The role of receptor MAS in microglia-driven retinal vascular development.

OBJECTIVE: The receptor MAS, encoded by Mas1, is expressed in microglia and its activation has been linked to anti-inflammatory actions. However, microglia are involved in several different processes in the central nervous system, including the promotion of angiogenesis. We therefore hypothesized that the receptor MAS also plays a role in angiogenesis via microglia. APPROACH AND RESULTS: To assess the role of MAS on vascular network development, flat-mounted retinas from 3-day-old wild-type (WT) and Mas1-/- mice were subjected to Isolectin B4 staining. The progression of the vascular front was reduced (- 24%, p < 0.0001) and vascular density decreased (- 38%, p < 0.001) in Mas1-/- compared to WT mice with no change in the junction density. The number of filopodia and filopodia bursts were decreased in Mas1-/- mice at the vascular front (- 21%, p < 0.05; - 29%, p < 0.0001, respectively). This was associated with a decreased number of vascular loops and decreased microglial density at the vascular front in Mas1-/- mice (-32%, p < 0.001; - 26%, p < 0.05, respectively). As the front of the developing vasculature is characterized by reduced oxygen levels, we determined the expression of Mas1 following hypoxia in primary microglia from 3-day-old WT mice. Hypoxia induced a 14-fold increase of Mas1 mRNA expression (p < 0.01). Moreover, stimulation of primary microglia with a MAS agonist induced expression of Notch1 (+ 57%, p < 0.05), Dll4 (+ 220%, p < 0.001) and Jag1 (+ 137%, p < 0.001), genes previously described to mediate microglia/endothelial cell interaction during angiogenesis. CONCLUSIONS: Our study demonstrates that the activation of MAS is important for microglia recruitment and vascular growth in the developing retina.

The angiotensin AT2-receptor mediates inhibition of cell proliferation in coronary endothelial cells.

Angiotensin II (ANG II) is known to be a potent growth promoting factor for vascular smooth muscle cells and fibroblasts but little is known about its influence on growth in endothelial cells. We studied the effects of ANG II on endothelial growth and the role of the angiotensin receptor subtypes involved. Proliferation of rat coronary endothelial cells (CEC) and rat vascular smooth muscle cells (VSMC) was determined by [3H]thymidine incorporation, the MTT-test and by directly counting cells in a coulter counter. Angiotensin AT1- and AT2-receptors were demonstrated by binding studies and by the presence of their respective mRNA through reverse transcription polymerase chain reaction (RT-PCR). In contrast to VSMC, which in culture only express the AT1-receptor, CEC express both, AT1- and AT2-receptors simultaneously up to the third passage. Whereas ANG II stimulated growth of quiescent VSMC, an effect abolished by pretreatment with the AT1-receptor antagonist, losartan, ANG II did not induce proliferation in quiescent CEC. However, after pretreatment of quiescent endothelial cells (< passage 4) with the AT2-receptor antagonist, PD 123177, ANG II induced proliferation. This effect was reversed by additional pretreatment with losartan. ANG II significantly inhibited the proliferation of bFGF-stimulated CEC in a dose-dependent manner by maximally 50%. This effect was prevented by PD 123177 while losartan was ineffective. The AT2-receptor agonist, CGP 42112, mimicked the antiproliferative actions of ANG II, confirming the specificity of the effect. Our results show that the growth modulating actions of ANG II depend on the type of angiotensin receptor present on a given cell. In coronary endothelial cells, the antiproliferative actions of the AT2-receptor offset the growth promoting effects mediated by the AT1-receptor.

Angiotensin receptor subtypes in the brain.

Development of specific angiotensin II receptor ligands has recently provided evidence for the existence of two angiotensin II receptor subtypes, termed AT1 and AT2, which differ in their signal transduction mechanisms and in the effects they mediate. In brain, both receptor subtypes are present. Most of the known central actions of angiotensin II, for example the regulation of blood pressure and of electrolyte and water balance, seem to be mediated by the AT1 receptor, while the role of the AT2 receptor is still an enigma. This review by Thomas Unger and colleagues summarizes the current knowledge and latest hypotheses in this rapidly developing field.

The AT2 receptor--a matter of love and hate.

In 1989, the development of specific angiotensin receptor antagonists which distinguish between two angiotensin receptor subtypes (AT1 and AT2) led to a breakthrough in angiotensin research. It turned out, that the AT1 receptor was almost entirely responsible for the "classical" actions of angiotensin II related to the regulation of blood pressure as well as volume and electrolyte balance. However, actions and signal transduction mechanisms coupled to the AT2 receptor remained enigmatic for a long time. The present review summarizes the current knowledge of AT2 receptor distribution, signaling and function with an emphasis on growth/anti-growth, differentiation and the regeneration of neuronal tissue.

