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Abstract Preeclampsia is caused by placental hypoxia and systemic inflammation and is associated with reduced placental growth factor (PlGF) and endothelial nitric oxide synthase (eNOS) levels. The molecular signaling axes involved in this process may play a role in the pathogenesis of preeclampsia. Here, we found that hypoxic exposure increased hypoxia-inducible factor-1α (HIF-1α)/Twist1-mediated miR-214-3p biogenesis in trophoblasts, suppressing PlGF production and trophoblast invasion. TNF-α stimulation increased NF-κB-dependent miR-214-3p expression in endothelial cells, impairing eNOS expression and causing endothelial dysfunction. Synthetic miR-214-3p administration to pregnant mice decreased PlGF and eNOS expression, resulting in preeclampsia-like symptoms, including hypertension, proteinuria, and fetal growth restriction. Conversely, miR-214-3p deletion maintained the PlGF and eNOS levels in hypoxic pregnant mice, alleviating preeclampsia-like symptoms and signs. These findings provide new insights into the role of HIF-1/Twist1- and NF-κB-responsive miR-214-3p-dependent PlGF and eNOS downregulation in the pathogenesis of preeclampsia and establish miR-214-3p as a therapeutic or preventive target for preeclampsia and its complications.

Abstract Regulated in development and DNA damage-response 1 (REDD1) is a stress-induced protein that controls various cellular functions, including metabolism, oxidative stress, autophagy, and cell fate, and contributes to the pathogenesis of metabolic and inflammatory disorders, neurodegeneration, and cancer. REDD1 usually exerts deleterious effects, including tumorigenesis, metabolic inflammation, neurodegeneration, and muscle dystrophy; however, it also exhibits protective functions by regulating multiple intrinsic cell activities through either an mTORC1-dependent or -independent mechanism. REDD1 typically regulates mTORC1 signaling, NF-κB activation, and cellular pro-oxidant or antioxidant activity by interacting with 14-3-3 proteins, IκBα, and thioredoxin-interacting protein or 75 kDa glucose-regulated protein, respectively. The diverse functions of REDD1 depend on cell type, cellular context, interaction partners, and cellular localization (e.g., mitochondria, endomembrane, or cytosol). Therefore, comprehensively understanding the molecular mechanisms and biological roles of REDD1 under pathophysiological conditions is of utmost importance. In this review, based on the published literature, we highlight and discuss the molecular mechanisms underlying the REDD1 expression and its actions, biological functions, and pathophysiological roles.

Abstract Background Acute lung injury (ALI) is a life-threatening condition that fundamentally results from inflammation and edema in the lung. There are no effective treatments available for clinical use. Previously, we found that as a leakage blocker CU06-1004 prevents endothelial barrier disruption and enhances endothelial cell survival under inflammatory conditions. In this study, we aimed to elucidate the effect of CU06-1004 in terms of prevention of inflammation and endothelial dysfunction in an ALI mouse model. Methods An ALI model was established that included intraperitoneal administration of LPS. Following LPS administration, survival rates and lung wet/dry ratios were assessed. Histological analysis was performed using hematoxylin and eosin staining. Scanning electron microscopy was used to examine alveolar and capillary morphology. Cytokines such as IL-1β, IL-6, and TNF-α were analyzed using an ELISA assay of bronchoalveolar lavage fluid (BALF) and serum. Neutrophil infiltration was observed in BALF using Wright-Giemsa staining, and myeloperoxidase (MPO) activity was assessed. Pulmonary vascular leakage was confirmed using Evans-blue dye, and the expression of junctional proteins was evaluated using immunofluorescent staining. Expression of adhesion molecules was observed using immunofluorescence staining. NF-κB activation was determined using immunohistochemistry and western blot analysis. Results Survival rates and pulmonary edema were ameliorated with CU06-1004 treatment. Administration of CU06-1004 normalized histopathological changes induced by LPS, and alveolar-capillary wall thickening was reduced. Compared with the LPS-challenged group, after CU06-1004 treatment, the infiltration of immune cells was decreased in the BALF, and MPO activity in lung tissue was reduced. Similarly, in the CU06-1004 treatment group, pro-inflammatory cytokines were significantly inhibited in both BALF and serum. Evans-blue leakage was reduced, and the expression of junctional proteins was recovered in the CU06-1004 group. Adhesion molecules were downregulated and NF-κB activation was inhibited after CU06-1004 treatment. Conclusions These results suggested that CU06-1004 had a therapeutic effect against LPS-induced ALI via alleviation of the inflammatory response and protection of vascular integrity.

