RESUMO
Importance: Chronic kidney disease (CKD) is an often-asymptomatic complication of type 2 diabetes (T2D) that requires annual screening to diagnose. Patient-level factors linked to inadequate screening and treatment can inform implementation strategies to facilitate guideline-recommended CKD care. Objective: To identify risk factors for nonconcordance with guideline-recommended CKD screening and treatment in patients with T2D. Design, Setting, and Participants: This retrospective cohort study was performed at 20 health care systems contributing data to the US National Patient-Centered Clinical Research Network. To evaluate concordance with CKD screening guidelines, adults with an outpatient clinician visit linked to T2D diagnosis between January 1, 2015, and December 31, 2020, and without known CKD were included. A separate analysis reviewed prescription of angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) and sodium-glucose cotransporter 2 (SGLT2) inhibitors in adults with CKD (estimated glomerular filtration rate [eGFR] of 30-90 mL/min/1.73 m2 and urinary albumin-to-creatinine ratio [UACR] of 200-5000 mg/g) and an outpatient clinician visit for T2D between October 1, 2019, and December 31, 2020. Data were analyzed from July 8, 2022, through June 22, 2023. Exposures: Demographics, lifestyle factors, comorbidities, medications, and laboratory results. Main Outcomes and Measures: Screening required measurement of creatinine levels and UACR within 15 months of the index visit. Treatment reflected prescription of ACEIs or ARBs and SGLT2 inhibitors within 12 months before or 6 months following the index visit. Results: Concordance with CKD screening guidelines was assessed in 316â¯234 adults (median age, 59 [IQR, 50-67] years), of whom 51.5% were women; 21.7%, Black; 10.3%, Hispanic; and 67.6%, White. Only 24.9% received creatinine and UACR screening, 56.5% received 1 screening measurement, and 18.6% received neither. Hispanic ethnicity was associated with lack of screening (relative risk [RR], 1.16 [95% CI, 1.14-1.18]). In contrast, heart failure, peripheral arterial disease, and hypertension were associated with a lower risk of nonconcordance. In 4215 patients with CKD and albuminuria, 3288 (78.0%) received an ACEI or ARB; 194 (4.6%), an SGLT2 inhibitor; and 885 (21.0%), neither therapy. Peripheral arterial disease and lower eGFR were associated with lack of CKD treatment, while diuretic or statin prescription and hypertension were associated with treatment. Conclusions and Relevance: In this cohort study of patients with T2D, fewer than one-quarter received recommended CKD screening. In patients with CKD and albuminuria, 21.0% did not receive an SGLT2 inhibitor or an ACEI or an ARB, despite compelling indications. Patient-level factors may inform implementation strategies to improve CKD screening and treatment in people with T2D.
Assuntos
Diabetes Mellitus Tipo 2 , Fidelidade a Diretrizes , Insuficiência Renal Crônica , Humanos , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/complicações , Estudos Retrospectivos , Idoso , Fidelidade a Diretrizes/estatística & dados numéricos , Guias de Prática Clínica como Assunto , Programas de Rastreamento/métodos , Programas de Rastreamento/normas , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Antagonistas de Receptores de Angiotensina/uso terapêutico , Fatores de Risco , Inibidores do Transportador 2 de Sódio-Glicose/uso terapêutico , Estados Unidos/epidemiologia , Taxa de Filtração GlomerularRESUMO
High endothelial venules (HEV) are specialized post-capillary venules that recruit naïve lymphocytes to lymph nodes. HEVs are essential for the development of adaptive immunity. HEVs can also develop in tumors where they are thought to be important for recruiting naïve T cells and B cells into the tumors and locally enhancing antitumor immunity by supporting the formation of tertiary lymphoid structures. Herein, we used comparative transcriptome analysis of human breast cancer to investigate genes differentially expressed between tumor-associated HEVs and the rest of the tumor vasculature. Tumor vessels highly expressing HEV-upregulated genes, such as the homeobox gene MEOX2 and the tetraspanin gene TSPAN7, were associated with extensive infiltration of T and B cells and the occurrence of tertiary lymphoid structures, which is known to predict therapeutic responses to immune-checkpoint inhibitors. Moreover, high transcript counts of these genes in clinical tumor specimens were associated with a significant survival benefit in advanced breast cancer. The molecular signature of HEVs identified herein may be useful for guiding immunotherapies and provides a new direction for investigating tumor-associated HEVs and their clinical significance. See related Spotlight by Gallimore, p. 371.
