The adaptive immune response and, in particular, T cells have been shown to be important in the genesis of hypertension. In the present study, we sought to determine how the interplay between ANG II, NADPH oxidase, and reactive oxygen species modulates T cell activation and ultimately causes hypertension. We determined that T cells express angiotensinogen, the angiotensin I-converting enzyme, and renin and produce physiological levels of ANG II. AT1 receptors were primarily expressed intracellularly, and endogenously produced ANG II increased T-cell activation, expression of tissue homing markers, and production of the cytokine TNF-alpha. Inhibition of T-cell ACE reduced TNF-alpha production, indicating endogenously produced ANG II has a regulatory role in this process. Studies with specific antagonists and T cells from AT1R and AT2R-deficient mice indicated that both receptor subtypes contribute to TNF-alpha production. We found that superoxide was a critical mediator of T-cell TNF-alpha production, as this was significantly inhibited by polyethylene glycol (PEG)-SOD, but not PEG-catalase. Thus, T cells contain an endogenous renin-angiotensin system that modulates T-cell function, NADPH oxidase activity, and production of superoxide that, in turn, modulates TNF-alpha production. These findings contribute to our understanding of how ANG II and T cells enhance inflammation in cardiovascular disease.

Background-Hypertensive target organ damage, especially cardiac hypertrophy with heart failure and arrhythmia, is a major source of morbidity and mortality. Angiotensin II, a major mediator of hypertension and cardiac damage, has proinflammatory properties. Inflammation and activation of the immune system play a pivotal role in pathogenesis of hypertensive target organ damage. However, the role of immunosuppressive CD4

Abstract-We tested the hypothesis that the renin inhibitor aliskiren ameliorates organ damage in rats transgenic for human renin and angiotensinogen genes (double transgenic rat [dTGR]). Six-week-old dTGR were matched by albuminuria (2 mg per day) and divided into 5 groups. Untreated dTGR were compared with aliskiren (3 and 0.3 mg/kg per day)-treated and valsartan (Val; 10 and 1 mg/kg per day)-treated rats. Treatment was from week 6 through week 9. At week 6, all groups had elevated systolic blood pressure (BP). Untreated dTGR showed increased BP (202Ϯ4 mm Hg), serum creatinine, and albuminuria (34Ϯ5.7 mg per day) at week 7. At week 9, both doses of aliskiren lowered BP (115Ϯ6 and 139Ϯ5 mm Hg) and albuminuria (0.4Ϯ0.1 and 1.6Ϯ0.6 mg per day) and normalized serum creatinine. Although high-dose Val lowered BP (148Ϯ4 mm Hg) and albuminuria (2.1Ϯ0.7 mg per day), low-dose Val reduced BP (182Ϯ3 mm Hg) and albuminuria (24Ϯ3.8 mg per day) to a lesser extent. Mortality was 100% in untreated dTGR and 26% in Val (1 mg/kg per day) treated rats, whereas in all other groups, survival was 100%. dTGR treated with low-dose Val had cardiac hypertrophy (4.4Ϯ0.1 mg/g), increased left ventricular (LV) wall thickness, and diastolic dysfunction. LV atrial natriuretic peptide and ␤-myosin heavy chain mRNA, albuminuria, fibrosis, and cell infiltration were also increased. In contrast, both aliskiren doses and the high-dose Val lowered BP to a similar extent and more effectively than low-dose Val. We conclude that in dTGR, equieffective antihypertensive doses of Val or aliskiren attenuated end-organ damage. Thus, renin inhibition compares favorably to angiotensin receptor blockade in reversing organ damage in dTGR. Key Words: renin Ⅲ rats, transgenic Ⅲ hypertrophy R enin is the rate-limiting step in the generation of angiotensin II (Ang II). 1 Thus, inhibiting this step reduces Ang II levels. Historically, renin inhibitors have not been clinically successful because of lack of potency or bioavailability. The new nonpeptidic renin inhibitor aliskiren is a potent human renin inhibitor (IC 50 ϭ0.6 nmol/L). 2 Because renin displays species specificity for its substrate, human renin inhibitors cannot be tested efficiently in conventional hypertensive rat models. To circumvent this problem, transgenic rats and mice were developed harboring the human renin and the human angiotensinogen genes. 3,4 Human renin does not effectively cleave rat angiotensinogen, and similarly, rat renin cleaves human angiotensinogen poorly. 5 Consequently, the single transgenic rats and mice (ie, transgenic for either human angiotensinogen or renin) are normotensive. However, when cross-bred, the double transgenic rat (dTGR) offspring develop hypertension with severe organ damage and do not live beyond the seventh or eighth week of age. We extensively studied these animals; the injury features nuclear factor B (NF-B) and activator protein-1 transcription factor activation, upregulation of surface adhesion molecules, cytokines, and the influx of inflammatory cells. 6 ...

