SA Mice Research Articles

Exercise training inhibits left ventricular collagen upregulation in mice with hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is estimated to affect 1:500, and is characterised by otherwise unexplained left ventricular (LV) hypertrophy, cardiomyocyte disarray, fibrosis, diastolic dysfunction, and ventricular arrhythmias. Historically, patients with HCM have been discouraged from participation in high intensity sports and exercise. However, the 2020 Sports Cardiology Guidelines recommend that patients with HCM should receive advice about exercise training (ET) based on individual risk assessment. To learn more about the effects of ET in HCM, we exposed mice carrying an HCM-causative sarcomere mutation (Myh6R403Q/+ (R403Q) mice) to high intensity interval training. Purpose To investigate the effect of exercise training on hypertrophic cardiomyopathy in mice. Methods R403Q mice were stratified to treadmill exercise (n=11) or sedentary behaviour (n=11). After 3 weeks, we induced HCM by giving CsA in the feed for 3 weeks, while the ET or sedentary behaviour continued for a total of 6 weeks. Each bout of treadmill running consisted of a 10-minute warm up, followed by 5 intervals of 8 minutes at high intensity (90% of VO2 max speed at week 0) and 2 minutes at medium intensity (60% of VO2 max speed at week 0). Every third day of the ET protocol we increased the high and medium intensity running speeds by 0.6 and 0.4 m/min, respectively. We performed echocardiography after 0, 3, and 6 weeks of the 6-week protocol. After completion of the protocol, we recorded lung and whole heart weight, and harvested LVs for molecular analyses. Results Confirming the expected HCM phenotype, R403Q mice that received CsA (R403Q SED+CsA) had a 1.3-fold increase in whole heart weight (p<0.0001), 1.5-fold increase in lung weight (p<0.001), and 2.4-fold increase in maximal left ventricular posterior wall (LVPW) thickness measured by echocardiography (p<0.0001) compared to sedentary wild type littermates given CsA (WT SED+CsA). Heart weight, lung weight, and maximum LVPW thickness were also increased 1.5- (p<0.0001), 1.6- (p<0.0001), and 2.0-(p<0.0001) fold, respectively, compared to R403Q not given CsA (R403Q SED-CsA). R403Q ET+CsA mice increased their running distance before exhaustion 2.0-fold compared to baseline (p=0.010), and ran 1.6-fold longer than R403Q SED+CsA (p=0.020). In R403Q ET+CsA mice, LV mRNA expression of Col1a2 was 51% (p=0.021), and Col3a1 49% (p=0.013) of R403Q SED+CsA expression levels. Compared to R403Q SED+CsA mice, exercise training did not affect heart weight, maximum LV posterior wall thickness or lung weight in R403Q ET+CsA mice. Conclusion Treadmill ET inhibited upregulation of LV collagen expression in mice with HCM, but did not affect hypertrophy. This could indicate that ET during early development of HCM attenuates development of fibrosis. Funding Acknowledgement Type of funding sources: Public Institution(s). Main funding source(s): South-Eastern Norway Regional Health Authority (Helse Sør-Øst RHF) and KG Jebsen Cardiac Research Center

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

During influenza A epidemics, bacterial coinfection is a major cause of increased morbidity and mortality. However, the roles of host factors in regulating influenza A virus (IAV)-triggered bacterial coinfection remain elusive. Cyclophilin A (CypA) is an important regulator of infection and immunity. Here, we show that IAV-induced CypA expression facilitates group A Streptococcus (GAS) coinfection both invitro and invivo. Upon IAV infection, CypA interacts with focal adhesion kinase (FAK) and inhibited E3 ligase cCbl-mediated, K48-linked ubiquitination of FAK, which positively regulates integrin α5 expression and actin rearrangement via the FAK/Akt signaling pathway to facilitate GAS colonization and invasion. Notably, CypA deficiency or inhibition by cyclosporine A significantly inhibits IAV-triggered GAS coinfection in mice. Collectively, thesefindings reveal that CypA is critical for GAS infection, and induction of CypA expression is another way for IAV to promote bacterial coinfection, suggesting that CypA is a promising therapeutic target for the secondary bacterial infection.

Phospho-ablation of cardiac sodium channel Nav1.5 mitigates susceptibility to atrial fibrillation and improves glucose homeostasis under conditions of diet-induced obesity

BackgroundAtrial fibrillation (AF) is the most common sustained arrhythmia, with growing evidence identifying obesity as an important risk factor for the development of AF. Although defective atrial myocyte excitability due to stress-induced remodeling of ion channels is commonly observed in the setting of AF, little is known about the mechanistic link between obesity and AF. Recent studies have identified increased cardiac late sodium current (INa,L) downstream of calmodulin-dependent kinase II (CaMKII) activation as an important driver of AF susceptibility.MethodsHere, we investigated a possible role for CaMKII-dependent INa,L in obesity-induced AF using wild-type (WT) and whole-body knock-in mice that ablates phosphorylation of the Nav1.5 sodium channel and prevents augmentation of the late sodium current (S571A; SA mice).ResultsA high-fat diet (HFD) increased susceptibility to arrhythmias in WT mice, while SA mice were protected from this effect. Unexpectedly, SA mice had improved glucose homeostasis and decreased body weight compared to WT mice. However, SA mice also had reduced food consumption compared to WT mice. Controlling for food consumption through pair feeding of WT and SA mice abrogated differences in weight gain and AF inducibility, but not atrial fibrosis, premature atrial contractions or metabolic capacity.ConclusionsThese data demonstrate a novel role for CaMKII-dependent regulation of Nav1.5 in mediating susceptibility to arrhythmias and whole-body metabolism under conditions of diet-induced obesity.