Assuntos
Neoplasias da Mama , Estruturas Linfoides Terciárias , Feminino , Humanos , Linfonodos/patologia , Linfócitos , Vênulas/patologiaRESUMO
Recruitment of naive T cells to lymph nodes is essential for the development of adaptive immunity. Upon pathogen infection, lymph nodes promptly increase the influx of naive T cells from the circulation in order to screen and prime the T cells. The precise contribution of the lymph node vasculature to the regulation of this process remains unclear. Here we show a role for the Ras GTPase, R-Ras, in the functional adaptation of high endothelial venules to increase naive T cell trafficking to the lymph nodes. R-Ras is transiently up-regulated in the endothelium of high endothelial venules by the inflammatory cytokine tumor necrosis factor (TNF) within 24 hours of pathogen inoculation. TNF induces R-Ras upregulation in endothelial cells via JNK and p38 mitogen-activated protein kinase but not NF-κB. Studies of T cell trafficking found that the loss of function of endothelial R-Ras impairs the rapid acceleration of naive T cell recruitment to the lymph nodes upon inflammation. This defect diminished the ability of naive OT-1 T cells to develop antitumor activity against ovalbumin-expressing melanoma. Proteomic analyses suggest that endothelial R-Ras facilitates TNF-dependent transendothelial migration (diapedesis) of naive T cells by modulating molecular assembly the at T cell-endothelial cell interface. These findings give new mechanistic insights into the functional adaptation of high endothelial venules to accelerate naive T cell recruitment to the lymph nodes.
Assuntos
Quimiotaxia de Leucócito/fisiologia , Linfócitos T/imunologia , Migração Transendotelial e Transepitelial/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas ras/metabolismo , Animais , Células Endoteliais/metabolismo , Humanos , Linfonodos/irrigação sanguínea , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos , Linfócitos T/metabolismo , Regulação para Cima , Vênulas/imunologia , Vênulas/metabolismoRESUMO
A close association between pericytes and endothelial cells (ECs) is crucial to the stability and function of capillary blood vessels and microvessels. The loss or dysfunction of pericytes results in significant disruption of these blood vessels as observed in pathological conditions, including cancer, diabetes, stroke, and Alzheimer's disease. Prostaglandin E2 (PGE2) is a lipid mediator of inflammation, and its tissue concentration is elevated in cancer and neurological disorders. Here, we show that the exposure to PGE2 switches pericytes to a fast-migrating, loosely adhered phenotype that fails to intimately interact with ECs. N-cadherin and connexin-43 in adherens junction and gap junction between pericytes and ECs are downregulated by EP-4 and EP-1-dependent mechanisms, leading to breakdown of the pericyte-EC interaction. Furthermore, R-Ras, a small GTPase important for vascular normalization and vessel stability, is transcriptionally repressed by PGE2 in an EP4-dependent manner. Mouse dermal capillary vessels lose pericyte coverage substantially upon PGE2 injection into the skin. Our results suggest that EP-mediated direct disruption of pericytes by PGE2 is a key process for vascular destabilization. Restoring pericyte-EC interaction using inhibitors of PGE2 signaling may offer a therapeutic strategy in cancer and neurological disorders, in which pericyte dysfunction contributes to the disease progression.
Assuntos
Dinoprostona/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Pericitos/efeitos dos fármacos , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Junções Aderentes/metabolismo , Animais , Caderinas/metabolismo , Movimento Celular , Células Cultivadas , Conexina 43/metabolismo , Regulação para Baixo , Junções Comunicantes/metabolismo , Células HEK293 , Células HT29 , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Pericitos/metabolismo , Pericitos/fisiologia , Proteínas ras/metabolismoRESUMO
Plant aerial organs are coated with cuticular waxes, a hydrophobic layer that primarily serves as a waterproofing barrier. Cuticular wax is a mixture of aliphatic very-long-chain molecules, ranging from 22 to 48 carbons, produced in the endoplasmic reticulum of epidermal cells. Among all wax components, alkanes represent up to 80% of total wax in Arabidopsis (Arabidopsis thaliana) leaves. Odd-numbered alkanes and their derivatives are produced through the alkane-forming pathway. Although the chemical reactions of this pathway have been well described, the enzymatic mechanisms catalyzing these reactions remain unclear. We previously showed that a complex made of Arabidopsis ECERIFERUM1 (CER1) and CER3 catalyzes the conversion of acyl-Coenzyme A's to alkanes with strict substrate specificity for compounds containing more than 29 carbons. To learn more about alkane biosynthesis in Arabidopsis, we characterized the biochemical specificity and physiological functions of a CER1 homolog, CER1-LIKE1. In a yeast strain engineered to produce very-long-chain fatty acids, CER1-LIKE1 interacted with CER3 and cytochrome B5 to form a functional complex leading to the production of alkanes that are of different chain lengths compared to that produced by CER1-containing complexes. Gene expression analysis showed that both CER1 and CER1-LIKE1 are differentially expressed in an organ- and tissue-specific manner. Moreover, the inactivation or overexpression of CER1-LIKE1 in Arabidopsis transgenic lines specifically impacted alkane biosynthesis and wax crystallization. Collectively, our study reports on the identification of a further plant alkane synthesis enzymatic component and supports a model in which several alkane-forming complexes with distinct chain-length specificities coexist in plants.