Abstract-We used rats transgenic for the human angiotensinogen (hAogen) gene and the human renin (hRen) gene and crossed the strains to produce a model of preeclampsia in the dams. The female (nϭ9) hAogen ϫ male hRen cross had severe (telemetry-measured) hypertension and albuminuria, which developed during the last trimester of pregnancy and subsided after delivery. The converse cross (nϭ9) and control (nϭ9) SD rats did not. We demonstrated that the female hAogen ϫ male hRen cross had agonistic antibodies capable of activating the angiotensin (Ang) II AT1 receptor (AT1R-AA) and defined the epitope on the receptor's second extracellular loop. The phenomenon also occurs in humans with preeclampsia. The rats displayed renal histology reminiscent of preeclampsia, including fibrin deposition confined to the glomeruli. The complement system was activated in glomeruli and IgG deposits were present that may represent AT1R-AA. Finally, we observed an atherosis-like lesion in the spiral arteries of the placental bed, which we called placental-bed arteriolosclerosis. Our model may be relevant to preeclampsia in humans. Key Words: preeclampsia Ⅲ rats, transgenic Ⅲ antibodies Ⅲ immune systems Ⅲ renin-angiotensin system P reeclampsia, proteinuria, and severe hypertension in the latter part of pregnancy affects Ϸ3% of women in industrialized nations and a much higher percentage of women in underdeveloped countries. 1 In all countries, preeclampsia represents the major cause of maternal and fetal morbidity and mortality. Constructing a suitable animal model has been difficult. Takimoto et al described hypertension induced in pregnant mice by placental renin and maternal angiotensinogen. 2 Mice were generated transgenic for the human renin (hRen) and human angiotensinogen (hAogen) genes. The rodent and human renin-angiotensinogen systems do not interact and single transgenic animals are normotensive. Severe hypertension develops in double-transgenic offspring. The investigators observed that hypertension developed in the latter third of pregnancy in hAogen dams mated with hRen males. They showed that secreted active hRen of placental origin was capable of reacting with hAogen in the dams to produce angiotensin (Ang) II. Takimoto et al suggested that the transgenic mice might offer a unique model of "genetically induced" preeclampsia. 2 We showed that the same phenomenon exists in a rat transgenic model. 3 We used telemetric blood pressure measurements in that study to document the precise timing of the model; however, we presented no functional or histological data. 3 We have also studied preeclamptic women and found that they produce an agonistic antibody to the Ang II receptor (AT1R). 4,5 These autoantibodies (AT1-AA) activate the receptor. The activation sets into motion a chain of signaling events that could be responsible for the clinical disease. 6 We have now restudied our transgenic animal model and have observed that the rats also have these novel autoantibodies. MethodsSprague-Dawley (SD) rats harboring the human Aogen...

