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Introduction: Vascular stiffening and inflammation are hallmarks of hypertension and contribute to the development of cardiovascular disease and cognitive impairment. Current treatments for hypertension may reduce blood pressure but do not target the associated pathological changes and end-organ damage. It is also inconclusive from clinical trials whether anti-hypertensive medication can improve cognitive impairment. Cell therapy has great therapeutic potential as unlike single pharmacological agents, cells can deliver multiple mediators which could more effectively target complex disease mechanisms. Human amnion epithelial cells (AECs) have many properties (eg. anti-inflammatory, anti-fibrotic, and immunologically inert) that make them attractive candidates for a cell-based therapy for vascular and brain pathology. Aim: To test the potential of AECs to treat vascular and brain pathology in angiotensin II-induced hypertension. Methods. Male C57Bl6 mice (8-12 weeks) were administered vehicle (saline; n=35) or angiotensin II (0.7 mg/kg/d, n=37) for 14 d via an osmotic minipump. After minipump implantation, a subset of mice were injected with 106 of AECs intravenously. Systolic blood pressure was measured using tail-cuff; pulse wave velocity was measured using ultrasound, inflammation was assessed using flow cytometry and RNA sequencing, markers of fibrosis using quantitative PCR and cognitive function using novel object recognition test. Results: Angiotensin II infusion increased blood pressure and aortic pulse wave velocity (n=6-7, P La Trobe University Start Up grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Hypertensive heart failure is characterised by cardiomyocyte hypertrophy, inflammation and fibrosis, leading to stiffening and diastolic dysfunction. There are currently no effective treatments for hypertensive heart failure, in part reflecting our limited knowledge of the molecular mechanisms. Interleukin-18 (IL-18) is a pro-inflammatory, inflammasome-derived cytokine that is elevated in the circulation of hypertensive patients; however, its role in promoting cardiac remodelling and dysfunction is unknown. Thus, we examined if IL-18-deficient ( Il18 -/- ) mice were protected from hypertensive cardiac remodelling and, if so, whether this occurs independently of changes in blood pressure. Hypertension was induced in male wild-type (WT) and Il18 -/- mice by angiotensin II (Ang II; 1.44 mg/kg/d, s.c. ) infusion for 28 d. Normotensive controls were saline-infused. Systolic blood pressure (SBP) was measured by tail cuff, gene expression was measured by single cell RNA sequencing (scRNA-seq), cardiac fibrosis by picrosirius red-staining, and cardiac hypertrophy/remodelling was assessed histologically and via heart weight:body weight. scRNAseq analysis of hearts isolated from Ang II-treated WT mice revealed that IL-18 was expressed by macrophages, granulocytes and epicardial cells. Baseline SBPs were not different between WT and Il18 -/- mice, and Ang II evoked a similar degree of hypertension in each genotype (160 ± 24 and 165 ± 16 mmHg in WT and Il18 -/- mice, respectively). Despite having no impact on pressor responses to Ang II, IL-18-deficiency provided marked protection against Ang II-induced cardiac hypertrophy (6.58 ± 0.53 cf. 7.87 ± 1.14 mg/g in Il18 -/- and WT, respectively; n≥7, P&lt0.01) and fibrosis (1.84 ± 0.18 cf. 4.06 ± 0.46 % in Il18 -/- and WT, respectively; n≥7, P&lt0.01). Furthermore, cardiac myocytes were enlarged, disorganised and displayed hyperchromatic nuclei in Ang II-treated WT mice, but not in Ang II-treated Il18 -/- mice. In conclusion, IL-18 deficiency protects against cardiac remodelling during hypertension, independent of an effect on SBP. These findings suggest that IL-18 is a driver of hypertensive cardiac remodelling and potential target for future therapies.

Metabolic syndrome (MetS) is a complex multifactorial disease that increases cardiovascular risk. Vascular dysfunction associated with MetS is strongly influenced by perivascular adipose tissue (PVAT) which acts as a reservoir for immune cells. Sexual dimorphisms of PVAT pathophysiology are poorly understood due to a lack of clinically relevant animal models of MetS that show metabolic disturbances in males and females. Using a new diet-induced model of MetS pathophysiology in both sexes, we characterised the aortic leukocyte profile and determined PVAT influence on vascular function. Six-week-old male and female C57BL/6 mice were fed either a high-fat diet (43% kcal) with high sugar and salt (10% high fructose and 0.9% NaCl; HFSS) in drinking water, or normal control diet (NCD) for 10 weeks. Physiological parameters were measured fortnightly. At endpoint, aorta with PVAT intact was isolated for leukocyte enumeration (flow cytometry) or for in vitro vascular reactivity (± PVAT; wire myography). Weight gain (NCD M: 29.3±0.5 g vs. MetS M: 37.5±1.0 g; NCD F: 23.6±0.7 g vs. MetS F: 28.9±1.2 g), fasting blood glucose (NCD M: 9.5±0.4 mmol/L vs. MetS M: 11.3 mmol/L; NCD F: 8.9±0.3 mmol/L vs. 10.4±0.5 mmol/L), blood cholesterol (NCD M: 125.0±3.0 mg/dL vs. MetS M: 177.5±6.5 mg/dL; NCD F: 96.7±2.9 mg/dL vs. MetS F: 130.7±3.4 mg/dL) and systolic blood pressure (NCD M: 121.0±1.8 mmHg vs. MetS M: 132.0±2.1 mmHg; NCD F: 122.3±2.2 mmHg vs. MetS F: 133.6±2.7 mmHg) were increased in both sexes of HFSS-fed mice (Phi+ ; M: 3.3±0.5 x 10 3 cells/aorta vs F: 1.6±0.4 x 10 3 cells/aorta) and neutrophils (Ly6G + ; M: 0.9±-0.2 x 10 3 cells/aorta vs F: 0.3+/0.1 x 10 3 cells/aorta); P

