DIX domain containing 1 (DIXDC1) modulates VEGFR2 level in vasculatures to regulate embryonic and postnatal retina angiogenesis.

BACKGROUND: In sprouting angiogenesis, VEGFR2 level is regulated via a fine-tuned process involving various signaling pathways. Other than VEGF/VEGFR2 signaling pathway, Wnt/ ß-catenin signaling is also important in vascular development. However, the crosstalk between these two signaling pathways is still unknown to date. In this study, we aimed to investigate the role of DIX domain containing 1 (DIXDC1) in vasculature, facilitating the crosstalk between VEGF/VEGFR2 and Wnt/ ß-catenin signaling pathways. RESULTS: In mice, DIXDC1 deficiency delayed angiogenesis at the embryonic stage and suppressed neovascularization at the neonatal stage. DIXDC1 knockdown inhibited VEGF-induced angiogenesis in endothelial cells in vitro by downregulating VEGFR2 expression. DIXDC1 bound Dishevelled Segment Polarity Protein 2 (Dvl2) and polymerized Dvl2 stabilizing VEGFR2 protein via its direct interaction. The complex formation and stability of VEGFR2 was potentiated by Wnt signaling. Moreover, hypoxia elevated DIXDC1 expression and likely modulated both canonical Wnt/ß-catenin signaling and VEGFR2 stability in vasculatures. Pathological angiogenesis in DIXDC1 knockout mice was decreased significantly in oxygen-induced retinopathy (OIR) and in wound healing models. These results suggest that DIXDC1 is an important factor in developmental and pathological angiogenesis. CONCLUSION: We have identified DIXDC1 as an important factor in early vascular development. These results suggest that DIXDC1 represents a novel regulator of sprouting angiogenesis that links Wnt signaling and VEGFR2 stability and may have a potential role in pathological neovascularization.

Beneficial and Detrimental Roles of Heme Oxygenase-1 in the Neurovascular System.

Heme oxygenase (HO) has both beneficial and detrimental effects via its metabolites, including carbon monoxide (CO), biliverdin or bilirubin, and ferrous iron. HO-1 is an inducible form of HO that is upregulated by oxidative stress, nitric oxide, CO, and hypoxia, whereas HO-2 is a constitutive form that regulates vascular tone and homeostasis. In brains injured by trauma, ischemia-reperfusion, or Alzheimer's disease (AD), the long-term expression of HO-1 can be detected, which can lead to cytotoxic ferroptosis via iron accumulation. In contrast, the transient induction of HO-1 in the peri-injured region may have regenerative potential (e.g., angiogenesis, neurogenesis, and mitochondrial biogenesis) and neurovascular protective effects through the CO-mediated signaling pathway, the antioxidant properties of bilirubin, and the iron-mediated ferritin synthesis. In this review, we discuss the dual roles of HO-1 and its metabolites in various neurovascular diseases, including age-related macular degeneration, ischemia-reperfusion injury, traumatic brain injury, Gilbert's syndrome, and AD.

Topical Administration of a Novel Acetylated Tetrapeptide Suppresses Vascular Permeability and Immune Responses and Alleviates Atopic Dermatitis in a Murine Model.

Although the pathogenesis of atopic dermatitis (AD) remains to be fully deciphered, skin barrier abnormality and immune dysregulation are known to be involved. Recently, the vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) system has also been implicated in the pathogenesis of this multifactorial chronic inflammatory skin disorder. Previously, we showed that a novel tetrapeptide, N-acetyl-Arg-Leu-Tyr-Glu (Ac-RLYE), inhibits angiogenesis and vascular permeability effectively by selectively antagonizing VEGFR-2. The current study aimed to investigate the pharmacological effect of Ac-RLYE on AD in vitro and in vivo. The in vitro experiments demonstrated that Ac-RLYE inhibited VEGF-induced vascular permeability in endothelial cells. Moreover, in an in vivo animal model of AD, Ac-RLYE relieved AD-like symptoms such as ear thickness and dermatitis severity scores and infiltration of immune cells, including mast cells and eosinophils. Ac-RLYE inhibited IgE secretion, restored the skin barrier protein filaggrin level, and markedly downregulated gene expression of AD-related Th1, Th2, and Th17 cytokines. Collectively, these findings suggest that Ac-RLYE would be useful for the treatment of AD and associated inflammatory skin disorders.

