ABSTRACT
Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) constitute an appealing tool for drug discovery, disease modeling, and cardiotoxicity screening. However, their physiological immaturity, resembling CMs in the late fetal stage, limits their utility. Herein, we have developed a novel, scalable cell culture medium designed to enhance the maturation of hPSC-CMs. This medium facilitates a metabolic shift towards fatty acid utilization and augments mitochondrial function by targeting Acetyl-CoA carboxylase 2 (ACC2) with a specific small molecule inhibitor. Our findings demonstrate that this maturation protocol significantly advances the metabolic, structural, molecular and functional maturity of hPSC-CMs at various stages of differentiation. Furthermore, it enables the creation of cardiac microtissues with superior structural integrity and contractile properties. Notably, hPSC-CMs cultured in this optimized maturation medium display increased accuracy in modeling a hypertrophic cardiac phenotype following acute endothelin-1 induction and show a strong correlation between in vitro and in vivo target engagement in drug screening efforts. This approach holds promise for improving the utility and translatability of hPSC-CMs in cardiac disease modeling and drug discovery.
Subject(s)
Acetyl-CoA Carboxylase , Cell Differentiation , Myocytes, Cardiac , Pluripotent Stem Cells , Humans , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Acetyl-CoA Carboxylase/metabolism , Acetyl-CoA Carboxylase/antagonists & inhibitors , Pluripotent Stem Cells/drug effects , Pluripotent Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Cell Differentiation/drug effects , Culture Media/pharmacology , Enzyme Inhibitors/pharmacology , AnimalsABSTRACT
Heart failure remains a leading cause of mortality. Therapeutic intervention for heart failure would benefit from targeted delivery to the damaged heart tissue. Here, we applied in vivo peptide phage display coupled with high-throughput Next-Generation Sequencing (NGS) and identified peptides specifically targeting damaged cardiac tissue. We established a bioinformatics pipeline for the identification of cardiac targeting peptides. Hit peptides demonstrated preferential uptake by human induced pluripotent stem cell (iPSC)-derived cardiomyocytes and immortalized mouse HL1 cardiomyocytes, without substantial uptake in human liver HepG2 cells. These novel peptides hold promise for use in targeted drug delivery and regenerative strategies and open new avenues in cardiovascular research and clinical practice.
Subject(s)
Induced Pluripotent Stem Cells , Myocytes, Cardiac , Peptides , Humans , Animals , Mice , Myocytes, Cardiac/metabolism , Peptides/metabolism , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Peptide Library , Hep G2 Cells , Cell Surface Display Techniques/methods , Drug Delivery Systems , High-Throughput Nucleotide Sequencing , Heart Failure/metabolism , Heart Failure/therapyABSTRACT
Small extracellular vesicles (sEV) derived from various cell sources have been demonstrated to enhance cardiac function in preclinical models of myocardial infarction (MI). The aim of this study was to compare different sources of sEV for cardiac repair and determine the most effective one, which nowadays remains limited. We comprehensively assessed the efficacy of sEV obtained from human primary bone marrow mesenchymal stromal cells (BM-MSC), human immortalized MSC (hTERT-MSC), human embryonic stem cells (ESC), ESC-derived cardiac progenitor cells (CPC), human ESC-derived cardiomyocytes (CM), and human primary ventricular cardiac fibroblasts (VCF), in in vitro models of cardiac repair. ESC-derived sEV (ESC-sEV) exhibited the best pro-angiogenic and anti-fibrotic effects in vitro. Then, we evaluated the functionality of the sEV with the most promising performances in vitro, in a murine model of MI-reperfusion injury (IRI) and analysed their RNA and protein compositions. In vivo, ESC-sEV provided the most favourable outcome after MI by reducing adverse cardiac remodelling through down-regulating fibrosis and increasing angiogenesis. Furthermore, transcriptomic, and proteomic characterizations of sEV derived from hTERT-MSC, ESC, and CPC revealed factors in ESC-sEV that potentially drove the observed functions. In conclusion, ESC-sEV holds great promise as a cell-free treatment for promoting cardiac repair following MI.