Cancer-associated fibroblasts downregulate type I interferon receptor to stimulate intratumoral stromagenesis.

Activation of cancer-associated fibroblasts (CAFs) and ensuing desmoplasia play an important role in the growth and progression of solid tumors. Here we demonstrate that, within colon and pancreatic ductal adenocarcinoma tumors, efficient stromagenesis relies on downregulation of the IFNAR1 chain of type I interferon (IFN1) receptor. Expression of the fibroblast activation protein (FAP) and accumulation of the extracellular matrix (ECM) was notably impaired in tumors grown in the Ifnar1S526A (SA) knock-in mice, which are deficient in IFNAR1 downregulation. Primary fibroblasts from these mice exhibited elevated levels of Smad7, a negative regulator of the tumor growth factor-beta (TGFβ) pathway. Knockdown of Smad7 alleviated deficient ECM production in SA fibroblasts in response to TGFβ. Analysis of human colorectal cancers revealed an inverse correlation between IFNAR1 and FAP levels. Whereas growth of tumors in SA mice was stimulated by co-injection of wild type but not SA fibroblasts, genetic ablation of IFNAR1 in fibroblasts also accelerated tumor growth. We discuss how inactivation of IFNAR1 in CAFs acts to stimulate stromagenesis and tumor growth.

Loss of β-adrenergic-stimulated phosphorylation of CaV1.2 channels on Ser1700 leads to heart failure.

L-type Ca2+ currents conducted by voltage-gated calcium channel 1.2 (CaV1.2) initiate excitation-contraction coupling in the heart, and altered expression of CaV1.2 causes heart failure in mice. Here we show unexpectedly that reducing β-adrenergic regulation of CaV1.2 channels by mutation of a single PKA site, Ser1700, in the proximal C-terminal domain causes reduced contractile function, cardiac hypertrophy, and heart failure without changes in expression, localization, or function of the CaV1.2 protein in the mutant mice (SA mice). These deficits were aggravated with aging. Dual mutation of Ser1700 and a nearby casein-kinase II site (Thr1704) caused accelerated hypertrophy, heart failure, and death in mice with these mutations (STAA mice). Cardiac hypertrophy was increased by voluntary exercise and by persistent β-adrenergic stimulation. PKA expression was increased, and PKA sites Ser2808 in ryanodine receptor type-2, Ser16 in phospholamban, and Ser23/24 in troponin-I were hyperphosphorylated in SA mice, whereas phosphorylation of substrates for calcium/calmodulin-dependent protein kinase II was unchanged. The Ca2+ pool in the sarcoplasmic reticulum was increased, the activity of calcineurin was elevated, and calcineurin inhibitors improved contractility and ameliorated cardiac hypertrophy. Cardio-specific expression of the SA mutation also caused reduced contractility and hypertrophy. These results suggest engagement of compensatory mechanisms, which initially may enhance the contractility of individual myocytes but eventually contribute to an increased sensitivity to cardiovascular stress and to heart failure in vivo. Our results demonstrate that normal regulation of CaV1.2 channels by phosphorylation of Ser1700 in cardiomyocytes is required for cardiovascular homeostasis and normal physiological regulation in vivo.

