No abstract

Inflammatory cytokines, including tumor necrosis factor-␣ (TNF␣), were elevated in patients with cardiovascular diseases and are also considered as crucial factors in the pathogenesis of preeclampsia; however, the underlying pathogenic mechanism has not been clearly elucidated. This study provides novel evidence that TNF␣ leads to endothelial dysfunction associated with hypertension and vascular remodeling in preeclampsia through down-regulation of endothelial nitric-oxide synthase (eNOS) by NF-Bdependent biogenesis of microRNA (miR)-31-5p, which targets eNOS mRNA. In this study, we found that miR-31-5p was up-regulated in sera from patients with preeclampsia and in human endothelial cells treated with TNF␣. TNF␣-mediated induction of miR-31-5p was blocked by an NF-B inhibitor and NF-B p65 knockdown but not by mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase inhibitors, indicating that NF-B is essential for biogenesis of miR-31-5p. The treatment of human endothelial cells with TNF␣ or miR-31-5p mimics decreased endothelial nitricoxide synthase (eNOS) mRNA stability without affecting eNOS promoter activity, resulting in inhibition of eNOS expression and NO/cGMP production through blocking of the functional activity of the eNOS mRNA 3-UTR. Moreover, TNF␣ and miR-31-5p mimic evoked endothelial dysfunction associated with defects in angiogenesis, trophoblastic invasion, and vasorelaxation in an ex vivo cultured model of human placental arterial vessels, which are typical features of preeclampsia. These results suggest that NF-B-responsive miR-31-5p elicits endothelial dysfunction, hypertension, and vascular remodeling via posttranscriptional down-regulation of eNOS and is a molecular risk factor in the pathogenesis and development of preeclampsia.

Regulated in development and DNA damage responses 1 (REDD-1), an inhibitor of mammalian target of rapamycin (mTOR), is induced by various cell stressors, including LPS, a major player in the pathogenesis of endotoxemic shock. However, the pathologic role of REDD-1 in endotoxemia is largely unknown. We found that LPS increased REDD-1 expression, nuclear transcription factor-κB (NF-κB) activation, and inflammation and that these responses were suppressed by REDD-1 knockdown and in REDD-1 macrophages. REDD-1 overexpression stimulated NF-κB-dependent inflammation without additional LPS stimulation. REDD-1-induced NF-κB activation was independent of 2 classic IKK-dependent NF-κB pathways and the mTOR signaling pathway; however, REDD-1, particularly its C-terminal region (178-229), interacted with and sequestered IκBα, to elicit atypical NF-κB activation during the delayed and persistent phases of inflammation after stimulation. Moreover, REDD-1 knockdown mitigated vascular inflammation and permeability in endotoxemic mice, resulting in decreases in immune cell infiltration, systemic inflammation, caspase-3 activation, apoptosis, and consequent mortality. We further confirmed the inflammatory and cytotoxic effects of REDD-1 in endotoxemic REDD-1 mice. Our data support the likelihood that REDD-1 exacerbates endotoxemic inflammation via atypical NF-κB activation by sequestering IκBα.-Lee, D.-K., Kim, J.-H., Kim, J., Choi, S., Park, M., Park, W., Kim, S., Lee, K.-S., Kim, T., Jung, J., Choi, Y. K., Ha, K.-S., Won, M.-H., Billiar, T. R., Kwon, Y.-G., Kim, Y.-M. REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation.