Abstract-We tested whether or not complement activation participates in angiotensin (Ang) II-induced vasculopathy. We used double transgenic rats harboring human renin and angiotensinogen genes (dTGR) with or without losartan or the human renin inhibitor aliskiren. Sprague-Dawley (SD) rats were controls. DTGR had increased blood pressure at week 5 that increased further by week 7. Albuminuria was absent at week 5 but increased markedly in weeks 6 and 7. C-reactive protein (CRP) elevation, macrophages, T cells, tumor necrosis factor (TNF)-␣, C1q, C3, C3c, and C5b-9 expression preceded albuminuria. C1q, C3, C3c, and C5b-9 were observed in the dTGR vessel media. C5b-9 colocalized with interleukin (IL)-6. Losartan and aliskiren reduced albuminuria and complement expression. We also studied vascular smooth muscle cells (VSMC) from dTGR compared VSMC from SD. C3 and IL-6 mRNA were analyzed after Ang II, TNF-␣, and CRP stimulation. VSMC from dTGR showed increased proliferation and C3 expression compared with SD. Ang II did not induce C3 mRNA in either VSMC type. However, TNF-␣ and CRP induced C3 mRNA slightly in SD VSMC but markedly in dTGR VSMC, whereas IL-6 induction was similar in both. Thus, complement activation and cell infiltration occurred before the onset of albuminuria in Ang II-mediated renal damage. TNF-␣ and CRP played a major role in C3 activation. VSMC from dTGR are more sensitive for C3 activation. Our data show that, in this Ang II-induced model, complement activation is a major participant and suggest that TNF-␣ and CRP may play a role in its induction. Key Words: angiotensin II Ⅲ complement Ⅲ immune system Ⅲ albuminuria and renal damage T he innate complement system eliminates invading pathogens, stimulates opsonization, enhances phagocytosis, cytolysis, chemotaxis, and solubilizes immune complexes. Complement forms a bridge between innate and acquired immunity. 1,2 On excessive activation or inappropriate deposition, complement can cause disease. 3 The classical alternative and lectin complement pathway merge at the level of C3, resulting in the generation of C5b-9, the membrane attack complex. Complement activation has been implicated in the pathogenesis of numerous proteinuric renal diseases including glomerulonephritis, transplant rejection, and ischemiareperfusion injury. [3][4][5][6][7] Pratt et al demonstrated that the absence of local C3 production modulates renal graft survival and regulates T-cell priming of donor antigens. 4 Very recently, Lin et al reported that young spontaneous hypertensive rats (SHR) that have not yet developed hypertension showed increased C3 expression and increased vascular smooth muscle cell (VSMC) proliferation. Both were blocked by C3 downregulation. 8 Several studies showed that angiotensin (Ang) II not only is a vasoconstrictor peptide but also promotes inflammation and renal damage. We showed recently that immunosuppression improved nonimmune Ang II-mediated renal damage. 9 The evidence that Ang II affects the complement system is indirect. Abbate et al demons...

Rats harboring the human renin and angiotensinogen genes (dTGR) feature angiotensin (ANG) II/ hypertension-induced cardiac damage and die suddenly between wk 7 and 8. We observed by electrocardiogram (ECG) telemetry that ventricular tachycardia (VT) is a common terminal event in these animals. Our aim was to investigate electrical remodeling. We used ECG telemetry, noninvasive cardiac magnetic field mapping (CMFM) at wk 5 and 7, and performed in vivo programmed electrical stimulation at wk 7. We also investigated whether or not losartan (Los; 30 mg ⅐ kg Ϫ1 ⅐ day Ϫ1 ) would prevent electrical remodeling. Cardiac hypertrophy and systolic blood pressure progressively increased in dTGR compared with Sprague-Dawley (SD) controls. Already by wk 5, untreated dTGR showed increased perivascular and interstitial fibrosis, connective tissue growth factor expression, and monocyte infiltration compared with SD rats, differences that progressed through time. Left-ventricular mRNA expression of potassium channel subunit Kv4.3 and gap-junction protein connexin 43 were significantly reduced in dTGR compared with Los-treated dTGR and SD. CMFM showed that depolarization and repolarization were prolonged and inhomogeneous. Los ameliorated all disturbances. VT could be induced in 88% of dTGR but only in 33% of Los-treated dTGR and could not be induced in SD. Untreated dTGR show electrical remodeling and probably die from VT. Los treatment reduces myocardial remodeling and predisposition to arrhythmias. ANG II target organ damage induces VT. magnetocardiography; noninvasive mapping; double-transgenic rat model; in vivo electrophysiological study ELECTRICAL REMODELING INVOLVES acquired changes in cardiac structure or function that promote the occurrence of atrial or ventricular cardiac arrhythmias (22). On the molecular level, electrical remodeling involves changes in function and expression of membrane ion channels, gap-junction proteins, Ca 2ϩ -cycling proteins, and extracellular matrix composition. All these factors predispose to arrhythmogenic mechanisms such as early and delayed afterdepolarizations and reentry (13). The multifactorial origin of electrical remodeling has been extensively studied in cardiac ischemia and heart failure. However, electrical remodeling in hypertension is less well defined. Patients with hypertension-induced left-ventricular hypertrophy are at increased risk for arrhythmias, which contribute to a twofold increase in cardiovascular mortality (7). Monitoring electrical remodeling is challenging. The standard 12-lead electrocardiogram (ECG), ECG-based body surface potential mapping, and signal-averaged ECG are correlated with an increased risk to develop malignant arrhythmias. However, the positive predictive accuracy is unacceptably low or not sufficiently tested in randomized trials (14).Multichannel cardiac magnetic field mapping (CMFM) reflects the magnetic fields generated by the myocardial electrical currents occurring during the cardiac cycle. CMFM signals have several advantages: 1) they are littl...