Targeting Neutrophil Aging and the Microbiota for the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is a genetic blood disorder characterized by repeated episodes of vaso-occlusion, in which neutrophils play a primary function through their recruitment in inflamed venules and interactions with sickle red blood cells (sRBCs) via activated αMβ2 integrin (Mac-1). Intravital microscopy analyses revealed considerable heterogeneity in Mac-1 activation on adherent neutrophils, suggesting that subsets of neutrophils differ markedly in their pro-inflammatory activity (Nat Med 2009; 15:384). Recently, we found that aged neutrophils, marked by CD62Llo CXCR4hi, represented an overly active subset exhibiting enhanced Mac-1 activation (Blood 2013; 122:324). To further characterize aged neutrophils, we compared their transcriptome with control and in vivo- activated neutrophils. Gene set enrichment analyses revealed that aged neutrophils up-regulated several pathways that were also enhanced during activation, including integrin, leukocyte adhesion, toll-like receptor (TLR), and NFκB signalling pathways (P < 0.05), suggesting a contribution by exogenous inflammatory mediators. Based on the expression profile, we hypothesized that microbiota-derived signals may influence neutrophil aging in the circulation. To test this idea, we analyzed aged neutrophil numbers in antibiotics (ABX)-treated and germ-free (GF) mice, in which the gut microbiota was largely depleted or deficient, respectively. We found significant reductions of aged neutrophil numbers in both models, and the numbers could be significantly restored by either lipopolysaccharide (LPS) gavage or fecal transplantation (Ctrl / ABX / ABX+LPS: 98±14 / 36±11 / 107±25 cells per μl blood; SPF / GF / GF-FT: 56±12 / 4±1 / 15±4 cells per μl blood; P < 0.05). We next investigated whether microbiota-driven neutrophil aging was mediated by TLRs and Myd88 signalling using chimeric mice reconstituted with a mixture of WT and Myd88, TLR4 or TLR2-deficient hematopoietic cells. Interestingly, we observed significant reductions in the aged subset of Myd88, TLR4 or TLR2-deficient neutrophils compared to WT neutrophils in the same mice, and in their capacity to capture fluosphere beads that specifically bound to activated Mac-1 (WT / LysM-cre/Myd88-flox: 10.4±0.3 / 7.0±0.6 %, 1.0±0.1 / 0.4±0.1 bead per cell; WT / Tlr4-/-: 10.3±0.2 / 6.0±0.2 %, 1.0±0.1 / 0.4±0.1 bead per cell; WT / Tlr2-/-: 10.0±0.4 / 6.8±0.5 %, 1.3±0.2 / 0.8±0.1 bead per cell; P < 0.01). Analysis of SCD mice (BERK) revealed that the aged neutrophil population was significantly increased (by >10-fold) compared to hemizygous (SA) mice, and the expansion was completely abrogated by microbiota depletion (SA / SCD (SS)-Ctrl / SS-ABX: 71±11 / 981±261 / 124±27 cells per μl blood, P < 0.01). We then challenged SA, untreated and ABX-treated SCD mice with TNF-α to induce acute vaso-occlusion. SCD mice exhibited significant increases in neutrophil adhesion, Mac-1 integrin activation and heterotypic interactions with RBCs compared to SA mice, all of which were markedly reduced by microbiota depletion (SA / SS-Ctrl / SS-ABX: 6.4±0.6 / 18.1±1.0 / 11.2±1.0 adherent neutrophil per vessel; 0.5±0.1 / 1.0±0.2 / 0.3±0.1 bead per cell; 0.1±0.1 / 0.5±0.1 / 0.2±0.1 RBC interaction per vessel per min; P < 0.05), resulting in significantly enhanced blood flow and prolonged survival in ABX-treated SCD mice (P < 0.05). Most interestingly, the splenomegaly of SCD mice was significantly reduced, and liver damage including fibrosis, necrosis and inflammation was dramatically alleviated in ABX-treated SCD mice (P < 0.05). Finally, we evaluated whether the numbers of circulating aged neutrophils were altered in patients with SCD. As Penicillin V antibiotic prophylaxis therapy is recommended for children < 5 years or older patients with immune defects to prevent life-threatening infections, we determined aged neutrophil numbers in this patient population. We found that SCD patients exhibited a marked increase in the numbers of circulating aged neutrophils compared to healthy controls, which were significantly reduced in patients on Penicillin V prophylaxis (Ctrl / SS / SS-PV: 261±40 / 1175±205 / 439±87 cells per μl blood, P < 0.05). Our results raise the possibility that targeting neutrophil aging and the microbiota may have broad implications in our understanding of sickle cell disease pathogenesis and its management that should be further studied in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Three Months of Human Haptoglobin Treatment Decreases Iron Deposition in the Kidneys of Townes Sickle Mice

Introduction: Haptoglobin (Hp), the scavenger for hemoglobin, and hemopexin (Hx), the scavenger for heme, are depleted in most patients with sickle cell disease due to chronic hemolysis. There is mounting evidence of the crucial role of free hemoglobin and/or free heme in mediating inflammatory and oxidative damage in sickle cell disease, including vaso-occlusion and acute chest syndrome. Purified Hp has been used in Japan for a variety of hemolytic conditions and has been proposed as a potential treatment for sickle cell disease. Although infusions of Hp or Hx have been shown to ameliorate vaso-occlusion, acute lung injury, and heme toxicity in sickle cell mouse models, no prior studies have examined the utility of chronic Hp treatment for amelioration of organ damage. We therefore studied the effect of 3 months of chronic Hp treatment in the Townes sickle mouse model.Methods: Male and female Townes mice (Stock number 013071, The Jackson Laboratory) were used for all experiments, starting at 1 or 3 months of age. SS genotype was confirmed by PCR and HPLC. Organ damage in the spleen, liver, and kidneys as previously described was confirmed. Human Hp solution was a kind gift from Bio Product Laboratory (BPL, Hertfordshire, UK). Hp or equivalent volume PBS control was administered intraperitoneally (IP) in the first cohort of 5 mice and then subcutaneously (SC) in the next two cohorts of 7 and 12 mice on a 48-72 hr dosing schedule of Monday, Wednesday, Friday for a period of 3 months. At the end of 3 months treatment, mice were evaluated by the following studies (with concurrent blinding to treatment group for most studies): plasma Hp (ELISA), plasma heme (QuantiChrom heme assay), urine osmolality (osmometer), urine albumin (ELISA), CBC (Advia 120), WBC differential (Advia 120 and manual count), red blood cell ektacyometry (ektacyometer), organ mass (percent of body weight), and organ histology.Results: Mouse Hp levels in SS Townes mice were confirmed to be markedly low compared to Townes AA mice (mean ± SD: SS 2 ± 1 versus AA 39 ± 4 ug/mL). Dose-finding experiments determined that a dose of 200-400 mg/kg IP or SC in SS mice resulted in a 24 hr peak concentration that was 5-14X supraphysiologic, variably physiologic at 48 hr, and absent or almost absent at 72 hr. Chronic dosing at the 400 mg/kg IP in SA mice showed no CBC or organ toxicity. Three successive cohorts of SS mice were treated with Hp (or equivalent volume of PBS): 200 mg/kg IP in 3-month old mice, 400 mg/kg SC in 3-month old mice, and 400 mg/kg SC in 1-month old mice. At the 400 mg/kg dosing levels, there was a significant decrease in iron deposition in the kidneys of both 4-month and 6-month old mice (treatment started at 1-month and 3 months, respectively) (Table 1). There was also a trend towards decreased liver infarction in 6-month old mice (Table 2).Discussion: Functional binding of the administered human Hp to the human Hb of the Townes mice likely occurred, as evidenced by the decrease in iron deposition in the kidneys, suggesting that formation of the complex prevents filtration of Hb into the kidneys. Surprisingly, kidney function as measured by urine osmolality or albumin excretion was not improved, which may be explained by continued heme-laden red cell microparticle filtration (Camus SM, Blood 2015). Encouragingly, however, a trend towards decreased liver infarction in older mice was observed. The less-than-expected effect of Hp on mouse disease severity may also be explained by: 1) continuous physiologic Hp concentrations not being maintained with the dosing frequency while continued hemolysis releases Hb every minute of the day, and 2) CD163-mediated uptake in mice seems to only account for a part of the Hb clearance as opposed to in humans (Etzerodt A, Antioxid Redox Signal 2013). Despite the limitations of the SCD mouse model, the current study suggests haptogobin infusions could be beneficial in SCD patients.Acknowledgment: The authors are grateful to Sandra Suzuka for performing the HPLC.Table 1Table 1. Kidney iron deposition (scale 1-10)Treatment group6-month old SS4-month old SS400 mg/kg Hp4.0 ± 1.41 ± 1.1PBS9.3 ± 0.65 ± 2.9p-value0.0020.02Table 2Liver infarction (scale 1-10)Treatment group6-month old SS4-month old SS400 mg/kg Hp2.6 ± 2.03.7 ± 2.8PBS6.3 ± 2.43.8 ± 2.3p-value0.070.91 DisclosuresBelcher:Biogen Idec: Research Funding; Seattle Genetics: Research Funding; CSL Behring: Research Funding. Vercellotti:CSL Behring: Research Funding; Seattle Genetics: Research Funding; Cydan: Research Funding; Biogen Idec: Research Funding.

