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1.
Nucleic Acid Ther ; 33(6): 381-385, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37782140

RESUMO

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused the current worldwide pandemic and the associated coronavirus disease 2019 with potentially lethal outcome. Although effective vaccines strongly contributed to reduce disease severity, establishing a toolbox to control current and newly emerging coronaviruses of epidemic concern requires the development of novel therapeutic compounds, to treat severely infected individuals and to prevent virus transmission. Here we present a therapeutic strategy targeting the SARS-CoV-2 RNA genome using antisense oligonucleotides (ASOs). We demonstrate that selected locked nucleic acid gapmers have the potency to reduce the in vitro intracellular viral load by up to 96%. Our promising results strongly support the case for further development of our preselected ASOs as therapeutic or prophylactic antiviral agents.


Assuntos
COVID-19 , Oligonucleotídeos Antissenso , Humanos , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , SARS-CoV-2/genética , RNA Viral/genética , COVID-19/genética , COVID-19/terapia
2.
Innate Immun ; 27(2): 118-132, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33241976

RESUMO

In response to inflammatory cytokines and chemokines, monocytes differentiate into macrophages. Comprehensive analysis of gene expression regulation of neuronal guidance cue (NGC) ligands and receptors in the monocyte-to-macrophage differentiation process is not available yet. We performed transcriptome profiling in both human primary PBMCs/PBMC-derived macrophages and THP-1 cells/THP-1-macrophages using microarray or RNA sequencing methods. Pathway analysis showed that the axonal guidance pathway is significantly regulated upon monocyte differentiation. We confirmed NGC ligands and receptors which were consistently regulated, including SEMA4D, SEMA7A, NRP1, NRP2, PLXNA1 and PLXNA3. The involvement of RNA-binding protein quaking (QKI) in the regulation of NGC expression was investigated using monocytes and macrophages from a QKI haplo-insufficient patient and her healthy sibling. This revealed a positive correlation of SEMA7A expression with QKI expression. In silico analysis of 3'UTRs of NGCs proposed the competitive binding of QKI to proximal microRNA targeting sites as the mechanism of QKI-dependent regulation of SEMA7A. RNA immunoprecipitation confirmed an interaction of QKI with the 3'UTR of SEMA7A. Loss of SEMA7A resulted in monocyte differentiation towards a more anti-inflammatory macrophage. Taken together, the axonal guidance pathway is regulated during monocyte-to-macrophage differentiation, and the regulation is in line with the necessary functional adaption for the specialised role of macrophages.


Assuntos
Regiões 3' não Traduzidas/genética , Macrófagos/fisiologia , MicroRNAs/genética , Monócitos/fisiologia , Proteínas de Ligação a RNA/genética , RNA/genética , Semaforinas/metabolismo , Orientação de Axônios/genética , Diferenciação Celular , Feminino , Perfilação da Expressão Gênica , Haploinsuficiência , Humanos , Cultura Primária de Células , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Semaforinas/genética , Irmãos , Células THP-1
3.
Int J Mol Sci ; 21(17)2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32887275

RESUMO

Atherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of large arteries is a key denominator of atherogenesis, making monocytes accountable for the development of atherosclerosis. With the development of high-throughput transcriptome profiling platforms and cytometric methods for circulating cells, it is now feasible to study in-depth the predicted functional change of circulating monocytes reflected by changes of gene expression in certain pathways and correlate the changes to disease outcome. Neuroimmune guidance cues comprise a group of circulating- and cell membrane-associated signaling proteins that are progressively involved in monocyte functions. Here, we employed the CIRCULATING CELLS study cohort to classify cardiovascular disease patients and healthy individuals in relation to their expression of neuroimmune guidance cues in circulating monocytes. To cope with the complexity of human datasets featured by noisy data, nonlinearity and multidimensionality, we assessed various machine-learning methods. Of these, the linear discriminant analysis, Naïve Bayesian model and stochastic gradient boost model yielded perfect or near-perfect sensibility and specificity and revealed that expression levels of the neuroimmune guidance cues SEMA6B, SEMA6D and EPHA2 in circulating monocytes were of predictive values for cardiovascular disease outcome.


