Ang II-treated Wild-type Mice Research Articles

Abstract 117: Interleukin-18 Is A Critical Mediator Of Cardiac Remodelling During Angiotensin II-induced Hypertension

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.

Abstract 13: P2X7 Receptor Knockout Attenuates Angiotensin II-Induced Hypertension, Vascular Injury And CD8 + T Cell Infiltration

Background: The P2X7 receptor (P2RX7) recognizes damage associated molecule patterns such as adenosine triphosphate (ATP), and triggers the activation of immune cells. Elevated plasma ATP levels have been observed in hypertensive patients, providing a potential mechanism for P2RX7 activation. Additionally, a hypomorphic polymorphism for P2X7 is correlated with a decreased risk for essential hypertension in Chinese post-menopausal women. However, it is unknown whether P2RX7 activation contributes to angiotensin (Ang) II-induced blood pressure (BP) elevation and vascular damage. We hypothesized that P2rx7 knockout would blunt Ang II-induced BP elevation, vascular injury, and infiltration of activated immune T cells into perivascular adipose tissue (PVAT). Methods: Ten-to-12-week-old male C57BL/6J male wild-type (WT) and P2rx7 -/- mice were infused or not with Ang II (1000ng/kg/min) for 14 days. BP was determined by telemetry, mesenteric artery function and remodeling using pressurized myography, aortic stiffening by ultrasound and infiltration of activated immune T cells in aortic PVAT by flow cytometry. Results: Ang II-infused P2rx7 -/- mice display a reduced systolic BP (164±3 vs. 176±2 mm Hg, P <0.05) and pulse pressure (37±4 vs. 53±3 mm Hg, P <0.001) in comparison to WT mice. Aortic stiffening occurred in WT mice treated with Ang II, demonstrated by an increased pulse wave velocity (7.7±0.7 vs. 5.9±0.3 m/s, P <0.05), accompanied by a 3.8-fold increased infiltration of activated CD8 + T cells in aortic PVAT (60±16 vs 16±3 cells/aortic PVAT, P <0.001), which were both absent in P2rx7 -/- mice (6.4±1.4 vs 5.5±1.1 m/s and 27±7 vs 16±3 cells/aortic PVAT). In addition, the frequency of IFN-γ producing CD8 + T cells in the spleen of Ang II-treated WT mice increased (2.6±0.2% vs 1.2±0.2%), which did not occur in P2rx7 -/- mice (1.7±0.3% vs 1.7±0.2%). Ang II-infusion induced mesenteric artery endothelial dysfunction in WT mice (61±7 vs 83±4% relaxation response to acetylcholine, P <0.05), which was absent in P2rx7 -/- mice (89±3 vs 90±3%). Conclusion: P2rx7 knockout attenuates Ang II-induced hypertension, vascular injury, and infiltration of activated CD8 + T cells into aortic PVAT.

Endothelial Dysfunction Induced by Mitochondrial Uncoupling is prevented by Retinol Binding Protein 7, a PPARγ Target Gene

