Genetically inducing renal lymphangiogenesis attenuates hypertension in mice.

BACKGROUND: Hypertension (HTN) is associated with renal proinflammatory immune cell infiltration and increased sodium retention. We reported previously that renal lymphatic vessels, which are responsible for trafficking immune cells from the interstitial space to draining lymph nodes, increase in density under hypertensive conditions. We also demonstrated that augmenting renal lymphatic density can prevent HTN in mice. Whether renal lymphangiogenesis can treat HTN in mice is unknown. We hypothesized that genetically inducing renal lymphangiogenesis after the establishment of HTN would attenuate HTN in male and female mice from three different HTN models. METHODS: Mice with inducible kidney-specific overexpression of VEGF-D (KidVD) experience renal lymphangiogenesis upon doxycycline administration. HTN was induced in KidVD+ and KidVD- mice by subcutaneous release of angiotensin II, administration of the nitric oxide synthase inhibitor L-NAME, or consumption of a 4% salt diet following a L-NAME priming and washout period. After a week of HTN stimuli treatment, doxycycline was introduced. Systolic blood pressure (SBP) readings were taken weekly. Kidney function was determined from urine and serum measures. Kidneys were processed for RT-qPCR, flow cytometry, and imaging. RESULTS: Mice that underwent renal-specific lymphangiogenesis had significantly decreased SBP and renal proinflammatory immune cells. Additionally, renal lymphangiogenesis was associated with a decrease in sodium transporter expression and increased fractional excretion of sodium, indicating improved sodium handling efficiency. CONCLUSIONS: These findings demonstrate that augmenting renal lymphangiogenesis can treat HTN in male and female mice by improving renal immune cell trafficking and sodium handling.

NF-κB mediated miR-130a modulation in lung microvascular cell remodeling: Implication in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive pulmonary vascular disease which is associated with pulmonary arterial endothelial cells (PAEC) dysfunction and pulmonary arterial smooth muscle cells proliferation. Moreover, inflammation is contributing a critical role in EC dysfunction and remains elusive. Nuclear factor-kappa B (NF-κB) is a master transcriptional regulator in various cardiovascular pathologies; but, NF-κB's role in EC dysfunction in unknown. Our previous study using cardiac and lung specific IκBα mutant mice (3M and IKBM) showed that PAH induced right ventricular hypertrophy (RVH) was prevented in monocrotaline (MCT) treated 3M and IKBM mice, compared to the wild-type mice. Recently, microRNAs (miRNAs) have emerged as a new class of post-transcriptional regulators in vascular remodeling; but, NF-κB regulated miRNA modulation in EC dysfunction is unknown. Using miRNA array analysis, we identified miR-130a which is upregulated in MCT-induced PAH mouse model, as a possible candidate to study. We showed that overexpressing miR-130a in lung microvascular endothelial cells (MVEC) promoted activation of α-smooth muscle actin, a critical component in endothelial-to-mesenchymal transition in EC remodeling. In this study, we demonstrated that bone morphogenetic protein receptor 2 (BMPR2) was a target for miR-130a and miR-130a was regulated by NF-κB which controlled apoptosis and vascular genes in MVEC. The findings reveal that NF-κB-mediated miR-130a modulation is critical in lung vascular remodeling.

Depletion of MHC class II invariant chain peptide or γ-δ T-cells ameliorates experimental preeclampsia.

Excessive innate immune system activation and inflammation during pregnancy can lead to organ injury and dysfunction and preeclampsia (PE); however, the molecular mechanisms involved are unknown. We tested the hypothesis that Toll-like receptor (TLR) activation induces major histocompatibility complex (MHC) class II invariant chain peptide (CLIP) expression on immune cells, makes them pro-inflammatory, and are necessary to cause PE-like features in mice. Treatment with VG1177, a competitive antagonist peptide for CLIP in the groove of MHC class II, was able to both prevent and treat PE-like features in mice. We then determined that γ-δ T cells are critical for the development of PE-like features in mice since γ-δ T-cell knockout mice, like CLIP deficient mice, are resistant to developing PE-like features. Placentas from women with PE exhibit significantly increased levels of γ-δ T cells. These preclinical data demonstrate that CLIP expression and activated γ-δ T cells are responsible for the development of immunologic PE-like features and that temporarily antagonizing CLIP and/or γ-δ T cells may be a therapeutic strategy for PE.

