RESUMEN
Identifying specific markers of adipose stem and progenitor cells (ASPCs) in vivo is crucial for understanding the biology of white adipose tissues (WAT). PDGFRα-positive perivascular stromal cells represent the best candidates for ASPCs. This cell lineage differentiates into myofibroblasts that contribute to the impairment of WAT function. However, ASPC marker protein(s) that are functionally crucial for maintaining WAT homeostasis are unknown. We previously identified Meflin as a marker of mesenchymal stem cells (MSCs) in bone marrow and tissue-resident perivascular fibroblasts in various tissues. We also demonstrated that Meflin maintains the undifferentiated status of MSCs/fibroblasts. Here, we show that Meflin is expressed in WAT ASPCs. A lineage-tracing experiment showed that Meflin+ ASPCs proliferate in the WAT of obese mice induced by a high-fat diet (HFD), while some of them differentiate into myofibroblasts or mature adipocytes. Meflin knockout mice fed an HFD exhibited a significant fibrotic response as well as increases in adipocyte cell size and the number of crown-like structures in WAT, accompanied by impaired glucose tolerance. These data suggested that Meflin expressed by ASPCs may have a role in reducing disease progression associated with WAT dysfunction.
RESUMEN
Pancreatic stellate cells (PSCs) are stromal cells in the pancreas that play an important role in pancreatic pathology. In chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDAC), PSCs are known to get activated to form myofibroblasts or cancer-associated fibroblasts (CAFs) that promote stromal fibroinflammatory reactions. However, previous studies on PSCs were mainly based on the findings obtained using ex vivo expanded PSCs, with few studies that addressed the significance of in situ tissue-resident PSCs using animal models. Their contributions to fibrotic reactions in CP and PDAC are also lesser-known. These limitations in our understanding of PSC biology have been attributed to the lack of specific molecular markers of PSCs. Herein, we established Meflin (Islr), a glycosylphosphatidylinositol-anchored membrane protein, as a PSC-specific marker in both mouse and human by using human pancreatic tissue samples and Meflin reporter mice. Meflin-positive (Meflin+ ) cells contain lipid droplets and express the conventional PSC marker Desmin in normal mouse pancreas, with some cells also positive for Gli1, the marker of pancreatic tissue-resident fibroblasts. Three-dimensional analysis of the cleared pancreas of Meflin reporter mice showed that Meflin+ PSCs have long and thin cytoplasmic protrusions, and are localised on the abluminal side of vessels in the normal pancreas. Lineage tracing experiments revealed that Meflin+ PSCs constitute one of the origins of fibroblasts and CAFs in CP and PDAC, respectively. In these diseases, Meflin+ PSC-derived fibroblasts showed a distinctive morphology and distribution from Meflin+ PSCs in the normal pancreas. Furthermore, we showed that the genetic depletion of Meflin+ PSCs accelerated fibrosis and attenuated epithelial regeneration and stromal R-spondin 3 expression, thereby implying that Meflin+ PSCs and their lineage cells may support tissue recovery and Wnt/R-spondin signalling after pancreatic injury and PDAC development. Together, these data indicate that Meflin may be a marker specific to tissue-resident PSCs and useful for studying their biology in both health and disease. © 2023 The Pathological Society of Great Britain and Ireland.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Pancreatitis Crónica , Animales , Humanos , Ratones , Carcinoma Ductal Pancreático/patología , Fibrosis , Páncreas/patología , Neoplasias Pancreáticas/patología , Células Estrelladas Pancreáticas/patología , Pancreatitis Crónica/genética , Pancreatitis Crónica/metabolismo , Pancreatitis Crónica/patología , RegeneraciónRESUMEN
BACKGROUND: The proliferation of cancer-associated fibroblasts (CAFs) hampers drug delivery and anti-tumor immunity, inducing tumor resistance to immune checkpoint blockade (ICB) therapy. However, it has remained a challenge to develop therapeutics that specifically target or modulate CAFs. METHODS: We investigated the involvement of Meflin+ cancer-restraining CAFs (rCAFs) in ICB efficacy in patients with clear cell renal cell carcinoma (ccRCC) and urothelial carcinoma (UC). We examined the effects of Am80 (a synthetic retinoid) administration on CAF phenotype, the tumor immune microenvironment, and ICB efficacy in cancer mouse models. RESULTS: High infiltration of Meflin+ CAFs correlated with ICB efficacy in patients with ccRCC and UC. Meflin+ CAF induction by Am80 administration improved ICB efficacy in the mouse models of cancer. Am80 exerted this effect when administered prior to, but not concomitant with, ICB therapy in wild-type but not Meflin-deficient mice. Am80-mediated induction of Meflin+ CAFs was associated with increases in antibody delivery and M1-like tumor-associated macrophage (TAM) infiltration. Finally, we showed the role of Chemerin produced from CAFs after Am80 administration in the induction of M1-like TAMs. CONCLUSION: Our data suggested that Am80 administration prior to ICB therapy increases the number of Meflin+ rCAFs and ICB efficacy by inducing changes in TAM phenotype.
