Katrin Wenzel

Hypertension in Response to Autoantibodies to the Angiotensin II Type I Receptor (AT1-AA) in Pregnant Rats: Role of Endothelin-1

Agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) and endothelin -1 (ET-1) are suggested to be important links between placental ischemia and hypertension during preeclampsia. Activation of the angiotensin II type 1 receptor (AT1R) increases endothelial cell production of ET-1; however, the importance of ET-1 in response to AT1-AA–mediated AT1 R activation during preeclampsia is unknown. Furthermore, the role of AT1-AA–mediated increases in blood pressure during pregnancy remains unclear. The objective of this study was to test the hypothesis that AT1-AA, increased to levels observed in preeclamptic women and placental ischemic rats, increases mean arterial pressure (MAP) by activation of the ET-1 system. Chronic infusion of purified rat AT1-AA into normal pregnant (NP) rats for 7 days increased AT1-AA from 0.68±0.5 to 10.88±1.1 chronotropic units (P<0.001). The increased AT1-AA increased MAP from 99±1 to 119±2 mm Hg (P<0.001). The hypertension was associated with significant increases in renal cortices (11-fold) and placental (4-fold) ET-1. To determine whether ET-1 mediates AT1-AA–induced hypertension, pregnant rats infused with AT1-AA and NP rats were treated with an ETA receptor antagonist. MAP was 100±1 mm Hg in AT1-AA+ETA antagonist-treated rats versus 98±2 mm Hg in ETA antagonist-treated rats. Collectively, these data support the hypothesis that one potential pathway whereby AT1-AAs increase blood pressure during pregnancy is by an ET-1–dependent mechanism.

The effect of immune factors, tumor necrosis factor-alpha, and agonistic autoantibodies to the angiotensin II type I receptor on soluble fms-like tyrosine-1 and soluble endoglin production in response to hypertension during pregnancy.

BACKGROUND Preeclampsia is considered a disease of immunological origin associated with abnormalities in inflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha), and activated lymphocytes secreting autoantibodies to the angiotensin II receptor (AT1-AA). Recent studies have also demonstrated that an imbalance of angiogenic factors, soluble fms-like tyrosine kinase (sFlt-1), and sEndoglin, exists in preeclampsia; however, the mechanisms that initiate their overproduction are unclear. METHODS To determine the role of immune regulation of these factors, circulating and placental sFlt-1 and/or sEndoglin was examined from pregnant rats chronically treated with TNF-alpha or AT1-AA. On day 19 of gestation blood pressure was analyzed and serum and tissues were collected. Placental villous explants were excised and cultured on matrigel coated inserts for 24 h and sFlt-1 and sEndoglin was measured from media. RESULTS In response to TNF-alpha-induced hypertension, sFlt-1 increased from 180 +/- 5 to 2,907 +/- 412 pg/ml. sFlt-1 was also increased from cultured placental explants of TNF-alpha induced hypertensive pregnant rats (n = 12) (2,544 +/- 1,132 pg/ml) vs. explants from normal pregnant (NP) rats (n = 12) (2,189 +/- 586 pg/ml) where as sEng was undetectable. Circulating sFlt-1 increased from 245 +/- 38 to 3,920 +/- 798 pg/ml in response to AT1-AA induced hypertension. sFlt-1 levels were higher (3,400 +/- 350 vs. 2,480 +/- 900 pg/ml) in placental explants from AT1-AA infused rats (n = 12) than NP rats (n = 7). In addition, sEndoglin increased from 30 +/- 2.7 to 44 +/- 3.3 pg/ml (P < 0.047) in AT1-AA infused rats but was undetectable in the media of the placental explants. CONCLUSIONS These data suggest that immune factors may serve as an important stimulus for both sFlt-1 and sEndoglin production in response to placental ischemia.

Angiotensin II type 1 receptor antibodies and increased angiotensin II sensitivity in pregnant rats.

