Regulatory T cells ameliorate angiotensin II-induced cardiac damage.

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+CD25+ regulatory T (Treg) cells in the pathogenesis of hypertensive target organ damage is unexplored. METHODS AND RESULTS: We conducted adoptive transfer of Treg cells into angiotensin II-infused hypertensive mice. Treg cell recipients exhibited improved cardiac hypertrophy and less cardiac fibrosis despite sustained hypertension. Amelioration of cardiac morphology was accompanied by an improvement in arrhythmogenic electric remodeling, indicating the functional significance of the enhanced cardiac morphology. Delocalization of the connexin 43 gap junction protein is one of the major pathomechanisms in electric remodeling. Pronounced connexin 43 immunoreactivity was found at the lateral borders of cardiomyocytes in angiotensin II-treated mice. In contrast, connexin 43 was restricted to the intercalated disk regions in sham controls. Surprisingly, angiotensin II+Treg-treated mice showed normal connexin 43 gap junction protein localization. Adoptive Treg cell transfer resulted in a marked reduction in cardiac CD4+, CD8+, and CD69+ cell and macrophage infiltration. CONCLUSIONS: Immunosuppressive effects of transferred Treg cells ameliorated cardiac damage and accounted for the improved electric remodeling independently of blood pressure-lowering effects. Our results provide new insights into the pathogenesis of hypertensive cardiac damage and could therefore lead to new therapeutic approaches that involve manipulation of the immune system.

Energy metabolism in human renin-gene transgenic rats: does renin contribute to obesity?

Renin initiates angiotensin II formation and has no other known functions. We observed that transgenic rats (TGR) overexpressing the human renin gene (hREN) developed moderate obesity with increased body fat mass and glucose intolerance compared with nontransgenic Sprague-Dawley (SD) rats. The metabolic changes were not reversed by an angiotensin-converting enzyme inhibitor, a direct renin inhibitor, or by (pro)renin receptor blocker treatment. The obese phenotype in TGR(hREN) originated from higher food intake, which was partly compensated by increases in resting energy expenditure, total thermogenesis (postprandial and exercise activity), and lipid oxidation during the first 8 weeks of life. Once established, the difference in body weight between TGR(hREN) and SD rats remained constant over time. When restricted to the caloric intake of SD, TGR(hREN) developed an even lower body weight than nontransgenic controls. We did not observe significant changes in the cocaine and amphetamine-regulated transcript, pro-opiomelanocortin, both anorexigenic, or neuropeptide Y, orexigenic, mRNA levels in TGR(hREN) versus SD controls. However, the mRNA level of the agouti-related peptide, orexigenic, was significantly reduced in TGR(hREN) versus SD controls at the end of the study, which indicates a compensatory mechanism. We suggest that the human renin transgene initiates a process leading to increased and early appetite, obesity, and metabolic changes not related to angiotensin II. The mechanisms are independent of any currently known renin-related effects.

Regulation of T-cell function by endogenously produced angiotensin II.

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.

Novel role for inhibitor of differentiation 2 in the genesis of angiotensin II-induced hypertension.

BACKGROUND: Angiotensin (Ang) II-induced target-organ damage involves innate and acquired immunity. Mice deficient for the helix-loop-helix transcription factor inhibitor of differentiation (Id2(-/-)) lack Langerhans and splenic CD8a+ dendritic cells, have reduced natural killer cells, and have altered CD8 T-cell memory. We tested the hypothesis that an alteration in the number and quality of circulating blood cells caused by Id2 deletion would ameliorate Ang II-induced target-organ damage. METHODS AND RESULTS: We used gene-deleted and transgenic mice. We conducted kidney and bone marrow transplants. In contrast to Ang II-infused Id2(+/-), Id2(-/-) mice infused with Ang II remained normotensive and failed to develop albuminuria or renal damage. Bone marrow transplant of Id2(+/-) bone marrow to Id2(-/-) mice did not restore the blunted blood pressure response to Ang II. Transplantation of Id2(-/-) kidneys to Id2(+/-) mice also could not prevent Ang II-induced hypertension and renal damage. We verified the Ang II resistance in Id2(-/-) mice in a model of local tissue Ang II production by crossing hypertensive mice transgenic for rat angiotensinogen with Id2(-/-) or Id2(+/-) mice. Angiotensinogen-transgenic Id2(+/-) mice developed hypertension, albuminuria, and renal injury, whereas angiotensinogen-transgenic Id2(-/-) mice did not. We also found that vascular smooth muscle cells from Id2(-/-) mice showed an antisenescence phenotype. CONCLUSIONS: Our bone marrow and kidney transplant experiments suggest that alterations in circulating immune cells or Id2 in the kidney are not responsible for Ang II resistance. The present studies identify a previously undefined role for Id2 in the pathogenesis of Ang II-induced hypertension.

