Therapeutic Assessment of Combination Therapy with a Neprilysin Inhibitor and Angiotensin Type 1 Receptor Antagonist on Angiotensin II-Induced Atherosclerosis, Abdominal Aortic Aneurysms, and Hypertension.

Combined neprilysin (NEP) inhibition (sacubitril) and angiotensin type 1 receptor (AT1R) antagonism (valsartan) is used in the treatment of congestive heart failure and is gaining interest for other angiotensin II (AngII)-related cardiovascular diseases. In addition to heart failure, AngII promotes hypertension, atherosclerosis, and abdominal aortic aneurysms (AAAs). Similarly, NEP substrates or products have broad effects on the cardiovascular system. In this study, we examined NEP inhibition (with sacubitril) and AT1R antagonism (with valsartan) alone or in combination on AngII-induced hypertension, atherosclerosis, or AAAs in male low-density lipoprotein receptor-deficient mice. Preliminary studies assessed drug delivery via osmotic minipumps for simultaneous release of sacubitril and/or valsartan with AngII over 28 days. Mice were infused with AngII (1000 ng/kg per minute) in the absence (vehicle) or presence of sacubitril (1, 6, or 9 mg/kg per day), valsartan (0.3, 0.5, 1, 6, or 20 mg/kg per day), or the combination thereof (1 and 0.3, or 9 or 0.5 mg/kg per day of sacubitril and valsartan, respectively). Plasma AngII and renin concentrations increased 4-fold at higher valsartan doses, indicative of removal of AngII negative feedback on renin. Sacubitril doubled plasma AngII concentrations at lower doses (1 mg/kg per day). Valsartan dose-dependently decreased systolic blood pressure, aortic atherosclerosis, and AAAs of AngII-infused mice, whereas sacubitril had no effect on atherosclerosis or AAAs but reduced blood pressure of AngII-infused mice. Combination therapy with sacubitril and valsartan did not provide additive benefits. These results suggest limited effects of combination therapy with NEP inhibition and AT1R antagonism against AngII-induced hypertension, atherosclerosis, or AAAs. SIGNIFICANCE STATEMENT: The combination of valsartan (angiotensin type 1 receptor antagonist) and sacubitril (neprilysin inhibitor) did not provide benefit above valsartan alone on AngII-induced hypertension, atherosclerosis, or abdominal aortic aneurysms in low-density lipoprotein receptor-deficient male mice. These results do not support this drug combination in therapy of these AngII-induced cardiovascular diseases.

Pseudomonas aeruginosa-derived pyocyanin reduces adipocyte differentiation, body weight, and fat mass as mechanisms contributing to septic cachexia.

Pseudomonas aeruginosa, a leading cause of sepsis, produces pyocyanin, a blue-pigmented virulence factor. Sepsis is associated with cachexia, but mechanisms are unknown and conventional nutrition approaches are not effective treatments. Pyocyanin has affinity for the aryl hydrocarbon receptor (AhR), which is expressed on adipocytes and regulates adipocyte differentiation. The purpose of this study was to define in vitro and in vivo effects of pyocyanin on adipocyte differentiation and body weight regulation as relates to septic cachexia. In 3T3-L1 preadipocytes, pyocyanin activated AhR and its downstream marker CYP1a1, and reduced differentiation. Administration of pyocyanin to male C57BL/6J mice acutely reduced body temperature with altered locomotion, but caused sustained weight loss. Chronic pyocyanin administration to male and female C57BL/6J mice resulted in sustained reductions in body weight and fat mass, with adipose-specific AhR activation. Pyocyanin-treated male mice had decreased energy expenditure and physical activity, and increased adipose explant lipolysis. In females, pyocyanin caused robust reductions in body weight, adipose-specific AhR activation, and increased expression of inflammatory cytokines in differentiated adipocytes. These results demonstrate that pyocyanin reduces adipocyte differentiation and decreases body weight and fat mass in male and female mice, suggesting that pyocyanin may play a role in septic cachexia.

Mas receptor deficiency augments angiotensin II-induced atherosclerosis and aortic aneurysm ruptures in hypercholesterolemic male mice.