Macrophage inhibitory cytokine-1 promotes angiogenesis by eliciting the GFRAL-mediated endothelial cell signaling.

Macrophage inhibitory cytokine-1 (MIC-1) is a cytokine with pleotropic actions and its expression is markedly increased by inflammation and cardiac injury and in cancers. In particular, MIC-1 production after cardiac ischemia injury is associated with enhanced cardiac angiogenesis as well as myocardial protection. However, it remains uncertain whether MIC-1 itself has proangiogenic activity. In this study, we tried to determine the precise role of MIC-1 in physiological and pathological angiogenesis. Human microvessel endothelial cells responded to MIC-1 with enhanced angiogenic behaviors. Employing various angiogenesis assays, MIC-1 was found to promote vessel formation and development with a potency similar to that of vascular endothelial growth factor (VEGF). MIC-1 transgenic (Tg) mice also displayed enhanced neovascularization in both developing embryos and neonatal mouse retinas, compared with wild-type mice. Furthermore, endothelial cells (ECs) isolated from MIC-1 Tg mouse lung exhibited higher angiogenic potential than ECs from wild-type lung. MIC-1-induced angiogenesis was also observed in the recovery or healing processes of injuries such as hindlimb ischemia and skin wounds in mice. However, unlike VEGF, MIC-1 induced neither endothelial inflammation nor increased vascular permeability. In ECs, the MIC-1 signal exerted proangiogenic actions via the MEK/extracellular signal-regulated kinase- and phosphatidylinositol 3-kinase/Akt-dependent pathways. Notably, these MIC-1 signaling events in ECs were abrogated by small interfering RNA-mediated knockdown of GFRAL, suggesting that GFRAL is an EC receptor for MIC-1. In summary, we here show a novel role of MIC-1 as a potent EC activator, which promotes both normal and injury-related angiogenesis.

Korean Red Ginseng Improves Astrocytic Mitochondrial Function by Upregulating HO-1-Mediated AMPKα-PGC-1α-ERRα Circuit after Traumatic Brain Injury.

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the peroxisome proliferator-activating receptor-γ coactivator-1α (PGC-1α)-estrogen-related receptor α (ERRα) pathway in astrocytes. However, the role of Korean red ginseng extract (KRGE) in HO-1-mediated mitochondrial function in traumatic brain injury (TBI) is not well-elucidated. We found that HO-1 was upregulated in astrocytes located in peri-injured brain regions after a TBI, following exposure to KRGE. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HO-1 levels highly correlated with increased AMP-activated protein kinase α (AMPKα) activation, which led to the PGC-1α-ERRα axis-induced increases in mitochondrial functions (detected based on expression of cytochrome c oxidase subunit 2 (MTCO2) and cytochrome c as well as O2 consumption and ATP production). Knockdown of ERRα significantly reduced the p-AMPKα/AMPKα ratio and PGC-1α expression, leading to AMPKα-PGC-1α-ERRα circuit formation. Inactivation of HO by injecting the HO inhibitor Sn(IV) protoporphyrin IX dichloride diminished the expression of p-AMPKα, PGC-1α, ERRα, MTCO2, and cytochrome c in the KRGE-administered peri-injured region of a brain subjected to TBI. These data suggest that KRGE enhanced astrocytic mitochondrial function via a HO-1-mediated AMPKα-PGC-1α-ERRα circuit and consequent oxidative phosphorylation, O2 consumption, and ATP production. This circuit may play an important role in repairing neurovascular function after TBI in the peri-injured region by stimulating astrocytic mitochondrial biogenesis.

