RESUMO
Tissue-resident macrophages are complementary to proinflammatory macrophages to promote the progression of atherosclerosis. The noninvasive detection of their presence and dynamic variation will be important to the understanding of their role in the pathogenesis of atherosclerosis. The goal of this study was to develop a targeted PET radiotracer for imaging CD163-positive (CD163+) macrophages in multiple mouse atherosclerosis models and assess the potential of CD163 as a biomarker for atherosclerosis in humans. Methods: CD163-binding peptide was identified using phage display and conjugated with a NODAGA chelator for 64Cu radiolabeling ([64Cu]Cu-ICT-01). CD163-overexpressing U87 cells were used to measure the binding affinity of [64Cu]Cu-ICT-01. Biodistribution studies were performed on wild-type C57BL/6 mice at multiple time points after tail vein injection. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages upregulated on the surface of atherosclerotic plaques were assessed in multiple mouse atherosclerosis models. Immunostaining, flow cytometry, and single-cell RNA sequencing were performed to characterize the expression of CD163 on tissue-resident macrophages. Human carotid atherosclerotic plaques were used to measure the expression of CD163+ resident macrophages and test the binding specificity of [64Cu]Cu-ICT-01. Results: [64Cu]Cu-ICT-01 showed high binding affinity to U87 cells. The biodistribution study showed rapid blood and renal clearance with low retention in all major organs at 1, 2, and 4 h after injection. In an ApoE-/- mouse model, [64Cu]Cu-ICT-01 demonstrated sensitive and specific detection of CD163+ macrophages and capability for tracking the progression of atherosclerotic lesions; these findings were further confirmed in Ldlr-/- and PCSK9 mouse models. Immunostaining showed elevated expression of CD163+ macrophages across the plaques. Flow cytometry and single-cell RNA sequencing confirmed the specific expression of CD163 on tissue-resident macrophages. Human tissue characterization demonstrated high expression of CD163+ macrophages on atherosclerotic lesions, and ex vivo autoradiography revealed specific binding of [64Cu]Cu-ICT-01 to human CD163. Conclusion: This work reported the development of a PET radiotracer binding CD163+ macrophages. The elevated expression of CD163+ resident macrophages on human plaques indicated the potential of CD163 as a biomarker for vulnerable plaques. The sensitivity and specificity of [64Cu]Cu-ICT-01 in imaging CD163+ macrophages warrant further investigation in translational settings.
Assuntos
Antígenos CD , Antígenos de Diferenciação Mielomonocítica , Aterosclerose , Macrófagos , Tomografia por Emissão de Pósitrons , Receptores de Superfície Celular , Animais , Camundongos , Tomografia por Emissão de Pósitrons/métodos , Antígenos de Diferenciação Mielomonocítica/metabolismo , Antígenos CD/metabolismo , Aterosclerose/diagnóstico por imagem , Aterosclerose/metabolismo , Macrófagos/metabolismo , Receptores de Superfície Celular/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Radioisótopos de Cobre , Distribuição Tecidual , Compostos Radiofarmacêuticos/farmacocinéticaRESUMO
Cardiac macrophages are essential mediators of cardiac development, tissue homeostasis, and response to injury. Cell-intrinsic shifts in metabolism and availability of metabolites regulate macrophage function. The human and mouse heart contain a heterogeneous compilation of cardiac macrophages that are derived from at least two distinct lineages. In this review, we detail the unique functional roles and metabolic profiles of tissue-resident and monocyte-derived cardiac macrophages during embryonic development and adult tissue homeostasis and in response to pathologic and physiologic stressors. We discuss the metabolic preferences of each macrophage lineage and how metabolism influences monocyte fate specification. Finally, we highlight the contribution of cardiac macrophages and derived metabolites on cell-cell communication, metabolic health, and disease pathogenesis.