Subject(s)
Extracellular Vesicles , Mesenchymal Stem Cells , Myocardial Infarction , Myocytes, Cardiac , Extracellular Vesicles/metabolism , Extracellular Vesicles/transplantation , Humans , Animals , Mice , Myocardial Infarction/therapy , Myocardial Infarction/metabolism , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/cytology , Mesenchymal Stem Cells/metabolism , Mesenchymal Stem Cells/cytology , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/cytology , Human Embryonic Stem Cells/metabolism , Fibroblasts/metabolism , Male , Myocardial Reperfusion Injury/therapy , Myocardial Reperfusion Injury/metabolism , Disease Models, Animal , Neovascularization, Physiologic , Cells, CulturedABSTRACT
BACKGROUND: Diabetic foot ulcers (DFU) pose a significant health risk in diabetic patients, with insufficient revascularization during wound healing being the primary cause. This study aimed to assess microvessel sprouting and wound healing capabilities using vascular endothelial growth factor (VEGF-A) and a modified fibroblast growth factor (FGF1). METHODS: An ex vivo aortic ring rodent model and an in vivo wound healing model in diabetic mice were employed to evaluate the microvessel sprouting and wound healing capabilities of VEGF-A and a modified FGF1 both as monotherapies and in combination. RESULTS: The combination of VEGF-A and FGF1 demonstrated increased vascular sprouting in the ex vivo mouse aortic ring model, and topical administration of a combination of VEGF-A and FGF1 mRNAs formulated in lipid nanoparticles (LNPs) in mouse skin wounds promoted faster wound closure and increased neovascularization seven days post-surgical wound creation. RNA-sequencing analysis of skin samples at day three post-wound creation revealed a strong transcriptional response of the wound healing process, with the combined treatment showing significant enrichment of genes linked to skin growth. CONCLUSION: f-LNPs encapsulating VEGF-A and FGF1 mRNAs present a promising approach to improving the scarring process in DFU.
Subject(s)
Diabetes Mellitus, Experimental , Diabetic Foot , Humans , Mice , Animals , Vascular Endothelial Growth Factor A/metabolism , Fibroblast Growth Factor 1 , Neovascularization, Physiologic/physiology , Wound Healing/physiology , Disease Models, AnimalABSTRACT
Lipid nanoparticles (LNPs) are currently used to transport functional mRNAs, such as COVID-19 mRNA vaccines. The delivery of angiogenic molecules, such as therapeutic VEGF-A mRNA, to ischemic tissues for producing new blood vessels is an emerging strategy for the treatment of cardiovascular diseases. Here, the authors deliver VEGF-A mRNA via LNPs and study stoichiometric quantification of their uptake kinetics and how the transport of exogenous LNP-mRNAs between cells is functionally extended by cells' own vehicles called extracellular vesicles (EVs). The results show that cellular uptake of LNPs and their mRNA molecules occurs quickly, and that the translation of exogenously delivered mRNA begins immediately. Following the VEGF-A mRNA delivery to cells via LNPs, a fraction of internalized VEGF-A mRNA is secreted via EVs. The overexpressed VEGF-A mRNA is detected in EVs secreted from three different cell types. Additionally, RNA-Seq analysis reveals that as cells' response to LNP-VEGF-A mRNA treatment, several overexpressed proangiogenic transcripts are packaged into EVs. EVs are further deployed to deliver VEGF-A mRNA in vitro and in vivo. Upon equal amount of VEGF-A mRNA delivery via three EV types or LNPs in vitro, EVs from cardiac progenitor cells are the most efficient in promoting angiogenesis per amount of VEGF-A protein produced. Intravenous administration of luciferase mRNA shows that EVs could distribute translatable mRNA to different organs with the highest amounts of luciferase detected in the liver. Direct injections of VEGF-A mRNA (via EVs or LNPs) into mice heart result in locally produced VEGF-A protein without spillover to liver and circulation. In addition, EVs from cardiac progenitor cells cause minimal production of inflammatory cytokines in cardiac tissue compared with all other treatment types. Collectively, the data demonstrate that LNPs transform EVs as functional extensions to distribute therapeutic mRNA between cells, where EVs deliver this mRNA differently than LNPs.