IL-18 (interleukin-18) is elevated in hypertensive patients, but its contribution to high blood pressure and end-organ damage is unknown. We examined the role of IL-18 in the development of renal inflammation and injury in a mouse model of low-renin hypertension. Hypertension was induced in male C57BL6/J (WT) and IL-18−/−mice by uninephrectomy, deoxycorticosterone acetate (2.4 mg/d, s.c.) and 0.9% drinking saline (1K/DOCA/salt). Normotensive controls received uninephrectomy and placebo (1K/placebo). Blood pressure was measured via tail cuff or radiotelemetry. After 21 days, kidneys were harvested for (immuno)histochemical, quantitative-PCR and flow cytometric analyses of fibrosis, inflammation, and immune cell infiltration. 1K/DOCA/salt-treated WT mice developed hypertension, renal fibrosis, upregulation of proinflammatory genes, and accumulation of CD3+T cells in the kidneys. They also displayed increased expression of IL-18 on tubular epithelial cells. IL-18−/−mice were profoundly protected from hypertension, renal fibrosis, and inflammation. Bone marrow transplantation between WT and IL-18−/−mice revealed that IL-18-deficiency in non-bone marrow-derived cells alone afforded equivalent protection against hypertension and renal injury as global IL-18 deficiency. IL-18 receptor subunits—interleukin-18 receptor 1 and IL-18R accessory protein—were upregulated in kidneys of 1K/DOCA/salt-treated WT mice and localized to T cells and tubular epithelial cells. T cells from kidneys of 1K/DOCA/salt-treated mice produced interferon-γ upon ex vivo stimulation with IL-18, whereas those from 1K/placebo mice did not. In conclusion, IL-18 production by tubular epithelial cells contributes to elevated blood pressure, renal inflammation, and fibrosis in 1K/DOCA/salt-treated mice, highlighting it as a promising therapeutic target for hypertension and kidney disease.

Aims Renal inflammation, leading to fibrosis and impaired function is a major contributor to the development of hypertension. The NLRP3 inflammasome mediates inflammation in several chronic diseases by processing the cytokines pro-interleukin (IL)-1β and pro-IL-18. In this study, we investigated whether MCC950, a recently-identified inhibitor of NLRP3 activity, reduces blood pressure (BP), renal inflammation, fibrosis and dysfunction in mice with established hypertension. Methods and results C57BL6/J mice were made hypertensive by uninephrectomy and treatment with deoxycorticosterone acetate (2.4 mg/day, s.c.) and 0.9% NaCl in the drinking water (1K/DOCA/salt). Normotensive controls were uninephrectomized and received normal drinking water. Ten days later, mice were treated with MCC950 (10 mg/kg/day, s.c.) or vehicle (saline, s.c.) for up to 25 days. BP was monitored by tail-cuff or radiotelemetry; renal function by biochemical analysis of 24-h urine collections; and kidney inflammation/pathology was assessed by real-time PCR for inflammatory gene expression, flow cytometry for leucocyte influx, and Picrosirius red histology for collagen. Over the 10 days post-surgery, 1K/DOCA/salt-treated mice became hypertensive, developed impaired renal function, and displayed elevated renal levels of inflammatory markers, collagen and immune cells. MCC950 treatment from day 10 attenuated 1K/DOCA/salt-induced increases in renal expression of inflammasome subunits (NLRP3, ASC, pro-caspase-1) and inflammatory/injury markers (pro-IL-18, pro-IL-1β, IL-17A, TNF-α, osteopontin, ICAM-1, VCAM-1, CCL2, vimentin), each by 25–40%. MCC950 reduced interstitial collagen and accumulation of certain leucocyte subsets in kidneys of 1K/DOCA/salt-treated mice, including CD206+ (M2-like) macrophages and interferon-gamma-producing T cells. Finally, MCC950 partially reversed 1K/DOCA/salt-induced elevations in BP, urine output, osmolality, [Na+], and albuminuria (each by 20–25%). None of the above parameters were altered by MCC950 in normotensive mice. Conclusion MCC950 was effective at reducing BP and limiting renal inflammation, fibrosis and dysfunction in mice with established hypertension. This study provides proof-of-concept that pharmacological inhibition of the NLRP3 inflammasome is a viable anti-hypertensive strategy.

Macrophages, which accumulate in tissues during inflammation, may be polarized toward pro-inflammatory (M1) or tissue reparative (M2) phenotypes. The balance between these phenotypes can have a substantial influence on the outcome of inflammatory diseases such as atherosclerosis. Improved biomarkers of M1 and M2 macrophages would be beneficial for research, diagnosis, and monitoring the effects of trial therapeutics in such diseases. To identify novel biomarkers, we have characterized the global proteomes of THP-1 macrophages polarized to M1 and M2 states in comparison with unpolarized (M0) macrophages. M1 polarization resulted in increased expression of numerous pro-inflammatory proteins including the products of 31 genes under the transcriptional control of interferon regulatory factor 1 (IRF-1). In contrast, M2 polarization identified proteins regulated by components of the transcription factor AP-1. Among the most highly upregulated proteins under M1 conditions were the three interferon-induced proteins with tetratricopeptide repeats (IFITs: IFIT1, IFIT2, and IFIT3), which function in antiviral defense. Moreover, IFIT1, IFIT2, and IFIT3 mRNA were strongly upregulated in M1 polarized human primary macrophages and IFIT1 was also expressed in a subset of macrophages in aortic sinus and brachiocephalic artery sections from atherosclerotic ApoE

