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BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.
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Citocinas , Pulmão , Microglia , Pneumonia , Citocinas/metabolismo , Pulmão/metabolismo , COVID-19 , Encéfalo , Autopsia , Humanos , Camundongos , Disfunção Cognitiva , Imunofluorescência , Pneumonia/metabolismo , Interleucina-1beta/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Background: To date, at least 20 different amyloidogenic proteins have been documented. Growing evidence suggests that despite being part of the universal amyloid proteome, apolipoprotein A-IV can be amyloidogenic, accounting for less than 1% of cases. Case summary: A 75-year-old woman was admitted for paroxysmal nocturnal dyspnoea and intermittent exertional shortness of breath and was found to be in acute heart failure. The patient underwent intravenous diuretic therapy and was discharged after decongestion. She then underwent a battery of outpatient tests to determine aetiology of her heart failure. Cardiac magnetic resonance imaging showed severe concentric left ventricular hypertrophy and diffuse late gadolinium enhancement, concerning for amyloidosis, but serologic evaluation for amyloidogenic light chain (AL) amyloidosis was negative. Tc 99m pyrophosphate (PYP) scan showed Grade 2 uptake at 1â h that was only moderately suggestive of transthyretin (TTR) amyloidosis. She ultimately received a right heart catheterization and endomyocardial biopsy, which showed apolipoprotein A-IV amyloid deposition within Congo red-positive areas of the endomyocardial specimen. The patient continues to report dyspnoea on exertion but has avoided additional heart failure admissions with intensification of her diuretic regimen. Discussion: In this case, nuclear PYP scan to evaluate for TTR amyloidosis demonstrated focal PYP uptake, but endomyocardial biopsy demonstrated apolipoprotein A-IV deposition without evidence of TTR amyloidosis. Our case increases knowledge of this rare form of amyloidosis, suggests that it may result in false positive nuclear PYP results, and highlights the importance of its evaluation, particularly in circumstances in which investigations do not reveal definitive evidence of AL or TTR amyloidosis.
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Neurological impairment is the most common finding in patients with post-acute sequelae of COVID-19. Furthermore, survivors of pneumonia from any cause have an elevated risk of dementia1-4. Dysfunction in microglia, the primary immune cell in the brain, has been linked to cognitive impairment in murine models of dementia and in humans5. Here, we report a transcriptional response in human microglia collected from patients who died following COVID-19 suggestive of their activation by TNF-α and other circulating pro-inflammatory cytokines. Consistent with these findings, the levels of 55 alveolar and plasma cytokines were elevated in a cohort of 341 patients with respiratory failure, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. While peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, cumulative cytokine exposure was higher in patients with COVID-19. Corticosteroid treatment, which has been shown to be beneficial in patients with COVID-196, was associated with lower levels of CXCL10, CCL8, and CCL2-molecules that sustain inflammatory circuits between alveolar macrophages harboring SARS-CoV-2 and activated T cells7. These findings suggest that corticosteroids may break this cycle and decrease systemic exposure to lung-derived cytokines and inflammatory activation of microglia in patients with COVID-19.
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Neoplasias Cardíacas/patologia , Leiomioma/patologia , Neoplasias Uterinas/secundário , Procedimentos Cirúrgicos Cardíacos , Feminino , Neoplasias Cardíacas/diagnóstico por imagem , Neoplasias Cardíacas/cirurgia , Humanos , Leiomioma/diagnóstico por imagem , Leiomioma/cirurgia , Neoplasias Pulmonares/diagnóstico por imagem , Neoplasias Pulmonares/secundário , Pessoa de Meia-Idade , Nódulos Pulmonares Múltiplos/diagnóstico por imagem , Nódulos Pulmonares Múltiplos/secundário , Resultado do TratamentoRESUMO
OBJECTIVES: The novel coronavirus, severe acute respiratory syndrome coronavirus 2, causing coronavirus disease 2019 (COVID-19) remains a global health threat and a significant source of human morbidity and mortality. While the virus primarily induces lung injury, it also has been reported to cause hepatic sequelae. METHODS: We aimed to detect the virus in formalin-fixed tissue blocks and document the liver injury patterns in patients with COVID-19 compared with a control group. RESULTS: We were able to detect viral RNA in the bronchioalveolar cell blocks (12/12, 100%) and formalin-fixed, paraffin-embedded tissue of the lung (8/8, 100%) and liver (4/9, 44%) of patients with COVID-19. Although the peak values of the main liver enzymes and bilirubin were higher in the patients with COVID-19 compared with the control group, the differences were not significant. The main histologic findings were minimal to focal mild portal tract chronic inflammation (7/8, 88%, P < .05) and mild focal lobular activity (6/8, 75%, P = .06). CONCLUSIONS: We found that most patients who died of COVID-19 had evidence of mild focal hepatitis clinically and histologically; however, the virus was detected in less than half of the cases.