Therapeutic Efficacy of a Novel Acetylated Tetrapeptide in Animal Models of Age-Related Macular Degeneration.

It has been shown previously that a novel tetrapeptide, Arg-Leu-Tyr-Glu (RLYE), derived from human plasminogen inhibits vascular endothelial growth factor (VEGF)-induced angiogenesis, suppresses choroidal neovascularization in mice by an inhibition of VEGF receptor-2 (VEGFR-2) specific signaling pathway. In this study, we report that a modified tetrapeptide (Ac-RLYE) showed improved anti-choroidal neovascularization (CNV) efficacy in a number of animal models of neovascular age-related macular degeneration (AMD) which include rat, rabbit, and minipig. The preventive and therapeutic in vivo efficacy of Ac-RLYE via following intravitreal administration was determined to be either similar or superior to that of ranibizumab and aflibercept. Assessment of the intraocular pharmacokinetic and toxicokinetic properties of Ac-RLYE in rabbits demonstrated that it rapidly reached the retina with minimal systemic exposure after a single intravitreal dose, and it did not accumulate in plasma during repetitive dosing (bi-weekly for 14 weeks). Our results suggested that Ac-RLYE has a great potential for an alternative therapeutics for neovascular (wet) AMD. Since the amino acids in human VEGFR-2 targeted by Ac-RLYE are conserved among the animals employed in this study, the therapeutic efficacies of Ac-RLYE evaluated in those animals are predicted to be observed in human patients suffering from retinal degenerative diseases.

CD200 Change Is Involved in Neuronal Death in Gerbil Hippocampal CA1 Field Following Transient Forebrain Ischemia and Postischemic Treatment with Risperidone Displays Neuroprotection without CD200 Change.

It has been reported that CD200 (Cluster of Differentiation 200), expressed in neurons, regulates microglial activation in the central nervous system, and a decrease in CD200 expression causes an increase in microglial activation and neuronal loss. The aim of this study was to investigate time-dependent changes in CD200 expression in the hippocampus proper (CA1, 2, and 3 fields) after transient forebrain ischemia for 5 min in gerbils. In this study, 5-min ischemia evoked neuronal death (loss) of pyramidal neurons in the CA1 field, but not in the CA2/3 fields, at 5 days postischemia. In the sham group, CD200 expression was found in pyramidal neurons of the CA1 field, and the immunoreactivity in the group with ischemia was decreased at 6 h postischemia, dramatically increased at 12 h postischemia, decreased (to level found at 6 h postischemia) at 1 and 2 days postischemia, and significantly increased again at 5 days postischemia. At 5 days postischemia, CD200 immunoreactivity was strongly expressed in microglia and GABAergic neurons. However, in the CA3 field, the change in CD200 immunoreactivity in pyramidal neurons was markedly weaker than that in the CA1 field, showing there was no expression of CD 200 in microglia and GABAergic neurons. In addition, treatment of 10 mg/kg risperidone (an atypical antipsychotic drug) after the ischemia hardly changed CD200 immunoreactivity in the CA1 field, showing that CA1 pyramidal neurons were protected from the ischemic injury. These results indicate that the transient ischemia-induced change in CD200 expression may be associated with specific and selective neuronal death in the hippocampal CA1 field following transient forebrain ischemia.

Increased Calbindin D28k Expression via Long-Term Alternate-Day Fasting Does Not Protect against Ischemia-Reperfusion Injury: A Focus on Delayed Neuronal Death, Gliosis and Immunoglobulin G Leakage.

