Pathological AT1R-B2R Protein Aggregation and Preeclampsia.

Preeclampsia is one of the most frequent and severe complications of pregnancy. Symptoms of preeclampsia usually occur after 20 weeks of pregnancy and include hypertension and kidney dysfunction with proteinuria. Up to now, delivery of the infant has been the most effective and life-saving treatment to alleviate symptoms of preeclampsia because a causative treatment does not exist, which could prolong a pregnancy complicated with preeclampsia. Preeclampsia is a complex medical condition, which is attributed to a variety of different risk factors and causes. Risk factors account for insufficient placentation and impaired vasculogenesis and finally culminate in this life-threatening condition of pregnancy. Despite progress, many pathomechanisms and causes of preeclampsia are still incompletely understood. In recent years, it was found that excessive protein complex formation between G-protein-coupled receptors is a common sign of preeclampsia. Specifically, the aberrant heteromerization of two vasoactive G-protein-coupled receptors (GPCRs), the angiotensin II AT1 receptor and the bradykinin B2 receptor, is a causative factor of preeclampsia symptoms. Based on this knowledge, inhibition of abnormal GPCR protein complex formation is an experimental treatment approach of preeclampsia. This review summarizes the impact of pathological GPCR protein aggregation on symptoms of preeclampsia and delineates potential new therapeutic targets.

Improvements of symptoms of Alzheimer`s disease by inhibition of the angiotensin system.

With ageing of the global society, the frequency of ageing-related neurodegenerative diseases such as Alzheimer`s disease (AD) is on the rise worldwide. Currently, there is no cure for AD, and the four drugs approved for AD only have very small effects on AD symptoms. Consequently, there are enormous efforts worldwide to identify new targets for treatment of AD. Approaches that interfere with classical neuropathologic features of AD, such as extracellular senile plaques formed of aggregated amyloid-beta (Abeta), and intracellular neurofibrillary tangles of hyperphosphorylated tau have not been successful so far. In search for a treatment approach of AD, we found that inhibition of the angiotensin-converting enzyme (ACE) by a centrally acting ACE inhibitor retards symptoms of neurodegeneration, Abeta plaque formation and tau hyperphosphorylation in experimental models of AD. Our approach is currently being investigated in a clinical setting. Initial evidence with AD patients shows that a brain-penetrating ACE inhibitor counteracts the process of neurodegeneration and dementia. Moreover, centrally acting ACE inhibitors given in addition to the standard therapy, cholinesterase inhibition, can improve cognitive function of AD patients for several months. This is one of the most promising results for AD treatment since more than a decade.

Discovery of Pathologic GPCR Aggregation.

The family of G-protein-coupled receptors (GPCRs) is one of the most important drug targets. Mechanisms underlying GPCR activation and signaling are therefore of great pharmacologic interest. It was long thought that GPCRs exist and function as monomers. This feature was considered to distinguish GPCRs from other membrane receptors such as receptor tyrosine kinases or cytokine receptors, which signal from dimeric receptor complexes. But during the last two decades it was increasingly recognized that GPCRs can undergo aggregation to form dimers and higher order oligomers, resulting in homomeric and/or heteromeric protein complexes with different stoichiometries. Moreover, this protein complex formation could modify GPCR signaling and function. We contributed to this paradigm shift in GPCR pharmacology by the discovery of the first pathologic GPCR aggregation, which is the protein complex formation between the angiotensin II AT1 receptor and the bradykinin B2 receptor. Increased AT1-B2 heteromerization accounts for the angiotensin II hypersensitivity of pregnant women with preeclampsia hypertension. Since the discovery of AT1-B2, other pathologic GPCR aggregates were found, which contribute to atherosclerosis, neurodegeneration and Alzheimer's disease. As a result of our findings, pathologic GPCR aggregation appears as an independent and disease-specific process, which is increasingly considered as a novel target for pharmacologic intervention.

Beta-Arrestin1 Prevents Preeclampsia by Downregulation of Mechanosensitive AT1-B2 Receptor Heteromers.

