Dementia has been shown to be associated with agonistic autoantibodies. The deleterious action of autoantibodies on the α1-adrenergic receptor for brain vasculature has been demonstrated in animal studies. In the current study, 169 patients with dementia were screened for the presence of agonistic autoantibodies. 47% of patients suffering from mild to moderate Alzheimer's disease and/or vascular dementia carried these autoantibodies. Eight patients positive for autoantibodies underwent immunoadsorption. Patients treated on four consecutive days were subsequently negative for autoantibodies and displayed stabilization of cognitive and mental condition during 12-18 months' follow-up. In patients treated for 2-3 days, autoantibodies were reduced by only 78%. They suffered a rebound of autoantibodies during follow-up, benefited from immunoadsorption too, but their mental parameters worsened. We provide first data on the clinical relevance of agonistic autoantibodies in dementia and show that immunoadsorption is safe and efficient in removing autoantibodies with overall benefits for patients.

Cerebrovascular abnormality is frequently accompanied by cognitive dysfunctions, such as dementia. Antibodies against the α1 -adrenoceptor (α1 -AR) can be found in patients with Alzheimer's disease with cerebrovascular disease, and have been shown to affect the larger vessels of the brain in rodents. However, the impact of α1 -AR antibodies on the cerebral vasculature remains unclear. In the present study, we established a neuroimaging method to measure the relative cerebral blood volume (rCBV) in small rodents with the ultimate goal to detect changes in blood vessel density and/or vessel size induced by α1 -AR antibodies. For this purpose, mapping of R2 * and R2 was performed using MRI at 9.4 T, before and after the injection of intravascular iron oxide particles (ferumoxytol). The change in the transverse relaxation rates (ΔR2 *, ΔR2 ) showed a significant rCBV decrease in the cerebrum, cortex and hippocampus of rats (except hippocampal ΔR2 ), which was more pronounced for ΔR2 * than for ΔR2 . Immunohistological analyses confirmed that the α1 -AR antibody induced blood vessel deficiencies. Our findings support the hypothesis that α1 -AR antibodies lead to cerebral vessel damage throughout the brain, which can be monitored by MRI-derived rCBV, a non-invasive neuroimaging method. This demonstrates the value of rCBV estimation by ferumoxytol-enhanced MRI at 9.4 T, and further underlines the significance of this antibody in brain diseases involving vasculature impairments, such as dementia.

BackgroundCirculating agonistic autoantibodies acting at G protein-coupled receptors have been associated with numerous sever pathologies in humans. Antibodies directed predominantly against the α(1)-adrenergig receptor were detected in patients suffering from widespread diseases such as hypertension and type 2 diabetes. Their deleterious action has been demonstrated for peripheral organs. We postulate that antibodies to the α(1)-adrenergig receptor are relevant pathomolecules in diseases of the central nervous system associated with vascular impairments.Methodology/principal findingsUsing a rat model we studied the long-term action of antibodies against the α(1)-adrenergig receptor either induced by immunization with a receptor peptide or applied by intravenous injection. The vasculature in the rat brains was investigated by time-of-flight magnetic resonance angiography using a 9.4 Tesla small animal MR imaging system. Visual examination of maximum-intensity-projections (MIPs) of brain angiographs revealed the development of vascular defects in antibody- exposed animals between three and eight months of treatment. Relative vascular areas were derived from representative MIP image sections by grayscale analysis and used to form an index of vascular circulation. Animals exposed to the action of α(1)-adrenergig receptor antibodies showed significantly reduced vascular areas (p<0.05). Calculated index values indicated attenuated blood flow in both antibody-treated cohorts compared to their respective controls reaching with (relative units ± standard error, n = 10) 0.839 ± 0.026 versus 0.919 ± 0.026 statistical significance (p<0.05) for peptide-immunized rats.Conclusion/significanceWe present evidence that antibodies to the α(1)-adrenergig receptor cause cerebrovascular impairments in the rat. Our findings suggest the pathological significance of these antibodies in pathologies of the human central nervous system linked to impairments of brain vasculature such as stroke and dementia.

