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1.
Commun Biol ; 6(1): 935, 2023 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-37704701

RESUMO

A recently emerged sub-lineage of Omicron, BA.5, together with BA.4, caused a fifth wave of coronavirus disease (COVID-19) in South Africa and subsequently emerged as a predominant strain globally due to its high transmissibility. The lethality of BA.5 infection has not been studied in an acute hACE2 transgenic (hACE2.Tg) mouse model. Here, we investigated tissue-tropism and immuno-pathology induced by BA.5 infection in hACE2.Tg mice. Our data show that intranasal infection of BA.5 in hACE2.Tg mice resulted in attenuated pulmonary infection and pathology with diminished COVID-19-induced clinical and pathological manifestations. BA.5, similar to Omicron (B.1.1.529), infection led to attenuated production of inflammatory cytokines, anti-viral response and effector T cell response as compared to the ancestral strain of SARS-CoV-2, Wuhan-Hu-1. We show that mice recovered from B.1.1.529 infection showed robust protection against BA.5 infection associated with reduced lung viral load and pathology. Together, our data provide insights as to why BA.5 infection escapes previous SARS-CoV-2 exposure induced-T cell immunity but may result in milder immuno-pathology and alleviated chances of re-infectivity in Omicron-recovered individuals.


Assuntos
COVID-19 , Camundongos , Animais , Camundongos Transgênicos , SARS-CoV-2 , Citocinas , Modelos Animais de Doenças
2.
Res Sq ; 2023 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-36909571

RESUMO

Breast cancer continues to be a major cause of death among women. The GATA3 gene is often overexpressed in breast cancer and is widely used to support a diagnosis. However, lower expression of GATA3 has been linked to poorer prognosis along with frequent gene mutations. Therefore, the role of GATA3 in breast cancer appears to be context specific. This study aims to identify a new downstream target of GATA3 to better understand its regulatory network. Clinical data analysis identified the prolyl 4-hydroxylase transmembrane protein (P4HTM) as one of the most highly co-expressed genes with GATA3. Immunohistochemical staining of breast tumors confirms co-expression between GATA3 and P4HTM at the protein level. Similar to GATA3, P4HTM expression levels are linked to patient prognosis, with lower levels indicating poorer survival. Genomics data found that GATA3 binds to the P4HTM locus, and that ectopic expression of GATA3 in basal breast cancer cells increases the P4HTM transcript level. These results collectively suggest that P4HTM is a novel downstream target of GATA3 in breast cancer and is involved in tumor progression.

3.
JCI Insight ; 3(11)2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29875324

RESUMO

Metabolic syndrome contributes to cardiovascular disease partly through systemic risk factors. However, local processes in the artery wall are becoming increasingly recognized to exacerbate atherosclerosis both in mice and humans. We show that arterial smooth muscle cell (SMC) glucose metabolism markedly synergizes with metabolic syndrome in accelerating atherosclerosis progression, using a low-density lipoprotein receptor-deficient mouse model. SMCs in proximity to atherosclerotic lesions express increased levels of the glucose transporter GLUT1. Cytokines, such as TNF-α produced by lesioned arteries, promote GLUT1 expression in SMCs, which in turn increases expression of the chemokine CCL2 through increased glycolysis and the polyol pathway. Furthermore, overexpression of GLUT1 in SMCs, but not in myeloid cells, accelerates development of larger, more advanced lesions in a mouse model of metabolic syndrome, which also exhibits elevated levels of circulating Ly6Chi monocytes expressing the CCL2 receptor CCR2. Accordingly, monocyte tracing experiments demonstrate that targeted SMC GLUT1 overexpression promotes Ly6Chi monocyte recruitment to lesions. Strikingly, SMC-targeted GLUT1 overexpression fails to accelerate atherosclerosis in mice that do not exhibit the metabolic syndrome phenotype or monocytosis. These results reveal a potentially novel mechanism whereby arterial smooth muscle glucose metabolism synergizes with metabolic syndrome to accelerate monocyte recruitment and atherosclerosis progression.


Assuntos
Aterosclerose/imunologia , Transportador de Glucose Tipo 1/metabolismo , Glicólise/imunologia , Síndrome Metabólica/complicações , Monócitos/imunologia , Animais , Artérias/citologia , Artérias/imunologia , Artérias/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Dicarbetoxi-Di-Hidrocolidina/administração & dosagem , Modelos Animais de Doenças , Progressão da Doença , Feminino , Glucose/metabolismo , Transportador de Glucose Tipo 1/genética , Humanos , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/imunologia , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/citologia , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Receptores de LDL/genética
4.
ACS Chem Biol ; 10(10): 2246-56, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26158404

