RESUMO
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.
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Quimiotaxia de Leucócito/imunologia , Neutrófilos/imunologia , Neutrófilos/metabolismo , Receptores de Formil Peptídeo/metabolismo , Receptores de Lipoxinas/metabolismo , Infecções Estafilocócicas/imunologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/imunologia , beta-Arrestinas/metabolismo , Biomarcadores , Interações Hospedeiro-Patógeno , Humanos , Imuno-Histoquímica , NADPH Oxidases/metabolismo , Ativação de Neutrófilo/imunologia , Espécies Reativas de Oxigênio/metabolismo , Infecções Estafilocócicas/microbiologiaRESUMO
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.
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Proliferação de Células/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/genética , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Animais , Células CHO , Diferenciação Celular/efeitos dos fármacos , Cricetulus , Feminino , Fatores de Crescimento de Fibroblastos/classificação , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica , Biblioteca Gênica , Ensaios de Triagem em Larga Escala , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cultura Primária de CélulasRESUMO
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.
Assuntos
Infecções por HIV/imunologia , HIV-1/imunologia , Imunoglobulina G/sangue , Imunoglobulina M/sangue , Lipoproteínas LDL/imunologia , Adulto , Idoso , Estudos de Casos e Controles , Colesterol/sangue , Colesterol/imunologia , Feminino , Infecções por HIV/sangue , Infecções por HIV/epidemiologia , Humanos , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Triglicerídeos/sangue , Triglicerídeos/imunologia , Adulto JovemRESUMO
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.
Assuntos
Diabetes Mellitus Experimental , Pé Diabético , Humanos , Camundongos , Animais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator 1 de Crescimento de Fibroblastos , Neovascularização Fisiológica/fisiologia , Cicatrização/fisiologia , Modelos Animais de DoençasRESUMO
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.
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Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Peptídeos , Humanos , Animais , Camundongos , Miócitos Cardíacos/metabolismo , Peptídeos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Biblioteca de Peptídeos , Células Hep G2 , Técnicas de Visualização da Superfície Celular/métodos , Sistemas de Liberação de Medicamentos , Sequenciamento de Nucleotídeos em Larga Escala , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/terapiaRESUMO
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.
Assuntos
Acetil-CoA Carboxilase , Diferenciação Celular , Miócitos Cardíacos , Células-Tronco Pluripotentes , Humanos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Acetil-CoA Carboxilase/metabolismo , Acetil-CoA Carboxilase/antagonistas & inibidores , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Diferenciação Celular/efeitos dos fármacos , Meios de Cultura/farmacologia , Inibidores Enzimáticos/farmacologia , AnimaisRESUMO
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.
Assuntos
Vesículas Extracelulares , Células-Tronco Mesenquimais , Infarto do Miocárdio , Miócitos Cardíacos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/transplante , Humanos , Animais , Camundongos , Infarto do Miocárdio/terapia , Infarto do Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Fibroblastos/metabolismo , Masculino , Traumatismo por Reperfusão Miocárdica/terapia , Traumatismo por Reperfusão Miocárdica/metabolismo , Modelos Animais de Doenças , Neovascularização Fisiológica , Células CultivadasRESUMO
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.
Assuntos
COVID-19 , Vesículas Extracelulares , Camundongos , Animais , RNA Mensageiro/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , COVID-19/metabolismo , Vesículas Extracelulares/metabolismoRESUMO
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.
Assuntos
Angiopoietina-2/sangue , Neoplasias da Próstata/irrigação sanguínea , Fator A de Crescimento do Endotélio Vascular/sangue , Idoso , Idoso de 80 Anos ou mais , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/sangue , Neovascularização Patológica/metabolismo , Orquiectomia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/cirurgia , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Adenocarcinoma/tratamento farmacológico , Inibidores da Angiogênese/uso terapêutico , Metástase Neoplásica/tratamento farmacológico , Orquiectomia , Neoplasias da Próstata/tratamento farmacológico , Quinolinas/uso terapêutico , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Camundongos , Camundongos Nus , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Quinolonas , Resultado do Tratamento , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Manuseio das Vias Aéreas , Intubação Intratraqueal , Animais , Gasometria , Gases , Hemodinâmica , Intubação Intratraqueal/efeitos adversos , Intubação Intratraqueal/métodos , RatosRESUMO
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.