Pregnant women who subsequently develop preeclampsia are highly sensitive to infused angiotensin (Ang) II; the sensitivity persists postpartum. Activating autoantibodies against the Ang II type 1 (AT1) receptor are present in preeclampsia. In vitro and in vivo data suggest that they could be involved in the disease process. We generated and purified activating antibodies against the AT1 receptor (AT1-AB) by immunizing rabbits against the AFHYESQ epitope of the second extracellular loop, which is the binding epitope of endogenous activating autoantibodies against AT1 from patients with preeclampsia. We then purified AT1-AB using affinity chromatography with the AFHYESQ peptide. We were able to detect AT1-AB both by ELISA and a functional bioassay. We then passively transferred AT1-AB into pregnant rats, alone or combined with Ang II. AT1-AB activated protein kinase C-&agr; and extracellular-related kinase 1/2. Passive transfer of AT1-AB alone or Ang II (435 ng/kg per minute) infused alone did not induce a preeclampsia-like syndrome in pregnant rats. However, the combination (AT1-AB plus Ang II) induced hypertension, proteinuria, intrauterine growth retardation, and arteriolosclerosis in the uteroplacental unit. We next performed gene-array profiling of the uteroplacental unit and found that hypoxia-inducible factor 1&agr; was upregulated by Ang II plus AT1-AB, which we then confirmed by Western blotting in villous explants. Furthermore, endothelin 1 was upregulated in endothelial cells by Ang II plus AT1-AB. We show that AT1-AB induces Ang II sensitivity. Our mechanistic study supports the existence of an “autoimmune-activating receptor” that could contribute to Ang II sensitivity and possible to preeclampsia.

β1-Adrenoceptor gene variations: a role in idiopathic dilated cardiomyopathy?

Abstract.A substantial body of evidence suggests involvement of the human β1-adrenoceptor (β1-AR) gene in the pathophysiology of dilated cardiomyopathy (DCM), a severe heart disease of significant public health impact. β1-AR-mediated signal transduction is dramatically altered due to downregulation, resulting in an impairment of myocardial response. The important role of genetic factors in idiopathic dilated cardiomyopathy (IDCM) recently recognized, we analyzed this prime candidate gene for genetic variation in carefully selected patients and controls. In this preliminary study, 18 single nucleotide polymorphisms were observed, 17 of which were located in the N-terminal and C-terminal region of the coding exon, resulting in 7 amino acid exchanges: Ser-49–Gly, Ala-59–Ser, Gly-389–Arg, Arg-399–Cys, His-402–Arg, Thr-404–Ala, and Pro-418–Ala. These mutations resulted in 11 different β1-AR genotypes. Importantly, the genotypes carrying the Ser-49–Gly mutation in the N-terminus of the molecule in a heterozygous or homozygous form were observed significantly more frequently in the group of IDCM patients. The present results may provide a clue on the molecular mechanisms involved in IDCM, and add moreover interesting information on nature, distribution, and evolutionary aspects of sequence variation in human adrenergic receptor genes.

Prevalence of Agonistic Autoantibodies Against the Angiotensin II Type 1 Receptor and Soluble fms-Like Tyrosine Kinase 1 in a Gestational Age–Matched Case Study

We showed earlier that activating autoantibodies against the angiotensin II type 1 (AT1) receptor (AT1-AA) circulate in preeclamptic women. They may be involved in the pathogenesis of preeclampsia. Protein alignment suggests that the binding site for AT1-AAs is highly homologous to the capsid protein VP2 of parvovirus B19. We performed a prospective, nested, case-control study of 30 gestational age–matched women with preeclampsia and 30 normotensive pregnant women. We measured AT1-AA, soluble fms-like tyrosine kinase 1 (sFlt-1), and serum immunoglobulin G against parvovirus B19 proteins. AT1-AAs were present in 70% of preeclamptic patients and absent in 80% of controls. Prediction by AT1-AA was improved in late-onset preeclampsia. The discrimination for sFlt-1 was 96%. We did not find an interaction between sFlt-1 and AT1-AA. A human monoclonal immunoglobulin G antibody against parvovirus B19 VP2-protein showed a positive reaction in the AT1-AA bioassay, which could be blocked by an AT1 receptor blocker, as well as by the epitope amino acid sequence. Immunoglobulin G against parvovirus B19 proteins was similarly distributed between preeclamptic patients and controls and had no significant importance. We detected significantly more AT1-AA in women with an immune response corresponding with parvovirus B19 infection corresponding with a distant viral infection associated with virus elimination. We concluded that AT1-AAs were common in patients with preeclampsia in a prospective case-control study, although sFlt-1 was a superior biomarker. AT1-AA may represent a better marker for late disease, whereas sFlt1 is a better marker for early onset disease.

Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is a frequent genetic cardiac disease and the most common cause of sudden cardiac death in young individuals. Most of the currently known HCM disease genes encode sarcomeric proteins. Previous studies have shown an association between CSRP3 missense mutations and either dilated cardiomyopathy (DCM) or HCM, but all these studies were unable to provide comprehensive genetic evidence for a causative role of CSRP3 mutations. We used linkage analysis and identified a CSRP3 missense mutation in a large German family affected by HCM. We confirmed CSRP3 as an HCM disease gene. Furthermore, CSRP3 missense mutations segregating with HCM were identified in four other families. We used a newly designed monoclonal antibody to show that muscle LIM protein (MLP), the protein encoded by CSRP3, is mainly a cytosolic component of cardiomyocytes and not tightly anchored to sarcomeric structures. Our functional data from both in vitro and in vivo analyses suggest that at least one of MLPs mutated forms seems to be destabilized in the heart of HCM patients harbouring a CSRP3 missense mutation. We also present evidence for mild skeletal muscle disease in affected persons. Our results support the view that HCM is not exclusively a sarcomeric disease and also suggest that impaired mechano-sensory stress signalling might be involved in the pathogenesis of HCM.

Potential Relevance of α1-Adrenergic Receptor Autoantibodies in Refractory Hypertension

Background Agonistic autoantibodies directed at the α1-adrenergic receptor (α1-AAB) have been described in patients with hypertension. We implied earlier that α1-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 α1-AAB; after immunoadsorption blood pressure was significantly reduced in these patients. Rabbits were immunized to generate α1-adrenergic receptor antibodies (α1-AB). Patient α1-AAB and rabbit α1-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 α1A-adrenergic receptor were incubated with patient α1-AAB and rabbit α1-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 α1-AAB and rabbit α1-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 α1-AAB and rabbit α1-AB can induce signaling pathways important for hypertension and cardiac remodeling. Our data provide evidence for a potential clinical relevance for α1-AAB in hypertensive patients, and the notion of immunity as a possible cause of hypertension.

Increased Susceptibility to Complement Attack due to Down-Regulation of Decay-Accelerating Factor/CD55 in Dysferlin-Deficient Muscular Dystrophy

Dysferlin is expressed in skeletal and cardiac muscles. However, dysferlin deficiency results in skeletal muscle weakness, but spares the heart. We compared intraindividual mRNA expression profiles of cardiac and skeletal muscle in dysferlin-deficient SJL/J mice and found down-regulation of the complement inhibitor, decay-accelerating factor/CD55, in skeletal muscle only. This finding was confirmed on mRNA and protein levels in two additional dysferlin-deficient mouse strains, A/J mice and Dysf−/− mice, as well as in patients with dysferlin-deficient muscular dystrophy. In vitro, the absence of CD55 led to an increased susceptibility of human myotubes to complement attack. Evidence is provided that decay-accelerating factor/CD55 is regulated via the myostatin-SMAD pathway. In conclusion, a novel mechanism of muscle fiber injury in dysferlin-deficient muscular dystrophy is demonstrated, possibly opening therapeutic avenues in this to date untreatable disorder.