AgAAB against the AR ±1 and ß2 were detected in ca.50% of patients with AD/VD (1). The agAAB has agonistic properties and induces a pathological receptor activation (2) which leads to vascular remodelling (3). We hypothesize that agAAB against AR play a pathogenic role in AD/VD. We investigated the feasibility of therapeutical apheresis (immunoglobulin G adsorption) for patients with mild AD/VD. IA was performed in 6 patients according to (4). We examined 6 patients (2 female, 4 male) with mild AD (MMSE 20-26), median age 68 years (-9/+7) with agAAB against AR ±1 and/or ß2 before and after immunoadsorption of immunoglobulin G (IA, 3-4 consecutive days). Patients have been under stable doses of antidementiva agents for 3-6 months before IA.Cognitive impairment and activities of daily living were diagnosed by ADAS-cog, ADAS-noncog, MMSE, SKT and CGI. MRI (before IA and after 6 months) was performed. IA reduces IgG>80% from blood and ag AAK against AR below the detection level without serious adverse events. 8 (-2/+2) months after IA, and under stable doses of antidementiva agents no decline in cognitive functions and activities of daily living could be observed. No infections or hospitalizations were reported. An update of the results will be presented. Our data support the concept to treat dementia associated with agAAB against AR by therapeutical apheresis. Cerebrovascular alterations mediated by these agAAB may act as pathogens and can be effectively removed by IA. For estimating the sustainability of IA and clinical outcome larger clinical trials are recommended.

Although primary causes of Alzheimer's and vascular dementia are unknown, the importance of preceding vascular lesions is widely accepted. Furthermore, there is strong evidence for the involvement of autoimmune mechanisms. Here, we report the presence of agonistic autoantibodies directed at adrenergic receptors in the circulation of patients with mild to moderate Alzheimer's and vascular dementia. In 59% of these patients, agonistic autoantibodies against the α(1) -adrenergic receptor and the β(2) -adrenergic receptor were identified. The majority of positive patients (66%) contained both types of autoantibodies in combination. In a control group of patients with neurological impairments others than Alzheimer's and vascular dementia, only 17% were found to harbour these autoantibodies. The autoantibodies to the α(1) -adrenergic receptor interacted preferably with the extracellular loop1 of the receptor. They were further studied in IgG preparations from the column regenerate of a patient who underwent immunoadsorption. The α(1) -adrenergic receptor autoantibodies specifically bound to the extracellular loop1 peptide of the receptor with an apparent EC(50) value of 30 nm. They mobilized intracellular calcium in a clonal cell line expressing the human form of the α(1) -adrenergic receptor. Our data support the notion that autoimmune mechanisms play a significant role in the pathogenesis of Alzheimer's and vascular dementia. We suggest that agonistic autoantibodies to the α(1) -adrenergic and the β(2) -adrenergic receptor may contribute to vascular lesions and increased plaque formation.

Hypertension is a major cause for hypertrophic remodelling of the myocardium. Agonistic autoantibodies to extracellular loops of the α1-adrenergic receptor (α1-AR) have been identified in patients with arterial hypertension. However, intracellular reactions elicited by these agonistic antibodies remain elusive. An anti-peptide antibody (anti-α1) was generated against the second extracellular loop of the α1-AR that bound to its peptide epitope with high affinity (K D ~50 nM). We studied anti-α1 effects on intracellular calcium (Cai), a key factor in cellular remodelling, and receptor-mediated cardiac protein phosphorylation. Anti-α1 induced pronounced but transient increases in Cai in CHO cells expressing the human α1-AR (CHO-α1) and in neonatal cardiomyocytes. Preincubation experiments failed to demonstrate a tonic effect of anti-α1 on Cai. However, preincubation with the antibody attenuated the effect of the α1-AR antagonist prazosin. In neonatal cardiomyocytes anti-α1 induced a robust phosphorylation of a 15-kDa protein that is involved in α1-AR signalling. Our data support the notion that elevation of Cai is a general feature of agonistic antibodies’ action and constitute an important pathogenic component of hypertension-associated autoantibodies. Furthermore, we suggest that agonistic antibodies to the α1-AR contribute to hypertrophic remodelling of cardiac myocytes, and that the cardiac 15-kDa protein is a relevant downstream target of their action.