Asunto(s)
Fibroblastos Asociados al Cáncer , Inhibidores de Puntos de Control Inmunológico , Macrófagos , Microambiente Tumoral , Animales , Fibroblastos Asociados al Cáncer/efectos de los fármacos , Fibroblastos Asociados al Cáncer/metabolismo , Inhibidores de Puntos de Control Inmunológico/farmacología , Inhibidores de Puntos de Control Inmunológico/uso terapéutico , Ratones , Humanos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Macrófagos/inmunología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Tetrahidronaftalenos/farmacología , Retinoides/farmacología , Retinoides/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/patología , Macrófagos Asociados a Tumores/efectos de los fármacos , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/inmunología , Neoplasias Renales/patología , Femenino , Línea Celular Tumoral , BenzoatosRESUMEN
Organoids are a three-dimensional (3D) culture system that simulate actual organs. Therefore, tumor organoids are expected to predict precise response to chemotherapy in patients. However, to date, few studies have studied the drug responses in organoids of malignant mesothelioma (MM). The poor prognosis of MM emphasizes the importance of establishing a protocol for generating MM-organoid for research and clinical use. Here, we established murine MM organoids from p53+/- or wild-type C57BL/6 strain by intraperitoneal injection either with crocidolite or carbon nanotube. Established MM-organoids proliferated in Matrigel as spheroids. Subcutaneous injection assays revealed that the MM-organoids mimicked actual tissue architecture and maintained the original histological features of the primary MM. RNA sequencing and pathway analyses revealed that the significant expressional differences between the 2D- and 3D-culture systems were observed in receptor tyrosine kinases, including IGF1R and EGFR, glycosylation and cholesterol/steroid metabolism. MM-organoids exhibited a more sensitive response to cisplatin through stable plasma membrane localization of a major cisplatin transporter, copper transporter 1/Slc31A1 (Ctr1) in comparison to 2D-cultures, presumably through glycosylation and lipidation. The Matrigel culture system facilitated the localization of CTR1 on the plasma membrane, which simulated the original MMs and the subcutaneous xenografts. These results suggest that the newly developed protocol for MM-organoids is useful to study strategies to overcome chemotherapy resistance to cisplatin.
Asunto(s)
Cisplatino , Transportador de Cobre 1 , Mesotelioma Maligno , Animales , Humanos , Ratones , Cisplatino/farmacología , Colágeno/metabolismo , Mesotelioma Maligno/metabolismo , Organoides/patología , Transportador de Cobre 1/metabolismoRESUMEN
Mesenchymal stem cells (MSCs) are the likely precursors of multiple lines of mesenchymal cells. The existence of bona fide MSCs with self-renewal capacity and differentiation potential into all mesenchymal lineages, however, has been unclear because of the lack of MSC-specific marker(s) that are not expressed by the terminally differentiated progeny. Meflin, a glycosylphosphatidylinositol-anchored protein, is an MSC marker candidate that is specifically expressed in rare stromal cells in all tissues. Our previous report showed that Meflin expression becomes down-regulated in bone marrow-derived MSCs cultured on plastic, making it difficult to examine the self-renewal and differentiation of Meflin-positive cells at the single-cell level. Here, we traced the lineage of Meflin-positive cells in postnatal and adult mice, showing that those cells differentiated into white and brown adipocytes, osteocytes, chondrocytes and skeletal myocytes. Interestingly, cells derived from Meflin-positive cells formed clusters of differentiated cells, implying the in situ proliferation of Meflin-positive cells or their lineage-committed progenitors. These results, taken together with previous findings that Meflin expression in cultured MSCs was lost upon their multilineage differentiation, suggest that Meflin is a useful potential marker to localize MSCs and/or their immature progenitors in multiple tissues.
