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Introduction: Cerebrovascular pathologies contribute to cognitive decline during aging, leading to vascular cognitive impairment and dementia (VCID). Levels of circulating insulin-like growth factor 1 (IGF-1), a vasoprotective hormone, decrease during aging. Decreased circulating IGF-1 in animal models leads to the development of VCID-like symptoms, but the cellular mechanisms underlying IGF-1-deficiency associated pathologies in the aged cerebrovasculature remain poorly understood. Here, we test the hypothesis that vascular smooth muscle cells (VSMCs) play an integral part in mediating the vasoprotective effects of IGF-1. Methods: We used a hypertension-based model of cerebrovascular dysfunction in mice with VSMC-specific IGF-1 receptor (Igf1r) deficiency and evaluated the development of cerebrovascular pathologies and cognitive dysfunction. Results: VSMC-specific Igf1r deficiency led to impaired cerebral myogenic autoregulation, independent of blood pressure changes, which was also associated with impaired spatial learning and memory function as measured by radial arm water maze and impaired motor learning measured by rotarod. In contrast, VSMC-specific IGF-1 receptor knockdown did not lead to cerebral microvascular rarefaction. Discussion: These studies suggest that VSMCs are key targets for IGF-1 in the context of cerebrovascular health, playing a role in vessel stability alongside other cells in the neurovascular unit, and that VSMC dysfunction in aging likely contributes to VCID.
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Cerebrovascular fragility and cerebral microhemorrhages (CMH) contribute to age-related cognitive impairment, mobility defects, and vascular cognitive impairment and dementia, impairing healthspan and reducing quality of life in the elderly. Insulin-like growth factor 1 (IGF-1) is a key vasoprotective growth factor that is reduced during aging. Circulating IGF-1 deficiency leads to the development of CMH and other signs of cerebrovascular dysfunction. Here our goal was to understand the contribution of IGF-1 signaling on vascular smooth muscle cells (VSMCs) to the development of CMH and associated gait defects. We used an inducible VSMC-specific promoter and an IGF-1 receptor (Igf1r) floxed mouse line (Myh11-CreERT2 Igf1rf/f) to knockdown Igf1r. Angiotensin II in combination with L-NAME-induced hypertension was used to elicit CMH. We observed that VSMC-specific Igf1r knockdown mice had accelerated development of CMH, and subsequent associated gait irregularities. These phenotypes were accompanied by upregulation of a cluster of pro-inflammatory genes associated with VSMC maladaptation. Collectively our findings support an essential role for VSMCs as a target for the vasoprotective effects of IGF-1, and suggest that VSMC dysfunction in aging may contribute to the development of CMH.
Assuntos
Hipertensão , Músculo Liso Vascular , Receptor IGF Tipo 1 , Idoso , Animais , Humanos , Camundongos , Marcha , Hipertensão/genética , Hipertensão/complicações , Fator de Crescimento Insulin-Like I/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Receptor IGF Tipo 1/genética , Transtornos Neurológicos da Marcha/genéticaRESUMO
OBJECTIVE: Vascular smooth muscle cell (VSMC) phenotypic switching is critical for normal vessel formation, vascular stability, and healthy brain aging. Phenotypic switching is regulated by mediators including platelet derived growth factor (PDGF)-BB, insulin-like growth factor (IGF-1), as well as transforming growth factor-ß (TGF-ß) and endothelin-1 (ET-1), but much about the role of these factors in microvascular VSMCs remains unclear. METHODS: We used primary rat microvascular VSMCs to explore PDGF-BB- and IGF-1-induced phenotypic switching. RESULTS: PDGF-BB induced an early proliferative response, followed by formation of polarized leader cells and rapid, directionally coordinated migration. In contrast, IGF-1 induced cell hypertrophy, and only a small degree of migration by unpolarized cells. TGF-ß and ET-1 selectively inhibit PDGF-BB-induced VSMC migration primarily by repressing migratory polarization and formation of leader cells. Contractile genes were downregulated by both growth factors, while other genes were differentially regulated by PDGF-BB and IGF-1. CONCLUSIONS: These studies indicate that PDGF-BB and IGF-1 stimulate different types of microvascular VSMC phenotypic switching characterized by different modes of cell migration. Our studies are consistent with a chronic vasoprotective role for IGF-1 in VSMCs in the microvasculature while PDGF is more involved in VSMC proliferation and migration in response to acute activities such as neovascularization. Better understanding of the nuances of the phenotypic switching induced by these growth factors is important for our understanding of a variety of microvascular diseases.