Inhibition of angiogenesis is considered a promising therapeutic approach for cancer treatment. Our previous genetic research showed that the use of a cell-penetrating peptide to inhibit the pleckstrin homology (PH) domain of 3-phosphoinositide-dependent kinase 1 (PDK1) was a viable approach to suppress pathological angiogenesis. Herein, we synthesized and characterized a novel small molecule, CU05-1189, based on our prior study and present evidence for the first time that this compound possesses antiangiogenic properties both in vitro and in vivo. The computational analysis showed that CU05-1189 can interact with the PH domain of PDK1, and it significantly inhibited vascular endothelial growth factor (VEGF)-induced proliferation, migration, invasion, and tube formation in human umbilical vein endothelial cells without apparent toxicity. Western blot analysis revealed that the Akt signaling pathway was specifically inhibited by CU05-1189 upon VEGF stimulation, without affecting other VEGF receptor 2 downstream molecules or cytosolic substrates of PDK1, by preventing translocation of PDK1 to the plasma membrane. We also found that CU05-1189 suppressed VEGF-mediated vascular network formation in a Matrigel plug assay. More importantly, CU05-1189 had a good pharmacokinetic profile with a bioavailability of 68%. These results led to the oral administration of CU05-1189, which resulted in reduced tumor microvessel density and growth in a xenograft mouse model. Taken together, our data suggest that CU05-1189 may have great potential and be a promising lead as a novel antiangiogenic agent for cancer treatment.

Abstract Background Age-related changes in the cerebrovasculature, including blood–brain barrier (BBB) disruption and vascular dementia, are emerging as potential risks for many neurodegenerative diseases. Therefore, the endothelial cells that constitute the cerebrovasculature may play key roles in preventing brain injury. Our previous study showed that CU06-1004, an endothelial cell dysfunction blocker, prevented vascular leakage, enhanced vascular integrity in ischemic reperfusion injury, and promoted the normalization of tumor vasculature. Here, we evaluated the effects of CU06-1004 on age-related cerebrovascular functional decline in the aged mouse brain. Results In this study, we investigated the protective effects of CU06-1004 against oxidative stress–induced damage in human brain microvascular endothelial cells (HBMECs). HBMECs were treated with hydrogen peroxide (H2O2) to establish an oxidative stress–induced model of cellular injury. Compared with H2O2 treatment alone, pretreatment of HBMECs with CU06-1004 considerably reduced oxidative stress–induced cytotoxicity, reactive oxygen species generation, senescence-associated β-galactosidase activity, senescence marker expression, and the expression levels of inflammatory proteins. Based on the observed cytoprotective effects of CU06-1004 in HBMECs, we examined whether CU06-1004 displayed protective effects against cerebrovascular aging in mice. Long-term administration of CU06-1004 alleviated age-associated cerebral microvascular rarefaction and cerebrovascular senescence in the aged mouse brain. CU06-1004 supplementation also reduced the extravasation of plasma IgG by improving BBB integrity in the aged mouse brain, associated with reductions in neuronal injury. A series of behavioral tests also revealed improved motor and cognitive functions in aged mice that received long-term CU06-1004 administration. Conclusions These findings suggest that CU06-1004 may represent a promising therapeutic approach for delaying age-related cerebrovascular impairment and improving cognitive function in old age.

The stress response protein REDD1 is regulates inflammation and energy metabolism. Here the authors report that global or adipocyte-specific deletion of REDD1 inhibits diet induced obesity, insulin resistance, liver steatosis and inflammation in mice, at least in part via reduced atypical NF-κB activation.

Background: Interleukin-2 (IL-2) is the first cancer therapeutic agent with an immunomodulatory function. Although it has been experimentally proven to be effective against metastatic renal cell carcinoma and metastatic melanoma, the clinical application of high-dose IL-2 (HDIL-2) has been limited because of its short half-life and severe side effects, such as vascular leakage syndrome (VLS) or capillary leaky syndrome (CLS). However, methods for overcoming this issue have not yet been identified.Methods: We discovered CU06-1004, an endothelial dysfunction blocker, through a previous study, and co-treated with IL-2 immunotherapy to confirm its inhibitory effect on HDIL-2-induced endothelial permeability. CU06-1004 was co-administered with HDIL-2 for 4 days in an in vivo mouse model. After drug injection, the mice were sacrificed, and Evans blue staining was performed.Results:In vitro, HDIL-2 treatment decreased HUVEC stability, which was rescued by co-treatment with CU06-1004. In our mouse model, co-administration of CU06-1004 and HDIL-2 prevented HDIL-2-induced vascular leakage by normalizing endothelial cells. Notably, the HDIL-2 and CU06-1004 combination therapy considerably reduced tumor growth in the B16F10 melanoma mouse model.Conclusion: Our data suggest that CU06-1004 acts as a potential anticancer drug candidate, not only by preventing HDIL-2-induced VLS but also by enhancing the anticancer effects of HDIL-2 immunotherapy.

