Brett M. Mitchell

Mice with the R176Q cardiac ryanodine receptor mutation exhibit catecholamine-induced ventricular tachycardia and cardiomyopathy

Mutations in the cardiac ryanodine receptor 2 (RyR2) have been associated with catecholaminergic polymorphic ventricular tachycardia and a form of arrhythmogenic right ventricular dysplasia. To study the relationship between RyR2 function and these phenotypes, we developed knockin mice with the human disease-associated RyR2 mutation R176Q. Histologic analysis of hearts from RyR2R176Q/+ mice revealed no evidence of fibrofatty infiltration or structural abnormalities characteristic of arrhythmogenic right ventricular dysplasia, but right ventricular end-diastolic volume was decreased in RyR2R176Q/+ mice compared with controls, indicating subtle functional impairment due to the presence of a single mutant allele. Ventricular tachycardia (VT) was observed after caffeine and epinephrine injection in RyR2R176Q/+, but not in WT, mice. Intracardiac electrophysiology studies with programmed stimulation also elicited VT in RyR2R176Q/+ mice. Isoproterenol administration during programmed stimulation increased both the number and duration of VT episodes in RyR2R176Q/+ mice, but not in controls. Isolated cardiomyocytes from RyR2R176Q/+ mice exhibited a higher incidence of spontaneous Ca2+ oscillations in the absence and presence of isoproterenol compared with controls. Our results suggest that the R176Q mutation in RyR2 predisposes the heart to catecholamine-induced oscillatory calcium-release events that trigger a calcium-dependent ventricular arrhythmia.

Interleukin-17 causes Rho-kinase-mediated endothelial dysfunction and hypertension

AIMS Elevated levels of pro-inflammatory cytokine interleukin-17A (IL-17) are associated with hypertensive autoimmune diseases; however, the connection between IL-17 and hypertension is unknown. We hypothesized that IL-17 increases blood pressure by decreasing endothelial nitric oxide production. METHODS AND RESULTS Acute treatment of endothelial cells with IL-17 caused a significant increase in phosphorylation of the inhibitory endothelial nitric oxide (NO) synthase residue threonine 495 (eNOS Thr495). Of the kinases known to phosphorylate eNOS Thr495, only inhibition of Rho-kinase prevented the IL-17-induced increase. IL-17 caused a threefold increase in the Rho-kinase activator RhoA, and this was prevented by an IL-17 neutralizing antibody. In isolated mouse aortas, IL-17 significantly increased eNOS Thr495 phosphorylation, induced RhoA expression, and decreased NO-dependent relaxation responses, all of which were prevented by either an IL-17 neutralizing antibody or inhibition of Rho-kinase. In mice, IL-17 treatment for 1 week significantly increased systolic blood pressure and this was associated with decreased aortic NO-dependent relaxation responses, increased eNOS Thr495 phosphorylation, and increased RhoA expression. Inhibition of Rho-kinase prevented the hypertension caused by IL-17. CONCLUSION These data demonstrate that IL-17 activates RhoA/Rho-kinase leading to endothelial dysfunction and hypertension. Inhibitors of IL-17 or Rho-kinase may prove useful as anti-hypertensive drugs in IL-17-associated autoimmune diseases.

Regulation of the Anti-Inflammatory Cytokines Interleukin-4 and Interleukin-10 during Pregnancy.

Inflammation mediated by both innate and adaptive immune cells is necessary for several important processes during pregnancy. Pro-inflammatory immune cell activation plays a critical role in embryo implantation, placentation, and parturition; however dysregulation of these cells can lead to detrimental pregnancy outcomes including spontaneous abortion, fetal growth restriction, maternal pathology including hypertensive disorders, or fetal and maternal death. The resolution of inflammation plays an important role throughout pregnancy and is largely mediated by immune cells that produce interleukin (IL)-4 and IL-10. The temporal and spatial aspects of reducing inflammation during pregnancy represent a complex process that if not functioning optimally can lead to persistent inflammation and pregnancy complications. In this review, we examine how immune cells that produce IL-4 and IL-10 are regulated throughout pregnancy as well as the effects that reduced IL-4 and IL-10 signaling has on fetal and maternal physiology.