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Fator de Crescimento Insulin-Like I , Ratos , Animais , Becaplermina/farmacologia , Proteínas Proto-Oncogênicas c-sis/farmacologia , Proteínas Proto-Oncogênicas c-sis/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Miócitos de Músculo Liso , Proliferação de Células , Movimento Celular , Células CultivadasRESUMO
Age-related cerebrovascular defects contribute to vascular cognitive impairment and dementia (VCID) as well as other forms of dementia. There has been great interest in developing biomarkers and other tools for studying cerebrovascular disease using more easily accessible tissues outside the brain such as the retina. Decreased circulating insulin-like growth factor 1 (IGF-1) levels in aging are thought to contribute to the development of cerebrovascular impairment, a hypothesis that has been supported by the use of IGF-1 deficient animal models. Here we evaluate vascular and other retinal phenotypes in animals with circulating IGF-1 deficiency and ask whether the retina mimics common age-related vascular changes in the brain such as the development of microhemorrhages. Using a hypertension-induced model, we confirm that IGF-1 deficient mice exhibited worsened microhemorrhages than controls. The retinas of IGF-1 deficient animals do not exhibit microhemorrhages but do exhibit signs of vascular damage and retinal stress such as patterns of vascular constriction and Müller cell activation. These signs of retinal stress are not accompanied by retinal degeneration or impaired neuronal function. These data suggest that the role of IGF-1 in the retina is complex, and while IGF-1 deficiency leads to vascular defects in both the brain and the retina, not all brain pathologies are evident in the retina.
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BACKGROUND: The discovery of TCF7L2 as a global type 2 diabetes (T2D) gene has sparked investigations to explore the clinical utility of its variants for guiding the development of new diagnostic and therapeutic strategies. However, interpreting the resulting associations into function still remains unclear. Canonical Wnt signaling regulates ß-catenin and its binding with TCF7L2, which in turn is critical for the production of glucagon-like peptide-1 (GLP-1). This study examines the role of a novel frame-shift insertion discovered in a conserved region of WNT16a, and it is proposed that this mutation affects T2D susceptibility in conjunction with gene variants in TCF7L2. RESULTS: Our results predicted that the insertion would convert the upstream open reading frame in the Wnt16a mRNA to an alternative, in-frame translation initiation site, resulting in the prevention of nonsense-mediated decay, leading to a consequent stabilization of the mutated WNT16a message. To examine the role of Wnt16a in the Wnt signaling pathway, DNA and serum samples from 2,034 individuals (48% with T2D) from the Sikh Diabetes Study were used in this investigation. Prevalence of Wnt16a insertion did not differ among T2D cases (33%) and controls (32%). However, there was a 3.2 fold increase in Wnt16a mRNA levels in pancreatic tissues from the insertion carriers and a significant increase (70%, p < 0.0001) in luciferase activity in the constructs carrying the insertion. The expression of TCF7L2 mRNA in pancreas was also elevated (~23-fold) among the insertion carriers (p=0.003). CONCLUSIONS: Our results suggest synergistic effects of WNT16a insertion and the at-risk 'T' allele of TCF7L2 (rs7903146) for elevating the expression of TCF7L2 in human pancreas which may affect the regulation of downstream target genes involved in the development of T2D through Wnt/ß-catenin/TCF7L2 signaling pathway. However, further studies would be needed to mechanistically link the two definitively.