Assuntos
Macrófagos , Miocárdio , Camundongos , Animais , Humanos , Miocárdio/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Coração , HomeostaseRESUMO
Solid organ transplant represents a potentially lifesaving procedure for patients suffering from end-stage heart, lung, liver, and kidney failure. However, rejection remains a significant source of morbidity and immunosuppressive medications have significant toxicities. Janus kinase (JAK) inhibitors are effective immunosuppressants in autoimmune diseases and graft versus host disease after allogeneic hematopoietic cell transplantation. Here we examine the role of JAK inhibition in preclinical fully major histocompatibility mismatched skin and heart allograft models. Baricitinib combined with cyclosporine A (CsA) preserved fully major histocompatibility mismatched skin grafts for the entirety of a 111-day experimental period. In baricitinib plus CsA treated mice, circulating CD4+T-bet+ T cells, CD8+T-bet+ T cells, and CD4+FOXP3+ regulatory T cells were reduced. Single cell RNA sequencing revealed a unique expression profile in immune cells in the skin of baricitinib plus CsA treated mice, including decreased inflammatory neutrophils and increased CCR2- macrophages. In a fully major histocompatibility mismatched mismatched heart allograft model, baricitinib plus CsA prevented graft rejection for the entire 28-day treatment period compared with 9 days in controls. Our findings establish that the combination of baricitinib and CsA prevents rejection in allogeneic skin and heart graft models and supports the study of JAK inhibitors in human solid organ transplantation.
Assuntos
Ciclosporina , Transplante de Coração , Humanos , Animais , Camundongos , Ciclosporina/uso terapêutico , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/efeitos adversos , SulfonamidasRESUMO
The recruitment of monocytes and their differentiation into immunosuppressive cells is associated with the low efficacy of preclinical nonconformal radiotherapy (RT) for tumors. However, nonconformal RT (non-CRT) does not mimic clinical practice, and little is known about the role of monocytes after RT modes used in patients, such as conformal RT (CRT). Here, we investigated the acute immune response induced by after CRT. Contrary to non-CRT approaches, we found that CRT induces a rapid and robust recruitment of monocytes to the tumor that minimally differentiate into tumor-associated macrophages or dendritic cells but instead up-regulate major histocompatibility complex II and costimulatory molecules. We found that these large numbers of infiltrating monocytes are responsible for activating effector polyfunctional CD8+ tumor-infiltrating lymphocytes that reduce tumor burden. Mechanistically, we show that monocyte-derived type I interferon is pivotal in promoting monocyte accumulation and immunostimulatory function in a positive feedback loop. We also demonstrate that monocyte accumulation in the tumor microenvironment is hindered when RT inadvertently affects healthy tissues, as occurs in non-CRT. Our results unravel the immunostimulatory function of monocytes during clinically relevant modes of RT and demonstrate that limiting the exposure of healthy tissues to radiation has a positive therapeutic effect on the overall antitumor immune response.
Assuntos
Interferon Tipo I , Neoplasias , Humanos , Monócitos , Neoplasias/radioterapia , Diferenciação Celular , Interferon Tipo I/farmacologia , Linfócitos do Interstício Tumoral , Microambiente TumoralRESUMO
Inflammation and tissue fibrosis co-exist and are causally linked to organ dysfunction. However, the molecular mechanisms driving immune-fibroblast crosstalk in human cardiac disease remains unexplored and there are currently no therapeutics to target fibrosis. Here, we performed multi-omic single-cell gene expression, epitope mapping, and chromatin accessibility profiling in 38 donors, acutely infarcted, and chronically failing human hearts. We identified a disease-associated fibroblast trajectory marked by cell surface expression of fibroblast activator protein (FAP), which diverged into distinct myofibroblasts and pro-fibrotic fibroblast populations, the latter resembling matrifibrocytes. Pro-fibrotic fibroblasts were transcriptionally similar to cancer associated fibroblasts and expressed high levels of collagens and periostin (POSTN), thymocyte differentiation antigen 1 (THY-1), and endothelin receptor A (EDNRA) predicted to be driven by a RUNX1 gene regulatory network. We assessed the applicability of experimental systems to model tissue fibrosis and demonstrated that 3 different in vivo mouse models of cardiac injury were superior compared to cultured human heart and dermal fibroblasts in recapitulating the human disease phenotype. Ligand-receptor analysis and spatial transcriptomics predicted that interactions between C-C chemokine receptor type 2 (CCR2) macrophages and fibroblasts mediated by interleukin 1 beta (IL-1ß) signaling drove the emergence of pro-fibrotic fibroblasts within spatially defined niches. This concept was validated through in silico transcription factor perturbation and in vivo inhibition of IL-1ß signaling in fibroblasts where we observed reduced pro-fibrotic fibroblasts, preferential differentiation of fibroblasts towards myofibroblasts, and reduced cardiac fibrosis. Herein, we show a subset of macrophages signal to fibroblasts via IL-1ß and rewire their gene regulatory network and differentiation trajectory towards a pro-fibrotic fibroblast phenotype. These findings highlight the broader therapeutic potential of targeting inflammation to treat tissue fibrosis and restore organ function.