About one-half of double transgenic rats (dTGR) overexpressing the human renin and angiotensinogen genes die by age 7 wk of terminal heart failure (THF); the other (preterminal) one-half develop cardiac damage but survive. Our study’s aim was to elucidate cardiac gene expression differences in dTGR-THF compared with dTGR showing compensated cardiac hypertrophy but not yet THF. dTGR treated with losartan (LOS) and nontransgenic rats (SD) served as controls. THF-dTGR body weight was significantly lower than for all other groups. At death, THF-dTGR had blood pressures of 228 ± 7 mmHg (cardiac hypertrophy index 6.2 ± 0.1 mg/g). Tissue Doppler showed reduced peak early (Ea) to late (Aa) diastolic expansion in THF-dTGR, indicating diastolic function. Preterminal dTGR had blood pressures of 197 ± 5 mmHg (cardiac hypertrophy index 5.1 ± 0.1 mg/g); Ea < Aa compared with LOS-dTGR (141 ± 6 mmHg; 3.7±0.1 mg/g; Ea > Aa) and SD (112 ± 4 mmHg; 3.6 ± 0.1 mg/g; Ea > Aa). Left ventricular RNA was isolated for the Affymetrix system and TaqMan RT-PCR. THF-dTGR and dTGR showed upregulation of hypertrophy markers and α/β-myosin heavy chain switch to the fetal isoform. THF-dTGR (vs. dTGR) showed upregulation of 239 and downregulation of 150 genes. Various genes of mitochodrial respiratory chain and lipid catabolism were reduced. In addition, genes encoding transcription factors (CEBP-β, c-fos, Fra-1), coagulation, remodeling/repair components (HSP70, HSP27, heme oxygenase), immune system (complement components, IL-6), and metabolic pathway were differentially expressed. In contrast, LOS-dTGR and SD had similar expression profiles. These data demonstrate that THF-dTGR show an altered expression profile compared with preterminal dTGR.

We investigated whether or not p38 mitogen-activated protein kinase inhibition ameliorates angiotensin II-induced target organ damage. We used double transgenic rats harboring both human renin and angiotensinogen genes (dTGRs). dTGR, with or without p38 inhibitor (BIRB796; 30 mg/kg per day in the diet), and nontransgenic Sprague-Dawley rats were studied in 2 protocols. In protocol 1 (week 7), systolic blood pressure of untreated dTGRs was 204+/-4 mm Hg, but partially reduced after BIRB796 treatment (166+/-7 mm Hg), whereas Sprague-Dawley rats were normotensive. The cardiac hypertrophy index was unchanged in untreated and BIRB796-treated dTGRs. The beta-myosin heavy chain expression of BIRB796-treated hearts was significantly lower in BIRB796 compared with dTGRs, indicating a delayed switch to the fetal isoform. BIRB796 treatment significantly reduced cardiac fibrosis, connective tissue growth factor, tumor necrosis factor-alpha, interleukin-6, and macrophage infiltration. Albuminuria was not reduced in BIRB796-treated dTGRs. Tubular and glomerular damage with tumor necrosis factor-alpha expression was unaltered, although serum creatinine and cystatin C were normalized. Renal macrophage infiltration, fibrosis, and vessel damage were reduced. In protocol 2 (week 8), we focused on mortality and arrhythmogenic electrical remodeling. Mortality of untreated dTGRs was 100% but was reduced to 10% in the BIRB796 group. Cardiac magnetic field mapping showed prolongation of depolarization and repolarization in untreated dTGRs compared with Sprague-Dawley rats with a partial reduction by BIRB796. Programmed electrical stimulation elicited ventricular tachycardias in 81% of untreated dTGRs but only in 48% of BIRB796-treated dTGRs. In conclusion, BIRB796 improved survival, target organ damage, and arrhythmogenic potential in angiotensin II-induced target organ damage.