The impact of chronic intermittent hypoxia on hematopoiesis and the bone marrow microenvironment

Abstract

Obstructive sleep apnea (OSA) is a highly prevalent sleep-related breathing disorder which is associated with patient morbidity and an elevated risk of developing hypertension and cardiovascular diseases. There is ample evidence for the involvement of bone marrow (BM) cells in the pathophysiology of cardiovascular diseases but a connection between OSA and modulation of the BM microenvironment had not been established. Here, we studied how chronic intermittent hypoxia (CIH) affected hematopoiesis and the BM microenvironment, in a rat model of OSA. We show that CIH followed by normoxia increases the bone marrow hypoxic area, increases the number of multipotent hematopoietic progenitors (CFU assay), promotes erythropoiesis, and increases monocyte counts. In the BM microenvironment of CIH-subjected animals, the number of VE-cadherin-expressing blood vessels, particularly sinusoids, increased, accompanied by increased smooth muscle cell coverage, while vWF-positive vessels decreased. Molecularly, we investigated the expression of endothelial cell-derived genes (angiocrine factors) that could explain the cellular phenotypes. Accordingly, we observed an increase in colony-stimulating factor 1, vascular endothelium growth factor, delta-like 4, and angiopoietin-1 expression. Our data shows that CIH induces vascular remodeling in the BM microenvironment, which modulates hematopoiesis, increasing erythropoiesis, and circulating monocytes. Our study reveals for the first time the effect of CIH in hematopoiesis and suggests that hematopoietic changes may occur in OSA patients.

Keywords: Bone marrow microenvironment; Chronic intermittent hypoxia; Hematopoiesis; Vascular niche.

Fig. 1

Chronic intermittent hypoxia affects the hypoxic state of the bone marrow and decreases body weight but does not affect bone marrow cell counts. a The extent of BM hypoxia was increased in animals exposed to CIH, as assessed by pimonidazole staining and b the significant increase in hypoxic area in CIH animals. c CIH also have increased Hif1α expression. d Rats subjected to CIH had a lower body weight than controls. e Total BM cell count shows that CIH does not modify BM cellularity. Results are represented as the mean ± SD of bone marrow sections from six male Wistar rats (*p < 0.05; **p < 0.01)

Fig. 2

Rats exposure to chronic intermittent hypoxia affects specific hematopoietic lineages and the commitment of bone marrow progenitor cells. a–d Representative plots of the flow cytometric analysis of bone marrow cells from normoxic (n = 5) and CIH (n = 5) exposed rats. Quantification of a’ CD90+/c-kit + stem and progenitor cells did not reveal a significant alteration in CIH animals. However, b’ CD11b⁺ myeloid- and d’ CD19⁺ B cells were increased as opposed to (c’) CD3 T lymphocytes that decreased upon CIH exposure. e Quantification of peripheral blood CD11b⁺ cells by flow cytometry also revealed an increase in the percentage of those cells in circulation. f Colony-forming unit counts from methylcellulose culture of 10⁵ BM cells reveal that CIH treatment induces an increased the number of HSPC, g with a particular increase in macrophage, granulocyte and erythroid (CFU-M, CFU-G and BFU-E) colonies. Results are represented as the mean ± SD of bone marrow cells from five male Wistar rats (*p < 0.05; **p < 0.01)

Fig. 3

Chronic intermittent hypoxia modulates circulating blood counts. a CIH may promote erythropoiesis. Erythrocyte, hemoglobin, and hematocrit, as well as mean corpuscular hemoglobin and mean cell hemoglobin concentration were assessed by peripheral blood cell counts. The erythrocyte count, hemoglobin, and hematocrit in CIH-exposed rats (n = 5) are significantly different from those in normoxia (n = 5) (*p < 0.05). b CIH increases circulating monocytes and decrease lymphocytes. However, peripheral blood cell counts showed no differences in neutrophils, eosinophils, or leukocytes. c Platelet count and mean platelet volume are not modified by exposure to CIH. Results are represented as the mean ± SD of blood samples from five male Wistar rats (*p < 0.05; **p < 0.01)

Fig. 4

Chronic intermittent hypoxia modifies the BM vascular structure. a’–e’ Representative images of femur bone marrow stained with vWF, CD105, VE-cadherin, SMA, and CD11b counterstained with hematoxylin. a”, c”, d” BM from CIH exposed rats (n = 6) has more VE-cadherin⁺ vessels and SMA coverage but less vWF⁺ sinusoids (400×, Leica DM2500). e’, e” Representative images of CD11b immunohistochemistry in femur BM show an increase in BM monocyte count in CIH exposed animals. (400×, Leica DM2500) a’, a”’, b’, b” No changes in the total number of vessels or in megakaryocyte count were observed, as accounted by CD105 and vWF staining, respectively. Results are represented as the mean ± SD of bone marrow sections from six male Wistar rats (*p < 0.05; **p < 0.01). f Representative images of femur bone marrow fluorescently immunostained for VE-cadherin show an increase in total VE-cadherin vessels and in VE-cadherin vessel coverage. Scale bar, 50 μm (insets magnified 2.5×). Images were acquired with a Zeiss LSM 510 META microscope

Fig. 5

Chronic intermittent hypoxia modulates bone marrow angiocrine gene expression. a Angiocrine gene modulation was assessed by relative quantification of mRNA of total BM samples from normoxia (n = 6) and CIH (n = 6) treated rats. As determined by RT-PCR, we observed an increase in Vegfa, Dll4, Angpt1, Dhh, and Csf1. b In addition, we also measured an increase in the expression of Flt4. Data are represented as mean ± SD of six male Wistar rats (*p < 0.05; **p < 0.01; ***p < 0.001)