A Novel Model of Tobacco Smoke–Mediated Aortic Injury

Objective Tobacco smoke exposure is a major risk factor for aortic aneurysm development. However, the initial aortic response to tobacco smoke, preceding aneurysm formation, is not well understood. We sought to create a model to determine the effect of solubilized tobacco smoke (STS) on the thoracic and abdominal aorta of mice as well as on cultured human aortic smooth muscle cells (HASMCs). Methods Tobacco smoke was solubilized and delivered to mice via implanted osmotic minipumps. Twenty male C57BL/6 mice received STS or vehicle infusion. The descending thoracic, suprarenal abdominal, and infrarenal abdominal segments of the aorta were assessed for elastic lamellar damage, smooth muscle cell phenotype, and infiltration of inflammatory cells. Cultured HASMCs grown in media containing STS were compared to cells grown in standard media in order to verify our in vivo findings. Results Tobacco smoke solution caused significantly more breaks in the elastic lamellae of the thoracic and abdominal aorta compared to control solution ( P< .0001) without inciting an inflammatory infiltrate. Elastin breaks occurred more frequently in the abdominal aorta than the thoracic aorta ( P < .01). Exposure to STS-induced aortic microdissections and downregulation of α-smooth muscle actin (α-SMA) by vascular smooth muscle cells (VSMCs). Treatment of cultured HASMCs with STS confirmed the decrease in α-SMA expression. Conclusion Delivery of STS via osmotic minipumps appears to be a promising model for investigating the early aortic response to tobacco smoke exposure. The initial effect of tobacco smoke exposure on the aorta is elastic lamellar damage and downregulation of (α-SMA) expression by VSMCs. Elastic lamellar damage occurs more frequently in the abdominal aorta than the thoracic aorta and does not seem to be mediated by the presence of macrophages or other inflammatory cells.

Abstract MP05: Pvn-specific Microgliosis And Inflammation Precedes Sympathoexcitation In gαI 2 Protein-dependent, Salt-sensitive Hypertension

Aim: Hypothalamic paraventricular nucleus (PVN) Gαi 2 proteins mediate sympathoinhibitory responses to a high salt (HS; 4% NaCl) diet. Failure to upregulate Gαi 2 proteins in response to a HS diet results in PVN inflammation and salt sensitive hypertension (SSHTN). We hypothesize that microglial-mediated PVN inflammation precedes sympathoexcitation in Gαi 2 protein-dependent SSHTN. Methods: Three-month-old male Sprague Dawley rats implanted with ICV cannulas fitted to osmotic minipumps to centrally infuse either a control scrambled (SCR) oligodeoxynucleotide (ODN) or a Gαi 2 targeted ODN, which downregulates CNS Gαi 2 proteins by ~85%, (25μg/5μl/day/ODN) were placed on a 1-7-day normal salt (NS; 0.6% NaCl) or HS diet (n=5/group) and underwent cardiac perfusion. Brain immunohistochemistry was used to assess PVN and subfornical organ microgliosis and qualitatively assess levels of PVN pro-inflammatory cytokines (PIC) IL-1β, IL-6, and TNFα. In additional groups, MAP was assessed via radiotelemetry, and whole blood and kidneys were obtained for ELISA measurement of plasma and renal norepinephrine (NE) as estimates of sympathetic tone. Results: By 24h in control SCR ODN infused rats a HS diet, which did not alter MAP or microglial activation, evoked sympathoinhibition. In contrast, in Gαi 2 ODN infused rats a HS diet did not result in sympathoinhibition and evoked significant increases in MAP, PVN microgliosis and PVN PIC expression within 24h, and elevated renal NE content by Day 3. Conclusions: Our data suggest that in the male Sprague-Dawley model of PVN Gαi 2 protein-dependent SSHTN PVN inflammation (microgliosis and PIC production) precedes sympathoexcitation.