Asunto(s)
Diferenciación Celular , Linaje de la Célula , Inmunoglobulinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Animales , Condrocitos/citología , Condrocitos/metabolismo , Inmunoglobulinas/genética , Células Madre Mesenquimatosas/citología , Ratones , Ratones Endogámicos C57BL , Células Musculares/citología , Células Musculares/metabolismo , Osteocitos/citología , Osteocitos/metabolismoRESUMEN
In the heart, fatty acid is a major energy substrate to fuel contraction under aerobic conditions. Ischemia downregulates fatty acid metabolism to adapt to the limited oxygen supply, making glucose the preferred substrate. However, the mechanism underlying the myocardial metabolic shift during ischemia remains unknown. Here, we show that lipoprotein lipase (LPL) expression in cardiomyocytes, a principal enzyme that converts triglycerides to free fatty acids and glycerol, increases during myocardial infarction (MI). Cardiomyocyte-specific LPL deficiency enhanced cardiac dysfunction and apoptosis following MI. Deficiency of aquaporin 7 (AQP7), a glycerol channel in cardiomyocytes, increased the myocardial infarct size and apoptosis in response to ischemia. Ischemic conditions activated glycerol-3-phosphate dehydrogenase 2 (GPD2), which converts glycerol-3-phosphate into dihydroxyacetone phosphate to facilitate adenosine triphosphate (ATP) synthesis from glycerol. Conversely, GPD2 deficiency exacerbated cardiac dysfunction after acute MI. Moreover, cardiomyocyte-specific LPL deficiency suppressed the effectiveness of peroxisome proliferator-activated receptor alpha (PPARα) agonist treatment for MI-induced cardiac dysfunction. These results suggest that LPL/AQP7/GPD2-mediated glycerol metabolism plays an important role in preventing myocardial ischemia-related damage.
Asunto(s)
Acuaporinas/metabolismo , Cardiomiopatías/prevención & control , Glicerol/metabolismo , Glicerolfosfato Deshidrogenasa/metabolismo , Hipoxia/fisiopatología , Isquemia/prevención & control , Lipoproteína Lipasa/fisiología , Proteínas Mitocondriales/metabolismo , Animales , Acuaporinas/genética , Cardiomiopatías/etiología , Cardiomiopatías/metabolismo , Cardiomiopatías/patología , Glicerolfosfato Deshidrogenasa/genética , Isquemia/etiología , Isquemia/metabolismo , Isquemia/patología , Masculino , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genéticaRESUMEN
RATIONALE: Myofibroblasts have roles in tissue repair following damage associated with ischemia, aging, and inflammation and also promote fibrosis and tissue stiffening, causing organ dysfunction. One source of myofibroblasts is mesenchymal stromal/stem cells that exist as resident fibroblasts in multiple tissues. We previously identified meflin (mesenchymal stromal cell- and fibroblast-expressing Linx paralogue), a glycosylphosphatidylinositol-anchored membrane protein, as a specific marker of mesenchymal stromal/stem cells and a regulator of their undifferentiated state. The roles of meflin in the development of heart disease, however, have not been investigated. OBJECTIVE: We examined the expression of meflin in the heart and its involvement in cardiac repair after ischemia, fibrosis, and the development of heart failure. METHODS AND RESULTS: We found that meflin has an inhibitory role in myofibroblast differentiation of cultured mesenchymal stromal/stem cells. Meflin expression was downregulated by stimulation with TGF (transforming growth factor)-ß, substrate stiffness, hypoxia, and aging. Histological analysis revealed that meflin-positive fibroblastic cells and their lineage cells proliferated in the hearts after acute myocardial infarction and pressure-overload heart failure mouse models. Analysis of meflin knockout mice revealed that meflin is essential for the increase in the number of cells that highly express type I collagen in the heart walls after myocardial infarction induction. When subjected to pressure overload by transverse aortic constriction, meflin knockout mice developed marked cardiac interstitial fibrosis with defective compensation mechanisms. Analysis with atomic force microscopy and hemodynamic catheterization revealed that meflin knockout mice developed stiff failing hearts with diastolic dysfunction. Mechanistically, we found that meflin interacts with bone morphogenetic protein 7, an antifibrotic cytokine that counteracts the action of TGF-ß and augments its intracellular signaling. CONCLUSIONS: These data suggested that meflin is involved in cardiac tissue repair after injury and has an inhibitory role in myofibroblast differentiation of cardiac fibroblastic cells and the development of cardiac fibrosis.
