Bone marrow endothelial dysfunction promotes myeloid cell expansion in cardiovascular disease

Abnormal hematopoiesis advances cardiovascular disease by generating excess inflammatory leukocytes that attack the arteries and the heart. The bone marrow niche regulates hematopoietic stem cell proliferation and hence the systemic leukocyte pool, but whether cardiovascular disease affects the hematopoietic organ’s microvasculature is unknown. Here we show that hypertension, atherosclerosis and myocardial infarction (MI) instigate endothelial dysfunction, leakage, vascular fibrosis and angiogenesis in the bone marrow, altogether leading to overproduction of inflammatory myeloid cells and systemic leukocytosis. Limiting angiogenesis with endothelial deletion of Vegfr2 (encoding vascular endothelial growth factor (VEGF) receptor 2) curbed emergency hematopoiesis after MI. We noted that bone marrow endothelial cells assumed inflammatory transcriptional phenotypes in all examined stages of cardiovascular disease. Endothelial deletion of Il6 or Vcan (encoding versican), genes shown to be highly expressed in mice with atherosclerosis or MI, reduced hematopoiesis and systemic myeloid cell numbers in these conditions. Our findings establish that cardiovascular disease remodels the vascular bone marrow niche, stimulating hematopoiesis and production of inflammatory leukocytes.

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Policy information about availability of computer code Data collection Flow cytometry data were acquired on an LSRII flow cytometer equipped with FACS Diva 6.1 software (BD Biosciences, San Jose, CA, USA).

Data analysis
Flow cytometry data were analyzed with FlowJo 10 software (BD, Franklin Lakes, NJ, USA). Light microscopy and immunofluorescence images were analyzed using ImageJ 1.51r software. 3D reconstruction of PET, CT, and MRI datasets was done with Amira 5.3.2 software (Thermo Fisher Scientific, Waltham, MA, USA). Confocal microscopy images were analyzed using AngioTool 0.5, ImageJ 1.51r, and MATLAB R2015b software (Mathworks, Natick, MA, USA). PET/MRI data were analyzed with MATLAB R2015b software. RNA-seq data were processed and analyzed using STAR aligner 2.7.3a, edgeR 3.28.0, GSEA 4.0.3., and clusterProfiler. GraphPad Prism 8 software (GraphPad, San Diego, CA, USA) was used for statistical analysis.
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March 2021
Data Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy RNA-seq data were deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession number GSE144498 (bone marrow endothelial cells).

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Population characteristics
Arterial hypertension patients had previously diagnosed arterial hypertension (based on office systolic blood pressure ≥140 mmHg and/or diastolic blood pressure ≥90 mmHg in repeated measurements) and chronic anti-hypertensive drug treatment. 83.33% of patients in this group had systolic blood pressure values ≥140 mmHg and/or diastolic blood pressure values ≥90 mmHg at the time of sample collection. Atherosclerosis and arterial hypertension patients had previously diagnosed coronary artery disease, cerebrovascular disease and/or peripheral artery disease combined with arterial hypertension. Sample collection in patients with acute myocardial infarction was performed between day 3 and day 7 after diagnosis of ST-segment elevation myocardial infarction (STEMI). Percutaneous coronary intervention (PCI) was performed in all patients on the day of onset of clinical symptoms. Healthy control subjects had no medical history for any of the aforementioned diagnoses and no chronic pharmacological treatment. Additional bone marrow samples from healthy controls were purchased from Stemcell Technologies (Cambridge, MA, USA) and HemaCare (Los Angeles, CA, USA). Age distribution in controls and patient cohorts

Recruitment
Patient recruitment was performed at University Hospital Germans Trias i Pujol, Amsterdam University Medical Center, and Massachusetts General Hospital. Patients were eligible for enrollment with a) one or more of the following previously diagnosed medical conditions or b) non of the following diagnoses and no chronic pharmacological treatment: 1) arterial hypertension (with chronic anti-hypertensive drug treatment), 2) atherosclerosis (defined as coronary artery disease, cerebrovascular disease, and/or peripheral artery disease), 3) acute ST-segment elevation myocardial infarction (STEMI).
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Flow Cytometry
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Sample preparation
Human bone marrow: Human bone marrow samples were immersed in ice-cold sterile PBS with 0.5% bovine serum albumin and passed through a 40μm cell strainer. Following centrifugation, the cell pellet was resuspended in 70% Dulbecco's Modified Eagle's Medium without phenol red (D1145, Sigma-Aldrich, St. Louis, MO, USA), 20% fetal calf serum, and 10% DMSO, transferred to cryovials and stored in liquid nitrogen. For flow cytometry, cells were thawed at 37°C, washed in PBS, and stained with the respective antibodies.
Mouse tissue: To assess bone marrow hematopoietic cells, mice were anesthetized and flushed with 20mL PBS in order to remove intravascular blood. Bones were dissected and the bone marrow was flushed out with FACS buffer (PBS containing 0.5% bovine serum albumin). A single-cell suspension was created by passing the bone marrow through a 40μm cell strainer. To investigate hematopoietic cells in the spleen, spleen tissue was minced and plunged through a 40μm cell strainer, before red blood cell lysis was performed with RBC lysis buffer (420301, BioLegend). Peripheral blood was collected by retro-orbital bleeding using heparinized capillary tubes (420316, BD, Sparks, MD, USA) and red blood cells were lysed in RBC lysis buffer (BioLegend). For bone marrow stroma cells, bone marrow was flushed out and enzymatically digested in Dulbecco's Modified Eagle's Medium (DMEM) without phenol red (D1145, Sigma-Aldrich) containing 1mg/mL Collagenase Type 4 (LS004188, Worthington Biochemical Corporation, Lakewood Township, NJ, USA) and 2mg/mL Dispase II (D4693, Sigma-Aldrich) for 30min at 37°C with gentle agitation. Spleen tissue was minced and subjected to the same digestion protocol for the analysis of stroma cells. Skeletal muscles were dissected, minced into small pieces and enzymatically digested in PBS containing 450U/mL Collagenase Type 1 (C0130), 125U/mL Collagenase Type 11 (C7657), 60U/mL DNase I (D5319) and 60U/mL hyaluronidase (H3506, all purchased from Sigma-Aldrich) for 60min at 37°C under constant agitation.

Instrument
Flow cytometry data were acquired on an LSRII flow cytometer (BD Biosciences, San Jose, CA, USA).
Software FACS Diva 6.1 software (BD Biosciences, San Jose, CA, USA) was used for data collection. Data were analyzed with FlowJo 10 software (BD, Franklin Lakes, NJ, USA).

Cell population abundance
Purity of sorted cells was >95% as assessed by confirmatory flow cytometry.