OBJECTIVE: Previous studies demonstrated that deficiency of angiotensin-converting enzyme 2 (ACE2) augmented angiotensin II (AngII)-induced atherosclerosis and abdominal aortic aneurysm (AAA) formation in hypercholesterolemic mice. Effects of ACE2 deficiency could arise from increased concentrations of its substrate, AngII, or decreased concentrations of its product, angiotensin-(1-7) [Ang-(1-7)]. Infusion of Ang-(1-7), a Mas receptor (MasR) ligand, to hypercholesterolemic male mice reduced AngII-induced atherosclerosis, suggesting a protective role of the Ang-(1-7)/MasR axis. However, it is unclear whether endogenous Ang-(1-7) acts at MasR to influence AngII-induced vascular diseases. The purpose of this study was to define the role of MasR deficiency in AngII-induced atherosclerosis and AAA formation and severity in hypercholesterolemic male mice. METHODS: MasR+/+ and MasR-/- male mice on a low-density lipoprotein receptor-deficient (Ldlr-/-) or apolipoprotein E-deficient (Apoe-/-) background were infused with AngII at either 600 or 1000 ng/kg/min by osmotic minipump for 28 days. Atherosclerosis was quantified at study end point as percentage lesion surface area of the aortic arch in Ldlr-/- mice. Abdominal aortic internal diameters were quantified by ultrasound, and maximal external AAA diameters were quantified at study end point. Blood pressure was quantified by radiotelemetry and a tail cuff-based technique. Serum cholesterol concentrations and vascular tissue characterization were examined at study end point. RESULTS: MasR deficiency did not influence body weight, systolic blood pressure at baseline and during AngII infusion, or serum cholesterol concentrations in either Apoe-/- or Ldlr-/- mice. MasR deficiency increased AngII-induced atherosclerosis in aortic arches of Ldlr-/- mice (P < .05), associated with increased oxidative stress and apoptosis in aortic root sections (P < .05). MasR deficiency also augmented internal and external AAA diameters and increased aortic ruptures of both Ldlr-/- and Apoe-/- mice (P < .05). These effects were associated with increased elastin breaks and T-lymphocyte and macrophage accumulation into abdominal aortas of AngII-infused MasR-deficient mice (P < .05). CONCLUSIONS: These results demonstrate that MasR deficiency augmented AngII-induced atherosclerosis and AAA rupture through mechanisms involving increased oxidative stress, inflammation, and apoptosis, suggesting that MasR activation may provide therapeutic efficacy against vascular diseases.

IKKß is a ß-catenin kinase that regulates mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs) can give rise to both adipocytes and osteoblasts, but the molecular mechanisms underlying MSC fate determination remain poorly understood. IκB kinase ß (IKKß), a central coordinator of inflammation and immune responses through activation of NF-κB, has been implicated as a critical molecular link between obesity and metabolic disorders. Here, we show that IKKß can reciprocally regulate adipocyte and osteoblast differentiation of murine and human MSCs through an NF-κB-independent mechanism. IKKß is a ß-catenin kinase that phosphorylates the conserved degron motif of ß-catenin to prime it for ß-TrCP-mediated ubiquitination and degradation, thereby increasing adipogenesis and inhibiting osteogenesis in MSCs. Animal studies demonstrated that deficiency of IKKß in BM mesenchymal stromal cells increased bone mass and decreased BM adipocyte formation in adult mice. In humans, IKKß expression in adipose tissue was also positively associated with increased adiposity and elevated ß-catenin phosphorylation. These findings suggest IKKß as a key molecular switch that regulates MSC fate, and they provide potentially novel mechanistic insights into the understanding of the cross-regulation between the evolutionarily conserved IKKß and Wnt/ß-catenin signaling pathways. The IKKß-Wnt axis we uncovered may also have important implications for development, homeostasis, and disease pathogenesis.

Blood Pressure Monitoring Using Radio Telemetry Method in Mice.

The TA11PA-C10 implantable transmitter (Data Sciences International, DSI) is designed to measure blood pressure (BP) and activity in freely moving laboratory mice. The fluid filled catheter is placed in the free flowing blood of the systemic artery (inserted into the left carotid artery and extended into the aorta), and the transmitter body is placed in a benign location for long-term biocompatibility. The transmitter can be used to monitor BP in mice (as small as 17 g) under normal physiological and unrestricted conditions 24 h a day while remaining free from stress associated with human interaction. Thus, telemetry is considered the gold standard for BP monitoring in small animals such as mice. However, this methodology does require a good understanding of the system as well as appropriate training to perform the delicate transmitter implantation surgery.

Reversal of Bone Marrow Mobilopathy and Enhanced Vascular Repair by Angiotensin-(1-7) in Diabetes.