Abstract 104: Cilnidipine Attenuated Angiotensin II-induced Abdominal Aortic Aneurysms in Apolipoprotein E-deficient Mice via Its Anti-oxidative Stress Effect

Objective: Chronic angiotensin II (AngII) infusion promotes development of abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Previous studies have shown that local inflammation and increased oxidative stress plays an important role on formation of AAAs. In addition, voltage-dependent N-type Ca 2+ channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by AngII in mice. Last year, we reported that cilnidipine, an N/L-type calcium channel blocker, attenuated AngII-induced AAAs in apolipoprotein E deficient mice. The purposed of this study was to determine the mechanism of cilnidipine reducing AngII-induced AAAs Methods and Results: Male apolipoprotein E deficient mice (8-12 weeks old) were fed a normal laboratory diet. Mice were infused subcutaneously with either cilnidipine or vehicle by osmotic minipumps. Three days later, mice were also infused subcutaneously with either AngII (1,000 ng/kg/min, n = 14-16) or saline (n = 4) by osmotic minipumps for 4 weeks. AngII increased systolic blood pressure. Cilnidipine decreased blood pressure in AngII-infused mice, but had not effect during saline infusion. Cilnidipine mildly restored plasma renin activity that were suppressed by AngII infusion. Cilnidipine mildly decreased plasma aldosterone concentrations that were increased by AngII infusion. Cilnidipine significantly reduced the incidence of AngII-induced AAAs (cilnidipine: 38 %, Vehicle: 88 %; P < 0.05). Elastica van Gieson staining demonstrated degeneration of elastic lamina by AngII was suppressed by cilnidipine administration. Immunohistochemical staining of CD68 revealed that cilnidipine administration attenuated accumulation of CD68 positive macrophage by AngII. In addition, cilnidipine reduced oxidative stress by AngII in 8-hydroxy-2’-deoxyguanosine and 4-Hydroxy-2-nonenal staining. Conclusion: Cilnidipine attenuated AngII-induced AAAs in male apolipoprotein E deficient mice by its anti-inflammation and anti-oxidative stress effect.

Abstract 461: Cilnidipine Attenuated Angiotensin II-induced Abdominal Aortic Aneurysms in Apolipoprotein E-deficient Mice

Objective: Chronic infusion of angiotensin II (AngII) promotes development of abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Previous studies have shown that local inflammation and increased oxidative stress plays an important role on formation of AAAs. In addition, it is reported that voltage-dependent N-type Ca2+ channels in endothelial cells contribute to oxidative stress-related endothelial dysfunction induced by AngII in mice. Therefore, we hypothesized that cilnidipine, an N/L-type calcium channel blocker, exerts vasoprotective effect by inhibiting inflammation and superoxide generation in mice. The purpose of this study was to evaluate whether cilnidipine influenced AngII-induced AAAs. Methods and Results: Male apolipoprotein E deficient mice (8-12 weeks old) were fed a normal laboratory diet. Mice were infused subcutaneously with either cilnidipine or vehicle by osmotic minipumps. Three days later, mice were also infused subcutaneously with either AngII (1,000 ng/kg/min, n = 14-16) or saline (n = 4) each by osmotic minipumps for 4 weeks. AngII increased systolic blood pressure. Cilnidipine decreased blood pressure in AngII-infused mice, but had not effect during saline infusion. AngII infusion did not alter serum cholesterol concentrations. However, cilnidipine slightly decreased serumcholesterol concentrations in AngII-infused mice. Cilnidipine had no effect on body weights, heart rates, and urine total protein, but mildly restored plasma renin activity that were suppressed by AngII infusion. Cilnidipine did not affect ex vivo measurement of maximal diameter of abdominal aorta (1.04 ± 0.09 mm vs 1.11 ± 0.06 mm, n.s.) in saline infused mice. AngII infusion significantly increased ex vivo maximal diameters of abdominal aortas, but was attenuated by cilnidipine (1.79 ± 0.59 mm vs 1.26 ± 0.38 mm, P < 0.05). In addition, cilnidipine significantly reduced the incidence of AngII-induced AAAs (Cilnidipine: 38 %, Vehicle: 88 %; P < 0.05). Furthermore, gelatin zymography demonstrated that cilnidipine diminished AngII-induced increase in aortic MMP-9 protein abundance. Conclusion: Cilnidipine attenuated AngII-induced AAAs in male apolipoprotein E deficient mice.

