recombinant Proteins/therapeutic use , Shock, Septic/etiology , Shock, Septic/immunologyABSTRACT
We examine whether increased oxidative stress in vivo promotes telomere shortening in CAST/Ei mice. We explored the effects of L-buthionine sulfoximine treatment (BSO) on telomere length. BSO shortened telomere length in white fat, brown fat, skin, tail, and testis in concert with diminished tissue glutathione content, increased tissue carbonyl content, and increased plasma advanced oxidized protein products. Telomerase activity was mainly detected in testis but no reduction of telomerase activity was observed in response to BSO. In conclusion, BSO-mediated increase in systemic oxidative stress shortens telomeres in several tissues of the mouse. The variable effect of BSO treatment on telomere length in different tissue may result from their different adaptive antioxidative capacity.
Subject(s)
Glutathione/metabolism , Oxidative Stress , Telomere/ultrastructure , Animals , Blood Pressure/drug effects , Buthionine Sulfoximine/pharmacology , Male , Mice , Organ Specificity , Protein Carbonylation , Telomerase/metabolism , Telomere/drug effects , Testis/metabolismABSTRACT
OBJECTIVE: To examine differences in telomere (terminal restriction fragment [TRF]) length and pulse wave velocity (PWV)--an index of arterial stiffness--in patients with type 2 diabetes with and without microalbuminuria (MA). RESEARCH DESIGN AND METHODS: A total of 84 men with type 2 diabetes, 40 with MA and 44 without MA (aged 63.5 +/- 9.0 vs. 61.2 +/- 9.8 years), were studied. TRF length was determined in white blood cells. MA was defined as albumin excretion rate (AER) in the range of 30-300 mg/24 h in at least two of three 24-h urine collections. PWV was assessed using applanation tonometry. Markers of oxidative stress were also measured. RESULTS: TRF length was shorter in patients with MA than in those without MA (6.64 +/- 0.74 vs. 7.23 +/- 1.01 kb, respectively, P = 0.004). PWV was significantly higher in the patients with MA. Multivariate linear regression analysis in the total sample demonstrated an independent association between TRF length and age (P = 0.02), MA status (P = 0.04) or AER (P = 0.002), and plasma nitrotyrosine levels (P = 0.02). AER was associated significantly with PWV (P < 0.01). CONCLUSIONS: Subjects with type 2 diabetes and MA have shorter TRF length and increased arterial stiffness than those without MA. Additionally, TRF length is associated with age, AER, and nitrosative stress. As shorter TRF length indicates older biological age, the increased arterial stiffness in patients with type 2 diabetes who have MA may be due to the more pronounced "aging " of these subjects.
Subject(s)
Albuminuria/physiopathology , Diabetes Mellitus, Type 2/physiopathology , Leukocytes/pathology , Telomere/ultrastructure , Aged , Albuminuria/genetics , Cellular Senescence , Diabetes Mellitus, Type 2/genetics , Diabetic Angiopathies/genetics , Diabetic Angiopathies/physiopathology , Diabetic Nephropathies/genetics , Diabetic Nephropathies/physiopathology , Humans , Leukocytes/physiology , Male , Middle Aged , Multivariate Analysis , Regression AnalysisABSTRACT
This review presents some of the recent technological developments in biomaterials used for the construction of synthetic cardiovascular vessels that are capable of simulating specific biological responses. However, with respect to the problems of stiffness, a major hypertensive risk factor, it is necessary to underline the important role of mechanical properties, such as vessel strength and composition, in vascular reconstructive surgery. Biomaterials occupy a central place in many cardiovascular disease treatments and they depend on the chemical nature of the polymers, on the biotechnology used, and also on cellular and gene therapy. Several methodologies using animal or human cells have emerged for constructing blood vessel replacements. Tissue-engineered blood vessel (TEBV) substitutes begin to motivate much work and have contributed to the restoration, maintenance, and/or improvement in tissue and organ function. Each methodology has it benefits, its promises, and holds many challenges in future biological, biomaterial and clinical research.