PPARγ is a nuclear receptor that prevents oxidative stress in the endothelium. Endothelium specific PPARγ target gene, retinol binding protein 7 (RBP7) is required for the protective effects of PPARγ. In response to a cardiovascular stressor (high fat diet or Ang II), transgenic mice carrying a dominant‐negative mutation in PPARγ in the endothelium (E‐V290M) and RBP7 knockout mice (RBP7KO) exhibited increased oxidative stress and endothelial dysfunction. We hypothesize that RBP7 has a protective role against endothelial dysfunction induced by the pro‐oxidant effects of mitochondrial uncoupling (MU). Acetylcholine (ACh) and sodium nitroprusside (SNP) induced vasodilation response were evaluated in carotid arteries from 4 and 8 month old RBP7KO mice, infused with either saline or a sub‐pressor dose of Ang II (120ng/kg/min) for 2 weeks. Ang II infusion did not alter systolic blood pressure either in wild type (WT) (4 months old: 116.2±4.2 vs 113.9±4.1 mmHg; 8 months old: 111.3±3.8 vs 109.5±2.9 mmHg) or RBP7KO (4 months old: 112.9±1.1 vs 111.8±5.2 mmHg; 8 months old: 112.2±4.2 vs 113.9±4.1 mmHg). Endothelium‐dependent relaxation to ACh was not affected by Ang II in either RBP7KO (4 month old: % relaxation ‐ saline, 89.7±1.3 vs Ang II, 93.8±2.0; 8 months old: % relaxation ‐ saline, 80.1±6.8 vs Ang II, 74.6±10.6) or WT littermates (4 months old: % relaxation ‐ saline, 98.1±0.3 vs Ang II, 94.5±1.7; 8 months old: % relaxation ‐ saline, 81.4±4.7 vs Ang II, 78.6±10.4). To test if RBP7 plays a role in MU, the vessels were pre‐incubated with the MU carbonyl cyanide m‐chlorophenyl hydrazone (CCCP; 1uM for 30 min). Pre‐incubation with CCCP did not alter the relaxation responses to ACh in 4 month old mice in all groups. However, in 8 month old mice, there is a trend towards impaired endothelial function in the carotid artery from Ang II‐infused RBP7KO compared with genotype‐matched saline‐treated mice (% relaxation: 51.9±3.8 vs 81.8±5.3, p=0.06) as well as Ang II‐treated WT mice (% relaxation: 51.9±3.8 vs 77.1±2.0 p=0.08). Giving that, RBP7 may play a role in MU, we analyzed the protein expression (western blot) of fusion (OPA1) and fission (DRP1) markers in RBP7 KO and WT mouse lung endothelial cells (MLEC) incubated with CCCP (10 uM) for 15, 30 and 60 minutes. At baseline, both, long (L) (3.62±0.19 vs 3.41±0.13 AU) and short (S) (1.01±0.07 vs 0.97±0.04) isoform of OPA1 were equally expressed in WT and RBP7 KO MLEC respectively. The expression of L OPA1 was almost inhibited in WT (0.12 ± 0.01 AU) and RBP7 KO (0.24 ± 0.01 AU) MLEC after 60 min, while there was no effect in S OPA1. We speculate that differences in L OPA 1 expression may be observed with a lower dose of CCCP or in a recovery experiment where the CCCP is replaced by new media for couple of hours. No differences were observed in neither, the phosphorylation or expression of DRP1.Although the role of RBP7 in fusion/fission process still unclear, we conclude that Ang II alone was not able to impair vascular function, however, in 8 month old mice, Ang II increased the susceptibility to CCCP‐dependent endothelial impairment, and RBP7 was required to mediate vascular protection against mitochondrial uncoupling.

Abstract P183: Retinol Binding Protein 7, a PPARγ Target Gene Protects Against Endothelial Dysfunction Induced by Mitochondrial Uncoupling

PPARγ is a nuclear receptor that prevents oxidative stress in the endothelium. Endothelium specific PPARγ target gene, retinol binding protein 7 (RBP7) is required for the protective effects of PPARγ. In response to a cardiovascular stressor (high fat diet or Ang II), transgenic mice carrying a dominant-negative mutation in PPARγ in the endothelium (E-V290M) and RBP7 knockout mice (RBP7KO) exhibited increased oxidative stress and endothelial dysfunction. We hypothesize that RBP7 has a protective role against endothelial dysfunction induced by the pro-oxidant effects of mitochondrial uncoupling (MU). Acetylcholine (ACh) and sodium nitroprusside (SNP) induced vasodilation response were evaluated in carotid arteries from 4 and 8 month old RBP7KO mice, infused with either saline or a sub-pressor dose of Ang II (120ng/kg/min) for 2 weeks. Ang II infusion did not alter systolic blood pressure either in wild type (WT) (4 months old: 116.2±4.2 vs 113.9±4.1 mmHg; 8 months old: 111.3±3.8 vs 109.5±2.9 mmHg) or RBP7KO (4 months old: 112.9±1.1 vs 111.8±5.2 mmHg; 8 months old: 112.2±4.2 vs 113.9±4.1 mmHg). Endothelium-dependent relaxation to ACh was not affected by Ang II in either RBP7KO (4 month old: % relaxation - saline, 89.7±1.3 vs Ang II, 93.8±2.0; 8 months old: % relaxation - saline, 80.1±6.8 vs Ang II, 74.6±10.6) or WT littermates (4 months old: % relaxation - saline, 98.1±0.3 vs Ang II, 94.5±1.7; 8 months old: % relaxation - saline, 81.4±4.7 vs Ang II, 78.6±10.4). To test if RBP7 plays a role in MU, the vessels were pre-incubated with the MU carbonyl cyanide m-chlorophenyl hydrazone (CCCP; 1uM for 30 min). Pre-incubation with CCCP did not alter the relaxation responses to ACh in 4 month old mice in all groups. However, in 8 month old mice, there is a trend towards impaired endothelial function in the carotid artery from Ang II-infused RBP7KO compared with genotype-matched saline-treated mice (% relaxation: 51.9±3.8 vs 81.8±5.3, p=0.06) as well as Ang II-treated WT mice (% relaxation: 51.9±3.8 vs 77.1±2.0 p=0.08). In conclusion, Ang II alone was not able to impair vascular function, however, in 8 month old mice, Ang II increased the susceptibility to CCCP-dependent endothelial impairment, and RBP7 was required to mediate vascular protection against mitochondrial uncoupling.