Calbindin-D28k (CB), a calcium-binding protein, mediates diverse neuronal functions. In this study, adult gerbils were fed a normal diet (ND) or exposed to intermittent fasting (IF) for three months, and were randomly assigned to sham or ischemia operated groups. Ischemic injury was induced by transient forebrain ischemia for 5 min. Short-term memory was examined via passive avoidance test. CB expression was investigated in the Cornu Ammonis 1 (CA1) region of the hippocampus via western blot analysis and immunohistochemistry. Finally, histological analysis was used to assess neuroprotection and gliosis (microgliosis and astrogliosis) in the CA1 region. Short-term memory did not vary significantly between ischemic gerbils with IF and those exposed to ND. CB expression was increased significantly in the CA1 pyramidal neurons of ischemic gerbils with IF compared with that of gerbils fed ND. However, the CB expression was significantly decreased in ischemic gerbils with IF, similarly to that of ischemic gerbils exposed to ND. The CA1 pyramidal neurons were not protected from ischemic injury in both groups, and gliosis (astrogliosis and microgliosis) was gradually increased with time after ischemia. In addition, immunoglobulin G was leaked into the CA1 parenchyma from blood vessels and gradually increased with time after ischemic insult in both groups. Taken together, our study suggests that IF for three months increases CB expression in hippocampal CA1 pyramidal neurons; however, the CA1 pyramidal neurons are not protected from transient forebrain ischemia. This failure in neuroprotection may be attributed to disruption of the blood-brain barrier, which triggers gliosis after ischemic insults.

Activity-expression profiling of glucose-6-phosphate dehydrogenase in tissues of normal and diabetic mice.

Glucose-6-phosphate dehydrogenase (G6PD) plays a principal role in the regulation of oxidative stress by modulating the nicotinamide adenine dinucleotide phosphate pool and is expected to be associated with metabolic diseases such as diabetes mellitus (DM). However, it is unclear whether hyperglycemia increases G6PD activity levels in DM because suitable assays for quantifying the activity in a high-throughput manner are lacking. Using liquid droplet arrays tailored to analyze tissue lysates, we performed G6PD activity profiling in eight tissues of normal and diabetic mice: brain, heart, kidney, liver, lung, muscle, spleen, and thyroid. Diabetic mice exhibited significantly higher G6PD activities in the kidney, liver, spleen, and thyroid than normal mice; no significant difference was found in the brain, heart, lung, or muscle. We also performed G6PD expression profiling in the eight tissues using Western blot analysis. Diabetic mice showed significantly elevated G6PD expression levels in the kidney, lung, spleen, and thyroid compared with normal mice; no significant difference was found in the brain, heart, liver, or muscle. An analysis of G6PD activity-expression profiles demonstrated tissue-specific changes in response to hyperglycemia. Thus, our approach would be helpful for understanding the role of G6PD in tissue-based pathogenesis of diabetic complications.

CLEC14A deficiency exacerbates neuronal loss by increasing blood-brain barrier permeability and inflammation.