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COVID-19/virologia , Formaldeído , Fígado/patologia , SARS-CoV-2/patogenicidade , Fixação de Tecidos , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Inflamação/patologia , Fígado/virologia , Pulmão/patologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Fixação de Tecidos/métodosAssuntos
Bloqueio Atrioventricular/diagnóstico , Cardiomiopatias/diagnóstico , Diagnóstico Diferencial , Células Gigantes/patologia , Granuloma/patologia , Miocardite/diagnóstico , Sarcoidose/diagnóstico , Bloqueio Atrioventricular/etiologia , Bloqueio Atrioventricular/fisiopatologia , Biópsia , Progressão da Doença , Ecocardiografia , Fluordesoxiglucose F18 , Bloqueio Cardíaco/diagnóstico , Bloqueio Cardíaco/etiologia , Bloqueio Cardíaco/fisiopatologia , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/cirurgia , Transplante de Coração , Humanos , Linfonodos/patologia , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Miocardite/complicações , Miocardite/patologia , Miocardite/fisiopatologia , Miocárdio/patologia , Peptídeo Natriurético Encefálico/sangue , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Compostos Radiofarmacêuticos , Volume Sistólico , Troponina I/sangueRESUMO
Some patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe pneumonia and acute respiratory distress syndrome1 (ARDS). Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from that in other types of pneumonia2. Here we investigate SARS-CoV-2 pathobiology by characterizing the immune response in the alveoli of patients infected with the virus. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens, and analysed them using flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA sequencing on 10 bronchoalveolar lavage fluid samples collected from patients with severe coronavirus disease 2019 (COVID-19) within 48 h of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-γ to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages containing SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.
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COVID-19/imunologia , COVID-19/virologia , Macrófagos Alveolares/imunologia , Pneumonia Viral/imunologia , Pneumonia Viral/virologia , SARS-CoV-2/patogenicidade , Linfócitos T/imunologia , Líquido da Lavagem Broncoalveolar/química , Líquido da Lavagem Broncoalveolar/imunologia , COVID-19/genética , Estudos de Coortes , Humanos , Interferon gama/imunologia , Interferons/imunologia , Interferons/metabolismo , Macrófagos Alveolares/metabolismo , Macrófagos Alveolares/virologia , Pneumonia Viral/genética , RNA-Seq , SARS-CoV-2/imunologia , Transdução de Sinais/imunologia , Análise de Célula Única , Linfócitos T/metabolismo , Fatores de TempoRESUMO
Lung transplantation can potentially be a life-saving treatment for patients with nonresolving COVID-19-associated respiratory failure. Concerns limiting lung transplantation include recurrence of SARS-CoV-2 infection in the allograft, technical challenges imposed by viral-mediated injury to the native lung, and the potential risk for allograft infection by pathogens causing ventilator-associated pneumonia in the native lung. Additionally, the native lung might recover, resulting in long-term outcomes preferable to those of transplant. Here, we report the results of lung transplantation in three patients with nonresolving COVID-19-associated respiratory failure. We performed single-molecule fluorescence in situ hybridization (smFISH) to detect both positive and negative strands of SARS-CoV-2 RNA in explanted lung tissue from the three patients and in additional control lung tissue samples. We conducted extracellular matrix imaging and single-cell RNA sequencing on explanted lung tissue from the three patients who underwent transplantation and on warm postmortem lung biopsies from two patients who had died from COVID-19-associated pneumonia. Lungs from these five patients with prolonged COVID-19 disease were free of SARS-CoV-2 as detected by smFISH, but pathology showed extensive evidence of injury and fibrosis that resembled end-stage pulmonary fibrosis. Using machine learning, we compared single-cell RNA sequencing data from the lungs of patients with late-stage COVID-19 to that from the lungs of patients with pulmonary fibrosis and identified similarities in gene expression across cell lineages. Our findings suggest that some patients with severe COVID-19 develop fibrotic lung disease for which lung transplantation is their only option for survival.