B-cell lymphoma/leukaemia 10 and angiotensin II-induced kidney injury.

B-cell lymphoma/leukaemia 10 (Bcl10) is a member of the CARMA-Bcl10-MALT1 signalosome, linking angiotensin (Ang) II, and antigen-dependent immune-cell activation to nuclear factor kappa-B signalling. We showed earlier that Bcl10 plays a role in Ang II-induced cardiac fibrosis and remodelling, independent of blood pressure. We now investigated the role of Bcl10 in Ang II-induced renal damage. Bcl10 knockout mice (Bcl10 KO) and wild-type (WT) controls were given 1% NaCl in the drinking water and Ang II (1.44 mg/kg/day) for 14 days. Additionally, Bcl10 KO or WT kidneys were transplanted onto WT mice that were challenged by the same protocol for 7 days. Kidneys of Ang II-treated Bcl10 KO mice developed less fibrosis and showed fewer infiltrating cells. Nevertheless, neutrophil gelatinase-associated lipocalin (Ngal) and kidney injury molecule (Kim)1 expression was higher in the kidneys of Ang II-treated Bcl10 KO mice, indicating exacerbated tubular damage. Furthermore, albuminuria was significantly higher in Ang II-treated Bcl10 KO mice accompanied by reduced glomerular nephrin expression and podocyte number. Ang II-treated WT mice transplanted with Bcl10 KO kidney showed more albuminuria and renal Ngal, compared to WT- > WT kidney-transplanted mice, as well as lower podocyte number but similar fibrosis and cell infiltration. Interestingly, mice lacking Bcl10 in the kidney exhibited less Ang II-induced cardiac hypertrophy than controls. Bcl10 has multi-faceted actions in Ang II-induced renal damage. On the one hand, global Bcl10 deficiency ameliorates renal fibrosis and cell infiltration; on the other hand, lack of renal Bcl10 aggravates albuminuria and podocyte damage. These data suggest that Bcl10 maintains podocyte integrity and renal function.

Abstract 038: The Dual Role of B-Cell Lymphoma/Leukemia 10 in Angiotensin II-Induced Renal Damage

Angiotensin (Ang) II activates NF-κB and thereby induces organ damage. B-cell Lymphoma/Leukemia 10 (Bcl10) is a member of the CARMA-Bcl10-MALT1 signalosome that links Ang II and antigen-dependent activation of immune cells to NF-κB signaling. We have shown earlier that Bcl10 plays a significant role in the generation of Ang II–induced cardiac fibrosis and electric remodeling in a blood pressure independent fashion. In our present study we investigated the role of Bcl10 in Ang II–induced renal damage. Bcl10 knockout mice (Bcl10 KO) and wild-type (WT) controls were given 1% NaCl in the drinking water and Ang II (1.44 mg/kg/d) for 14 days. Additionally, Bcl10 KO or WT kidneys were transplanted onto WT mice and were challenged by the same protocol for 7 days. Kidneys of Bcl10 KO mice developed less fibrosis and showed reduced number of infiltrating macrophages and T cells. Nevertheless, renal expression of neutrophil gelatinase-associated lipocalin (Ngal) and kidney injury molecule 1 expression was higher after Ang II treatment in Bcl10 KO mice compared to WT mice indicating exacerbated tubular damage (4.94±1.56 vs 1.44±0.44 and 1.14±0.22 vs 2.87±1.05 AU respectively). In addition, albuminuria was significantly higher in Ang II-treated Bcl10 KO mice than in controls (1194±238 vs. 377±112 μg/day) accompanied by reduced glomerular nephrin expression and reduced podocyte number (5.9±0.4 vs. 8.2±0.5 Wilms’ tumor-1 protein (WT1) positive cells/glomerulus). Ang II-treated WT mice transplanted with Bcl10 KO kidney showed significantly higher albuminuria (6003±1988 vs. 1779±831 μg/day) and renal Ngal expression (1.75±4.08 vs 0.71±0.57 AU) compared to WT->WT kidney transplanted mice, as well as lower podocyte number (5.4±0.3 vs. 4.9±0.2 WT1 positive cells/glomerulus) but similar extent of fibrosis and cell infiltration. Lacking Bcl10 in the kidney only was protective from Ang II-induced cardiac hypertrophy. Bcl10 has a multi-faceted action in hypertensive renal damage. While global Bcl10 deficiency ameliorates renal fibrosis and cell infiltration, the lack of renal Bcl10 aggravates albuminuria and podocyte damage. These data indicate that Bcl10 plays a crucial role in maintaining podocyte integrity and renal function.