Asunto(s)
Diástole , Inmunoglobulinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Infarto del Miocardio/metabolismo , Miofibroblastos/metabolismo , Regeneración , Animales , Células CHO , Células Cultivadas , Colágeno/genética , Colágeno/metabolismo , Cricetinae , Cricetulus , Células HEK293 , Humanos , Inmunoglobulinas/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Infarto del Miocardio/genética , Infarto del Miocardio/fisiopatología , Miofibroblastos/fisiología , Unión ProteicaRESUMEN
OBJECTIVE: Angiocrine factors, mediating the endothelial-mural cell interaction in vascular wall construction as well as maintenance, are incompletely characterized. This study aims to investigate the role of endothelial cell-derived FSTL1 (follistatin-like protein 1) in vascular homeostasis. Approach and Results: Using conditional knockout mouse models, we show that loss of FSTL1 in endothelial cells (Fstl1ECKO) led to an increase of pulmonary vascular resistance, resulting in the heart regurgitation especially with tricuspid valves. However, this abnormality was not detected in mutant mice with Fstl1 knockout in smooth muscle cells or hematopoietic cells. We further showed that there was excessive αSMA (α-smooth muscle actin) associated with atrial endocardia, heart valves, veins, and microvessels after the endothelial FSTL1 deletion. There was also an increase in collagen deposition, as demonstrated in livers of Fstl1ECKO mutants. The SMAD3 (mothers against decapentaplegic homolog 3) phosphorylation (pSMAD3) was significantly enhanced, and pSMAD3 staining was colocalized with αSMA in vein walls, suggesting the activation of TGFß (transforming growth factor ß) signaling in vascular mural cells of Fstl1ECKO mice. Consistently, treatment with a TGFß pathway inhibitor reduced the abnormal association of αSMA with the atria and blood vessels in Fstl1ECKO mutant mice. CONCLUSIONS: The findings imply that endothelial FSTL1 is critical for the homeostasis of vascular walls, and its insufficiency may favor cardiovascular fibrosis leading to heart failure.
Asunto(s)
Endotelio Vascular/fisiopatología , Fibrosis/fisiopatología , Proteínas Relacionadas con la Folistatina/fisiología , Proteína smad3/fisiología , Actinas/metabolismo , Animales , Modelos Animales de Enfermedad , Células Endoteliales/fisiología , Proteínas Relacionadas con la Folistatina/metabolismo , Homeostasis , Humanos , Ratones Noqueados , Fosforilación , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/fisiología , Insuficiencia de la Válvula Tricúspide/fisiopatología , Resistencia VascularRESUMEN
BACKGROUND: Heart failure is a complex syndrome that results from structural or functional impairment of ventricular filling or blood ejection. Protein phosphorylation is a major and essential intracellular mechanism that mediates various cellular processes in cardiomyocytes in response to extracellular and intracellular signals. The RHOA-associated protein kinase (ROCK/Rho-kinase), an effector regulated by the small GTPase RHOA, causes pathological phosphorylation of proteins, resulting in cardiovascular diseases. RHOA also activates protein kinase N (PKN); however, the role of PKN in cardiovascular diseases remains unclear. METHODS: To explore the role of PKNs in heart failure, we generated tamoxifen-inducible, cardiomyocyte-specific PKN1- and PKN2-knockout mice by intercrossing the αMHC-CreERT2 line with Pkn1flox/flox and Pkn2flox/flox mice and applied a mouse model of transverse aortic constriction- and angiotensin II-induced heart failure. To identify a novel substrate of PKNs, we incubated GST-tagged myocardin-related transcription factor A (MRTFA) with recombinant GST-PKN-catalytic domain or GST-ROCK-catalytic domain in the presence of radiolabeled ATP and detected radioactive GST-MRTFA as phosphorylated MRTFA. RESULTS: We demonstrated that RHOA activates 2 members of the PKN family of proteins, PKN1 and PKN2, in cardiomyocytes of mice with cardiac dysfunction. Cardiomyocyte-specific deletion of the genes encoding Pkn1 and Pkn2 (cmc-PKN1/2 DKO) did not affect basal heart function but protected mice from pressure overload- and angiotensin II-induced cardiac dysfunction. Furthermore, we identified MRTFA as a novel substrate of PKN1 and PKN2 and found that MRTFA phosphorylation by PKN was considerably more effective than that by ROCK in vitro. We confirmed that endogenous MRTFA phosphorylation in the heart was induced by pressure overload- and angiotensin II-induced cardiac dysfunction in wild-type mice, whereas cmc-PKN1/2 DKO mice suppressed transverse aortic constriction- and angiotensin II-induced phosphorylation of MRTFA. Although RHOA-mediated actin polymerization accelerated MRTFA-induced gene transcription, PKN1 and PKN2 inhibited the interaction of MRTFA with globular actin by phosphorylating MRTFA, causing increased serum response factor-mediated expression of cardiac hypertrophy- and fibrosis-associated genes. CONCLUSIONS: Our results indicate that PKN1 and PKN2 activation causes cardiac dysfunction and is involved in the transition to heart failure, thus providing unique targets for therapeutic intervention for heart failure.