RESUMO

Bromodomain-containing protein dysregulation is linked to cancer, diabetes, and inflammation. Selective inhibition of bromodomain function is a newly proposed therapeutic strategy. We describe a (19)F NMR dual screening method for small molecule discovery using fluorinated tryptophan resonances on two bromodomain-containing proteins. The chemical shift dispersion of (19)F resonances within fluorine-labeled proteins enables the simultaneous analysis of two fluorinated bromodomains by NMR. A library of 229 small molecules was screened against the first bromodomain of Brd4 and the BPTF bromodomain. We report the first small molecule selective for BPTF over Brd4, termed AU1. The Kd = 2.8 µM for AU1, which is active in a cell-based reporter assay. No binding is detected with Brd4. Three new Brd4 inhibitors with submicromolar affinity were also discovered. Brd4 hits were validated in a thermal stability assay and potency determined via fluorescence anisotropy. The speed, ease of interpretation, and low protein concentration needed for protein-observed (19)F NMR experiments in a multiprotein format offers a new method to discover and characterize selective ligands for bromodomain-containing proteins.


Assuntos
Antígenos Nucleares/química , Proteínas do Tecido Nervoso/química , Proteínas Nucleares/química , Fatores de Transcrição/química , Ligação Competitiva , Proteínas de Ciclo Celular , Linhagem Celular , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Flúor/química , Humanos , Concentração Inibidora 50 , Imageamento por Ressonância Magnética , Estrutura Molecular , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Temperatura
5.
J Cardiovasc Transl Res ; 8(3): 158-63, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25788147

RESUMO

Vascular smooth muscle cells (VSMCs) play important roles in cardiovascular disorders and biology. Outlined in this paper is a step-by-step procedure for isolating aortic VSMCs from adult C57BL6J male mice by enzymatic digestion of the aorta using collagenase. The plating, culturing, and subculturing of the isolated cells are discussed in detail along with techniques to characterize VSMC phenotype by gene expression and immunofluorescence. Traction force microscopy was used to characterize contractility of single subcultured VSMCs at baseline.


Assuntos
Técnicas de Cultura de Células/normas , Separação Celular/normas , Músculo Liso Vascular/fisiologia , Miócitos de Músculo Liso/fisiologia , Actinas/genética , Actinas/metabolismo , Animais , Aorta/citologia , Aorta/metabolismo , Aorta/fisiologia , Biomarcadores/metabolismo , Proliferação de Células , Separação Celular/métodos , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Regulação da Expressão Gênica , Masculino , Camundongos Endogâmicos C57BL , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fenótipo , Vasoconstrição
6.
Circ Cardiovasc Genet ; 8(2): 294-304, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25628389

RESUMO

BACKGROUND: Genome-wide association studies are powerful tools for nominating pathogenic variants, but offer little insight as to how candidate genes affect disease outcome. Such is the case for SH2B adaptor protein 3 (SH2B3), which is a negative regulator of multiple cytokine signaling pathways and is associated with increased risk of myocardial infarction (MI), but its role in post-MI inflammation and fibrosis is completely unknown. METHODS AND RESULTS: Using an experimental model of MI (left anterior descending artery occlusion/reperfusion injury) in wild-type and Sh2b3 knockout rats (Sh2b3(em2Mcwi)), we assessed the role of Sh2b3 in post-MI fibrosis, leukocyte infiltration, angiogenesis, left ventricle contractility, and inflammatory gene expression. Compared with wild-type, Sh2b3(em2Mcwi) rats had significantly increased fibrosis (2.2-fold; P<0.05) and elevated leukocyte infiltration (>2-fold; P<0.05), which coincided with decreased left ventricle fractional shortening (-Δ11%; P<0.05) at 7 days post left anterior descending artery occlusion/reperfusion injury. Despite an increased angiogenic potential in Sh2b3(em2Mcwi) rats (1.7-fold; P<0.05), we observed no significant differences in left ventricle capillary density between wild-type and Sh2b3(em2Mcwi) rats. In total, 12 genes were significantly elevated in the post left anterior descending artery occluded/reperfused hearts of Sh2b3(em2Mcwi) rats relative to wild-type, of which 3 (NLRP12, CCR2, and IFNγ) were significantly elevated in the left ventricle of heart failure patients carrying the MI-associated rs3184504 [T] SH2B3 risk allele. CONCLUSIONS: These data demonstrate for the first time that SH2B3 is a crucial mediator of post-MI inflammation and fibrosis.


