A segmental approach from molecular profiling to medical imaging to study bicuspid aortic valve aortopathy

Bicuspid aortic valve (BAV) patients develop ascending aortic (AAo) dilation. The pathogenesis of BAV aortopathy (genetic vs. haemodynamic) remains unclear. This study aims to identify regional changes around the AAo wall in BAV patients with aortopathy, integrating molecular data and clinical imaging. BAV patients with aortopathy (n = 15) were prospectively recruited to surgically collect aortic tissue and measure molecular markers across the AAo circumference. Dilated (anterior/right) vs. non-dilated (posterior/left) circumferential segments were profiled for whole-genomic microRNAs (next-generation RNA sequencing, miRCURY LNA PCR), protein content (tandem mass spectrometry), and elastin fragmentation and degeneration (histomorphometric analysis). Integrated bioinformatic analyses of RNA sequencing and proteomic datasets identified five microRNAs (miR-128-3p, miR-210-3p, miR-150-5p, miR-199b-5p, and miR-21-5p) differentially expressed across the AAo circumference. Among them, three miRNAs (miR-128-3p, miR-150-5p, and miR-199b-5p) were predicted to have an effect on eight common target genes, whose expression was dysregulated, according to proteomic analyses, and involved in the vascular-endothelial growth-factor signalling, Hippo signalling, and arachidonic acid pathways. Decreased elastic fibre levels and elastic layer thickness were observed in the dilated segments. Additionally, in a subset of patients n = 6/15, a four-dimensional cardiac magnetic resonance (CMR) scan was performed. Interestingly, an increase in wall shear stress (WSS) was observed at the anterior/right wall segments, concomitantly with the differentially expressed miRNAs and decreased elastic fibres. This study identified new miRNAs involved in the BAV aortic wall and revealed the concomitant expressional dysregulation of miRNAs, proteins, and elastic fibres on the anterior/right wall in dilated BAV patients, corresponding to regions of elevated WSS

Microvesicles from indoxyl sulfate-treated endothelial cells induce vascular calcification in vitro

Vascular calcification (VC), an unpredictable pathophysiological process and critical event in patients with cardiovascular diseases (CVDs), is the leading cause of morbi-mortality and disability in chronic kidney disease (CKD) patients worldwide. Currently, no diagnostic method is available for identifying patients at risk of VC development; the pathology is detected when the process is irreversible. Extracellular vesicles (EVs) from endothelial cells might promote VC. Therefore, their evaluation and characterization could be useful for designing new diagnostic tools. The aim of the present study is to investigate whether microvesicles (MVs) from endothelial cells damaged by uremic toxin and indoxyl sulfate (IS) could induce calcification in human vascular smooth muscle cells (VMSCs). Besides, we have also analyzed the molecular mechanisms by which these endothelial MVs can promote VC development. Endothelial damage has been evaluated according to the percentage of senescence in endothelial cells, differential microRNAs in endothelial cells, and the amount of MVs released per cell. To identify the role of MVs in VC, VSMCs were treated with MVs from IS-treated endothelial cells. Calcium, inflammatory gene expression, and procalcification mediator levels in VSMCs were determined. IS-treated endothelial cells underwent senescence and exhibited modulated microRNA expression and an increase in the release of MVs. VSMCs exposed to these MVs modulated the expression of pro-inflammatory genes and some mediators involved in calcification progression. MVs produced by IS-treated endothelial cells promoted calcification in VSMCs

Synthetic microparticles conjugated with VEGF165 improve the survival of endothelial progenitor cells via microRNA-17 inhibition

Several cell-based therapies are under pre-clinical and clinical evaluation for the treatment of ischemic diseases. Poor survival and vascular engraftment rates of transplanted cells force them to work mainly via time-limited paracrine actions. Although several approaches, including the use of soluble vascular endothelial growth factor (sVEGF)-VEGF165, have been developed in the last 10 years to enhance cell survival, they showed limited efficacy. Here, we report a pro-survival approach based on VEGF-immobilized microparticles (VEGF-MPs). VEGF-MPs prolong VEGFR-2 and Akt phosphorylation in cord blood-derived late outgrowth endothelial progenitor cells (OEPCs). In vivo, OEPC aggregates containing VEGF-MPs show higher survival than those treated with sVEGF. Additionally, VEGF-MPs decrease miR-17 expression in OEPCs, thus increasing the expression of its target genes CDKN1A and ZNF652. The therapeutic effect of OEPCs is improved in vivo by inhibiting miR-17. Overall, our data show an experimental approach to improve therapeutic efficacy of proangiogenic cells for the treatment of ischemic diseases.Soluble vascular endothelial growth factor (VEGF) enhances vascular engraftment of transplanted cells but the efficacy is low. Here, the authors show that VEGF-immobilized microparticles prolong survival of endothelial progenitors in vitro and in vivo by downregulating miR17 and upregulating CDKN1A and ZNF652