Abstract Background In sprouting angiogenesis, VEGFR2 level is regulated via a fine-tuned process involving various signaling pathways. Other than VEGF/VEGFR2 signaling pathway, Wnt/ β-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/ β-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/β-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.

Background: Korean Red ginseng extract (KRGE) has beneficial effects on the cardiovascular system by improving endothelial cell function. However, its pharmacological effect on endothelial cell senescence has not been clearly elucidated. Therefore, we examined the effect and molecular mechanism of KRGE on the senescence of human umbilical vein endothelial cells (HUVECs). Methods: HUVECs were grown in normal or KRGE-supplemented medium. Furthermore, they were transfected with heme oxygenase-1 (HO-1) gene or treated with its inhibitor, a NF-κB inhibitor, and a miR-155-5p mimic or inhibitor. Senescence-associated characteristics of endothelial cells were determined by biochemical and immunohistochemical analyses. Results: Treatment of HUVECs with KRGE resulted in delayed onset and progression of senescence-associated characteristics, such as increased lysosomal acidic β-galactosidase and decreased telomerase activity, angiogenic dysfunction, and abnormal cell morphology. KRGE preserved the levels of anti-senescent factors, such as eNOS-derived NO, MnSOD, and cyclins D and A: however, it decreased the levels of senescence-promoting factors, such as ROS, activated NF-κB, endothelial cell inflammation, and p21 expression. The beneficial effects of KRGE were due to the induction of HO-1 and the inhibition of NF-κB-dependent biogenesis of miR-155-5p that led to the downregulation of eNOS. Moreover, treatment with inhibitors of HO-1, NF-κB, and miR-155-5p abolished the anti-senescence effects of KRGE. Conclusion: KRGE delayed or prevented HUVEC senescence through a signaling cascade involving the induction of HO-1, the inhibition of NF-κB-dependent miR-155-5p biogenesis, and the maintenance of the eNOS/NO axis activity, suggesting that it may protect against vascular diseases associated with endothelial senescence.

Inflammatory vascular disease: miRNAs in blood vessel damage miRNA molecules that inhibit the activity of specific genes are implicated in a cellular control network involved in some of the damaging effects of inflammation on blood vessels. Researchers in South Korea led by Young-Myeong Kim at Kangwon National University School of Medicine, Chuncheon, identified the link by studying cells from patients with the inflammatory condition pre-eclampsia, characterized by hypertension. They found that two miRNAs, miR-31 and miR-155, are involved in molecular signaling processes that impair the production of endothelial progenitor cells lining blood vessels, which is essential for maintenance and repair. The research also identified the key protein eNOS involved in the miRNA molecules’ mechanism of action. Understanding these miRNAs and the protein involved in their production and action may help researchers develop new treatments for blood vessel diseases associated with inflammation.

Abstract 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.

Vascular disease: Small RNA as a possible culprit The overexpression of a microRNA molecule adversely affects the functioning of vascular smooth muscle cells (VSMCs) and may contribute to the development of artherosclerosis and preeclampsia. The interactions between VSMCs and the cells lining blood vessels (endothelium) are crucial for maintaining the healthy phenotype and relaxation of blood vessels. Disruption to these interactions via inflammation, for example, can trigger serious vascular diseases. Young-Myeong Kim at Kangwon National University, Chungcheon, South Korea, and co-workers demonstrated that expression levels of a microRNA-155 are elevated in patients with artherosclerosis and preeclampsia, while an enzyme found in VSMCs called soluble guanylyl cyclase is considerably reduced. Using human and mice tissues, the team showed that miR-155 impairs the contractile phenotype and relaxation of VSMCs by reducing guanylyl cyclase expression. Their findings may inform new therapies for vascular diseases.

Endothelial barrier integrity is important for vascular homeostasis, and hyperpermeability participates in the progression of many pathological states, such as diabetic retinopathy, ischemic stroke, chronic bowel disease, and inflammatory disease. Here, using drug repositioning, we discovered that primaquine diphosphate (PD), previously known as an antimalarial drug, was a potential blocker of vascular leakage. PD inhibited the linear pattern of vascular endothelial growth factors (VEGF)-induced disruption at the cell boundaries, blocked the formation of VEGF-induced actin stress fibers, and stabilized the cortactin actin rings in endothelial cells. PD significantly reduced leakage in the Miles assay and mouse model of streptozotocin (STZ)-induced diabetic retinopathy. Targeted prediction programs and deubiquitinating enzyme activity assays identified a potential mechanism of action for PD and demonstrated that this operates via ubiquitin specific protease 1 (USP1). USP1 inhibition demonstrated a conserved barrier function by inhibiting VEGF-induced leakage in endothelial permeability assays. Taken together, these findings suggest that PD could be used as a novel drug for vascular leakage by maintaining endothelial integrity.

