Angiotensin II as an Inflammatory Mediator

Akira Matsumori
Kyoto University Graduate School of Medicine, Kyoto, Japan

Heart failure is an inflammatory disease and Angiotensin II (Ang II) plays a role as an inflammatory mediator. This is a new hypothesis by this group of investigators, and the speaker presented data from their research to support their theory.

Inflammation has been shown by recent studies to be an important aspect of cardiovascular (CV) disease and infection. A viral infection, hypertension, or myocardial infarction (MI) induces an inflammatory response in the heart and this inflammation comprises cell infiltration, such as macrophages, mast cells, and lymphocytes, which produce cytokines and may lead to the development of heart failure.

Mast cells induce allergic inflammation and are activated by IgE, thrombin, and neuropeptides such as Substance P and complements. Activated mast cells release granules that increase histamine, heparin, cytokines, growth factors, metalloproteinases, and proteases. These factors play an important role in angiogenesis, inflammation, and fibrosis, which in turn play a critical role in heart failure and cardiomyopathies.

Evidence from their research

Mast-cell deficient mice did not develop heart failure in pressure overload-induced hypertrophy by aortic banding, in experiments by this group. Chamber dilation and decreased systolic function were seen in wild-type (WT) mice, but not in mast-cell deficient mice (WW).

In the murine model of dilated cardiomyopathy (DCM) induced by the encephalomyocarditis viral (EMCV) infection, heart failure developed after 2 weeks of infection and dilatation and hypertrophy developed 3 months after infection. In this animal model, mast cell-mediators, such as mast cell protease 4 (mMCP-4) and mMCP-5, both chymases, and mMCP-7, a tryptase, are upregulated in the heart failure stage with myocarditis. Membrane type MMP-2 (MT-MMP-2), MMP-9, and Type I procollagen are upregulated in the heart failure stage with myocarditis.

In further studies, they showed that survival was better in the WW mast cell-deficient mice than in the WT mice (about 85% vs about 35%), and there was less cell infiltration (1.0 vs 0.1 histologic score, respectively; p<0.01) and myocardial necrosis (0.7 vs 0.1 histologic score, respectively; p<0.01).

In other studies, Ang II was present in human mast cells (HMC), which are already preformed in the cytoplasm of mast cells. In Ang II HMC lines, mast cells from the human lung and umbilical cord blood contain Ang II. Stimulation of the mast cells by calcitonin, a gene-related peptide (CGRP), caused Ang II release into the supernatant in the cultured mast cells. CGRP induced angiotensinogen expression in these mast cell lines. The genes of angiotensinogen and renin were found, but ACE was not found, in these cell lines. Therefore, it is likely that in this mast cell line, Ang II was formed by chymase.

Recent studies by other investigators have shown that Ang II induces gene transcription through cell-type-dependent effects on NF-kB, that NF-kB is activated through AT1 and AT2 receptors in vascular smooth muscle cells, and the transcription factor for NF-kB is necessary for the upregulation of type 1 angiotensin II receptor mRNA in rat cardiac fibroblasts treated with tumor necrosis factor- and interleukin 1ß.   

Angiotensin II activates NF-kB through the AT1 receptor, and this activation in hepatocytes probably induces angiotensinogen, and this creates a positive feedback circuit and can lead to remodeling of the heart (Figure 1).

As shown by this group, Ang II injection activates NF-kB in WT mice, but does not activate the AT1 receptor in knockout mice. Also, circulating Ang II is elevated in the heart failure stage in the animal model of heart failure due to myocarditis, and the presence of circulating Ang II precedes heart failure. Ang II and cytokines become present at about the same time.

Improved survival in AT1-receptor deficient mice infected with the EMCV myocarditis, compared to WT mice (40% vs 20%, respectively; p<0.05) was found in other experiments. Further, NF-kB activity was increased in the WT mice but not in the AT-1 receptor knockout mice. TNF- and IL-1ß expression was enhanced in the WT mice, but was lower in the AT-1 receptor knockout.  Also, iNOS expression was decreased in the knockout mice. Candesartan inhibited expression of TNF-, IL-1ß, and NF-kB. 