Deficiency of the Angiotensinase Aminopeptidase A Increases Susceptibility to Glomerular Injury.

Aminopeptidase A (APA) is expressed in glomerular podocytes and tubular epithelia and metabolizes angiotensin II (AngII), a peptide known to promote glomerulosclerosis. In this study, we tested whether APA expression changes in response to progressive nephron loss or whether APA exerts a protective role against glomerular damage and during AngII-mediated hypertensive kidney injury. At advanced stages of FSGS, fawn-hooded hypertensive rat kidneys exhibited distinctly increased APA staining in areas of intact glomerular capillary loops. Moreover, BALB/c APA-knockout (KO) mice injected with a nephrotoxic serum showed persistent glomerular hyalinosis and albuminuria 96 hours after injection, whereas wild-type controls achieved virtually full recovery. We then tested the effect of 4-week infusion of AngII (400 ng/kg per minute) in APA-KO and wild-type mice. Although we observed no significant difference in achieved systolic BP, AngII-treated APA-KO mice developed a significant rise in albuminuria not observed in AngII-treated wild-type mice along with increased segmental and global sclerosis and/or collapse of juxtamedullary glomeruli, microcystic tubular dilation, and tubulointerstitial fibrosis. In parallel, AngII treatment significantly increased the kidney AngII content and attenuated the expression of podocyte nephrin in APA-KO mice but not in wild-type controls. These data show that deficiency of APA increases susceptibility to glomerular injury in BALB/c mice. The augmented AngII-mediated kidney injury observed in association with increased intrarenal AngII accumulation in the absence of APA suggests a protective metabolizing role of APA in AngII-mediated glomerular diseases.

Abstract 074: An Obligatory Role for B Cells in the Development of Angiotensin II-dependent Hypertension

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.

TRPV4 contributes to renal injury induced by angiotensin II in mice

To determine whether deletion of TRPV4 attenuates angiotensin II (Ang II)‐induced renal injury, Ang II was infused systemically into wild type (WT) and TRPV4‐null mutant (TRPV4‐/‐) mice for 4 weeks. Systolic blood pressure increased in Ang II‐treated WT or TRPV4‐/‐mice with the similar degree (P>0.05). Ang II treatment increased urinary excretion of albumin and 8‐isoprostane, and decreased creatinine clearance in both WT and TRPV4‐/‐mice with a greater magnitude in the former strain (P<0.05). Periodic acid‐Schiff and Masson's trichrome staining showed that kidneys of Ang II‐treated WT mice exhibited more severe glomerulosclerosis and tubulointerstitial injury compared with Ang II‐treated TRPV4‐/‐mice (P<0.05). Hydroxyproline assay and F4/80‐staining showed that renal collagen levels and monocyte/macrophage infiltration were greater in Ang II‐treated WT mice compared with TRPV4‐/‐mice (P<0.05). Thus, our data show that deletion of TRPV4 alleviates renal injury in the absence of alteration in blood pressure in Ang II‐hypertensive mice, indicating that TRPV4 could serve as a novel target for treating renal injury associated with hypertension. [This work was supported by grants from the National Natural Science Foundation of China (No.81170243) and Henan Provincial Science and Technology Innovative Program for Outstanding Scholarship (No. 124200510007)].

Induction of Atrial Fibrillation by Neutrophils Critically Depends on CD11b/CD18 Integrins

BackgroundRecent observational clinical and ex-vivo studies suggest that inflammation and in particular leukocyte activation predisposes to atrial fibrillation (AF). However, whether local binding and extravasation of leukocytes into atrial myocardium is an essential prerequisite for the initiation and propagation of AF remains elusive. Here we investigated the role of atrial CD11b/CD18 mediated infiltration of polymorphonuclear neutrophils (PMN) for the susceptibility to AF.Methods and ResultsC57bl/6J wildtype (WT) and CD11b/CD18 knock-out (CD11b−/−) mice were treated for 14 days with subcutaneous infusion of angiotensin II (Ang II), a known stimulus for PMN activation. Atria of Ang II-treated WT mice were characterized by increased PMN infiltration assessed in immunohistochemically stained sections. In contrast, atrial sections of CD11b−/− mice lacked a significant increase in PMN infiltration upon Ang II infusion. PMN infiltration was accompanied by profoundly enhanced atrial fibrosis in Ang II treated WT as compared to CD11b−/− mice. Upon in-vivo electrophysiological investigation, Ang II treatment significantly elevated the susceptibility for AF in WT mice if compared to vehicle treated animals given an increased number and increased duration of AF episodes. In contrast, animals deficient of CD11b/CD18 were entirely protected from AF induction. Likewise, epicardial activation mapping revealed decreased electrical conduction velocity in atria of Ang II treated WT mice, which was preserved in CD11b−/− mice. In addition, atrial PMN infiltration was enhanced in atrial appendage sections of patients with persistent AF as compared to patients without AF.ConclusionsThe current data critically link CD11b-integrin mediated atrial PMN infiltration to the formation of fibrosis, which promotes the initiation and propagation of AF. These findings not only reveal a mechanistic role of leukocytes in AF but also point towards a potential novel avenue of treatment in AF.