Assuntos
Proteínas Musculares/metabolismo , Infarto do Miocárdio/metabolismo , Miocardite/metabolismo , Proteínas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Modelos Animais de Doenças , Fibrose , Proteínas Musculares/genética , Infarto do Miocárdio/complicações , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Miocardite/etiologia , Miocardite/genética , Miocardite/patologia , Proteínas/genética , Ratos , Ratos Mutantes
7.
J Mol Cell Cardiol ; 79: 287-94, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25528964

RESUMO

Atherosclerosis, a syndrome with abnormal arterial walls, is one of the major causes that lead to the development of various cardiovascular diseases. The key initiator of atherosclerosis is cholesterol accumulation. The uncontrolled cholesterol deposition, mainly involving low-density lipoprotein (LDL), causes atheroma plaque formation, which initiates chronic inflammation due to the recruitment of inflammatory cells such as macrophages. Macrophages scavenge excess peripheral cholesterol and transport intracellular cholesterol to high-density lipoprotein (HDL) for excretion or storage. Cholesterol-laden macrophage-derived foam cell formation is the main cause of atherogenesis. It is critical to understand the regulatory mechanism of cholesterol homeostasis in the macrophage in order to prevent foam cells formation and further develop novel therapeutic strategies against atherosclerosis. Here we identified a protein, RIP140 (receptor interacting protein 140), which enhances macrophage-derived foam cell formation by reducing expression of reverse cholesterol transport genes, A TP-binding membrane cassette transporter A-1 (ABCA1) and ATP-binding membrane cassette transporter G-1 (ABCG1). In animal models, we found that reducing RIP140 levels by crossing macrophage-specific RIP140 knockdown (MϕRIP140KD) mice with ApoE null mice effectively ameliorates high-cholesterol diet-induced atherosclerosis. Our data suggest that reducing RIP140 levels in macrophages significantly inhibits atherosclerosis, along with markers of inflammation and the number of macrophages in a western diet fed ApoE null mouse. This study provides a proof-of-concept for RIP140 as a risk biomarker of, and a therapeutic target for, atherosclerosis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Aterosclerose/metabolismo , Aterosclerose/patologia , Colesterol/metabolismo , Células Espumosas/metabolismo , Homeostase , Proteínas Nucleares/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Acetilação/efeitos dos fármacos , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Transporte Biológico/efeitos dos fármacos , Dieta Ocidental , Células Espumosas/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Homeostase/efeitos dos fármacos , Lipoproteínas LDL/farmacologia , Receptores X do Fígado , Lisina/metabolismo , Camundongos Transgênicos , Proteína 1 de Interação com Receptor Nuclear , Receptores Nucleares Órfãos/metabolismo
8.
PLoS One ; 9(7): e101509, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25033200

RESUMO

RATIONALE: The rationale was to utilize a bioinformatics approach to identify miRNA binding sites in genes with single nucleotide mutations (SNPs) to discover pathways in heart failure (HF). OBJECTIVE: The objective was to focus on the genes containing miRNA binding sites with miRNAs that were significantly altered in end-stage HF and in response to a left ventricular assist device (LVAD). METHODS AND RESULTS: BEDTools v2.14.3 was used to discriminate SNPs within predicted 3'UTR miRNA binding sites. A member of the miR-15/107 family, miR-16, was decreased in the circulation of end-stage HF patients and increased in response to a LVAD (p<0.001). MiR-16 decreased Vacuolar Protein Sorting 4a (VPS4a) expression in HEK 293T cells (p<0.01). The SNP rs16958754 was identified in the miR-15/107 family binding site of VPS4a which abolished direct binding of miR-16 to the 3'UTR of VPS4a (p<0.05). VPS4a was increased in the circulation of end-stage HF patients (p<0.001), and led to a decrease in the number of HEK 293T cells in vitro (p<0.001). CONCLUSIONS: We provide evidence that miR-16 decreases in the circulation of end-stage HF patients and increases with a LVAD. Modeling studies suggest that miR-16 binds to and decreases expression of VPS4a. Overexpression of VPS4a decreases cell number. Together, these experiments suggest that miR-16 and VPS4a expression are altered in end-stage HF and in response to unloading with a LVAD. This signaling pathway may lead to reduced circulating cell number in HF.


Assuntos
Regiões 3' não Traduzidas/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Insuficiência Cardíaca/genética , MicroRNAs/genética , ATPases Vacuolares Próton-Translocadoras/genética , ATPases Associadas a Diversas Atividades Celulares , Idoso , Sítios de Ligação/genética , Linhagem Celular , Complexos Endossomais de Distribuição Requeridos para Transporte/biossíntese , Complexos Endossomais de Distribuição Requeridos para Transporte/sangue , Feminino , Células HEK293 , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/terapia , Coração Auxiliar , Humanos , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Miocárdio/patologia , Polimorfismo de Nucleotídeo Único , ATPases Vacuolares Próton-Translocadoras/biossíntese , ATPases Vacuolares Próton-Translocadoras/sangue
9.
Mol Cell Biochem ; 385(1-2): 225-38, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24101444