RESUMO
Adipocytes transfer mitochondria to macrophages in white and brown adipose tissues to maintain metabolic homeostasis. In obesity, adipocyte-to-macrophage mitochondria transfer is impaired, and instead, adipocytes release mitochondria into the blood to induce a protective antioxidant response in the heart. We found that adipocyte-to-macrophage mitochondria transfer in white adipose tissue is inhibited in murine obesity elicited by a lard-based high-fat diet, but not a hydrogenated-coconut-oil-based high-fat diet, aging, or a corn-starch diet. The long-chain fatty acids enriched in lard suppress mitochondria capture by macrophages, diverting adipocyte-derived mitochondria into the blood for delivery to other organs, such as the heart. The depletion of macrophages rapidly increased the number of adipocyte-derived mitochondria in the blood. These findings suggest that dietary lipids regulate mitochondria uptake by macrophages locally in white adipose tissue to determine whether adipocyte-derived mitochondria are released into systemic circulation to support the metabolic adaptation of distant organs in response to nutrient stress.
Assuntos
Tecido Adiposo Branco , Antioxidantes , Adipócitos/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Antioxidantes/metabolismo , Dieta Hiperlipídica , Ácidos Graxos/metabolismo , Macrófagos/metabolismo , Camundongos , Mitocôndrias/metabolismo , Obesidade/metabolismo , Amido/metabolismoRESUMO
BACKGROUND: Cellular rejection after heart transplantation imparts significant morbidity and mortality. Current immunosuppressive strategies are imperfect, target recipient T cells, and have adverse effects. The innate immune response plays an essential role in the recruitment and activation of T cells. Targeting the donor innate immune response would represent the earliest interventional opportunity within the immune response cascade. There is limited knowledge about donor immune cell types and functions in the setting of cardiac transplantation, and no current therapeutics exist for targeting these cell populations. METHODS: Using genetic lineage tracing, cell ablation, and conditional gene deletion, we examined donor mononuclear phagocyte diversity and macrophage function during acute cellular rejection of transplanted hearts in mice. We performed single-cell RNA sequencing on donor and recipient macrophages and monocytes at multiple time points after transplantation. On the basis of our imaging and single-cell RNA sequencing data, we evaluated the functional relevance of donor CCR2+ (C-C chemokine receptor 2) and CCR2- macrophages using selective cell ablation strategies in donor grafts before transplant. Last, we performed functional validation that donor macrophages signal through MYD88 (myeloid differentiation primary response protein 88) to facilitate cellular rejection. RESULTS: Donor macrophages persisted in the rejecting transplanted heart and coexisted with recipient monocyte-derived macrophages. Single-cell RNA sequencing identified donor CCR2+ and CCR2- macrophage populations and revealed remarkable diversity among recipient monocytes, macrophages, and dendritic cells. Temporal analysis demonstrated that donor CCR2+ and CCR2- macrophages were transcriptionally distinct, underwent significant morphologic changes, and displayed unique activation signatures after transplantation. Although selective depletion of donor CCR2- macrophages reduced allograft survival, depletion of donor CCR2+ macrophages prolonged allograft survival. Pathway analysis revealed that donor CCR2+ macrophages are activated through MYD88/nuclear factor kappa light chain enhancer of activated B cells signaling. Deletion of MYD88 in donor macrophages resulted in reduced antigen-presenting cell recruitment, reduced ability of antigen-presenting cells to present antigen to T cells, decreased emergence of allograft-reactive T cells, and extended allograft survival. CONCLUSIONS: Distinct populations of donor and recipient macrophages coexist within the transplanted heart. Donor CCR2+ macrophages are key mediators of allograft rejection, and deletion of MYD88 signaling in donor macrophages is sufficient to suppress rejection and extend allograft survival. This highlights the therapeutic potential of donor heart-based interventions.