Background: Agonistic autoantibodies directed at the alpha(1)-adrenergic receptor (alpha(1)-AAB) have been described in patients with hypertension. We implied earlier that alpha(1)-AAB might have a mechanistic role and could represent a therapeutic target.Methodology/Principal Findings: To pursue the issue, we performed clinical and basic studies. We observed that 41 of 81 patients with refractory hypertension had alpha(1)-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate alpha(1)-adrenergic receptor antibodies (alpha(1)-AB). Patient alpha(1)-AAB and rabbit alpha(1)-AB were purified using affinity chromatography and characterized both by epitope mapping and surface plasmon resonance measurements. Neonatal rat cardiomyocytes, rat vascular smooth muscle cells (VSMC), and Chinese hamster ovary cells transfected with the human alpha(1A)-adrenergic receptor were incubated with patient alpha(1)-AAB and rabbit alpha(1)-AB and the activation of signal transduction pathways was investigated by Western blot, confocal laser scanning microscopy, and gene expression. We found that phospholipase A2 group IIA (PLA2-IIA) and L-type calcium channel (Cacna1c) genes were upregulated in cardiomyocytes and VSMC after stimulation with both purified antibodies. We showed that patient alpha(1)-AAB and rabbit alpha(1)-AB result in protein kinase C alpha activation and transient extracellular-related kinase (EKR1/2) phosphorylation. Finally, we showed that the antibodies exert acute effects on intracellular Ca2+ in cardiomyocytes and induce mesentery artery segment contraction.Conclusions/Significance: Patient alpha(1)-AAB and rabbit alpha(1)-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for alpha(1)-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.

In contrast to immortal cell lines, primary cells are hardly susceptible to intracellular delivery methods such as transfection. In this study, we evaluated the direct delivery of several cell-permeable peptides under noninvasive conditions into living primary adult rat cardiomyocytes. We specifically monitored the functional effects of a cell-permeable peptide containing the 15 amino acid N-terminal peptide from human ventricular light chain-1 (VLC-1) on contraction and intracellular Ca2+ signals after electrical stimulation in primary adult cardiomyocytes. The transducible VLC-1 variant was taken up by cardiomyocytes within 5 min with more than 95% efficiency and localized to sarcomeric structures. Analysis of the functional effects of the cell-permeable VLC-1 revealed an enhancement of the intrinsic contractility of cardiomyocytes without affecting the intracellular Ca2+. Therefore, peptide transduction mediated by cell-penetrating peptides represents not only a unique strategy to enhance heart muscle function with no secondary effect on intracellular Ca2+ but also an invaluable tool for the modulation and manipulation of protein interactions in general and in primary cells.

The causal relationship between obesity and heart failure is broadly acknowledged; however, the pathophysiological mechanisms involved remain unclear. In this study, we investigated whether human adipocytes secrete cardioactive substances that may affect cardiomyocyte contractility. We cultivated adipocytes obtained from human white adipose tissue, and incubated isolated rat adult cardiomyocytes with adipocyte-conditioned or control medium. This is the first report to demonstrate that human adipocytes exhibit cardiodepressant activity with a direct and acute effect on cardiomyocyte contraction. This adipocyte-derived negative inotropic activity directly depresses shortening amplitude as well as intracellular systolic peak Ca2+ in cardiomyocytes within a few minutes. The adipocyte-derived cardiodepressant activity was dose-dependent, and was completely blunted by heating. Filtration of adipocyte-conditioned medium based on molecular mass characterized the cardiodepressant activity at between 10 and 30 kDa. In summary, adipose tissue exerts highly potent activity with acute depressant effect directly on cardiomyocytes, which may well contribute to increased heart failure risk in overweight patients.

In this study we investigated whether the expression of N-terminal myosin light chain-1 (MLC-1) peptides could improve the intrinsic contractility of the whole heart. We generated transgenic rats (TGR) that overexpressed minigenes encoding the N-terminal 15 amino acids of human atrial MLC-1 (TGR/hALC-1/1-15, lines 7475 and 3966) or human ventricular MLC-1 (TGR/hVLC-1/1-15, lines 6113 and 6114) isoforms in cardiomyocytes. Synthetic N-terminal peptides revealed specific actin binding, with a significantly (P<0.01) lower dissociation constant (K(D)) for the hVLC-1/1-15-actin complex compared with the K(D) value of the hALC-1/1-15-actin complex. Using synthetic hVLC-1/1-15 as a TAT fusion peptide labeled with the fluorochrome TAMRA, we observed specific accumulation of the N-terminal MLC-1 peptide at the sarcomere predominantly within the actin-containing I-band, but also within the actin-myosin overlap zone (A-band) in intact adult cardiomyocytes. For the first time we show that the expression of N-terminal human MLC-1 peptides in TGR (range: 3-6 muM) correlated positively with significant (P<0.001) improvements of the intrinsic contractile state of the isolated perfused heart (Langendorff mode): systolic force generation, as well as the rates of both force generation and relaxation, rose in TGR lines that expressed the transgenic human MLC-1 peptide, but not in a TGR line with undetectable transgene expression levels. The positive inotropic effect of MLC-1 peptides occurred in the absence of a hypertrophic response. Thus, expression of N-terminal domains of MLC-1 represent a valuable tool for the treatment of the failing heart.

