Chronic sympathetic driven hypertension promotes atherosclerosis by enhancing hematopoiesis

Hypertension is a major, independent risk factor for atherosclerotic cardiovascular disease. However, this pathology can arise through multiple pathways, which could influence vascular disease through distinct mechanisms. An overactive sympathetic nervous system is a dominant pathway that can precipitate in elevated blood pressure. We aimed to determine how the sympathetic nervous system directly promotes atherosclerosis in the setting of hypertension. We used a mouse model of sympathetic nervous system-driven hypertension on the atherosclerotic-prone apolipoprotein E-deficient background. When mice were placed on a western type diet for 16 weeks, we showed the evolution of unstable atherosclerotic lesions. Fortuitously, the changes in lesion composition were independent of endothelial dysfunction, allowing for the discovery of alternative mechanisms. With the use of flow cytometry and bone marrow imaging, we found that sympathetic activation caused deterioration of the hematopoietic stem and progenitor cell niche in the bone marrow, promoting the liberation of these cells into the circulation and extramedullary hematopoiesis in the spleen. Specifically, sympathetic activation reduced the abundance of key hematopoietic stem and progenitor cell niche cells, sinusoidal endothelial cells and osteoblasts. Additionally, sympathetic bone marrow activity prompted neutrophils to secrete proteases to cleave the hematopoietic stem and progenitor cell surface receptor CXCR4. All these effects could be reversed using the β-blocker propranolol during the feeding period. These findings suggest that elevated blood pressure driven by the sympathetic nervous system can influence mechanisms that modulate the hematopoietic system to promote atherosclerosis and contribute to cardiovascular events.

. Bone marrow microenvironment alteration in sympathetic activation. A) Real time qPCR was used to assess mRNA expression levels of BM Cxcl12. B-D) BloodSpot (Online software) generation of a hierarchal myeloid tree depicting the expression of the adrenergic receptors b-1, b-2, b-3. E) BPH/Apoe +/+ mice were treated with the β2 specific antagonist ICI-118551 for 2 weeks and blood HSPCs assessed via flow cytometry. F) Latent MMP9 levels assessed by gelatin zymography. G) mRNA expression levels of BM Cxcr4. H) BM neutrophils were stimulated with NA ± ICI-118551 and the harvested supernatant was incubated with BM HSPCs to assess CXCR4 cleavage via flow cytometry. Data are presented as mean ± SEM where *p<0.05 measured by a One-Way ANOVA. A) n=5-9, E) n=4, F) n= 4, G) n=5-9, H) n=6.

Methods
Animal Models: Apoe -/mice were purchased from Jackson Laboratories and bred at the AMREP Animal centre. To generate hypertensive Apoe -/mice, BPH/2J mice were crossed with Apoe -/mice to produce BPH/2J x Apoe -/-(BPH/Apoe -/-) mice. At 6 weeks of age, male Apoe -/and BPH/ Apoe -/mice were placed on a western type diet (WTD -SF00-219, Specialty Feeds, Australia. 21% fat, 0.15% cholesterol) for 16 weeks. In the first cohort of mice, age-matched mice Apoe -/and BPH/Apoe -/were placed on a WTD for 16 weeks for end-point analysis. In a second cohort of mice, obtained from a new set of breeders, three groups of aged-matched mice were employed: 1) Apoe -/-, 2) BPH/Apoe -/and 3) BPH/ Apoe -/-+ propranolol (0.5g/L; administered via drinking water for the duration of the WTD feeding).
For the propranolol group, mice consumed on average 2.5ml of water amounting to an average daily dose of 35-40mg/kg/daily of propranolol.
To determine the effect of specific β2-adrenoreceptor blockade on HSPC mobilization and blood pressure we used BPH mice on an Apoe +/+ background. The mice were injected daily with ICI-118551 (5mg/kg; Abcam, AUS) for 2 weeks.
All animal experiments were approved by the AMREP Animal Ethics Committee and conducted in accordance with the Australian code of practice for the care and use of animals for scientific purposes as stipulated by the National Health and Medical Research Council of Australia. All mice were housed in a normal light and dark cycle and had ad libitum access to food and water.
Lesion Analysis: Mice were perfused with saline before heart and aorta collection. The aorta was dissociated from the heart at the point of entry between the highest point of the atria and fixed in PFA for further analysis (described below). The heart was dissected so that the upper region of the heart containing the aortic sinus was retained and frozen in optimal cutting temperature (OCT) compound. Serial 6μm sections of the proximal aorta were harvested on a cryostat at -20°C. Images were captured on an Olympus BX61 microscope, whereas collagen was imaged using polarised light. Image quantification was performed using Adobe Photoshop CC.
Aortic Arch Lipid Analysis: Lipid content, as a readout of atherosclerotic plaques in the aortic arch was measured by en face analysis. Dissected aortas were fixed in PFA, with fat and connective tissue removed from the outer layers of the vessel prior to staining. The aorta was then cut longitudinally and stained with ORO. After washing, the stained aortas were mounting on a silicone coated dish. Aortas were viewed on an Olympus SZX10 and captured using Q-Capture Pro 7 (QImaging) software. Quantification of ORO staining was performed off-line using Adobe Photoshop CC5.
Total Plasma Cholesterol: The Wako total cholesterol kit was used to measure plasma cholesterol as previously described 26 .