Assuntos
Transplante de Coração , Animais , Rejeição de Enxerto/prevenção & controle , Transplante de Coração/efeitos adversos , Humanos , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Fator 88 de Diferenciação Mieloide/genética , Doadores de TecidosRESUMO
Cardiac macrophages represent a heterogeneous cell population with distinct origins, dynamics, and functions. Recent studies have revealed that C-C Chemokine Receptor 2 positive (CCR2+) macrophages derived from infiltrating monocytes regulate myocardial inflammation and heart failure pathogenesis. Comparatively little is known about the functions of tissue resident (CCR2-) macrophages. Herein, we identified an essential role for CCR2- macrophages in the chronically failing heart. Depletion of CCR2- macrophages in mice with dilated cardiomyopathy accelerated mortality and impaired ventricular remodeling and coronary angiogenesis, adaptive changes necessary to maintain cardiac output in the setting of reduced cardiac contractility. Mechanistically, CCR2- macrophages interacted with neighboring cardiomyocytes via focal adhesion complexes and were activated in response to mechanical stretch through a transient receptor potential vanilloid 4 (TRPV4)-dependent pathway that controlled growth factor expression. These findings establish a role for tissue-resident macrophages in adaptive cardiac remodeling and implicate mechanical sensing in cardiac macrophage activation.
Assuntos
Cardiomiopatia Dilatada/metabolismo , Ativação de Macrófagos/fisiologia , Macrófagos/metabolismo , Remodelação Ventricular/fisiologia , Animais , Cardiomiopatia Dilatada/genética , Cardiomiopatia Dilatada/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Mutação , Miocárdio/metabolismo , Troponina T/genéticaRESUMO
Since the 1960s, heart and lung transplantation has remained the optimal therapy for patients with end-stage disease, extending and improving quality of life for thousands of individuals annually. Expanding donor organ availability and immunologic compatibility is a priority to help meet the clinical demand for organ transplant. While effective, current immunosuppression is imperfect as it lacks specificity and imposes unintended adverse effects such as opportunistic infections and malignancy that limit the health and longevity of transplant recipients. In this review, we focus on donor macrophages as a new target to achieve allograft tolerance. Donor organ-directed therapies have the potential to improve allograft survival while minimizing patient harm related to global suppression of recipient immune responses. Topics highlighted include the role of ontogenically distinct donor macrophage populations in ischemia-reperfusion injury and rejection, including their interaction with allograft-infiltrating recipient immune cells and potential therapeutic approaches. Ultimately, a better understanding of how donor intrinsic immunity influences allograft acceptance and survival will provide new opportunities to improve the outcomes of transplant recipients.