Subject(s)
Arteries/cytology , Blood Vessel Prosthesis , Tissue Engineering/methods , Biocompatible Materials , Biomechanical Phenomena , HumansABSTRACT
BACKGROUND: We examined the effects of early high salt diet (HSD) and angiotensin II type 1 (AT1) receptor antagonist valsartan (Val) on mortality and carotid distensibility in surviving spontaneously hypertensive rats (SHRs). METHODS: The HSD was initiated either early (week 4 after birth) or late (week 10), continued until 20 weeks of age, and compared to normal salt diet (NSD) groups. Valsartan was given from the fourth week after birth. RESULTS: Eighty-six percent of the rats died in early HSD on placebo, 70% in early HSD on Val-3 mg, 35% in early HSD on Val-30 mg, and 13% in late HSD on placebo. Mean arterial pressure (MAP) was higher in the early HSD and late HSD groups on placebo compared with NSD. The Val-30 mg reduced MAP in all except early HSD groups. Distensibility at MAP (operational distensibility) was lower in late HSD on placebo than in NSD placebo groups. The Val-30 mg increased distensibility in NSD groups. There was no effect of Val in late HSD and early HSD groups. Operational distensibility was negatively correlated with MAP and salt and positively correlated with Val treatment. All animals receiving HSD showed a higher isobaric distensibility in early HSD than in late HSD groups and a smaller distensibility in rats treated with Val. CONCLUSIONS: Our results showed that administration of early HSD in SHR was associated together with a high mortality, a protective action of Val that increased longevity, and an increased level of isobaric distensibility. Survival in HSD groups suggest a direct role of angiotensin II in salt-induced cardiovascular mortality. This role is associated with MAP independent of changes in carotid stiffness.
Subject(s)
Angiotensin II Type 1 Receptor Blockers/pharmacology , Antihypertensive Agents/pharmacology , Cardiovascular Diseases/prevention & control , Carotid Arteries/drug effects , Hypertension/drug therapy , Sodium Chloride, Dietary/administration & dosage , Tetrazoles/pharmacology , Valine/analogs & derivatives , Angiotensin II Type 1 Receptor Blockers/therapeutic use , Animals , Antihypertensive Agents/therapeutic use , Blood Pressure/drug effects , Cardiovascular Diseases/etiology , Carotid Arteries/physiopathology , Compliance/drug effects , Disease Models, Animal , Hypertension/complications , Hypertension/physiopathology , Male , Rats , Rats, Inbred SHR , Tetrazoles/therapeutic use , Time Factors , Valine/pharmacology , Valine/therapeutic use , ValsartanABSTRACT
Stiffening of large arteries is considered as an independent predictor of cardiovascular events. This article summarizes recent theories on the mechanisms contributing to arterial stiffness involving extracellular matrix proteins, endothelial and smooth muscle cells, cell-matrix interactions, and genetic background. Despite the important role of genetic factors in essential hypertension, little is known about the genetic of arterial stiffness. In the future, candidate genes approaches will allow to determine the cellular and molecular mechanisms of arterial stiffness. A review of different strategies aimed at lowering arterial stiffness and potentially reducing cardiovascular risk are presented. Lifestyle changes and antihypertensive drugs have showed beneficial effects in reversing stiffness. New emerging tools as gene therapy and molecules involved in matrix attachments or crosslink of collagen may be promising. Therapeutic trials using such strategies will be necessary to demonstrate their impact on morbidity and mortality.