RESUMO

Heparan sulfate proteoglycans act as co-receptors for many chemokines and growth factors. The sulfation pattern of the heparan sulfate chains is a critical regulatory step affecting the binding of chemokines and growth factors. N-deacetylase-N-sulfotransferase1 (Ndst1) is one of the first enzymes to catalyze sulfation. Previously published work has shown that HSPGs alter tangent moduli and stiffness of tissues and cells. We hypothesized that loss of Ndst1 in smooth muscle would lead to significant changes in heparan sulfate modification and the elastic properties of arteries. In line with this hypothesis, the axial tangent modulus was significantly decreased in aorta from mice lacking Ndst1 in smooth muscle (SM22αcre(+)Ndst1(-/-), p < 0.05, n = 5). The decrease in axial tangent modulus was associated with a significant switch in myosin and actin types and isoforms expressed in aorta and isolated aortic vascular smooth muscle cells. In contrast, no changes were found in the compliance of smaller thoracodorsal arteries of SM22αcre(+)Ndst1(-/-) mice. In summary, the major findings of this study were that targeted ablation of Ndst1 in smooth muscle cells results in altered biomechanical properties of aorta and differential expression of myosin and actin types and isoforms.


Assuntos
Deleção de Genes , Músculo Liso Vascular/fisiopatologia , Sulfotransferases/deficiência , Animais , Artérias/fisiopatologia , Fenômenos Biomecânicos , Complacência (Medida de Distensibilidade) , Regulação para Baixo/genética , Técnicas In Vitro , Camundongos , Proteínas dos Microfilamentos/metabolismo , Proteínas Musculares/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Reprodutibilidade dos Testes , Coloração e Rotulagem , Estresse Mecânico , Sulfotransferases/metabolismo , Regulação para Cima/genética , Vasoconstrição
10.
PLoS One ; 8(10): e77951, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24205042

RESUMO

INTRODUCTION: Continuous-flow left ventricular assist devices (LVADs) are an established therapy for patients with end-stage heart failure. The short- and long-term impact of these devices on peripheral blood gene expression has not been characterized, and may provide insight into the molecular pathways mediated in response to left ventricular remodeling and an improvement in overall systemic circulation. We performed RNA sequencing to identify genes and pathways influenced by these devices. METHODS: RNA was extracted from blood of 9 heart failure patients (8 male) prior to LVAD implantation, and at 7 and 180 days postoperatively. Libraries were sequenced on an Illumina HiSeq2000 and sequences mapped to the human Ensembl GRCh37.67 genome assembly. RESULTS: A specific set of genes involved in regulating cellular immune response, antigen presentation, and T cell activation and survival were down-regulated 7 days after LVAD placement. 6 months following LVAD placement, the expression levels of these genes were significantly increased; yet importantly, remained significantly lower than age and sex-matched samples from healthy controls. CONCLUSIONS: In summary, this genomic analysis identified a significant decrease in the expression of genes that promote a healthy immune response in patients with heart failure that was partially restored 6 months following LVAD implant.


Assuntos
Biomarcadores/sangue , Perfilação da Expressão Gênica , Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/genética , Transplante de Coração/efeitos adversos , Coração Auxiliar/efeitos adversos , Idoso , Western Blotting , Feminino , Insuficiência Cardíaca/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo
11.
J Cardiovasc Transl Res ; 5(3): 274-9, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22555965

RESUMO

Heparan sulfate proteoglycans are abundant matrix and membrane molecules. Smooth muscle specific deletion of one heparan sulfate biosynthetic enzyme, N-deacetylase-N-sulfotransferase1 leads to decreased vascular smooth muscle cell proliferation, and vascular wall thickness. We hypothesized that this may lead to changes in blood pressure in conscious mice. Blood pressure was measured via telemetry in SM22αCre(+)Ndst1(-/-)(n = 4) and wild type (n = 8) mice. Aorta and thoracodorsal artery luminal area is significantly smaller in SM22αCre(+)Ndst1(-/-) (n = 4-8, P = 0.02, P = 0.0002) compared to wild type (n = 7) mice. Diurnal differences were observed in both cohorts for systolic, diastolic, mean arterial blood pressure, and heart rate (P < 0.001 from T test). No significant differences were found in the above parameters between the cohorts in either light or dark times using a linear mixed model. In conclusion, deletion of N-deacetylase-N-sulfotransferase1 in smooth muscle did not influence any of the blood pressure parameters measured despite significant decrease in aorta and thoracodorsal artery luminal area.