Repression of c-fos and c-jun gene expression is not part of AT2 receptor coupled signal transduction.

The signal transduction mechanism coupled to angiotensin AT2 receptors is still a matter of debate. Based on the findings that AT2 receptor stimulation causes inhibition of proliferation, and that other antiproliferative agents such as transforming growth factor-beta, retinoic acid, and MyoD act via repression of immediate early gene (IEG) expression, this study was aimed at elucidating whether downregulation of IEG expression is also part of the AT2 receptor coupled signaling mechanism. Stimulation of angiotensin AT2 receptors in the rat pheochromocytoma cell line PC12 W following pretreatment with growth factors was able to counteract growth factor induced proliferation but not to repress growth factor induced c-fos and c-jun expression; neither did AT2 receptor stimulation cause an induction of c-fos expression. We conclude that, in contrast to other growth-inhibiting agents, the antiproliferative effect of angiotensin II via the AT2 receptor is not mediated by repression of the immediate early genes c-fos and c-jun.

Selective activation of angiotensin AT2 receptors attenuates progression of pulmonary hypertension and inhibits cardiopulmonary fibrosis.

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) is a devastating disease characterized by increased pulmonary arterial pressure, which progressively leads to right-heart failure and death. A dys-regulated renin angiotensin system (RAS) has been implicated in the development and progression of PH. However, the role of the angiotensin AT2 receptor in PH has not been fully elucidated. We have taken advantage of a recently identified non-peptide AT2 receptor agonist, Compound 21 (C21), to investigate its effects on the well-established monocrotaline (MCT) rat model of PH. EXPERIMENTAL APPROACH: A single s.c. injection of MCT (50 mg·kg(-1) ) was used to induce PH in 8-week-old male Sprague Dawley rats. After 2 weeks of MCT administration, a subset of animals began receiving either 0.03 mg·kg(-1) C21, 3 mg·kg(-1) PD-123319 or 0.5 mg·kg(-1) A779 for an additional 2 weeks, after which right ventricular haemodynamic parameters were measured and tissues were collected for gene expression and histological analyses. KEY RESULTS: Initiation of C21 treatment significantly attenuated much of the pathophysiology associated with MCT-induced PH. Most notably, C21 reversed pulmonary fibrosis and prevented right ventricular fibrosis. These beneficial effects were associated with improvement in right heart function, decreased pulmonary vessel wall thickness, reduced pro-inflammatory cytokines and favourable modulation of the lung RAS. Conversely, co-administration of the AT2 receptor antagonist, PD-123319, or the Mas antagonist, A779, abolished the protective actions of C21. CONCLUSIONS AND IMPLICATIONS: Taken together, our results suggest that the AT2 receptor agonist, C21, may hold promise for patients with PH.

The angiotensin II AT2 receptor inhibits proliferation and promotes differentiation in PC12W cells.

Angiotensin II (ANG II) has been implicated in cell growth and differentiation. We investigated the effect of AT2 receptor stimulation on proliferation and morphological differentiation in cells of neuronal origin by using the pheochromocytoma derived cell line, PC12W. ANG II (10(-8)-10(-6) M) inhibited fetal calf serum (FCS)-induced cell proliferation in a concentration dependent manner. In half of the experiments, the epidermal growth factor (EGF) exerted a mitogenic action which was concentration-dependently inhibited by ANG II. In the other half of the experiments, EGF had an antimitogenic effect which was further enhanced by ANG II (maximally at 10(-6) M). Treatment with nerve growth factor (NGF) induced an inhibition of [3H]thymidine incorporation, which was enhanced by ANG II, maximally 25% at the highest concentration. The effects of ANG II on [3H]thymidine incorporation were reflected by those on cell number and were prevented by the AT2 receptor antagonist, PD123177, but not influenced by the AT1 receptor antagonist, losartan. The ANG II-induced inhibition of cell proliferation was paralleled by morphological differentiation in response to daily treatment with ANG II. ANG II also enhanced low-dose NGF-induced neurite formation. Again, these effects of ANG II were abolished by the AT2 receptor antagonist, PD123177. Our data in PC12W cells show that the AT2 receptor not only inhibits growth factor-induced proliferation and enhances the NGF-mediated growth arrest but also induces morphological differentiation in cells of neuronal origin. These findings strongly support the hypothesis that the AT2 receptor promotes differentiation in neuronal cells.

Mortality and morbidity in different immunization protocols for experimental autoimmune myocarditis in rats.