Reports from other investigators showed that a mineralocorticoid receptor activated NF-kB in double transgenic rats for human renin and angiotensinogen genes. Also, that aldosterone induces inflammation in the vasculature, heart, and kidneys. These data indicate that the renin-angiotensin-aldosterone system may play a role in inflammation in heart failure.

Conclusion

Thus, they hypothesize that in addition to the direct myocytolysis and immunological damage to cytotoxic lymphocytes and cytokines, the EMCV infection also induces mast cell recruitment to the heart and might induce mast cell degranulation, which induces mast cell proteases and release of cytokines. This may play an important role in myocardial damage, inflammation, and remodeling.

Figure 2 illustrates their hypothesis. Infection, hypertension, or MI induces inflammation in the heart. Cytokines play a major role in the development of heart failure. This group has shown that the PDE inhibitor pimobendan and a new NF-kB inhibitor (SUNC8079) prevented the development of heart failure by inhibiting the production of cytokines. Ang II also induces inflammation and the ARB candesartan prevented the development of inflammation in heart failure. Mast cells play a very important role in inducing Ang II and inflammation, and the mast cell stabilizer tranilast and the histamine 1 blocker cetirizine prevents this inflammation and heart failure. Anti-inflammatory therapy is a promising future treatment of heart failure.

Role of Angiotensin II Type 1 Receptor in the Regulation of Angiogenesis

Toyoaki Murohara
Nagoya University Graduate School of Medicine, Nagoya, Japan

The regulation of angiogenesis through the Angiotensin (Ang) II Type 1 receptor is a newly understood mechanism. Work by Murohara and colleagues presented in this lecture showed that the Ang II type 1 receptor pathway functions as a promoter of the ischemia-induced neovascularization in vivo by supporting inflammatory mononuclear cell infiltration. Further, angiogenesis was mediated by the Ang II type 1a receptor in both hindlimb ischemia and tumor implantation models in mice.
The enhanced angiogenesis was likely mediated at least in part by Ang II-induced inflammatory cell infiltration, which can release a variety of angiogenic cytokines.

Study background

Previous research by other investigators showed that an ACE inhibitor can protect against the risk of cancer in patients with hypertension, and basic research showed that the ACE inhibitor captopril inhibited the basic VEGF-induced angiogenesis in a rat colony micropocket assay, but other work showed an ACE inhibitor increased angiogenesis in the hind leg ischemia model.

ACE inhibitors block the conversion of Ang I to Ang II and inhibit the breakdown of bradykinin. The increased bradykinin stimulates endothelial cells to release nitric oxide and prostacyclin, molecules known to modulate angiogenesis. Thus, ACE inhibitors have a limited ability to regulate angiogenesis because of the bradykinin-nitric oxide pathway.

Therefore, this group examined the role of the AT1 receptor, a major effective receptor for vascular or cardiac tissue by Ang II in 1) ischemia-induced angiogenesis (mouse hindlimb ischemia), and 2) tumor angiogenesis (mouse tumor implantation). The Ang II type 1a receptor (AT1a-/-) knockout mice model, with reduced systemic blood pressure, was used.

Ischemia-induced angiogenesis

A similar marked reduction of blood perfusion in the ischemic limb, after resection of the left femoral artery and veins, was found in both the AT1a-/- knockout mice (KO) model and wild type (WT) mice. However, the recovery of blood flow in the ischemic hindlimb was much greater in the AT1a-/- KO mice than in the WT mice. The recovery of blood perfusion in the ischemic limb was much greater in the WT mice than in the KO mice. These data suggest that angiogenesis after hindlimb ischemia might be reduced in the AT1a-/- KO mice.

Micovascular angiogenesis by capillary density and arteriogenesis identified by angiography and functional blood flow were all reduced in the KO mice compared to WT mice. On day 14, the angiographic score was reduced in the KO mice compared to the WT mice. In isolated ischemic skeletal muscle immunostained with anti-CD31 monoclonal antibody, at day 14 the capillary density was decreased in the KO mice compared to the WT mice (about 18/field vs 28/field, respectively).