Assuntos
Biomarcadores/sangue , Doenças Cardiovasculares/diagnóstico , Efrinas/sangue , Aprendizado de Máquina , Monócitos/metabolismo , Netrina-1/sangue , Semaforinas/sangue , Adulto , Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/genética , Estudos de Casos e Controles , Estudos de Coortes , Efrinas/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Netrina-1/genética , Semaforinas/genética , Transcriptoma
4.
Epigenomes ; 4(1)2020 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-34968236

RESUMO

In the pathophysiologic setting of acute and chronic kidney injury, the excessive activation and recruitment of blood-borne monocytes prompts their differentiation into inflammatory macrophages, a process that leads to progressive glomerulosclerosis and interstitial fibrosis. Importantly, this differentiation of monocytes into macrophages requires the meticulous coordination of gene expression at both the transcriptional and post-transcriptional level. The transcriptomes of these cells are ultimately determined by RNA-binding proteins such as QUAKING (QKI), that define their pre-mRNA splicing and mRNA transcript patterns. Using two mouse models, namely (1) quaking viable mice (qkv) and (2) the conditional deletion in the myeloid cell lineage using the lysozyme 2-Cre (QKIFL/FL;LysM-Cre mice), we demonstrate that the abrogation of QKI expression in the myeloid cell lineage reduces macrophage infiltration following kidney injury induced by unilateral urethral obstruction (UUO). The qkv and QKIFL/FL;LysM-Cre mice both showed significant diminished interstitial collagen deposition and fibrosis in the UUO-damaged kidney, as compared to wild-type littermates. We show that macrophages isolated from QKIFL/FL;LysM-Cre mice are associated with defects in pre-mRNA splicing. Our findings demonstrate that reduced expression of the alternative splice regulator QKI in the cells of myeloid lineage attenuates renal interstitial fibrosis, suggesting that inhibition of this splice regulator may be of therapeutic value for certain kidney diseases.

5.
Biomaterials ; 194: 47-56, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30580195

RESUMO

Tissue engineered blood vessels (TEBVs) hold great promise for clinical use in patients with end stage renal disease (ESRD) requiring vascular access for hemodialysis. A promising way to make TEBVs is to exploit foreign body response (FBR) of polymeric rods used as templates. However, since the FBR predominantly involves bone-marrow (BM) derived cells and ESRD coincides with impaired function of BM, it is important to assess the generation of TEBVs in conditions of renal failure. To this end, we implanted polymer rods in the subcutis of rats after BM-transplantation with GFP-labeled BM cells in a model of chronic kidney disease (CKD). At 3 weeks after implantation, rods were encapsulated by tissue capsule (TC) composed of collagen, myofibroblasts and macrophages. On average, 13% of CD68+ macrophages were GFP+, indicating BM origin. Macrophage-to-myofibroblasts differentiation appeared to play an important role in TC formation as 26% of SMA+/GFP+ myofibroblasts co-expressed the macrophage marker CD68. Three weeks after rod implantation, the cellular response changed towards tissue repair, characterized by 40% increase in CD68+/CD163+ repair associated macrophages and 95% increase in TGFß and IL10 gene expression as compared to TCs harvested at 1 week. These results show that both BM derived and tissue resident cells, contribute to TC formation, whereas macrophages serve as precursors of myofibroblasts in mature TCs. Finally, the presence of CKD did not significantly alter the process of TC formation, which holds the potential to support our approach for future clinical use in ESRD patients.


Assuntos
Transplante de Medula Óssea , Corpos Estranhos/etiologia , Rim/patologia , Insuficiência Renal Crônica/terapia , Animais , Células da Medula Óssea/citologia , Transplante de Medula Óssea/efeitos adversos , Transplante de Medula Óssea/métodos , Modelos Animais de Doenças , Corpos Estranhos/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Insuficiência Renal Crônica/patologia , Engenharia Tecidual/métodos
6.
FASEB J ; : fj201800437R, 2018 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-29882709