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COVID-19/cirurgia , Transplante de Pulmão , Pulmão/cirurgia , Fibrose Pulmonar/cirurgia , Adulto , Idoso de 80 Anos ou mais , COVID-19/diagnóstico , COVID-19/fisiopatologia , COVID-19/virologia , Teste de Ácido Nucleico para COVID-19 , Bases de Dados Factuais , Progressão da Doença , Feminino , Humanos , Hibridização in Situ Fluorescente , Pulmão/fisiopatologia , Pulmão/virologia , Masculino , Pessoa de Meia-Idade , Fibrose Pulmonar/diagnóstico , Fibrose Pulmonar/fisiopatologia , Fibrose Pulmonar/virologia , RNA-Seq , Recuperação de Função Fisiológica , Estudos Retrospectivos , Índice de Gravidade de Doença , Análise de Célula Única , Resultado do TratamentoRESUMO
Lung transplantation can potentially be a life-saving treatment for patients with non-resolving COVID-19 acute respiratory distress syndrome. Concerns limiting transplant include recurrence of SARS-CoV-2 infection in the allograft, technical challenges imposed by viral-mediated injury to the native lung, and potential risk for allograft infection by pathogens associated with ventilator-induced pneumonia in the native lung. Additionally, the native lung might recover, resulting in long-term outcomes preferable to transplant. Here, we report the results of the first two successful lung transplantation procedures in patients with non-resolving COVID-19 associated acute respiratory distress syndrome in the United States. We performed smFISH to detect both positive and negative strands of SARS-CoV-2 RNA in the explanted lung tissue, extracellular matrix imaging using SHIELD tissue clearance, and single cell RNA-Seq on explant and warm post-mortem lung biopsies from patients who died from severe COVID-19 pneumonia. Lungs from patients with prolonged COVID-19 were free of virus but pathology showed extensive evidence of injury and fibrosis which resembled end-stage pulmonary fibrosis. Single cell RNA-Seq of the explanted native lungs from transplant and paired warm post-mortem autopsies showed similarities between late SARS-CoV-2 acute respiratory distress syndrome and irreversible end-stage pulmonary fibrosis requiring lung transplantation. There was no recurrence of SARS-CoV-2 or pathogens associated with pre-transplant ventilator associated pneumonias following transplantation in either patient. Our findings suggest that some patients with severe COVID-19 develop fibrotic lung disease for which lung transplantation is the only option for survival. SINGLE SENTENCE SUMMARY: Some patients with severe COVID-19 develop end-stage pulmonary fibrosis for which lung transplantation may be the only treatment.
