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1.
Nat Biomed Eng ; 5(8): 880-896, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34426676

RESUMO

Fibroblasts can be directly reprogrammed into cardiomyocytes, endothelial cells or smooth muscle cells. Here we report the reprogramming of mouse tail-tip fibroblasts simultaneously into cells resembling these three cell types using the microRNA mimic miR-208b-3p, ascorbic acid and bone morphogenetic protein 4, as well as the formation of tissue-like structures formed by the directly reprogrammed cells. Implantation of the formed cardiovascular tissue into the infarcted hearts of mice led to the migration of reprogrammed cells to the injured tissue, reducing regional cardiac strain and improving cardiac function. The migrated endothelial cells and smooth muscle cells contributed to vessel formation, and the migrated cardiomyocytes, which initially displayed immature characteristics, became mature over time and formed gap junctions with host cardiomyocytes. Direct reprogramming of somatic cells to make cardiac tissue may aid the development of applications in cell therapy, disease modelling and drug discovery for cardiovascular diseases.


Assuntos
Células Endoteliais/transplante , Coração/fisiologia , Infarto do Miocárdio/terapia , Miócitos de Músculo Liso/transplante , Regeneração , Animais , Ácido Ascórbico/farmacologia , Proteína Morfogenética Óssea 4/farmacologia , Reprogramação Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Junções Comunicantes/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Neovascularização Fisiológica , Transcriptoma
2.
Sci Rep ; 11(1): 3630, 2021 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-33574435

RESUMO

Preeclampsia (PE) is a prevalent pregnancy disorder that leads to high maternal and fetal morbidity and mortality. While defective vascular development and angiogenesis in placenta are known as crucial pathological findings, its pathophysiological mechanism remains elusive. To better understand the effects of PE on angio-vasculogenesis and inflammatory networks in the fetus and to identify their biological signatures, we investigated the quantitative and functional characteristics of cord blood-derived mononuclear cells (CB-MNCs) and CD31-positive MNCs. Flow cytometry analysis demonstrated that the CB-MNCs from the severe PE group had significantly decreased number of cells expressing CD3, CD11b, CD14, CD19, KDR, and CD31 compared with the normal group. Quantitative real time PCR (qRT-PCR) shows down-regulation of the major angiogenic factor VEGFA in MNCs and CD31+ MNCs in severe PE. The major inflammatory cytokines IL1 was highly upregulated in CD31+ CB-MNCs in the severe PE patients. Mild PE patients, however, did not display any significant difference in expression of all measured angiogenic genes and most inflammatory genes. These findings show distinct angiogenic and inflammatory signatures from severe PE, and they may play a significant role in the pathogenesis of vascular defects in placenta of severe PE.

3.
Circulation ; 136(20): 1939-1954, 2017 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-28972000

RESUMO

BACKGROUND: Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel. METHODS: We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3ß inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix-mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2-degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5+ cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription-polymerase chain reaction, and histological analysis. RESULTS: The resultant hPSC-derived CDH5+ cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS-, or human umbilical vein EC-injected groups. However, the group receiving the PA-RGDS-encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC-injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months. CONCLUSIONS: We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS-mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs.


Assuntos
Células Endoteliais da Veia Umbilical Humana/transplante , Isquemia/terapia , Metaloproteinase 2 da Matriz/administração & dosagem , Nanoestruturas/administração & dosagem , Oligopeptídeos/administração & dosagem , Células-Tronco Pluripotentes/transplante , Animais , Diferenciação Celular/fisiologia , Terapia Baseada em Transplante de Células e Tecidos/métodos , Células Cultivadas , Células Endoteliais/fisiologia , Células Endoteliais/transplante , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Isquemia/fisiopatologia , Masculino , Camundongos , Camundongos Nus , Células-Tronco Pluripotentes/fisiologia , Distribuição Aleatória , Resultado do Tratamento
4.
Circ Res ; 120(5): 848-861, 2017 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-28003219