Abstract 641: Expression Of GLUT4 In Vascular Smooth Muscle Affects Endothelial Function In Hypertension.

Expression of GLUT4 is decreased in arterial smooth muscle of hypertensive rats and mice, and total body overexpression of GLUT4 in mice prevents enhanced arterial reactivity. To demonstrate that the effect on vascular response to GLUT4 overexpression is vascular rather than systemic in origin we utilized smooth muscle-specific GLUT4 transgenic mice (SMG4). GLUT4 expression in aortae of SMG4 compared to WT mice was increased 2-3 fold. Adult wild-type (WT) and SMG4 mice were made hypertensive or not through implantation of angiotensin II (AngII; 1.4mg/kg/d for 2 wks) or vehicle containing osmotic mini-pumps. Both WT and SMG4 mice AngII-treated mice exhibited significantly increased systolic blood pressure. In AngII-treated WT mice (WT-AngII) aortic GLUT4 expression was significantly decreased, whereas GLUT4 expression in aortae of AngII-treated SMG4 mice (SMG4-AngII) was maintained. The phosphorylation of ERM and MYPT1(Thr850) were significantly increased in aortae of WT-AngII compared to WT-Sham and SMG4-AngII mice. Responsiveness to the contractile agonists, phenylephrine, 5-HT, and PGF 2 was significantly increased in endothelium-intact aortic rings from WT-AngII mice, but remained normal in aortae of SMG4-AngII mice. Following pretreatment with Rho-kinase inhibitor Y-27632, relative inhibition of contractility to 5-HT was equal in aortae from WT-AngII and SMG4-AngII-treated mice. With endothelial denudation, contractility to 5-HT was equally enhanced in aortae of WT-AngII and SMG4-AngII-treated mice. Interestingly, whereas acetylcholine stimulated relaxation was significantly decreased in aortic rings of WT-AngII mice, relaxation in rings from SMG4-AngII mice was not significantly different from WT or SMG4. These results demonstrate an interesting phenomenon whereby decreased expression of GLUT4 in vascular smooth muscle leads to an endothelial dysfunction that not only impairs relaxation, but also enhances contractility.

GW24-e0525 The role of caspase-associated recruitment domain 9 in cardiac inflammation and fibrosis caused by hypertension

ObjectivesThe aim of this study is to examine the role of CARD9 in inflammation and cardiac fibrosis induced by Ang II infusion, and to elucidate the mechanism that is responsible...

Determination of an Angiotensin II-regulated Proteome in Primary Human Kidney Cells by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC)

Angiotensin II (AngII), the major effector of the renin-angiotensin system, mediates kidney disease progression by signaling through the AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) to identify potential AngII activity markers in the kidney. We utilized stable isotope labeling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of the 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by a different mass spectrometry technique called selected reaction monitoring. Both SILAC and selected reaction monitoring revealed heme oxygenase-1 (HO-1) as the most significantly up-regulated protein in response to AngII stimulation. AngII-dependent regulation of the HO-1 gene and protein was further verified in PTECs. To extend these in vitro observations, we overlaid a network of significantly enriched gene ontology terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five gene ontology terms were enriched in both datasets and included HO-1. Furthermore, HO-1 kidney expression and urinary excretion were reduced in AngII-treated mice with PTEC-specific AT-1R deletion compared with AngII-treated wild-type mice, thus confirming AT-1R-mediated regulation of HO-1. Our in vitro approach identified novel molecular markers of AngII activity, and the animal studies demonstrated that these markers are relevant in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models.