BACKGROUND: Ischemic stroke is a main cause of mortality. Blood-brain barrier (BBB) breakdown appears to play a critical role in inflammation in patients with ischemic stroke and acceleration of brain injury. The BBB has a protective function and is composed of endothelial cells, pericytes, and astrocytes. In ischemic stroke treatments, regulation of vascular endothelial growth factor (VEGF)-A and vascular endothelial growth factor receptor (VEGFR)-2 is a crucial target despite adverse effects. Our previous study found that loss of C-type lectin family 14 member A (CLEC14A) activated VEGF-A/VEGFR-2 signaling in developmental and tumoral angiogenesis. Here, we evaluate the effects of BBB impairment caused by CLEC14A deficiency in ischemia-reperfusion injury. METHODS: In vitro fluorescein isothiocyanate (FITC)-dextran permeability, transendothelial electrical resistance (TEER) assay, and immunostaining were used to evaluate endothelial integrity. BBB permeability was assessed using Evans blue dye and FITC-dextran injection in Clec14a-/- (CLEC14A-KO) mice and wild-type mice. Middle cerebral artery occlusion surgery and behavioral assessments were performed to evaluate the neurologic damage. The change of tight junctional proteins, adhesion molecules, pro-inflammatory cytokines, and microglial were confirmed by immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction of brain samples. RESULTS: In endothelial cells, knockdown of CLEC14A increased FITC-dextran permeability and decreased transendothelial electrical resistance; the severity of this effect increased with VEGF treatment. Immunofluorescence staining revealed that tight junctional proteins were attenuated in the CLEC14A knockdown endothelial cells. Consistent with the in vitro results, CLEC14A-KO mice that were injected with Evans blue dye had cerebral vascular leakage at postnatal day 8; wild-type mice had no leakage. We used a middle cerebral artery occlusion model and found that CLEC14A-KO mice had severe infarcted brain and neurological deficits with upregulated VEGFR-2 expression. FITC-dextran leakage was present in CLEC14A-KO mice after ischemia-reperfusion, and the numbers of tight junctional molecules were significantly decreased. Loss of CLEC14A increased the pro-inflammatory response through adhesion molecule expression, and glial cells were activated. CONCLUSIONS: These results suggest that activation of VEGFR-2 in CLEC14A-KO mice aggravates ischemic stroke by exacerbating cerebral vascular leakage and increasing neuronal inflammation after ischemia-reperfusion injury.

SALM4 regulates angiogenic functions in endothelial cells through VEGFR2 phosphorylation at Tyr1175.

Angiogenesis depends on VEGF-mediated signaling. However, the regulatory mechanisms and functions of individual VEGF receptor 2 (VEGFR2) phosphorylation sites remain unclear. Here, we report that synaptic adhesion-like molecule 4 (SALM4) regulates a specific VEGFR2 phosphorylation site. SALM4 silencing in HUVECs and Salm4 knockout (KO) in lung endothelial cells (ECs) of Salm4-/- mice suppressed phosphorylation of VEGFR2 tyrosine (Y) 1175 (Y1173 in mice) and downstream signaling upon VEGF-A stimulation. However, VEGFR2 phosphorylation at Y951 (Y949 in mice) and Y1214 (Y1212 in mice) remained unchanged. Knockdown and KO of SALM4 inhibited VEGF-A-induced angiogenic functions of ECs. SALM4 depletion reduced endothelial leakage, sprouting, and migratory activities. Furthermore, in an ischemia and reperfusion (I/R) model, brain injury was attenuated in Salm4-/- mice compared with wild-type (WT) mice. In brain lysates after I/R, VEGFR2 phosphorylation at Y949, Y1173, and Y1212 were induced in WT brains, but only Y1173 phosphorylation of VEGFR2 was reduced in Salm4-/- brains. Taken together, our results demonstrate that SALM4 specifically regulates VEGFR2 phosphorylation at Y1175 (Y1173 in mice), thereby fine-tuning VEGF signaling in ECs.-Kim, D. Y., Park, J. A., Kim, Y., Noh, M., Park, S., Lie, E., Kim, E., Kim, Y.-M., Kwon, Y.-G. SALM4 regulates angiogenic functions in endothelial cells through VEGFR2 phosphorylation at Tyr1175.

The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory-induced endothelial dysfunction.

Clinical trials suggested that the vascular system can remember episodes of poor glycemic control through a phenomenon known as hyperglycemic memory (HGM). HGM is associated with long-term diabetic vascular complications in type 1 and type 2 diabetes, although the molecular mechanism of that association is not clearly understood. We hypothesized that transglutaminase 2 (TGase2) and intracellular reactive oxygen species (ROS) play a key role in HGM-induced vascular dysfunction. We found that hyperglycemia induced persistent oxidative stress, expression of inflammatory adhesion molecules, and apoptosis in the aortic endothelium of HGM mice whose blood glucose levels had been normalized by insulin supplementation. TGase2 activation and ROS generation were in a vicious cycle in the aortic endothelium of HGM mice and also in human aortic endothelial cells after glucose normalization, which played a key role in the sustained expression of inflammatory adhesion molecules and apoptosis. Our findings suggest that the TGase2-ROS vicious cycle plays an important role in HGM-induced endothelial dysfunction.-Lee, J.-Y., Lee, Y.-J., Jeon, H.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The vicious cycle between transglutaminase 2 and reactive oxygen species in hyperglycemic memory-induced endothelial dysfunction.

Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

C-peptide has a beneficial effect against diabetic complications, but its role in hyperglycemia-induced metastasis is unknown. We investigated hyperglycemia-mediated pulmonary vascular leakage and metastasis and C-peptide inhibition of these molecular events using human pulmonary microvascular endothelial cells (HPMVECs) and streptozotocin-induced diabetic mice. VEGF, which is elevated in the lungs of diabetic mice, activated transglutaminase 2 (TGase2) in HPMVECs by sequential elevation of intracellular Ca2+ and reactive oxygen species (ROS) levels. VEGF also induced vascular endothelial (VE)-cadherin disruption and increased the permeability of endothelial cells, both of which were prevented by the TGase inhibitors monodansylcadaverine and cystamine or TGM2-specific small interfering RNA. C-peptide prevented VEGF-induced VE-cadherin disruption and endothelial cell permeability through inhibiting ROS-mediated activation of TGase2. C-peptide supplementation inhibited hyperglycemia-induced ROS generation and TGase2 activation and prevented vascular leakage and metastasis in the lungs of diabetic mice. The role of TGase2 in hyperglycemia-induced pulmonary vascular leakage and metastasis was further demonstrated in diabetic Tgm2-/- mice. These findings demonstrate that hyperglycemia induces metastasis, and C-peptide prevents the hyperglycemia-induced metastasis in the lungs of diabetic mice by inhibiting VEGF-induced TGase2 activation and subsequent vascular leakage.-Jeon, H.-Y., Lee, Y.-J., Kim, Y.-S., Kim, S.-Y., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. Proinsulin C-peptide prevents hyperglycemia-induced vascular leakage and metastasis of melanoma cells in the lungs of diabetic mice.

Experimental Pretreatment with Chlorogenic Acid Prevents Transient Ischemia-Induced Cognitive Decline and Neuronal Damage in the Hippocampus through Anti-Oxidative and Anti-Inflammatory Effects.

Chlorogenic acid (CGA), an ester of caffeic acid and quinic acid, is among the phenolic acid compounds which can be naturally found in green coffee extract and tea. CGA has been studied since it displays significant pharmacological properties. The aim of this study was to investigate the effects of CGA on cognitive function and neuroprotection including its mechanisms in the hippocampus following transient forebrain ischemia in gerbils. Memory and learning following the ischemia was investigated by eight-arm radial maze and passive avoidance tests. Neuroprotection was examined by immunohistochemistry for neuronal nuclei-specific protein and Fluoro-Jade B histofluorescence staining. For mechanisms of the neuroprotection, alterations in copper, zinc-superoxide dismutase (SOD1), SOD2 as antioxidant enzymes, dihydroethidium and 4-hydroxy-2-nonenal as indicators for oxidative stress, and anti-inflammatory cytokines (interleukin (IL)-4 and IL-13) and pro-inflammatory cytokines (tumor necrosis factor α (TNF-α) and IL-2) were examined by Western blotting and/or immunohistochemistry. As a result, pretreatment with 30 mg/kg CGA attenuated cognitive impairment and displayed a neuroprotective effect against transient forebrain ischemia (TFI). In Western blotting, the expression levels of SOD2 and IL-4 were increased due to pretreatment with CGA and, furthermore, 4-HNE production and IL-4 expressions were inhibited by CGA pretreatment. Additionally, pretreated CGA enhanced antioxidant enzymes and anti-inflammatory cytokines and, in contrast, attenuated oxidative stress and pro-inflammatory cytokine expression. Based on these results, we suggest that CGA can be a useful neuroprotective material against ischemia-reperfusion injury due to its antioxidant and anti-inflammatory efficacies.