Assuntos
Transplante de Coração , Transplante de Pulmão , Rejeição de Enxerto/etiologia , Humanos , Transplante de Pulmão/efeitos adversos , Macrófagos , Qualidade de Vida , Doadores de TecidosRESUMO
RATIONALE: Paradigm shifting studies have revealed that the heart contains functionally diverse populations of macrophages derived from distinct embryonic and adult hematopoietic progenitors. Under steady-state conditions, the heart is largely populated by CCR2- (C-C chemokine receptor type 2) macrophages of embryonic descent. After tissue injury, a dramatic shift in macrophage composition occurs whereby CCR2+ monocytes are recruited to the heart and differentiate into inflammatory CCR2+ macrophages that contribute to heart failure progression. Currently, there are no techniques to noninvasively detect CCR2+ monocyte recruitment into the heart and thus identify patients who may be candidates for immunomodulatory therapy. OBJECTIVE: To develop a noninvasive molecular imaging strategy with high sensitivity and specificity to visualize inflammatory monocyte and macrophage accumulation in the heart. METHODS AND RESULTS: We synthesized and tested the performance of a positron emission tomography radiotracer (68Ga-DOTA [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid]-ECL1i [extracellular loop 1 inverso]) that allosterically binds to CCR2. In naive mice, the radiotracer was quickly cleared from the blood and displayed minimal retention in major organs. In contrast, biodistribution and positron emission tomography demonstrated strong myocardial tracer uptake in 2 models of cardiac injury (diphtheria toxin induced cardiomyocyte ablation and reperfused myocardial infarction). 68Ga-DOTA-ECL1i signal localized to sites of tissue injury and was independent of blood pool activity as assessed by quantitative positron emission tomography and ex vivo autoradiography. 68Ga-DOTA-ECL1i uptake was associated with CCR2+ monocyte and CCR2+ macrophage infiltration into the heart and was abrogated in CCR2-/- mice, demonstrating target specificity. Autoradiography demonstrated that 68Ga-DOTA-ECL1i specifically binds human heart failure specimens and with signal intensity associated with CCR2+ macrophage abundance. CONCLUSIONS: These findings demonstrate the sensitivity and specificity of 68Ga-DOTA-ECL1i in the mouse heart and highlight the translational potential of this agent to noninvasively visualize CCR2+ monocyte recruitment and inflammatory macrophage accumulation in patients.
Assuntos
Coração/diagnóstico por imagem , Macrófagos/fisiologia , Monócitos/fisiologia , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/patologia , Animais , Movimento Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Imagem Molecular , Tomografia por Emissão de Pósitrons , Receptores CCR2/análiseRESUMO
Macrophages are integral components of cardiac tissue and exert profound effects on the healthy and diseased heart. Paradigm shifting studies using advanced molecular techniques have revealed significant complexity within these macrophage populations that reside in the heart. In this final of a 4-part review series covering the macrophage in cardiovascular disease, the authors review the origins, dynamics, cell surface markers, and respective functions of each cardiac macrophage subset identified to date, including in the specific scenarios of myocarditis and after myocardial infarction. Looking ahead, a deeper understanding of the diverse and often dichotomous functions of cardiac macrophages will be essential for the development of targeted therapies to mitigate injury and orchestrate recovery of the diseased heart. Moreover, as macrophages are critical for cardiac healing, they are an emerging focus for therapeutic strategies aimed at minimizing cardiomyocyte death, ameliorating pathological cardiac remodeling, and for treating heart failure and after myocardial infarction.
Assuntos
Doenças Cardiovasculares/fisiopatologia , Homeostase/fisiologia , Macrófagos/fisiologia , Neovascularização Fisiológica/fisiologia , Animais , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/patologia , Coração/embriologia , Coração/fisiologia , HumanosRESUMO
Neuregulin-1 (NRG1) ligand and its epidermal growth factor receptor (EGFR)/ERBB family regulate normal cellular proliferation and differentiation in many tissues including the cochlea. Aberrant NRG1 and ERBB signaling cause significant hearing impairment in mice. Dysregulation of the same signaling pathway in humans is involved in certain types of cancers such as breast cancer or non-small cell lung cancer (NSCLC). A new irreversible pan-ERBB inhibitor, canertinib, has been tested in clinical trials for the treatment of refractory NSCLC. Its possible ototoxicity was unknown. In this study, a significant dose-dependent canertinib ototoxicity was observed in a zebrafish model. Canertinib ototoxicity was further confirmed in two mouse models with different genetic backgrounds. The data strongly suggested an evolutionally preserved ERBB molecular mechanism underlying canertinib ototoxicity. Thus, these results imply that clinical monitoring of hearing loss should be considered for clinical testing of canertinib or other pan-ERBB inhibitors.