Aging is commonly associated with chronic low-grade inflammation and hypertension but it is unknown whether a cause-effect relationship exists between them. We compared the sensitivity of young adult (8-12 w) and aged (23-31 mo) male C57Bl6J mice to develop hypertension in response to a slow-pressor dose of angiotensin II (Ang II; 0.28 mg/kg/d; 28 d). In young mice, the pressor response to Ang II was gradual and increased to 142±8 mmHg over 28 d. However, in aged mice, Ang II promptly increased SBP and reached 155±12 mmHg by 28 d. Aging increased renal but not brain expression of Ang II receptors (At1ar and At2r) and elevated AT1R:AT2R expression ratio in mesenteric artery. Maximal contractile responses of mesenteric arteries to Ang II were enhanced in aged mice and were not affected by L-NAME, indomethacin or tempol. Mesenteric arteries and thoracic aortae from aged mice exhibited higher Nox2-dependent superoxide production. Despite having higher renal expression of Nlrp3, Casp-1 and Il-1β, Ang II-induced hypertension (SBP: 139±7 mmHg) was unaffected by co-infusion of the NLRP3 inflammasome inhibitor, MCC950 (10 mg/kg/d; SBP: 145±10 mmHg). Thus, increased vascular AT1R:AT2R expression, rather than NLRP3 inflammasome activation, may contribute to enhanced responses to Ang II in aging.

Although hypertension may involve underlying inflammation, it is unknown whether advanced atherosclerosis − a chronic inflammatory condition − can by itself promote hypertension. We thus tested if advanced atherosclerosis in chronically hypercholesterolemic mice is associated with systemic and end-organ inflammation, vascular dysfunction and oxidative stress, and whether blood pressure is higher than in control mice. Male ApoE −/− and wild-type (C57Bl6J) mice were placed on a high fat or chow diet, respectively, from 5 to 61 weeks of age. Expression of several cytokines (including IL-6, TNF-α, IFN-γ and/or IL-1β) was elevated in plasma, brain, and aorta of ApoE −/− mice. Aortic superoxide production was ∼3.5-fold greater, and endothelium-dependent relaxation was markedly reduced in aorta and mesenteric artery of ApoE −/− versus wild-type mice. There was no difference in blood pressure of aged ApoE −/− (104 ± 3 mmHg, n = 13) and wild-type mice (113 ± 1 mmHg, n = 18). To clarify any effects of aging alone, findings from 61 week-old wild-type mice were compared with those from young (8–12 weeks old) chow-fed wild-type mice. The data indicate that aging alone increased renal and aortic expression of numerous cytokines (including CCL2, CCL7 and IL-1β). Aging had no effect on blood pressure, systemic inflammation, oxidative stress or endothelial function. Despite systemic and end-organ inflammation, oxidative stress and endothelial dysfunction, advanced atherosclerosis does not necessarily result in elevated blood pressure.

Supplemental Digital Content is available in the text., Background: Cardiac fibrosis is a key antecedent to many types of cardiac dysfunction including heart failure. Physiological factors leading to cardiac fibrosis have been recognized for decades. However, the specific cellular and molecular mediators that drive cardiac fibrosis, and the relative effect of disparate cell populations on cardiac fibrosis, remain unclear. Methods: We developed a novel cardiac single-cell transcriptomic strategy to characterize the cardiac cellulome, the network of cells that forms the heart. This method was used to profile the cardiac cellular ecosystem in response to 2 weeks of continuous administration of angiotensin II, a profibrotic stimulus that drives pathological cardiac remodeling. Results: Our analysis provides a comprehensive map of the cardiac cellular landscape uncovering multiple cell populations that contribute to pathological remodeling of the extracellular matrix of the heart. Two phenotypically distinct fibroblast populations, Fibroblast-Cilp and Fibroblast-Thbs4, emerged after induction of tissue stress to promote fibrosis in the absence of smooth muscle actin–expressing myofibroblasts, a key profibrotic cell population. After angiotensin II treatment, Fibroblast-Cilp develops as the most abundant fibroblast subpopulation and the predominant fibrogenic cell type. Mapping intercellular communication networks within the heart, we identified key intercellular trophic relationships and shifts in cellular communication after angiotensin II treatment that promote the development of a profibrotic cellular microenvironment. Furthermore, the cellular responses to angiotensin II and the relative abundance of fibrogenic cells were sexually dimorphic. Conclusions: These results offer a valuable resource for exploring the cardiac cellular landscape in health and after chronic cardiovascular stress. These data provide insights into the cellular and molecular mechanisms that promote pathological remodeling of the mammalian heart, highlighting early transcriptional changes that precede chronic cardiac fibrosis.