Changes in circulating microRNA levels can be identified as early as day 8 of pregnancy in cattle

<div><p>Poor reproductive performance remains a major issue in the dairy industry, with low conception rates having a significant impact on milk production through extended calving intervals. A major limiting factor is the lack of reliable methods for early pregnancy diagnosis. Identification of animals within a herd that fail to conceive within 3 weeks after insemination would allow early re-insemination and shorten calving intervals. In a previous study, we found an increase in plasma miR-26a levels in Day 16-pregnant relative to non-pregnant heifers, however changes in miRNA levels that early during pregnancy were very small which likely prevented the identification of robust biomarkers. In this study, we extended our analyses to a wider interval during pregnancy (Days 8 to 60, n = 11 heifers) with the rationale that this may facilitate the identification of additional early pregnancy miRNA biomarkers. Using small RNA sequencing we identified a total of 77 miRNAs that were differentially expressed on Day 60 relative to Day 0 of pregnancy. We selected 14 miRNAs for validation by RT-qPCR and confirmed significant differences in the expression of let-7f, let-7c, miR-30c, miR-101, miR-26a, miR-205 and miR-143 between Days 0 and 60. RT-qPCR profiling throughout Days 0, 8, 16 and 60 of pregnancy showed a distinct increase in circulating levels of miR-26a (3.1-fold, P = 0.046) as early as Day 8 of pregnancy. In summary, in contrast to earlier stages of pregnancy (≤ Day 24), marked differences in the levels of multiple miRNAs can be detected in circulation by Day 60 in cattle. Retrospective analyses showed miR-26a levels to be increased in circulation as early as Day 8, sooner than previously reported in any species, suggesting a biological role for this miRNA in the very early events of pregnancy.</p></div

Extracellular Vesicle miRNAs in the Promotion of Cardiac Neovascularisation

Cardiovascular disease (CVD) is the leading cause of mortality worldwide claiming almost 17. 9 million deaths annually. A primary cause is atherosclerosis within the coronary arteries, which restricts blood flow to the heart muscle resulting in myocardial infarction (MI) and cardiac cell death. Despite substantial progress in the management of coronary heart disease (CHD), there is still a significant number of patients developing chronic heart failure post-MI. Recent research has been focused on promoting neovascularisation post-MI with the ultimate goal being to reduce the extent of injury and improve function in the failing myocardium. Cardiac cell transplantation studies in pre-clinical models have shown improvement in cardiac function; nonetheless, poor retention of the cells has indicated a paracrine mechanism for the observed improvement. Cell communication in a paracrine manner is controlled by various mechanisms, including extracellular vesicles (EVs). EVs have emerged as novel regulators of intercellular communication, by transferring molecules able to influence molecular pathways in the recipient cell. Several studies have demonstrated the ability of EVs to stimulate angiogenesis by transferring microRNA (miRNA, miR) molecules to endothelial cells (ECs). In this review, we describe the process of neovascularisation and current developments in modulating neovascularisation in the heart using miRNAs and EV-bound miRNAs. Furthermore, we critically evaluate methods used in cell culture, EV isolation and administration

Mecanismos de contracción-relajación vascular y celular: papel central de la guanilato ciclasa soluble y de