Blocking the immune evasion mechanism of tumor cells has become an attractive means for treating cancers. However, the usage of a drug such as nivolumab (αPD-1), which blocks programmed cell death protein 1 (PD-1), turned out to be only effective against certain types of cancer. Especially, vascular abnormal structures of which deter delivery route by leakage and cause the poor perfusion were considered to be environment unfavorable to T cells and immune checkpoint blockade (ICB) delivery within the tumor microenvironment (TME). Herein, we report stabilization of tumor blood vessels by endothelial dysfunctional blocker CU06-1004, which modified the TME and showed synergistic effects with immunotherapy anti-PD-1 antibody. CU06-1004 combination therapy consistently prolonged the survival of tumor-bearing mice by decreasing tumor growth. T-cell infiltration increased in the tumors of the combination group, with cytotoxic CD8+ T cell activity within the tumor parenchyma upregulated compared with anti-PD-1 monotherapy. Tumor inhibition was associated with reduced hypoxia and reduced vessel density in the central region of the tumor. These effects correlated significantly with enhanced expression of IFN gamma and PD-L1 in tumors. Taken together, our findings suggest that CU06-1004 is a potential candidate drug capable of improving therapeutic efficacy of anti-PD-1 through beneficial changes in the TME.

[This corrects the article DOI: 10.1371/journal.pone.0243497.].

Inflammatory bowel disease is an autoimmune disease that causes chronic inflammation of the gastrointestinal tract. Endothelial dysfunction, defined by a reduced endothelial barrier and an increase in the expression of adhesion molecules, is part of the pathology of inflammatory bowel disease. In this study, we assessed the therapeutic effect of CU06-1004, an endothelial dysfunction blocker that reduces vascular hyperpermeability and inflammation in a mouse model of colitis. Acute colitis was induced in mice using 3% (w/v) dextran sodium sulfate added to their drinking water for 7 days. Twenty-four hours after the addition of dextran sodium sulfate, either mesalazine or CU06-1004 was administered orally each day. Administration of CU06-1004 significantly reduced the clinical manifestations (weight loss, diarrhea, and bloody stool) and histological changes (epithelium loss, inflammatory cell infiltration, and crypt destruction) induced by dextran sodium sulfate. Proinflammatory cytokines were also reduced, indicating that inflammation was ameliorated. From a vascular perspective, CU06-1004 reduced interrupted and tortuous vessels, enhanced junction protein expression, and reduced inflammatory adhesion molecules, indicating a broad improvement of endothelial dysfunction. Endothelial protection induced epithelial barrier restoration and decreased epithelial inflammation. Blocking endothelial dysfunction with CU06-1004 significantly ameliorated the progression of inflammatory bowel disease. Therefore, CU06-1004 may represent a potential therapeutic agent for the treatment of inflammatory bowel disease as well as other inflammatory diseases.

Non-alcoholic steatohepatitis (NASH) is a severe, advanced form of non-alcoholic fatty liver disease (NAFLD) that is associated with features of metabolic syndrome and characterized by hepatic steatosis, inflammation, and fibrosis. In addition, NASH is associated with endothelial dysfunction within the hepatic vasculature. Treatment with CU06-1004 (previously called Sac-1004) ameliorates endothelial dysfunction by inhibiting hyperpermeability and inflammation. In this study, we investigated the protective effects of CU06-1004 in a choline-deficient L-amino acid (CDAA)-induced mouse model of NASH for 3 or 6 weeks. Specifically, we evaluated the effects of CU06-1004 on lipid accumulation, inflammation, hepatic fibrosis, and liver sinusoidal endothelial cell (LSEC) capillarization through biochemical analysis, immunohistochemistry, and real-time PCR. We found that the administration of CU06-1004 to mice improved liver triglyceride (TG) and serum alanine aminotransferase (ALT) in this CDAA-induced model of NASH for 6 weeks. In groups of NASH induced mice for both 3 and 6 weeks, CU06-1004 significantly reduced the hepatic expression of genes related to lipogenesis, inflammation, and cell adhesion. However, expression of genes related to hepatic fibrosis and vascular endothelial changes were only decreased in animals with mild NASH. These results suggest that the administration of CU06-1004 suppresses hepatic steatosis, inflammation, fibrosis, and LSEC capillarization in a CDAA-induced mouse model of NASH. This suggests that CU06-1004 has therapeutic potential for the treatment of mild NASH.