Hydralazine, given to the WT mice to reduce blood pressure to a level similar to the KO mice, had no effect on angiogensis or blood flow recovery in WT mice. However, after treatment with the AT receptor blocker TCV-116, the ischemic/normal hindlimb blood flow ratio in the WT mice was more similar to the KO mice.

Ang II induces an inflammatory response, and plays an important role in the infiltration of macrophages or lymphocytes in the ischemic hindlimb. In the KO mice, infiltrated T-lymphocytes or macrophages were reduced. Interestingly, the infiltrated cells intensely expressed VEGF, on double immunofluorescence staining. Thus, VEGF released by the infiltrated inflammatory cells might contribute to angiogenesis in ischemic tissue. Therefore, the ischemia-induced angiogenesis was markedly reduced in the KO mice.

The implantation of WT mast-derived mononuclear cells directly into ischemic tissue of KO mice restored the ischemic/normal laser Doppler blood flow ratio after hindlimb ischemia, compared to non-implanted KO mice and WT mice.

Tumor angiogenesis

The growth curves for implanted B16-F1 melanoma cells and QRsP-11 fibrosarcoma cells were much reduced in KO mice compared to WT mice. Consistently, the survival rate was much greater in the KO than the WT mice.

Analysis of angiogenesis within and around the tissue surrounding the tumor showed angiogenic evidence was reduced in the KO mice compared to WT mice. VEGF-expressing macrophage infiltration was reduced in the KO mice compared to the WT mice in the tissue surrounding tumors. Recent studies by other investigators have shown that tumor-associated macrophages are released by angiogenic cytokines including VEGF. These cytokines can induce tumor-related angiogenesis.

Clinical implications

These investigators hypothesize that marked inhibition of the AT1 receptor early after ischemia may have an adverse effect on subsequent ischemic tissue injury by inhibiting physiological angiogenesis.
The effects of the Ang II type 1 receptor blocker on the incidence and mortality in cancer patients warrants further clinical investigation.

An ARB alone did not improve prognosis after a myocardial infarction (MI) in the VALIANT study. But, ACE inhibitor therapy plus an ARB improved prognosis in patients with heart failure in the VAL-HeFT and CHARM trials. Therefore, in patients with an acute MI, especially early after the infarction, the marked inhibition of the AT1 receptor may reduce the subsequent angiogenic response. Thus, these investigators believe that the addition of an ARB to an ACE inhibitor may be recommended at 4-6 months after the onset of infarction.

Local Renin-Angiotensin System Induces Ventricular Remodeling through Oxidant Stress-mediated Breakdown of Extracellular Matrix in Rats with Salt-Sensitive Hypertension

Yasuki Kihara
Kyoto University Graduate School of Medicine, Kyoto, Japan

Although the renin-angiotensin system (RAS) may play a major role in left ventricular (LV) remodeling, the precise mechanisms are not fully characterized. To address this question, these investigators conducted experiments in Dahl salt-sensitive (DS) rats, a relevant model for LV remodeling, and Dahl salt-resistant controls (DR). The DR controls maintain their normotensive state, normal LV function, and normal LV versus body weight ratio. In contrast, the DS rats are hypertensive, with significant concentric hypertrophy at age 11 weeks, normal LV function and a calculated LV wall stress within the normal limit. Thus, the left ventricle is well compensated against overload at 11 weeks. But, at about 15-17 weeks, eccentric hypertrophy and hypofunction of the LV is seen. The rats die of pulmonary congestion very rapidly. Thus, this is a good model to see the process from a compensated left ventricle to LV remodeling or heart failure.

In the so-called low renin, high Angiotensin II (Ang II) models, these investigators previously showed that LV tissue angiotensinogen, ACE, and Ang II is upregulated at the stage of LV hypertrophy.

They also showed in the DR and DS models that the extracellular metalloproteinase (MMP) is markedly activated during LV remodeling. MMP plays an important role in the degradation of extracellular matrix. MMP is at a normal level in both the DS and DR rats in the LVH phase, but is markedly increased in the DS rats after the transition to heart failure (from 0.9 at 11 weeks to 1.7 at 17 weeks; p<0.05). A tight, linear relation between MMP activation and LV diameter and LV systolic wall stress was found.