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To systematically investigate the reproducibility of global and segmental left ventricular (LV) velocities derived from tissue phase mapping (TPM). Breath held and ECG synchronized TPM data (spatial/temporal resolution = 2 × 2 mm(2)/20.8 ms) were acquired in 18 healthy volunteers. To analyze scan-rescan variability, TPM was repeated in all subjects during a second visit separated by 16 ± 5 days. Data analysis included LV segmentation, and quantification of global and regional (AHA 16-segment modal) metrics of LV function [velocity-time curves, systolic and diastolic peak and time-to-peak (TTP) velocities] for radial (Vr), long-axis (Vz) and circumferential (VΦ) LV velocities. Mean velocity time curves in basal, mid-ventricular, and apical locations showed highly similar LV motion patterns for all three velocity components (Vr, VΦ, Vz) for scan and rescan. No significant differences for both systolic and diastolic peak and TTP myocardial velocities were observed. Segmental analysis revealed similar regional peak Vr and Vz during both systole and diastole except for three LV segments (p = 0.045, p = 0.033, and p = 0.009). Excellent (p < 0.001) correlations between scans and rescan for peak Vr (R(2) = 0.92), peak Vz (R(2) = 0.90), radial TTP (R(2) = 0.91) and long-axis TTP (R(2) = 0.88) confirmed good agreement. Bland-Altman analysis demonstrated excellent intra-observer and good inter-observer analysis agreement but increased variability for long axis peak velocities. TPM based analysis of global and regional myocardial velocities can be performed with good reproducibility. Robustness of regional quantification of long-axis velocities was limited but spatial velocity distributions across the LV could reliably be replicated.
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Ventrículos do Coração/diagnóstico por imagem , Imagem Cinética por Ressonância Magnética/métodos , Contração Miocárdica , Função Ventricular Esquerda , Adulto , Suspensão da Respiração , Eletrocardiografia , Feminino , Voluntários Saudáveis , Humanos , Interpretação de Imagem Assistida por Computador , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Fatores de TempoRESUMO
A 56-year-old woman with hypertension-induced end stage renal disease presented with skin thickening and mottled discoloration. Cutaneous biopsy showed increased dermal fibroblasts embedded in fibromyxoid stroma with scattered perivascular and interstitial mononuclear cells. Immunohistochemistry revealed prominent CD34+ dendritic cells in septal spaces, consistent with Nephrogenic Systemic Fibrosis (NSF). Seven years and two years prior she had received a gadolinium-based contrast agent (GBCA). She died due to NSF. Gross autopsy revealed a thickened and stenotic superior vena cava (SVC). Extensive fibrosis of the SVC, dermis, and subcutaneous tissue was noted, together with hyalinized collagen fibers within the muscular wall of the intestines and dura mater. These findings support the importance of skin changes in the recognition of life threatening extracutaneous tissue involvement in NSF.
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Dermopatia Fibrosante Nefrogênica/complicações , Síndrome da Veia Cava Superior/etiologia , Meios de Contraste/administração & dosagem , Meios de Contraste/efeitos adversos , Evolução Fatal , Feminino , Gadolínio/administração & dosagem , Gadolínio/efeitos adversos , Humanos , Imuno-Histoquímica , Falência Renal Crônica , Pessoa de Meia-Idade , Dermopatia Fibrosante Nefrogênica/etiologia , Dermopatia Fibrosante Nefrogênica/fisiopatologia , Síndrome da Veia Cava Superior/fisiopatologiaRESUMO
Recombinant EF-hand domain of phospholipase C δ1 has a moderate affinity for anionic phospholipids in the absence of Ca(2+) that is driven by interactions of cationic and hydrophobic residues in the first EF-hand sequence. This region of PLC δ1 is missing in the crystal structure. The relative orientation of recombinant EF with respect to the bilayer, established with NMR methods, shows that the N-terminal helix of EF-1 is close to the membrane interface. Specific mutations of EF-1 residues in full-length PLC δ1 reduce enzyme activity but not because of disturbing partitioning of the protein onto vesicles. The reduction in enzymatic activity coupled with vesicle binding studies are consistent with a role for this domain in aiding substrate binding in the active site once the protein is transiently anchored at its target membrane.
