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1.
Genes Dev ; 33(9-10): 524-535, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30862660

RESUMO

The balance between proliferation and differentiation of muscle stem cells is tightly controlled, ensuring the maintenance of a cellular pool needed for muscle growth and repair. We demonstrate here that the transcriptional regulator Hes1 controls the balance between proliferation and differentiation of activated muscle stem cells in both developing and regenerating muscle. We observed that Hes1 is expressed in an oscillatory manner in activated stem cells where it drives the oscillatory expression of MyoD. MyoD expression oscillates in activated muscle stem cells from postnatal and adult muscle under various conditions: when the stem cells are dispersed in culture, when they remain associated with single muscle fibers, or when they reside in muscle biopsies. Unstable MyoD oscillations and long periods of sustained MyoD expression are observed in differentiating cells. Ablation of the Hes1 oscillator in stem cells interfered with stable MyoD oscillations and led to prolonged periods of sustained MyoD expression, resulting in increased differentiation propensity. This interfered with the maintenance of activated muscle stem cells, and impaired muscle growth and repair. We conclude that oscillatory MyoD expression allows the cells to remain in an undifferentiated and proliferative state and is required for amplification of the activated stem cell pool.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Proteína MyoD/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição HES-1/metabolismo , Animais , Células Cultivadas , Camundongos , Proteína MyoD/genética , Receptores Notch/metabolismo , Transdução de Sinais , Fatores de Transcrição HES-1/genética
2.
Am J Physiol Lung Cell Mol Physiol ; 324(1): L64-L75, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36410022

RESUMO

Influenza-A virus (IAV) infects yearly an estimated one billion people worldwide, resulting in 300,000-650,000 deaths. Preventive vaccination programs and antiviral medications represent the mainstay of therapy, but with unacceptably high morbidity and mortality rates, new targeted therapeutic approaches are urgently needed. Since inflammatory processes are commonly associated with measurable changes in the cell membrane potential (Em), we investigated whether Em hyperpolarization via TREK-1 (K2P2.1) K+ channel activation can protect against influenza-A virus (IAV)-induced pneumonia. We infected mice with IAV, which after 5 days caused 10-15% weight loss and a decrease in spontaneous activity, representing a clinically relevant infection. We then started a 3-day intratracheal treatment course with the novel TREK-1 activating compounds BL1249 or ML335. We confirmed TREK-1 activation with both compounds in untreated and IAV-infected primary human alveolar epithelial cells (HAECs) using high-throughput fluorescent imaging plate reader (FLIPR) assays. In mice, TREK-1 activation with BL1249 and ML335 counteracted IAV-induced histological lung injury and decrease in lung compliance and improved BAL fluid total protein levels, cell counts, and inflammatory IL-6, IP-10/CXCL-10, MIP-1α, and TNF-α levels. To determine whether these anti-inflammatory effects were mediated by activation of alveolar epithelial TREK-1 channels, we studied the effects of BL1249 and ML335 in IAV-infected HAEC, and found that TREK-1 activation decreased IAV-induced inflammatory IL-6, IP-10/CXCL10, and CCL-2 secretion. Dissection of TREK-1 downstream signaling pathways and construction of protein-protein interaction (PPI) networks revealed NF-κB1 and retinoic acid-inducible gene-1 (RIG-1) cascades as the most likely targets for TREK-1 protection. Therefore, TREK-1 activation may represent a novel therapeutic approach against IAV-induced lung injury.


Assuntos
Lesão Pulmonar Aguda , Vírus da Influenza A , Influenza Humana , Infecções por Orthomyxoviridae , Canais de Potássio de Domínios Poros em Tandem , Animais , Humanos , Camundongos , Lesão Pulmonar Aguda/patologia , Quimiocina CXCL10/metabolismo , Influenza Humana/patologia , Interleucina-6/metabolismo , Pulmão/metabolismo , Infecções por Orthomyxoviridae/patologia , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo
3.
Int J Mol Sci ; 24(4)2023 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-36835507

RESUMO

Elevated TNF-α levels in serum and broncho-alveolar lavage fluid of acute lung injury patients correlate with mortality rates. We hypothesized that pharmacological plasma membrane potential (Em) hyperpolarization protects against TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells through inhibition of inflammatory Ca2+-dependent MAPK pathways. Since the role of Ca2+ influx in TNF-α-mediated inflammation remains poorly understood, we explored the role of L-type voltage-gated Ca2+ (CaV) channels in TNF-α-induced CCL-2 and IL-6 secretion from human pulmonary endothelial cells. The CaV channel blocker, Nifedipine, decreased both CCL-2 and IL-6 secretion, suggesting that a fraction of CaV channels is open at the significantly depolarized resting Em of human microvascular pulmonary endothelial cells (-6 ± 1.9 mV), as shown by whole-cell patch-clamp measurements. To further explore the role of CaV channels in cytokine secretion, we demonstrated that the beneficial effects of Nifedipine could also be achieved by Em hyperpolarization via the pharmacological activation of large conductance K+ (BK) channels with NS1619, which elicited a similar decrease in CCL-2 but not IL-6 secretion. Using functional gene enrichment analysis tools, we predicted and validated that known Ca2+-dependent kinases, JNK-1/2 and p38, are the most likely pathways to mediate the decrease in CCL-2 secretion.


