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SNS driven hypertension enhances hematopoiesis
unclear.2 The most frequently targeted pathway in reduc- ing blood pressure is the renin-angiotensin system (RAS). The contribution of the RAS to hypertension and athero- sclerosis is not exclusive, as angiotensin II (AngII) can also accelerate atherogenesis independent of hypertension.3,4 Another major determinant of hypertension is an overac- tive sympathetic nervous system (SNS).5,6 There are indi- cations that autonomic input into the bone marrow (BM) may be altered in the setting of hypertension.7-10 However, the mechanisms promoting atherogenesis with SNS acti- vation associated hypertension are not completely eluci- dated. While there is an overlap in some atherosclerosis promoting mechanisms between the RAS and SNS, a dis- tinct subset of events is also likely to be evoked by the SNS, which requires further investigation.
Atherosclerosis is a disease driven by the infiltration of immune cells, in particular monocytes, into the plaque.11-13 It is also well established that the abundance of circulating monocytes predicts cardiovascular (CV) events and is directly linked to atherogenesis.14,15 Interestingly, the SNS plays a direct role in regulating the hematopoietic system from which immune cells, including monocytes, arise.16-19 In the context of CVD, the mobilization of hematopoietic stem and progenitor cells (HSPCs) from the BM to extramedullary tissues such as the spleen results in the generation of atherogenic monocytes that abundantly enter into the atherosclerotic plaque.20 Mobilization of HSPCs can be mediated by sympathetic signaling within the BM, particularly in response to granulocyte-colony stimulating factor (G-CSF). The SNS synergizes with G- CSF to promote the breakdown of the HSPC BM microen- vironment, which decreases the abundance of key HSPC retention factors and results in the liberation of HSPCs into the circulation.16 This pathway has also been shown to be activated following a myocardial infarction (MI).21 Sympathetic activation, along with raised G-CSF levels that are observed in apolipoprotein E knockout (Apoe-/-) mice, caused HSPC mobilization from the BM and hom- ing to the spleen where monocytes were subsequently produced that infiltrated atherosclerotic lesions. Interestingly, this promoted an unstable plaque pheno- type, prone to rupturing and thus provides a plausible explanation for primary heart attack survivors being high- ly prone to a secondary, often fatal, CV event.21,22 Importantly, the involvement of the SNS in driving aber- rant hematopoiesis is not restricted to complications fol- lowing a MI, as similarities in other models of stress and ischemic stroke are evident, suggesting this to be a more general mechanism. The augmented hematopoietic response in these pathologies caused by overactivation of the SNS were inhibited by administration of b-blockers or genetic deletion of b-adrenergic receptors.21,23-25
There appears to be an important role of the SNS in reg- ulating hematopoiesis in acute stressors (i.e., MI, stroke, variable stress). However, it remains unknown if chronic sympathetic activation invokes this same atherogenic process. Thus, it is plausible that chronic sympathetic acti- vation present in some cases of hypertension could play an important role in regulating atherogenesis by altering hematopoiesis. To address this question, we employed the Schlager hypertensive mice which were crossed onto an Apoe-/- background to produce hypertensive atheroscle- rosis-prone mice. The Schlager mouse was chosen as it represents a model of hypertension that is almost entirely driven by the SNS, with minimal contribution by the
RAS.26 We sought to characterize the contribution of SNS activation associated hypertension to the development of atherosclerosis, with the aim of understanding whether this form of hypertension was also associated with alter- ations to the hematopoietic system. Moreover, we aimed to investigate whether targeting the SNS could inhibit atherogenesis and, in turn, reveal an additional mecha- nism of hypertension associated atherosclerosis.
Methods
Detailed methods are available in Online supplementary 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 2-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 sci- entific 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. Mice were randomly assigned to treatment and end-point analysis was blinded.
Statistics
Data are presented as mean ± SEM (unless stated otherwise) and were analysed using the two-tailed Student t-test or One-way ANOVA where appropriate. Analysis of baseline and final blood pressure between strains was performed using a two-way ANOVA with the factors strain (Pstrain) and time (Ptime) followed by a Sidak post-hoc test to account for multiple comparisons. A P<0.05 was considered significant. All tests were performed using the Prism software (GraphPad Software, Inc., La Jolla, CA, USA).
Results
Hypertension associated with chronic sympathetic activation promotes an unstable atherosclerotic phenotype
To determine the contribution of chronic sympathetic activation in hypertension to atherosclerosis we crossed Schlager hypertensive mice with Apoe-/- mice (BPH/Apoe-/-) and compared these to normotensive Apoe-/- mice. Mice were fed a high fat, high cholesterol western type diet (WTD) for 16 weeks. We preferenced this model over con-
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