RESUMO

The pathophysiology of arteriovenous fistula (AVF) maturation failure is not completely understood but impaired outward remodeling (OR) and intimal hyperplasia are thought to be contributors. This adverse vascular response after AVF surgery results from interplay between vascular smooth muscle cells (VSMCs), the extracellular matrix (ECM), and inflammatory cells. Relaxin (RLN) is a hormone that acts on the vasculature via interaction with RLN/insulin-like peptide family receptor 1 (RXFP1), resulting in vasodilatation, ECM remodeling, and decreased inflammation. In the present study, we evaluated the consequences of RXFP1 knockout ( Rxfp1-/-) on AVF maturation in a murine model of AVF failure. Rxfp1-/- mice showed a 22% decrease in vessel size at the venous outflow tract 14 d after AVF surgery. Furthermore, a 43% increase in elastin content was observed in the lesions of Rxfp1-/- mice and coincided with a 41% reduction in elastase activity. In addition, Rxfp1-/- mice displayed a 6-fold increase in CD45+ leukocytes, along with a 2-fold increase in monocyte chemoattractant protein 1 (MCP1) levels, when compared with wild-type mice. In vitro, VSMCs from Rxfp1-/- mice exhibited a synthetic phenotype, as illustrated by augmentation of collagen, fibronectin, TGF-ß, and platelet-derived growth factor mRNA. In addition, VSMCs derived from Rxfp1-/- mice showed a 5-fold increase in cell migration. Finally, RXFP1 and RLN expression levels were increased in human AVFs when compared with unoperated cephalic veins. In conclusion, RXFP1 deficiency hampers elastin degradation and results in induced vascular inflammation after AVF surgery. These processes impair OR in murine AVF, suggesting that the RLN axis could be a potential therapeutic target for promoting AVF maturation.-Bezhaeva, T., de Vries, M. R., Geelhoed, W. J., van der Veer, E. P., Versteeg, S., van Alem, C. M. A., Voorzaat, B. M., Eijkelkamp, N., van der Bogt, K. E., Agoulnik, A. I., van Zonneveld, A.-J., Quax, P. H. A., Rotmans, J. I. Relaxin receptor deficiency promotes vascular inflammation and impairs outward remodeling in arteriovenous fistulas.

7.
Am J Physiol Renal Physiol ; 315(4): F1129-F1138, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-29846108

RESUMO

Fine-tuning of the body's water balance is regulated by vasopressin (AVP), which induces the expression and apical membrane insertion of aquaporin-2 water channels and subsequent water reabsorption in the kidney. Here we demonstrate that silencing of microRNA-132 (miR-132) in mice causes severe weight loss due to acute diuresis coinciding with increased plasma osmolality, reduced renal total and plasma membrane expression of aquaporin-2, and abrogated increase in AVP levels. Infusion with synthetic AVP fully reversed the antagomir-132-induced diuresis, and low-dose intracerebroventricular administration of antagomir-132 similarly caused acute diuresis. Central and intracerebroventricular antagomir-132 injection both decreased hypothalamic AVP mRNA levels. At the molecular level, antagomir-132 increased the in vivo and in vitro mRNA expression of methyl-CpG-binding protein-2 (MECP2), which is a miR-132 target and which blocks AVP gene expression by binding its enhancer region. In line with this, treatment of hypothalamic N6 cells with a high-salt solution increased its miR-132 levels, whereas it attenuated endogenous Mecp2 mRNA levels. In conclusion, we identified miR-132 as a first miRNA regulating the osmotic balance by regulating the hypothalamic AVP gene mRNA expression.


Assuntos
Arginina Vasopressina/metabolismo , Homeostase/fisiologia , Proteína 2 de Ligação a Metil-CpG/genética , MicroRNAs/genética , Vasopressinas/metabolismo , Animais , Aquaporina 2/metabolismo , Expressão Gênica/genética , Hipotálamo/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Concentração Osmolar , Receptores de Vasopressinas/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia
8.
Am J Transl Res ; 10(3): 816-826, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29636871