RESUMO

RATIONALE: Direct conversion or reprogramming of human postnatal cells into endothelial cells (ECs), bypassing stem or progenitor cell status, is crucial for regenerative medicine, cell therapy, and pathophysiological investigation but has remained largely unexplored. OBJECTIVE: We sought to directly reprogram human postnatal dermal fibroblasts to ECs with vasculogenic and endothelial transcription factors and determine their vascularizing and therapeutic potential. METHODS AND RESULTS: We utilized various combinations of 7 EC transcription factors to transduce human postnatal dermal fibroblasts and found that ER71/ETV2 (ETS variant 2) alone best induced endothelial features. KDR+ (kinase insert domain receptor) cells sorted at day 7 from ER71/ETV2-transduced human postnatal dermal fibroblasts showed less mature but enriched endothelial characteristics and thus were referred to as early reprogrammed ECs (rECs), and did not undergo maturation by further culture. After a period of several weeks' transgene-free culture followed by transient reinduction of ER71/ETV2, early rECs matured during 3 months of culture and showed reduced ETV2 expression, reaching a mature phenotype similar to postnatal human ECs. These were termed late rECs. While early rECs exhibited an immature phenotype, their implantation into ischemic hindlimbs induced enhanced recovery from ischemia. These 2 rECs showed clear capacity for contributing to new vessel formation through direct vascular incorporation in vivo. Paracrine or proangiogenic effects of implanted early rECs played a significant role in repairing hindlimb ischemia. CONCLUSIONS: This study for the first time demonstrates that ER71/ETV2 alone can directly reprogram human postnatal cells to functional, mature ECs after an intervening transgene-free period. These rECs could be valuable for cell therapy, personalized disease investigation, and exploration of the reprogramming process.


Assuntos
Técnicas de Reprogramação Celular/métodos , Células Endoteliais/fisiologia , Fibroblastos/fisiologia , Fatores de Transcrição/biossíntese , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Membro Posterior/irrigação sanguínea , Membro Posterior/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Isquemia/metabolismo , Masculino , Camundongos , Camundongos Nus , Neovascularização Fisiológica/fisiologia , Fatores de Transcrição/genética
5.
Stem Cell Reports ; 5(6): 1239-1249, 2015 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-26651608

RESUMO

Isolation of ventricular cardiomyocytes (vCMs) has been challenging due to the lack of specific surface markers. Here we show that vCMs can be purified from differentiating mouse embryonic stem cells (mESCs) using molecular beacons (MBs) targeting specific intracellular mRNAs. We designed MBs (IRX4 MBs) to target mRNA encoding Iroquois homeobox protein 4 (Irx4), a transcription factor specific for vCMs. To purify mESC vCMs, IRX4 MBs were delivered into cardiomyogenically differentiating mESCs, and IRX4 MBs-positive cells were FACS-sorted. We found that, of the cells isolated, ~98% displayed vCM-like action potentials by electrophysiological analyses. These MB-purified vCMs continuously maintained their CM characteristics as verified by spontaneous beating, Ca(2+) transient, and expression of vCM-specific proteins. Our study shows the feasibility of isolating pure vCMs via cell sorting without modifying host genes. The homogeneous and functional ventricular CMs generated via the MB-based method can be useful for disease investigation, drug discovery, and cell-based therapies.


Assuntos
Separação Celular/métodos , Células-Tronco Embrionárias/citologia , Ventrículos do Coração/citologia , Proteínas de Homeodomínio/genética , Miócitos Cardíacos/citologia , Potenciais de Ação , Animais , Sequência de Bases , Diferenciação Celular , Células Cultivadas , Citometria de Fluxo , Camundongos , Sondas de Oligonucleotídeos/genética , RNA Mensageiro/genética
6.
Sci Rep ; 5: 11019, 2015 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-26066093

RESUMO

Human pluripotent stem cells (hPSCs) have emerged as an important source for cell therapy. However, to date, no studies demonstrated generation of purified hPSC-derived lymphatic endothelial cells (LECs) and tested their therapeutic potential in disease models. Here we sought to differentiate hPSCs into the LEC lineage, purify them with LEC markers, and evaluate their therapeutic effects. We found that an OP9-assisted culture system reinforced by addition of VEGF-A, VEGF-C, and EGF most efficiently generated LECs, which were then isolated via FACS-sorting with LYVE-1 and PODOPLANIN. These hPSC-derived LYVE-1(+)PODOPLANIN(+)cells showed a pure committed LEC phenotype, formed new lymphatic vessels, and expressed lymphangiogenic factors at high levels. These hPSC-derived LECs enhanced wound healing through lymphangiogenesis and lymphvasculogenesis. Here we report, for the first time, that LECs can be selectively isolated from differentiating hPSCs, and that these cells are potent for lymphatic vessel formation in vivo and wound healing. This system and the purified hPSC-derived LECs can serve as a new platform for studying LEC development as well as for cell therapy.