Asunto(s)
Actinas/metabolismo , Insuficiencia Cardíaca/enzimología , Miocitos Cardíacos/enzimología , Proteína Quinasa C/metabolismo , Transactivadores/metabolismo , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/fisiopatología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/patología , Fosforilación , Unión Proteica , Proteína Quinasa C/deficiencia , Proteína Quinasa C/genética , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Introduction: The aim of this study was to determine the role of Notch in indoxyl sulfate (IS)-induced vascular calcification (VC). Materials and methods: VC and expression of Notch-related and osteogenic molecules were examined in Dahl salt-sensitive (DS), DS hypertensive (DH), and DH IS-treated rats (DH+IS). The effects of IS on expression of Notch receptors, apoptotic activity, and calcification were examined in cultured aortic smooth muscle cells (SMCs). Results: Medial calcification was noted only in aortas and coronary arteries of DH+IS rats. Notch1, Notch3, and Hes-1 were expressed in aortic SMCs of all rats, but only weakly in the central areas of the media and around the calcified lesions in DH+IS rats. RT-PCR and western blotting of DH+IS rat aortas showed downregulation of Notch ligands, Notch1 and Notch3, downstream transcriptional factors, and SM22, and conversely, overexpression of osteogenic markers. Expression of Notch1 and Notch3 in aortic SMCs was highest in incubation under 500 µM IS for 24hrs, and then decreased time- and dose-dependently. Coupled with this decrease, IS increased caspase 3/7 activity and TUNEL-positive aortic SMCs. In addition, pharmacological Notch signal inhibition with DAPT induced apoptosis in aortic SMCs. ZVAD, a caspase inhibitor abrogated IS-induced and DAPT-induced in vitro vascular calcification. Knockdown of Notch1 and Notch3 cooperatively increased expression of osteogenic transcriptional factors and decreased expression of SM22. Conclusion: Our results suggested that IS-induced VC is mediated through suppression of Notch activity in aortic SMCs, induction of osteogenic differentiation and apoptosis.
Asunto(s)
Indicán/toxicidad , Miocitos del Músculo Liso/patología , Receptores Notch/metabolismo , Calcificación Vascular/patología , Animales , Aorta/citología , Aorta/patología , Calcio/análisis , Línea Celular , Dipéptidos/farmacología , Técnicas de Silenciamiento del Gen , Indicán/administración & dosificación , Miocitos del Músculo Liso/efectos de los fármacos , Ratas , Receptores Notch/antagonistas & inhibidores , Receptores Notch/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Calcificación Vascular/inducido químicamente , Calcificación Vascular/diagnósticoRESUMEN
Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinase-mediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophy-related gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart.
Asunto(s)
Miocardio/enzimología , Proteómica , Transducción de Señal , Quinasas Asociadas a rho/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/metabolismo , Animales , Encéfalo/metabolismo , Células Cultivadas , Cromatografía de Afinidad , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Inmunohistoquímica , Espectrometría de Masas , Microscopía Fluorescente , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Fosforilación , Unión Proteica , Ratas , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Especificidad por Sustrato , Quinasas Asociadas a rho/químicaRESUMEN
OBJECTIVE: Vascular endothelial growth factor (VEGF) signaling is a major regulator of physiological and pathological angiogenesis. VEGF receptor activity is strongly controlled by endocytosis, which can terminate or enhance signal transduction in the angiogenic endothelium, but the exact molecular regulation of these processes remains incompletely understood. We have therefore examined the function of Numb family clathrin-associated sorting proteins in angiogenesis. APPROACH AND RESULTS: We show that Numb proteins are expressed by endothelial cells during retinal angiogenesis in mice. Inducible inactivation of the Numb/Numbl genes in the postnatal endothelium led to impaired vessel growth, reduced endothelial proliferation and sprouting, and decreased VEGF receptor activation. Biochemistry and cell biology experiments established that Numb can interact with VEGFR2 and VEGFR3 and controls VEGF receptor activation in response to ligand stimulation. Experiments in cultured endothelial cells showed that Numb proteins counteract VEGF receptor degradation and promote VEGFR2 recycling back to the plasma membrane. CONCLUSIONS: Numb proteins control VEGF receptor endocytosis, signaling, and recycling in endothelial cells, which promotes the angiogenic growth of blood vessels.