Heme oxygenase-1-derived carbon monoxide prevents inflammatory vascular disorders. To date, there is no clear evidence that HO-1/CO prevents endothelial dysfunction associated with the downregulation of endothelial NO synthesis in human endothelial cells stimulated with TNF-α. Here, we found that the CO-releasing compound CORM-2 prevented TNF-α-mediated decreases in eNOS expression and NO/cGMP production, without affecting eNOS promoter activity, by maintaining the functional activity of the eNOS mRNA 3′-untranslated region. By contrast, CORM-2 inhibited MIR155HG expression and miR-155-5p biogenesis in TNF-α-stimulated endothelial cells, resulting in recovery of the 3′-UTR activity of eNOS mRNA, a target of miR-155-5p. The beneficial effect of CORM-2 was blocked by an NF-κB inhibitor, a miR-155-5p mimic, a HO-1 inhibitor and siRNA against HO-1, indicating that CO rescues TNF-α-induced eNOS downregulation through NF-κB-responsive miR-155-5p expression via HO-1 induction; similar protective effects of ectopic HO-1 expression and bilirubin were observed in endothelial cells treated with TNF-α. Moreover, heme degradation products, except iron and N-acetylcysteine prevented H2O2-mediated miR-155-5p biogenesis and eNOS downregulation. These data demonstrate that CO prevents TNF-α-mediated eNOS downregulation by inhibiting redox-sensitive miR-155-5p biogenesis through a positive forward circuit between CO and HO-1 induction. This circuit may play an important preventive role in inflammatory endothelial dysfunction associated with human vascular diseases.

Vascular smooth muscle cells (VSMCs) play an important role in maintaining vascular function. Inflammation-mediated VSMC dysfunction leads to atherosclerotic intimal hyperplasia and preeclamptic hypertension; however, the underlying mechanisms are not clearly understood. We analyzed the expression levels of microRNA-155 (miR-155) in cultured VSMCs, mouse vessels, and clinical specimens and then assessed its role in VSMC function. Treatment with tumor necrosis factor-α (TNF-α) elevated miR-155 biogenesis in cultured VSMCs and vessel segments, which was prevented by NF-κB inhibition. MiR-155 expression was also increased in high-fat diet-fed ApoE−/− mice and in patients with atherosclerosis and preeclampsia. The miR-155 levels were inversely correlated with soluble guanylyl cyclase β1 (sGCβ1) expression and nitric oxide (NO)-dependent cGMP production through targeting the sGCβ1 transcript. TNF-α-induced miR-155 caused VSMC phenotypic switching, which was confirmed by the downregulation of VSMC-specific marker genes, suppression of cell proliferation and migration, alterations in cell morphology, and NO-induced vasorelaxation. These events were mitigated by miR-155 inhibition. Moreover, TNF-α did not cause VSMC phenotypic modulation and limit NO-induced vasodilation in aortic vessels of miR-155−/− mice. These findings suggest that NF-κB-induced miR-155 impairs the VSMC contractile phenotype and NO-mediated vasorelaxation by downregulating sGCβ1 expression. These data suggest that NF-κB-responsive miR-155 is a novel negative regulator of VSMC functions by impairing the sGC/cGMP pathway, which is essential for maintaining the VSMC contractile phenotype and vasorelaxation, offering a new therapeutic target for the treatment of atherosclerosis and preeclampsia.

cGMP-dependent protein kinase 1 (PKG1) plays an important role in nitric oxide (NO)/cGMP-mediated maintenance of vascular smooth muscle cell (VSMC) phenotype and vasorelaxation. Inflammatory cytokines, including tumor necrosis factor-α (TNFα), have long been understood to mediate several inflammatory vascular diseases. However, the underlying mechanism of TNFα-dependent inflammatory vascular disease is unclear. Here, we found that TNFα treatment decreased PKG1 expression in cultured VSMCs, which correlated with NF-κB-dependent biogenesis of miR-155-5p that targeted the 3'-UTR of PKG1 mRNA. TNFα induced VSMC phenotypic switching from a contractile to a synthetic state through the down-regulation of VSMC marker genes, suppression of actin polymerization, alteration of cell morphology, and elevation of cell proliferation and migration. All of these events were blocked by treatment with an inhibitor of miR-155-5p or PKG1, whereas transfection with miR-155-5p mimic or PKG1 siRNA promoted phenotypic modulation, similar to the response to TNFα. In addition, TNFα-induced miR-155-5p inhibited the vasorelaxant response of de-endothelialized mouse aortic vessels to 8-Br-cGMP by suppressing phosphorylation of myosin phosphatase and myosin light chain, both of which are downstream signal modulators of PKG1. Moreover, TNFα-induced VSMC phenotypic alteration and vasodilatory dysfunction were blocked by NF-κB inhibition. These results suggest that TNFα impairs NO/cGMP-mediated maintenance of the VSMC contractile phenotype and vascular relaxation by down-regulating PKG1 through NF-κB-dependent biogenesis of miR-155-5p. Thus, the NF-κB/miR-155-5p/PKG1 axis may be crucial in the pathogenesis of inflammatory vascular diseases, such as atherosclerotic intimal hyperplasia and preeclamptic hypertension.

