Hans Imboden

A novel angiotensin II type 2 receptor signaling pathway: possible role in cardiac hypertrophy

We describe a novel signaling mechanism mediated by the G‐protein‐coupled receptor (GPCR) angiotensin II (Ang II) type 2 receptor (AT2). Yeast two‐hybrid studies and affinity column binding assay show that the isolated AT2 C‐terminus binds to the transcription factor promyelocytic zinc finger protein (PLZF). Cellular studies employing confocal microscopy show that Ang II stimulation induces cytosolic PLZF to co‐localize with AT2 at the plasma membrane, then drives AT2 and PLZF to internalize. PLZF slowly emerges in the nucleus whereas AT2 accumulates in the perinuclear region. Nuclear PLZF binds to a consensus sequence of the phosphatidylinositol‐3 kinase p85α subunit (p85α PI3K) gene. AT2 enhances expression of p85α PI3K followed by enhanced p70S6 kinase, essential to protein synthesis. An inactive mutant of PLZF abolishes this effect. PLZF is expressed robustly in the heart in contrast to many other tissues. This cardiac selective pathway involving AT2, PLZF and p85α PI3K may explain the absence of a cardiac hypertrophic response in AT2 gene‐deleted mice.

Angiotensin II AT1 Receptor Blockade Reverses Pathological Hypertrophy and Inflammation in Brain Microvessels of Spontaneously Hypertensive Rats

Background and Purpose— The spontaneously hypertensive rat (SHR) is vulnerable to brain ischemia and stress and exhibits a chronically stimulated brain angiotensin II system, cerebrovascular hypertrophy, and inflammation. Pretreatment with angiotensin II type 1 (AT1) receptor antagonists protects from brain ischemia and from stress and prevents the development of stress-induced gastric ulcers in part by reducing inflammation in the gastric mucosa. We studied whether AT1 receptor antagonists could exert antiinflammatory effects in the brain vasculature as a mechanism for their protective effects against ischemia. Methods— Ten-week-old SHR and normotensive Wistar-Kyoto male rats received the AT1 receptor antagonist candesartan (0.3 mg/kg per day) or vehicle for 28 days via osmotic minipumps. We studied AT1 receptors, intercellular adhesion molecule-1 (ICAM-1), endothelial nitric oxide synthase (eNOS), and number of macrophages by immunohistochemistry and Western blots. Results— We found increased endothelial AT1 receptor expression of brain microvessels and middle cerebral artery of SHR. Brain AT1 receptor inhibition reversed the pathological vascular hypertrophy, increased and normalized eNOS expression, and decreased ICAM-1 expression and the number of adherent and infiltrating macrophages in cerebral vessels of SHR. Conclusions— The antiinflammatory effects of AT1 receptor antagonists may be an important mechanism in protecting against ischemia.

Organ-specific distribution of ACE2 mRNA and correlating peptidase activity in rodents

Abstract Biochemical analysis revealed that angiotensin-converting enzyme related carboxy-peptidase (ACE2) cleaves angiotensin (Ang) II to Ang-(1–7), a heptapeptide identified as an endogenous ligand for the G protein-coupled receptor Mas. No data are currently available that systematically describe ACE2 distribution and activity in rodents. Therefore, we analyzed the ACE2 expression in different tissues of mice and rats on mRNA (RNase protection assay) and protein levels (immunohistochemistry, ACE2 activity, western blot). Although ACE2 mRNA in both investigated species showed the highest expression in the ileum, the mouse organ exceeded rat ACE2, as also demonstrated in the kidney and colon. Corresponding to mRNA, ACE2 activity was highest in the ileum and mouse kidney but weak in the rat kidney, which was also confirmed by immunohistochemistry. Contrary to mRNA, we found weak activity in the lung of both species. Our data demonstrate a tissue- and species-specific pattern for ACE2 under physiological conditions.