Assuntos
Alcanos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Carbono-Carbono Liases , Regulação da Expressão Gênica de Plantas , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Folhas de Planta/genética , Folhas de Planta/metabolismo , Caules de Planta/genética , Caules de Planta/metabolismo , Plantas Geneticamente Modificadas , Saccharomyces cerevisiae/genética , Nicotiana/genética , Ceras/química , Ceras/metabolismoRESUMO
Obesity promotes a chronic inflammatory and hypercoagulable state that drives cardiovascular disease, type 2 diabetes, fatty liver disease, and several cancers. Elevated thrombin activity underlies obesity-linked thromboembolic events, but the mechanistic links between the thrombin/fibrin(ogen) axis and obesity-associated pathologies are incompletely understood. In this work, immunohistochemical studies identified extravascular fibrin deposits within white adipose tissue and liver as distinct features of mice fed a high-fat diet (HFD) as well as obese patients. Fibγ390-396A mice carrying a mutant form of fibrinogen incapable of binding leukocyte αMß2-integrin were protected from HFD-induced weight gain and elevated adiposity. Fibγ390-396A mice had markedly diminished systemic, adipose, and hepatic inflammation with reduced macrophage counts within white adipose tissue, as well as near-complete protection from development of fatty liver disease and glucose dysmetabolism. Homozygous thrombomodulin-mutant ThbdPro mice, which have elevated thrombin procoagulant function, gained more weight and developed exacerbated fatty liver disease when fed a HFD compared with WT mice. In contrast, treatment with dabigatran, a direct thrombin inhibitor, limited HFD-induced obesity development and suppressed progression of sequelae in mice with established obesity. Collectively, these data provide proof of concept that targeting thrombin or fibrin(ogen) may limit pathologies in obese patients.
Assuntos
Fibrina/metabolismo , Inflamação/metabolismo , Obesidade/metabolismo , Obesidade/terapia , Trombina/metabolismo , Tecido Adiposo/metabolismo , Adiposidade , Motivos de Aminoácidos , Animais , Glicemia/metabolismo , Composição Corporal , Peso Corporal , Coagulantes/farmacologia , Dabigatrana/farmacologia , Dieta Hiperlipídica , Fígado Gorduroso/metabolismo , Feminino , Genótipo , Homozigoto , Fígado/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Aumento de PesoRESUMO
Thrombin activates its G-coupled seven transmembrane protease-activated receptor (PAR-1) by cleaving the receptor's N-terminal end. In several human cancers, PAR1 expression and activation correlates with tumor progression and metastatization. This provides compelling evidence for the effectiveness of an appropriate antithrombin agent for the adjuvant treatment of patients with cancer. Dabigatran is a selective direct thrombin inhibitor that reversibly binds to thrombin. In this study, we aimed to explore if dabigatran may affect mechanisms favoring tumor growth by interfering with thrombin-induced PAR-1 activation. We confirmed that exposure of tumor cells to thrombin significantly increased cell proliferation and this was coupled with downregulation of p27 and concomitant induction of cyclin D1. Dabigatran was consistently effective in antagonizing thrombin-induced proliferation as well as it restored the baseline pattern of cell cycle protein expression. Thrombin significantly upregulated the expression of proangiogenetic proteins like Twist and GRO-α in human umbilical vascular endothelial cells (HUVEC) cells and their expression was significantly brought down to control levels when dabigatran was added to culture. We also found that the chemoattractant effect of thrombin on tumor cells was lost in the presence of dabigatran, and that the thrombin antagonist was effective in dampening vascular tube formation induced by thrombin. Our data support a role of thrombin in inducing the proliferation, migration, and proangiogenetic effects of tumor cells in vitro. Dabigatran has activity in antagonizing all these effects, thereby impairing tumor growth and progression. In vivo models may help to understand the relevance of this pathway.