Subject(s)
COVID-19 , Extracellular Vesicles , Mice , Animals , RNA, Messenger/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , COVID-19/metabolism , Extracellular Vesicles/metabolismABSTRACT
Heart regeneration is an unmet clinical need, hampered by limited renewal of adult cardiomyocytes and fibrotic scarring. Pluripotent stem cell-based strategies are emerging, but unravelling cellular dynamics of host-graft crosstalk remains elusive. Here, by combining lineage tracing and single-cell transcriptomics in injured non-human primate heart biomimics, we uncover the coordinated action modes of human progenitor-mediated muscle repair. Chemoattraction via CXCL12/CXCR4 directs cellular migration to injury sites. Activated fibroblast repulsion targets fibrosis by SLIT2/ROBO1 guidance in organizing cytoskeletal dynamics. Ultimately, differentiation and electromechanical integration lead to functional restoration of damaged heart muscle. In vivo transplantation into acutely and chronically injured porcine hearts illustrated CXCR4-dependent homing, de novo formation of heart muscle, scar-volume reduction and prevention of heart failure progression. Concurrent endothelial differentiation contributed to graft neovascularization. Our study demonstrates that inherent developmental programmes within cardiac progenitors are sequentially activated in disease, enabling the cells to sense and counteract acute and chronic injury.
Subject(s)
Nerve Tissue Proteins , Pluripotent Stem Cells , Animals , Cell Differentiation , Cicatrix/pathology , Cicatrix/prevention & control , Fibrosis , Humans , Myocardium/pathology , Myocytes, Cardiac/pathology , Pluripotent Stem Cells/pathology , Receptors, Immunologic , SwineABSTRACT
The dysregulated physical interaction between two intracellular membrane proteins, the sarco/endoplasmic reticulum Ca2+ ATPase and its reversible inhibitor phospholamban, induces heart failure by inhibiting calcium cycling. While phospholamban is a bona-fide therapeutic target, approaches to selectively inhibit this protein remain elusive. Here, we report the in vivo application of intracellular acting antibodies (intrabodies), derived from the variable domain of camelid heavy-chain antibodies, to modulate the function of phospholamban. Using a synthetic VHH phage-display library, we identify intrabodies with high affinity and specificity for different conformational states of phospholamban. Rapid phenotypic screening, via modified mRNA transfection of primary cells and tissue, efficiently identifies the intrabody with most desirable features. Adeno-associated virus mediated delivery of this intrabody results in improvement of cardiac performance in a murine heart failure model. Our strategy for generating intrabodies to investigate cardiac disease combined with modified mRNA and adeno-associated virus screening could reveal unique future therapeutic opportunities.
Subject(s)
Calcium-Binding Proteins , Heart Failure , Animals , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Dependovirus/genetics , Dependovirus/metabolism , Heart , Mice , RNA, MessengerABSTRACT
Inflammatory diseases are often characterised by excessive neutrophil infiltration from the blood stream to the site of inflammation, which damages healthy tissue and prevents resolution of inflammation. Development of anti-inflammatory drugs is hindered by lack of in vitro and in vivo models which accurately represent the disease microenvironment. In this study, we used the OrganoPlate to develop a humanized 3D in vitro inflammation-on-a-chip model to recapitulate neutrophil transmigration across the endothelium and subsequent migration through the extracellular matrix (ECM). Human umbilical vein endothelial cells formed confluent vessels against collagen I and geltrex mix, a mix of basement membrane extract and collagen I. TNF-α-stimulation of vessels upregulated inflammatory cytokine expression and promoted neutrophil transmigration. Intriguingly, major differences were found depending on the composition of the ECM. Neutrophils transmigrated in higher number and further in geltrex mix than collagen I, and did not require an N-formyl-methionyl-leucyl-phenylalanine (fMLP) gradient for transmigration. Inhibition of neutrophil proteases inhibited neutrophil transmigration on geltrex mix, but not collagen I. These findings highlight the important role of the ECM in determining cell phenotype and response to inhibitors. Future work could adapt the ECM composition for individual diseases, producing accurate models for drug development.