Angiotensin II‐induced impairment of vasodilation in mouse mesenteric arteries: role of endothelial TRPV4 channels

Up‐regulation of angiotensin II (Ang II) signaling causes endothelial dysfunction and altered production of endothelial relaxing factors. Endothelial TRPV4 is a Ca2+entry channel critically involved in endothelium‐mediated dilation in various species and vascular beds; however, the importance of this channel in disease remains largely unknown. This study investigated the contribution of TRPV4 to Ang II‐induced impairment of vasodilation in mice. The study was performed in TRPV4‐deficient (TRPV4−/−) and wild‐type (WT) mice implanted with an osmotic mini‐pump for continuous infusion of Ang II (600 ng/kg/min) for 14 days. After Ang II infusion, mean arterial pressure was similar in TRPV4−/− (143 ± 4 mmHg) and WT (137 ± 7 mmHg). In isolated mesenteric arteries from control WT mice, acetylcholine (ACh) elicited potent endothelium‐dependent dilation (dilation at 10−5 M, 92 ± 2%; n=9). The dilation was markedly reduced in Ang II‐treated WT mice (71 ± 5%; n=7). Ang II also reduced the dilation to TRPV4 agonist GSK1016790A (dilation at 10−8 M, 14 ± 5% vs. 77 ± 7% of control). Surprisingly, Ang II did not affect ACh‐induced dilation in TRPV4−/− mice (71 ± 3% vs. 69 ± 9% of control; n=9 and 5, respectively). In conclusion, Ang II reduces endothelium‐mediated dilation in mouse mesenteric arteries from normal but not TRPV4−/− mice. The precise role of TRPV4 in Ang II‐induced endothelial dysfunction remains to be determined.

Abstract 443: Peroxisome Proliferator Activated Receptor-α Gene Deletion Exacerbates the Progression of Hypertension and Inflammation in Chronic Angiotensin II-Hypertensive Mice

The anti-inflammatory properties of peroxisome proliferator activated receptor- (PPAR-α) contribute to the incidence of hypertension and its associated cardiovascular disease. Fenofibrate, a PPAR-α agonist has been demonstrated to reduce angiotensin II (Ang II)-induced hypertension via inducing cytochrome P450 4A (CYP4A) and increasing 20-hydroxyeicosatetraenoic acid (20-HETE) production. The current study determines whether deletion of PPAR-α gene exacerbates the elevation in blood pressure, plasma interleukin-6 (IL-6) levels, and renal inflammatory markers during infusion of a slow-pressor dose of Ang II (400 ng/kg/min). Ten to twelve week old male PPAR-α knockout (KO) mice and their B129s wild-type (WT) controls were implanted with telemetry devices and infused with Ang II for 12 days. There was no difference in basal mean arterial pressure (MAP) between WT and PPAR-α KO mice compared to WT (113 ± 1 vs. 111 ± 2 mmHg). On day 14 of Ang II infusion, MAP was elevated in PPAR-α KO mice compared to WT (160 ± 3 vs. 142 ± 4 mmHg, P < 0.05) and fenofibrate administration significantly reduced the elevation in MAP in WT + Ang II mice (134 ± 7 mmHg, P < 0.05) with no significant effect on PPAR-α KO + Ang II mice (158 ± 8 mmHg). Plasma IL-6 levels were significantly higher in PPAR-α KO mice when compared to WT on day 12 of Ang II infusion (30 ± 4 vs. 8 ± 2 pg/ml, P < 0.05) and fenofibrate significantly reduced plasma IL-6 in Ang II-treated WT mice (10 ± 3 pg/ml, P < 0.05) with no significant effect in Ang II-treated PPAR-α KO mice (28 ± 4 pg/ml). Fenofibrate significantly increased renal expression of CYP4A and restored the decrease in renal CYP2J expression in WT + Ang II treated mice with no effect on PPAR-α KO + Ang II. Fenofibrate also significantly reduced the elevation in renal expression of the inflammatory markers intracellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1) and cyclooxygenase-2 (COX-2) in WT + Ang II mice. Our results demonstrate that PPAR-α KO mice have a greater presser response during Ang II hypertension and activation of PPAR-α attenuates Ang II-induced hypertension possibly via up-regulation of renal CYP4A and CYP2J and anti-inflammatory mechanisms involving reducing the inflammatory markers IL-6, MCP-1, ICAM-1 and COX-2.