Critical illness myopathy and GLUT4 - significance of insulin and muscle contraction

RATIONALE Critical illness myopathy (CIM) has no known cause and no treatment. Immobilization and impaired glucose metabolism are implicated. OBJECTIVES We assessed signal transduction in skeletal muscle of patients at risk for CIM. We also investigated the effects of evoked muscle contraction. METHODS In a prospective observational and interventional pilot study, we screened 874 mechanically ventilated patients with a sepsis-related organ-failure assessment score greater than or equal to 8 for 3 consecutive days in the first 5 days of intensive care unit stay. Thirty patients at risk for CIM underwent euglycemic-hyperinsulinemic clamp, muscle microdialysis studies, and muscle biopsies. Control subjects were healthy. In five additional patients at risk for CIM, we performed corresponding analyses after 12-day, daily, unilateral electrical muscle stimulation with the contralateral leg as control. MEASUREMENTS AND MAIN RESULTS We performed successive muscle biopsies and assessed systemic insulin sensitivity and signal transduction pathways of glucose utilization at the mRNA and protein level and glucose transporter-4 (GLUT4) localization in skeletal muscle tissue. Skeletal muscle GLUT4 was trapped at perinuclear spaces, most pronounced in patients with CIM, but resided at the sarcolemma in control subjects. Glucose metabolism was not stimulated during euglycemic-hyperinsulinergic clamp. Insulin signal transduction was competent up to p-Akt activation; however, p-adenosine monophosphate-activated protein kinase (p-AMPK) was not detectable in CIM muscle. Electrical muscle stimulation increased p-AMPK, repositioned GLUT4, locally improved glucose metabolism, and prevented type-2 fiber atrophy. CONCLUSIONS Insufficient GLUT4 translocation results in decreased glucose supply in patients with CIM. Failed AMPK activation is involved. Evoked muscle contraction may prevent muscle-specific AMPK failure, restore GLUT4 disposition, and diminish protein breakdown. Clinical trial registered with http://www.controlled-trials.com (registration number ISRCTN77569430).

Hypertension in Response to AT1-AA: Role of Reactive Oxygen Species in Pregnancy-Induced Hypertension

BACKGROUND Agonistic autoantibodies to the angiotensin II type I receptor (AT1-AA) and reactive oxygen species (ROS) are implicated in the pathophysiology of preeclampsia. The objective of this study was to determine the role of AT1-AA to stimulate placental oxidative stress in vivo and role ROS in mediating hypertension in response to AT1-AA during pregnancy. METHODS To achieve these goals, blood pressure (mean arterial pressure (MAP)) and ROS were analyzed in AT1-AA-induced hypertensive pregnant rats in the presence and absence of a superoxide dismutase mimetic, tempol. Rat AT1-AA (1:50) and tempol (30 mg/kg/day) were administered to pregnant rats beginning on day 12 of gestation. On day 19, MAP was analyzed and tissues collected for ROS analysis via lucigenin chemiluminescence. RESULTS MAP increased from 101 ± 2 normal pregnant (NP) rats to 116 ± 2 mm Hg in chronic AT1-AA infused rats (P = 0.002). Placental basal and NADPH oxidase stimulated ROS was 29 ± 6 and 92 ± 10 relative light units (RLUs) in NP rats. These levels increased to 159 ± 29 (P < 0.0001) and 287 ± 60 RLUs (P < 0.006) in AT1-AA infused rats. MAP in AT1-AA + tempol rats was 109 ± 2 mm Hg, no difference than tempol-treated controls (109 ± 3 mm Hg). Administration of tempol decreased basal and NADPH-stimulated placental ROS in AT1-AA-treated rats (121 ± 13; 262 ± 21 RLUs). Basal and NADPH-stimulated ROS in tempol-treated controls were 69 ± 24; 141 ± 33 RLUs. CONCLUSION This study indicates that AT1-AAs contribute to placental oxidative stress; one mechanism whereby the AT1-AA mediates hypertension during pregnancy.