Preeclampsia is the most frequent pregnancy-related complication worldwide with no cure. While a number of molecular features have emerged, the underlying causal mechanisms behind the disorder remain obscure. Here, we find that increased complex formation between angiotensin II AT1 and bradykinin B2, two G protein-coupled receptors with opposing effects on blood vessel constriction, triggers symptoms of preeclampsia in pregnant mice. Aberrant heteromerization of AT1-B2 led to exaggerated calcium signaling and high vascular smooth muscle mechanosensitivity, which could explain the onset of preeclampsia symptoms at late-stage pregnancy as mechanical forces increase with fetal mass. AT1-B2 receptor aggregation was inhibited by beta-arrestin-mediated downregulation. Importantly, symptoms of preeclampsia were prevented by transgenic ARRB1 expression or a small-molecule drug. Because AT1-B2 heteromerization was found to occur in human placental biopsies from pregnancies complicated by preeclampsia, specifically targeting AT1-B2 heteromerization and its downstream consequences represents a promising therapeutic approach.

Inhibition of ACE Retards Tau Hyperphosphorylation and Signs of Neuronal Degeneration in Aged Rats Subjected to Chronic Mild Stress.

With increasing life expectancy, Alzheimer's disease (AD) and other types of age-associated dementia are on the rise worldwide. Treatment approaches for dementia are insufficient and novel therapies are not readily available. In this context repurposing of established drugs appears attractive. A well-established class of cardiovascular drugs, which targets the angiotensin II system, is such a candidate, which currently undergoes a paradigm shift with regard to the potential benefit for treatment of neurodegenerative symptoms. In search for additional evidence, we subjected aged rats to chronic unpredictable mild stress, which is known to enhance the development of AD-related neuropathological features. We report here that four weeks of chronic mild stress induced a strong upregulation of the hippocampal angiotensin-converting enzyme (Ace) at gene expression and protein level. Concomitantly, tau protein hyperphosphorylation developed. Signs of neurodegeneration were detected by the significant downregulation of neuronal structure proteins such as microtubule-associated protein 2 (Map2) and synuclein-gamma (Sncg). Ace was involved in neurodegenerative symptoms because treatment with the brain-penetrating ACE inhibitor, captopril, retarded tau hyperphosphorylation and signs of neurodegeneration. Moreover, ACE inhibitor treatment could counteract glutamate neurotoxicity by preventing the downregulation of glutamate decarboxylase 2 (Gad2). Taken together, ACE inhibition targets neurodegeneration triggered by environmental stress.

Vasopressor meets vasodepressor: The AT1-B2 receptor heterodimer.

The AT1 receptor for the vasopressor angiotensin II is one of the most important drug targets for the treatment of cardiovascular diseases. Sensitization of the AT1 receptor system is a common feature contributing to the pathogenesis of many cardiovascular disorders but underlying mechanisms are not fully understood. More than a decade ago, evidence was provided for control of AT1R activation by heterodimerization with the B2 receptor for the vasodepressor peptide, bradykinin, a physiological counterpart of the vasoconstrictor angiotensin II. AT1-B2 receptor heterodimerization was shown to enhance AT1R-stimulated signaling under pathophysiological conditions such as experimental and human pregnancy hypertension. Notably, AT1R signal sensitization of patients with preeclampsia hypertension was attributed to AT1R-B2R heterodimerization. Vice versa, transgenic mice lacking the AT1-B2 receptor heterodimer due to targeted deletion of the B2R gene showed a significantly reduced AT1R-stimulated vasopressor response compared to transgenic mice with abundant AT1R-B2R heterodimerization. Biophysical methods such as BRET and FRET confirmed those data by demonstrating efficient AT1-B2 receptor heterodimerization in transfected cells and transgenic mice. Recently, a study on AT1R-specific biased agonism directed the focus to the AT1-B2 receptor heterodimer again. The ß-arrestin-biased [Sar1,Ile4,Ile8]-angiotensin II promoted not only the recruitment of ß-arrestin to the AT1R but also stimulated the down-regulation of the AT1R-associated B2 receptor by co-internalization. Thereby specific targeting of the AT1R-B2R heterodimer became feasible and could open the way to a new class of drugs, which specifically interfere with pathological angiotensin II-AT1 receptor system activation.

ACE inhibition with captopril retards the development of signs of neurodegeneration in an animal model of Alzheimer's disease.