The angiotensin (ANG)-(1-7)/Mas receptor (MasR) pathway activates vascular repair-relevant functions of bone marrow progenitor cells. We tested the effects of ANG-(1-7) on mobilization and vasoreparative functions of progenitor cells that are impaired in diabetes. The study was performed in streptozotocin-induced diabetic (db/db) mice. Diabetes resulted in a decreased number of Lineage-Sca-1+c-Kit+ (LSK) cells in the circulation, which was normalized by ANG-(1-7). Diabetes-induced depletion of LSK cells in the bone marrow was reversed by ANG-(1-7). ρ-Kinase (ROCK) activity was increased specifically in bone marrow LSK cells by ANG-(1-7) in diabetes, and the beneficial effects of ANG-(1-7) were prevented by fasudil. ANG-(1-7) increased Slit3 levels in the bone marrow supernatants, which activated ROCK in LSK cells and sensitized them for stromal-derived factor-1α (SDF)-induced migration. Diabetes prevented the mobilization of LSK cells in response to ischemia and impaired the recovery of blood flow, both of which were reversed by ANG-(1-7) in both models of diabetes. Genetic ablation of MasR prevented ischemia-induced mobilization of LSK cells and impaired blood flow recovery, which was associated with decreased proliferation and migration of LSK cells in response to SDF or vascular endothelial growth factor. These results suggest that MasR is a promising target for the treatment of diabetic bone marrow mobilopathy and vascular disease.

Administration of 17ß-estradiol to ovariectomized obese female mice reverses obesity-hypertension through an ACE2-dependent mechanism.

We recently demonstrated that female mice are resistant to the development of obesity-induced hypertension through a sex hormone-dependent mechanism that involved adipose angiotensin-converting enzyme 2 (ACE2). In this study, we hypothesized that provision of 17ß-estradiol (E2) to ovariectomized (OVX) high-fat (HF)-fed female hypertensive mice would reverse obesity-hypertension through an ACE2-dependent mechanism. Pilot studies defined dose-dependent effects of E2 in OVX female mice on serum E2 concentrations and uterine weights. An E2 dose of 36 µg/ml restored normal serum E2 concentrations and uterine weights. Therefore, HF-fed OVX female Ace2(+/+) and Ace2(-/-) mice were administered vehicle or E2 (36 µg/ml) for 16 wk. E2 administration significantly decreased body weights of HF-fed OVX female Ace2(+/+) and Ace2(-/-) mice of either genotype. At 15 wk, E2 administration decreased systolic blood pressure (SBP) of OVX HF-fed Ace2(+/+) but not Ace2(-/-) females during the light but not the dark cycle. E2-mediated reductions in SBP in Ace2(+/+) females were associated with significant elevations in adipose ACE2 mRNA abundance and activity and reduced plasma ANG II concentrations. In contrast to females, E2 administration had no effect on any parameter quantified in HF-fed male hypertensive mice. In 3T3-L1 adipocytes, E2 promoted ACE2 mRNA abundance through effects at estrogen receptor-α (ERα) and resulted in ERα-mediated binding at the ACE2 promoter. These results demonstrate that E2 administration to OVX females reduces obesity-induced elevations in SBP (light cycle) through an ACE2-dependent mechanism. Beneficial effects of E2 to decrease blood pressure in OVX obese females may result from stimulation of adipose ACE2.

Deficiency of ACE2 in Bone-Marrow-Derived Cells Increases Expression of TNF-α in Adipose Stromal Cells and Augments Glucose Intolerance in Obese C57BL/6 Mice.

Deficiency of ACE2 in macrophages has been suggested to promote the development of an inflammatory M1 macrophage phenotype. We evaluated effects of ACE2 deficiency in bone-marrow-derived stem cells on adipose inflammation and glucose tolerance in C57BL/6 mice fed a high fat (HF) diet. ACE2 activity was increased in the stromal vascular fraction (SVF) isolated from visceral, but not subcutaneous adipose tissue of HF-fed mice. Deficiency of ACE2 in bone marrow cells significantly increased mRNA abundance of F4/80 and TNF-α in the SVF isolated from visceral adipose tissue of HF-fed chimeric mice, supporting increased presence of inflammatory macrophages in adipose tissue. Moreover, deficiency of ACE2 in bone marrow cells modestly augmented glucose intolerance in HF-fed chimeric mice and increased blood levels of glycosylated hemoglobin. In summary, ACE2 deficiency in bone marrow cells promotes inflammation in adipose tissue and augments obesity-induced glucose intolerance.