Assuntos
Pressão Sanguínea , Estado de Consciência , Deleção de Genes , Músculo Liso Vascular/enzimologia , Sulfotransferases/deficiência , Animais , Aorta/enzimologia , Monitorização Ambulatorial da Pressão Arterial , Ritmo Circadiano , Genótipo , Frequência Cardíaca , Integrases/genética , Modelos Lineares , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Fenótipo , Sulfotransferases/genética , Telemetria , Artérias Torácicas/enzimologia , Fatores de Tempo
12.
J Cardiovasc Transl Res ; 4(3): 313-20, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21468773

RESUMO

Proteoglycan core proteins are linked to four different classes of linear sugar chains referred to as glycosaminoglycans. Heparan sulfate constitutes one of these classes of glycosaminoglycans, and has been shown to be important in developmental processes as well as disease. We designed a low-density gene expression array to identify expression levels of heparan sulfate biosynthetic enzymes and proteoglycan core proteins in the aorta of late stage embryos (E18.5) and adult mice (12 weeks). Significant changes were found in mRNA expression of proteoglycan core proteins syndecan, glypican, decorin, perlecan, and versican from development to adulthood (n = 8, p < 0.05). Immunohistochemistry revealed a striking localization of both decorin and perlecan staining to the subendothelium in adult vessels, which differed from consistent staining of the endothelium, smooth muscle, and adventitia in development. Significant differences were also identified in the expression of the heparan sulfate modifying enzymes, glururonyl C5 epimerase, 2-O and 6-O sulfotransferases, and N-deacetylase/N sulfotransferases 1-3 (n = 8, p < 0.05). In conclusion, proteoglycan core proteins and heparan sulfate biosynthetic enzymes in the aorta undergo significant changes in their expression from development to adulthood. These findings may have important biological significance in the specific cell-defined roles of proteoglycan and heparan sulfate related targets in vascular development, maintenance, and response to various perturbations.


Assuntos
Envelhecimento/metabolismo , Aorta/enzimologia , Proteoglicanas de Heparan Sulfato/biossíntese , Proteoglicanas/biossíntese , Fatores Etários , Envelhecimento/genética , Análise de Variância , Animais , Aorta/embriologia , Aorta/crescimento & desenvolvimento , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica no Desenvolvimento , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , Proteoglicanas de Heparan Sulfato/genética , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Proteoglicanas/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa
13.
Arterioscler Thromb Vasc Biol ; 31(1): 86-94, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20947823

RESUMO

OBJECTIVE: The goal of this study was to test the contributing role of increasing glucose uptake in vascular smooth muscle cells (VSMCs) in vascular complications and disease. METHODS AND RESULTS: A murine genetic model was established in which glucose trasporter 1 (GLUT1), the non-insulin-dependent glucose transporter protein, was overexpressed in smooth muscle using the sm22α promoter. Overexpression of GLUT1 in smooth muscle led to significant increases in glucose uptake (n=3, P<0.0001) as measured using radiolabeled 2-deoxyglucose. Fasting blood glucose, insulin, and nonesterified fatty acids were unchanged. Contractility in aortic ring segments was decreased in sm22α-GLUT1 mice (n=10, P<0.04). In response to vascular injury, sm22α-GLUT1 mice exhibited a proinflammatory phenotype, including a significant increase in the percentage of neutrophils in the lesion (n=4, P<0.04) and an increase in monocyte chemoattractant protein-1 (MCP-1) immunofluorescence. Circulating haptoglobin and glutathione/total glutathione were significantly higher in the sm22α-GLUT1 mice postinjury compared with controls (n=4, P<0.05), suggesting increased flux through the pentose phosphate pathway. sm22α-GLUT1 mice exhibited significant medial hypertrophy following injury that was associated with a significant increase in the percentage of VSMCs in the media staining positive for nuclear phosphoSMAD2/3 (n=4, P<0.003). CONCLUSIONS: In summary, these findings suggest that increased glucose uptake in VSMCs impairs vascular contractility and accelerates a proinflammatory, neutrophil-rich lesion in response to injury, as well as medial hypertrophy, which is associated with enhanced transforming growth factor-ß activity.


Assuntos
Transportador de Glucose Tipo 1/metabolismo , Inflamação/etiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição , Animais , Aorta/metabolismo , Aorta/fisiopatologia , Glicemia/metabolismo , Células Cultivadas , Quimiocina CCL2/metabolismo , Desoxiglucose/metabolismo , Modelos Animais de Doenças , Ácidos Graxos não Esterificados/sangue , Artéria Femoral/lesões , Artéria Femoral/metabolismo , Transportador de Glucose Tipo 1/genética , Glutationa/sangue , Haptoglobinas/metabolismo , Humanos , Hipertrofia , Inflamação/metabolismo , Inflamação/patologia , Insulina/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas dos Microfilamentos/genética , Proteínas Musculares/genética , Músculo Liso Vascular/lesões , Músculo Liso Vascular/patologia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/patologia , Infiltração de Neutrófilos , Fosforilação , Regiões Promotoras Genéticas , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Regulação para Cima
14.
J Clin Invest ; 120(11): 4141-54, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20972335