AIM: We aimed to investigate the histological and clinical presentations of experimental autoimmune myocarditis (EAM) induced by different immunization schemes. METHODS: Male young Lewis rats were divided into five groups immunized by porcine myocardial myosin: subcutaneously (SC) 2 mg (in two 1-mg doses on day 0 and 7), 0 mg (sham group) subcutaneously into rear footpads (RF), 0.25 mg RF, 0.5 mg RF or 1 mg RF (all RF once on day 0). On day 21, left ventricular (LV) function was assessed by cardiac magnetic resonance imaging and cardiac catheterization. The type and degree of myocardial inflammatory infiltrates were determined by conventional histology and immunohistochemistry. RESULTS: In the SC immunized rats and in the RF sham group, we observed 0% mortality, while in the actively RF immunized rats, mortality was 20, 20 and 44% for the 0.25 mg, 0.5 mg and 1 mg myosin doses respectively. Morbidity as defined by inflammatory infiltrates on haematoxylin and eosin (HE) staining was 22% in the SC immunized rats, 0% in the RF sham group and 100% in all actively RF immunized groups. We observed augmented relative ventricle weight and spleen weight, increased LV end-diastolic pressure, reduced LV developed pressure and reduced LV ejection fraction in all with myosin-immunized RF groups without any systematic dose effect. CONCLUSION: Subcutaneous immunization to the neck and flanks did not induce a reproducible EAM, while RF myosin administration reliably led to EAM. Lower myosin doses seem to induce the complete histological and clinical picture of EAM while being associated with lower mortality, non-specific symptoms and animal distress.

Differential expression of angiotensin receptors in human cutaneous wound healing.

BACKGROUND: Angiotensin AT1 and AT2 receptors are expressed in human skin. Furthermore, AT2 receptors have been reported to be upregulated during tissue repair and remodelling in various noncutaneous human tissues. OBJECTIVES: Detection of alterations in angiotensin II receptor expression during wound healing in human skin. METHODS: Three models were employed. (i) Primary human keratinocytes were razor scraped in culture flasks and alterations in the expression of angiotensin receptor mRNA determined by semiquantitative reverse transcription-polymerase chain reaction for 1-12 h thereafter. (ii) Early wound healing (48 h after cutting) was studied in punch biopsies from human skin ex vivo by means of immunohistochemical staining using polyclonal antibodies against the AT1 or AT2 receptor. (iii) In vivo wound healing was studied in sections of human cutaneous scars by immunohistochemistry to determine receptor expression early (2 days) and late (2 weeks-3 months) after surgery. RESULTS: In all experimental settings, an upregulation of both receptor subtypes was noticed after wounding. Immunohistochemically stained skin sections showed a stronger expression of AT2 than of AT1 receptors within the area of scarring. Enhanced receptor expression was detectable as early as 24 h after injury and lasted for up to 3 months. CONCLUSIONS: From these data, we conclude that angiotensin AT1 and AT2 receptors are upregulated in human cutaneous wounds, giving further support to the concept that angiotensin II plays a role even at an early stage during cutaneous wound healing.

The renin-angiotensin-system in the skin. Evidence for its presence and possible functional implications.

Among the several hormonal systems regulating body functions, the renin-angiotensin-system has long been considered a classical endocrine system with angiotensin II, its effector hormone, being synthesized in and subsequently distributed by the circulation to act on its numerous, mainly renal and cardiovascular target organs throughout the body. Angiotensin II has long been regarded to be primarily responsible for the regulation of blood-pressure and of volume- and electrolyte-homeostasis. Recent evidence suggests that it also affects cellular proliferation and differentiation via the so-called local or tissue-renin-angiotensin-systems. Such trophic actions have already been observed in tissues not belonging to the renal or cardiovascular systems such as cultured cells of neuronal origin. Evidence for a rôle of angiotensin II in the skin is so far scanty and mainly based on the demonstration of angiotensin receptors on cultured human keratinocytes and in subcutaneous tissue of rats. Although almost every single component of the renin-angiotensin-system has already been identified in skin of one or another species, comprehensive data regarding the skin renin-angiotensin-system as a whole within one particular species, especially in man, are still lacking. The present manuscript reviews novel recent data regarding the renin-angiotensin-system particularly in skin, and it discusses a possible functional rôle of the cutaneous renin-angiotensin-system on the basis of these findings.

Angiotensin-converting enzyme inhibitors as inducers of adverse cutaneous reactions.