Subject(s)
Lab-On-A-Chip Devices , Neutrophils , Collagen , Endothelium , Extracellular Matrix , Human Umbilical Vein Endothelial Cells , Humans , Inflammation , Neutrophils/physiologyABSTRACT
Open chest surgery in rodents requires assisted breathing and the most common approach for ventilation is via an endotracheal tube. Even with well-trained operators the endotracheal intubation is technically challenging and may lead to prolonged procedures and endotracheal intubation complications. Nose cone ventilation is a simpler procedure compared to endotracheal intubation and has the potential to improve animal welfare by reducing procedure time and endotracheal intubation associated complications. Rats are obligate nose breathers, and therefore replacing intubation with air supply from a nose cone would be an advantage and a more natural way of breathing. Here, we compared the values for several blood gases, blood pressure and heart rate from rats that were nose cone ventilated with rats that underwent endotracheal intubation at 12 timepoints equally distributed across three surgical stages: baseline, open chest and closed chest. Throughout the monitoring period the hemodynamic and blood gas values for both methods of ventilation were within published, normal ranges for the rat and were biologically equivalent (equivalence test p value ≤ 0.05). Our data showed that nose cone ventilation-maintained blood gases and hemodynamic homeostasis equivalent to endotracheal intubation. Nose cone ventilation can be recommended as an alternative to endotracheal intubation in rat experiments where investigators require airway control.
Subject(s)
Airway Management , Intubation, Intratracheal , Animals , Blood Gas Analysis , Gases , Hemodynamics , Intubation, Intratracheal/adverse effects , Intubation, Intratracheal/methods , RatsABSTRACT
Objective: After myocardial infarction (MI), the non-infarcted left ventricle (LV) ensures appropriate contractile function of the heart. Metabolic disturbance in this region greatly exacerbates post-MI heart failure (HF) pathology. This study aimed to provide a comprehensive understanding of the metabolic derangements occurring in the non-infarcted LV that could trigger cardiovascular deterioration. Methods and Results: We used a pig model that progressed into chronic HF over 3 months following MI induction. Integrated gene and metabolite signatures revealed region-specific perturbations in amino acid- and lipid metabolism, insulin signaling and, oxidative stress response. Remote LV, in particular, showed impaired glutamine and arginine metabolism, altered synthesis of lipids, glucose metabolism disorder, and increased insulin resistance. LPIN1, PPP1R3C, PTPN1, CREM, and NR0B2 were identified as the main effectors in metabolism dysregulation in the remote zone and were found differentially expressed also in the myocardium of patients with ischemic and/or dilated cardiomyopathy. In addition, a simultaneous significant decrease in arginine levels and altered PRCP, PTPN1, and ARF6 expression suggest alterations in vascular function in remote area. Conclusions: This study unravels an array of dysregulated genes and metabolites putatively involved in maladaptive metabolic and vascular remodeling in the non-infarcted myocardium and may contribute to the development of more precise therapies to mitigate progression of chronic HF post-MI.
ABSTRACT
FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to ß-arrestin recruitment. ß-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of ß-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent ß-arrestin/AP2 interaction and ß-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/ß-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of ß-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in ß-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of ß-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of ß-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/ß-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils.