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Mutação da Fase de Leitura , Triagem de Portadores Genéticos , Pâncreas/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteínas Wnt/genética , Adulto , Idoso , Sequência de Bases , Estudos de Casos e Controles , Primers do DNA , Diabetes Mellitus Tipo 2/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da PolimeraseRESUMO
Cell migration is fundamental to many biological processes, including development, normal tissue remodeling, wound healing, and many pathologies. However, cell migration is a complex process, and understanding its regulation in health and disease requires the ability to manipulate and measure this process quantitatively under controlled conditions. This report describes a simple in vitro assay for quantitative analysis of cell migration in two-dimensional cultures that is an inexpensive alternative to the classic "scratch" assay. The method described utilizes flexible silicone masks fabricated in the lab according to the research demands of the specific experiment to create a cell-free area for cells to invade, followed by quantitative analysis based on widely available microscopic imaging tools. This experimental approach has the important advantage of visualizing cell migration in the absence of the cellular damage and disruption of the substrate that occurs when the "wound" is created in the scratch assay. This approach allows the researcher to study the intrinsic migratory characteristics of cells in the absence of potentially confounding contributions from cellular responses to injury and disruption of cell-substrate interactions. This assay has been used with vascular smooth muscle cells, fibroblasts, and epithelial cell types, but should be applicable to the study of practically any type of cultured cell. Furthermore, this method can be easily adapted for use with fluorescence microscopy, molecular biological, or pharmacological manipulations to explore the molecular mechanisms of cell migration, live cell imaging, fluorescence microscopy, and correlative immunolabeling.
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Movimento Celular/genética , Fibroblastos/metabolismo , Elastômeros de Silicone/química , Bioensaio , Células Cultivadas , Humanos , Elastômeros de Silicone/metabolismo , Cicatrização/genéticaRESUMO
During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-ß, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-ß, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation.
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Fibroblastos/enzimologia , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Miofibroblastos/enzimologia , Actinas/química , Actinas/metabolismo , Animais , Técnicas de Cultura de Células/métodos , Linhagem Celular , Movimento Celular/genética , Movimento Celular/fisiologia , Fibroblastos/citologia , Fibroblastos/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Fator de Crescimento Insulin-Like I/farmacologia , Modelos Biológicos , Miofibroblastos/citologia , Miofibroblastos/fisiologia , Fenótipo , Multimerização Proteica , Interferência de RNA , Ratos , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
During platelet-derived growth factor (PDGF)-BB-mediated recruitment to neovascular sprouts, vascular smooth muscle cells (VSMCs) dedifferentiate from a contractile to a migratory phenotype. This involves the downregulation of contractile markers such as smooth muscle (SM) alpha-actin and the upregulation of promigration genes such as matrix metalloproteinase (MMP)-2. The regulation of MMP-2 in response to PDGF-BB is complex and involves both stimulatory and inhibitory signaling pathways, resulting in a significant delay in upregulation. Here, we provide evidence that the delay in MMP-2 upregulation may be due to the autocrine expression and activation of transforming growth factor (TGF)-beta, which is known to promote the contractile phenotype in VSMCs. Whereas PDGF-BB could induce the loss of stress fibers and focal adhesions, TGF-beta was able to block or reverse this transition to a noncontractile state. TGF-beta did not, however, suppress early signaling events stimulated by PDGF-BB. Over time, though PDGF-BB induced increased TGF-beta1 levels, it suppressed TGF-beta2 and TGF-beta3 expression, leading to a net decrease in the total TGF-beta pool, resulting in the upregulation of MMP-2. Together, these findings indicate that MMP-2 expression is suppressed by a threshold level of active TGF-beta, which in turn promotes a contractile VSMC phenotype that prevents the upregulation of MMP-2.