Angiotensin converting enzyme 2 contributes to sex differences in the development of obesity hypertension in C57BL/6 mice.

OBJECTIVE: Obesity promotes hypertension, but it is unclear if sex differences exist in obesity-related hypertension. Angiotensin converting enzyme 2 (ACE2) converts angiotensin II (AngII) to angiotensin-(1-7) (Ang-[1-7]), controlling peptide balance. We hypothesized that tissue-specific regulation of ACE2 by high-fat (HF) feeding and sex hormones contributes to sex differences in obesity-hypertension. METHODS AND RESULTS: HF-fed females gained more body weight and fat mass than males. HF-fed males exhibiting reduced kidney ACE2 activity had increased plasma angiotensin II levels and decreased plasma Ang-(1-7) levels. In contrast, HF-fed females exhibiting elevated adipose ACE2 activity had increased plasma Ang-(1-7) levels. HF-fed males had elevated systolic and diastolic blood pressure that were abolished by losartan. In contrast, HF-fed females did not exhibit increased systolic blood pressure until females were administered the Ang-(1-7) receptor antagonist, D-Ala-Ang-(1-7). Deficiency of ACE2 increased systolic blood pressure in HF-fed males and females, which was abolished by losartan. Ovariectomy of HF-fed female mice reduced adipose ACE2 activity and plasma Ang-(1-7) levels, and promoted obesity-hypertension. Finally, estrogen, but not other sex hormones, increased adipocyte ACE2 mRNA abundance. CONCLUSIONS: These results demonstrate that tissue-specific regulation of ACE2 by diet and sex hormones contributes to sex differences in obesity-hypertension.

Myosin light chain kinase/actin interaction in phorbol dibutyrate-stimulated smooth muscle cells.

Previous work has suggested that in addition to its kinase activity, myosin light chain kinase (MLCK) exhibits non-kinase properties within its N-terminus that could influence cytoskeletal organization of smooth muscle cells (A. Nakamura et al. Biochem Biophys Res Commun. 2008;369:135-143). Myosin ATPase activity measurements indicate that the 26-41 peptide of MLCK significantly decreases ATPase activity as the concentration of this peptide increases. Sliding velocity of actin-filaments on myosin and stress responses in skinned smooth muscle tissue are also inhibited. Peptide-mediated uptake and the microinjection technique in cells indicate that the peptide was necessary for actin-filament stabilization. Fluorescence resonance energy transfer analysis indicated that in the presence of MLCK, α-actin but not ß-actin remodeled during phorbol 12,13-dibutyrate (PDBu)-induced contractions. PDBu also induced podosomes in the cell. When MLCK expression was down-regulated by introduction of RNAi for MLCK by lentivirus vector into the cells, we failed to observe the podosome induction upon PDBu stimulation. Rescue experiments indicate that the non-kinase activity of MLCK plays an important role in maintaining actin stress fibers and in the PDBu-induced reorganization of actin-filaments in smooth muscle cells.

Angiotensin-converting enzyme 2 deficiency in whole body or bone marrow-derived cells increases atherosclerosis in low-density lipoprotein receptor-/- mice.

OBJECTIVE: The renin-angiotensin system contributes to atherosclerotic lesion formation. Angiotensin-converting enzyme 2 (ACE2) catabolizes angiotensin II (Ang II) to angiotensin 1-7 (Ang-(1-7)) to limit effects of the renin-angiotensin system. The purpose of this study was to define the role of ACE2 in atherosclerosis. METHODS AND RESULTS: Male Ace2(-/y) mice in an low-density lipoprotein receptor-deficient background were fed a high-fat diet for 3 months. ACE2 deficiency increased atherosclerotic area (Ace2(+/y), 17 ± 1; Ace2(-/y), 23 ± 2 mm(2), P < 0.002). This increase was blunted by losartan. To determine whether leukocytic ACE2 influenced atherosclerosis, irradiated low-density lipoprotein receptor-deficient male mice were repopulated with bone marrow-derived cells from Ace2(+/y) or Ace2(-/y) mice and fed a high-fat diet for 3 months. ACE2 deficiency in bone marrow-derived cells increased atherosclerotic area (Ace2(+/y), 1.6 ± 0.3; Ace2(-/y), 2.8 ± 0.3 mm(2); P < 0.05). Macrophages from Ace2(-/y) mice exhibited increased Ang II secretion and elevated expression of inflammatory cytokines. Conditioned media from mouse peritoneal macrophages of Ace2(-/y) mice increased monocyte adhesion to human umbilical vein endothelial cells. Incubation of human umbilical vein endothelial cells with Ang II promoted monocyte adhesion, which was blocked by Ang-(1-7). Coinfusion of Ang-(1-7) with Ang II reduced atherosclerosis. CONCLUSIONS: These results demonstrate that ACE2 deficiency in bone marrow-derived cells promotes atherosclerosis through regulation of Ang II/Ang-(1-7) peptides.