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Bicamadas Lipídicas/química , Fosfolipase C delta/química , Fosfolipídeos/química , Animais , Cálcio/química , Cálcio/metabolismo , Domínio Catalítico , Bicamadas Lipídicas/metabolismo , Mutação , Ressonância Magnética Nuclear Biomolecular , Fosfolipase C delta/genética , Fosfolipase C delta/metabolismo , Fosfolipídeos/metabolismo , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Many membrane-associated enzymes, including those of the phospholipase C (PLC) superfamily, are regulated by specific interactions with lipids. Previously, we have shown that the C2 domain of PLC δ1 is required for phosphatidylserine (PS)-dependent enzyme activation and that activation requires the presence of Ca(2+). To identify the site of interaction and the role of Ca(2+) in the activation mechanism, we mutagenized three highly conserved Ca(2+) binding residues (Asp-653, Asp-706, and Asp-708) to Gly in the C2 domain of PLC δ1. The PS-dependent Ca(2+) binding affinities of the mutant enzymes D653G, D706G, and D708G were reduced by 1 order of magnitude, and the maximal level of Ca(2+) binding was reduced to half of that of the native enzyme. The level of Ca(2+)-dependent PS binding was also reduced in the mutant enzymes. Under basal conditions, the Ca(2+) dependence and the maximal level of hydrolysis of phosphatidylinositol 4,5-bisphosphate were not altered in the mutants. However, the Ca(2+)-dependent PS stimulation was severely defective. PS reduces the K(m) of the native enzyme almost 20-fold, but far less for the mutants. Replacing Asp-653, Asp-706, and Asp-708 simultaneously with glycine in the C2 domain of PLC δ1 leads to a complete and selective loss of the stimulation and binding by PS. These results show that D653, D706, and D708 are required for Ca(2+) binding in the C2 domain and demonstrate a mechanism by which C2 domains can mediate regulation of enzyme activity by specific lipid ligands.
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Cálcio/metabolismo , Fosfatidilserinas/metabolismo , Fosfolipase C delta/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação , Hidrólise , Cinética , Ligantes , Modelos Moleculares , Fosfatidilinositóis/metabolismo , Fosfatidilserinas/química , Fosfolipase C delta/metabolismoRESUMO
BACKGROUND: Atrial fibrillation (AF) is commonly associated with congestive heart failure (CHF). The autonomic nervous system is involved in the pathogenesis of both AF and CHF. We examined the role of autonomic remodeling in contributing to AF substrate in CHF. METHODS AND RESULTS: Electrophysiological mapping was performed in the pulmonary veins and left atrium in 38 rapid ventricular-paced dogs (CHF group) and 39 control dogs under the following conditions: vagal stimulation, isoproterenol infusion, ß-adrenergic blockade, acetylcholinesterase (AChE) inhibition (physostigmine), parasympathetic blockade, and double autonomic blockade. Explanted atria were examined for nerve density/distribution, muscarinic receptor and ß-adrenergic receptor densities, and AChE activity. In CHF dogs, there was an increase in nerve bundle size, parasympathetic fibers/bundle, and density of sympathetic fibrils and cardiac ganglia, all preferentially in the posterior left atrium/pulmonary veins. Sympathetic hyperinnervation was accompanied by increases in ß(1)-adrenergic receptor R density and in sympathetic effect on effective refractory periods and activation direction. ß-Adrenergic blockade slowed AF dominant frequency. Parasympathetic remodeling was more complex, resulting in increased AChE activity, unchanged muscarinic receptor density, unchanged parasympathetic effect on activation direction and decreased effect of vagal stimulation on effective refractory period (restored by AChE inhibition). Parasympathetic blockade markedly decreased AF duration. CONCLUSIONS: In this heart failure model, autonomic and electrophysiological remodeling occurs, involving the posterior left atrium and pulmonary veins. Despite synaptic compensation, parasympathetic hyperinnervation contributes significantly to AF maintenance. Parasympathetic and/or sympathetic signaling may be possible therapeutic targets for AF in CHF.