Study design

The goals of the present study were to 1) elucidate the roles of the local RAS that may induce LV remodeling, 2) trace the signaling process between Ang II and MMP activation, and 3) test the hypothesis that a direct inhibition of MMPs could block LV remodeling in a manner independent of tissue RAS activation.

DS rats were fed an 8% high-salt diet after 6 weeks of age, and after 11 weeks the DS rats were divided into 4 groups for chronic pharmacologic interventions: 1) control group, given 0.5% CMC solvent twice daily, 2) ARB group, given telmisartan 5 mg/kg once daily, 3) MMPi group, given the MMP inhibitor ONO-4817 (100 mg/kg twice daily), and 4) combined ARB and MMPi group. As a reference, the DR rats were fed the same diet.

Assessments were animal survival for each group, serial in-vivo echocardiographic study to quantify LV size and function, electron microscopic study to visualize the extracellular matrix (ECM) degradation, and immunohistochemical staining to determine the LV tissue damage by oxidant stress. Quantitative, real-time PCR and Western blotting were performed to measure the activation of oxidant stress signaling.

Study results

Improved survival with the MMP inhibitor was found in the animals with heart failure.  The animals in the control group died quire rapidly, by 20 weeks of age, while the MMP inhibitor initiated at 11 weeks extended survival to about 25 weeks in the heart failure animals (Figure 1). Animal survival was also about 25 weeks when the MMP inhibitor was initiated at 15 weeks. Thus, the MMP inhibitor acts along with the activation of MMP at the heart failure transition phase.

A substantial improvement in survival to nearly 30 weeks was also seen with the ARB, with no reduction in blood pressure. Importantly, combined ARB and MMPi significantly improved survival to about 35 weeks (Figure 2). This suggests that the ARB and MMPi could be working via different cellular actions, and worked synergistically to improve survival.

Figure 3 summarizes the echocardiographic study. In the control animals, the LV diameter and LV end systolic diameter began to increase at 15 weeks of age and continued to 17 weeks, whereas the increase in LV wall stress began at 11 weeks, and there was a decrease in LV fractional shortening. In the ARB group and the ARB plus MMPi group there was a similar trend from 11 weeks in LV diameter, LV end systolic diameter, LV wall stress, and fractional shortening. These 2 groups maintained their LV shape and function at normal levels.

In the LVH stage, there was a very dense network between the cardiomyocytes on electron photomicrography. Surprisingly, the ECM became very thin after the heart failure transition. The ECM degradation was lessened with the ARB, and combined ARB plus MMPi maintained ECM.

Marked activation of oxidative stress was seen during heart failure transition, while none was seen in the LVH stage, based on findings from the immunofluorescent staining with HNE (4-hydroxy-2-nonenal), the fingerprint of oxidant stress on membrane proteins, and with 8-OhdG (8-hydroxy-2’-deoxyduanosine), the fingerprint of oxidant stress on nuclear proteins. Notably, the ARB markedly reduced the oxidative stress, but the MMPi did not block oxidative stress.

NAD(P)H could be the critical mediator of the oxidative stress in the presence of Ang II activation. Measurement of NAD(P)H oxidized activation in mRNA and Western blotting showed that the protein levels of P^47phox were substantially increased in heart failure compared to LVH, but in the presence of the ARB, the P^47phox level was normal. But, the activation of P^47phox was not blocked by the MMPi. Thus, the subcellular mechanism for the ARB and the MMPi are completely different.

Summary

In an animal model of the process from ventricular remodeling to heart failure, these investigators showed that 1) chronic administration of an ARB and a MMP inhibitor improved animal survival, LV shape and function, 2) this improvement was associated with preservation of ECM, 3) tissue Ang II affected ECM degradation through activation of NAD(P)H oxidase-mediated oxidant stress, and 4) tissue MMP activation directly caused ECM degradation independent of tissue Ang II activation.