Localization of angiotensin-converting enzyme, angiotensin II, angiotensin II receptor subtypes, and vasopressin in the mouse hypothalamus.

The hypothalamic angiotensin II (Ang II) system plays an important role in pituitary hormone release. Little is known about this system in the mouse brain. We studied the distribution of angiotensin-converting-enzyme (ACE), Ang II, Ang II receptor subtypes, and vasopressin in the hypothalamus of adult male mice. Autoradiography of binding of the ACE inhibitor [125I]351A revealed low levels of ACE throughout the hypothalamus. Ang II- and vasopressin-immunoreactive neurons and fibers were detected in the paraventricular, accessory magnocellulary, and supraoptic nuclei, in the retrochiasmatic part of the supraoptic nucleus and in the median eminence. Autoradiography of Ang II receptors was performed using [125I]Sar1-Ang II binding. Ang II receptors were present in the paraventricular, suprachiasmatic, arcuate and dorsomedial nuclei, and in the median eminence. In all areas [125I]Sar1-Ang II binding was displaced by the AT1 receptor antagonist losartan, indicating the presence of AT1 receptors. In the paraventricular nucleus [125I]Sar1-Ang II binding was displaced by Ang II (Ki = 7.6 X 10(-9)) and losartan (Ki = 1.4 X 10(-7)) but also by the AT2 receptor ligand PD 123319 (Ki = 5.0 X 10(-7)). In addition, a low amount of AT2 receptor binding was detected in the paraventricular nucleus using [125I]CGP42112 as radioligand, and the binding was displaced by Ang II (Ki = 2.4 X 10(-9)), CGP42112 (Ki = 7.9 x 10(-10)), and PD123319 (Ki = 2.2 x 10(-7)). ACE, Ang II, and AT1 as well as AT2 receptor subtypes are present in the mouse hypothalamus. Our data are the basis for further studies on the mouse brain Ang II system.

Rescue of a severe mouse model for spinal muscular atrophy by U7 snRNA-mediated splicing modulation

In spinal muscular atrophy (SMA), the leading genetic cause of early childhood death, the survival motor neuron 1 gene (SMN1) is deleted or inactivated. The nearly identical SMN2 gene has a silent mutation that impairs the utilization of exon 7 and the production of functional protein. It has been hypothesized that therapies boosting SMN2 exon 7 inclusion might prevent or cure SMA. Exon 7 inclusion can be stimulated in cell culture by oligonucleotides or intracellularly expressed RNAs, but evidence for an in vivo improvement of SMA symptoms is lacking. Here, we unambiguously confirm the above hypothesis by showing that a bifunctional U7 snRNA that stimulates exon 7 inclusion, when introduced by germline transgenesis, can efficiently complement the most severe mouse SMA model. These results are significant for the development of a somatic SMA therapy, but may also provide new means to study pathophysiological aspects of this devastating disease.

Effects of angiotensin analogues and angiotensin receptor antagonists on paraventricular neurones

In a previous study we observed that most neurones in the paraventricular nucleus are excited by angiotensin-(1-7). In comparison with angiotensin III this excitatory action was significantly delayed. The aim of the present microiontophoretic study of angiotensin II-sensitive rat paraventricular neurones was to compare the effect of the angiotensin-analogues angiotensin-(1-7), angiotensin-(2-7), angiotensin II and angiotensin III on the spontaneous activity of these neurones and to test angiotensin receptor subtype 1 antagonists (CGP 46027 or DuP 753) and subtype 2 selective antagonists (CGP 42112A and PD 123177) in order to acquire more evidence of the receptor subtype present. As previously observed angiotensin II, angiotensin III and angiotensin-(1-7) excited most neurones. The effect of angiotensin-(1-7) was usually weaker than that of angiotensin II, and in contrast to angiotensin III the latencies were not significantly different. Angiotensin-(1-7) seemed to be active by itself, because its effect was antagonised by angiotensin receptor antagonists. Angiotensin-(2-7) was mostly inactive, although a few cells were excited. Whereas the excitatory effects of angiotensin-(1-7), angiotensin II and angiotensin III could always be inhibited with both angiotensin receptor subtype antagonists 1 and 2, that produced by angiotensin-(2-7) was only weakly antagonised, if at all. Subtype 1 selective antagonists were effective at lower concentrations than selective subtype 2 antagonists.