Assuntos
Dispositivos Lab-On-A-Chip , Neutrófilos , Colágeno , Endotélio , Matriz Extracelular , Células Endoteliais da Veia Umbilical Humana , Humanos , Inflamação , Neutrófilos/fisiologiaRESUMO
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.
Assuntos
Proteínas de Ligação ao Cálcio , Insuficiência Cardíaca , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Coração , Camundongos , RNA MensageiroRESUMO
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.
Assuntos
Proteínas do Tecido Nervoso , Células-Tronco Pluripotentes , Animais , Diferenciação Celular , Cicatriz/patologia , Cicatriz/prevenção & controle , Fibrose , Humanos , Miocárdio/patologia , Miócitos Cardíacos/patologia , Células-Tronco Pluripotentes/patologia , Receptores Imunológicos , SuínosRESUMO
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.
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BACKGROUND: Decreased expression of the angiogenesis inhibitor ADAMTS1 (ADAM metallopeptidase with thrombospondin type 1 motif, 1) has previously been reported during prostate cancer progression. The aim of this study was to investigate the function of ADAMTS1 in prostate tumors. METHODS: ADAMTS1 was downregulated by shRNA technology in the human prostate cancer cell line LNCaP (androgen-dependent), originally expressing ADAMTS1, and was upregulated by transfection in its subline LNCaP-19 (androgen-independent), expressing low levels of ADAMTS1. Cells were implanted subcutaneously in nude mice and tumor growth, microvessel density (MVD), blood vessel morphology, pericyte coverage and thrombospondin 1 (TSP1) were studied in the tumor xenografts. RESULTS: Modified expression of ADAMTS1 resulted in altered blood vessel morphology in the tumors. Low expression levels of ADAMTS1 were associated with small diameter blood vessels both in LNCaP and LNCaP-19 tumors, while high levels of ADAMTS1 were associated with larger vessels. In addition, TSP1 levels in the tumor xenografts were inversely related to ADAMTS1 expression. MVD and pericyte coverage were not affected. Moreover, upregulation of ADAMTS1 inhibited tumor growth of LNCaP-19, as evidenced by delayed tumor establishment. In contrast, downregulation of ADAMTS1 in LNCaP resulted in reduced tumor growth rate. CONCLUSIONS: The present study demonstrates that ADAMTS1 is an important regulatory factor of angiogenesis and tumor growth in prostate tumors, where modified ADAMTS1 expression resulted in markedly changed blood vessel morphology, possibly related to altered TSP1 levels.
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Proteínas ADAM/metabolismo , Vasos Sanguíneos/enzimologia , Neovascularização Patológica/enzimologia , Neoplasias da Próstata/irrigação sanguínea , Neoplasias da Próstata/enzimologia , Trombospondina 1/metabolismo , Proteínas ADAM/genética , Proteína ADAMTS1 , Animais , Vasos Sanguíneos/patologia , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/genética , Pericitos/enzimologia , Neoplasias da Próstata/genética , Interferência de RNA , Fatores de Tempo , Transfecção , Carga TumoralRESUMO
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.
RESUMO
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.