Assuntos
Células Epiteliais Alveolares , Quimiocina CCL2 , Canais de Potássio Ativados por Cálcio de Condutância Alta , Pneumonia , Fator de Necrose Tumoral alfa , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Alta/agonistas , Nifedipino/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Pneumonia/metabolismo , Pneumonia/prevenção & controle , Quimiocina CCL2/metabolismo
4.
Am J Respir Cell Mol Biol ; 64(2): 224-234, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33217242

RESUMO

We recently established a role for the stretch-activated two-pore-domain K+ (K2P) channel TREK-1 (K2P2.1) in inflammatory cytokine secretion using models of hyperoxia-, mechanical stretch-, and TNF-α-induced acute lung injury. We have now discovered the expression of large conductance, Ca2+-activated K+ (BK) channels in human pulmonary microvascular endothelial cells and primary human alveolar epithelial cells using semiquantitative real-time PCR, IP and Western blot, and investigated their role in inflammatory cytokine secretion using an LPS-induced acute lung injury model. As expected, LPS induced IL-6 and CCL-2 secretion from pulmonary endothelial and epithelial cells. BK activation with NS1619 decreased LPS-induced CCL-2 but not IL-6 secretion from endothelial cells and had no effect on epithelial cells, although fluorometric assays revealed that BK activation hyperpolarized the plasma membrane potential (Em) of both cell types. Interestingly, BK inhibition (Paxilline) did not alter cytokine secretion or the Em in either cell type. Furthermore, LPS treatment by itself did not affect the Em or intracellular Ca2+ concentrations. Therefore, we propose BK channel activation as a novel targeted approach to counteract LPS-induced CCL-2 secretion from endothelial cells. This protective effect appears to occur via Em hyperpolarization but independent of intracellular Ca2+ concentrations.


Assuntos
Células Epiteliais Alveolares/metabolismo , Quimiocina CCL2/metabolismo , Células Endoteliais/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Pulmão/metabolismo , Células A549 , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Cálcio/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Citocinas/metabolismo , Células Endoteliais/efeitos dos fármacos , Células HEK293 , Humanos , Hiperóxia/induzido quimicamente , Hiperóxia/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Interleucina-6/metabolismo , Lipopolissacarídeos/farmacologia , Pulmão/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo
5.
Sci Rep ; 10(1): 22011, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33319831

RESUMO

No targeted therapies exist to counteract Hyperoxia (HO)-induced Acute Lung Injury (HALI). We previously found that HO downregulates alveolar K2P2.1 (TREK-1) K+ channels, which results in worsening lung injury. This decrease in TREK-1 levels leaves a subset of channels amendable to pharmacological intervention. Therefore, we hypothesized that TREK-1 activation protects against HALI. We treated HO-exposed mice and primary alveolar epithelial cells (AECs) with the novel TREK-1 activators ML335 and BL1249, and quantified physiological, histological, and biochemical lung injury markers. We determined the effects of these drugs on epithelial TREK-1 currents, plasma membrane potential (Em), and intracellular Ca2+ (iCa) concentrations using fluorometric assays, and blocked voltage-gated Ca2+ channels (CaV) as a downstream mechanism of cytokine secretion. Once-daily, intra-tracheal injections of HO-exposed mice with ML335 or BL1249 improved lung compliance, histological lung injury scores, broncho-alveolar lavage protein levels and cell counts, and IL-6 and IP-10 concentrations. TREK-1 activation also decreased IL-6, IP-10, and CCL-2 secretion from primary AECs. Mechanistically, ML335 and BL1249 induced TREK-1 currents in AECs, counteracted HO-induced cell depolarization, and lowered iCa2+ concentrations. In addition, CCL-2 secretion was decreased after L-type CaV inhibition. Therefore, Em stabilization with TREK-1 activators may represent a novel approach to counteract HALI.


Assuntos
Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/metabolismo , Hiperóxia/complicações , Ativação do Canal Iônico , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Substâncias Protetoras/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Líquido da Lavagem Broncoalveolar , Cálcio/metabolismo , Linhagem Celular , Citocinas/metabolismo , Mediadores da Inflamação/metabolismo , Espaço Intracelular/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Tetra-Hidronaftalenos/farmacologia , Tetrazóis/farmacologia
6.
J Vis Exp ; (148)2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-31259892

RESUMO

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (αSMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.