Subject(s)
Arteries/pathology , Vascular Diseases/physiopathology , Vascular Diseases/therapy , Vascular Resistance/genetics , Animals , Antihypertensive Agents/pharmacology , Cell Adhesion , Collagen/chemistry , Endothelial Cells/cytology , Extracellular Matrix/metabolism , Genetic Predisposition to Disease , Humans , Hypertension/genetics , Models, Biological , Models, GeneticABSTRACT
1. Smooth muscle cells (SMC) participate in both inflammatory and dedifferentiation processes during atherosclerosis, as well as during mechanical injury following angioplasty. In the latter, we studied medial SMC differentiation and inflammation processes implicated early after de-endothelialization in relation to mechanical stresses. We hypothesized that activation of a subpopulation of SMC within the media plays a crucial role in the early phase of neointimal formation. 2. For this purpose, we used a rabbit model of balloon injury to study activation and differentiation of medial SMC in the early time after denudation and just before neointima thickening. Inflammation was evaluated by the expression of vascular cell adhesion molecule (VCAM)-1, integrin alpha4beta1 and nuclear factor (NF)-kB. Myosin isoforms and 2P1A2 antigen, a membrane protein expressed by rabbit dedifferentiated SMC, were used as markers of differentiation. 3. On day 2 after de-endothelialization, VCAM-1, alpha4beta1 and NF-kB were coexpressed by a well-defined subpopulation of SMC of the internal part of the media, in the vicinity of the blood stream. At the same time, the majority of SMC throughout the media expressed non-muscle myosin heavy chain-B (nm-MHC-B) and 2P1A2 antigen. On day 7, when intimal thickening appeared, SMC of the media were no longer activated, whereas some intimal SMC expressed the activation markers. Thus, after de-endothelialization, early dedifferentiation occurs in most of the medial SMC, whereas activation concerned only a subpopulation of SMC located in the internal media. Using the T-type voltage-operated calcium channel blocker mibefradil (0.1-1 micromol/L) in SMC culture, we showed that this agent exhibited an antiproliferative effect in a dose-dependent manner only on undifferentiated cells. 4. In conclusion, the results suggest that the activated SMC represent cells that are potentially able to migrate and participate in the intimal thickening process. Thus, the medial SMC inflammatory process, without any contribution of inflammatory cells, may represent a major mechanism underlying the development of intimal thickening following mechanical stress. In humans, inhibition of T-type calcium channels may be a tool to prevent the early proliferation step leading to neointimal formation.
Subject(s)
Aorta/metabolism , Myocytes, Smooth Muscle/metabolism , Tunica Media/metabolism , Animals , Aorta/drug effects , Aorta/injuries , Catheterization/adverse effects , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression/drug effects , Humans , Immunohistochemistry , In Situ Hybridization , Inflammation/metabolism , Inflammation/pathology , Integrin alpha4beta1/analysis , Mibefradil/pharmacology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Muscle, Smooth, Vascular/pathology , Myocytes, Smooth Muscle/drug effects , Myocytes, Smooth Muscle/pathology , Myosin Heavy Chains/analysis , Myosin Heavy Chains/genetics , NF-kappa B/analysis , Rabbits , Time Factors , Tunica Media/drug effects , Tunica Media/pathology , Vascular Cell Adhesion Molecule-1/analysis , Vasodilator Agents/pharmacologyABSTRACT
PURPOSE: In rats, chronic aldosterone administration with high diet intake increases aortic stiffness independent of mechanical stress. In hypertensive humans, enhanced plasma aldosterone and arterial stiffness are positively associated. Whether the aldosterone synthase gene polymorphism (ASGP) CYP11B2 influences the age-related changes in blood pressure (BP) and arterial stiffness in hypertensive subjects has never been investigated. METHODS: In 425 untreated hypertensive men and women, ASGP was evaluated together with aortic pulse wave velocity (PWV). In 191 of these subjects, cardiac haemodynamics were measured using echo-Doppler techniques. RESULTS: In the overall population, independently of sex, the TC and CC genotypes of ASPG had significantly higher heart rate (HR) (P < 0.05) and lower stroke index (P < 0.01) than the TT genotype, but did not affect BP. In men, the adjusted slopes of the curves relating age to PWV and HR were significantly steeper (P = 0.04; P = 0.002) for the TC and CC than for the TT genotype. Such gene-related differences were not observed for the age-systolic BP relationship. CONCLUSION: In hypertensive subjects, the TC and CC genotypes of ASGP involve, by comparison with the TT genotype, significantly higher HR and reduced stroke index. In men with the C allele, the reduced stroke index (cardiac effect) compensates for the steep increase of PWV with age (arterial effect), thus modulating the cardiovascular phenotype and explaining the lack of increased incidence of systolic hypertension. The results are consistent with a local role of endogenous aldosterone on both heart and vessels.