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Metaloproteinase 2 da Matriz/genética , Músculo Liso Vascular/enzimologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacos , Actinas/genética , Animais , Becaplermina , Primers do DNA , Regulação para Baixo , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genes fos , Humanos , Fator Estimulador de Colônias de Macrófagos/farmacologia , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Reação em Cadeia da Polimerase , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Endogâmicos WKY , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-2/genéticaRESUMO
PURPOSE: Cellular retinaldehyde-binding protein (CRALBP), transcribed from the RLBP1 gene, is a 36-kDa water-soluble protein with 316 amino acids found in the retinal pigment epithelium (RPE) and in retinal Müller cells. It is thought to play a critical role in the visual cycle by functioning as an acceptor of 11-cis-retinol from the isomerohydrolase reaction. The goal here was to evaluate the functional promoter of this gene. METHODS: 5' RACE analysis, promoter-reporter assays, and semiquantitative PCR with exon-specific primers were performed using human-derived RPE cells (ARPE-19 and D407) in culture to evaluate the 5' sequence flanking the RLBP1 gene. In addition, the murine, bovine, and porcine RLBP1 genes were evaluated in silico to identify likely proximal promoter/exon 1 sequences similar to the human gene. RESULTS: 5' RACE analysis revealed the presence of a previously undescribed exon in the RLBP1 gene. This was confirmed by analysis of the GenBank Human EST database, which revealed the presence of 18 sequences matching exon 1. Exon-specific PCR revealed that most CRALBP transcripts expressed in ARPE-19 cells contain both exon 1 and the final exon, suggesting that the primary promoter of CRALBP exists 5' of the newly identified exon 1. Highly homologous sequences in the murine, bovine, and porcine genes were also identified. Finally, promoter-reporter constructs revealed a minimal sequence necessary for promoter function and indicated significantly greater promoter activity compared with previously described RLBP1 promoters. CONCLUSIONS: The findings presented here suggest that CRALBP transcripts in RPE cells contain a noncoding exon in addition to a newly described promoter and, by definition, an additional intron. This finding sets the stage for a mechanistic understanding of the high degree of cell type-specific expression of RLBP1.
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Proteínas de Transporte/genética , Epitélio Pigmentado Ocular/fisiologia , Regiões Promotoras Genéticas/genética , Visão Ocular/genética , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Bovinos , Linhagem Celular , Elementos Facilitadores Genéticos/genética , Éxons/genética , Humanos , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Epitélio Pigmentado Ocular/citologia , SuínosRESUMO
In response to growth factors, vascular smooth muscle cells (VSMCs) undergo a phenotypic modulation from a contractile, non-proliferative state to an activated, migratory state. This transition is characterized by changes in their gene expression profile, particularly by a significant down-regulation of contractile proteins. Platelet-derived growth factor (PDGF)-BB has long been known to initiate VSMC de-differentiation and mitogenesis. Insulin-like growth factor (IGF)-I, on the other hand, has differing effects depending on the model studied. Here, we report that both IGF-I and PDGF-BB stimulated VSMC de-differentiation of rat heart-derived SMCs in culture, although only PDGF-BB was capable of inducing proliferation. Although both PDGF-BB and IGF-I stimulation resulted in decreased smooth muscle alpha-actin expression and increased matrix metalloproteinase (MMP)-2 expression, the response to IGF-I was significantly more rapid. The increased MMP-2 expression in response to both growth factors was due to increased transcription rates and was dependent on the action of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. Both PDGF-BB and IGF-I activated PI3K/Akt to similar degrees; however, only PDGF-BB concomitantly stimulated an inhibitory signaling pathway that antagonized the effects of Akt but did not alter the extent or duration of Akt activation. Together, these findings suggest that changes in MMP-2 expression are part of the program of VSMC phenotypic modulation and that both PDGF-BB and IGF-I, despite their different abilities to induce proliferation in this model, are capable of inducing VSMC activation.
