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.

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.

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.

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.

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.

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.

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-4 deficiency induces mild preeclampsia in mice.

OBJECTIVE: Inflammation is necessary for successful pregnancy; however, excessive inflammation plays a central role in the development of the pregnancy-specific hypertensive disorder preeclampsia. Numerous anti-inflammatory cytokines are decreased in women with preeclampsia but the role of individual cytokines in blood pressure regulation during pregnancy is unknown. Therefore, we examined whether the lack of the potent anti-inflammatory cytokine interleukin-4 (IL-4) would be sufficient to elicit a preeclampsia-like syndrome in mice, and when coupled with immune system activation that these symptoms would be further augmented. METHODS: Measures of splenic immune cells, placental inflammation, blood pressure, endothelial function, and urinary protein excretion were performed in pregnant IL-4-deficient mice as well as in pregnant IL-4-deficient mice treated with the Toll-like receptor 3 agonist polyinosinic:polycytidylic (poly I:C). RESULTS: Pregnant IL-4-deficient mice exhibited altered splenic immune cell subsets, increased levels of pro-inflammatory cytokines, placental inflammation, mild hypertension, endothelial dysfunction, and proteinuria compared to pregnant control mice. Compared to pregnant control mice treated with poly I:C which exhibit preeclampsia-like symptoms, poly I:C-treated pregnant IL-4-deficient mice exhibited a further increase in pro-inflammatory cytokine levels, which was associated with augmented SBP and endothelial dysfunction. CONCLUSION: Collectively, these data show that the absence of IL-4 is sufficient to induce mild preeclampsia-like symptoms in mice due to excessive inflammation. Thus, the anti-inflammatory effects of IL-4 are important in preventing hypertension during pregnancy.

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.

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.

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.

Pin1 deficiency causes endothelial dysfunction and hypertension.

Pin1 is a peptidyl prolyl cis-trans isomerase that only binds to and isomerizes phosphorylated serine/threonine-proline motifs, inducing conformational changes that alter target protein function and phosphorylation. We have shown previously that deficiency of another peptidyl prolyl isomerase, FK506 binding protein 12/12.6, alters endothelial NO synthase phosphorylation and causes endothelial dysfunction and hypertension. Endothelial NO synthase contains the Pin1 binding sequence at (p)serine 116-proline 117 and phosphorylation of endothelial NO synthase serine 116 inhibits NO production; however, whether Pin1 deficiency alters vascular function and blood pressure is unknown. We hypothesized that Pin1 isomerizes p-endothelial NO synthase serine 116, which enables dephosphorylation and stimulates NO production. Immunoprecipitation of endothelial NO synthase and probing for Pin1 in rat aortic endothelial cells confirmed the interaction between the two. Pin1 knockdown via small interfering RNA or inhibition by juglone increased endothelial NO synthase serine 116 phosphorylation and prevented vascular endothelial growth factor-induced serine 116 dephosphorylation in endothelial cells. Acute treatment of isolated mouse aortas with juglone increased endothelial NO synthase serine 116 phosphorylation and decreased NO production and relaxation responses. Mice treated with juglone for 2 weeks, as well as Pin1 knockout mice, exhibited increased aortic endothelial NO synthase serine 116 phosphorylation, endothelial dysfunction, and hypertension. These data demonstrate that Pin1 binds endothelial NO synthase and enables dephosphorylation of serine 116, which increases NO production and endothelium-dependent dilation, leading to blood pressure maintenance.

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.

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-ß receptor activation, Tie2-Cre-mediated deletion of FKBP12 increased transforming growth factor-ß 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-γ, 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-ß 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.

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.

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.