Increased generation of reactive oxygen species (ROS) is a significant pathological feature in the brains of patients with Alzheimer's disease (AD). Experimental evidence indicates that inhibition of brain ROS could be beneficial in slowing the neurodegenerative process triggered by amyloid-beta (Abeta) aggregates. The angiotensin II AT1 receptor is a significant source of brain ROS, and AD patients have an increased brain angiotensin-converting enzyme (ACE) level, which could account for an excessive angiotensin-dependent AT1-induced ROS generation. Therefore, we analyzed the impact of ACE inhibition on signs of neurodegeneration of aged Tg2576 mice as a transgenic animal model of AD. Whole genome microarray gene expression profiling and biochemical analyses demonstrated that the centrally active ACE inhibitor captopril normalized the excessive hippocampal ACE activity of AD mice. Concomitantly, the development of signs of neurodegeneration was retarded by six months of captopril treatment. The neuroprotective profile triggered by captopril was accompanied by reduced amyloidogenic processing of the amyloid precursor protein (APP), and decreased hippocampal ROS, which is known to enhance Abeta generation by increased activation of beta- and gamma-secretases. Taken together, our data present strong evidence that ACE inhibition with a widely used cardiovascular drug could interfere with Abeta-dependent neurodegeneration.

Up-regulation of the cardiac lipid metabolism at the onset of heart failure.

Chronic pressure overload and atherosclerosis are primary etiologic factors for cardiac hypertrophy and failure. However, mechanisms underlying the transition from hypertrophy to heart failure are incompletely understood. We analyzed the development of heart failure in mice with chronic pressure overload induced by aortic constriction and compared the results with aged apolipoprotein E-deficient mice suffering from advanced atherosclerosis. We combined cardiac function analysis by echocardiography and invasive hemodynamics with a comprehensive microarray gene expression study (GSE25765-8). The microarray data showed that the onset of heart failure induced by pressure overload or advanced atherosclerosis was accompanied by a strong up-regulation of key lipid metabolizing enzymes involved in fat synthesis, storage and oxidation. Cardiac lipid overload may be involved in the progression of heart failure by enhancing cardiomyocyte death. Up-regulation of the cardiac lipid metabolism was related to oxygen and ATP depletion of failing hearts because anti-ischemic treatment with ranolazine normalized the cardiac lipid metabolism and improved cardiac function. Vice versa, inhibition of cellular respiration and ATP generation by mild thiol-blocking with cystamine triggered the cardiac lipid metabolism and caused signs of heart failure. Cardiac tissue specimens of patients with heart failure also showed high protein levels of key fat metabolizing enzymes as well as lipid accumulation. Taken together, our data strongly indicate that up-regulation of the cardiac lipid metabolism and myocardial lipid overload are underlying the development of heart failure.

A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor.

Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1-EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor's amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer.

Dominant negative AT2 receptor oligomers induce G-protein arrest and symptoms of neurodegeneration.

Neurodegeneration in Alzheimer's disease (AD) correlates with dysfunction of signaling mediated by Galphaq/11. Nondissociable angiotensin II AT2 receptor oligomers are linked to the impaired Galphaq/11-stimulated signaling of AD patients and transgenic mice with AD-like symptoms. To further analyze the role of AT2 receptor oligomers, we induced the formation of AT2 oligomers in an in vitro cell system. Similarly as in vivo, sequential oxidative and transglutaminase-dependent cross-linking steps triggered the formation of AT2 oligomers in vitro. Elevated reactive oxygen species mediated oxidative cross-linking of AT2 monomers to dimers involving tyrosine residues located at putative interreceptor contact sites of the cytoplasmic loop connecting transmembrane helices III/IV. Cross-linked AT2 dimers were subsequently a substrate of activated transglutaminase-2, which targeted the carboxyl terminus of AT2 dimers, as assessed by truncated and chimeric AT2 receptors, respectively. AT2 oligomers acted as dominant negative receptors in vitro by mediating Galphaq/11 protein sequestration and Galphaq/11 protein arrest. The formation of AT2 oligomers and G-protein dysfunction could be suppressed in vitro and in vivo by an AT2 receptor mutant. Inhibition of AT2 oligomerization upon stereotactic expression of the AT2 receptor mutant revealed that Galphaq/11-sequestering AT2 oligomers enhanced the development of neurodegenerative symptoms in the hippocampus of transgenic mice with AD-like pathology. Thus, AT2 oligomers inducing Galphaq/11 arrest are causally involved in inducing symptoms of neurodegeneration.

Angiotensin II AT2 receptor oligomers mediate G-protein dysfunction in an animal model of Alzheimer disease.