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Fator de Crescimento Insulin-Like I/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Músculo Liso Vascular , Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/fisiologia , Animais , Becaplermina , Biomarcadores/metabolismo , Diferenciação Celular , Células Cultivadas , Meios de Cultura Livres de Soro , Genes Reporter , Humanos , Metaloproteinase 2 da Matriz/genética , Músculo Liso Vascular/citologia , Músculo Liso Vascular/enzimologia , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-sis , Ratos , Ratos Endogâmicos WKY , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: In a previous study, we reported that keratinocyte growth factor (KGF) produced a rapid increase in the motility of ER-positive breast cancer cells. Others have demonstrated that KGF treatment in rodent species produces rapid mammary ductal hyperplasia. Epithelial cells do not produce KGF; thus, in the present study, MCF-7 cells were stably transfected with a KGF-expressing vector and the motility and morphology of the transfected, non-transfected and empty vector cell lines compared. MATERIALS AND METHODS: A mammalian expression vector containing a KGF cDNA was transfected into MCF-7/beta cells, and two stable clones (MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9) were identified. Western blotting of conditioned medium from these clones was used to confirm the expression of KGF. The motility of wild-type and KGF-transfected MCF-7 cells was compared using time-lapse videomicroscopy and a cell culture wounding model which examined cell migration over a period of 1-3 days. RESULTS: The Western blots demonstrated that the expression of KGF in both the MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9 cell lines was higher than the wild-type and MCF-7/beta cell lines. The cell proliferation and migration distance was significantly greater for both KGF-transfected MCF-7 cell lines than the wild-type and MCF-7/beta cell lines under the same experimental conditions. Further, changes in motile morphology were observed in both the MCF-7/beta/KGF-T8 and MCF-7/beta/KGF-T9 cell lines. In addition, the MCF-7/beta/KGF-T8 clone was found to produce much larger tumors than both the MCF-7/beta/KGF-T9 and EV clones in mouse xenografts. These results indicated that autocrine production of KGF in the KGF-transfected MCF-7 cell lines enhanced cell migration, migration-related morphology and xenograft tumor growth. CONCLUSION: KGF-transfected MCF-7 cells displayed a much greater motility than non-transfected cells, confirming the KGF motility enhancement effect which we previously reported. The use of KGF-transfected breast cancer cells in the xenograft model may help to study the mechanism of KGF-mediated cell motility and to identify specific KGF antagonists that may be used to prevent or impede KGF-mediated metastatic progression.
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Neoplasias da Mama/patologia , Movimento Celular/fisiologia , Fator 7 de Crescimento de Fibroblastos/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Fator 7 de Crescimento de Fibroblastos/biossíntese , Fator 7 de Crescimento de Fibroblastos/genética , Fator 7 de Crescimento de Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Transfecção , Transplante HeterólogoRESUMO
Angiopoietins play a significant role in vascular development and angiogenesis. Both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) bind the receptor tyrosine kinase Tie2. However, while Ang1 signaling results in the stabilization of vessel structure, Ang2 has been linked to vascular instability. The ratio of these two Tie2 ligands is thus critical for vascular stability and remodeling. This study identifies a mechanism of growth factor-mediated reduction in Ang2 expression in vascular smooth muscle cells (VSMCs). In response to PDGF, VSMCs downregulated Ang2 mRNA levels by 75% within 4 h, with a subsequent decrease in Ang2 protein levels. Quantitation of endogenous transcription rates revealed that PDGF stimulation did not alter Ang2 transcription rates, but instead induced a posttranscriptional mechanism of rapid Ang2 mRNA destabilization. The Ang2 mRNA half-life was reduced by at least 50% after PDGF treatment. The PDGF-induced mRNA turnover mechanism was dependent on several MAPK pathways, including ERK and JNK. In contrast, IGF-I, which did not significantly activate ERK or JNK, stimulated increased Ang2 expression through transcriptional activation. These findings demonstrate that VSMCs adjust Ang2 expression through multiple mechanisms, including changes in transcription as well as posttranscriptional mRNA destabilization.