Asunto(s)
Células Endoteliales/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de la Membrana/metabolismo , Neovascularización Fisiológica , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Neovascularización Retiniana/metabolismo , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Endocitosis , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas de la Membrana/genética , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Transporte de Proteínas , Interferencia de ARN , Neovascularización Retiniana/genética , Neovascularización Retiniana/fisiopatología , Transducción de Señal , TransfecciónRESUMEN
Axon formation is one of the most important events in neuronal polarization and is regulated by signaling molecules involved in cytoskeletal rearrangement and protein transport. We previously found that Partition-defective 3 (Par3) is associated with KIF3A (kinesin-2) and is transported into the nascent axon in a KIF3A-dependent fashion. Par3 interacts with the Rac-specific guanine nucleotide-exchange factors (GEFs) Tiam1/2, which activate Rac1, and participates in axon formation in cultured hippocampal neurons. However, the regulatory mechanism of the Par3-KIF3A interaction is poorly understood, and the role of Par3 in neuronal polarization in vivo remains elusive. Here, we found that extracellular signal-regulated kinase 2 (ERK2) directly interacts with Par3, that ERK2 phosphorylates Par3 at Ser-1116, and that the phosphorylated Par3 accumulates at the axonal tips in a manner dependent upon ERK2 activity. The phosphorylation of Par3 by ERK2 inhibited the interaction of Par3 with KIF3A but not with the other Par3 partners, including Par6 and aPKC. The phosphomimic mutant of Par3 (Par3-S1116D) showed less binding activity with the KIF3s and slower transport in the axons. The knockdown of Par3 by RNA interference impaired neuronal polarization, which was rescued with RNAi-resistant Par3, but not with the phosphomimic Par3 mutant, in cultured rat hippocampal neurons and mouse cortical projection neurons in vivo. These results suggest that ERK2 phosphorylates Par3 and inhibits its binding with KIF3A, thereby controlling Par3 transport and neuronal polarity.
Asunto(s)
Proteínas Portadoras/metabolismo , Polaridad Celular/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Neuronas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Inmunoprecipitación , Cinesinas/metabolismo , Ratones , Ratones Endogámicos ICR , Proteínas del Tejido Nervioso , Neuronas/citología , Fosforilación , Transporte de Proteínas/fisiología , ARN Interferente Pequeño , Ratas , Ratas Wistar , Transfección , XenopusRESUMEN
Extracellular vesicles (EVs) are complex structures that transport various DNA, RNA, and protein. Recently, new EV secretion mechanisms have been identified through the iron regulatory system in mammalian cells. We revealed that ferroptosis increases EV secretion, which is named ferroptosis-dependent EVs (FedEVs). Here, we describe a step-by-step procedure to isolate GFP-expressing FedEVs for in vitro analysis. The FedEVs are further analyzed by imaging and flow cytometry analysis. For complete details on the use and execution of this protocol, please refer to Ito et al.1.
Asunto(s)
Vesículas Extracelulares , Ferroptosis , Animales , Ferroptosis/genética , Proteínas/metabolismo , Técnicas de Cultivo de Célula , Vesículas Extracelulares/química , MamíferosRESUMEN
The adult mammalian heart has extremely limited cardiac regenerative capacity. Most cardiomyocytes live in a state of permanent cell-cycle arrest and are unable to re-enter the cycle. Cardiomyocytes switch from cell proliferation to a maturation state during neonatal development. Although several signaling pathways are involved in this transition, the molecular mechanisms by which these inputs coordinately regulate cardiomyocyte maturation are not fully understood. Retinoic acid (RA) plays a pivotal role in development, morphogenesis, and regeneration. Despite the importance of RA signaling in embryo heart development, little is known about its function in the early postnatal period. We found that mRNA expression of aldehyde dehydrogenase 1 family member A2 (Aldh1a2), which encodes the key enzyme for synthesizing all-trans retinoic acid (ATRA) and is an important regulator for RA signaling, was transiently upregulated in neonatal mouse ventricles. Single-cell transcriptome analysis and immunohistochemistry revealed that Aldh1a2 expression was enriched in cardiac fibroblasts during the early postnatal period. Administration of ATRA inhibited cardiomyocyte proliferation in cultured neonatal rat cardiomyocytes and human cardiomyocytes. RNA-seq analysis indicated that cell proliferation-related genes were downregulated in prenatal rat ventricular cardiomyocytes treated with ATRA, while cardiomyocyte maturation-related genes were upregulated. These findings suggest that RA signaling derived from cardiac fibroblasts is one of the key regulators of cardiomyocyte proliferation and maturation during neonatal heart development.