Assuntos
Antitrombinas/farmacologia , Neoplasias Encefálicas/metabolismo , Dabigatrana/farmacologia , Glioblastoma/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Neoplasias Encefálicas/genética , Neoplasias da Mama , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/genética , Humanos , Trombina/antagonistas & inibidores , Trombina/farmacologiaRESUMO
OBJECTIVE: Dabigatran etexilate is a direct thrombin inhibitor approved for use in patients with non-valvular atrial fibrillation. There is no currently available pharmacological therapy to reverse this renally cleared anticoagulant. Dabigatran has a low level of plasma protein binding and has been considered dialyzable. We used a pig model with renal artery ligation to exclude intrinsic drug excretion to examine the efficacy of ultrafiltration (UF) during cardiopulmonary bypass (CPB) for dabigatran removal. METHOD: Dabigatran was intravenously infused (20 mg) in Yorkshire pigs (male, n=7, 70±1 kg) following renal artery ligation. CPB with UF was initiated after heparinization and continued until a total volume of 6 liters of UF effluent was removed. Serial labs, including dabigatran concentration, activated coagulation times (ACT), hematocrit and creatinine were drawn at intervals before the start of CPB and then incrementally during UF (0, 2, 4 and 6 L removed). Hemodialysis (HD) was performed on one animal following UF. RESULTS: Dabigatran concentration (ng/mL) rose from undetectable levels at baseline to 296±70 (p<0.05) at the conclusion of infusion, but dropped significantly upon administration of heparin (178±40, p<0.05). A further decrement in dabigatran concentration was observed from the administration of heparin to the start of CPB (to 135±28, p<0.05). Once on CPB, dabigatran remained stable, with the end UF (eUF) dabigatran concentration being 133±34. Dabigatran concentration in the UF effluent was measured in one animal and was 98.8, with 6 L of effluent having been removed. The total recovery of dabigatran was calculated to be less than 5%. Dabigatran concentrations also did not decrease appreciably with HD on CPB following UF. CONCLUSIONS: UF in conjunction with CPB was ineffective at removing dabigatran. Heparin demonstrated a dabigatran-lowering effect, suggesting a possible drug interaction or assay impairment. Based on these findings, emergent cardiac surgery with UF on cardiopulmonary bypass to remove dabigatran is not advisable. Alternative forms of drug removal or reversal must be identified.
Assuntos
Antitrombinas/sangue , Ponte Cardiopulmonar , Dabigatrana/sangue , Ultrafiltração , Animais , Dabigatrana/isolamento & purificação , Heparina/farmacologia , Masculino , Suínos , Tempo de Coagulação do Sangue TotalRESUMO
Cancer is often associated with an increased risk of thrombotic events which are exacerbated by treatment with chemotherapeutics such as cyclosphosphamide (CP). Evidence suggests that thrombin can stimulate tumor progression via formation of fibrin and activation of protease-activated receptors (PARs) and platelets. We examined the effect of co-treatment with CP and dabigatran etexilate, a direct inhibitor of thrombin, using the murine orthotopic 4T1 tumor model. Mice receiving co-treatment with both low dose CP and dabigatran etexilate had significantly smaller mammary tumors and fewer lung metastases than mice treated with CP or dabigratran etexilate alone. Co-treatment with dabigatran etexilate and low dose CP also significantly decreased the number of arginase(+)Gr-1(+)CD11b(+) myeloid derived suppressor cells as well as levels of TGF-ß in spleens from tumor bearing mice. 4T1 tumors express procoagulant tissue factor (TF) and spontaneously release TF(+) microparticles which are potent procoagulant factors that promote thrombin generation. Treatment with dabigatran etexilate alone prevented tumor-induced increases in circulating TF(+) microparticles and also decreased the numbers of tumor-induced activated platelets by 40%. These results show that co-treatment with dabigatran etexilate and CP synergistically inhibits growth and metastasis of mammary tumors, suggesting that oral administration of the thrombin inhibitor dabigatran etexilate may be beneficial in not only preventing thrombotic events in cancer patients but also in treating malignant tumors themselves.