Interleukin-10 reduces inflammation, endothelial dysfunction, and blood pressure in hypertensive pregnant rats

Hypertensive disorders of pregnancy are characterized by systemic and placental inflammation; however, treatment for these conditions has remained elusive. We tested whether administration of the anti-inflammatory cytokine interleukin-10 (IL-10) during pregnancy would attenuate the hypertension, endothelial dysfunction, proteinuria, and inflammation seen in pregnant DOCA/saline-treated (PDS) rats. Normal pregnant (NP) rats and PDS were given daily intraperitoneal injections of recombinant IL-10 from gestational day 13 until death on day 20. Systolic blood pressure, aortic endothelium-dependent relaxation responses, and urinary protein excretion were measured on days 13 and 20 of gestation. Fetal number and development, plasma endothelin-1 levels, serum and placental levels of IFNgamma and IL-10, and aortic and placental levels of platelet endothelial cell adhesion molecule (PECAM) were assessed on gestational day 20. Systolic blood pressure, aortic endothelial dysfunction, and urinary protein excretion were significantly increased at gestational day 13 in PDS rats. However, all of these were restored to NP levels following IL-10 treatment in PDS rats. IL-10 treatment also significantly increased the number of pups per litter in PDS rats and did not further affect fetal development. The beneficial effects of IL-10 in PDS rats were likely mediated by the decreased plasma levels of endothelin-1, decreased levels of circulating and placental IFNgamma, as well as decreased aortic and placental expression of PECAM. These data demonstrate that exogenous IL-10 can normalize blood pressure and endothelial function in pregnancy-induced hypertensive rats and may be beneficial in women with hypertensive disorders of pregnancy.

Placental Toll-Like Receptor 3 and Toll-Like Receptor 7/8 Activation Contributes to Preeclampsia in Humans and Mice

Preeclampsia (PE) is a pregnancy-specific hypertensive syndrome characterized by excessive maternal immune system activation, inflammation, and endothelial dysfunction. Toll-like receptor (TLR) 3 activation by double-stranded RNA (dsRNA) and TLR7/8 activation by single-stranded RNA (ssRNA) expressed by viruses and/or released from necrotic cells initiates a pro-inflammatory immune response; however it is unknown whether viral/endogenous RNA is a key initiating signal that contributes to the development of PE. We hypothesized that TLR3/7/8 activation will be evident in placentas of women with PE, and sufficient to induce PE-like symptoms in mice. Placental immunoreactivity and mRNA levels of TLR3, TLR7, and TLR8 were increased significantly in women with PE compared to normotensive women. Treatment of human trophoblasts with the TLR3 agonist polyinosine-polycytidylic acid (poly I:C), the TLR7-specific agonist imiquimod (R-837), or the TLR7/8 agonist CLO97 significantly increased TLR3/7/8 levels. Treatment of mice with poly I:C, R-837, or CLO97 caused pregnancy-dependent hypertension, endothelial dysfunction, splenomegaly, and placental inflammation. These data demonstrate that RNA-mediated activation of TLR3 and TLR7/8 plays a key role in the development of PE.

Toll-Like Receptor 3 Activation During Pregnancy Elicits Preeclampsia-Like Symptoms in Rats