Aims The G protein-coupled estrogen receptor 1 (GPER) may modulate some effects of aldosterone. In addition, G-1 (a GPER agonist) can lower blood pressure (BP) and promote T cell-mediated anti-inflammatory responses. This study aimed to test the effects of G-1 and G-15 (a GPER antagonist) on aldosterone-induced hypertension in mice and to examine the cellular mechanisms involved. Methods and results C57Bl/6 (wild-type, WT), RAG1-deficient and GPER-deficient mice were infused with vehicle, aldosterone (0.72 mg/kg/day S.C. plus 0.9% NaCl for drinking) ± G-1 (0.03 mg/kg/day S.C.) ± G-15 (0.3 mg/kg/day S.C.) for 14 days. G-1 attenuated aldosterone-induced hypertension in male WT but not male GPER-deficient mice. G-15 alone did not alter hypertension but it prevented the anti-hypertensive effect of G-1. In intact female WT mice, aldosterone-induced hypertension was markedly delayed and suppressed compared with responses in males, with BP remaining unchanged until after Day 7. In contrast, co-administration of aldosterone and G-15 fully increased BP within 7 days in WT females. Similarly, aldosterone robustly increased BP by Day 7 in ovariectomized WT females, and in both sexes of GPER-deficient mice. Whereas aldosterone had virtually no effect on BP in RAG1-deficient mice, adoptive transfer of T cells from male WT or male GPER-deficient mice into male RAG1-deficient mice restored the pressor response to aldosterone. This pressor effect could be attenuated by G-1 in RAG1-deficient mice that were reconstituted with either WT or GPER-deficient T cells, suggesting that G-1 does not act via T cells to lower BP. Conclusion Our findings indicate that although aldosterone-induced hypertension is largely mediated by T cells, it can be attenuated by activation of GPER on non-T cells, which accounts for the sex difference in sensitivity to the pressor effect.

Aims. To date, the ROS-generating capacities of macrophages in different activation states have not been thoroughly compared. This study is aimed at determining the nature and levels of ROS generated following stimulation with common activators of M1 and M2 macrophages and investigating the potential for this to impact fibrosis.Results. Human primary and THP-1 macrophages were treated with IFN-γ+LPS or IL-4-activating stimuli, and mRNA expression of established M1 (CXCL11, CCR7, IL-1β) and M2 (MRC-1, CCL18, CCL22) markers was used to confirm activation. Superoxide generation was assessed by L-012-enhanced chemiluminescence and was increased in both M(IFN-γ+LPS) and M(IL-4) macrophages, as compared to unpolarised macrophages (MΦ). This signal was attenuated with NOX2 siRNA. Increased expression of the p47phox and p67phox subunits of the NOX2 oxidase complex was evident in M(IFN-γ+LPS) and M(IL-4) macrophages, respectively. Amplex Red and DCF fluorescence assays detected increased hydrogen peroxide generation following stimulation with IL-4, but not IFN-γ+LPS. Coculture with human aortic adventitial fibroblasts revealed that M(IL-4), but not M(IFN-γ+LPS), enhanced fibroblast collagen 1 protein expression. Macrophage pretreatment with the hydrogen peroxide scavenger, PEG-catalase, attenuated this effect.Conclusion. We show that superoxide generation is not only enhanced with stimuli associated with M1 macrophage activation but also with the M2 stimulus IL-4. Macrophages activated with IL-4 also exhibited enhanced hydrogen peroxide generation which in turn increased aortic fibroblast collagen production. Thus, M2 macrophage-derived ROS is identified as a potentially important contributor to aortic fibrosis.

The essential role of the Y chromosome in male sex determination has largely overshadowed the possibility that it may exert other biologic roles. Here, we show that Y-chromosome lineage is a strong determinant of perivascular and renal T-cell infiltration in the stroke-prone spontaneously hypertensive rat, which, in turn, may influence vascular function and blood pressure (BP). We also show, for the first time to our knowledge, that augmented perivascular T-cell levels can directly instigate vascular dysfunction, and that the production of reactive oxygen species that stimulate cyclo-oxygenase underlies this. We thus provide strong evidence for the consideration of Y-chromosome lineage in the diagnosis and treatment of male hypertension, and point to the modulation of cardiovascular organ T-cell infiltration as a possible mechanism that underpins Y- chromosome regulation of BP.-Khan, S. I., Andrews, K. L., Jackson, K. L., Memon, B., Jefferis, A.-M., Lee, M. K. S., Diep, H., Wei, Z., Drummond, G. R., Head, G. A., Jennings, G. L., Murphy, A. J., Vinh, A., Sampson, A. K., Chin-Dusting, J. P. F. Y-chromosome lineage determines cardiovascular organ T-cell infiltration in the stroke-prone spontaneously hypertensive rat.

Clinical hypertension is associated with raised serum IgG antibodies. However, whether antibodies are causative agents in hypertension remains unknown. We investigated whether hypertension in mice is associated with B-cell activation and IgG production and moreover whether B-cell/IgG deficiency affords protection against hypertension and vascular remodeling. Angiotensin II (Ang II) infusion (0.7 mg/kg per day; 28 days) was associated with (1) a 25% increase in the proportion of splenic B cells expressing the activation marker CD86, (2) an 80% increase in splenic plasma cell numbers, (3) a 500% increase in circulating IgG, and (4) marked IgG accumulation in the aortic adventitia. In B-cell–activating factor receptor–deficient (BAFF-R −/− ) mice, which lack mature B cells, there was no evidence of Ang II–induced increases in serum IgG. Furthermore, the hypertensive response to Ang II was attenuated in BAFF-R −/− (Δ30±4 mm Hg) relative to wild-type (Δ41±5 mm Hg) mice, and this response was rescued by B-cell transfer. BAFF-R −/− mice displayed reduced IgG accumulation in the aorta, which was associated with 80% fewer aortic macrophages and a 70% reduction in transforming growth factor-β expression. BAFF-R −/− mice were also protected from Ang II–induced collagen deposition and aortic stiffening (assessed by pulse wave velocity analysis). Finally, like BAFF-R deficiency, pharmacological depletion of B cells with an anti-CD20 antibody attenuated Ang II–induced hypertension by ≈35%. Hence, these studies demonstrate that B cells/IgGs are crucial for the development of Ang II–induced hypertension and vessel remodeling in mice. Thus, B-cell–targeted therapies—currently used for autoimmune diseases—may hold promise as future treatments for hypertension.