Assuntos
Diferenciação Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Endoteliais/metabolismo , Linfangiogênese , Cicatrização , Animais , Células Endoteliais/citologia , Células Endoteliais/transplante , Fator de Crescimento Epidérmico/farmacologia , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Células-Tronco Pluripotentes , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator C de Crescimento do Endotélio Vascular/farmacologia
7.
Biomaterials ; 63: 158-67, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26102992

RESUMO

Various stem cells and their progeny have been used therapeutically for vascular regeneration. One of the major hurdles for cell-based therapy is low cell retention in vivo, and to improve cell survival several biomaterials have been used to encapsulate cells before transplantation. Vascular regeneration involves new blood vessel formation which consists of two processes, vasculogenesis and angiogenesis. While embryonic stem cell (ESC)-derived endothelial cells (ESC-ECs) have clearer vasculogenic potency, adult cells exert their effects mainly through paracrine angiogenic activities. While these two cells have seemingly complementary advantages, there have not been any studies to date combining these two cell types for vascular regeneration. We have developed a novel chitosan-based hydrogel construct that encapsulates both CD31-expressing BM-mononuclear cells (BM-CD31(+) cells) and ESC-ECs, and is loaded with VEGF-releasing microtubes. This cell construct showed high cell survival and minimal cytotoxicity in vitro. When implanted into a mouse model of hindlimb ischemia, it induced robust cell retention, neovascularization through vasculogenesis and angiogenesis, and efficiently induced recovery of blood flow in ischemic hindlimbs. This chitosan-based hydrogel encapsulating mixed adult and embryonic cell derivatives and containing VEGF can serve as a novel platform for treating various cardiovascular diseases.


Assuntos
Quitosana/química , Células-Tronco Embrionárias/transplante , Células Endoteliais/transplante , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Tecidos Suporte/química , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Células Cultivadas , Células-Tronco Embrionárias/citologia , Células Endoteliais/citologia , Membro Posterior/patologia , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Isquemia/patologia , Masculino , Camundongos , Neovascularização Fisiológica , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Fator A de Crescimento do Endotélio Vascular/farmacologia
8.
ACS Nano ; 8(10): 10815-25, 2014 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-25210842

RESUMO

A significant barrier to the therapeutic use of stem cells is poor cell retention in vivo. Here, we evaluate the therapeutic potential and long-term engraftment of cardiomyocytes (CMs) derived from mouse embryonic stem cells (mESCs) encapsulated in an injectable nanomatrix gel consisting of peptide amphiphiles incorporating cell adhesive ligand Arg-Gly-Asp-Ser (PA-RGDS) in experimental myocardial infarction (MI). We cultured rat neonatal CMs in PA-RGDS for 7 days and found that more than 90% of the CMs survived. Next, we intramyocardially injected mouse CM cell line HL-1 CMs with or without PA-RGDS into uninjured hearts. Histologic examination and flow cytometry analysis of digested heart tissues showed approximately 3-fold higher engraftment in the mice that received CMs with PA-RGDS compared to those without PA-RGDS. We further investigated the therapeutic effects and long-term engraftment of mESC-CMs with PA-RGDS on MI in comparison with PBS control, CM-only, and PA-RGDS only. Echocardiography demonstrated that the CM-only and CM+PA-RGDS groups showed higher cardiac function at week 2 compared to other groups. However, from 3 weeks, higher cardiac function was maintained only in the CM+PA-RGDS group; this was sustained for 12 weeks. Confocal microscopic examination of the cardiac tissues harvested at 14 weeks demonstrated sustained engraftment and integration of mESC-CMs into host myocardium in the CM+PA-RGDS group only. This study for the first time demonstrated that PA-RGDS encapsulation can enhance survival of mESC-derived CMs and improve cardiac function post-MI. This nanomatrix gel-mediated stem cell therapy can be a promising option for treating MI.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Embrionárias/citologia , Coração/fisiopatologia , Miócitos Cardíacos/citologia , Nanoestruturas , Animais , Ratos
9.
Circulation ; 128(17): 1897-909, 2013 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-23995537

RESUMO

BACKGROUND: Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell-derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. METHOD AND RESULTS: Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but <3% of 4 noncardiomyocyte cell types in flow cytometry analysis, which indicates that MHC1-MB is specific for identifying cardiomyocytes. We delivered MHC1-MB into cardiomyogenically differentiated pluripotent stem cells through nucleofection. The detection rate of cardiomyocytes was similar to the percentages of cardiac troponin T- or cardiac troponin I-positive cardiomyocytes, which supports the specificity of MBs. Finally, MHC1-MB-positive cells were sorted by fluorescence-activated cell sorter from mouse and human pluripotent stem cell differentiating cultures, and ≈97% cells expressed cardiac troponin T or cardiac troponin I as determined by flow cytometry. These MB-based sorted cells maintained their cardiomyocyte characteristics, which was verified by spontaneous beating, electrophysiological studies, and expression of cardiac proteins. When transplanted in a myocardial infarction model, MB-based purified cardiomyocytes improved cardiac function and demonstrated significant engraftment for 4 weeks without forming tumors. CONCLUSIONS: We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.