Progressive neurodegeneration and decline of cognitive functions are major hallmarks of Alzheimer disease (AD). Neurodegeneration in AD correlates with dysfunction of diverse signal transduction mechanisms, such as the G-protein-stimulated phosphoinositide hydrolysis mediated by Galphaq/11. We report here that impaired Galphaq/11-stimulated signaling in brains of AD patients and mice correlated with the appearance of cross-linked oligomeric angiotensin II AT2 receptors sequestering Galphaq/11. Amyloid beta (Abeta) was causal to AT2 oligomerization, because cerebral microinjection of Abeta triggered AT2 oligomerization in the hippocampus of mice in a dose-dependent manner. Abeta induced AT2 oligomerization by a two-step process of oxidative and transglutaminase-dependent cross-linking. The induction of AT2 oligomers in a transgenic mouse model with AD-like symptoms was associated with Galphaq/11 dysfunction and enhanced neurodegeneration. Vice versa, stereotactic inhibition of AT2 oligomers by RNA interference prevented the impairment of Galphaq/11 and delayed Tau phosphorylation. Thus, Abeta induces the formation of cross-linked AT2 oligomers that contribute to the dysfunction of Galphaq/11 in an animal model of Alzheimer disease.

Mesangial AT1/B2 receptor heterodimers contribute to angiotensin II hyperresponsiveness in experimental hypertension.

Angiotensin II plays a central role in the pathogenesis of hypertension and of related cardiovascular disorders by binding to and activating angiotensin II receptors (AT1 receptors). Sensitization to the vasopressor response of angiotensin II is a key feature in many cardiovascular disorders. However, underlying mechanisms responsible for angiotensin II hypersensitivity are barely understood. Because angiotensin II responsiveness of AT1 receptors can be specifically modified by AT1/B2 receptor dimerization, we determined the AT1 receptor dimerization status in an experimental model of hypertension. AT1/B2 receptor heterodimers were abundant on renal mesangial cells isolated from spontaneously hypertensive rats compared with that on cells from normotensive controls. Heterodimerization of AT1 with B2 receptors was correlated with high levels of B2 receptor protein on kidneys and on mesangial cells of hypertensive rats, as determined in immunoblot with receptor-specific antibodies. Specific inhibition of AT1/B2 receptor heterodimers revealed that these receptor heterodimers mediated an enhanced angiotensin II-stimulated Galphaq/11 activation and an increased endothelin-1 secretion of mesangial cells from hypertensive rats. Thus, AT1/B2 receptor heterodimerization contributes to angiotensin II hyperresponsiveness of mesangial cells in experimental hypertension.

Factor XIIIA transglutaminase crosslinks AT1 receptor dimers of monocytes at the onset of atherosclerosis.

Many G protein-coupled receptors form dimers in cells. However, underlying mechanisms are barely understood. We report here that intracellular factor XIIIA transglutaminase crosslinks agonist-induced AT1 receptor homodimers via glutamine315 in the carboxyl-terminal tail of the AT1 receptor. The crosslinked dimers displayed enhanced signaling and desensitization in vitro and in vivo. Inhibition of angiotensin II release or of factor XIIIA activity prevented formation of crosslinked AT1 receptor dimers. In agreement with this finding, factor XIIIA-deficient individuals lacked crosslinked AT1 dimers. Elevated levels of crosslinked AT1 dimers were present on monocytes of patients with the common atherogenic risk factor hypertension and correlated with an enhanced angiotensin II-dependent monocyte adhesion to endothelial cells. Elevated levels of crosslinked AT1 receptor dimers on monocytes could sustain the process of atherogenesis, because inhibition of angiotensin II generation or of intracellular factor XIIIA activity suppressed the appearance of crosslinked AT1 receptors and symptoms of atherosclerosis in ApoE-deficient mice.

AT1 receptor heterodimers and angiotensin II responsiveness in preeclampsia.

Preeclampsia is a pregnancy-specific hypertensive disorder with unknown etiology, which affects 5% to 10% of all pregnancies. Increased sensitivity to the vasoconstrictor angiotensin II is a common feature of preeclampsia, although underlying mechanisms are barely understood. Recent data reveal a potential mechanism for the increased angiotensin II responsiveness in preeclampsia: increased levels of heterodimers between the vasopressor receptor AT1 and the vasodepressor receptor B2. The receptor heterodimers display increased sensitivity toward angiotensin II and are found in platelets and in omental vessels of preeclamptic women. Moreover, AT1/B2 receptor heterodimers are resistant to inactivation by reactive oxygen species, which is elevated in normal and preeclamptic pregnancies. Thus, a major symptom of preeclampsia is the result of complex formation between two G-protein-coupled receptors.