Human placenta-derived stromal cells decrease inflammation, placental injury and blood pressure in hypertensive pregnant mice.

Pre-eclampsia, the development of hypertension and proteinuria or end-organ damage during pregnancy, is a leading cause of both maternal and fetal morbidity and mortality, and there are no effective clinical treatments for pre-eclampsia aside from delivery. The development of pre-eclampsia is characterized by maladaptation of the maternal immune system, excessive inflammation and endothelial dysfunction. We have reported that detection of extracellular RNA by the Toll-like receptors (TLRs) 3 and 7 is a key initiating signal that contributes to the development of pre-eclampsia. PLacental eXpanded (PLX-PAD) cells are human placenta-derived, mesenchymal-like, adherent stromal cells that have anti-inflammatory, proangiogenic, cytoprotective and regenerative properties, secondary to paracrine secretion of various molecules in response to environmental stimulation. We hypothesized that PLX-PAD cells would reduce the associated inflammation and tissue damage and lower blood pressure in mice with pre-eclampsia induced by TLR3 or TLR7 activation. Injection of PLX-PAD cells on gestational day 14 significantly decreased systolic blood pressure by day 17 in TLR3-induced and TLR7-induced hypertensive mice (TLR3 144-111 mmHg; TLR7 145-106 mmHg; both P<0.05), and also normalized their elevated urinary protein:creatinine ratios (TLR3 5.68-3.72; TLR7 5.57-3.84; both P<0.05). On gestational day 17, aortic endothelium-dependent relaxation responses improved significantly in TLR3-induced and TLR7-induced hypertensive mice that received PLX-PAD cells on gestational day 14 (TLR3 35-65%; TLR7 37-63%; both P<0.05). In addition, markers of systemic inflammation and placental injury, increased markedly in both groups of TLR-induced hypertensive mice, were reduced by PLX-PAD cells. Importantly, PLX-PAD cell therapy had no effects on these measures in pregnant control mice or on the fetuses. These data demonstrate that PLX-PAD cell therapy can safely reverse pre-eclampsia-like features during pregnancy and have a potential therapeutic role in pre-eclampsia treatment.

Endothelial cell transforming growth factor-ß receptor activation causes tacrolimus-induced renal arteriolar hyalinosis.

Arteriolar hyalinosis is a common histological finding in renal transplant recipients treated with the calcineurin inhibitor tacrolimus; however, the pathophysiologic mechanisms remain unknown. In addition to increasing transforming growth factor (TGF)-ß levels, tacrolimus inhibits calcineurin by binding to FK506-binding protein 12 (FKBP12). FKBP12 alone also inhibits TGF-ß receptor activation. Here we tested whether tacrolimus binding to FKBP12 removes an inhibition of the TGF-ß receptor, allowing ligand binding, ultimately leading to receptor activation and arteriolar hyalinosis. We found that specific deletion of FKBP12 from endothelial cells was sufficient to activate endothelial TGF-ß receptors and induce renal arteriolar hyalinosis in these knockout mice, similar to that induced by tacrolimus. Tacrolimus-treated and knockout mice exhibited significantly increased levels of aortic TGF-ß receptor activation as evidenced by SMAD2/3 phosphorylation, along with increased collagen and fibronectin expression compared to controls. Treatment of isolated mouse aortas with tacrolimus increased TGF-ß receptor activation and collagen and fibronectin expression. These effects were independent of calcineurin, absent in endothelial denuded aortic rings, and could be prevented by the small molecule TGF-ß receptor inhibitor SB-505124. Thus, endothelial cell TGF-ß receptor activation is sufficient to cause vascular remodeling and renal arteriolar hyalinosis.