Assuntos
Transplante de Células/métodos , Infarto do Miocárdio/terapia , Miócitos Cardíacos/citologia , Células-Tronco Pluripotentes/citologia , RNA Mensageiro/isolamento & purificação , Potenciais de Ação/fisiologia , Animais , Biomarcadores , Diferenciação Celular/fisiologia , Linhagem Celular , Células Cultivadas , Citometria de Fluxo/métodos , Humanos , Camundongos , Miócitos Cardíacos/fisiologia , Cadeias Pesadas de Miosina/genética , Nanotecnologia , Conformação de Ácido Nucleico , Células-Tronco Pluripotentes/fisiologia , Sondas RNA/química , Sondas RNA/isolamento & purificação , RNA Mensageiro/química , Troponina I/genética , Troponina T/genética
10.
Int J Cardiol ; 168(1): 41-52, 2013 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-23044428

RESUMO

BACKGROUND: Human pluripotent stem cells (hPSCs) hold great promise for treating ischemic heart disease. However, current protocols for differentiating hPSCs either result in low yields or require expensive cytokines. METHODS: Here we developed a novel two dimensional (2D) stepwise differentiation system that generates a high yield of cardiomyocytes (CMs) from hPSCs without using special cytokines. Initially, undifferentiated hPSCs were transferred onto Matrigel-coated plates without forming embryoid bodies (EBs) for a few days and were cultured in bFGF-depleted human embryonic stem cells (hESCs) medium. When linear cell aggregation appeared in the margins of the hPSC colonies, the medium was changed to DMEM supplemented with 10% fetal bovine serum (FBS). Thereafter when cell clusters became visible, the medium was changed to DMEM with 20% FBS. RESULTS AND CONCLUSIONS: At about two weeks of culture, contracting clusters began to appear and the number of contracting clusters continuously increased, reaching approximately 70% of all clusters. These clusters were dissociated by two-step enzyme treatment to monolayered CMs, of which ~90% showed CM phenotypes confirmed by an α-myosin heavy chain reporter system. Electrophysiologic studies demonstrated that the hPSC-derived CMs showed three major CM action potential types with 61 to 78% having a ventricular-CM phenotype. This differentiation system showed a clear spatiotemporal role of the surrounding endodermal cells for differentiation of mesodermal cell clusters into CMs. In conclusion, this system provides a novel platform to generate CMs from hPSCs at high yield without using cytokines and to study the development of hPSCs into CMs.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Pluripotentes Induzidas/transplante , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/transplante , Cultura Primária de Células/métodos , Animais , Humanos , Masculino , Infarto do Miocárdio/patologia , Infarto do Miocárdio/cirurgia , Células-Tronco Pluripotentes/fisiologia , Células-Tronco Pluripotentes/transplante , Cultura Primária de Células/tendências , Ratos Nus
11.
PLoS Genet ; 7(6): e1002154, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21731508

RESUMO

Covalent modification of DNA distinguishes cellular identities and is crucial for regulating the pluripotency and differentiation of embryonic stem (ES) cells. The recent demonstration that 5-methylcytosine (5-mC) may be further modified to 5-hydroxymethylcytosine (5-hmC) in ES cells has revealed a novel regulatory paradigm to modulate the epigenetic landscape of pluripotency. To understand the role of 5-hmC in the epigenomic landscape of pluripotent cells, here we profile the genome-wide 5-hmC distribution and correlate it with the genomic profiles of 11 diverse histone modifications and six transcription factors in human ES cells. By integrating genomic 5-hmC signals with maps of histone enrichment, we link particular pluripotency-associated chromatin contexts with 5-hmC. Intriguingly, through additional correlations with defined chromatin signatures at promoter and enhancer subtypes, we show distinct enrichment of 5-hmC at enhancers marked with H3K4me1 and H3K27ac. These results suggest potential role(s) for 5-hmC in the regulation of specific promoters and enhancers. In addition, our results provide a detailed epigenomic map of 5-hmC from which to pursue future functional studies on the diverse regulatory roles associated with 5-hmC.