Subject(s)
Endocytosis/genetics , Pyrimidines/pharmacology , Receptors, Formyl Peptide/genetics , Receptors, Lipoxin/genetics , beta-Arrestin 1/genetics , Adaptor Protein Complex 2/chemistry , Adaptor Protein Complex 2/genetics , Clathrin/chemistry , Endocytosis/drug effects , HEK293 Cells , Humans , NADPH Oxidases/genetics , Neutrophils/drug effects , Protein Binding/drug effects , Pyrimidines/chemistry , Reactive Oxygen Species/metabolism , Receptors, Formyl Peptide/chemistry , Receptors, G-Protein-Coupled/genetics , Receptors, Lipoxin/chemistry , Signal Transduction/drug effects , beta-Arrestin 1/chemistryABSTRACT
PURPOSE: Targeted alpha therapy (TAT) is a promising treatment for micrometastatic and minimal residual cancer. We evaluated systemic α-radioimmunotherapy (α-RIT) of metastatic castration-resistant prostate cancer (mCRPC) using the α-particle emitter 211At-labeled to the anti-PSCA A11 minibody. A11 is specific for prostate stem cell antigen (PSCA), a cell surface glycoprotein which is overexpressed in more than 90% of both localized prostate cancer and bone metastases. METHODS: PC3-PSCA cells were implanted subcutaneously (s.c.) and intratibially (i.t) in nude mice. Efficacy of α-RIT (two fractions-14-day interval) was studied on s.c. macrotumors (0, 1.5 and 1.9 MBq) and on i.t. microtumors (~100-200 µm; 0, 0.8 or 1.5 MBq) by tumor-volume measurements. The injected activities for therapies were estimated from separate biodistribution and myelotoxicity studies. RESULTS: Tumor targeting of 211At-A11 was efficient and the effect on s.c. macrotumors was strong and dose-dependent. At 6 weeks, the mean tumor volumes for the treated groups, compared with controls, were reduced by approximately 85%. The separate myelotoxicity study following one single fraction showed reduced white blood cells (WBC) for all treated groups on day 6 after treatment. For the 0.8 and 1.5 MBq, the WBC reductions were transient and followed by recovery at day 13. For 2.4 MBq, a clear toxicity was observed and the mice were sacrificed on day 7. In the long-term follow-up of the 0.8 and 1.5 MBq-groups, blood counts on day 252 were normal and no signs of radiotoxicity observed. Efficacy on i.t. microtumors was evaluated in two experiments. In experiment 1, the tumor-free fraction (TFF) was 95% for both treated groups and significantly different (p < 0.05) from the controls at a TFF of 66%). In experiment 2, the difference in TFF was smaller, 32% for the treated group versus 20% for the controls. However, the difference in microtumor volume in experiment 2 was highly significant, 0.010 ± 0.003 mm3 versus 3.79 ± 1.24 mm3 (treated versus controls, respectively), i.e., a 99.7% reduction (p < 0.001). The different outcome in experiment 1 and 2 is most likely due to differences in microtumor sizes at therapy, or higher tumor-take in experiment 2 (where more cells were implanted). CONCLUSION: Evaluating fractionated α-RIT with 211At-labeled anti-PSCA A11 minibody, we found clear growth inhibition on both macrotumors and intratibial microtumors. For mice treated with multiple fractions, we also observed radiotoxicity manifested by progressive loss in body weight at 30 to 90 days after treatment. Our findings are conceptually promising for a systemic TAT of mCRPC and warrant further investigations of 211At-labeled PSCA-directed vectors. Such studies should include methods to improve the therapeutic window, e.g., by implementing a pretargeted regimen of α-RIT or by altering the size of the targeting vector.
ABSTRACT
Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naïve cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.