PPAR‐α regulates NOX2 and SOD expression in the heart during Angiotensin II hypertension

Previous reports suggest peroxisome proliferator‐activated receptor‐α (PPAR‐α) agonist attenuates hypertension by suppressing oxidative stress and increasing superoxide dismutase (SOD) activity. We tested the hypothesis that the absence of PPAR‐α would increase NOX2 and decrease SOD expression in mice hearts during Angiotensin II (ANG II) hypertension. Ten to twelve week old male PPAR‐α knockout (KO) mice and their wild‐type (WT) controls were implanted with biotelemetry devices and infused with ANG II (400 ng/kg/min) for twelve days. Separate groups of PPAR‐α KO and WT mice were treated with ANG II plus apocynin (1 g/1L) and ANG II + fenofibrate (500 mg/kg/day). Mean arterial pressure (MAP) and heart rates (HR) were similar between PPAR‐α KO and WT mice during the control period. On day twelve of ANG II treatment, MAP was significantly higher in PPAR‐α KO than WT, 161 ± 5 and 145 ± 4 mmHG, respectively. Fenofibrate significantly reduced MAP in ANG II treated WT mice (134 ± 5 mmHg) and apocynin reduced MAP in both ANG II treated PPAR‐α KO and WT mice. NOX2 expression was significantly increased in ANG II‐treated PPAR‐α KO mice hearts with no significant changes in ANG II‐treated WT mice. Apocynin significantly reduced NOX2 expression in ANG II‐treated PPAR‐α KO mice with no changes in WT. SOD expression in ANG II + fenofibrate was significantly higher than ANG II‐treated WT mice, with no differences between ANG II + fenofibrate and ANG II‐treated PPAR‐α KO. Our results suggest that increased NOX2 and decreased SOD expression in PPAR‐α KO mice hearts contributes to the significant increases in MAP during ANG II hypertension.

Podocytes of AT2 Receptor Knockout Mice Are Protected from Angiotensin II-Mediated RAGE Induction

Background/Aims: The interaction of ‘advanced glycation end products’ (AGEs) and their receptor ‘RAGE’ plays an important role in diabetic nephropathy. We have previously found that in cultured differentiated podocytes, angiotensin II (ANG II) induces RAGE expression via an AT2 receptor-mediated pathway. Methods: To further confirm our results in an in vivo study, AT2 receptor knockout mice (AT2^–/–) and wild-type mice were infused with ANG II by osmotic minipumps for 14 days. Results: As shown by immunohistochemistry, ANG II treatment of wild-type animals (C57BL6) allowed a significantly increased RAGE expression in renal podocytes in comparison to sham-operated C57BL6 mice. In contrast, RAGE expression in podocytes of ANG II-treated knockout mice (AT2^–/–) was only moderately higher than in control animals, but significantly lower than in ANG II-treated wild-type mice. For the AGE species NΕ-carboxymethyllysine, a similar immunohistochemical staining pattern was found. There was no significant change in glomerular AT1a receptor expression. However, no difference in RAGE mRNA expression could be found between ANG II-infused wild-type and AT2^–/– animals by real-time PCR using whole kidney mRNA, presumably due to the low abundance of podocyte mRNA in these preparations. No effects were seen on glomerular apoptosis. Conclusion: These data support the fact that ANG II-mediated RAGE induction in podocytes occurs via AT2 receptors. The present findings may suggest that not all ANG II-mediated changes in diabetic nephropathy can be treated with AT1 receptor blockers.

Angiotensin II Impairs Endothelial Progenitor Cell Number and Function In Vitro and In Vivo

Endothelial progenitor cells (EPCs) contribute to endothelial regeneration. Angiotensin II (Ang II) through Ang II type 1 receptor (AT(1)-R) activation plays an important role in vascular damage. The effect of Ang II on EPCs and the involved molecular mechanisms are incompletely understood. Stimulation with Ang II decreased the number of cultured human early outgrowth EPCs, which express both AT(1)-R and Ang II type 2 receptor, mediated through AT(1)-R activation and induction of oxidative stress. Ang II redox-dependently induced EPC apoptosis through increased apoptosis signal-regulating kinase 1, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase phosphorylation; decreased Bcl-2 and increased Bax expression; and activation of caspase 3 but had no effect on the low cell proliferation. In addition, Ang II impaired colony-forming and migratory capacities of early outgrowth EPCs. Ang II infusion diminished numbers and functional capacities of EPCs in wild-type (WT) but not AT(1)a-R knockout mice (AT(1)a(-/-)). Reendothelialization after focal carotid endothelial injury was decreased during Ang II infusion. Salvage of reendothelialization by intravenous application of spleen-derived progenitor cells into Ang II-treated WT mice was pronounced with AT(1)a(-/-) cells compared with WT cells, and transfusion of Ang II-pretreated WT cells into WT mice without Ang II infusion was associated with less reendothelialization. Transplantation of AT(1)a(-/-) bone marrow reduced atherosclerosis development in cholesterol-fed apolipoprotein E-deficient mice compared with transplantation of apolipoprotein E-deficient or WT bone marrow. Randomized treatment of patients with stable coronary artery disease with the AT(1)-R blocker telmisartan significantly increased the number of circulating CD34/KDR-positive EPCs. Ang II through AT(1)-R activation, oxidative stress, and redox-sensitive apoptosis signal-regulating kinase 1-dependent proapoptotic pathways impairs EPCs in vitro and in vivo, resulting in diminished vascular regeneration.