Vascular endothelial growth factor (VEGF) plays a pivotal role in pathologic ocular neovascularization and vascular leakage via activation of VEGF receptor 2 (VEGFR2). This study was undertaken to evaluate the therapeutic mechanisms and effects of the tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a VEGFR2 inhibitor, in the development of vascular permeability and choroidal neovascularization (CNV). In cultured human retinal microvascular endothelial cells (HRMECs), treatment with RLYE blocked VEGF-A-induced phosphorylation of VEGFR2, Akt, ERK, and endothelial nitric oxide synthase (eNOS), leading to suppression of VEGF-A-mediated hyper-production of NO. Treatment with RLYE also inhibited VEGF-A-stimulated angiogenic processes (migration, proliferation, and tube formation) and the hyperpermeability of HRMECs, in addition to attenuating VEGF-A-induced angiogenesis and vascular permeability in mice. The anti-vascular permeability activity of RLYE was correlated with enhanced stability and positioning of the junction proteins VE-cadherin, β-catenin, claudin-5, and ZO-1, critical components of the cortical actin ring structure and retinal endothelial barrier, at the boundary between HRMECs stimulated with VEGF-A. Furthermore, intravitreally injected RLYE bound to retinal microvascular endothelium and inhibited laser-induced CNV in mice. These findings suggest that RLYE has potential as a therapeutic drug for the treatment of CNV by preventing VEGFR2-mediated vascular leakage and angiogenesis.

Serum deprivation or withdrawal induces apoptosis in endothelial cells, resulting in endothelial cell dysfunction that is associated with cardiovascular disease. However, there is still limited information on the role of miRNA in serum deprivation-induced apoptosis. Here we found that serum deprivation increased caspase-dependent apoptosis through miRNA-101-3p downregulation, without altering expression of its host gene RNA 3′-terminal phosphate cyclase-like 1, which was highly correlated with suppressed expression levels of Dicer and Argonaute 2 (Ago2), indicating that miR-101-3p is post-transcriptionally elevated in serum-deprived conditions. The decreased miR-101-3p caused elevated Bim expression by targeting its 3′-untranslated region (3′-UTR). This resulted in activation of the intrinsic pathway of apoptosis via interaction with Bcl-2, decreased mitochondrial membrane potential, cytochrome c release, mitochondrial reactive oxygen species (ROS) production, and caspase activation. These events were abrogated by miR-101-3p mimic and the proapoptotic Bim siRNA, which suggest a determinant role of the miR-101-3p/Bim axis in serum deprivation-induced apoptosis. The apoptosis induced by miR-101-3p-mediated Bim expression is mediated by both caspase-3 and -1, which are activated by two distinct intrinsic mechanisms, cytochrome c release and ROS-induced inflammasome activation, respectively. In other words, the antioxidant inhibited endothelial cell death mediated by caspase-1 that activated caspase-7, but not caspase-3. These findings provide mechanistic insight into a novel function of miR-101-3p in serum withdrawal-induced apoptosis triggered by activating two different intrinsic or mitochondrial apoptosis pathways, implicating miR-101-3p as a therapeutic target that limits endothelial cell death associated with vascular disorders.