Time restricted feeding decreases renal innate immune cells and blood pressure in hypertensive mice.

BACKGROUND: Renal innate immune cell accumulation and inflammation are associated with hypertension. Time restricted feeding (TRF) has been reported to decrease inflammation and blood pressure. Whether TRF can decrease blood pressure by decreasing renal innate immune cells in hypertension is unknown. METHODS AND RESULTS: We determined whether TRF can decrease blood pressure in two separate mouse models of hypertension, N(G)-nitro-L-arginine methyl ester hydrochloride-induced hypertension (LHTN) and salt-sensitive hypertension (SSHTN). Once hypertension was established after 2 days, TRF (12-h food/12-h no food) for 4 weeks significantly decreased systolic blood pressure in both LHTN and SSHTN mice despite no differences in the amount of food eaten or body weight between groups. Activated macrophages and dendritic cells in the kidneys of both LHTN and SSHTN mice were decreased significantly in mice that underwent TRF. This was associated with an improvement in kidney function (decreased serum creatinine, decreased fractional excretion of sodium, and increased creatinine clearance) which achieved significance in LHTN mice and trended towards improvement in SSHTN mice. CONCLUSIONS: Our findings demonstrate that TRF can significantly decrease renal innate immune cells and blood pressure in two mouse models of hypertension.

Knockout of the neurokinin-1 receptor reduces cholangiocyte proliferation in bile duct-ligated mice.

In bile duct-ligated (BDL) rats, cholangiocyte proliferation is regulated by neuroendocrine factors such as α-calcitonin gene-related peptide (α-CGRP). There is no evidence that the sensory neuropeptide substance P (SP) regulates cholangiocyte hyperplasia. Wild-type (WT, (+/+)) and NK-1 receptor (NK-1R) knockout (NK-1R(-/-)) mice underwent sham or BDL for 1 wk. Then we evaluated 1) NK-1R expression, transaminases, and bilirubin serum levels; 2) necrosis, hepatocyte apoptosis and steatosis, and the number of cholangiocytes positive by CK-19 and terminal deoxynucleotidyl transferase biotin-dUTP nick-end labeling in liver sections; 3) mRNA expression for collagen 1α and α-smooth muscle (α-SMA) actin in total liver samples; and 4) PCNA expression and PKA phosphorylation in cholangiocytes. In cholangiocyte lines, we determined the effects of SP on cAMP and D-myo-inositol 1,4,5-trisphosphate levels, proliferation, and PKA phosphorylation. Cholangiocytes express NK-1R with expression being upregulated following BDL. In normal NK-1R(-/-) mice, there was higher hepatocyte apoptosis and scattered hepatocyte steatosis compared with controls. In NK-1R (-)/(-) BDL mice, there was a decrease in serum transaminases and bilirubin levels and the number of CK-19-positive cholangiocytes and enhanced biliary apoptosis compared with controls. In total liver samples, the expression of collagen 1α and α-SMA increased in BDL compared with normal mice and decreased in BDL NK-1R(-/-) compared with BDL mice. In cholangiocytes from BDL NK-1R (-)/(-) mice there was decreased PCNA expression and PKA phosphorylation. In vitro, SP increased cAMP levels, proliferation, and PKA phosphorylation of cholangiocytes. Targeting of NK-1R may be important in the inhibition of biliary hyperplasia in cholangiopathies.

Protein kinase CbetaII-mediated phosphorylation of endothelial nitric oxide synthase threonine 495 mediates the endothelial dysfunction induced by FK506 (tacrolimus).