N-Terminal Modification of the Tetrapeptide Arg-Leu-Tyr-Glu, a Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) Antagonist, Improves Antitumor Activity by Increasing its Stability against Serum Peptidases.

The tetrapeptide Arg-Leu-Tyr-Glu (RLYE), a vascular endothelial growth factor (VEGF) receptor-2 antagonist, has been used previously either alone or in combination with chemotherapeutic drugs for treating colorectal cancer in a mouse model. We analyzed the half-life of the peptide and found that because of degradation by aminopeptidases B and N, it had a short half-life of 1.2 hours in the serum. Therefore, to increase the stability and potency of the peptide, we designed the modified peptide, N-terminally acetylated RLYE (Ac-RLYE), which had a strongly stabilized half-life of 8.8 hours in serum compared with the original parent peptide. The IC50 value of Ac-RLYE for VEGF-A-induced endothelial cell migration decreased to approximately 37.1 pM from 89.1 pM for the parent peptide. Using a mouse xenograft tumor model, we demonstrated that Ac-RLYE was more potent than RLYE in inhibiting tumor angiogenesis and growth, improving vascular integrity and normalization through enhanced endothelial cell junctions and pericyte coverage of the tumor vasculature, and impeding the infiltration of macrophages into tumor and their polarization to the M2 phenotype. Furthermore, combined treatment of Ac-RLYE and irinotecan exhibited synergistic effects on M1-like macrophage activation and apoptosis and growth inhibition of tumor cells. These findings provide evidence that the N-terminal acetylation augments the therapeutic effect of RLYE in solid tumors via inhibition of tumor angiogenesis, improvement of tumor vessel integrity and normalization, and enhancement of the livery and efficacy of the coadministered chemotherapeutic drugs. SIGNIFICANCE STATEMENT: The results of this study demonstrate that the N-terminal acetylation of the tetrapeptide RLYE (Ac-RLYE), a novel vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitor, significantly improves its serum stability, antiangiogenic activity, and vascular normalizing potency, resulting in enhanced therapeutic effect on solid tumors. Furthermore, the combined treatment of Ac-RLYE with the chemotherapeutic drug, irinotecan, synergistically enhanced its antitumor efficacy by improving the perfusion and delivery of the drug into the tumors and stimulating the conversion of the tumor-associated macrophages to an immunostimulatory M1-like antitumor phenotype.

NF-κB-responsive miRNA-31-5p elicits endothelial dysfunction associated with preeclampsia via down-regulation of endothelial nitric-oxide synthase.

Inflammatory cytokines, including tumor necrosis factor-α (TNFα), 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 TNFα leads to endothelial dysfunction associated with hypertension and vascular remodeling in preeclampsia through down-regulation of endothelial nitric-oxide synthase (eNOS) by NF-κB-dependent 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 TNFα. TNFα-mediated induction of miR-31-5p was blocked by an NF-κB inhibitor and NF-κB p65 knockdown but not by mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase inhibitors, indicating that NF-κB is essential for biogenesis of miR-31-5p. The treatment of human endothelial cells with TNFα or miR-31-5p mimics decreased endothelial nitric-oxide 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, TNFα 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-κB-responsive miR-31-5p elicits endothelial dysfunction, hypertension, and vascular remodeling via post-transcriptional down-regulation of eNOS and is a molecular risk factor in the pathogenesis and development of preeclampsia.

TNF-α elicits phenotypic and functional alterations of vascular smooth muscle cells by miR-155-5p-dependent down-regulation of cGMP-dependent kinase 1.

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.

The metastasis suppressor CD82/KAI1 represses the TGF-ß 1 and Wnt signalings inducing epithelial-to-mesenchymal transition linked to invasiveness of prostate cancer cells.