Background and Purpose Inflammasomes are multimeric complexes that facilitate caspase-1-mediated processing of the pro-inflammatory cytokines IL-1β and IL-18. Clinical hypertension is associated with renal inflammation and elevated circulating levels of IL-1β and IL-18. Therefore, we investigated whether hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and if inhibition of inflammasome activity reduces BP, markers of renal inflammation and fibrosis. Experimental Approach Wild-type and inflammasome-deficient ASC−/− mice were uninephrectomized and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt). Control mice were uninephrectomized but received a placebo pellet and water. BP was measured by tail cuff; renal expression of inflammasome subunits and inflammatory markers was measured by real-time PCR and immunoblotting; macrophage and collagen accumulation was assessed by immunohistochemistry. Key Results 1K/DOCA/salt-induced hypertension in mice was associated with increased renal mRNA expression of inflammasome subunits NLRP3, ASC and pro-caspase-1, and the cytokine, pro-IL-1β, as well as protein levels of active caspase-1 and mature IL-1β. Following treatment with 1K/DOCA/salt, ASC−/− mice displayed blunted pressor responses and were also protected from increases in renal expression of IL-6, IL-17A, CCL2, ICAM-1 and VCAM-1, and accumulation of macrophages and collagen. Finally, treatment with a novel inflammasome inhibitor, MCC950, reversed hypertension in 1K/DOCA/salt-treated mice. Conclusions and Implications Renal inflammation, fibrosis and elevated BP induced by 1K/DOCA/salt treatment are dependent on inflammasome activity, highlighting the inflammasome/IL-1β pathway as a potential therapeutic target in hypertension. Linked Articles This article is part of a themed section on Inflammation: maladies, models, mechanisms and molecules. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2016.173.issue-4

Nicotinamide adenine dinucleotide phosphate oxidases (NOX) are enzymes that generate reactive oxygen species (ROS). NOX2 activity in the vascular wall is elevated in hypercholesterolemia, and contributes to oxidative stress and atherogenesis. Here we examined the role of another NOX isoform, NOX1, in atherogenesis in apolipoprotein E-knockout (APOE(-/-)) mice fed a Western diet for 14 weeks. Although NOX1 mRNA expression was unchanged in aortas from APOE(-/-) versus wild-type mice, expression of the NOX1-specific organizer, NOXO1, was diminished, consistent with an overall reduction in NOX1 activity in APOE(-/-) mice. To examine the impact of a further reduction in NOX1 activity, APOE(-/-) mice were crossed with NOX1(-/y) mice to generate NOX1(-/y)/APOE(-/-) double-knockouts. NOX1 deficiency in APOE(-/-) mice was associated with 30-50% higher plasma very-low-density lipoprotein (VLDL)/LDL and triglyceride levels (P < 0.01). Vascular ROS levels were also elevated by twofold in NOX1(-/y)/APOE(-/-) versus APOE(-/-) mice (P < 0.05), despite no changes in expression of other NOX subunits. Although en face analysis of the descending aorta revealed no differences in plaque area between NOX1(-/y)/APOE(-/-) and APOE(-/-) mice, intimal thickening in the aortic sinus was increased by 40% (P < 0.05) in the double-knockouts. Moreover, NOX1 deficiency was associated with a less stable plaque phenotype; aortic sinus lesions contained 60% less collagen (P < 0.01), 40% less smooth muscle (P < 0.01), and 2.5-fold higher levels of matrix metalloproteinase-9 (P < 0.001) than lesions in APOE(-/-) mice. Thus, these data, which suggest a protective role for NOX1 against hyperlipidemia and atherosclerosis in APOE(-/-) mice, highlight the complex and contrasting roles of different NOX isoforms (e.g., NOX2 versus NOX1) in vascular pathology.

To determine whether a clinically-utilised IL-1 receptor antagonist, anakinra, reduces renal inflammation, structural damage and blood pressure (BP) in mice with established hypertension.Hypertension was induced in male mice by uninephrectomy, deoxycorticosterone acetate (2.4mg/d,s.c.) and replacement of drinking water with saline (1K/DOCA/salt). Control mice received uninephrectomy, a placebo pellet and normal drinking water. 10days post-surgery, mice commenced treatment with anakinra (75mg/kg/d, i.p.) or vehicle (0.9% saline, i.p.) for 11days. Systolic BP was measured by tail cuff while qPCR, immunohistochemistry and flow cytometry were used to measure inflammatory markers, collagen and immune cell infiltration in the kidneys.By 10days post-surgery, 1K/DOCA/salt-treated mice displayed elevated systolic BP (148.3±2.4mmHg) compared to control mice (121.7±2.7mmHg; n=18, P0.05). Anakinra reduced renal collagen content (n=6, P