BACKGROUND Preeclampsia (PE), a pregnancy-specific hypertensive syndrome, is one of the leading causes of premature births as well as fetal and maternal death. There is strong evidence that maternal immune system activation, of which Toll-like receptors (TLRs) play a major role, contributes to the development of PE. Viral infections, sensed by TLR3, are associated with hypertensive disorders of pregnancy. We tested the hypothesis that TLR3 activation during pregnancy would cause hypertension, endothelial dysfunction, proteinuria, and intrauterine growth restriction in normal pregnant rats. METHODS We treated pregnant and nonpregnant rats with the viral mimetic polyinosinic:polycytidylic acid (poly I:C) or vehicle every other day beginning at day 10 of gestation and measured systolic blood pressure, aortic vasodilation, urinary protein concentration, fetal growth, and serum and placental cytokine levels. RESULTS Pregnant rats treated with poly I:C displayed significantly elevated systolic blood pressures compared to pregnant rats and nonpregnant rats treated with poly I:C on day 18 of gestation. Poly I:C-treated pregnant rats also exhibited significantly decreased aortic vasodilation, significantly increased urinary protein concentrations, and had more malformed pups/litter. Additionally, poly I:C-treated rats exhibited a significant increase in placental TLR3 expression and proinflammatory/anti-inflammatory cytokine ratio compared to vehicle-treated rats. Poly I:C treatment of nonpregnant control rats had no effect on systolic blood pressure, aortic vasodilation, or urinary protein concentrations. CONCLUSION These findings demonstrate that sustained maternal immune system activation via TLR3 during pregnancy causes PE-like symptoms in rats and suggest that viral infection during pregnancy may contribute to the development of PE.

Interleukin 10 Deficiency Exacerbates Toll-Like Receptor 3–Induced Preeclampsia-Like Symptoms in Mice

Preeclampsia may result from overactivation of the maternal immune system and is characterized by endothelial dysfunction and excessive inflammation. Given the importance of maternal immune system regulation and anti-inflammatory cytokines in normotensive pregnancies, we hypothesized that maternal immune system activation via Toll-like receptor 3 during pregnancy would cause preeclampsia-like symptoms in mice, which would be made worse by deficiency of the anti-inflammatory cytokine interleukin 10. The Toll-like receptor 3 agonist polyinosine-polycytidylic acid (poly I:C) caused hypertension, endothelial dysfunction, and proteinuria in mice only when pregnant. In the absence of poly I:C, pregnant interleukin 10 knockout mice exhibited a significant increase in systolic blood pressure, endothelial dysfunction, and serum proinflammatory cytokines, as well as aortic and placental platelet-endothelial cell adhesion molecule expression compared with pregnant wild-type mice. Deficiency of interleukin 10 further augmented these measures in poly I:C–treated pregnant mice. In addition, sera from poly I:C-treated pregnant wild-type mice significantly decreased relaxation responses and increased platelet-endothelial cell adhesion molecule expression in isolated aortas from nonpregnant wild-type mice, and these effects were augmented by sera from poly I:C-treated interleukin 10 knockout mice. Coincubation with recombinant interleukin 10 normalized relaxation responses and platelet-endothelial cell adhesion molecule expression in all of the groups. Collectively, Toll-like receptor 3 activation during pregnancy causes preeclampsia-like symptoms, which are exacerbated by the absence of interleukin 10. Exogenous interleukin 10 treatment had beneficial effects on endothelial function and may be beneficial in women with preeclampsia.

The endocannabinoid anandamide inhibits cholangiocarcinoma growth via activation of the noncanonical Wnt signaling pathway

Cholangiocarcinomas are cancers that have poor prognosis and limited treatment options. The noncanonical Wnt pathway is mediated predominantly by Wnt 5a, which activates a Ca(2+)-dependent pathway involving protein kinase C, or a Ca(2+)-independent pathway involving the orphan receptor Ror2 and subsequent activation of Jun NH(2)-terminal kinase (JNK). This pathway is associated with growth-suppressing effects in numerous cell types. We have shown that anandamide decreases cholangiocarcinoma growth in vitro. Therefore, we determined the effects of anandamide on cholangiocarcinoma tumor growth in vivo using a xenograft model and evaluated the effects of anandamide on the noncanonical Wnt signaling pathways. Chronic administration of anandamide decreased tumor growth and was associated with increased Wnt 5a expression in vitro and in vivo. Treatment of cholangiocarcinoma cells with recombinant Wnt 5a decreased cell proliferation in vitro. Neither anandamide nor Wnt 5a affected intracellular calcium release, but both increased the JNK phosphorylation. Stable knockdown of Wnt 5a or Ror2 expression in cholangiocarcinoma cells abolished the effects of anandamide on cell proliferation and JNK activation. Modulation of the endocannabinoid system may be important in cholangiocarcinoma treatment. The antiproliferative actions of the noncanonical Wnt signaling pathway warrants further investigation to dissect the mechanism by which this may occur.