RESUMO

Adaptive changes to oxygen availability are critical for cell survival and tissue homeostasis. Prolonged oxygen deprivation due to reduced blood flow to cardiac or peripheral tissues can lead to myocardial infarction and peripheral vascular disease, respectively. Mammalian cells respond to hypoxia by modulating oxygen-sensing transducers that stabilize the transcription factor hypoxia-inducible factor 1α (HIF-1α), which transactivates genes governing angiogenesis and metabolic pathways. Oxygen-dependent changes in HIF-1α levels are regulated by proline hydroxylation and proteasomal degradation. Here we provide evidence for what we believe is a novel mechanism regulating HIF-1α levels in isolated human ECs during hypoxia. Hypoxia differentially increased microRNA-424 (miR-424) levels in ECs. miR-424 targeted cullin 2 (CUL2), a scaffolding protein critical to the assembly of the ubiquitin ligase system, thereby stabilizing HIF-α isoforms. Hypoxia-induced miR-424 was regulated by PU.1-dependent transactivation. PU.1 levels were increased in hypoxic endothelium by RUNX-1 and C/EBPα. Furthermore, miR-424 promoted angiogenesis in vitro and in mice, which was blocked by a specific morpholino. The rodent homolog of human miR-424, mu-miR-322, was significantly upregulated in parallel with HIF-1α in experimental models of ischemia. These results suggest that miR-322/424 plays an important physiological role in post-ischemic vascular remodeling and angiogenesis.


Assuntos
Células Endoteliais/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Hipóxia/metabolismo , MicroRNAs/metabolismo , Neovascularização Fisiológica , Isoformas de Proteínas/metabolismo , Animais , Linhagem Celular , Células Endoteliais/citologia , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Camundongos , Camundongos Nus , MicroRNAs/genética , Análise em Microsséries , Oxigênio/metabolismo , Isoformas de Proteínas/genética , Ratos , Ratos Sprague-Dawley
15.
Mol Cell Biochem ; 342(1-2): 57-62, 2010 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-20461448

RESUMO

The mitogen activated protein kinase (MAPK) signaling pathway regulates multiple events leading to heart failure including ventricular remodeling, contractility, hypertrophy, apoptosis, and fibrosis. The regulation of conserved intrinsic inhibitors of this pathway is poorly understood. We recently identified an up-regulation of Sprouty1 (Spry1) in a targeted approach for novel inhibitors of the MAPK signaling pathway in failing human hearts following reverse remodeling. The goal of this study was to test the hypothesis that up-regulated expression of Spry1 in cardiac myocytes would be sufficient to inhibit ERK1/2 activation and tissue remodeling. We established a murine model with up-regulated Spry1 expression in cardiac myocytes using the alpha-myosin heavy chain promoter (alpha-MHC). Heart weight and cardiac myocyte morphology were unchanged in adult male alpha-MHC-Spry1 mice compared to control mice. Ventricular function of alpha-MHC-Spry1 mice was unaltered at 8 weeks or 1 year of age. These findings were consistent with the lack of an effect of Spry1 on ERK1/2 activity. In summary, targeted up-regulation of Spry1 in cardiac myocytes is not sufficient to alter cell or tissue remodeling consistent with the lack of an effect on ERK1/2 activity.


Assuntos
Proteínas de Membrana/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Miocárdio/citologia , Miócitos Cardíacos/metabolismo , Fosfoproteínas/fisiologia , Remodelação Ventricular/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Animais , Apoptose , Western Blotting , Feminino , Expressão Gênica/fisiologia , Coração/crescimento & desenvolvimento , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/genética , Miocárdio/metabolismo , Miócitos Cardíacos/citologia , Cadeias Pesadas de Miosina/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Regulação para Cima
16.
J Mol Cell Cardiol ; 49(2): 287-93, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20206635

RESUMO

Heparan sulfate proteoglycans are abundant molecules in the extracellular matrix and at the cell surface. Heparan sulfate chains are composed of groups of disaccharides whose side chains are modified through a series of enzymatic reactions. Deletion of these enzymes alters heparan sulfate fine structure and leads to changes in cell proliferation and tissue development. The role of heparan sulfate modification has not been explored in the vessel wall. The goal of this study was to test the hypothesis that altering heparan sulfate fine structure would impact vascular smooth muscle cell (VSMC) proliferation, vessel structure, and remodeling in response to injury. A heparan sulfate modifying enzyme, N-deacetylase N-sulfotransferase1 (Ndst1) was deleted in smooth muscle resulting in decreased N- and 2-O sulfation of the heparan sulfate chains. Smooth muscle specific deletion of Ndst1 led to a decrease in proliferating VSMCs and the circumference of the femoral artery in neonatal and adult mice. In response to vascular injury, mice lacking Ndst1 exhibited a significant reduction in lesion formation. Taken together, these data provide new evidence that modification of heparan sulfate fine structure through deletion of Ndst1 is sufficient to decrease VSMC proliferation and alter vascular remodeling.