AT1 Receptor Blockade Regulates the Local Angiotensin II System in Cerebral Microvessels From Spontaneously Hypertensive Rats

Background and Purpose— Blockade of angiotensin II AT1 receptors in cerebral microvessels protects against brain ischemia and inflammation. In this study, we tried to clarify the presence and regulation of the local renin-angiotensin system (RAS) in brain microvessels in hypertension. Methods— Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) controls were treated with an AT1 receptor antagonist (candesartan, 0.3 mg/kg per day) via subcutaneous osmotic minipumps for 4 weeks. The expression and localization of RAS components and the effect of AT1 receptor blockade were assessed by Affymetrix microarray, qRT-PCR, Western blots, immunohistochemistry and immunofluorescence. Results— We found transcripts of most of RAS components in our microarray database, and confirmed their expression by qRT-PCR. Angiotensinogen (Aogen), angiotensin-converting enzyme (ACE) and AT1 receptors were localized to the endothelium. There was no evidence of AT2 receptor localization in the microvascular endothelium. In SHR, (pro)renin receptor mRNA and AT1 receptor mRNA and protein expression were higher, whereas Aogen, ACE mRNA and AT2 receptor mRNA and protein expression were lower than in WKY rats. Candesartan treatment increased Aogen, ACE and AT2 receptor in SHR, and increased ACE and decreased Aogen in WKY rats, without affecting the (pro)renin and AT1 receptors. Conclusions— Increased (pro)renin and AT1 receptor expression in SHR substantiates the importance of the local RAS overdrive in the cerebrovascular pathophysiology in hypertension. AT1 receptor blockade and increased AT2 receptor stimulation after administration of candesartan may contribute to the protection against brain ischemia and inflammation.

Identification of noncytotoxic and IL-10-producing CD8+AT2R+ T cell population in response to ischemic heart injury.

Emerging evidence suggests a cardioprotective role of the angiotensin AT2R, albeit the underlying cellular mechanisms are not well understood. We aimed in this article to elucidate a potential role of cardiac angiotensin AT2R in regulating cellular immune response to ischemic heart injury. Seven days after myocardial infarction in rats, double-immunofluorescence staining showed that AT2R was detected in a fraction of CD8+ T cells infiltrating in the peri-infarct myocardium. We developed a method that allowed the isolation of myocardial infiltrating CD8+AT2R+ T cells using modified MACS, and further characterization and purification with flow cytometry. Although the CD8+AT2R− T cells exhibited potent cytotoxicity to both adult and fetal cardiomyocytes (CMs), the CD8+AT2R+ T cells were noncytotoxic to these CMs. The CD8+AT2R+ T cells were characterized by upregulated IL-10 and downregulated IL-2 and INF-γ expression when compared with CD8+AT2R− T cells. We further showed that IL-10 gene expression was enhanced in CD8+ T cells on in vitro AT2R stimulation. Importantly, in vivo AT2R activation engendered an increment of CD8+AT2R+ T cells and IL-10 production in the ischemic myocardium. In addition, intramyocardial transplantation of CD8+AT2R+ T cells (versus CD8+AT2R−) led to reduced ischemic heart injury. Moreover, the CD8+AT2R+ T cell population was also demonstrated in human peripheral blood. Thus, we have defined the cardioprotective CD8+AT2R+ T cell population, which increases during ischemic heart injury and contributes to maintaining CM viability and providing IL-10, hence revealing an AT2R-mediated cellular mechanism in modulating adaptive immune response in the heart.