Assuntos
Separação Celular/métodos , Células Epiteliais/citologia , Aparelho Lacrimal/citologia , Glândula Submandibular/citologia , Animais , Molécula de Adesão da Célula Epitelial/metabolismo , Regulação da Expressão Gênica , Camundongos
7.
Sci Rep ; 8(1): 9919, 2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29967327

RESUMO

The purpose of the present studies was to investigate the impact of chronic inflammation of the lacrimal gland, as occurs in Sjögren's syndrome, on the morphology and function of myoepithelial cells (MECs). In spite of the importance of MECs for lacrimal gland function, the effect of inflammation on MECs has not been well defined. We studied changes in MEC structure and function in two animal models of aqueous deficient dry eye, NOD and MRL/lpr mice. We found a statistically significant reduction in the size of MECs in diseased compared to control lacrimal glands. We also found that oxytocin receptor was highly expressed in MECs of mouse and human lacrimal glands and that its expression was strongly reduced in diseased glands. Furthermore, we found a significant decrease in the amount of two MEC contractile proteins, α-smooth muscle actin (SMA) and calponin. Finally, oxytocin-mediated contraction was impaired in lacrimal gland acini from diseased glands. We conclude that chronic inflammation of the lacrimal gland leads to a substantial thinning of MECs, down-regulation of contractile proteins and oxytocin receptor expression, and therefore impaired acini contraction. This is the first study highlighting the role of oxytocin mediated MEC contraction on lacrimal gland function.


Assuntos
Células Acinares/fisiologia , Aparelho Lacrimal/fisiopatologia , Contração Muscular , Receptores de Ocitocina/metabolismo , Síndrome de Sjogren/fisiopatologia , Células Acinares/metabolismo , Animais , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Feminino , Humanos , Inflamação/metabolismo , Inflamação/fisiopatologia , Aparelho Lacrimal/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos MRL lpr , Camundongos Endogâmicos NOD , Células Musculares/metabolismo , Células Musculares/fisiologia , Síndrome de Sjogren/metabolismo
8.
Invest Ophthalmol Vis Sci ; 58(13): 5654-5665, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29098296

RESUMO

Purpose: Sjögren's syndrome is a systemic chronic autoimmune inflammatory disease that primarily targets the salivary and lacrimal glands (LGs). Currently there is no cure; therefore, cell-based regenerative therapy may be a viable option. LG inflammation is facilitated by extracellular ATP and mediated by the Pannexin-1 (Panx1) membrane channel glycoprotein. We propose that suppression of inflammation through manipulation of Panx1 activity can stimulate epithelial cell progenitor (EPCP) engraftment. Methods: The expression of pannexins in the mouse and human LG was assayed by qRT-PCR and immunostaining. Acute LG inflammation was induced by interleukin-1α (IL1α) injection. Prior to EPCP transplantation, IL1α-injured or chronically inflamed LGs of thrombospondin-1-null mice (TSP-1-/-) were treated with the Panx1-specific blocking peptide (10panx) or the self-deliverable RNAi (sdRNAi). The efficacy of cell engraftment and the area of inflammation were analyzed by microscopy. Results: Panx1 and Panx2 were detected in the mouse and human LGs. Panx1 and proinflammatory factors were upregulated during acute inflammation at days 1 to 3 after the IL1α injection. The analysis of EPCP engraftment demonstrated a significant and reproducible positive correlation between the 10panx peptide or Panx1 sdRNAi treatment and the number of engrafted cells. Similarly, treatment of the LG of the TSP-1-/- mouse (mouse model of chronic LG inflammation) by either Panx1 or Caspase-4 (also known as Casp11) sdRNAi showed a significant decrease in expression of proinflammatory markers and the lymphocyte infiltration. Conclusions: Our results suggest that blocking Panx1 and/or Casp4 activities is a beneficial strategy to enhance donor cell engraftment and LG regeneration through the reduction of inflammation.


Assuntos
Conexinas/genética , Células Epiteliais/transplante , Regulação da Expressão Gênica , Aparelho Lacrimal/metabolismo , Proteínas do Tecido Nervoso/genética , Síndrome de Sjogren/genética , Transplante de Células-Tronco/métodos , Células-Tronco/citologia , Idoso , Idoso de 80 Anos ou mais , Animais , Western Blotting , Conexinas/biossíntese , Modelos Animais de Doenças , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Imuno-Histoquímica , Aparelho Lacrimal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/biossíntese , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Síndrome de Sjogren/metabolismo , Síndrome de Sjogren/terapia , Células-Tronco/metabolismo
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