A Simple Method to AssessIn VivoProliferation in Lung Vasculature with EdU: The Case of MMC-Induced PVOD in Rat

Abstract

5-Ethynyl-2′-deoxyuridine (EdU) incorporation is becoming the gold standard method for in vitro and in vivo visualization of proliferating cells. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration. It can be used to easily detect DNA replication in large tissue samples or organ explants with low proliferation and turnover of cells formerly believed to be in a “terminal” state of differentiation. Here we describe a protocol for the localization and identification of proliferating cells in quiescent or injured pulmonary vasculature, in a model of pulmonary veno-occlusive disease (PVOD). PVOD is an uncommon form of pulmonary hypertension characterized by progressive obstruction of small pulmonary veins. We previously reported that mitomycin-C (MMC) therapy is associated with PVOD in human. We demonstrated that MMC can induce PVOD in rats, which currently represents the sole animal model that recapitulates human PVOD lesions. Using the EdU assay, we demonstrated that MMC-exposed lungs displayed areas of exuberant microvascular endothelial cell proliferation which mimics pulmonary capillary hemangiomatosis, one of the pathologic hallmarks of human PVOD. In vivo pulmonary cell proliferation measurement represents an interesting methodology to investigate the potential efficacy of therapies aimed at normalizing pathologic angioproliferation.

Patient-specific and gene-corrected induced pluripotent stem cell-derived endothelial cells elucidate single-cell phenotype of pulmonary veno-occlusive disease

“…These data clearly indicate that EIF2AK4 compound heterozygous variants regulate EC proliferation. This is consistent with a previous finding, in which mitomycin (MMC)-exposed PVOD rat lungs presented areas of exuberant microvascular endothelial cell proliferation ( Fabrice et al., 2015 ). Tube formation assay is one of the most widely used in vitro assays to model the reorganization of angiogenesis, which plays a critical role in microvascular growth ( Fabrice et al., 2015 ).…”

“…This is consistent with a previous finding, in which mitomycin (MMC)-exposed PVOD rat lungs presented areas of exuberant microvascular endothelial cell proliferation ( Fabrice et al., 2015 ). Tube formation assay is one of the most widely used in vitro assays to model the reorganization of angiogenesis, which plays a critical role in microvascular growth ( Fabrice et al., 2015 ). As shown in Figure 2 E, PVOD-iPSC-ECs had a more aggressive tube formation capacity, when compared with the other lines, indicating that the angiogenesis involved in the pathogenesis of PVOD was caused by the EIF2AK4 compound heterozygous variants.…”

MUS81 is associated with cell proliferation and cisplatin sensitivity in serous ovarian cancer

“…2B) in both transduced SKOV3 and A2780 cells (P < 0.05). 5-Ethynyl-2 0 -deoxyuridine (EdU) incorporation is becoming the gold standard method for in vitro and in vivo visualization of proliferating cells [19], thus we further performed this experiment to verify the proliferation of transduced cells. As expected, compared with shCtrl group, the percentage of EdU þ cells in shMUS81-1/shMUS81-2 group decreased significantly (Fig.…”

In vivo miR-138-5p inhibition alleviates monocrotaline-induced pulmonary hypertension and normalizes pulmonary KCNK3 and SLC45A3 expression

“…Cell proliferation in the rats was analysed by quantifying the pulmonary cells that incorporated the exogenously supplied 5-ethynyl-2′-deoxyuridine (EdU) to identify the cells undergoing DNA replication. EdU was intraperitoneally injected into the rats at 50 μg/g 24 h before sacrifice [ 21 ]. EdU staining was performed using the Click-iT EdU imaging kit (C10337, Life Technologies) on frozen sections according to the manufacturer’s instructions.…”