Introduction: The prevalence of hypertension increases with age. Chronic low-grade inflammation commonly occurs with ageing, and inflammasomes are important initiators of inflammatory responses. We tested whether aged mice have enhanced pressor responses to angiotensin II (Ang II) and whether this is associated with exaggerated pro-inflammatory and vascular contractile responses. We also tested whether MCC950, a NLRP3 inflammasome inhibitor, reduces blood pressure (BP) in Ang II-treated aged mice. Methods: Young (8-12 weeks) and aged (24-30 months) male C57Bl6 mice were left untreated, or either treated with vehicle or a slow-pressor dose of Ang II (0.28 mg/kg/day) for 28 days. Another group of aged mice were treated with either Ang II + saline or Ang II + MCC950 (10 mg/kg/day) for 10 days. We measured systolic BP, mRNA expression of inflammatory markers and components of the renin-angiotensin system, and vascular contractile responses. Results: In young mice, Ang II caused a gradual increase in BP (final BP: 141.6 ± 8.3 mmHg), whereas BP increased by ~20 mmHg from baseline in aged mice, and continued to increase for 28 days (final BP: 155 ± 12.4 mmHg) (n=8-9, P0.05). Conclusions: Aged mice have enhanced pressor responses to Ang II and this is associated with exacerbated pro-inflammatory and vascular contractile responses. The NLRP3 inflammasome does not appear to contribute to Ang II-induced hypertension in aged mice.

Background: Clinical hypertension is associated with raised serum antibody levels. However, no studies have examined whether B cells and antibodies play causative roles in the pathogenesis of hypertension. We investigated whether experimental hypertension is similarly associated with elevated IgG production, and if B cell/antibody deficiency affords protection against hypertension and associated vascular remodeling. Methods and Results: Ang II-dependent hypertension in mice was associated with B cell activation/maturation as revealed by a: (1) a 25% increase in the proportion of splenic B cells that expressed the activation marker CD86; (2) an 80% increase in numbers of splenic plasma cells, (3) a 500% increase in serum IgG levels; and (4) marked accumulation of IgG deposits in the aortic adventitia. Indicative of a causative role for B cells, B cell activating factor-receptor-deficient (BAFF-R-/-) mice, which lack mature B cells, displayed blunted hypertensive responses to chronic angiotensin II infusion (Δ30±4 mmHg) compared to wild-type (WT) mice (Δ41±5 mmHg). Importantly, adoptive transfer of B cells into BAFF-R-/- mice restored the hypertensive response. BAFF-R-/- mice showed no evidence of Ang II-induced increases in aortic IgG accumulation. They also had 80% fewer F4/80+ macrophages, 60% fewer CD4+ T cells, and 70% reduction in TGF-β expression and collagen in their aortas compared to Ang II-treated wild-type mice. Ang II doubled aortic pulse-wave velocity, a measure of vessel stiffening, whereas BAFF-R-/- mice were largely protected (70% reduced) from this effect. Finally, pharmacological depletion of B cells with an anti-CD20 antibody blunted Ang II-induced hypertension (Δ37±9 mmHg vs Δ57±6 mmHg). Conclusions: We provide the first evidence that B cells are activated during development of hypertension and contribute to vessel remodeling and elevated blood pressure. These findings suggest B cells and/or the IgG antibodies they produce could be targeted for anti-hypertensive therapy.

Introduction: Evidence suggests that hypertension involves underlying inflammation, however whether atherosclerosis - a chronic inflammatory condition - can cause hypertension is unknown. We tested whether blood pressure (BP) is higher in high-fat fed ApoE-/- vs chow-fed wild-type (WT) mice, and whether advanced atherosclerosis is associated with systemic and end-organ inflammation, oxidative stress and endothelial dysfunction. Methods: Male ApoE-/- and WT mice were placed on high fat and chow diets, respectively, from 5-56 weeks. To clarify the effects of ageing alone, aged WT mice were compared to young chow-fed WT mice (8-12 week old). We measured systolic BP, plasma cytokine levels, mRNA expression of inflammatory markers, vascular superoxide and endothelial function. Results: There was no difference in BP of aged ApoE-/- (104.2 ± 2.9 mmHg) and age-matched WT mice (113.2 ± 1.3 mmHg) (n=13-18, P>0.05). However, plasma IL-6, TNF-α and IFN-γ were elevated in ApoE-/- by more than 2-fold vs age-matched WT (n=9-10, all P Conclusions: Despite the systemic and end-organ inflammation, oxidative stress and endothelial dysfunction, advanced atherosclerosis does not result in elevated BP.

Macrophages accumulate in blood vessels during hypertension. However, their contribution to vessel remodeling is unknown. In the present study, we examined the polarization state of macrophages (M1/M2) in aortas of mice during hypertension and investigated whether antagonism of chemokine receptors involved in macrophage accumulation reduces vessel remodeling and blood pressure (BP). Mice treated with ANG II (0.7 mg·kg−1·day−1, 14 days) had elevated systolic BP (158 ± 3 mmHg) compared with saline-treated animals (122 ± 3 mmHg). Flow cytometry revealed that ANG II infusion increased numbers of CD45+CD11b+Ly6Chi monocytes and CD45+CD11b+F4/80+ macrophages by 10- and 2-fold, respectively. The majority of macrophages were positive for the M2 marker CD206 but negative for the M1 marker inducible nitric oxide synthase. Expression of other M2 genes (arginase-1, Fc receptor-like S scavenger receptor, and receptor-1) was elevated in aortas from ANG II-treated mice, whereas M1 genes [TNF and chemokine (C-X-C motif) ligand 2] were unaltered. A PCR array to identify chemokine receptor targets for intervention revealed chemokine (C-C motif) receptor 2 (CCR2) to be upregulated in aortas from ANG II-treated mice, while flow cytometry identified Ly6Chi monocytes as the main CCR2-expressing cell type. Intervention with a CCR2 antagonist (INCB3344; 30 mg·kg−1·day−1), 7 days after the commencement of ANG II infusion, reduced aortic macrophage numbers. INCB334 also reduced aortic collagen deposition, elastin loss, and BP in ANG II-treated mice. Thus, ANG II-dependent hypertension in mice is associated with Ly6Chi monocyte and M2 macrophage accumulation in the aorta. Inhibition of macrophage accumulation with a CCR2 antagonist prevents ANG II-induced vessel fibrosis and elevated BP, highlighting this as a promising approach for the future treatment of vessel remodeling/stiffening in hypertension.