Subject(s)
Cell Proliferation/drug effects , Fibroblast Growth Factors/genetics , Induced Pluripotent Stem Cells/drug effects , Myocytes, Cardiac/drug effects , Animals , CHO Cells , Cell Differentiation/drug effects , Cricetulus , Female , Fibroblast Growth Factors/classification , Fibroblast Growth Factors/metabolism , Fibroblast Growth Factors/pharmacology , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , Gene Library , High-Throughput Screening Assays , Humans , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mice , Mice, Inbred C57BL , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Primary Cell CultureABSTRACT
Formyl peptide receptor 2 (FPR2) is a G protein-coupled pattern recognition receptor sensing both mitochondrial- and bacterial-derived formylated peptides, including the PSMα toxins secreted by community-associated methicillin-resistant Staphylococcus aureus strains. Similar to many other FPR2 agonistic peptides, nanomolar concentrations of both PSMα2 and PSMα3 activate neutrophils to increase the cytosolic concentration of Ca2+ and release NADPH oxidase-derived reactive oxygen species. In addition, the PSMα peptides induce FPR2 homologous desensitization, actin polymerization, and neutrophil reactivation through a receptor cross-talk mechanism. However, in contrast to conventional FPR2 agonistic peptides, including the host-derived formyl peptide MCT-ND4, we found that the PSMα peptides lacked the ability to recruit ß-arrestin and induce neutrophil chemotaxis, supporting the previous notion that ß-arrestin translocation is of importance for cell migration. Despite the lack of ß-arrestin recruitment, the PSMα peptides induced an FPR2-dependent ERK1/2 phosphorylation and internalization. Furthermore, structure-activity relationship analysis with PSMα2 derivatives revealed critical roles of the first 3 aa linked to N-fMet as well as the C terminus of PSMα2 in promoting FPR2 to recruit ß-arrestin. In summary, our data demonstrate a novel neutrophil activation pattern upon FPR2 sensing of PSMα peptides, signified by the ability to induce increased intracellular Ca2+, ERK1/2 phosphorylation, internalization, and NADPH oxidase activity, yet lack of ß-arrestin recruitment and neutrophil chemoattraction. These novel features adopted by the PSMα peptides could be of importance for S. aureus virulence and might facilitate identification of new therapeutic strategies for treating S. aureus infections.
Subject(s)
Chemotaxis, Leukocyte/immunology , Neutrophils/immunology , Neutrophils/metabolism , Receptors, Formyl Peptide/metabolism , Receptors, Lipoxin/metabolism , Staphylococcal Infections/immunology , Staphylococcal Infections/metabolism , Staphylococcus aureus/immunology , beta-Arrestins/metabolism , Biomarkers , Host-Pathogen Interactions , Humans , Immunohistochemistry , NADPH Oxidases/metabolism , Neutrophil Activation/immunology , Reactive Oxygen Species/metabolism , Staphylococcal Infections/microbiologyABSTRACT
mRNA can direct dose-dependent protein expression in cardiac muscle without genome integration, but to date has not been shown to improve cardiac function in a safe, clinically applicable way. Herein, we report that a purified and optimized mRNA in a biocompatible citrate-saline formulation is tissue specific, long acting, and does not stimulate an immune response. In small- and large-animal, permanent occlusion myocardial infarction models, VEGF-A 165 mRNA improves systolic ventricular function and limits myocardial damage. Following a single administration a week post-infarction in mini pigs, left ventricular ejection fraction, inotropy, and ventricular compliance improved, border zone arteriolar and capillary density increased, and myocardial fibrosis decreased at 2 months post-treatment. Purified VEGF-A mRNA establishes the feasibility of improving cardiac function in the sub-acute therapeutic window and may represent a new class of therapies for ischemic injury.
ABSTRACT
BACKGROUND: Type 2 diabetes is associated with macro- and microvascular complications in man. Microvascular dysfunction affects both cardiac and renal function and is now recognized as a main driver of cardiovascular mortality and morbidity. However, progression of microvascular dysfunction in experimental models is often obscured by macrovascular pathology and consequently demanding to study. The obese type 2 diabetic leptin-deficient (ob/ob) mouse lacks macrovascular complications, i.e. occlusive atherosclerotic disease, and may therefore be a potential model for microvascular dysfunction. The present study aimed to test the hypothesis that these mice with an insulin resistant phenotype might display microvascular dysfunction in both coronary and renal vascular beds. METHODS AND RESULTS: In this study we used non-invasive Doppler ultrasound imaging to characterize microvascular dysfunction during the progression of diabetes in ob/ob mice. Impaired coronary flow velocity reserve was observed in the ob/ob mice at 16 and 21 weeks of age compared to lean controls. In addition, renal resistivity index as well as pulsatility index was higher in the ob/ob mice at 21 weeks compared to lean controls. Moreover, plasma L-arginine was lower in ob/ob mice, while asymmetric dimethylarginine was unaltered. Furthermore, a decrease in renal vascular density was observed in the ob/ob mice. CONCLUSION: In parallel to previously described metabolic disturbances, the leptin-deficient ob/ob mice also display cardiac and renal microvascular dysfunction. This model may therefore be suitable for translational, mechanistic and interventional studies to improve the understanding of microvascular complications in type 2 diabetes.