Tissue Ang II activation causes ECM degradation and plays critical roles in the process of ventricular remodeling. ARBs, such as telmisartan, effectively suppress this detrimental process. Exogenous administration of MMP Inhibitors, such as ONO-4817, may serve as an adjuvant therapy to further suppress the process of ventricular remodeling in combination with the angiotensin blockade.

Effect of Eplerenone, a Novel Selective Aldosterone Receptor Antagonist in Salt-Sensitive Hypertension

Yoshiyu Takeda
Kanazawa University, Kanazawa, Japan

The reported deleterious effects of aldosterone include myocardial fibrosis, vascular injury and inflammation, endothelial dysfunction, and progressive renal injury. Although the RALES trial showed a 30% risk reduction for death in heart failure patients, the side effects profile of spironolactone is problematic. The specific aldosterone receptor antagonist eplerenone reduced cardiovascular death by 21% in post-AMI heart failure patients.

About 50-60% of Japanese patients with hypertension are salt-sensitive. The Dahl salt-sensitive rat (DS) is a good model for salt-sensitive hypertension and heart failure. This group previously reported that in DS rats, a high sodium diet increased blood pressure, reduced plasma renin activity, reduced plasma aldosterone, and reduced vascular 11ß-HSD11 activity and mRNA expression. The expression of mineralocorticoid receptor RNA was increased by a high sodium diet. Therefore, these investigators hypothesized that a local excess of aldosterone because of reduced 11ß-HSD11 activity is a major cause of salt-sensitive hypertension in rats.

Study design

This study sought to clarify the mechanisms responsible for the anti-hypertensive and anti-hypertrophic effects of eplerenone in salt-sensitive hypertension.

DS rats (n=20) were treated with a low salt diet, high salt diet or high salt diet plus eplerenone (100mg/kg/day) for 12 weeks. Blood pressure, plasma renin activity (PRA), plasma aldosterone, heart weight, the expression of mRNA of type I angiotensin receptor (AT1R), angiotensinogen, angiotensin converting enzyme (ACE) and endothelial nitric oxide synthase (eNOS) were measured. Real-time PCR methods were used to quantify the mRNA of each gene.

Study results

In DS rats, a high-salt diet increased blood pressure to about 250 mm Hg, but eplerenone treatment prevented the increase (160 mm Hg). Body weight was similar in each group (Figure 1). 

The high sodium diet significantly decreased the PRA and plasma aldosterone levels. Treatment with eplerenone slightly increased PRA but did not affect the aldosterone concentration (Figure 2). As illustrated in Figure 3, eplerenone improved the survival ratio.

Acetylcholine-induced relaxation was reduced by a high sodium diet, but treatment with eplerenone partially improved this relaxation (Figure 4). Aortic eNOS mRNA expression was reduced by a high salt diet, but this was attenuated with eplerenone. The heart weight/body weight ratio and collagen II mRNA were increased by a high sodium diet, while eplerenone treatment improved the heart weight and cardiac fibrosis (Figure 5).

Cardiac calcineurin mRNA and cardiac Angiotensin type 1 receptor mRNA, cardiac ACE mRNA, and cardiac Ang mRNA were increased by a high sodium diet, and were improved by eplerenone (Figure 6). So, salt-sensitive hypertension is a type of local aldosterone excess state, and the specific aldosterone receptor blocker eplerenone improved hypertension and cardiac hypertrophy and fibrosis (Figure 7).

Blockade of Cardiac Aldosterone Production as a New Therapeutic Strategy of Heart Failure

Michiro Yoshimura
Kumamoto University, Kumamoto, Japan

Production of aldosterone in the human heart

Recent evidence showed that aldosterone blockade using either spironolactone or eplerenone improved the prognosis of patients with heart failure. Based on this evidence, it can be speculated that there are unknown effects of aldosterone and that there is the possibility of extra-adrenal synthesis of aldosterone.