Assuntos
Antineoplásicos/efeitos adversos , Receptores ErbB/antagonistas & inibidores , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Perda Auditiva/induzido quimicamente , Audição/efeitos dos fármacos , Morfolinas/efeitos adversos , Animais , Antineoplásicos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Orelha , Eletrofisiologia , Feminino , Neoplasias Pulmonares/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Morfolinas/farmacologia , Neuregulina-1/metabolismo , Transdução de Sinais/efeitos dos fármacos , Peixe-ZebraRESUMO
BACKGROUND: Hearing restoration through hair cell regeneration will require revealing the dynamic interactions between proliferation and differentiation during development to avoid the limited viability of regenerated hair cells. Pax2-Cre N-Myc conditional knockout (CKO) mice highlighted the need of N-Myc for proper neurosensory development and possible redundancy with L-Myc. The late-onset hair cell death in the absence of early N-Myc expression could be due to mis-regulation of genes necessary for neurosensory formation and maintenance, such as Neurod1, Atoh1, Pou4f3, and Barhl1. RESULTS: Pax2-Cre N-Myc L-Myc double CKO mice show that proliferation and differentiation are linked together through Myc and in the absence of both Mycs, altered proliferation and differentiation result in morphologically abnormal ears. In particular, the organ of Corti apex is re-patterned into a vestibular-like organization and the base is truncated and fused with the saccule. CONCLUSIONS: These data indicate that therapeutic approaches to restore hair cells must take into account a dynamic interaction of proliferation and differentiation regulation of basic Helix-Loop-Helix transcription factors in attempts to stably replace lost cochlear hair cells. In addition, our data indicate that Myc is an integral component of the evolutionary transformation process that resulted in the organ of Corti development.
Assuntos
Diferenciação Celular/fisiologia , Proliferação de Células , Orelha Interna/embriologia , Células Ciliadas Auditivas/fisiologia , Proteínas Proto-Oncogênicas c-myc/genética , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Estimulação Acústica , Animais , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Marcha/fisiologia , Genótipo , Imuno-Histoquímica , Hibridização In Situ , Integrases , Camundongos , Camundongos Knockout , Microscopia Confocal/métodos , Fator de Transcrição PAX2/metabolismo , Compostos de Fenilureia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de TempoRESUMO
Sensorineural hearing loss results from damage to the hair cells of the organ of Corti and is irreversible in mammals. While hair cell regeneration may prove to be the ideal therapy after hearing loss, prevention of initial hair cell loss could provide even more benefit at a lower cost. Previous studies have shown that the deletion of Atoh1 results in embryonic loss of hair cells while the absence of Barhl1, Gfi1, and Pou4f3 leads to the progressive loss of hair cells in newborn mice. We recently reported that in the early embryonic absence of N-Myc (using Pax2-Cre), hair cells in the organ of Corti develop and remain until at least seven days after birth, with subsequent progressive loss. Thus, N-Myc plays a role in hair cell viability; however, it is unclear if this is due to its early expression in hair cell precursors and throughout the growing otocyst as it functions through proliferation or its late expression exclusively in differentiated hair cells. Furthermore, the related family member L-Myc is mostly co-expressed in the ear, including in differentiated hair cells, but its function has not been studied and could be partially redundant to N-Myc. To test for a long-term function of the Mycs in differentiated hair cells, we generated nine unique genotypes knocking out N-Myc and/or L-Myc after initial formation of hair cells using the well-characterized Atoh1-Cre. We tested functionality of the auditory and vestibular systems at both P21 and four months of age and under the administration of the ototoxic drug cisplatin. We conclude that neither N-Myc nor L-Myc is likely to play important roles in long-term hair cell maintenance. Therefore, it is likely that the late-onset loss of hair cells resulting from early deletion of the Mycs leads to an unsustainable developmental defect.