RESUMO

The pathophysiology of ischemia/reperfusion (I/R) injury is complex and poorly understood. Animal studies imply platelet activation as an initiator of the inflammatory response upon reperfusion. However, it remains unclear whether and how these results translate to clinical I/R. This study evaluates putative platelet activation in the context of two forms of clinical I/R (heart valve surgery with aortic-cross clamping, n = 39 and kidney transplantation, n = 34). The technique of sequential selective arteriovenous (AV) measurements over the reperfused organs was applied to exclude the influence of systemic changes occurring during surgery while simultaneously maximizing sensitivity. Platelet activation and degranulation was evaluated by assessing the expression levels of established markers, i.e. RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted), ß-thromboglobulin (ß-TG), platelet-derived growth factor (PDGF)-BB and CXCL8 (known as interleukin-8), and by employing an in-vitro assay that specifically tests for platelet excitability. Moreover, a histological analysis was performed by means of CD41 staining. Results show stable RANTES, ß-TG, PDGF-BB and CXCL8 AV-concentrations within the first half hour over the reperfused organs, suggesting that myocardial and renal I/R are not associated with platelet activation. Results from the platelet excitability assay were in line with these findings and indicated reduced and stable platelet excitability following renal and myocardial reperfusion, respectively. Histological analysis yield evidence of platelet marginalization in the reperfused organs. In conclusion, results from this study do not support a role for platelet activation in early phases of clinical I/R injury.

9.
Nephrol Dial Transplant ; 33(1): 44-53, 2018 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-28992069

RESUMO

BACKGROUND: Treatment of inflammatory kidney diseases with systemic high-dose glucocorticoids (GCs) has severe side effects. Liposomal encapsulation could facilitate local delivery of GCs to the inflamed kidney, as liposomes encapsulate their payload until extravasation at sites of inflammation, potentially resulting in local bioactivity. Our aim was to evaluate the ability of liposomes to accumulate locally after renal ischaemia-reperfusion injury in the rat and to study its effect on macrophages. METHODS: In vitro, human macrophages were incubated with fluorescent liposomes, liposomal prednisolone, prednisolone, empty liposomes or saline. Uptake was studied microscopically and treatment effect was assessed by interkeukin 6 (IL-6) enzyme-linked immunosorbent assay. The mechanism of action was evaluated by analysing GC receptor activation by microscopy and quantitative polymerase chain reaction (qPCR). In vivo, rats were subjected to ischaemia-reperfusion injury and were injected intravenously with fluorescent liposomes, liposomal prednisolone, prednisolone, empty liposomes or saline. Uptake was measured by the FLARE camera and the treatment effect by immunohistochemistry for myeloid cells and qPCR for inflammatory markers. RESULTS: In vitro, macrophages internalized liposomes after 8 hours. Prednisolone or liposomal prednisolone treatment reduced IL-6 production and both compounds induced translocation of the GC receptor to the nucleus and upregulation of PER1 messenger RNA (mRNA), indicating a similar mechanism of action. In vivo, fluorescent liposomes accumulated in the inflamed kidney. Liposomal prednisolone treatment increased the presence of ED2-positive anti-inflammatory macrophages and both prednisolone and liposomal prednisolone reduced monocyte chemoattractant protein-1 (MCP-1) mRNA production, indicating a reduced pro-inflammatory profile in the kidney. CONCLUSIONS: Liposomal encapsulation is a promising strategy for local delivery of glucocorticoids to the inflamed kidney.


Assuntos
Anti-Inflamatórios/uso terapêutico , Sistemas de Liberação de Medicamentos , Mediadores da Inflamação/metabolismo , Inflamação/prevenção & controle , Lipossomos/administração & dosagem , Prednisolona/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Animais , Células Cultivadas , Humanos , Inflamação/metabolismo , Lipossomos/química , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Masculino , Ratos , Ratos Endogâmicos Lew , Traumatismo por Reperfusão/metabolismo
10.
Cardiovasc Res ; 114(2): 210-225, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29186452

RESUMO

Cardiovascular disease (CVD) is the primary cause of death among men and women worldwide. Nevertheless, our comprehension of how CVD progresses in women and elicits clinical outcomes is lacking, leading CVD to be under-diagnosed and under-treated in women. A clear example of this differential presentation of CVD pathophysiologies in females is the strikingly higher prevalence of heart failure with preserved ejection fraction (HFpEF). Women with a history of pre-eclampsia or those who present with co-morbidities such as obesity, hypertension, and diabetes mellitus are at increased risk of developing HFpEF. Long understood to be a critical CVD risk factor, our understanding of how gender differentially affects the development of CVD has been greatly expanded by extensive genomic and transcriptomic studies. These studies uncovered a pivotal role for differential microRNA (miRNA) expression in response to systemic inflammation, where their co-ordinated expression forms a post-transcriptional regulatory network that instigates microcirculation defects. Importantly, the potential sex-biased expression of the given miRNAs may explain sex-specific cardiovascular pathophysiologies in women, such as HFpEF. Sex-biased miRNAs are regulated by oestrogen (E2) in their transcription and processing or are expressed from loci on the X-chromosome due to incomplete X-chromosome inactivation. Interestingly, while E2-induced miRNAs predominantly appear to serve protective functions, it could be argued that many X-linked miRNAs have been found to challenge microvascular and myocardial integrity. Therefore, menopausal E2 deficiency, resulting in protective miRNA loss, and the augmentation of X-linked miRNA expression, may well contribute to the molecular mechanisms that underlie the female-specific cardiovascular aetiology in HFpEF.