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Fibrilação Atrial/fisiopatologia , Sistema Nervoso Autônomo/fisiopatologia , Mapeamento Potencial de Superfície Corporal/métodos , Átrios do Coração/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Veias Pulmonares/fisiopatologia , Animais , Fibrilação Atrial/etiologia , Estimulação Cardíaca Artificial , Modelos Animais de Doenças , Cães , Insuficiência Cardíaca/complicações , Veias Pulmonares/inervaçãoRESUMO
The mechanism for stem cell-mediated improvement following acute myocardial infarction has been actively debated. We support hypotheses that the stem cell effect is primarily paracrine factor-linked. We used a heparin-presenting injectable nanofibre network to bind and deliver paracrine factors derived from hypoxic conditioned stem cell media to mimic this stem cell paracrine effect. Our self-assembling peptide nanofibres presenting heparin were capable of binding paracrine factors from a medium phase. When these factor-loaded materials were injected into the heart following coronary artery ligation in a mouse ischaemia-reperfusion model of acute myocardial infarction, we found significant preservation of haemodynamic function. Through media manipulation, we were able to determine that crucial factors are primarily < 30 kDa and primarily heparin-binding. Using recombinant VEGF- and bFGF-loaded nanofibre networks, the effect observed with conditioned media was recapitulated. When evaluated in another disease model, a chronic rat ischaemic hind limb, our factor-loaded materials contributed to extensive limb revascularization. These experiments demonstrate the potency of the paracrine effect associated with stem cell therapies and the potential of a biomaterial to bind and deliver these factors, pointing to a potential therapy based on synthetic materials and recombinant factors as an acellular therapy.
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Doenças Cardiovasculares/terapia , Heparina/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Nanofibras/química , Comunicação Parácrina/efeitos dos fármacos , Animais , Meios de Cultivo Condicionados/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Ventrículos do Coração/fisiopatologia , Membro Posterior/irrigação sanguínea , Membro Posterior/efeitos dos fármacos , Membro Posterior/patologia , Transplante de Células-Tronco Mesenquimais , Camundongos , Peso Molecular , Contração Miocárdica/efeitos dos fármacos , Infarto do Miocárdio/fisiopatologia , Peptídeos/química , Peptídeos/metabolismo , Ratos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
This study investigated the effects on cardiomyocyte differentiation of embryonic stem cells by the overexpression of the transcription factor, Pitx2c, and examined the effects of transplantation of these differentiated cells on cardiac function in a mouse model of myocardial infarction. Pitx2c overexpressing embryonic stem cells were characterized for cardiac differentiation by immunocytochemistry, RNA analysis, and electrophysiology. Differentiated cells were transplanted by directed injection into the infarcted murine myocardium and functional measurements of blood pressure, contractility, and relaxation were performed. Histochemistry and FISH analysis performed on these mice confirmed the engraftment and cardiac nature of the transplanted cells. Pitx2c overexpressing embryonic stem cells robustly differentiated into spontaneously contracting cells which acquired cardiac protein markers and exhibited action potentials resembling that of cardiomyocytes. These cells could also be synchronized to an external pacemaker. Significant improvements (P < 0.01) in blood pressure (56%), contractility (57%), and relaxation (59%) were observed in infarcted mice with transplants of these differentiated cells but not in mice which were transplanted with control cells. The Pitx2c overexpressing cells secrete paracrine factors which when adsorbed onto a heparinated gel and injected into the infarcted myocardium produce a comparable and significant (P < 0.01) functional recovery. Pitx2c overexpression is a valuable method for producing cardiomyocytes from embryonic stem cells, and transplantation of these cardiomyocytes into infracted myocardium restores cardiac function through multiple mechanisms.
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Células-Tronco Embrionárias/transplante , Coração/fisiologia , Infarto do Miocárdio/cirurgia , Fatores de Transcrição/análise , Potenciais de Ação , Animais , Cálcio/análise , Diferenciação Celular , Células Cultivadas , Células-Tronco Embrionárias/química , Células-Tronco Embrionárias/fisiologia , Feminino , Imuno-Histoquímica , Camundongos , Infarto do Miocárdio/fisiopatologia , Miocárdio/citologia , RNA/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alicerces TeciduaisRESUMO
Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non-receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP(2)) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP(2), although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection.