Hyperhomocysteinemia potentiates megakaryocyte differentiation and thrombopoiesis via GH-PI3K-Akt axis

Abstract

Hyperhomocysteinemia (HHcy) is closely associated with thrombotic diseases such as myocardial infarction and stroke. Enhanced platelet activation was observed in animals and humans with HHcy. However, the influence of HHcy on thrombopoiesis remains largely unknown. Here, we reported increased platelet count (PLT) in mice and zebrafish with HHcy. In hypertensive patients (n = 11,189), higher serum level of total Hcy was observed in participants with PLT ≥ 291 × 10⁹/L (full adjusted β, 0.59; 95% CI 0.14, 1.04). We used single-cell RNA sequencing (scRNA-seq) to characterize the impact of Hcy on transcriptome, cellular heterogeneity, and developmental trajectories of megakaryopoiesis from human umbilical cord blood (hUCB) CD34⁺ cells. Together with in vitro and in vivo analysis, we demonstrated that Hcy promoted megakaryocytes (MKs) differentiation via growth hormone (GH)-PI3K-Akt axis. Moreover, the effect of Hcy on thrombopoiesis is independent of thrombopoietin (TPO) because administration of Hcy also led to a significant increase of PLT in homozygous TPO receptor (Mpl) mutant mice and zebrafish. Administration of melatonin effectively reversed Hcy-induced thrombopoiesis in mice. ScRNA-seq showed that melatonin abolished Hcy-facilitated MK differentiation and maturation, inhibited the activation of GH-PI3K-Akt signaling. Our work reveals a previously unrecognized role of HHcy in thrombopoiesis and provides new insight into the mechanisms by which HHcy confers an increased thrombotic risk.Trial Registration clinicaltrials.gov Identifier: NCT00794885.

Keywords: Growth hormone; Hyperhomocysteinemia; Megakaryocyte; ScRNA-seq; Thrombopoiesis.

Fig. 1

Hcy facilitates MKs differentiation and platelet production. A Peripheral PLT in C57BL/6J mice. Significance according to two-tailed unpaired t test (n = 8). B Representative images for staining and C quantification of mpl:eGFP⁺ cells (green) in zebrafish Tg(mpl:eGFP)smu4 larvae caudal hematopoietic tissue (CHT) region. Scale bars, 50 μm. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 10). D PLT recovery after platelet depletion by monoclonal rat anti-mouse CD42b antibody (Anti-CD42b MoAb). Significance according to two-tailed unpaired t test (n ≥ 5). E PLT in splenectomized mice. Significance according to two-tailed unpaired t test (n = 8). F PLT in C57BL/6J-Mpl^hlb219/J mice. Significance according to two-tailed unpaired t test (n ≥ 5). G Representative images for staining and H quantification of mpl:eGFP⁺ cells (green) at mpl-mutational zebrafish Tg(mpl:eGFP)smu4;mpl^smu3 larvae CHT region. Scale bars, 50 μm. Significance according to Welch ANOVA test with Dunnett T3 multiple comparisons test (n = 10). I 13 cell clusters were displayed by UMAP. Colors indicate cell types. CMP, common myeloid progenitor; GMP, granulocyte-monocyte progenitor; MEMP, megakaryocyte-erythroid-mast cell progenitor; MK, megakaryocyte. J Bar diagram showing the representative GO biological process terms of MKs subpopulations. K Bar diagram showing the percentage of each MKs subpopulation number to all cells. L Venn diagram visualizing the up-regulated biological processes of each MKs subpopulation in Hcy group compared with control group. M Representative images of PPF detected by phase contrast imaging (left) and confocal microscopy (right). β1-tubulin (green) and DAPI (blue) were stained; Scale bars, 20 μm. N Histogram showing the number of PPF-MKs. Significance according to two-tailed unpaired t test (n = 6). O Representative images and P quantification of CD41⁺ MKs (green) in mice femurs bone marrow by immunofluorescence staining. Scale bars, 50 μm. Significance according to two-tailed unpaired t test (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant

Fig. 2

Hcy promotes MKs differentiation via GH-PI3K-Akt axis. A Venn diagram visualizing the elevated Pathway Interaction Database pathways of MKs subpopulations in Hcy group. B Heatmap showing the relative GSVA scores for each gene set based on bulk RNA-seq of BM MKs (n = 4). C and D Western blot analysis of p-PI3K and p-AKT (Ser473) in Meg-01 cells after exposure to Hcy (100 μM) for indicated time period. Total PI3K, AKT and GAPDH were used as loading control. Significance according to one-way ANOVA with LSD multiple comparisons test (n = 3). E Culture-derived MKs and F platelets were analyzed by flow cytometry. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 3). G PLT in Ghr^−/− mice. Significance according to two-tailed unpaired t test (n = 6). H Quantification of CD41⁺ MKs in femurs bone marrow of Ghr^−/− mice. Significance according to Mann–Whitney test (n = 6). I and J Western blot analysis of p-PI3K and p-AKT (Ser473) in the bone marrow MKs after exposure to Hcy (100 μM) for 30 min. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 6). K Bar diagram showing the percentage of MK2 to all cells. L Peripheral PLT. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 8). M Representative images and N quantification of CD41⁺ MKs (green) in femurs bone marrow of mice. Scale bars, 50 μm. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 8). O Bar chart showing the significantly down-regulated GO-BP terms and P PID pathways in MKs. T values are from the linear model in the limma package. Q–R Western blot analysis the level of p-PI3K and p-AKT (Ser473) in Meg-01 cells after exposure to Hcy (100 μM) with or without MT (1 μM) for 30 min. Significance according to one-way ANOVA with Tukey multiple comparisons test (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns not significant