This group previously reported that BNP is secreted from the ventricles and that ACE is activated in the ventricles of patients with heart failure. Aldosterone is also secreted from the ventricles of the patients with heart failure, but not from the ventricles of control subjects. Another experiment confirmed the expression of the CYP11B2 gene and aldosterone production in the human heart; in autopsied human hearts expression of the CYP11B2 gene was higher than in patients who died of cancer without heart failure.

Aldosterone actions currently proved

Several reports have shown that aldosterone induces inflammation in many organs including the heart and blood vessels. This group hypothesized that aldosterone has many actions, including ACE gene expression, creating a circular cascade within the cardiac renin-angiotensin-aldosterone system (RAAS).

In experiments in cultured rat neonatal cardiomyocytes using real-time PCR, this group showed that aldosterone increases ACE gene expression, which was completely suppressed by spironolactone, a mineralocorticoid receptor antagonist. This result confirms the presence of the circular cascade with cardiac RAAS via the mineralocorticoid receptor. Interestingly, they found this action is cell-specific and is species different. Nevertheless, this action can be seen in the left ventricle of patients with heart failure, based on preliminary data by this group.

Inhibitory effect of natriuretic peptides on cardiac aldosterone production

The natriuretic peptide family comprises ANP, BNP, and CNP, which have many important actions. It is well known that ANP and BNP secrete from the failing heart

Angiotensin II did not increase CYP11B2 gene expression in cultured rat neonatal cardiomyocytes. However, pre-treatment of the cells with HS1421, a GC-A receptor antagonist suppressing endogenous effects of natriuretic peptide, Angiotensin II significantly increased the CYP11B2 expression. This results confirms the inhibitory action of natriuretic peptide on cardiac aldosterone production and suggests that if there were no natriuretic peptides in the failing heart, it could produce considerable aldosterone.

Production of dehydroepiandrosterone (DHEA) in the human heart

Considering the presence of the cascade to produce aldosterone, they hypothesized this cascade could produce CYP17 in the human heart. Real-time PCR of the enzymes required for steroid synthesis of human hearts obtained at autopsy confirmed the presence of CYP17. Then, during cardiac catheterization they performed direct sampling of DHEA and aldosterone in patients with heart failure and control subjects. They showed that DHEA is secreted from the heart of control subjects, but not from the heart of patients with heart failure. In contrast, aldosterone is secreted from the heart of patients with heart failure, but not from the heart of control subjects. The DHEA/aldosterone ratio showed that the value was significantly lower in patients with heart failure compared to the control subjects, especially in the coronary sinus.

Aldosterone is thought to be a hormone for oxidation and inflammation, whereas DHEA is thought to a hormone for anti-oxidation and anti-inflammation. Thus, they examined the possible inhibitory action of low-dose DHEA on cardiac hypertrophy induced by ET-1 in cultured rat cardiomyocytes. Pre-treatment with DHEA suppressed cardiac hypertrophy induced by ET-1, both for cell size and BNP gene expression. So, even though the receptor for DHEA has not been elucidated, it appears that DHEA plays an important role in cardiac protection.

Production of adrenocorticotropic hormone (ACTH)

Based on the previous evidence, they thought that ACTH may be also secreted from the human heart. During cardiac catheterization, direct sampling was performed in patients with hypertension and in control subjects. ACTH was secreted from the ventricles of patients with hypertension, but not from the ventricles of the control subjects.

They found a close relationship between cardiac ACTH and cardiac aldosterone. This result implies that ACTH produced in the ventricle continuously induces aldosterone synthesis in the ventricle. It is interesting to see this action of ACTH, because it is thought to suppress aldosterone synthesis in the adrenal gland in the chronic phase, thus the action of ACTH would be in both the heart and the adrenal gland. They hypothesize that ACTH and Angiotensin II collectively induces aldosterone synthesis in the heart.

Aldosterone is stimulated by Angiotensin II in the failing heart. ACTH produced in the heart stimulates aldosterone synthesis in the failing heart. Natriuretic peptides suppress aldosterone synthesis. Interestingly, there is an unstable balance between aldosterone and DHEA. They have studied only part of the cascade of RAAS, ACTH, and steroids in the human heart. To understand this complex cascade, the many possible relationships among these must be investigated.