Assuntos
Vasos Sanguíneos/patologia , Vasos Sanguíneos/fisiopatologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/enzimologia , Miócitos de Músculo Liso/patologia , Sulfotransferases/deficiência , Envelhecimento/patologia , Animais , Animais Recém-Nascidos , Vasos Sanguíneos/enzimologia , Proliferação de Células , Artéria Femoral/enzimologia , Artéria Femoral/patologia , Deleção de Genes , Testes de Função Cardíaca , Heparitina Sulfato/metabolismo , Camundongos , Tamanho do Órgão , Sulfotransferases/metabolismo , Túnica Íntima/enzimologia , Túnica Íntima/patologia , Túnica Íntima/fisiopatologia , Túnica Média/enzimologia , Túnica Média/patologia , Túnica Média/fisiopatologia
17.
Mol Cell Biochem ; 338(1-2): 255-61, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20054616

RESUMO

Sprouty1 (Spry1) is a conserved antagonist of FGF signaling. The goal of this study was to further explore the downstream mechanisms governing Spry1 inhibition of endothelial cell proliferation. Up-regulation of Spry1 in HUVECs inhibited tube formation on Matrigel (n = 6, P < 0.001). This was associated with decreased proliferation as measured by BrdU incorporation (n = 6, P < 0.001) and increased protein expression of the cyclin-dependent kinase inhibitor 1A (CDKN1A), p21 and cyclin-dependent kinase inhibitor 1B (CDKN1B), p27. A transcriptional analysis using a targeted human angiogenesis array following up-regulation of Spry1 demonstrated a >2-fold increase in an anti-angiogenic factor, serpin peptidase inhibitor, clad F (Serpinf1), and a >2-fold decrease in pro-angiogenic factors fms-related tyrosine kinase 1 (FLT1), angiopoietin2 (Ang-2), and placental growth factor (PGF) (n = 2). To define upstream mechanisms that may regulate endogenous Spry1, we performed a search for responsive elements upstream of the promoter region. This search resulted in the identification of multiple degenerate hypoxia responsive elements. Exposure to hypoxia resulted in a significant increase in Spry1 expression (n = 8, P < 0.01). These findings shed new light on downstream signaling pathways associated with Spry1 anti-proliferative responses, and provide new evidence that hypoxia stimulates Spry1 expression.


Assuntos
Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Proteínas de Membrana/metabolismo , Neovascularização Fisiológica , Fosfoproteínas/metabolismo , Proliferação de Células , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p27/genética , Células Endoteliais/citologia , Células Endoteliais/fisiologia , Humanos , Hipóxia/metabolismo , Proteínas de Membrana/genética , Fosfoproteínas/genética , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Regulação para Cima
18.
Hum Mol Genet ; 18(20): 3795-804, 2009 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-19602480

RESUMO

Common variants in the transcription factor 7-like 2 (TCF7L2) gene have been identified as the strongest genetic risk factors for type 2 diabetes (T2D). However, the mechanisms by which these non-coding variants increase risk for T2D are not well-established. We used 13 expression assays to survey mRNA expression of multiple TCF7L2 splicing forms in up to 380 samples from eight types of human tissue (pancreas, pancreatic islets, colon, liver, monocytes, skeletal muscle, subcutaneous adipose tissue and lymphoblastoid cell lines) and observed a tissue-specific pattern of alternative splicing. We tested whether the expression of TCF7L2 splicing forms was associated with single nucleotide polymorphisms (SNPs), rs7903146 and rs12255372, located within introns 3 and 4 of the gene and most strongly associated with T2D. Expression of two splicing forms was lower in pancreatic islets with increasing counts of T2D-associated alleles of the SNPs: a ubiquitous splicing form (P = 0.018 for rs7903146 and P = 0.020 for rs12255372) and a splicing form found in pancreatic islets, pancreas and colon but not in other tissues tested here (P = 0.009 for rs12255372 and P = 0.053 for rs7903146). Expression of this form in glucose-stimulated pancreatic islets correlated with expression of proinsulin (r(2) = 0.84-0.90, P < 0.00063). In summary, we identified a tissue-specific pattern of alternative splicing of TCF7L2. After adjustment for multiple tests, no association between expression of TCF7L2 in eight types of human tissue samples and T2D-associated genetic variants remained significant. Alternative splicing of TCF7L2 in pancreatic islets warrants future studies. GenBank Accession Numbers: FJ010164-FJ010174.