Es un hecho ampliamente demostrado que las alteraciones en la vía de señalización del óxido nítrico?guanilato soluble?proteína kinasa G (NO-GCs-PKG) están relacionadas con la génesis de la disfunción endotelial. Previamente se ha demostrado que la expresión de la eNOS y GCs depende de la interacción de las células con la matriz extracelular a través de las integrinas, pero no se han descrito efectos similares en la expresión de PKG. El principal objetivo de este estudio fue evaluar si el contenido celular de PKG dependía de la activación de integrinas y testar la posibilidad de que GCs y PKG pudiesen ser moduladas por la estimulación de integrinas in vivo. La administración de tirofiban en ratas incrementó el contenido de GCs (ß1) en la pared vascular. Como consecuencia de este aumento, también incrementó la respuesta farmacológica a donadores de NO, mejoró la tolerancia asociada con el tratamiento prolongado con nitritos e incrementó la capacidad de reducir la presión arterial sistólica de los donadores de NO en un modelo genético de hipertensión. Por otro lado, la fibronectina incrementó el contenido proteico de PKG I¿ en células mesangiales humanas a través de la interacción con la integrina b1. Akt está implicado en la modulación de la expresión de PKG I¿ por fibronectina. La administración de tirofiban en ratas produjo un aumento del contenido proteico de PKG I¿ en la pared vascular y además, el tirofiban favoreció la respuesta hipotensora del dibutiril GMPc (dbGMPc) en estos animales, lo cual sugiere la relevancia funcional de los cambios detectados en el contenido de PKG I¿ en vaso. La dilucidación de este nuevo mecanismo proporciona una base para posibles farmacoterapias aplicables a determinadas enfermedades vasculares. Estudios previos han demostrado que la sobreexpresión de H-Ras en animales modificados genéticamente produce hipertensión e hipertrofia cardiaca, lo cual sugiere que H-Ras tiene un papel importante en la regulación de la presión arterial. El segundo objetivo de este trabajo fue estudiar el efecto de la ausencia de H-Ras en la presión arterial en ratones deficientes en la isoforma H-Ras. Los ratones H-Ras -/- presentaron una disminución de la presión arterial y un aumento en la respuesta a diferentes agonistas del sistema NO-GCs-PKG, concretamente nitroprusiato sódico (SNP) y dbGMPc. El tratamiento con antagonistas del sistema del NO revertió la hipotensión en los ratones H-Ras -/-. Además, estos animales presentaron un mayor contenido proteico y actividad de eNOS, GCs (¿) y PKG I en pared vascular que los ratones control. Los resultados obtenidos sugieren que H-Ras se podría considerar una diana terapéutica en el campo del tratamiento para la hipertensión

Mecanismos de contracción-relajación vascular y celular: papel central de la guanilato ciclasa soluble y de

Es un hecho ampliamente demostrado que las alteraciones en la vía de señalización del óxido nítrico?guanilato soluble?proteína kinasa G (NO-GCs-PKG) están relacionadas con la génesis de la disfunción endotelial. Previamente se ha demostrado que la expresión de la eNOS y GCs depende de la interacción de las células con la matriz extracelular a través de las integrinas, pero no se han descrito efectos similares en la expresión de PKG. El principal objetivo de este estudio fue evaluar si el contenido celular de PKG dependía de la activación de integrinas y testar la posibilidad de que GCs y PKG pudiesen ser moduladas por la estimulación de integrinas in vivo. La administración de tirofiban en ratas incrementó el contenido de GCs (ß1) en la pared vascular. Como consecuencia de este aumento, también incrementó la respuesta farmacológica a donadores de NO, mejoró la tolerancia asociada con el tratamiento prolongado con nitritos e incrementó la capacidad de reducir la presión arterial sistólica de los donadores de NO en un modelo genético de hipertensión. Por otro lado, la fibronectina incrementó el contenido proteico de PKG I¿ en células mesangiales humanas a través de la interacción con la integrina b1. Akt está implicado en la modulación de la expresión de PKG I¿ por fibronectina. La administración de tirofiban en ratas produjo un aumento del contenido proteico de PKG I¿ en la pared vascular y además, el tirofiban favoreció la respuesta hipotensora del dibutiril GMPc (dbGMPc) en estos animales, lo cual sugiere la relevancia funcional de los cambios detectados en el contenido de PKG I¿ en vaso. La dilucidación de este nuevo mecanismo proporciona una base para posibles farmacoterapias aplicables a determinadas enfermedades vasculares. Estudios previos han demostrado que la sobreexpresión de H-Ras en animales modificados genéticamente produce hipertensión e hipertrofia cardiaca, lo cual sugiere que H-Ras tiene un papel importante en la regulación de la presión arterial. El segundo objetivo de este trabajo fue estudiar el efecto de la ausencia de H-Ras en la presión arterial en ratones deficientes en la isoforma H-Ras. Los ratones H-Ras -/- presentaron una disminución de la presión arterial y un aumento en la respuesta a diferentes agonistas del sistema NO-GCs-PKG, concretamente nitroprusiato sódico (SNP) y dbGMPc. El tratamiento con antagonistas del sistema del NO revertió la hipotensión en los ratones H-Ras -/-. Además, estos animales presentaron un mayor contenido proteico y actividad de eNOS, GCs (¿) y PKG I en pared vascular que los ratones control. Los resultados obtenidos sugieren que H-Ras se podría considerar una diana terapéutica en el campo del tratamiento para la hipertensión