Assuntos
Disparidades nos Níveis de Saúde , Insuficiência Cardíaca/genética , MicroRNAs/genética , Volume Sistólico , Função Ventricular Esquerda , Fatores Etários , Animais , Cromossomos Humanos X , Comorbidade , Estradiol/metabolismo , Feminino , Regulação da Expressão Gênica , Insuficiência Cardíaca/epidemiologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Humanos , Masculino , Menopausa/genética , Menopausa/metabolismo , MicroRNAs/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Prevalência , Medição de Risco , Fatores de Risco , Fatores Sexuais , Remodelação Ventricular , Inativação do Cromossomo X
11.
Sci Rep ; 7(1): 10269, 2017 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-28860634

RESUMO

Arteriovenous access dysfunction is a major cause of morbidity for hemodialysis patients. The pathophysiology of arteriovenous fistula (AVF) maturation failure is associated with inflammation, impaired outward remodeling (OR) and intimal hyperplasia. RP105 is a critical physiologic regulator of TLR4 signaling in numerous cell types. In the present study, we investigated the impact of RP105 on AVF maturation, and defined cell-specific effects of RP105 on macrophages and vascular smooth muscle cells (VSMCs). Overall, RP105-/- mice displayed a 26% decrease in venous OR. The inflammatory response in RP105-/- mice was characterized by accumulation of anti-inflammatory macrophages, a 76% decrease in pro- inflammatory macrophages, a 70% reduction in T-cells and a 50% decrease in MMP-activity. In vitro, anti-inflammatory macrophages from RP105-/- mice displayed increased IL10 production, while MCP1 and IL6 levels secreted by pro-inflammatory macrophages were elevated. VSMC content in RP105-/- AVFs was markedly decreased. In vitro, RP105-/- venous VSMCs proliferation was 50% lower, whereas arterial VSMCs displayed a 50% decrease in migration, relative to WT. In conclusion, the impaired venous OR in RP105-/- mice could result from of a shift in both macrophages and VSMCs towards a regenerative phenotype, identifying a novel relationship between inflammation and VSMC function in AVF maturation.


Assuntos
Antígenos CD/genética , Fístula Arteriovenosa/genética , Fístula Arteriovenosa/fisiopatologia , Deleção de Genes , Remodelação Vascular/genética , Animais , Antígenos CD/metabolismo , Fístula Arteriovenosa/patologia , Biomarcadores , Biópsia , Proliferação de Células , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Imunofluorescência , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Macrófagos/patologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Knockout , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo
12.
Eur Heart J ; 38(18): 1380-1388, 2017 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-28064149

RESUMO

The cardiovascular system comprises multiple cell types that possess the capacity to modulate their phenotype in response to acute or chronic injury. Transcriptional and post-transcriptional mechanisms play a key role in the regulation of remodelling and regenerative responses to damaged cardiovascular tissues. Simultaneously, insufficient regulation of cellular phenotype is tightly coupled with the persistence and exacerbation of cardiovascular disease. Recently, RNA-binding proteins such as Quaking, HuR, Muscleblind, and SRSF1 have emerged as pivotal regulators of these functional adaptations in the cardiovascular system by guiding a wide-ranging number of post-transcriptional events that dramatically impact RNA fate, including alternative splicing, stability, localization and translation. Moreover, homozygous disruption of RNA-binding protein genes is commonly associated with cardiac- and/or vascular complications. Here, we summarize the current knowledge on the versatile role of RNA-binding proteins in regulating the transcriptome during phenotype switching in cardiovascular health and disease. We also detail existing and potential DNA- and RNA-based therapeutic approaches that could impact the treatment of cardiovascular disease in the future.