Assuntos
Endocitose/genética , Pirimidinas/farmacologia , Receptores de Formil Peptídeo/genética , Receptores de Lipoxinas/genética , beta-Arrestina 1/genética , Complexo 2 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/genética , Clatrina/química , Endocitose/efeitos dos fármacos , Células HEK293 , Humanos , NADPH Oxidases/genética , Neutrófilos/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Pirimidinas/química , Espécies Reativas de Oxigênio/metabolismo , Receptores de Formil Peptídeo/química , Receptores Acoplados a Proteínas G/genética , Receptores de Lipoxinas/química , Transdução de Sinais/efeitos dos fármacos , beta-Arrestina 1/químicaRESUMO
BACKGROUND: Interactions between prostate cancer cells and their surrounding stroma play an important role in the growth and maintenance of prostate tumors. To elucidate this further, we investigated how growth of androgen-dependent (AD) LNCaP and androgen-independent (AI) LNCaP-19 prostate tumors was affected by different microenvironments and androgen levels. METHODS: Tumor cells were implanted subcutaneously and orthotopically in intact and castrated immunodeficient mice. Orthotopic tumor growth was followed by magnetic resonance imaging (MRI). Gene expression in the tumors was evaluated by means of microarray analysis and microvessel density (MVD) was analyzed using immunohistochemistry. RESULTS: The results showed that LNCaP-19 tumors grew more rapidly at the subcutaneous site than in the prostate, where tumors were obviously inhibited. Castration of the mice did not affect ectopic tumors but did result in increased tumor growth in the prostatic environment. This effect was reversed by testosterone treatment. In contrast to LNCaP-19, the LNCaP cells grew rapidly in the prostate and castration reduced tumor development. Gene expression analysis of LNCaP-19 tumors revealed an upregulation of genes, inhibiting tumor growth (including ADAMTS1, RGS2 and protocadherin 20) and a downregulation of genes, promoting cell adhesion and metastasis (including N-cadherin and NRCAM) in the slow-growing orthotopic tumors from intact mice. CONCLUSIONS: The results show that the prostatic environment has a varying impact on AD and AI tumor xenografts. Data indicate that the androgen-stimulated prostatic environment limits growth of orthotopic AI tumors through induction of genes that inhibit tumor growth and suppression of genes that promote cell adhesion and metastasis.
Assuntos
Adenocarcinoma/patologia , Androgênios/fisiologia , Proliferação de Células/efeitos dos fármacos , Próstata/fisiologia , Neoplasias da Próstata/patologia , Testosterona/farmacologia , Transplante Heterólogo/patologia , Proteínas ADAM/metabolismo , Proteína ADAMTS1 , Adenocarcinoma/metabolismo , Adenocarcinoma/fisiopatologia , Animais , Caderinas/metabolismo , Castração , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Proteínas RGS/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
OBJECTIVE: To investigate the expression of 'ADAM metallopeptidase with thrombospondin type I motif, 1' (ADAMTS1) in human prostate cancer, and to study its relationship to microvessel density (MVD) and metastasis. ADAMTS1 has been described as an anti-angiogenic and antitumour factor, but its function in prostate cancer is unknown. PATIENTS AND METHODS: ADAMTS1 expression was evaluated by immunohistochemistry in specimens obtained by transurethral resection of the prostate from patients with hormone-naïve and hormone-refractory prostate tumours, including adjacent benign tissue. A semiquantitative scoring system was used for evaluating the staining. MVD was quantified by counting the number of CD34-positive blood vessels. RESULTS: ADAMTS1 was strongly expressed in the luminal epithelial cells in benign prostate glands, whereas expression was significantly lower in prostate cancer cells. There was no obvious difference between hormone-naïve and hormone-refractory tumours, and ADAMTS1 expression did not correlate with Gleason score. However, in hormone-refractory tumours from patients with metastatic disease, the expression of ADAMTS1 was significantly lower than in tumours from patients without metastases. Furthermore, the MVD was higher in hormone-refractory than in hormone-naïve tumours and benign tissue, and MVD correlated with Gleason score. There was no association between ADAMTS1 and MVD in the hormone-naïve tumours, while hormone-refractory tumours with low ADAMTS1 expression had a higher MVD than those with moderate/high expression. CONCLUSION: ADAMTS1 expression is decreased in prostate cancer, and might be involved in the early steps of prostate cancer development. Further, ADAMTS1 might have an anti-angiogenic and antimetastatic role in hormone-refractory prostate cancer, where low ADAMTS1 expression is associated with a high MVD and metastasis.