Macrophages accumulate in the vascular wall during hypertension. However, their contribution to vascular remodeling remains to be established. In the present study we examined the polarization state of macrophages (M1/M2) in the aorta during experimental hypertension. We also investigated whether pharmacological antagonism of the chemokine receptors involved in macrophage accumulation reduces vascular remodeling and/or blood pressure (BP). Mice treated with angiotensin II (Ang II; 0.7 mg/kg/d) displayed elevated systolic BP (158±3 mmHg) compared with saline-treated animals (122±3 mmHg; P+ ) numbers (P

Arterial fibrosis and stiffening are hallmarks of hypertension and contribute to elevated systolic blood pressure (BP), heart failure and end organ damage. Hypertension is also associated with accumulation of macrophages in the vascular wall; however, the phenotype of these cells (M1/M2) and their contribution to vascular fibrosis/stiffening remain to be established. Chronic angiotensin II-infusion in mice (0.7 mg/kg/d, 28 d) caused a marked increase in systolic BP (169±6 mmHg vs 118±3 mmHg in saline-infused mice). Flow cytometric analysis of aortic cell suspensions revealed that angiotensin II infusion also caused a 2-fold increase in F4/80 + macrophage numbers. Moreover, these cells were positive for the M2 marker, CD206. Quantitative PCR confirmed that aortic expression of CD206 and other M2 markers (arginase-1, Fc receptor-like S, scavenger receptor) were elevated in angiotensin II-infused mice, whereas levels of M1 markers (inducible nitric oxide synthase, CXCL2, tumour necrosis factor) were unchanged. Twice-weekly treatment of angiotensin II-infused mice with clodronate-encapsulated liposomes reduced numbers of circulating monocytes (i.e. macrophage precursors) by ~70%, whereas saline-containing liposomes had no effect. Clodronate treatment prevented M2 macrophage accumulation in the aorta, and also in the kidneys, and importantly reduced collagen deposition in these tissues. Clodronate treatment also improved arterial compliance and lowered BP by ~20 mmHg in angiotensin II-treated mice. In conclusion, M2 macrophages contribute to tissue fibrosis and aortic stiffening during angiotensin II-induced hypertension in mice. Thus, strategies that limit macrophage accumulation in the vessel wall and/or prevent their polarization towards an M2 phenotype may hold promise as future therapies for hypertension.

Inflammasomes are signalling complexes comprised of a NOD-like receptor protein (NLRP), an adapter protein (ASC) and caspase-1. Inflammasomes detect host-derived danger signals and induce inflammation via activation of caspase-1, which in turn cleaves the cytokines pro-interleukin(IL)-1β and pro-IL-18 into their active, pro-inflammatory forms. Hypertension is associated with chronic renal inflammation, but the role of the inflammasome in this process is not known. Hence, we tested whether deoxycorticosterone acetate (DOCA)/salt-induced hypertension in mice is associated with increased expression and/or activation of the inflammasome in the kidney, and assessed the impact of inhibition of inflammasome activity on blood pressure (BP) and markers of renal inflammation and fibrosis. Male C57BL6/J (wild type) and ASC -/- mice were uninephrectomised, implanted with a DOCA pellet (2.4 mg/d, 21 d, s.c. ) and had their drinking water replaced with 1% saline (1K/DOCA/salt). Control mice had a kidney removed but received a placebo pellet and normal drinking water. 1K/DOCA/salt-treated mice had elevated systolic BP (146±4 mmHg) compared to control mice (115±2 mmHg; n=13-16; P-/- mice, which lack an active inflammasome complex, displayed blunted hypertensive responses to 1K/DOCA/salt-treatment (140±3 mmHg) compared to wild types (155±8 mmHg; n=8-9; P-/- mice were also protected from 1K/DOCA/salt-induced increases in renal expression of the inflammatory genes IL-6, IL-17a, CCL2, ICAM-1 and VCAM-1, and accumulation of collagen. Thus, renal inflammation, fibrosis and elevated BP in response to 1K/DOCA/salt-treatment are critically dependent on inflammasome activity, highlighting this signalling complex and its cytokine products as potential therapeutic targets to treat hypertension.

Infiltration of macrophages into the artery wall plays detrimental roles during hypertension by promoting vascular inflammation and endothelial dysfunction, and it occurs via a chemo-attractant action of chemokines on macrophage cytokine receptors. We sought to identify the key chemokine receptors associated with macrophage infiltration into the vascular wall during deoxycorticosterone acetate (DOCA)/salt-induced hypertension in mice and to evaluate the impact of pharmacological inhibition of these receptors on blood pressure and leukocyte accumulation. Mice treated with DOCA/salt for 21 days displayed markedly elevated systolic blood pressure (158±2 versus 114±5 mm Hg in sham group; P 2-fold versus sham; P + CD11b + F4/80 + cells) in the aortic wall of DOCA/salt versus sham-treated mice. Intervention with a CCR2 antagonist, INCB3344 (30 mg/kg per day, IP), 10 days after the induction of hypertension with DOCA/salt treatment, reduced the aortic expression of CCR2 mRNA and completely reversed the DOCA/salt-induced influx of macrophages. Importantly, INCB3344 substantially reduced the elevated blood pressure in DOCA/salt-treated mice. Hence, our findings highlight CCR2 as a promising therapeutic target to reduce both macrophage accumulation in the vascular wall and blood pressure in hypertension.