Androgen-Androgen Receptor System Protects against Angiotensin II-Induced Vascular Remodeling

Age-related andropause promotes cardiovascular disease in males. Although we had previously reported that the androgen-androgen receptor (AR) system plays important roles in cardiac growth and remodeling, the system's involvement in vascular remodeling remains unclear. To clarify this role, 25-wk-old male AR knockout (ARKO) mice and littermate male wild-type (WT) mice were divided into two groups with and without angiotensin II (Ang II) administration (2.0 mg/kg . d) for 14 d, respectively. No morphological differences in the coronary artery and thoracic aorta were observed between the groups without Ang II. Ang II stimulation markedly increased medial thickness and perivascular fibrosis in ARKO mice, with enhanced TGF-beta1, collagen type I, and collagen type III gene expression in the aorta. Ang II stimulation also prominently increased superoxide production, lipid peroxidation, and gene expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase components in ARKO mice compared with WT mice. In addition, phosphorylation of c-Jun N-terminal kinase (JNK) and phosphorylated (Smad2/3) was remarkably enhanced in Ang II-treated ARKO mice compared with Ang II-treated WT mice. Notably, daily urinary nitric oxide (NO) metabolites excretion as a marker of NO bioavailability, aortic endothelial NO synthase expression and phosphorylation, and Akt phosphorylation were significantly reduced in ARKO mice compared with WT mice, regardless of Ang II stimulation. In conclusion, the androgen-AR system is required for the preservation of NO bioavailability through Akt-endothelial NO synthase system activation and exerts protective effects against Ang II-induced vascular remodeling by regulating oxidative stress, c-Jun N-terminal kinase (JNK) signaling, and the TGF-beta-phosphorylated Smad pathway.

Alternative Activation

The calcium/calmodulin (Ca 2+ /CaM)-dependent protein kinase II (CaMKII) is activated through the binding of Ca 2+ /CaM to its regulatory domain and plays important roles in the nervous system and the heart. CaMKII activity is also increased under pro-oxidant conditions, so Erickson et al . investigated whether reactive oxygen species (ROS) might be involved. Exposure of purified CaMKII to hydrogen peroxide (H 2 O 2 ) in the presence, but not absence, of Ca 2+ /CaM resulted in prolonged enzyme activity compared with that of untreated CaMKII. Ca 2+ /CaM-dependent autophosphorylation of Thr 287 is known to prevent the reassociation of the enzyme’s autoinhibitory region with its catalytic domain and so enable prolonged enzyme activity. However, the effect of H 2 O 2 on enzyme activity still occurred with a mutant form of CaMKII in which Thr 287 was mutated to alanine. Mutation of Met 281 and Met 282 in the regulatory domain of CaMKII to valines blocked H 2 O 2 -dependent activation, although Thr 287 autophosphorylation was unaffected. This suggests that Thr 287 phosphorylation and Met 281 /Met 282 oxidation are distinct mechanisms that lead to prolonged CaMKII activation in the presence of Ca 2+ /CaM. The authors developed an antibody against oxidized Met 281 /Met 282 residues and found that the hormone angiotensin II (AngII) increased the abundance of oxidized CaMKII in the hearts of wild-type mice compared with that in untreated mice. AngII induced ROS production and apoptosis in treated cardiomyocytes, which was blocked when CaMKII was knocked down by short hairpin RNA. Finally, the abundance of oxidized CaMKII and the incidence of apoptotic cells were higher in the hearts of AngII-treated mice deficient in MsrA, an enzyme that reverses methionine oxidation, than in AngII-treated wild-type mice. As Griffith notes in commentary, the discovery that ROS directly activate CaMKII may help in our understanding of oxidant-dependent diseases of the heart and nervous system. J. R. Erickson, M.-l. A. Joiner, X. Guan, W. Kutschke, J. Yang, C. V. Oddis, R. K. Bartlett, J. S. Lowe, S. E. O’Donnell, N. Aykin-Burns, M. C. Zimmerman, K. Zimmerman, A.-J. L. Ham, R. M. Weiss, D. R. Spitz, M. A. Shea, R. J. Colbran, P. J. Mohler, M. E. Anderson, A dynamic pathway for calcium-independent activation of CaMKII by methionine oxidation. Cell 133 , 462-474 (2008). [PubMed] L. C. Griffith, CaMKII: New tricks for an old dog. Cell 133 , 397-399 (2008). [PubMed]