Assuntos
Antineoplásicos Alquilantes/farmacologia , Antitrombinas/farmacologia , Ciclofosfamida/farmacologia , Neoplasias/patologia , Trombina/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Micropartículas Derivadas de Células/metabolismo , Dabigatrana/farmacologia , Modelos Animais de Doenças , Progressão da Doença , Sinergismo Farmacológico , Feminino , Camundongos , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Metástase Neoplásica , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/metabolismo , Ativação Plaquetária/efeitos dos fármacos , Tromboplastina/metabolismoRESUMO
Thrombin, the key effector protease of the coagulation cascade, drives fibrin deposition and activates human platelets through protease-activated receptor-1 (PAR1). These processes are critical to the progression of thrombotic diseases. Thrombin is the main target of anticoagulant therapy, and major efforts have led to the discovery of new oral direct inhibitors of thrombin. Dabigatran is the first oral anticoagulant licensed for the prevention of thromboembolisms associated with orthopedic surgery and stroke prevention in atrial fibrillation. Dabigatran is a direct thrombin inhibitor that effectively blocks thrombin's catalytic activity but does not preclude thrombin's exosites and binding to fibrinogen. Thus, we hypothesized that catalytically inactive thrombin retains the capacity to bind to PAR1 through exosite-I and may modulate its function independent of receptor cleavage and activation. Here, we report that dabigatran at clinically relevant concentrations is an effective and acute inhibitor of thrombin-induced PAR1 cleavage, activation, internalization, and ß-arrestin recruitment in vitro. Interestingly, prolonged exposure to catalytic inactive thrombin incubated with dabigatran at 20-fold higher therapeutic concentration resulted in increased PAR1 cell-surface expression, which correlated with higher detectable levels of ubiquitinated receptor. These findings are consistent with ubiquitin function as a negative regulator of PAR1 constitutive internalization. Increased PAR1 expression also enhanced agonist-induced phosphoinositide hydrolysis and endothelial barrier permeability. Thus, catalytically inactive thrombin appears to modulate PAR1 function in vitro by stabilizing receptor cell-surface expression; but given the high clearance rate of thrombin, the high concentration of dabigatran required to achieve this effect the in vivo physiologic relevance is unknown.
Assuntos
Antitrombinas/farmacologia , Arrestinas/metabolismo , Benzimidazóis/farmacologia , Receptor PAR-1/metabolismo , Trombina/metabolismo , beta-Alanina/análogos & derivados , Dabigatrana , Regulação da Expressão Gênica/efeitos dos fármacos , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos , beta-Alanina/farmacologia , beta-ArrestinasRESUMO
Thromboembolic disease is a major cause of mortality and morbidity in the developed world and is caused by an excessive stimulation of coagulation. Thrombin is a key serine protease in the coagulation cascade and numerous efforts have been made to develop safe and effective orally active direct thrombin inhibitors (DTIs). Current anticoagulant therapy includes the use of indirect thrombin inhibitors (e.g., heparins, low-molecular-weight-heparins) and vitamin K antagonists such as warfarin. However there are several caveats in the clinical use of these agents including narrow therapeutic window, parenteral delivery, and food- and drug-drug interactions. Dabigatran is a synthetic, reversible DTI with high affinity and specificity for its target binding both free and clot-bound thrombin, and offers a favorable pharmacokinetic profile. Large randomized clinical trials have demonstrated that dabigatran provides comparable or superior thromboprophylaxis in multiple thromboembolic disease indications compared to standard of care. This minireview will highlight the discovery and development of dabigatran, the first in a class of new oral anticoagulant agents to be licensed worldwide for the prevention of thromboembolism in the setting of orthopedic surgery and stroke prevent in atrial fibrillation.
RESUMO
Smooth muscle in the lung is thought to derive from the developing lung mesenchyme. Smooth muscle formation relies upon coordination of both autocrine and paracrine signaling between the budding epithelium and adjacent mesenchyme to govern its proliferation and differentiation. However, the pathways initiating the earliest aspects of smooth muscle specification and differentiation in the lung are poorly understood. Here, we identify the Wnt2 ligand as a critical regulator of the earliest aspects of lung airway smooth muscle development. Using Wnt2 loss and gain of function models, we show that Wnt2 signaling is necessary and sufficient for activation of a transcriptional and signaling network critical for smooth muscle specification and differentiation including myocardin/Mrtf-B and the signaling factor Fgf10. These studies place Wnt2 high in a hierarchy of signaling molecules that promote the earliest aspects of lung airway smooth muscle development.