Assuntos
Citosina/análogos & derivados , Células-Tronco Embrionárias/citologia , Epigenômica , Genoma Humano , 5-Metilcitosina/metabolismo , Sítios de Ligação , Linhagem Celular , Mapeamento Cromossômico , Citosina/metabolismo , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Biblioteca Gênica , Heterocromatina/química , Histonas/metabolismo , Humanos , Immunoblotting , Metáfase , Regiões Promotoras Genéticas , Alinhamento de Sequência , Fatores de Transcrição/metabolismo
12.
Eur J Neurosci ; 23(10): 2543-50, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16817857

RESUMO

Mechanosensitive (MS) channels are ion channels gated by different types of mechanical stimuli. MS channels in sensory neurons are thought to be molecular transducers for somatic sensations such as touch, pressure, proprioception and pain. Previously, we reported that two types of MS channels are present in sensory neurons. These channels are termed low threshold (LT) and high threshold (HT) MS channels based on their pressure threshold for activation. Here, we report another type of MS channel present in sensory neurons. The channel is activated by low pressure applied to a patch (threshold approximately 20 mmHg, similar to that in the LT channel). However, because this channel has a smaller single-channel conductance than that of the LT channel, the newly classified MS channel is now called a low threshold small conductance (LTSC) channel. Unlike the LT channel, which has outwardly rectifying currents, the current-voltage relationship of the LTSC is linear. The LTSC was permeable to monovalent cations and Ca2+, and reversibly blocked by gadolinium, a blocker of MS channels. Unlike the LT channel, the LTSC was sensitized by prostaglandin E2, an inflammatory mediator that is known to sensitize nociceptors to mechanical stimuli. LTSC channels were found mostly in small cultured sensory neurons. Thus, these results suggest that the LTSC is a distinct type of MS channel that is different from the LT and HT channels in sensory neurons, and that LTSCs might play a role in mediating somatosensations, including pain.


Assuntos
Canais Iônicos/metabolismo , Mecanorreceptores/metabolismo , Mecanotransdução Celular/fisiologia , Neurônios Aferentes/metabolismo , Amilorida/farmacologia , Animais , Tamanho Celular , Dinoprostona/farmacologia , Gadolínio/farmacologia , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Canais Iônicos/efeitos dos fármacos , Mecanorreceptores/efeitos dos fármacos , Mecanotransdução Celular/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Bloqueadores dos Canais de Sódio/farmacologia
13.
J Neurosci ; 26(9): 2403-12, 2006 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-16510717

RESUMO

TRPV1, a cloned capsaicin receptor, is a molecular sensor for detecting adverse stimuli and a key element for inflammatory nociception and represents biophysical properties of native channel. However, there seems to be a marked difference between TRPV1 and native capsaicin receptors in the pharmacological response profiles to vanilloids or acid. One plausible explanation for this overt discrepancy is the presence of regulatory proteins associated with TRPV1. Here, we identify Fas-associated factor 1 (FAF1) as a regulatory factor, which is coexpressed with and binds to TRPV1 in sensory neurons. When expressed heterologously, FAF1 reduces the responses of TRPV1 to capsaicin, acid, and heat, to the pharmacological level of native capsaicin receptor in sensory neurons. Furthermore, silencing FAF1 by RNA interference augments capsaicin-sensitive current in native sensory neurons. We therefore conclude that FAF1 forms an integral component of the vanilloid receptor complex and that it constitutively modulates the sensitivity of TRPV1 to various noxious stimuli in sensory neurons.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neurônios Aferentes/fisiologia , Canais de Cátion TRPV/fisiologia , Ácidos/farmacologia , Análise de Variância , Animais , Animais Recém-Nascidos , Proteínas Reguladoras de Apoptose , Biotinilação/métodos , Western Blotting/métodos , Western Blotting/estatística & dados numéricos , Capsaicina/farmacologia , Células Cultivadas , Clonagem Molecular/métodos , Relação Dose-Resposta a Droga , Estimulação Elétrica/métodos , Gânglios Espinais/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica/métodos , Imunoprecipitação/métodos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos da radiação , Mutação , Neurônios Aferentes/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Estrutura Terciária de Proteína/fisiologia , RNA Interferente Pequeno/farmacologia , Ensaio Radioligante/métodos , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Temperatura , Transfecção/métodos , Ubiquitina/metabolismo
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