Subject(s)
Coronary Circulation , Diabetes Mellitus, Type 2/diagnostic imaging , Leptin/deficiency , Renal Circulation , Animals , Diabetes Mellitus, Type 2/genetics , Laser-Doppler Flowmetry , Leptin/genetics , Male , Mice , UltrasonographyABSTRACT
BACKGROUND: Chronic HIV infection is associated with increased risk of cardiovascular disease caused by atherosclerosis. Oxidized forms of low-density lipoprotein (LDL) are present in atherosclerotic lesions and constitute major epitopes for natural antibodies. IgM has been shown to be protective against atherosclerosis, whereas the role of corresponding IgG is less clear. The objective of this study was to determine if HIV + individuals have disturbed levels of IgM and IgG directed against oxidized forms of LDL as compared to HIV- individuals. METHODS: Ninety-one HIV + patients and 92 HIV- controls were included in this retrospective study. Circulating levels of IgG and IgM directed against two forms of oxidized LDL; copper oxidized (OxLDL) and malondialdehyde modified (MDA-LDL), total IgM and IgG, C-reactive protein (CRP), soluble CD14, and apolipoproteins A1 and B were determined. RESULTS: HIV + individuals had slightly lower levels of IgM against MDA-LDL and higher levels of IgG against MDA-LDL, OxLDL, and total IgG, than HIV- controls. Anti-MDA-LDL and Anti-OxLDL IgG displayed a positive correlation with viral load and a negative correlation with the CD4+ T-cell count. HIV + individuals also displayed elevated CRP and soluble CD14 levels compared to HIV- individuals, but there were no correlations between CRP or soluble CD14 and specific antibodies. CONCLUSIONS: HIV infection is associated with higher levels of IgG including specific IgG against oxidized forms of LDL, and lower IgM against the same epitope. In addition to dyslipidemia, immune activation, HIV-replication and an accumulation of risk factors for atherosclerosis, this adverse antibody profile may be of major importance for the increased risk of cardiovascular disease in HIV + individuals.
Subject(s)
HIV Infections/immunology , HIV-1/immunology , Immunoglobulin G/blood , Immunoglobulin M/blood , Lipoproteins, LDL/immunology , Adult , Aged , Case-Control Studies , Cholesterol/blood , Cholesterol/immunology , Female , HIV Infections/blood , HIV Infections/epidemiology , Humans , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Male , Middle Aged , Retrospective Studies , Triglycerides/blood , Triglycerides/immunology , Young AdultABSTRACT
Castration-resistant prostate cancer (CRPC) is strongly associated with sclerotic bone metastases and poor prognosis. Models that mimic human CRPC are needed to identify the mechanisms for prostate cancer (PC) growth in bone and to develop new therapeutic strategies. We characterize a new model, LNCaP-19, and investigate the interaction between tumor cells and osteoblasts in the sclerotic tumor response of CRPC. Osteogenic profiling of PC cell lines (LNCaP-19, LNCaP, C4-2B4, and PC-3) was performed by gene expression arrays and mineral staining. Conditioned medium from MC3T3-E1 was used for osteoblast stimulation of CRPC cells. The capacity of LNCaP-19 cells to induce sclerotic lesions was assessed in intratibial xenografts and verified by serum markers, histological analysis and bone mineral density (BMD) measurements. The CRPC cell line LNCaP-19 expresses a pronounced osteogenic profile compared to its parental androgen-dependent cell line LNCaP. Osteoblast-derived factors further increase the expression of genes known to enhance metastatic progression of PC. LNCaP-19 forms sclerotic tumors in tibia of castrated mice as evident by increased total BMD (P < 0.01). There was a strong correlation between serum osteocalcin and BMD (total: R (2) 0.811, P < 0.01, trabecular: R (2) 0.673, P < 0.05). For the first time we demonstrate that a CRPC cell line generated in vitro has osteogenic capacity and that osteomimicry can be an inherent feature of these cells. Osteoblast-derived factors further promote the osteogenic and metastatic phenotype in CRPC cells. Altogether, our model demonstrates that both tumor cells and osteoblasts are mediators of the bone forming process of CRPC.