Angiotensin II type 1 receptor blocker losartan prevents and rescues cerebrovascular, neuropathological and cognitive deficits in an Alzheimer's disease model.

Angiotensin II (AngII) receptor blockers that bind selectively AngII type 1 (AT1) receptors may protect from Alzheimers disease (AD). We studied the ability of the AT1 receptor antagonist losartan to cure or prevent AD hallmarks in aged (~18months at endpoint, 3months treatment) or adult (~12months at endpoint, 10months treatment) human amyloid precursor protein (APP) transgenic mice. We tested learning and memory with the Morris water maze, and evaluated neurometabolic and neurovascular coupling using [(18)F]fluoro-2-deoxy-D-glucose-PET and laser Doppler flowmetry responses to whisker stimulation. Cerebrovascular reactivity was assessed with on-line videomicroscopy. We measured protein levels of oxidative stress enzymes (superoxide dismutases SOD1, SOD2 and NADPH oxidase subunit p67phox), and quantified soluble and deposited amyloid-β (Aβ) peptide, glial fibrillary acidic protein (GFAP), AngII receptors AT1 and AT2, angiotensin IV receptor AT4, and cortical cholinergic innervation. In aged APP mice, losartan did not improve learning but it consolidated memory acquisition and recall, and rescued neurovascular and neurometabolic coupling and cerebrovascular dilatory capacity. Losartan normalized cerebrovascular p67phox and SOD2 protein levels and up-regulated those of SOD1. Losartan attenuated astrogliosis, normalized AT1 and AT4 receptor levels, but failed to rescue the cholinergic deficit and the Aβ pathology. Given preventively, losartan protected cognitive function, cerebrovascular reactivity, and AT4 receptor levels. Like in aged APP mice, these benefits occurred without a decrease in soluble Aβ species or plaque load. We conclude that losartan exerts potent preventive and restorative effects on AD hallmarks, possibly by mitigating AT1-initiated oxidative stress and normalizing memory-related AT4 receptors.

Cardiac c‐kit+AT2+ Cell Population is Increased in Response to Ischemic Injury and Supports Cardiomyocyte Performance

The expression pattern of angiotensin AT2 receptors with predominance during fetal life and upregulation under pathological conditions during tissue injury/repair process suggests that AT2 receptors may exert an important action in injury/repair adaptive mechanisms. Less is known about AT2 receptors in acute ischemia‐induced cardiac injury. We aimed here to elucidate the role of AT2 receptors after acute myocardial infarction. Double immunofluorescence staining showed that cardiac AT2 receptors were mainly detected in clusters of small c‐kit+ cells accumulating in peri‐infarct zone and c‐kit+AT2+ cells increased in response to acute cardiac injury. Further, we isolated cardiac c‐kit+AT2+ cell population by modified magnetic activated cell sorting and fluorescence activated cell sorting. These cardiac c‐kit+AT2+ cells, represented ∼0.19% of total cardiac cells in infarcted heart, were characterized by upregulated transcription factors implicated in cardiogenic differentiation (Gata‐4, Notch‐2, Nkx‐2.5) and genes required for self‐renewal (Tbx‐3, c‐Myc, Akt). When adult cardiomyocytes and cardiac c‐kit+AT2+ cells isolated from infarcted rat hearts were cocultured, AT2 receptor stimulation in vitro inhibited apoptosis of these cocultured cardiomyocytes. Moreover, in vivo AT2 receptor stimulation led to an increased c‐kit+AT2+ cell population in the infarcted myocardium and reduced apoptosis of cardiomyocytes in rats with acute myocardial infarction. These data suggest that cardiac c‐kit+AT2+ cell population exists and increases after acute ischemic injury. AT2 receptor activation supports performance of cardiomyocytes, thus contributing to cardioprotection via cardiac c‐kit+AT2+ cell population. STEM CELLS 2009;27:2488–2497