Assuntos
Fator 10 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica , Pulmão/embriologia , Músculo Liso/embriologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética , Proteína Wnt2/fisiologia , Animais , Células Cultivadas , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Pulmão/metabolismo , Mesoderma/embriologia , Camundongos , Músculo Liso/metabolismo , Reação em Cadeia da Polimerase , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , beta Catenina/fisiologiaRESUMO
Although mutations in Kras are present in 21% of lung tumors, there is a high level of heterogeneity in phenotype and outcome among patients with lung cancer bearing similar mutations, suggesting that other pathways are important. Wnt/ß-catenin signaling is a known oncogenic pathway that plays a well-defined role in colon and skin cancer; however, its role in lung cancer is unclear. We have shown here that activation of Wnt/ß-catenin in the bronchiolar epithelium of the adult mouse lung does not itself promote tumor development. However, concurrent activation of Wnt/ß-catenin signaling and expression of a constitutively active Kras mutant (KrasG12D) led to a dramatic increase in both overall tumor number and size compared with KrasG12D alone. Activation of Wnt/ß-catenin signaling altered the KrasG12D tumor phenotype, resulting in a phenotypic switch from bronchiolar epithelium to the highly proliferative distal progenitors found in the embryonic lung. This was associated with decreased E-cadherin expression at the cell surface, which may underlie the increased metastasis of tumors with active Wnt/ß-catenin signaling. Together, these data suggest that activation of Wnt/ß-catenin signaling can combine with other oncogenic pathways in lung epithelium to produce a more aggressive tumor phenotype by imposing an embryonic distal progenitor phenotype and by decreasing E-cadherin expression.
Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , Células-Tronco/citologia , Proteínas Wnt/metabolismo , Animais , Brônquios/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Epiteliais/citologia , Humanos , Pulmão/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Fenótipo , Transdução de SinaisRESUMO
Little is understood about the molecular mechanisms underlying the morphogenesis of the posterior pole of the heart. Here we show that Wnt2 is expressed specifically in the developing inflow tract mesoderm, which generates portions of the atria and atrio-ventricular canal. Loss of Wnt2 results in defective development of the posterior pole of the heart, resulting in a phenotype resembling the human congenital heart syndrome complete common atrio-ventricular canal. The number and proliferation of posterior second heart field progenitors is reduced in Wnt2(-/-) mutants. Moreover, these defects can be rescued in a temporally restricted manner through pharmacological inhibition of Gsk-3beta. We also show that Wnt2 works in a feedforward transcriptional loop with Gata6 to regulate posterior cardiac development. These data reveal a molecular pathway regulating the posterior cardiac mesoderm and demonstrate that cardiovascular defects caused by loss of Wnt signaling can be rescued pharmacologically in vivo.
Assuntos
Coração Fetal/embriologia , Coração Fetal/fisiologia , Fator de Transcrição GATA6/fisiologia , Proteína Wnt2/fisiologia , Animais , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Feminino , Coração Fetal/efeitos dos fármacos , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase Quinase 3 beta , Cardiopatias Congênitas/embriologia , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/fisiopatologia , Humanos , Cloreto de Lítio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Mutantes , Modelos Cardiovasculares , Fenótipo , Gravidez , Transdução de Sinais , Proteína Wnt2/deficiência , Proteína Wnt2/genéticaRESUMO
Patterning of the primitive foregut promotes appropriate organ specification along its anterior-posterior axis. However, the molecular pathways specifying foregut endoderm progenitors are poorly understood. We show here that Wnt2/2b signaling is required to specify lung endoderm progenitors within the anterior foregut. Embryos lacking Wnt2/2b expression exhibit complete lung agenesis and do not express Nkx2.1, the earliest marker of the lung endoderm. In contrast, other foregut endoderm-derived organs, including the thyroid, liver, and pancreas, are correctly specified. The phenotype observed is recapitulated by an endoderm-restricted deletion of beta-catenin, demonstrating that Wnt2/2b signaling through the canonical Wnt pathway is required to specify lung endoderm progenitors within the foregut. Moreover, activation of canonical Wnt/beta-catenin signaling results in the reprogramming of esophagus and stomach endoderm to a lung endoderm progenitor fate. Together, these data reveal that canonical Wnt2/2b signaling is required for the specification of lung endoderm progenitors in the developing foregut.