Abstract P113: Angiotensin Converting Enzyme 2 Modulates Bradykinin B1 Receptor Function During DOCA-Salt Hypertension

DOCA-salt hypertension is associated with reduced angiotensin converting enzyme 2 (ACE2) and increased bradykinin B1 receptor (B1R) expression in the brain. ACE2 hydrolyzes des-Arg(9)-bradykinin, the endogenous B1R agonist, into inactive metabolites. Therefore, we hypothesized that ACE2 overexpression or deletion modulates B1R function in the brain during neurogenic hypertension. To test this hypothesis, we used mice overexpressing ACE2 in neurons (SA), ACE2 knockout and B1R knockout mice. Blood pressure (BP) was monitored using telemetry probes in conscious animals. While baseline BP was not different between strains, DOCA-salt treatment (1 mg/g body weight DOCA, 1% NaCl for 3 weeks) resulted in a significantly lower BP in B1R knockout mice (121 ±2 mmHg, n=5) compared to wildtype (WT) mice (138 ±3 mmHg, n=8). DOCA-salt hypertension resulted in 27% decrease (74 ±6 vs. 54 ±2 Fluorescence Units (FU)/min/μg of protein, p&lt;0.05) in ACE2 activity in the hypothalamus, but not in B1R knockout mice with DOCA (69 ±6 vs. 65 ±3 FU/min/μg of protein). In DOCA-treated WT mice, B1R mRNA (Real time PCR) and protein expression (Western blot) in the hypothalamus were increased by 3 and 2 fold (n=6, p&lt;0.01), respectively. This increased B1R expression was blunted in SA mice with DOCA (3.2 ±0.4 vs. 0.9 ±0.1 fold, p&lt;0.001) but not in ACE2 knockout mice with DOCA (3.2 ±0.4 vs. 3.1 ±0.9 fold). Moreover, DOCA-salt treatment resulted in an increased expression of pro-inflammatory ADAM17 in the brain by 2.2 fold (n=6, p&lt;0.01 vs. WT). ACE2 overexpression or B1R knockdown blunted this ADAM17 expression (1.4 ±0.1 and 1.1 ±0.6 fold, respectively, n=3, p&lt;0.01 vs. WT+DOCA), while it was remain increased in ACE2 knockout mice (2.6 ±0.7 fold, n=3, p&lt;0.01) compared to control mice. Together, our data provide novel evidence to support a role for ACE2 in the modulation of central B1R function in the development of DOCA-salt hypertension.

Apelin Icv Infusion Blunts Brain ACE2 Overexpression‐mediated Metabolic Abnormalities in Mice