FK506 [tacrolimus; hexadecahydro-5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycyclohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-c][1,4]oxa-azacyclotricosine-1,7,20,21(4H,23H)-tetrone] is used clinically to reduce the incidence of allograft rejection; however, chronic administration leads to endothelial dysfunction and hypertension. We have previously shown that FK506 activates Ca(2+)/diacylglycerol-dependent conventional protein kinase C (cPKC), which phosphorylates endothelial nitric oxide synthase (eNOS) at one of its inhibitory sites, Thr495. However, which cPKC isoform is responsible for phosphorylating eNOS Thr495 is unknown. The aim of the current study was to determine the cPKC isoform that is activated by FK506, leading to decreased endothelial function. FK506 reduced endothelium-dependent relaxation responses, yet had no effect on endothelium-independent relaxation responses in aortas from control mice. Of the various cPKC isoforms, only the administration of a PKCß(II) isoform-specific peptide inhibitor restored aortic relaxation responses to that of controls. In aortic endothelial cells, FK506 significantly increased PKCß(II) activation compared with vehicle-treated controls, and this was prevented by a PKCß(II) isoform-specific peptide inhibitor. In addition, a PKCß(II) isoform-specific peptide inhibitor prevented the increase in eNOS Thr495 phosphorylation induced by FK506. Taken together, our results indicate that ß(II) is the cPKC isoform responsible for phosphorylating eNOS at the inhibitory site Thr495 in response to FK506. PKCß(II) inhibition could prove beneficial in ameliorating the endothelial dysfunction and hypertension in patients treated with FK506.

Knockout of secretin receptor reduces large cholangiocyte hyperplasia in mice with extrahepatic cholestasis induced by bile duct ligation.

UNLABELLED: During bile duct ligation (BDL), the growth of large cholangiocytes is regulated by the cyclic adenosine monophosphate (cAMP)/extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and is closely associated with increased secretin receptor (SR) expression. Although it has been suggested that SR modulates cholangiocyte growth, direct evidence for secretin-dependent proliferation is lacking. SR wild-type (WT) (SR(+/+)) or SR knockout (SR(-/-)) mice underwent sham surgery or BDL for 3 or 7 days. We evaluated SR expression, cholangiocyte proliferation, and apoptosis in liver sections and proliferating cell nuclear antigen (PCNA) protein expression and ERK1/2 phosphorylation in purified large cholangiocytes from WT and SR(-/-) BDL mice. Normal WT mice were treated with secretin (2.5 nmoles/kg/day by way of osmotic minipumps for 1 week), and biliary mass was evaluated. Small and large cholangiocytes were used to evaluate the in vitro effect of secretin (100 nM) on proliferation, protein kinase A (PKA) activity, and ERK1/2 phosphorylation. SR expression was also stably knocked down by short hairpin RNA, and basal and secretin-stimulated cAMP levels (a functional index of biliary growth) and proliferation were determined. SR was expressed by large cholangiocytes. Knockout of SR significantly decreased large cholangiocyte growth induced by BDL, which was associated with enhanced apoptosis. PCNA expression and ERK1/2 phosphorylation were decreased in large cholangiocytes from SR(-/-) BDL compared with WT BDL mice. In vivo administration of secretin to normal WT mice increased ductal mass. In vitro, secretin increased proliferation, PKA activity, and ERK1/2 phosphorylation of large cholangiocytes that was blocked by PKA and mitogen-activated protein kinase kinase inhibitors. Stable knockdown of SR expression reduced basal cholangiocyte proliferation. SR is an important trophic regulator sustaining biliary growth. CONCLUSION: The current study provides strong support for the potential use of secretin as a therapy for ductopenic liver diseases.

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.

Tacrolimus reduces nitric oxide synthase function by binding to FKBP rather than by its calcineurin effect.