Assuntos
Processamento Alternativo , Diabetes Mellitus Tipo 2/genética , Especificidade de Órgãos , Fatores de Transcrição TCF/genética , Adolescente , Adulto , Linhagem Celular , Colo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Feminino , Expressão Gênica , Humanos , Ilhotas Pancreáticas/metabolismo , Fígado/metabolismo , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Pâncreas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição TCF/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição , Adulto Jovem
19.
J Cardiovasc Transl Res ; 2(2): 182-90, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20559986

RESUMO

The purpose of this study was to determine the effects of chronic treatment with the beta 2 adrenergic receptor agonist clenbuterol on endothelial progenitor cells (EPC) in a well-characterized model of heart failure, the muscle LIM protein knockout (MLP(-/-)) mouse. MLP(-/-) mice were treated daily with clenbuterol (2 mg/kg) or saline subcutaneously for 6 weeks. Clenbuterol led to a 30% increase in CD31(+) cells in the bone marrow of MLP(-/-) heart failure mice (p < 0.004). Clenbuterol did not improve ejection fraction. Clenbuterol treatment in MLP(-/-) mice was associated with significant changes in the following circulating factors: tissue inhibitor of metalloproteinase-type 1, leukemia inhibitory factor 1, C-reactive protein, apolipoprotein A1, fibroblast growth factor 2, serum glutamic oxaloacetic transaminase, macrophage-derived chemokine, and monocyte chemoattractant protein-3. Clenbuterol treatment in the MLP(-/-) model of heart failure did not rescue heart function, yet did increase CD31(+) cells in the bone marrow. This is the first evidence that a beta 2 agonist increases EPC proliferation in the bone marrow in a preclinical model of heart failure.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2 , Agonistas Adrenérgicos beta/farmacologia , Biomarcadores/sangue , Cardiomiopatias/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Clembuterol/farmacologia , Células Endoteliais/efeitos dos fármacos , Miocárdio/patologia , Células-Tronco/efeitos dos fármacos , Agonistas Adrenérgicos beta/administração & dosagem , Animais , Apolipoproteína A-I/sangue , Aspartato Aminotransferases/sangue , Proteína C-Reativa/metabolismo , Carboxipeptidases A/sangue , Cardiomiopatias/sangue , Cardiomiopatias/genética , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Quimiocina CCL22/sangue , Clembuterol/administração & dosagem , Modelos Animais de Doenças , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Fator 2 de Crescimento de Fibroblastos/sangue , Regulação da Expressão Gênica , Injeções Subcutâneas , Proteínas com Domínio LIM , Fator Inibidor de Leucemia/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Miocárdio/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/metabolismo , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Células-Tronco/imunologia , Células-Tronco/metabolismo , Volume Sistólico , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/sangue , Função Ventricular Esquerda
20.
J Cardiovasc Transl Res ; 1(3): 236-40, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19396363

RESUMO

Heparan sulfate (HS) is ubiquitous throughout the human body. The backbone of HS is composed of many types of sugars. HS serves as a docking site for a vast array of protein ligands. Recent evidence suggests a unique diversity in HS structure that alters protein binding and protein function. This diversity in HS structure has been overlooked till now. The goal of this study was to determine whether femoral artery wire injury modified HS structure. Femoral artery wire injury was performed in 16-week-old male C57BL6 mice. Transcript levels of a panel of enzymes that regulate HS fine structure, including N-deacetylase-N-sulfotransferases (Ndst) 1 and 2, exostoses (Ext) 1 and 2, C5 epimerase, and 2-O and 6-O sulfotransferases, were quantified with real-time quantitative polymerase chain reaction at 7 and 14 days post injury. All enzymes showed significant alterations in messenger RNA expression in response to injury. Ndst1, the most prevalent isoform, exhibited a 20-fold increase in response to injury. Injury induced significant alterations in fine structure specially increases in N-sulfated disaccharides at 14 days post injury. Vascular injury invokes transcriptional regulation of the enzymes that regulate HS structure, as well as changes in the pattern of HS chains in the vessel wall 14 days post injury. These findings may be important as the foundation of altered growth factor and chemokine binding in the process of vascular remodeling.


Assuntos
Artéria Femoral/lesões , Artéria Femoral/metabolismo , Heparitina Sulfato/química , Animais , Cromatografia Líquida de Alta Pressão , Sistemas Computacionais , Dissacarídeos/metabolismo , Heparitina Sulfato/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , RNA Mensageiro/metabolismo , Sulfotransferases/genética , Transcrição Gênica , Ferimentos e Lesões/metabolismo
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