We previously reported that transgenic mice overexpressing ACE2 in the brain (SA) exhibit age‐dependent impaired metabolic function. Apelin is involved in the central regulation of energy and glucose homeostasis. ACE2 cleaves Apelin‐13 and 36 in vitro, however, the effect in vivo is unknown. We hypothesized that ACE2 overexpression alters apelin signaling in the brain, leading to metabolic dysfunction. To test this, hypothalamus Apelin levels, daily food intake, body weight, fasting blood glucose (16 h fasting) and glucose tolerance (measure blood glucose at 0, 15, 30, 60, 120 min after IP injection of glucose 2 g/kg) were determined in SA and wildtype mice (24wks old, n=5). In another set of experiments, SA mice were infused with Apelin‐13 (icv, 40 ng/d, osmotic pump) for 4 wks, metabolic parameters were measured before and after infusion. Interestingly, a significant reduction in hypothalamus Apelin level was observed in SA mice compared to wildtype (1.2±0.16 vs. 2.0±0.14 fmol/mg). This was concomitant with increased food intake (0.12±0.01 vs.0.09±0.01 g/g), higher body weight (39±1.5 vs. 31±0.8 g), elevated fasting blood glucose (147.8±13 vs.106.5±7 mg/dl) and impaired glucose tolerance (AUC: 51.6±2 vs. 41.5±1.7) in these animals (P&lt;0.05). Following Apelin‐13 infusion, although food intake in SA mice was not affected (P&gt;0.05), body weight was significantly reduced compared to pre‐infusion (38±1.1 vs. 44±1.1 g, P&lt;0.05). In addition, Apelin‐13 infusion significantly decreased fasting blood glucose level (113±7 vs. 168±22 mg/dl) and improved glucose tolerance (AUC: 43±8 vs. 72±4) in SA mice (P&lt;0.05 vs. Pre‐infusion). These data suggest that central ACE2‐overexpression decreases Apelin levels in the brain, thus contributing to metabolic abnormalities. (NIH P20 GM103514)

Entrapment into nanoemulsions potentiates the anticancer activity of tocotrienols against the highly malignant (+SA) mouse mammary epithelial cells.

The highly malignant +SA mouse mammary epithelial cells were used as the model cell line over the years to establish the anticancer activity of tocotrienols. Tocotrienols, however, have poor oral bioavailability and were therefore entrapped into parenteral nanoemulsions for parenteral administration. The objective of this work was to test whether the activity of tocotrienols in lipid nanoemulsions against the +SA cells was retained. A secondary objective was to test whether stabilizing the nanoemulsions with poloxamer or sodium oleate would affect their activity. Nanoemulsions were found to be significantly more potent than tocotrienol/albumin conjugate. The IC50 values of the poloxamer and sodium oleate nanoemulsions were 3 and 6 microM, respectively, whereas the IC50 value of the conjugate was 10 microM. The antiproliferative activity of the nanoemulsions was also found to inversely correlate with particle size. No activity was observed with nanoemulsions loaded with alpha-tocopherol or vehicle, which confirmed the cytotoxic activity of tocotrienols and the potential use of nanoemulsions in cancer therapy.

Brain ACE2 overexpression reduces DOCA‐salt hypertension independently of endoplasmic reticulum stress (875.3)

Endoplasmic reticulum (ER) stress was recently reported to contribute to hypertension. We previously observed that ACE2 in the paraventricular nucleus (PVN) is pivotal for blunting DOCA‐salt hypertension. To assess the contribution of ER stress, the PVN was collected from non‐transgenic (NT, n=4) and transgenic mice overexpressing brain ACE2 (SA, n=4), submitted to sham or 3 wk DOCA‐salt. ER stress (Bip, ATF4) and autophagy (LC3, LAMP2) markers were measured by Western blot. Baseline expression of Bip (1±0.07 vs. 0.96±0.11), ATF4 (1±0.06 vs. 0.99±0.02), LC3 II/I (1±0.02 vs. 1.16±0.11) and LAMP2 (1±0.08 vs. 0.97±0.05) were similar in NT and SA mice. Surprisingly, DOCA‐salt treatment did not alter these markers in NT (1.13±0.13; 1.14±0.05; 1.14±0.09; 1.13±0.09, respectively) or SA (1.04±0.04; 1.19±0.09; 1.1±0.07; 1.18±0.06, respectively) mice, despite a significant increase in blood pressure (BP, telemetry) in NT and a smaller rise in SA mice. To further assess the role of ER stress in DOCA‐salt hypertension, NT mice (n=8) were infused icv with TUDCA (5 µg/day, 21 days), an ER stress inhibitor, during DOCA‐salt treatment. DOCA‐salt alone significantly increased BP (127±5 vs. 101±2 mmHg, P&lt;0.05), however, this increase was not affected by TUDCA (123±4 mmHg). Our data suggest that ER stress does not contribute to DOCA‐salt hypertension and that ACE2 blunts this neurogenic hypertension independently of ER stress.Grant Funding Source: Supported by AHA (12EIA830004)

Brain ACE2 overexpression stimulates food intake and promotes glucose intolerance in mice (1126.15)

The brain Renin‐Angiotensin System (RAS) plays an important role in the regulation of metabolic function. While ACE2 has been reported to modulate brain RAS activity, its role in the central regulation of metabolism is unknown. To determine the role of central ACE2 in metabolism, Body weight and intake behaviors, assessed using metabolic cages, were measured in 24 wk old non‐transgenic (NT, n=8) and transgenic mice overexpressing ACE2 in the brain (SA, n=8). Fasting blood glucose was measured after a 14 h overnight fasting. Glucose tolerance test was performed by measuring blood glucose at 15, 30, 60 and 120 min after IP injection of glucose (2 g/kg). Plasma insulin level was measured by ELISA. Interestingly, body weight was significantly higher in SA than NT mice (39±1.5 vs. 31±0.8 g, P&lt;0.05). In addition, SA mice exhibited increased daily food intake (0.12±0.01 vs. 0.09±0.01 g/g body weight), water intake (0.14±0.01 vs. 0.08±0.01 ml/g body weight), fasting blood glucose (147.8±13 vs.106.5±7 mg/dl), impaired glucose tolerance (AUC: 51.6±2 vs.41.5±1.7) and increased plasma insulin level (5.5±1.2 vs.1.7±0.3 ng/ml), compared to NT mice (P&lt;0.05). These data suggest that ACE2 is involved in the central regulation of energy and glucose homeostasis. Brain ACE2 over‐expression leads to increased body weight, fasting glycemia and glucose intolerance, possibly through stimulating energy intake and insulin resistance.Grant Funding Source: Supported by NIH (NIGMS)

