RESUMO
BACKGROUND: Endothelial colony-forming cells (ECFCs) derived from patients can be used to investigate pathogenic mechanisms of vascular diseases like von Willebrand disease. Considerable phenotypic heterogeneity has been observed between ECFC clones derived from healthy donors. This heterogeneity needs to be well understood in order to use ECFCs as endothelial models for disease. OBJECTIVES: Therefore, we aimed to determine phenotypic and gene expression differences between control ECFCs. METHODS: A total of 34 ECFC clones derived from 16 healthy controls were analyzed. The transcriptome of a selection of ECFC clones (n = 15) was analyzed by bulk RNA sequencing and gene set enrichment analysis. Gene expression was measured in all ECFC clones by quantitative polymerase chain reaction. Phenotypic profiling was performed and migration speed of the ECFCs was measured using confocal microscopy, followed by automated quantification of cell morphometrics and migration speed. RESULTS: Through hierarchical clustering of RNA expression profiles, we could distinguish 2 major clusters within the ECFC cohort. Major differences were associated with proliferation and migration in cluster 1 and inflammation and endothelial-to-mesenchymal transition in cluster 2. Phenotypic profiling showed significantly more and smaller ECFCs in cluster 1, which contained more and longer Weibel-Palade bodies. Migration speed in cluster 1 was also significantly higher. CONCLUSION: We observed a range of different RNA expression patterns between ECFC clones, mostly associated with inflammation and clear differences in Weibel-Palade body count and structure. We developed a quantitative polymerase chain reaction panel that can be used for the characterization of ECFC clones, which is essential for the correct analysis of pathogenic mechanisms in vascular disorders.
Assuntos
Movimento Celular , Perfilação da Expressão Gênica , Inflamação , Fenótipo , Transcriptoma , Humanos , Inflamação/genética , Células Cultivadas , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Transição Epitelial-Mesenquimal , Proliferação de Células , Células Progenitoras Endoteliais/metabolismo , Células Progenitoras Endoteliais/patologia , Masculino , Feminino , Pessoa de Meia-Idade , Adulto , Transcrição GênicaRESUMO
INTRODUCTION: Von Willebrand factor (VWF) plays a pathophysiological role in hemostatic disorders. Partial inhibition of the VWF gene through small interfering RNA (siRNA)-mediated allele-selective silencing could be a promising therapeutic strategy. For von Willebrand disease, allele-selectively inhibiting dominant-negative VWF-alleles might ameliorate the phenotype. For thrombotic disorders, partial VWF reduction can lower thrombotic risk, while avoiding bleeding. Previously, we demonstrated the feasibility of Vwf-silencing in homozygous C57BL/6J (B6) or 129S1/SvImJ (129S) mice. The present study investigated allele-selective Vwf-silencing in a complex heterozygous setting of crossed B6 and 129S mice and its subsequent hemostatic impact. MATERIALS AND METHODS: Heterozygous B6.129S mice were treated with siRNAs targeting Vwf expressed from either B6- (siVwf.B6) or 129S-alleles (siVwf.129S). Plasma VWF and lung Vwf mRNA were determined. siVwf.B6-treated B6.129S mice were subjected to ferric chloride-induced mesenteric vessel thrombosis and tail-bleeding. RESULTS: In B6.129S mice, siVwf.B6 reduced Vwf mRNA of the targeted B6-allele by 72% vs. only 12% of the non-targeted 129S-allele (41% total mRNA reduction), lowering plasma VWF by 46%. Oppositely, siVwf.129S reduced Vwf mRNA by 45%, now selectively inhibiting the 129S-allele over the B6-allele (58% vs. 9%), decreasing plasma VWF by 43%. The allele-selective VWF reduction by siVwf.B6 coincided with decreased thrombus formation in mesenteric arterioles, without prolonging tail-bleeding times. CONCLUSIONS: This study demonstrates the feasibility of allele-selective Vwf-silencing in a heterozygous setting, achieving a controlled close to 50% reduction of plasma VWF. The observed thromboprotection and absence of prolonged bleeding times underline the potential of allele-selective Vwf-silencing as a therapeutic strategy in hemostatic disorders.
Assuntos
Transtornos Hemostáticos , Fator de von Willebrand , Animais , Camundongos , Alelos , Hemorragia/genética , Camundongos Endogâmicos C57BL , RNA Mensageiro , Trombose/genética , Doenças de von Willebrand , Fator de von Willebrand/genéticaRESUMO
One of the most used and versatile methods to study number, dimensions, content and localization of secretory organelles is confocal microscopy analysis. However, considerable heterogeneity exists in the number, size and shape of secretory organelles that can be present in the cell. One thus needs to analyze large numbers of organelles for valid quantification. Properly evaluating these parameters requires an automated, unbiased method to process and quantitatively analyze microscopy data. Here, we describe two pipelines, run by CellProfiler software, called OrganelleProfiler and OrganelleContentProfiler. These pipelines were used on confocal images of endothelial colony forming cells (ECFCs), which contain unique secretory organelles called Weibel-Palade bodies (WPBs), and on early endosomes in ECFCs and human embryonic kidney 293T (HEK293T) cells. Results show that the pipelines can quantify the cell count, size, organelle count, organelle size, shape, relation to cells and nuclei, and distance to these objects in both endothelial and HEK293T cells. Additionally, the pipelines were used to measure the reduction in WPB size after disruption of the Golgi and to quantify the perinuclear clustering of WPBs after triggering of cAMP-mediated signaling pathways in ECFCs. Furthermore, the pipeline is able to quantify secondary signals located in or on the organelle or in the cytoplasm, such as the small WPB GTPase Rab27A. Cell profiler measurements were checked for validity using Fiji. To conclude, these pipelines provide a powerful, high-processing quantitative tool for the characterization of multiple cell and organelle types. These pipelines are freely available and easily editable for use on different cell types or organelles.