FK506 Binding Protein 12/12.6 Depletion Increases Endothelial Nitric Oxide Synthase Threonine 495 Phosphorylation and Blood Pressure

Chronic treatment with the immunosuppressive drug rapamycin leads to hypertension; however, the mechanisms are unknown. Rapamycin binds FK506 binding protein 12 and its related isoform 12.6 (FKBP12/12.6) and displaces them from intracellular Ca2+ release channels (ryanodine receptors) eliciting a Ca2+ leak from the endoplasmic/sarcoplasmic reticulum. We tested whether this Ca2+ leak promotes conventional protein kinase C–mediated endothelial NO synthase phosphorylation at Thr495, which reduces production of the vasodilator NO. Rapamycin treatment of control mice for 7 days, as well as genetic deletion of FKBP12.6, increased systolic arterial pressure significantly compared with controls. Untreated aortas from FKBP12.6−/− mice and in vitro rapamycin-treated control aortas had similarly decreased endothelium-dependent relaxation responses and NO production and increased endothelial NO synthase Thr495 phosphorylation and protein kinase C activity. Inhibition of either conventional protein kinase C or ryanodine receptor restored endothelial NO synthase Thr495 phosphorylation and endothelial function to control levels. Rapamycin induced a small increase in basal intracellular Ca2+ levels in isolated endothelial cells, and rapamycin or FKBP12.6 gene deletion decreased acetylcholine-induced intracellular Ca2+ release, all of which were reversed by ryanodine. These data demonstrate that displacement of FKBP12/12.6 from ryanodine receptors induces an endothelial intracellular Ca2+ leak and increases conventional protein kinase C–mediated endothelial NO synthase Thr495 phosphorylation leading to decreased NO production and endothelial dysfunction. This molecular mechanism may, in part, explain rapamycin-induced hypertension.

Fk506 Binding Protein 12 Deficiency in Endothelial and Hematopoietic Cells Decreases Regulatory T Cells and Causes Hypertension

Patients treated with the immunosuppressive drug tacrolimus (FK506), which binds FK506 binding protein 12 (FKBP12) and then inhibits the calcium-dependent phosphatase calcineurin, exhibit decreased regulatory T cells, endothelial dysfunction, and hypertension; however, the mechanisms and whether altered T-cell polarization play a role are unknown. Tacrolimus treatment of mice for 1 week dose-dependently decreased splenic CD4+/FoxP3+ (regulatory T cells), increased splenic CD4+/IL-17+ (T-helper 17) cells, and caused endothelial dysfunction and hypertension. To determine the mechanisms, we crossed floxed FKBP12 mice with Tie2-Cre mice to generate offspring lacking FKBP12 in endothelial and hematopoietic cells only (FKBP12EC knockout [KO]). Given the role of FKBP12 in inhibiting transforming growth factor-&bgr; receptor activation, Tie2-Cre–mediated deletion of FKBP12 increased transforming growth factor-&bgr; receptor activation and SMAD2/3 signaling. FKBP12EC KO mice exhibited increased vascular expression of genes and proteins related to endothelial cell activation and inflammation. Serum levels of the proinflammatory cytokines IL-2, IL-6, interferon-&ggr;, IL-17a, IL-21, and IL-23 were increased significantly, suggesting a T-helper 17 cell-mediated inflammatory state. Flow cytometry studies confirmed this, because splenic levels of CD4+/IL-17+ cells were increased significantly, whereas CD4+/FoxP3+ cells were decreased in FKBP12EC KO mice. Furthermore, spleens from FKBP12EC KO mice showed increased signal transducer and activator of transcription 3 activation, involved in T-helper 17 cell induction, and decreased signal transducer and activator of transcription 5 activation, involved in regulatory T-cell induction. FKBP12EC KO mice also exhibited endothelial dysfunction and hypertension. These data suggest that tacrolimus, through its activation of transforming growth factor-&bgr; receptors in endothelial and hematopoietic cells, may cause endothelial dysfunction and hypertension by activating endothelial cells, reducing regulatory T cells, and increasing T-helper 17 cell polarization and inflammation.