Angiotensin Converting Enzyme 2/Ang‐(1–7)/Mas Axis Protects Brain from Ischemic Injury with a Tendency of Age‐dependence

The angiotensin (Ang) converting enzyme 2 (ACE2)/Ang-(1-7)/Mas receptor pathway is an important component of the renin-angiotensin system and has been suggested to exert beneficial effects in ischemic stroke. This study explored whether the ACE2/Ang-(1-7)/Mas pathway has a protective effect on cerebral ischemic injury and whether this effect is affected by age. We used three-month and eight-month transgenic mice with neural over-expression of ACE2 (SA) and their age-matched nontransgenic (NT) controls. Neurological deficits and ischemic stroke volume were determined following middle cerebral artery occlusion (MCAO). In oxygen and glucose deprivation (OGD) experiments on brain slices, the effects of the Mas receptor agonist (Ang1-7) or antagonist (A779) on tissue swelling, Nox2/Nox4 expression reactive oxygen species (ROS) production and cell death were measured. (1) Middle cerebral artery occlusion -induced ischemic injury and neurological deficit were reduced in SA mice, especially in eight-month animals; (2) OGD-induced tissue swelling and cell death were decreased in SA mice with a greater reduction seen in eight-month mice; (3) Ang-(1-7) and A779 had opposite effects on OGD-induced responses, which correlated with changes in Nox2/Nox4 expression and ROS production. Angiotensin converting enzyme 2/Ang-(1-7)/Mas axis protects brain from ischemic injury via the Nox/ROS signaling pathway, with a greater effect in older animals.

Abstract 179: ACE2 Overexpression Prevents DOCA-Salt Hypertension by Modulating Cyclooxygenase Mediated Neuro-inflammation in the Central Nervous System

DOCA-salt hypertension, a low-renin hypertension model, is mediated through activation of the renin angiotensin system within the brain and is mediated by inflammation. We previously reported that central Angiotensin Converting Enzyme 2 (ACE2) overexpression prevents the development of hypertension induced by DOCA-salt in a transgenic mouse model (syn-hACE2; SA) with human ACE2 targeted selectively to neurons. The present study examined whether ACE2 modulates cyclooxygenase (COX)-mediated neuro-inflammation in DOCA-salt-mediated hypertension. DOCA-salt treatment (1 mg/g body weight DOCA, 1% saline in drinking water, 3 weeks) of non-transgenic (NT) and SA mice produced a significant hypertension (MAP; telemetry) in NT (138 ±3 mmHg, n=8, p&lt;0.05) that was blunted in SA mice (122 ±3 mmHg, n=12, p&lt;0.05). NT mice treated with DOCA-salt showed increased inflammation as indicated by increased gene expression of TNF (3 fold, n=9, p&lt;0.001 vs. NT), IL-1beta (8 fold, n=9, p&lt;0.001 vs. NT) and IL-6 (5 fold, n=9, p&lt;0.01 vs. NT) in the hypothalamic paraventricular nucleus (PVN) which was attenuated in SA mice. Furthermore, DOCA-salt hypertension resulted in increased expression of COX-1 (2.5 fold, n=9, p&lt;0.001 vs. NT) and COX-2 in the PVN (5 fold, n=9, p&lt;0.05 vs. NT) and this increase was prevented by ACE2 overexpression. ACE2 overexpression also prevented DOCA-salt induced increases in EP1 (1.5 fold, n=9, p&lt;0.01 vs. NT) and EP3 (1.5 fold, n=9, p&lt;0.01 vs. NT) receptors expression in the PVN. Taken together, these data provide evidence that brain ACE2 prevents COX mediated neuro-inflammation, thereby preventing the development of DOCA-salt hypertension.

In Utero Exposure to Second-Hand Smoke Aggravates Adult Responses to Irritants

In utero exposure to second-hand smoke (SHS) is associated with exacerbated asthmatic responses in children. We tested the hypothesis that in utero SHS will aggravate the lung responses of young adult mice re-exposed to SHS. We exposed Balb/c mice in utero to SHS (S) or filtered air (AIR; A), and re-exposed the male offspring daily from 11-15 weeks of age to either SHS (AS and SS) or AIR (AA and SA). After the adult exposures, we analyzed samples of bronchoalveolar lavage fluid (BALF), examined the results of histopathology, and assessed pulmonary function and gene expression changes in lung samples. In SS mice, compared with the other three groups (AA, AS, and SA), we found decreases in breathing frequency and increases in airway hyperresponsiveness (AHR), as well as low but significantly elevated concentrations of BALF proinflammatory cytokines (IL-1b, IL-6, and keratinocyte-derived chemokine). Lung morphometric analyses revealed enlarged airspaces and arteries in SA and SS mice compared with their in utero AIR counterparts, as well as increased collagen deposition in AS and SS mice. Unique gene expression profiles were found for in utero, adult, and combined exposures, as well as for mice with elevated AHR responses. The profibrotic metalloprotease genes, Adamts9 and Mmp3, were up-regulated in the SS and AHR groups, suggesting a role for in utero SHS exposure on the adult development of chronic obstructive pulmonary disease. Our results indicate that in utero exposures to environmentally relevant concentrations of SHS alter lung structure more severely than do adult SHS exposures of longer duration. These in utero exposures also aggravate AHR and promote a profibrotic milieu in adult lungs.