BACKGROUND: The epithelial-to-mesenchymal transition (EMT) is closely associated with cancer invasion and metastasis. Since the transforming growth factor ß (TGF-ß) and Wnt signals induce EMT in various epithelial cell types, we examined whether and how the CD82/KAI1 metastasis suppressor affects the TGF-ß and Wnt signal-dependent EMT in human prostate cancer cells. METHODS: The invasiveness of cancer cells was evaluated by examining their ability to pass through the basement membrane matrigel. The subcellular localizations of Smad4 and ß-catenin proteins were respectively examined by confocal microscopy following immunofluorescence antibody staining and immunoblotting analysis following subcellular fractionation. The transcriptional activities of the TGF-ß1 -responsive TRE and Wnt-responsive Tcf/Lef promoters were determined by a luciferase reporter assay following transfection of the recombinant reporter vector into the cell. RESULTS: TGF-ß1 and Wnt3a treatments of human prostate cancer cells without CD82 expression resulted in not only increased invasiveness but also EMT involving the development of motile structures, downregulation of E-cadherin, and upregulation of the mesenchymal proteins. However, in the cells with high levels of CD82, the TGF-ß1 and Wnt3a stimulations neither elevated invasiveness nor induced EMT. Furthermore, the TGF-ß1 signaling events occurring in the CD82-deficient cells, such as phosphorylation of Smad2, nuclear translocation of Smad4, and transactivation of the TRE promoter, did not take place in the high CD82-expressing cells. Further, high CD82 expression interfered with the Wnt signal-dependent alterations in the phosphorylation pattern of glycogen synthase kinase 3ß (GSK-3ß) in prostate cancer cells, which allowed GSK-3ß to continue phosphorylating ß-catenin, thereby attenuating the Wnt signaling effects on the nuclear translocation of ß-catenin and subsequent transactivation of the Tcf/Lef promoter. CONCLUSIONS: The results of the present study suggest that CD82/KAI1 functions in suppressing TGF-ß1 - and Wnt-induced EMT in prostate cancer cells by inhibiting the TGF-ß1 /Smad and Wnt/ß-catenin pathways. Therefore, loss or decrease of CD82 expression is likely to render prostate cancer cells prone to respond to the TGF-ß1 and Wnt signals with EMT, resulting in the development of a motile and invasive mesenchymal phenotype related to the initiation of the metastatic cascade.

REDD-1 aggravates endotoxin-induced inflammation via atypical NF-κB activation.

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.

The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.

We investigated the beneficial effects of midazolam against vascular endothelial growth factor (VEGF)-induced vascular leakage and its molecular mechanism of action in human retinal endothelial cells (HRECs) and the retinas of diabetic mice. Midazolam inhibited VEGF-induced elevation of intracellular Ca2+, generation of reactive oxygen species (ROS), and transglutaminase activation in HRECs; these effects were reversed by the GABA, type A (GABAA) receptor antagonist flumazenil but not by the translocator protein antagonist PK11195. Midazolam also prevented VEGF-induced disassembly of adherens junctions and in vitro permeability. Intravitreal injection of midazolam prevented hyperglycemia-induced ROS generation, transglutaminase activation, and subsequent vascular leakage in the retinas of diabetic mice, and those effects were reversed by flumazenil. The roles of flumazenil were further supported by identifying GABAA receptors in mouse retinas. Thus, midazolam prevents hyperglycemia-induced vascular leakage by inhibiting VEGF-induced intracellular events in the retinas of diabetic mice.-Lee, Y.-J., Kim, M., Lee, J.-Y., Jung, S.-H., Jeon, H.-Y., Lee, S.-A., Kang, S., Han, E.-T., Park, W. S., Hong, S.-H., Kim, Y.-M., Ha, K.-S. The benzodiazepine anesthetic midazolam prevents hyperglycemia-induced microvascular leakage in the retinas of diabetic mice.