Abstract
Although thrombosis has been extensively studied using various animal models, our understanding of the underlying mechanism remains elusive. Here, using zebrafish model, we demonstrated that smarca5-deficient red blood cells (RBCs) formed blood clots in the caudal vein plexus. We further used the anti-thrombosis drugs to treat smarca5zko1049a embryos and found that a thrombin inhibitor, argatroban, partially prevented blood clot formation in smarca5zko1049a. To explore the regulatory mechanism of smarca5 in RBC homeostasis, we profiled the chromatin accessibility landscape and transcriptome features in RBCs from smarca5zko1049a and their siblings and found that both the chromatin accessibility at the keap1a promoter and expression of keap1a were decreased. Keap1 is a suppressor protein of Nrf2, which is a major regulator of oxidative responses. We further identified that the expression of hmox1a, a downstream target of Keap1-Nrf2 signaling pathway, was markedly increased upon smarca5 deletion. Importantly, overexpression of keap1a or knockdown of hmox1a partially rescued the blood clot formation, suggesting that the disrupted Keap1-Nrf2 signaling is responsible for the RBC aggregation in smarca5 mutants. Together, our study using zebrafish smarca5 mutants characterizes a novel role for smarca5 in RBC aggregation, which may provide a new venous thrombosis animal model to support drug screening and pre-clinical therapeutic assessments to treat thrombosis.
Highlights
The erythrocytes, or red blood cells (RBCs), are highly differentiated cells produced during erythropoiesis
There was no difference in the distribution of myeloid cells labelled by transgenic line 105 (Tg) or Tg in caudal hematopoietic tissue (CHT) between smarca5zko1049a and their siblings, and we did not observe accumulation of myeloid cells in the blood clots of smarca5zko1049a (Figure 1-figure supplement 1A). To further determine whether smarca5 is involved in the development of primitive hematopoiesis, we examined the expression level of gata1 and pu.1, which are the erythrocyte and myeloid marker genes, respectively, in smarca5zko1049a and their siblings
whole mount in situ hybridization (WISH) and quantitative PCR analyses showed that the expression level of gata1 and pu.1 was comparable between smarca5zko1049a and their siblings at 33 hours post fertilization (Figure 1D-E and supplemental Figure 1B-C)
Summary
The erythrocytes, or red blood cells (RBCs), are highly differentiated cells produced during erythropoiesis. Epigenetic regulation of RBC-related genes is fundamental for normal development and maintenance of RBCs (Hewitt, Sanalkumar, Johnson, Keles, & Bresnick, 2014). Phenylhydrazine causes externalization of phosphatidylserine on plasma of RBC membrane and generates oxidative radicals, thereafter, resulting in the thrombosis formation. These studies in animal models shed light on the understanding and treatment of vaso-occlusion phenotype in patients with RBC defects. We develop a new zebrafish RBC aggregation model with a deletion of smarca, loss of which leads to the formation of blood clots in the caudal vein plexus (CVP). Our smarca5-deficient zebrafish model may serve as a new venous thrombosis model for drug screening in clinical therapy
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