Hypertension develops in many patients receiving the immunosuppressive drug tacrolimus (FK506). One possible mechanism for hypertension is a reduction in vasodilatory nitric oxide. We found that tacrolimus and a calcineurin autoinhibitory peptide significantly decreased vascular calcineurin activity; however, only tacrolimus altered intracellular calcium release in mouse aortic endothelial cells. In mouse aortas, incubation with tacrolimus increased protein kinase C activity and basal endothelial nitric oxide synthase phosphorylation at threonine 495 but reduced basal and agonist-induced endothelial nitric oxide synthase phosphorylation at serine 1177, a mechanism known to inhibit synthase activity. While this decreased nitric oxide production and endothelial function, the calcineurin autoinhibitory peptide had no such effects. Inhibition of ryanodine receptor opening or protein kinase C blocked the effects of tacrolimus. Since it is known that the FK506 binding protein (FKBP12/12.6) interacts with the ryanodine receptor to regulate calcium release, we propose this as the mechanism by which tacrolimus alters intracellular calcium and endothelial nitric oxide synthase rather than by its effect on calcineurin. Our study shows that prevention of the tacrolimus-induced intracellular calcium leak may attenuate endothelial dysfunction and the consequent hypertension.

Deficiency of MicroRNA miR-1954 Promotes Cardiac Remodeling and Fibrosis.

Background Cardiac fibrosis occurs because of disruption of the extracellular matrix network leading to myocardial dysfunction. Angiotensin II (AngII) has been implicated in the development of cardiac fibrosis. Recently, microRNAs have been identified as an attractive target for therapeutic intervention in cardiac pathologies; however, the underlying mechanism of microRNAs in cardiac fibrosis remains unclear. Next-generation sequencing analysis identified a novel characterized microRNA, miR-1954, that was significantly reduced in AngII-infused mice. The finding led us to hypothesize that deficiency of miR-1954 triggers cardiac fibrosis. Methods and Results A transgenic mouse was created using α-MHC (α-myosin heavy chain) promoter and was challenged with AngII infusion. AngII induced cardiac hypertrophy and remodeling. The in vivo overexpression of miR-1954 showed significant reduction in cardiac mass and blood pressure in AngII-infused mice. Further analysis showed significant reduction in cardiac fibrotic genes, hypertrophy marker genes, and an inflammatory gene and restoration of a calcium-regulated gene (Atp2a2 [ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2]; also known as SERCA2), but no changes were observed in apoptotic genes. THBS1 (thrombospondin 1) is indicated as a target gene for miR-1954. Conclusions Our findings provide evidence, for the first time, that miR-1954 plays a critical role in cardiac fibrosis by targeting THBS1. We conclude that promoting the level of miR-1954 would be a promising strategy for the treatment of cardiac fibrosis.

Both maternal and placental toll-like receptor activation are necessary for the full development of proteinuric hypertension in mice.

OBJECTIVE: Innate immune system activation and excessive inflammation contributes to hypertension during pregnancy (HTN-preg). Activation of Toll-like receptors (TLRs), the primary innate immune system sensor, is evident in women with HTN-preg and is sufficient to induce pregnancy-dependent, proteinuric hypertension in animals. However, whether HTN-preg is a maternal disease, a placental disease, or both is unclear. We hypothesized that activation of TLR3, the double-stranded RNA sensor, in both maternal systemic and placental cells would be necessary for the full development of HTN-preg in mice. STUDY DESIGN: Various mating schemes generated pregnant mice that lacked TLR3 in maternal cells, paternally-derived placental cells, and both. Mice were then injected with a TLR3 agonist on days 13, 15, and 17 of pregnancy. MAIN OUTCOME MEASURES: Blood pressure, urinary protein excretion, fetal development, maternal vascular endothelial function, and immune system activation were all assessed and compared between groups. RESULTS: Pregnant mice lacking TLR3 in maternal cells as well as pregnant mice lacking TLR3 in placental cells had significantly attenuated increases in systolic blood pressure, urinary protein excretion, fetal demise, and endothelial dysfunction compared to wild-type pregnant mice following TLR3 activation. Pregnant mice lacking TLR3 in both maternal systemic and placental cells were completely resistant to the hypertension, proteinuria, fetal demise, endothelial dysfunction, splenomegaly, and increases in pro-inflammatory immune cells induced by TLR3 activation. CONCLUSIONS: These data suggest that both maternal and placental TLR3 activation are crucial for the full development of HTN-preg and that TLR3 antagonists may be beneficial in some women with HTN-preg.