Reactive oxygen species (ROS) generated by Nox2 oxidase are reported to contribute to infarct damage following cerebral ischemia–reperfusion. Here we have examined for the first time the role of Nox2 expression in outcomes following permanent focal cerebral ischemia. Ischemia was induced by middle cerebral artery filament occlusion (MCAO) for 24 h in wild-type (WT) and Nox2 −/y mice. Neurological deficit and the hanging wire test were assessed, and infarct and edema volumes were estimated using thionin-stained brain sections. Genetic deletion of Nox2 had no effect on any outcome measures at 24 h after permanent MCAO. Our data therefore suggest that ROS production by Nox2 oxidase activity plays no significant role in the pathophysiology of cerebral ischemia in the absence of reperfusion.

Cerebral infarct volume is typically smaller in premenopausal females than in age-matched males after ischemic stroke, but the underlying mechanisms are poorly understood. In this study we provide evidence in mice that this gender difference only occurs when the ischemic brain is reperfused. The limited tissue salvage achieved by reperfusion in male mice is associated with increased expression of proinflammatory proteins, including cyclooxygenase-2 (Cox-2), Nox2, and vascular cell adhesion molecule-1 (VCAM-1), and infiltration of Nox2-containing T lymphocytes into the infarcted brain, whereas such changes are minimal in female mice after ischemia-reperfusion (I-R). Infarct volume after I-R was no greater at 72 h than at 24 h in either gender. Infarct development was Nox2 dependent in male but not in female mice, and Nox2 within the infarct was predominantly localized in T lymphocytes. Stroke resulted in an approximately 15-fold increase in Nox2-dependent superoxide production by circulating, but not spleen-derived, T lymphocytes in male mice, and this was approximately sevenfold greater than in female mice. These circulating immune cells may thus represent a major and previously unrecognized source of superoxide in the acutely ischemic and reperfused brain of males (and potentially in postmenopausal females). Our findings provide novel insights into mechanisms that could be therapeutically targeted in acute ischemic stroke patients who receive thrombolysis therapy to induce cerebral reperfusion.

The Nox family NADPH oxidases are reactive oxygen species (ROS)-generating enzymes that are strongly implicated in atherogenesis. However, no studies have examined which Nox isoform(s) are involved. Here we investigated the role of the Nox2-containing NADPH oxidase in atherogenesis in apolipoprotein E-null (ApoE−/−) mice. Wild-type (C57Bl6/J), ApoE−/−, and Nox2−/y/ApoE−/−mice were maintained on a high-fat (21%) diet from 5 wk of age until they were 12 or 19 wk old. Mice were euthanized and their aortas removed for measurement of Nox2 expression (Western blot analysis and immunohistochemistry), ROS production (L012-enhanced chemiluminescence), nitric oxide (NO) bioavailability (contractions to Nω-nitro-l-arginine), and atherosclerotic plaque development along the aorta and in the aortic sinus. Nox2 expression was upregulated in the aortic endothelium of ApoE−/−mice before the appearance of lesions, and this was associated with elevated ROS levels. Within developing plaques, macrophages were also a prominent source of Nox2. The absence of Nox2 in Nox2−/y/ApoE−/−double-knockout mice had minimal effects on plasma lipids or lesion development in the aortic sinus in animals up to 19 wk of age. However, an en face examination of the aorta from the arch to the iliac bifurcation revealed a 50% reduction in lesion area in Nox2−/y/ApoE−/−versus ApoE−/−mice, and this was associated with a marked decrease in aortic ROS production and an increased NO bioavailability. In conclusion, this is the first demonstration of a role for Nox2-NADPH oxidase in vascular ROS production, reduced NO bioavailability, and early lesion development in ApoE−/−mice, highlighting this Nox isoform as a potential target for future therapies for atherosclerosis.

Hypertension remains the leading risk factor for cardiovascular disease (CVD). Experimental hypertension is associated with increased T cell infiltration into blood pressure-controlling organs, such as the aorta and kidney; importantly in absence of T cells of the adaptive immune system, experimental hypertension is significantly blunted. However, the function and phenotype of these T cell infiltrates remains speculative and undefined in the setting of hypertension. The current study compared T cell-derived cytokine and reactive oxygen species (ROS) production from normotensive and hypertensive mice. Splenic, blood, aortic, kidney and brain T cells were isolated from C57BL/6J mice following 14-day vehicle or angiotensin (Ang) II (0.7 mg/kg/day, s.c.) infusion. T cell infiltration was increased in aorta, kidney and brain from hypertensive mice. Cytokine analysis in stimulated T cells indicated an overall Th1 pro-inflammatory phenotype, but a similar proportion (flow cytometry) and quantity (cytometric bead array) of IFN-γ, TNF-α, IL-4 and IL-17 between vehicle- and Ang II- treated groups. Strikingly, elevated T cell-derived production of a chemokine, chemokine C-C motif ligand 2 (CCL2), was observed in aorta (∼6-fold) and kidney in response to Ang II, but not in brain, spleen or blood. Moreover, T cell-derived ROS production in aorta was elevated ∼3 -fold in Ang II-treated mice (n = 7; P