Blood Pressure Measurements:
Tail Cuff method: Systolic blood pressure (SBP) was measured in all mice (prior to WTD administration) and after 16 weeks of feeding using the non-invasive tail cuff plethysmography technique. A MC4000 blood pressure analysis system (Hatteras Instruments) was used with a 37 o C pre-heated stage. Mice were placed in restrainers with tails placed through a cuff and onto a heart rate monitor. Mice remained in restraints for 5 mins to allow acclimatisation after which 5 preliminary BP measurement cycles were run to ensure BP was detectable. Ten measurement cycles were then performed to ensure correct readings, the average of the readings for systolic, diastolic and mean arterial blood pressure were recorded.

Blood pressure telemetry:
Under isoflurane open circuit anaesthesia (1.5-2.5%) Apoe -/and BPH/Apoe -/mice were implanted with radiotelemetry devices weighing 1.4g and approximately 10mm in length (TA11PA-C10; DataSciences International (DSI), St Paul, USA) as previously described. 27 Following a 10-day recovery from surgery, 1 min averages of pulsatile arterial blood pressure readings and locomotor activity were recorded continuously over a 72-hour period sampled at 100 Hz as previously described. 28 Mean arterial pressure and heart rate were analysed in Labview. 29 Vessel reactivity: Preparation of vessels: Mice were ethuanased via CO2 asphyxiation which was confirmed by cessation of corneal and pedal reflexes. The thoracic aorta was removed and placed in ice-cold Krebs solution (composition in mM: NaCl 119, KCl 4.7, KH2PO4 1.18, MgSO4 1.17, NaHCO3 25, CaCl2 2.5, EDTA 0.026 and glucose 5.5). Perivascular fat and adhering connective tissue were removed from the aorta and it was cut into rings 2 mm in length. Each ring was mounted onto two parallel stainless-steel wires (200 µm in diameter) in a 5 ml chamber of a 4 channel myograph (Mulvaney Myograph, model 610M, JP trading), containing oxygenated Krebs solution and maintained at 37 o C. Changes in tension were recorded using a PowerLab 8/35 (ADI Instruments Inc) connected to a computer.
Myograph: Tissues were equilibrated at resting tension for 30mins before undergoing a "normalisation" procedure to determine optimal resting tension and vessel diameter. During the normalisation procedure, tissues are subjected to a series of stretches beginning at wire touch and progressing to tensions of 30mN. Once tissues were set at their optimal resting tension, they were equilibrated for a further 30 min before their viability was tested with a high potassium physiological salt solution, (KPSS, composition in mM: KCl 123, MgSO4 1.17, KH2PO4 2.37, CaCl2 2.5, EDTA 0.02 and glucose 5.5). Once the contractile response had plateaued, the chamber was rinsed three times with normal Krebs solution and the tissue was allowed to return to baseline. For relaxation curves, tissues were pre-constricted to 50% of the KPSS contraction using PE. Once the contraction had plateaued, cumulative concentrationresponse curves were acquired to ACh (1nM-10µM) or SNP (0.1nM-10µM) in the presence or absence of L-NAME. Only one protocol was completed in each aortic ring to avoid tolerance.
White Blood Cell Counts: White blood cell (WBC) counts were obtained from blood collected via cardiac puncture immediately following euthanasia. The counts were quantified on a Sysmex XS-1000i automated hematology analyser (Japan).
Flow Cytometry: All samples were run and analyzed on a BD FACS Canto II flow cytometer or Fortessa X-20 as stated below. Flow cytometry antibodies were all used at 1:400 dilutions and details of antibodies are available in Supplementary table 1.

T-Cell Populations:
Aortas were harvested from the mice following cardiac puncture and flushing with saline. Aortas were digested in Liberase (Sigma-Aldrich, USA, Cat # 5401160001) for 45 mins at 37 o C after which the cells were spun down, washed and resuspended in FACS Buffer (1x HBSS + 0.5mM EDTA and 0.05% BSA). Viable cells were gated using a live/dead stain (Ghost violet 510 viability dye, Tonbo Biosciences) on the Canto II where CD4 -T cells were gated as CD45 + CD3 + CD4and CD4 + T cells as CD45 + CD3 + CD4 + .
Monocyte Populations: Bone marrow and spleen cells were obtained by mashing whole spleen through a 40µm strainer and then lysed using 1x RBC lysis buffer. Blood was collected via cardiac puncture into EDTA-lined tubes and placed directly on ice to prevent leukocyte activation and loss of cell surface CD115. The samples remained on ice or at 4 o C for the remainder of the procedure. Red blood cells (RBCs) were lysed, and the washed cells were then stained with CD45, CD115 and Gr1 (Ly6-C/G) for 30 min on ice. The cells were washed, resuspended in FACS buffer and run on a Canto II flow cytometer (Figure 3) or a Fortessa X-20 ( Figure 5). Leukocyte populations were identified as previously described. 20,26,30 Briefly, viable, single cells were selected on the basis of forward and side scatter characteristics, from which CD45 + leukocytes were selected. Monocytes were identified as being CD115 + with Ly6-C hi monocytes identified as Gr1 + and Ly6-C lo monocytes identified as Gr1 -. Neutrophils were identified as CD115 -Gr1 + .