Abstract 71: STAT3 Affects Myofibrillar Structure and Its Loss May Contribute to Heart Failure in Hypertension

How hypertension causes heart failure is not known. Since patients with heart failure have reduced cardiac STAT3 and STAT3 KO mice develop heart failure with age, we tested the hypothesis that reduced STAT3 transcriptional activity contributes at an early stage to remodeling that precedes heart failure in hypertension using SA mice with a STAT3 S727A mutation. SA and wild type (WT) mice received angiotensin (A) II (1000 ng/kg/min) or saline (S) for 17 days. Hearts of WT and SA mice had similar levels of STAT3-induced protective proteins Bcl-xL and SOD2, and unlike STAT3 KO mice, cardiac miR-199a levels were not increased in SA mice. AII increased systolic blood pressure measured by telemetry in SA (124 ± 1 to 167 ± 3) and WT (122 ± 3 to 162 ± 3) mice to the same extent. AII increased cardiac levels of cytokines (pg/μg protein) associated with heart failure in both WT and SA mice, but significantly less so (P&lt;0.05) in SA mice; IL-6, 13.6 ± 1.4 vs. 9.1 ± 0.6; TGFβ, 56 ± 4 vs. 38 ± 3 and MCP1 35 ± 2 vs. 22 ± 2. Compared to WT mice, hearts of SA mice showed signs of developing systolic dysfunction with AII as seen by a significant (P&lt;0.05) reduction in ejection fraction (63.7 ± 7.1 to 51.7 ± 6.9) and fractional shortening (34.3 ± 4.9 to 26.4 ± 4.3). AII caused fibrosis in the left ventricle of both WT and SA mice characterized by cardiac myocyte loss and increased % collagen: WT+S, 5.59 ± 0.34; WT+AII, 15.70 ± 1.87; SA+S, 6.70 ± 0.40; SA+AII, 16.50 ± 1.91. In WT+AII mice there was a nonsignificant trend towards a loss of myofibrillar content of cardiac myocytes, but an increase in the mass of the myofibrils (IOD/myofibrillar area). In contrast, cardiac myocytes of SA+AII mice had a significant (P&lt;0.001) % loss in myofibrils (5.71 ± 0.28) compared to SA+S (0.75 ± 0.07), WT+S (0.80 ± 0.06) and WT+AII (1.54 ± 0.10) mice. In addition, the mass of the myofibrils in SA+AII mice (6.01 ± 0.07) was significantly less (P&lt;0.001) than those of SA+S mice (6.46 ± 0.04), although greater than WT+S (4.85 ± 0.06) or WT+AII (5.27 ± 0.08) mice. Our findings reveal that STAT3 transcriptional activity is important for proper morphology of the myofibrils of cardiac myocytes. Loss of STAT3 activity may impair cardiac function in the hypertensive heart due to defective myofibrillar structure and remodeling that may lead to heart failure.

Abstract 103: ACE2 Overexpression Prevents DOCA-Salt Hypertension by Modulating Oxidative Stress and Nitric Oxide in the Central Nervous System

Elevated reactive oxygen species (ROS) in the central nervous system (CNS) through NADPH oxidase and diminished Nitric oxide (NO) levels are involved in the pathogenesis of hypertension. We previously reported that central Angiotensin Converting Enzyme 2 (ACE2) overexpression prevents the development of hypertension induced by DOCA-salt in a transgenic mouse model (syn-hACE2; SA) with human ACE2 targeted selectively to neurons in the CNS. While baseline blood pressure (BP; telemetry) was not different among genotypes, DOCA-salt treatment (1mg/g body wt DOCA, 1% saline in drinking water for 3 weeks) resulted in significantly lower BP level in SA mice (122 ±3 mmHg, n=12) compared to non-transgenic (NT) littermates (138 ±3 mmHg, n=8). To elucidate the mechanisms involved in this response, we investigated the paraventricular nucleus (PVN) expression of Nox-2 (catalytic subunit of NADPH oxidase), 3-nitrotyrosine, and endothelial nitric oxide synthase (eNOS) and anti-oxidant enzymes superoxide dismutase (SOD) and catalase in the hypothalamus. DOCA-salt treatment resulted in decreased catalase (95.2 ±5.6 vs. 113.8 ±17.6 mmol/min/ml, p&lt;0.05) and SOD (4.1 ±0.4 vs. 5.9 ±0.2 U/ml, p&lt;0.01) activities in hypothalamic homogenates of NT mice, which was prevented by ACE2 overexpression (141.8 ±9.9 vs. 142.1 ±9.2 mmol/min/ml and 5.9 ±0.3 vs. 7.9 ±0.2 U/ml, respectively). NT mice treated with DOCA-salt showed increased oxidative stress as indicated by increased expression of Nox-2 (61 ±5 % increase, n=9, p&lt;0.001 vs. NT) and 3-nitrotyrosine (89 ±32 % increase, n=9, p&lt;0.01 vs. NT) in the PVN which was attenuated in SA mice. Furthermore, DOCA-salt hypertension resulted in decreased phosphorylation of eNOS-ser1177 in the PVN (33 ±5 % decrease, n=9, p&lt;0.05 vs NT) and this decrease was prevented by ACE2 overexpression. Taken together, these data provide evidence that brain ACE2 regulates the balance between NO and ROS levels, thereby preventing the development of DOCA-salt hypertension.