Asunto(s)
Familia de Aldehído Deshidrogenasa 1 , Proliferación Celular , Miocitos Cardíacos , Retinal-Deshidrogenasa , Transducción de Señal , Tretinoina , Animales , Tretinoina/farmacología , Tretinoina/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Ratones , Familia de Aldehído Deshidrogenasa 1/metabolismo , Familia de Aldehído Deshidrogenasa 1/genética , Retinal-Deshidrogenasa/metabolismo , Retinal-Deshidrogenasa/genética , Proliferación Celular/efectos de los fármacos , Ratas , Humanos , Regulación hacia Arriba , Animales Recién Nacidos , Ciclo Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Corazón/efectos de los fármacos , Corazón/crecimiento & desarrollo , Células CultivadasRESUMEN
Alveologenesis is a spatially coordinated morphogenetic event, during which alveolar myofibroblasts surround the terminal sacs constructed by epithelial cells and endothelial cells (ECs), then contract to form secondary septa to generate alveoli in the lungs. Recent studies have demonstrated the important role of alveolar ECs in this morphogenetic event. However, the mechanisms underlying EC-mediated alveologenesis remain unknown. Herein, we show that ECs regulate alveologenesis by constructing basement membranes (BMs) acting as a scaffold for myofibroblasts to induce septa formation through activating mechanical signaling. Rap1, a small GTPase of the Ras superfamily, is known to stimulate integrin-mediated cell adhesions. EC-specific Rap1-deficient (Rap1iECKO) mice exhibit impaired septa formation and hypo-alveolarization due to the decreased mechanical signaling in myofibroblasts. In Rap1iECKO mice, ECs fail to stimulate integrin ß1 to recruit Collagen type IV (Col-4) into BMs required for myofibroblast-mediated septa formation. Consistently, EC-specific integrin ß1-deficient mice show hypo-alveolarization, defective mechanical signaling in myofibroblasts, and disorganized BMs. These data demonstrate that alveolar ECs promote integrin ß1-mediated Col-4 recruitment in a Rap1-dependent manner, thereby constructing BMs acting as a scaffold for myofibroblasts to induce mechanical signal-mediated alveologenesis. Thus, this study unveils a mechanism of organ morphogenesis mediated by ECs through intrinsic functions.
Asunto(s)
Células Endoteliales , Miofibroblastos , Animales , Ratones , Membrana Basal , Integrina beta1/genética , MorfogénesisRESUMEN
RATIONALE: It is well established that the migration and proliferation of vascular smooth muscle cells (VSMCs) have major roles in the vascular remodeling process. Our previous study showed that the Akt substrate Girdin, which is expressed in VSMCs and endothelial cells, is essential for postnatal angiogenesis. However, the function of Girdin and its Akt-mediated phosphorylation in VSMCs and their in vivo roles in vascular remodeling remain to be elucidated. OBJECTIVE: We investigated the function of Girdin and its Akt-mediated phosphorylation using cultured VSMCs and animal models of vascular remodeling. METHODS AND RESULTS: The depletion of Girdin by RNA interference disrupted the rearrangement of the actin cytoskeleton in VSMCs, resulting in impaired cell migration. The depletion of Girdin also inhibited VSMC proliferation. Girdin expression was highly upregulated and its serine at position 1416 was phosphorylated in the neointima of carotid arteries after balloon injury in a rat model. The introduction of an adenovirus harboring short hairpin RNA against Girdin attenuated the proliferation of VSMCs and neointima formation without affecting reendothelialization. Furthermore, we found that neointima formation after femoral wire injury was significantly attenuated in Girdin S1416A knock-in mice, in which the Akt phosphorylation site of Girdin was mutated, thus indicating a major role for Girdin phosphorylation in vascular remodeling. CONCLUSIONS: These findings indicate that Girdin and its Akt-mediated phosphorylation have major roles in the migration and proliferation of VSMCs and vascular remodeling, making the Akt/Girdin signaling pathway a potential target for the development of new therapeutics for vascular diseases.
Asunto(s)
Arteria Femoral/lesiones , Arteria Femoral/metabolismo , Proteínas de Microfilamentos/fisiología , Modelos Animales , Neointima/metabolismo , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas de Transporte Vesicular/fisiología , Animales , Traumatismos de las Arterias Carótidas/etiología , Traumatismos de las Arterias Carótidas/patología , Movimiento Celular/genética , Proliferación Celular , Células Cultivadas , Arteria Femoral/patología , Técnicas de Sustitución del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Neointima/genética , Neointima/patología , Fosforilación/genética , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/genética , Proteínas de Transporte Vesicular/deficiencia , Proteínas de Transporte Vesicular/genéticaRESUMEN
OBJECTIVE: Sirolimus-eluting stent (SES) has shown a significant efficacy in reducing restenosis after percutaneous coronary interventions. However, an increase in total number of SES use along with targeting more complex lesions generated a large number of SES restenosis. This study aimed to investigate the clinical and angiographic outcomes of different revascularization strategies for SES restenosis. METHODS AND RESULTS: A total of 176 lesions in 149 patients were included in the study. Fifteen patients underwent coronary artery bypass graft surgery (CABG group) and the remaining patients were treated with percutaneous coronary intervention (PCI). Stent reimplantation was performed in 88 patients (Stent group), whereas 46 patients received balloon therapy (Balloon group). Among 176 lesions, major cardiac adverse event (MACE) occurred in 41 lesions (23.3%) during a median follow-up of 310 days (interquartile range: 146-517 days). The Kaplan-Meier method with a log-rank test revealed no significant difference in MACE rates between the three groups (6%, 25%, 26%, p = 0.13; CABG group, Stent group, Balloon group, respectively). However, when the Balloon group and Stent group were combined together as a PCI group, PCI group had a significantly higher rate of MACE compared with the CABG group (p = 0.04). In addition, angiographic restenosis was significantly less prevalent in the CABG group when compared with the other two groups (8%, 57%, 46%, p = 0.006; CABG group, Stent group, Balloon group, respectively). CONCLUSIONS: CABG surgery for patients with SES restenosis is associated with the better clinical outcomes as well as better angiographic outcomes when compared with that of PCI.