Assuntos
Células Ciliadas Auditivas Internas/metabolismo , Órgão Espiral/embriologia , Órgão Espiral/crescimento & desenvolvimento , Proteínas Proto-Oncogênicas c-myc/metabolismo , Animais , Embrião de Mamíferos , Células Ciliadas Auditivas Internas/citologia , Imuno-Histoquímica , Hibridização In Situ , Camundongos , Camundongos Knockout , Microscopia Confocal , Órgão Espiral/metabolismoRESUMO
Reconstructing a functional organ of Corti is the ultimate target towards curing hearing loss. Despite the impressive technical gains made over the last few years, many complications remain ahead for the two main restoration avenues: in vitro transformation of pluripotent cells into hair cell-like cells and adenovirus-mediated gene therapy. Most notably, both approaches require a more complete understanding of the molecular networks that ensure specific cell types form in the correct places to allow proper function of the restored organ of Corti. Important to this understanding are the basic helix-loop-helix (bHLH) transcription factors (TFs) that are highly diverse and serve to increase functional complexity but their evolutionary implementation in the inner ear neurosensory development is less conspicuous. To this end, we review the evolutionary and developmentally dynamic interactions of the three bHLH TFs that have been identified as the main players in neurosensory evolution and development, Neurog1, Neurod1 and Atoh1. These three TFs belong to the neurogenin/atonal family and evolved from a molecular precursor that likely regulated single sensory cell development in the ectoderm of metazoan ancestors but are now also expressed in other parts of the body, including the brain. They interact extensively via intracellular and intercellular cross-regulation to establish the two main neurosensory cell types of the ear, the hair cells and sensory neurons. Furthermore, the level and duration of their expression affect the specification of hair cell subtypes (inner hair cells vs. outer hair cells). We propose that appropriate manipulation of these TFs through their characterized binding sites may offer a solution by itself, or in conjunction with the two other approaches currently pursued by others, to restore the organ of Corti.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Orelha Interna/crescimento & desenvolvimento , Animais , Orelha Interna/citologia , Orelha Interna/metabolismo , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento , Perda Auditiva/genética , Perda Auditiva/metabolismo , Humanos , Órgão Espiral/citologia , Órgão Espiral/crescimento & desenvolvimento , Órgão Espiral/metabolismoRESUMO
Due to the multisensory input into the balance system, the loss of one input, such as an ear, can generally be compensated for. However, when a mismatch or incomplete loss of inputs occurs, the ability to compensate for the stimulus misrepresentation may be compromised. The inner ear and cerebellum are important input and processing centers for balance but no genetic models have been generated to assess balance or compensation in the abnormal development of both these organs/brain areas. Important to their formation is regulation of proliferation mediated by the proto-oncogene N-Myc. Conditional knockouts (CKOs) of N-Myc using Tg(Pax2-Cre) have a misshapen and smaller ear with a fused utricle, saccule, and cochlea and absent horizontal canal, aberrant cochlear and vestibular innervations, and a size reduction in the cerebellum. CKOs are viable with obvious behavioral deficits, including circling behavior and unstable gait. To test the degree of ataxia and possible compensation of vestibular defects in these mutant mice, we use the Noldus Catwalk System to assess the gait of Tg(Pax2-Cre) N-Myc CKOs over five months. N-Myc CKOs perform worse than control littermates, in particular, in step regularity. We show that disrupting one member of the Myc family during embryonic development coincides with a differential loss of function in the cochlea compared to the vestibular apparatus. In addition, we show that the distortion in the ear morphology combined with a reduction of the cerebellum, rather than a complete loss of the vestibular-cerebellar pathway, leads to partial behavioral compensation that remains unchanged over time.