Assuntos
Doenças Cardiovasculares/genética , Proteínas de Ligação a RNA/fisiologia , Processamento Alternativo/genética , Doenças Cardiovasculares/terapia , Células Endoteliais/fisiologia , Terapia Genética/métodos , Humanos , Músculo Liso Vascular/fisiologia , Miócitos Cardíacos/fisiologia , Fenótipo , RNA/genética , Células Estromais/fisiologia , Transcriptoma/genética
13.
Tissue Eng Part C Methods ; 22(10): 923-931, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27604583

RESUMO

Elastin, critical for its structural and regulatory functions, is a missing link in vascular tissue engineering. Several elastin-inducting compounds have previously been reported, but their relative efficiency in promoting elastogenesis by adult arterial and venous vascular smooth muscle cells (VSMCs) and fibroblasts, four main vascular and elastogenic cells, has not been described. In addition to elasto-inductive substances, microRNA-29a was recently established as a potent post-transcriptional inhibitor of elastogenesis. Here, we explored if stimulating positive regulators or blocking inhibitors of elastogenesis could maximize elastin production. We tested whether the elasto-inducing compounds IGF-1, TGF-ß1, and minoxidil could indeed augment elastin production, and whether microRNA-29a antagonism could block elastin production in adult arterial and venous fibroblasts and VSMCs. The effects on elastin, lysyl oxidase, and fibrillin-1 mRNA expression levels and tropoelastin protein were determined. IGF-1 and minoxidil exerted little effect on tropoelastin mRNA expression levels in all cell types, while TGF-ß1 predominantly enhanced mRNA tropoelastin levels, but this mRNA increase did not impact tropoelastin protein abundance. In contrast, microRNA29a inhibition resulted in the upregulation of tropoelastin mRNA in all cell types, but most pronounced in venous VSMCs. Importantly, microRNA-29a-antagonism also enhanced lysyl oxidase and fibrillin-1 mRNA expression, and revealed a dose-dependent increase in tropoelastin protein expression in venous VSMCs. Our studies suggest that the elastogenic potential of microRNA-29a inhibition in vascular cells is superior to that of established elastin-stimulating compounds IGF-1, TGF-ß1, and minoxidil. Thus, microRNA-29a antagonism could serve as an attractive means of enhancing elastin synthesis in tissue-engineered blood vessels.


Assuntos
Artérias/metabolismo , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Engenharia Tecidual/métodos , Tropoelastina/metabolismo , Veias/metabolismo , Animais , Artérias/citologia , Células Cultivadas , Feminino , Fibroblastos/citologia , Fator de Crescimento Insulin-Like I/farmacologia , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Minoxidil/farmacologia , Músculo Liso Vascular/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Suínos , Fator de Crescimento Transformador beta1/farmacologia , Tropoelastina/genética , Vasodilatadores/farmacologia , Veias/citologia
14.
Kidney Int ; 89(6): 1268-80, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27165825

RESUMO

Chronic kidney disease is associated with progressive renal fibrosis, where perivascular cells give rise to the majority of α-smooth muscle actin (α-SMA) positive myofibroblasts. Here we sought to identify pericytic miRNAs that could serve as a target to decrease myofibroblast formation. Kidney fibrosis was induced in FoxD1-GC;Z/Red-mice by unilateral ureteral obstruction followed by FACS sorting of dsRed-positive FoxD1-derivative cells and miRNA profiling. MiR-132 selectively increased 21-fold during pericyte-to-myofibroblast formation, whereas miR-132 was only 2.5-fold up in total kidney lysates (both in obstructive and ischemia-reperfusion injury). MiR-132 silencing during obstruction decreased collagen deposition (35%) and tubular apoptosis. Immunohistochemistry, Western blot, and qRT-PCR confirmed a similar decrease in interstitial α-SMA(+) cells. Pathway analysis identified a rate-limiting role for miR-132 in myofibroblast proliferation that was confirmed in vitro. Indeed, antagomir-132-treated mice displayed a reduction in the number of proliferating Ki67(+) interstitial myofibroblasts. Interestingly, this was selective for the interstitial compartment and did not impair the reparative proliferation of tubular epithelial cells, as evidenced by an increase in Ki67(+) epithelial cells, as well as increased phospho-RB1, Cyclin-A and decreased RASA1, p21 levels in kidney lysates. Additional pathway and gene expression analyses suggest miR-132 coordinately regulates genes involved in TGF-ß signaling (Smad2/Smad3), STAT3/ERK pathways, and cell proliferation (Foxo3/p300). Thus, silencing miR-132 counteracts the progression of renal fibrosis by selectively decreasing myofibroblast proliferation and could potentially serve as a novel antifibrotic therapy.