Myeloid-Derived Suppressor Cells Ameliorate Cyclosporine A-Induced Hypertension in Mice.

The calcineurin inhibitor cyclosporine A (CsA) suppresses the immune system but promotes hypertension, vascular dysfunction, and renal damage. CsA decreases regulatory T cells and this contributes to the development of hypertension. However, CsA's effects on another important regulatory immune cell subset, myeloid-derived suppressor cells (MDSCs), is unknown. We hypothesized that augmenting MDSCs would ameliorate the CsA-induced hypertension and vascular and renal injury and dysfunction and that CsA reduces MDSCs in mice. Daily interleukin-33 treatment, which increased MDSC levels, completely prevented CsA-induced hypertension and vascular and renal toxicity. Adoptive transfer of MDSCs from control mice into CsA-treated mice after hypertension was established dose-dependently reduced blood pressure and vascular and glomerular injury. CsA treatment of aortas and kidneys isolated from control mice for 24 hours decreased relaxation responses and increased inflammation, respectively, and these effects were prevented by the presence of MDSCs. MDSCs also prevented the CsA-induced increase in fibronectin in microvascular and glomerular endothelial cells. Last, CsA dose-dependently reduced the number of MDSCs by inhibiting calcineurin and preventing cell proliferation, as other direct calcineurin signaling pathway inhibitors had the same dose-dependent effect. These data suggest that augmenting MDSCs can reduce the cardiovascular and renal toxicity and hypertension caused by CsA.

MicroRNAs: New Players in the Pathobiology of Preeclampsia.

Our understanding of how microRNAs (miRNAs) regulate gene networks and affect different molecular pathways leading to various human pathologies has significantly improved over the years. In contrary, the role of miRNAs in pregnancy-related hypertensive disorders such as preeclampsia (PE) is only beginning to emerge. Recent papers highlight that adverse pregnancy outcomes are associated with aberrant expression of several miRNAs. Presently, efforts are underway to determine the biologic function of these placental miRNAs which can shed light on their contribution to these pregnancy-related disease conditions. The discovery that miRNAs are stable in circulation coupled with the fact that the placenta is capable of releasing them to the circulation in exosomes generates a lot of enthusiasm to use them as biomarkers. In this review, we will summarize the recent findings of our understanding of miRNA regulation in relation to PE, a hypertensive disorder of pregnancy. Particular emphasis will be given to the role of key miRNA molecules such as miR-210 and miR-155 that are known to be consistently dysregulated in women with PE.

Regulatory T-Cell Augmentation or Interleukin-17 Inhibition Prevents Calcineurin Inhibitor-Induced Hypertension in Mice.

The immunosuppressive calcineurin inhibitors cyclosporine A and tacrolimus alter T-cell subsets and can cause hypertension, vascular dysfunction, and renal toxicity. We and others have reported that cyclosporine A and tacrolimus decrease anti-inflammatory regulatory T cells and increase proinflammatory interleukin-17-producing T cells; therefore, we hypothesized that inhibition of these effects using noncellular therapies would prevent the hypertension, endothelial dysfunction, and renal glomerular injury induced by calcineurin inhibitor therapy. Daily treatment of mice with cyclosporine A or tacrolimus for 1 week significantly decreased CD4+/FoxP3+ regulatory T cells in the spleen and lymph nodes, as well as induced hypertension, vascular injury and dysfunction, and glomerular mesangial expansion in mice. Daily cotreatment with all-trans retinoic acid reported to increase regulatory T cells and decrease interleukin-17-producing T cells, prevented all of the detrimental effects of cyclosporine A and tacrolimus. All-trans retinoic acid also increased regulatory T cells and prevented the hypertension, endothelial dysfunction, and glomerular injury in genetically modified mice that phenocopy calcineurin inhibitor-treated mice (FKBP12-Tie2 knockout). Treatment with an interleukin-17-neutralizing antibody also increased regulatory T-cell levels and prevented the hypertension, endothelial dysfunction, and glomerular injury in cyclosporine A-treated and tacrolimus-treated mice and FKBP12-Tie2 knockout mice, whereas an isotype control had no effect. Augmenting regulatory T cells and inhibiting interleukin-17 signaling using noncellular therapies prevents the cardiovascular and renal toxicity of calcineurin inhibitors in mice.