Since adverse effects due to angiotensin-converting enzyme (ACE) inhibitors frequently occur in cutaneous locations, this review summarizes the spectrum of expected and unexpected adverse effects of these drugs, possible associated mechanisms, and their basic functions for dermatologists. ACE inhibitors block the activity of the metalloproteinase ACE by binding to its active site, thus displacing angiotensin I and preventing its conversion to vasopressive angiotensin II. Furthermore, ACE degrades bradykinin, substance P, enkephalins and some of the reproductive peptide hormones. The overall incidence of adverse effects to ACE inhibitors is estimated at 28%, approximately half of which occurs in the skin. General reactions are first-dose hypotension, hyperkalaemia and renal failure. Cutaneous reactions comprise life-threatening angioedema, pruritus, bullous eruptions, urticaria, other generalized rashes, photosensitivity and hair loss. ACE inhibitors thus mimic a broad variety of skin diseases, why these drugs should be thought of when sudden, unexplainable skin eruptions are observed.

Angiotensin II stimulates proliferation of primary human keratinocytes via a non-AT1, non-AT2 angiotensin receptor.

Angiotensin II is a hormone with long known cardiovascular actions. Recent studies revealed an additional role for angiotensin II in the regulation of cell proliferation. This study was performed to clarify whether skin is a target organ for these novel angiotensin actions. Radioligand binding studies identified a high-affinity angiotensin receptor on human primary keratinocytes in vitro with a Kd of 4.5 nM and a Bmax of 0.12 nM. Competition experiments with losartan and PD 123177 revealed that this receptor was not of the AT1- nor the AT2-subtype. Stimulation of human keratinocytes with angiotensin II (10(-10) to 10(-5) M) led to a dose dependent increase in 3H-thymidine incorporation, indicating that the keratinocyte receptor mediates a mitogenic effect. This effect is comparable at 10(-9) M to stimulation of keratinocytes by EGF (50ng/ml) and FGF (50ng/ml). These results demonstrate for the first time a possible physiological role for angiotensin II in human skin involving the regulation of keratinocyte proliferation.

The renin-angiotensin system in the brain. Localization and functional significance.

Many neuropeptides have been localized in brain areas involved in cardiovascular regulation. In some cases, regulatory systems which have been originally described in other organs have been found in the brain. A prominent member of this group is the renin-angiotensin system. All of its known components have been localized in the central nervous system and angiotensin II has numerous actions in the central regulation of cardiovascular homeostasis. In addition there are actions which go beyond an active role in the control of blood pressure; these include behavioural actions and the regulation of thirst. The brain renin angiotensin system interacts with other neurotransmitters such as the catecholaminergic system and may act as a neuromodulator.

Angiotensin II receptor subtypes: characterization, signalling mechanisms, and possible physiological implications.

Thanks to the recent discovery of angiotensin II (ANG II) receptor subtypes linked to different signalling pathways, research in the different areas related to this peptide has regained a strong interest. In the following review, we first describe the biochemistry and actions of angiotensin peptides formed both in the circulation and locally at the tissue and organ level. Evidence for the existence and distribution of ANG II receptor subtypes in mammalian as well as in nonmammalian species and lower organisms is presented. The changes in receptor subtype expression during development and disease are described. The signal transduction mechanisms and biological actions of ANG II mediated by the recently cloned AT1 receptor are reviewed and the recent data concerning the signalling pathways linked to the AT2 receptor are discussed. Finally, based upon their molecular pharmacology, we present evidence and also speculate upon the physiological function of the ANG II receptor subtypes.

Mast cells and their mediators in cutaneous wound healing--active participants or innocent bystanders?

Mast cells are traditionally viewed as effector cells of immediate type hypersensitivity reactions. There is, however, a growing body of evidence that the cells might play an important role in the maintenance of tissue homeostasis and repair. We here present our own data and those from the literature elucidating the possible role of mast cells during wound healing. Studies on the fate of mast cells in scars of varying ages suggest that these cells degranulate during wounding, with a marked decrease of chymase-positive cells, although the total number of cells does not decrease, based on SCF-receptor staining. Mast cells contain a plethora of preformed mediators like heparin, histamine, tryptase, chymase, VEGF and TNF-alpha which, on release during the initial stages of wound healing, affect bleeding and subsequent coagulation and acute inflammation. Various additional vasoactive and chemotactic, rapidly generated mediators (C3a, C5a, LTB4, LTC4, PAF) will contribute to these processes, whereas mast cell-derived proinflammatory and growth promoting peptide mediators (VEGF, FGF-2, PDGF, TGF-beta, NGF, IL-4, IL-8) contribute to neoangiogenesis, fibrinogenesis or re-epithelization during the repair process. The increasing number of tryptase-positive mast cells in older scars suggest that these cells continue to be exposed to specific chemotactic, growth- and differentiation-promoting factors throughout the process of tissue remodelling. All these data indicate that mast cells contribute in a major way to wound healing. their role as potential initiators of or as contributors to this process, compared to other cell types, will however have to be further elucidated.