Assuntos
Proliferação de Células/genética , Rim/patologia , MicroRNAs/genética , Miofibroblastos/fisiologia , Insuficiência Renal Crônica/patologia , Actinas/metabolismo , Animais , Antagomirs/genética , Apoptose , Linhagem Celular , Colágeno/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Fibrose , Humanos , Imuno-Histoquímica , Túbulos Renais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Miofibroblastos/metabolismo , Pericitos/metabolismo , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/genética , Fator de Crescimento Transformador beta
15.
Nat Commun ; 7: 10846, 2016 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-27029405

RESUMO

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function.


Assuntos
Macrófagos/fisiologia , Monócitos/fisiologia , Splicing de RNA , Proteínas de Ligação a RNA/fisiologia , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Diferenciação Celular , Células Espumosas/citologia , Células Espumosas/metabolismo , Regulação da Expressão Gênica , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Modelos Biológicos , Modelos Genéticos , Monócitos/citologia , Monócitos/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo
16.
Nucleic Acids Res ; 44(9): e83, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-26837572

RESUMO

Recent studies show that RNA-binding proteins (RBPs) and microRNAs (miRNAs) function in coordination with each other to control post-transcriptional regulation (PTR). Despite this, the majority of research to date has focused on the regulatory effect of individual RBPs or miRNAs. Here, we mapped both RBP and miRNA binding sites on human 3'UTRs and utilized this collection to better understand PTR. We show that the transcripts that lack competition for HuR binding are destabilized more after HuR depletion. We also confirm this finding for PUM1(2) by measuring genome-wide expression changes following the knockdown of PUM1(2) in HEK293 cells. Next, to find potential cooperative interactions, we identified the pairs of factors whose sites co-localize more often than expected by random chance. Upon examining these results for PUM1(2), we found that transcripts where the sites of PUM1(2) and its interacting miRNA form a stem-loop are more stabilized upon PUM1(2) depletion. Finally, using dinucleotide frequency and counts of regulatory sites as features in a regression model, we achieved an AU-ROC of 0.86 in predicting mRNA half-life in BEAS-2B cells. Altogether, our results suggest that future studies of PTR must consider the combined effects of RBPs and miRNAs, as well as their interactions.


Assuntos
MicroRNAs/genética , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/metabolismo , Regiões 3' não Traduzidas/genética , Sítios de Ligação/genética , Linhagem Celular Tumoral , Mapeamento Cromossômico , Biologia Computacional/métodos , Células HEK293 , Meia-Vida , Células HeLa , Humanos , Células MCF-7 , Conformação de Ácido Nucleico , Interferência de RNA , RNA Interferente Pequeno/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica/genética
17.
Sci Rep ; 6: 21643, 2016 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-26905650

RESUMO

Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis, and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells, and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and ß-catenin and can induce mRNA translation mediated by the 3'UTR of these genes. Reduced quaking levels attenuated VE-cadherin and ß-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity.


Assuntos
Antígenos CD/genética , Caderinas/genética , Células Endoteliais da Veia Umbilical Humana/fisiologia , Proteínas de Ligação a RNA/fisiologia , beta Catenina/genética , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Permeabilidade Capilar , Feminino , Expressão Gênica , Células HEK293 , Humanos , Fatores de Transcrição Kruppel-Like/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ativação Transcricional , beta Catenina/metabolismo
18.
Microrna ; 3(3): 144-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25541911