Obesity promotes inflammation in periaortic adipose tissue and angiotensin II-induced abdominal aortic aneurysm formation.

OBJECTIVE: Obesity promotes macrophage infiltration into adipose tissue and is associated with increases in several cardiovascular diseases. Infusion of angiotensin II (AngII) to mice induces formation of abdominal aortic aneurysms (AAAs) with profound medial and adventitial macrophage infiltration. We sought to determine whether obesity promotes macrophage infiltration and proinflammatory cytokines in periaortic adipose tissue surrounding abdominal aortas and increases AngII-induced AAAs. METHODS AND RESULTS: Hypertrophied white adipocytes surrounded abdominal aortas, whereas brown adipocytes surrounded thoracic aortas of obese mice. mRNA abundance of macrophage proinflammatory chemokines and their receptors were elevated with obesity to a greater extent in abdominal compared to thoracic periaortic adipose tissue. Periaortic adipose tissue explants surrounding abdominal aortas of obese mice released greater concentrations of MCP-1 and promoted more macrophage migration than explants from thoracic aortas. Male C57BL/6 mice were fed a high-fat (HF) diet for 1, 2, or 4 months and then infused with AngII (1000 ng/kg/min) for 28 days. AAA incidence increased progressively with the duration of HF feeding (18%, 36%,and 60%, respectively). Similarly, AngII-infused ob/ob mice exhibited increased AAAs compared to lean controls (76% compared to 32%, respectively, P<0.05). Infusion of AngII to obese mice promoted further macrophage infiltration into periaortic and visceral adipose tissue, and obese mice exhibiting AAAs had greater macrophage content in visceral adipose tissue than mice not developing AAAs. CONCLUSIONS: Increased macrophage accumulation in periaortic adipose tissue surrounding abdominal aortas of AngII-infused obese mice is associated with enhanced AAA formation.

Blebbistatin inhibits the chemotaxis of vascular smooth muscle cells by disrupting the myosin II-actin interaction.

Blebbistatin is a myosin II-specific inhibitor. However, the mechanism and tissue specificity of the drug are not well understood. Blebbistatin blocked the chemotaxis of vascular smooth muscle cells (VSMCs) toward sphingosylphosphorylcholine (IC(50) = 26.1 +/- 0.2 and 27.5 +/- 0.5 microM for GbaSM-4 and A7r5 cells, respectively) and platelet-derived growth factor BB (IC(50) = 32.3 +/- 0.9 and 31.6 +/- 1.3 muM for GbaSM-4 and A7r5 cells, respectively) at similar concentrations. Immunofluorescence and fluorescent resonance energy transfer analysis indicated a blebbistatin-induced disruption of the actin-myosin interaction in VSMCs. Subsequent experiments indicated that blebbistatin inhibited the Mg(2+)-ATPase activity of the unphosphorylated (IC(50) = 12.6 +/- 1.6 and 4.3 +/- 0.5 microM for gizzard and bovine stomach, respectively) and phosphorylated (IC(50) = 15.0 +/- 0.6 microM for gizzard) forms of purified smooth muscle myosin II, suggesting a direct effect on myosin II motor activity. It was further observed that the Mg(2+)-ATPase activities of gizzard myosin II fragments, heavy meromyosin (IC(50) = 14.4 +/- 1.6 microM) and subfragment 1 (IC(50) = 5.5 +/- 0.4 microM), were also inhibited by blebbistatin. Assay by in vitro motility indicated that the inhibitory effect of blebbistatin was reversible. Electron-microscopic evaluation showed that blebbistatin induced a distinct conformational change (i.e., swelling) of the myosin II head. The results suggest that the site of blebbistatin action is within the S1 portion of smooth muscle myosin II.