Chronic lipopolysaccharide infusion fails to induce depressive-like behaviour in adult male rats

BackgroundChronic inflammation is implicated in numerous diseases, including major depression and type 2 diabetes mellitus (T2DM). Since depression and T2DM often co-exist, inflammatory pathways are suggested as a possible link. Hence, the establishment of an immune-mediated animal model would shed light on mechanisms possibly linking depression and metabolic alterations.ObjectiveIn this study we investigated a behavioural and metabolic paradigm following chronic infusion with low doses of lipopolysaccharide (LPS) using osmotic minipumps in male rats.MethodsBehavioural testing consisted of evaluating activity level in the open field and depressive-like behaviour in the forced swim test. Metabolic assessment included measurement of body weight, food and water intake, and glucose and insulin levels during an oral glucose tolerance test.ResultsLPS-infused rats showed acute signs of sickness behaviour, but chronic LPS infusion did not induce behavioural or metabolic changes.ConclusionThese results suggest that although inflammation is immediately induced as indicated by acute sickness, 4 weeks of chronic LPS administration via osmotic minipumps did not result in behavioural changes. Therefore, this paradigm may not be a suitable model for studying the underlying mechanisms that link depression and T2DM.

Abstract 231: Angiotensin-(1-9) Antagonises Cardiac Remodelling in a Mouse Model of Angiotensin Ii-induced Hypertension.

The identification of angiotensin converting enzyme-2 (ACE2), generating angiotensin-(1-7) [Ang-(1-7)] which signals via the Mas receptor to inhibit pathophysiological effects in cardiovascular disease has led to the definition of a counter-regulatory axis of the renin angiotensin system (RAS). Recently, we showed that another angiotensin peptide, Ang-(1-9), may also be part of this counter-regulatory axis as it prevents cardiomyocyte hypertrophy via the angiotensin type 2 receptor (AT2R). Furthermore, infusion of Ang-(1-9) via osmotic minipumps into stroke-prone spontaneously hypertensive rats reduced cardiac fibrosis via the AT2R. Here, we investigated Ang-(1-9) in an acute model of AngII-induced hypertension in C57BL/6J mice. Ang-(1-9), Ang-(1-9)+PD123,319 (AT2R antagonist), or water (control) were co-infused with AngII for 2 weeks via osmotic minipumps. Blood pressure was monitored via radiotelemetry. Significant increases in mean arterial pressure were observed in AngII infused mice compared to control (control 108.8±5.7 mmHg; AngII 125.1± 8.4 mmHg; p>0.05) however co-infusion of Ang-(1-9) or Ang-(1-9) and PD123,319 did not affect AngII-induced hypertension (AngII+ Ang-(1-9) 122.4±10.3 mmHg; AngII+Ang-(1-9)+PD123,319 118.3±mmHg; n=6 mice). When assessing cardiac hypertrophy by heart weight/tibia length ratio Ang-(1-9) reduced AngII-induced cardiac hypertrophy (ratio heart weight/tibia length: control 9.4 ± 1.2; AngII 11.9 ± 0.6; AngII+Ang-(1-9) 8.2 ± 0.7; p<0.01). Sections of heart were stained with wheat germ agglutinin and cardiomyocyte size measured supporting an anti-hypertrophic effect of Ang-(1-9) via the AT2R (control 25.7±3.8 μm; AngII 28.8±3.8 μm; AngII+Ang-(1-9) 24.6±4.8 μm; AngII+Ang-(1-9)+PD123,319 27.7±3.2μm; p<0.05). Furthermore, quantification of fibrosis following staining with picrosirius red also showed a significant reduction in animals co-infused with Ang-(1-9) and AngII compared to AngII-infusion (p<0.05). Further studies are ongoing to analyse cardiac effects of Ang-(1-9) at gene and protein level. These data support a direct biological role for Ang-(1-9) in cardiac remodeling and highlights Ang-(1-9) as a potential new therapeutic target in cardiovascular disease.