RESUMO

Integrity of the capillary network in the kidney is essential in the recovery from ischemia/ reperfusion injury (IRI), a phenomenon central to kidney transplantation and acute kidney injury. MicroRNA- 126 (miR-126) is known to be important in maintaining vascular homeostasis by facilitating vascular regeneration and modulating the mobilization of vascular progenitor cells. Stromal cell-derived factor 1 (SDF-1), important in the mobilization of vascular progenitor cells, is a direct target of miR-126 and modulation of miR-126 was previously shown to affect the number of circulating Sca-1(+)/Lin(-) vascular progenitor cells in a mouse model for hind limb ischemia. Here, we assessed the in vivo contribution of miR-126 to progenitor cell mobilization and kidney function following IRI in mice. A three day follow up of blood urea levels following kidney IRI demonstrated that systemic antagomir silencing of miR-126 did not impact the loss or subsequent restoration of kidney function. However, whole kidney lysates displayed elevated gene expression levels of Sdf-1, Vegf-A and eNOS after IRI as a result of systemic silencing of miR-126. Furthermore, FACS-analysis on whole blood three days after surgery revealed a marked up regulation of the number of circulating Sca-1(+)/Lin(-) progenitor cells in the antagomir-126 treated mice, in an ischemia dependent manner. Our data indicate that silencing of miR-126 can enhance renal expression of Sdf-1 after IRI, leading to the mobilization of vascular progenitor cells into the circulation.


Assuntos
Antígenos Ly/metabolismo , Quimiocina CXCL12/metabolismo , Células Progenitoras Endoteliais/metabolismo , Rim/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Antígenos Ly/genética , Quimiocina CXCL12/genética , Células Progenitoras Endoteliais/citologia , Inativação Gênica , Rim/irrigação sanguínea , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
J Am Soc Nephrol ; 25(8): 1710-22, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24610930

RESUMO

Ischemia/reperfusion injury (IRI) is a central phenomenon in kidney transplantation and AKI. Integrity of the renal peritubular capillary network is an important limiting factor in the recovery from IRI. MicroRNA-126 (miR-126) facilitates vascular regeneration by functioning as an angiomiR and by modulating mobilization of hematopoietic stem/progenitor cells. We hypothesized that overexpression of miR-126 in the hematopoietic compartment could protect the kidney against IRI via preservation of microvascular integrity. Here, we demonstrate that hematopoietic overexpression of miR-126 increases neovascularization of subcutaneously implanted Matrigel plugs in mice. After renal IRI, mice overexpressing miR-126 displayed a marked decrease in urea levels, weight loss, fibrotic markers, and injury markers (such as kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin). This protective effect was associated with a higher density of the peritubular capillary network in the corticomedullary junction and increased numbers of bone marrow-derived endothelial cells. Hematopoietic overexpression of miR-126 increased the number of circulating Lin(-)/Sca-1(+)/cKit(+) hematopoietic stem and progenitor cells. Additionally, miR-126 overexpression attenuated expression of the chemokine receptor CXCR4 on Lin(-)/Sca-1(+)/cKit(+) cells in the bone marrow and increased renal expression of its ligand stromal cell-derived factor 1, thus favoring mobilization of Lin(-)/Sca-1(+)/cKit(+) cells toward the kidney. Taken together, these results suggest overexpression of miR-126 in the hematopoietic compartment is associated with stromal cell-derived factor 1/CXCR4-dependent vasculogenic progenitor cell mobilization and promotes vascular integrity and supports recovery of the kidney after IRI.


Assuntos
Injúria Renal Aguda/prevenção & controle , Células-Tronco Hematopoéticas/fisiologia , Rim/irrigação sanguínea , MicroRNAs/fisiologia , Neovascularização Fisiológica/fisiologia , Traumatismo por Reperfusão/prevenção & controle , Injúria Renal Aguda/metabolismo , Injúria Renal Aguda/patologia , Animais , Movimento Celular/fisiologia , Quimiocina CXCL12/metabolismo , Rim/metabolismo , Rim/patologia , Masculino , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia
20.
Circ Res ; 113(9): 1065-75, 2013 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-23963726

RESUMO

RATIONALE: RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. OBJECTIVE: We sought to determine the role of QKI in regulating adult VSMC function and plasticity. METHODS AND RESULTS: We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. CONCLUSIONS: We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.


Assuntos
Proliferação de Células , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Lesões das Artérias Carótidas/metabolismo , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Movimento Celular , Reestenose Coronária/metabolismo , Reestenose Coronária/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Quaking , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Interferência de RNA , Proteínas de Ligação a RNA/genética , Transativadores/genética , Transativadores/metabolismo , Transfecção
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