Assuntos
Cerebelo/fisiopatologia , Cóclea/fisiopatologia , Marcha Atáxica/fisiopatologia , Doenças Vestibulares/fisiopatologia , Vestíbulo do Labirinto/fisiopatologia , Animais , Cerebelo/anormalidades , Cerebelo/embriologia , Cóclea/anormalidades , Cóclea/embriologia , Marcha , Marcha Atáxica/embriologia , Genes myc , Camundongos , Camundongos Knockout , Doenças Vestibulares/embriologia , Doenças Vestibulares/genética , Vestíbulo do Labirinto/embriologiaRESUMO
Atonal homolog1 (Atoh1) is a bHLH transcription factor essential for inner ear hair cell differentiation. Targeted expression of Atoh1 at various stages in development can result in hair cell differentiation in the ear. However, the level and duration of Atoh1 expression required for proper hair cell differentiation and maintenance remain unknown. We generated an Atoh1 conditional knockout (CKO) mouse line using Tg(Atoh1-cre), in which the cre expression is driven by an Atoh1 enhancer element that is regulated by Atoh1 protein to "self-terminate" its expression. The mutant mice show transient, limited expression of Atoh1 in all hair cells in the ear. In the organ of Corti, reduction and delayed deletion of Atoh1 result in progressive loss of almost all the inner hair cells and the majority of the outer hair cells within three weeks after birth. The remaining cells express hair cell marker Myo7a and attract nerve fibers, but do not differentiate normal stereocilia bundles. Some Myo7a-positive cells persist in the cochlea into adult stages in the position of outer hair cells, flanked by a single row of pillar cells and two to three rows of disorganized Deiters cells. Gene expression analyses of Atoh1, Barhl1 and Pou4f3, genes required for survival and maturation of hair cells, reveal earlier and higher expression levels in the inner compared to the outer hair cells. Our data show that Atoh1 is crucial for hair cell mechanotransduction development, viability, and maintenance and also suggest that Atoh1 expression level and duration may play a role in inner vs. outer hair cell development. These genetically engineered Atoh1 CKO mice provide a novel model for establishing critical conditions needed to regenerate viable and functional hair cells with Atoh1 therapy.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Células Ciliadas Auditivas/metabolismo , Órgão Espiral/metabolismo , Animais , Animais Recém-Nascidos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Sobrevivência Celular/genética , Cóclea/embriologia , Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/ultraestrutura , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Imuno-Histoquímica , Hibridização In Situ , Masculino , Mecanotransdução Celular/genética , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Varredura , Miosina VIIa , Miosinas/genética , Miosinas/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Órgão Espiral/embriologia , Órgão Espiral/crescimento & desenvolvimento , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Estereocílios/genética , Estereocílios/metabolismo , Fator de Transcrição Brn-3C/genética , Fator de Transcrição Brn-3C/metabolismoRESUMO
Ear development requires interactions of transcription factors for proliferation and differentiation. The proto-oncogene N-Myc is a member of the Myc family that regulates proliferation. To investigate the function of N-Myc, we conditionally knocked out N-Myc in the ear using Tg(Pax2-Cre) and Foxg1(KiCre). N-Myc CKOs had reduced growth of the ear, abnormal morphology including fused sensory epithelia, disrupted histology, and disorganized neuronal innervation. Using Thin-Sheet Laser Imaging Microscopy (TSLIM), 3D reconstruction and quantification of the cochlea revealed a greater than 50% size reduction. Immunochemistry and in situ hybridization showed a gravistatic organ-cochlear fusion and a "circularized" apex with no clear inner and outer hair cells. Furthermore, the abnormally developed cochlea had cross innervation from the vestibular ganglion near the basal tip. These findings are put in the context of the possible functional relationship of N-Myc with a number of other cell proliferative and fate determining genes during ear development.