Cotreatment with interleukin 4 and interleukin 10 modulates immune cells and prevents hypertension in pregnant mice.

BACKGROUND: Excessive maternal immune system activation plays a central role in the development of the hypertensive disorder of pregnancy preeclampsia (PE). The immunomodulatory cytokines interleukin 4 (IL-4) and interleukin 10 (IL-10) are dysregulated during PE; therefore we hypothesized that treatment with both recombinant IL-4 and IL-10 during pregnancy could prevent the development of PE in mice. METHODS: Using our mouse model of PE in which immune system activation is induced by the double-stranded RNA receptor agonist poly I:C, we gave daily injections of IL-4, IL-10, or both on days 13-17 of pregnancy. Mice were then killed on day 18. RESULTS: Poly I:C caused a significant increase in systolic blood pressure in pregnant (P-PIC) mice compared with vehicle-treated pregnant (P) mice. All 3 treatments significantly decreased blood pressure in P-PIC mice to P levels, ameliorated the endothelial dysfunction, and decreased placental TLR3 levels in P-PIC mice. However, only IL-4/IL-10 cotreatment prevented the proteinuria and increased incidence of fetal demise in P-PIC mice; IL-4 or IL-10 alone had no effect. Additionally, only IL-4/IL-10 cotreatment prevented the significant increase in CD3(+)/γδ(+) T cells and CD11c(+) dendritic cells and significant decrease in CD11b(+)/CD14(-) suppressor monocytes, as well as completely prevented placental necrosis, in P-PIC mice. Importantly, IL-4/IL-10 cotreatment in P mice had no detrimental effects. CONCLUSIONS: Taken together, these data demonstrate that exogenous IL-4 and IL-10 administration concurrently during pregnancy can normalize immune cell subsets and prevent PE induced by maternal immune system activation.

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.

TLR3-induced placental miR-210 down-regulates the STAT6/interleukin-4 pathway.

Several clinical studies have reported increased placental miR-210 expression in women with PE compared to normotensive women, but whether miR-210 plays a role in the etiology of PE is unknown. We reported that activation of TLR3 produces the PE-like symptoms of hypertension, endothelial dysfunction, and proteinuria in mice only when pregnant, but whether TLR3 activation in pregnant mice and human cytotrophoblasts (CTBs) increases miR-210 and modulates its targets related to inflammation are unknown. Placental miR-210 levels were increased significantly in pregnant mice treated with the TLR3 agonist poly I:C (P-PIC). Both HIF-1α and NF-κBp50, known to bind the miR-210 promoter and induce its expression, were also increased significantly in placentas of P-PIC mice. Target identification algorithms and gene ontology predicted STAT6 as an inflammation-related target of miR-210 and STAT6 was decreased significantly in placentas of P-PIC mice. IL-4, which is regulated by STAT6 and increases during normotensive pregnancy, failed to increase in serum of P-PIC mice. P-PIC TLR3 KO mice did not develop hypertension and placental HIF-1α, NF-κBp50, miR-210, STAT6, and IL-4 levels were unchanged. To determine the placental etiology, treatment of human CTBs with poly I:C significantly increased HIF-1α, NF-κBp50, and miR-210 levels and decreased STAT6 and IL-4 levels. Overexpression of miR-210 in CTBs decreased STAT6 and IL-4 while inhibition of miR-210 increased STAT6 and IL-4. These findings demonstrate that TLR3 activation induces placental miR-210 via HIF-1α and NF-κBp50 leading to decreased STAT6 and IL-4 levels and this may contribute to the development of PE.

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.