Mas receptor in the RVLM mediates sympatho‐inhibitory effect in mice with ACE2 overexpression during heart failure

Elevated central Ang II signaling contributes to the sustained increase of sympathetic outflow during chronic heart failure (CHF). This is associated with downregulation of central angiotensin‐converting enzyme 2 (ACE2). We have previously shown that sympathetic outflow was attenuated in mice with neuron‐selective ACE2 overexpression (SYN‐hACE2, SA) during CHF. However, it is not clear whether this effect is due to Ang‐(1‐7) and mas receptor signaling in the pre‐sympathetic neurons in these mice. We hypothesized that knock‐down of the mas receptor in the RVLM increases sympathetic drive in SA mice during CHF. Five weeks after coronary artery ligation, mean arterial pressure (MAP) and heart rate (HR) were recorded with telemetry for 3 days. Lentivirus encoding MAS1 shRNA (mas KD) or scrambled shRNA (scrb) were microinjected into the RVLM bilaterally, and MAP and HR were recorded after 7 days of recovery. Although a decrease in MAP and increase in HR were observed as cardiac function deteriorated, mas KD had no effect in wild type mice. In SA mice, HR was maintained after control microinjection (590±4 bpm vs. 588±6 bpm), however mas receptor KD increased HR (595±1 bpm vs. 618±5 bpm, p&lt;0.05). MAP was decreased similarly in two groups. These data suggest that the cardiac sympatho‐inhibitory effect of central ACE2 overexpression is mediated through mas receptor in the RVLM.

Brain-Selective Overexpression of Angiotensin-Converting Enzyme 2 Attenuates Sympathetic Nerve Activity and Enhances Baroreflex Function in Chronic Heart Failure

Angiotensin-converting enzyme 2 (ACE2) has been suggested to be involved in the central regulation of autonomic function. During chronic heart failure (CHF), elevated central angiotensin II signaling contributes to the sustained increase of sympathetic outflow. This is accompanied by a downregulation of ACE2 in the brain. We hypothesized that central overexpression of ACE2 decreases sympathetic outflow and enhances baroreflex function in CHF. Transgenic mice overexpressing human ACE2 selectively in the brain (SYN-hACE2 [SA]) and wild-type littermates (WT) were used. CHF was induced by permanent coronary artery ligation. Four weeks after coronary artery ligation, both WT and SA mice exhibited a significant decrease in left ventricular ejection fraction (<40%). A slight decrease in mean arterial pressure was found only in SA mice. Compared with WT mice with CHF, brain-selective ACE2 overexpression attenuated left ventricular end-diastolic pressure; decreased urinary norepinephrine excretion; baseline renal sympathetic nerve activity (WT CHF: 71.6±7.6% max versus SA CHF: 49.3±6.1% max); and enhanced baroreflex sensitivity (maximum slope: WT sham: 1.61±0.16%/mm Hg versus SA CHF: 1.51±0.17%/mm Hg). Chronic subcutaneous blockade of mas receptor increased renal sympathetic nerve activity in SA mice with CHF (A779: 67.3±5.8% versus vehicle: 46.4±3.6% of max). An upregulation in angiotensin II type 1 receptor expression was detected in medullary nuclei in WT CHF mice, which was significantly attenuated in SA mice with CHF. These data suggest that central ACE2 overexpression exerts a potential protective effect in CHF through attenuating sympathetic outflow. The mechanism for this effect involves angiotensin (1-7) mas signaling, as well as a decrease in angiotensin II type 1 receptor signaling in the medulla.

Synergistic anticancer effects of combined γ-tocotrienol with statin or receptor tyrosine kinase inhibitor treatment

Systemic chemotherapy is the only current method of treatment that provides some chance for long-term survival in patients with advanced or metastatic cancer. γ-Tocotrienol is a natural form of vitamin E found in high concentrations in palm oil and displays potent anticancer effects, but limited absorption and transport of by the body has made it difficult to obtain and sustain therapeutic levels in the blood and target tissues. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMGCoA) reductase and are an example of a promising cancer chemotherapeutic agent whose clinical usefulness has been limited due to high-dose toxicity. Similarly, erlotinib and gefitinib are anticancer agents that inhibit the activation of individual HER/ErbB receptor subtypes, but have shown limited clinical success because of heterodimerization between different EGF receptor family members that can rescue cancer cells from agents directed against a single receptor subtype. Recent studies have investigated the anticancer effectiveness of low-dose treatment of various statins or EGF receptor inhibitors alone and in combination with γ-tocotrienol on highly malignant +SA mouse mammary epithelial cells in vitro. Combined treatment with subeffective doses of γ-tocotrienol with these other chemotherapeutic agents resulted in a synergistic inhibition of +SA cell growth and viability. These findings strongly suggest that combined treatment of γ-tocotrienol with other anticancer agents may not only provide an enhanced therapeutic response but also provide a means to avoid the toxicity, low bioavailability, or limited therapeutic action associated with high-dose monotherapy.