The causal relationship between obesity and heart failure is broadly acknowledged; however, the pathophysiological mechanisms involved remain unclear. In this study we investigated whether human adipocytes secrete cardioactive substances that may affect cardiomyocyte contractility. We cultivated adipocytes obtained from human white adipose tissue and incubated isolated rat adult cardiomyocytes with adipocyte-conditioned or control medium. This is the first report to demonstrate that human adipocytes exhibit cardiodepressant activity with a direct and acute effect on cardiomyocyte contraction. This adipocyte-derived negative inotropic activity directly depresses shortening amplitude as well as intracellular systolic peak Ca2+ in cardiomyocytes within a few minutes. The adipocyte-derived cardiodepressant activity was dose-dependent and was completely blunted by heating or by trypsin digestion. Filtration of adipocyte-conditioned medium based on molecular mass characterized the cardiodepressant activity at between 10 and 30 kDa. In summary, adipose tissue exerts highly potent activity with an acute depressant effect directly on cardiomyocytes, which may well contribute to increased heart failure risk in overweight patients.

We investigated expression regulation of the human atrial myosin light chain 1 (hALC-1) gene using a cardiomyocyte H9c2 cell line stably transfected with a construct consisting of the human ALC-1 promoter cloned in front of the luciferase gene (H9c2T1). H9c2T1 cells were stimulated with vasopressin, which is known to induce cardiomyocyte hypertrophy and to activate a panel of signaling pathways. Those pathways involved in hALC-1 promoter activity regulation were dissected by using pharmacological inhibitor substances. Stimulation with vasopressin was associated with nuclear NFAT translocation and significantly increased human ALC-1 promoter activity. Inhibition of calcineurin by cyclosporin A blocked the effects of vasopressin on ALC-1 promoter activity to approximately 50%. This suggests that the Ca2+-calmodulin-calcineurin-NFAT pathway is involved in human ALC-1 promoter activation. However, inhibition of multifunctional Ca2+-calmodulin-dependent protein kinases (CaMK) by KN-93 decreased human ALC-1 promoter activity to almost basal levels. CaMK regulation of ALC-1 promoter activity effect could well be mediated by CaMKIV, which accumulated in the nucleus upon vasopressin stimulation. Inhibition of protein kinase C (PKC) isoforms by bisindolylmaleimide had no significant influence on human ALC-1 promoter activity. Thus, our results demonstrate a dominant role of Ca2+-calmodulin-dependent signaling pathways in the regulation of human ALC-1 expression.

We investigated expression regulation of the human atrial myosin light chain 1 (hALC-1) gene using a cardiomyocyte H9c2 cell line stably transfected with a construct consisting of the human ALC-1 promoter cloned in front of the luciferase gene (H9c2T1). H9c2T1 cells were stimulated with vasopressin, which is known to induce cardiomyocyte hypertrophy and to activate a panel of signaling pathways. Those pathways involved in hALC-1 promoter activity regulation were dissected by using pharmacological inhibitor sub-stances. Stimulation with vasopressin was associated with nuclear NFAT translocation and significantly increased human ALC-1 promoter activity. Inhibition of calcineurin by cyclosporin A blocked the effects of vasopressin on ALC-1 promoter activity to (cid:1) 50%. This suggests that the Ca 2 (cid:1) -calmodulin-calcineurin-NFAT pathway is involved in human ALC-1 promoter activation. However, inhibition of multifunctional Ca 2 (cid:1) -cal-modulin-dependent protein kinases (CaMK) by KN-93 decreased human ALC-1 promoter activity to almost basal levels. CaMK regulation of ALC-1 promoter activity effect could well be mediated by CaMKIV, which accumulated in the 9 to 12 measurements/treatment). Significance analysis was performed using the unpaired Stu- dent’s t test or ANOVA with Bonferroni as post test. Promoter sequence analysis to detect consensus binding sites was per- formed using Genomatix Matinspector.