Low-dose renin inhibitor and low-dose AT(1)-receptor blocker therapy ameliorate target-organ damage in rats harbouring human renin and angiotensinogen genes.

We studied the effects of extremely low-dose human renin inhibition (aliskiren) with low angiotensin II receptor blockade (losartan) in a novel double-transgenic rat model harbouring both human renin and angiotensinogen genes. We found that low-dose aliskiren and low-dose losartan effectively reduced mortality and target-organ damage with minimal, non-significant, effects on blood pressure (BP). Our data suggest that renin-angiotensin system (RAS) inhibition ameliorates target-organ damage in an Ang II-driven model of hypertension. Direct renin inhibition is equally efficacious in this regard. Our study does not fully answer the question of BP-lowering versus RAS inhibition. This question is important and was at least partially addressed with our low-dose model.

Angiotensin II-induced sudden arrhythmic death and electrical remodeling.

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 x kg(-1) x day(-1)) 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.

p38 mitogen-activated protein kinase inhibition ameliorates angiotensin II-induced target organ damage.

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.

Complement activation in angiotensin II-induced organ damage.

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)-alpha, 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-alpha, 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-alpha 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-alpha 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-alpha and CRP may play a role in its induction.

Aliskiren, a human renin inhibitor, ameliorates cardiac and renal damage in double-transgenic rats.

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 (202+/-4 mm Hg), serum creatinine, and albuminuria (34+/-5.7 mg per day) at week 7. At week 9, both doses of aliskiren lowered BP (115+/-6 and 139+/-5 mm Hg) and albuminuria (0.4+/-0.1 and 1.6+/-0.6 mg per day) and normalized serum creatinine. Although high-dose Val lowered BP (148+/-4 mm Hg) and albuminuria (2.1+/-0.7 mg per day), low-dose Val reduced BP (182+/-3 mm Hg) and albuminuria (24+/-3.8 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 (4.4+/-0.1 mg/g), increased left ventricular (LV) wall thickness, and diastolic dysfunction. LV atrial natriuretic peptide and beta-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.

Agonistic autoantibodies to the AT1 receptor in a transgenic rat model of preeclampsia.

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 (n=9) hAogen x 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 (n=9) and control (n=9) SD rats did not. We demonstrated that the female hAogen x 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.

Cardiac gene expression profile in rats with terminal heart failure and cachexia.

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 alpha/beta-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-beta, 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.

Differential gene expression in the periprosthetic membrane: lubricin as a new possible pathogenetic factor in prosthesis loosening.

About 10% of hip endoprostheses will loosen after 10 years. Prosthesis loosening is caused by two different pathomechanisms: aseptic loosening (AL) and septic loosening (SL). This study evaluated differences in gene expression in AL and SL. Eight hybridizations were performed on PIQOR cDNA arrays. Objects of the study were periprosthetic interface tissue samples from two patients with SL and three patients with AL. Tissue parts directly adjacent to the site of RNA isolation were analyzed immuno/histopathologically in order to overcome the problem of tissue heterogeneity. Thirty-three genes were found constantly differentially expressed, among which were cd11b, cd18, cd68, osteopontin and ferritin heavy-chain upregulated in AL and collagen types 1alpha-1, 3alpha-1, integrin alpha-1, thrombospondin2 and nidogen upregulated in SL. The most striking finding was the strong upregulation (from 20-fold to 323-fold) of megakaryocyte stimulating factor (msf) in all aseptic cases and one of the two septic cases, which was confirmed by real-time reverse transcription-polymerase chain reaction. In this study, msf is linked to prosthesis loosening for the first time. The upregulation in AL suggests an important pathogenetic role: the msf splice product lubricin is responsible for the lubrication of healthy joints, but its excellent lubrication ability may disturb the tight interaction between bone and prosthesis and thereby contribute to prosthesis loosening.