Asunto(s)
Angioplastia Coronaria con Balón , Puente de Arteria Coronaria , Reestenosis Coronaria/terapia , Stents Liberadores de Fármacos , Inmunosupresores/uso terapéutico , Sirolimus/uso terapéutico , Anciano , Reestenosis Coronaria/diagnóstico por imagen , Reestenosis Coronaria/mortalidad , Reestenosis Coronaria/prevención & control , Muerte , Femenino , Estudios de Seguimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Infarto del Miocardio/epidemiología , Infarto del Miocardio/etiología , Intervención Coronaria Percutánea/instrumentación , Complicaciones Posoperatorias/epidemiología , Radiografía , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
To maintain and restore skeletal muscle mass and function is essential for healthy aging. We have found that myonectin acts as a cardioprotective myokine. Here, we investigate the effect of myonectin on skeletal muscle atrophy in various male mouse models of muscle dysfunction. Disruption of myonectin exacerbates skeletal muscle atrophy in age-associated, sciatic denervation-induced or dexamethasone (DEX)-induced muscle atrophy models. Myonectin deficiency also contributes to exacerbated mitochondrial dysfunction and reduces expression of mitochondrial biogenesis-associated genes including PGC1α in denervated muscle. Myonectin supplementation attenuates denervation-induced muscle atrophy via activation of AMPK. Myonectin also reverses DEX-induced atrophy of cultured myotubes through the AMPK/PGC1α signaling. Furthermore, myonectin treatment suppresses muscle atrophy in senescence-accelerated mouse prone (SAMP) 8 mouse model of accelerated aging or mdx mouse model of Duchenne muscular dystrophy. These data indicate that myonectin can ameliorate skeletal muscle dysfunction through AMPK/PGC1α-dependent mechanisms, suggesting that myonectin could represent a therapeutic target of muscle atrophy.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Músculo Esquelético , Animales , Masculino , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Ratones Endogámicos mdx , Músculo Esquelético/metabolismo , Atrofia Muscular/prevención & control , Atrofia Muscular/inducido químicamente , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismoRESUMEN
The small GTP-binding protein Rho plays a crucial role in a wide variety of cellular functions through various effector proteins. Rho-kinase is a key effector protein of Rho, which is composed of two isoforms, ROCK1 and ROCK2. To clarify the site of action of ROCK1 and ROCK2, we performed immunofluorescence and immunoelectron microscopic analyses using isoform-specific antibodies in mouse tissues. In the large and small intestines, ROCK1 immunoreactivity was predominantly identified in epithelial cells, and ROCK2 immunoreactivity was negligible. In these epithelial cells, ROCK1 immunoreactivity was distributed on plasma membranes, while ROCK1 immunogold signals were localized at cell-cell contacts and cell adhesion sites, especially at the adherens junctions at the ultrastructural level. In the bladder epithelium, however, ROCK1 and ROCK2 signals were identified at intermediate filaments, and ROCK2 signals were also observed in nuclei. In the three types of muscular cells-smooth, cardiac, and skeletal muscle cells-ROCK1 and ROCK2 also showed differential distribution. ROCK1 signals were localized at actin filaments, plasma membranes, and vesicles near plasma membranes in smooth muscle cells; at the lysosomes in skeletal muscle cells; and were undetectable in cardiac muscle cells. ROCK2 signals were localized at actin filaments and centrosomes in smooth muscle cells, at intercalated discs in cardiac muscle cells, and at Z-discs and sarcoplasmic reticulum in skeletal muscle cells. In the brain, ROCK1 immunoreactivity was distributed in glia, whereas ROCK2 immunoreactivity was observed in neurons. These results indicate that the two isoforms of Rho-kinase distribute differentially to accomplish their specific functions.