Assuntos
Sistema Digestório , Pulmão , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Proteínas Wnt/metabolismo , Proteína Wnt2/metabolismo , beta Catenina/metabolismo , Animais , Padronização Corporal/fisiologia , Sistema Digestório/anatomia & histologia , Sistema Digestório/embriologia , Endoderma/citologia , Endoderma/fisiologia , Pulmão/anatomia & histologia , Pulmão/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco/citologia , Proteínas Wnt/genética , Proteína Wnt2/genética , beta Catenina/genéticaRESUMO
Epithelial organs, including the lung, are known to possess regenerative abilities through activation of endogenous stem cell populations, but the molecular pathways regulating stem cell expansion and regeneration are not well understood. Here we show that Gata6 regulates the temporal appearance and number of bronchioalveolar stem cells (BASCs) in the lung, its absence in Gata6-null lung epithelium leading to the precocious appearance of BASCs and concurrent loss in epithelial differentiation. This expansion of BASCs was the result of a pronounced increase in canonical Wnt signaling in lung epithelium upon loss of Gata6. Expression of the noncanonical Wnt receptor Fzd2 was downregulated in Gata6 mutants and increased Fzd2 or decreased beta-catenin expression rescued, in part, the lung epithelial defects in Gata6 mutants. During lung epithelial regeneration, canonical Wnt signaling was activated in the niche containing BASCs and forced activation of Wnt signaling led to a large increase in BASC numbers. Moreover, Gata6 was required for proper lung epithelial regeneration, and postnatal loss of Gata6 led to increased BASC expansion and decreased differentiation. Together, these data demonstrate that Gata6-regulated Wnt signaling controls the balance between progenitor expansion and epithelial differentiation required for both lung development and regeneration.
Assuntos
Fator de Transcrição GATA6/fisiologia , Regeneração/genética , Mucosa Respiratória/fisiologia , Células-Tronco/fisiologia , Proteínas Wnt/fisiologia , Animais , Proliferação de Células , Embrião de Mamíferos , Células Epiteliais/fisiologia , Receptores Frizzled/fisiologia , Fator de Transcrição GATA6/genética , Perfilação da Expressão Gênica , Pulmão/embriologia , Pulmão/metabolismo , Pulmão/fisiologia , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Receptores Acoplados a Proteínas G/fisiologia , Regeneração/fisiologia , Mucosa Respiratória/metabolismo , Fenômenos Fisiológicos Respiratórios , Transdução de Sinais/fisiologia , Células-Tronco/metabolismo , beta Catenina/antagonistas & inibidoresRESUMO
Parthenogenesis is the biological phenomenon by which embryonic development is initiated without male contribution. Whereas parthenogenesis is a common mode of reproduction in lower organisms, the mammalian parthenote fails to produce a successful pregnancy. We herein describe in vitro parthenogenetic development of monkey (Macaca fascicularis) eggs to the blastocyst stage, and their use to create a pluripotent line of stem cells. These monkey stem cells (Cyno-1 cells) are positive for telomerase activity and are immunoreactive for alkaline phosphatase, octamer-binding transcription factor 4 (Oct-4), stage-specific embryonic antigen 4 (SSEA-4), tumor rejection antigen 1-60 (TRA 1-60), and tumor rejection antigen 1-81 (TRA 1-81) (traditional markers of human embryonic stem cells). They have a normal chromosome karyotype (40 + 2) and can be maintained in vitro in an undifferentiated state for extended periods of time. Cyno-1 cells can be differentiated in vitro into dopaminergic and serotonergic neurons, contractile cardiomyocyte-like cells, smooth muscle, ciliated epithelia, and adipocytes. When Cyno-1 cells were injected into severe combined immunodeficient mice, teratomas with derivatives from all three embryonic germ layers were obtained. When grown on fibronectin/laminin-coated plates and in neural progenitor medium, Cyno-1 cells assume a neural precursor phenotype (immunoreactive for nestin). However, these cells remain proliferative and express no functional ion channels. When transferred to differentiation conditions, the nestin-positive precursors assume neuronal and epithelial morphologies. Over time, these cells acquire electrophysiological characteristics of functional neurons (appearance of tetrodotoxin-sensitive, voltage-dependent sodium channels). These results suggest that stem cells derived from the parthenogenetically activated nonhuman primate egg provide a potential source for autologous cell therapy in the female and bypass the need for creating a competent embryo.