Subject(s)
Neoplasm Metastasis/pathology , Osteogenesis , Prostatic Neoplasms, Castration-Resistant/pathology , Animals , Bone Neoplasms/genetics , Bone Neoplasms/secondary , Cell Line, Tumor , Disease Models, Animal , Gene Expression Regulation, Neoplastic , Humans , Male , Mice , Osteoblasts/cytology , Osteoblasts/metabolism , Prostate-Specific Antigen/blood , Prostatic Neoplasms, Castration-Resistant/blood , Prostatic Neoplasms, Castration-Resistant/etiology , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that has completed phase II clinical trial in patients with castration resistant prostate cancer, showing promising inhibiting effects on the occurrence of metastasis and delayed disease progression. Its mechanism of action is not fully elucidated, but previous studies show anti-angiogenic effects and strong interaction with the S100A9 protein. METHODS: This study was performed to evaluate if tasquinimod inhibits prostate cancer metastasis, by using both orthotopic and intratibial xenograft models. Animals were treated with tasquinimod, and tumor growth characteristics as well as molecular markers for metastasis and angiogenesis were analyzed. RESULTS: The results show that formation of lung and lymph node metastases from orthotopic castration resistant prostate tumors was inhibited by tasquinimod treatment. Importantly, establishment of tumors in the bone after intratibial implantation was suppressed by tasquinimod. In addition, establishment and growth of subcutaneous tumors were affected. Both in primary tumors and serum from treated mice an upregulation of thrombospondin 1 was observed. Further, downregulation of the hypoxia driven genes VEGF, CXCR4, and LOX was detected in the primary tasquinimod-treated tumors and decreased expression of chemotactic ligand SDF-1 was demonstrated in the lungs. Thus, these molecular changes could contribute to the anti-angiogenic and anti-metastatic effects of tasquinimod. CONCLUSIONS: In conclusion, this study and clinical data show that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Therefore, tasquinimod is an interesting treatment option for patients with prostate cancer prone to metastasis.
Subject(s)
Adenocarcinoma/drug therapy , Angiogenesis Inhibitors/therapeutic use , Neoplasm Metastasis/drug therapy , Orchiectomy , Prostatic Neoplasms/drug therapy , Quinolines/therapeutic use , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Cell Line, Tumor , Down-Regulation , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Male , Mice , Mice, Nude , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Quinolones , Treatment Outcome , Up-Regulation , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: Angiogenesis is important for the progression of prostate cancer and may be a target for treatment in castration resistant (CR) disease. This study was performed to investigate blood vessel stabilization and expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and Angiopoietin-2 (Ang-2) in CR and hormone naïve (HN) prostate cancer. The effect of androgen deprivation therapy (ADT) on these parameters was also studied. METHODS: VEGF and Ang-2, as well as pericyte coverage of blood vessels were studied in HN and CR prostate tumors by immunohistochemistry. The effects of ADT on VEGF expression and microvessel density (MVD) were investigated in biopsies at diagnosis, 3 months after starting ADT and at tumor relapse. Plasma was also analyzed for VEGF and Ang-2 with ELISA. RESULTS: CR tumors had higher levels of VEGF and Ang-2 as well as increased blood vessel stabilization compared to HN tumors. Three months after initiated ADT an increase of VEGF but not MVD in the tumors was observed. In contrast, plasma levels of VEGF decreased after ADT, and increased again at time of tumor relapse. Ang-2 levels were unaffected. CONCLUSIONS: CR prostate cancer is associated with elevated levels of VEGF and Ang-2, indicating that these factors could be used as targets for anti-angiogenic treatment. Still, the observed increase in blood vessel stabilization in CR tumors could influence the outcome of anti-angiogenic treatment. Furthermore, increased VEGF expression after 3 months of ADT justifies the use of VEGF-based anti-angiogenic drugs in combination with ADT for the treatment of advanced prostate cancer.