Abstract Penile erection is a neurovascular event and neurologic or vascular disturbances are major causes of erectile dysfunction (ED). Radical prostatectomy for prostate cancer not only induces cavernous nerve injury (CNI) but also results in cavernous angiopathy, which is responsible for poor responsiveness to oral phosphodiesterase-5 inhibitors. Dickkopf2 (DKK2) is known as a Wnt signaling antagonist and is reported to promote mature and stable blood vessel formation. Here, we demonstrated in CNI mice that overexpression of DKK2 by administering DKK2 protein or by using DKK2-Tg mice successfully restored erectile function: this recovery was accompanied by enhanced neural regeneration through the secretion of neurotrophic factors, and restoration of cavernous endothelial cell and pericyte content. DKK2 protein also promoted neurite outgrowth in an ex vivo major pelvic ganglion culture experiment and enhanced tube formation in primary cultured mouse cavernous endothelial cells and pericytes co-culture system in vitro. In light of critical role of neuropathy and angiopathy in the pathogenesis of radical prostatectomy-induced ED, reprogramming of damaged erectile tissue toward neurovascular repair by use of a DKK2 therapeutic protein may represent viable treatment option for this condition.

Abstract Background Blood–brain barrier (BBB) breakdown and inflammation are critical events in ischemic stroke, contributing to aggravated brain damage. The BBB mainly consists of microvascular endothelial cells sealed by tight junctions to protect the brain from blood-borne substances. Thus, the maintenance of BBB integrity may be a potential target for neuroprotection. Sac-1004, a pseudo-sugar derivative of cholesterol, enhances the endothelial barrier by the stabilization of the cortical actin ring. Results Here, we report on the protective effects of Sac-1004 on cerebral ischemia-reperfusion (I/R) injury. Treatment with Sac-1004 significantly blocked the interleukin-1β-induced monolayer hyperpermeability of human brain microvascular endothelial cells (HBMECs), loss of tight junctions, and formation of actin stress fiber. Sac-1004 suppressed the expression of adhesion molecules, adhesion of U937 cells, and activation of nuclear factor-κB in HBMECs. Using a rat model of transient focal cerebral ischemia, it was shown that Sac-1004 effectively ameliorated neurological deficits and ischemic damage. In addition, Sac-1004 decreased BBB leakage and rescued tight junction-related proteins. Moreover, the staining of CD11b and glial fibrillary acidic protein showed that Sac-1004 inhibited glial activation. Conclusions Taken together, these results demonstrate that Sac-1004 has neuroprotective activities through maintaining BBB integrity, suggesting that it is a great therapeutic candidate for stroke.

Preeclampsia (PE) is a pregnancy-specific disorder associated with hypertension and proteinuria. Since there is no proven method to treat PE, early prediction and accurate diagnosis are essential for appropriate management of the disease. Thus, reliable biomarkers for diagnosing PE need to be identified and evaluated. We analyzed serum-soluble factors and miRNAs in 92 patients with PE and an equal number of healthy controls to identify new useful biomarkers for PE. Serum miR-31-5p, miR-155-5p, and miR-214-3p levels were significantly elevated in these patients and highly correlated with clinical symptoms of hypertension and proteinuria, whereas the miR-1290-3p level was decreased. The increased miRNAs were upregulated in an NF-κB-dependent manner and suppressed endothelial nitric oxide synthase (eNOS) and placental growth factor (PlGF) expression. The level of each miRNA had greater than 90% diagnostic accuracy, which was further increased by analyzing its ratio relative to that of miR-1290-3p. Taken together, the ratios of miR-31-5p, miR-155-5p, or miR-214-3p to miR-1290-3p may serve as reliable diagnostic or prognostic tools for PE, thereby providing evidence that these miRNAs are promising mechanism-based targets for designing therapeutic and preventive strategies for the clinical management of PE.

Background Arginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase (eNOS). We tested the hypotheses that arginase II activity participates in the regulation of Ca2+/Ca2+/calmodulin‐dependent kinase II/eNOS activation, and this process is dependent on mitochondrial p32. Methods and Results Downregulation of arginase II increased the concentration of cytosolic Ca2+ ([Ca2+]c) and decreased mitochondrial Ca2+ ([Ca2+]m) in microscopic and fluorescence‐activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca2+/Ca2+/calmodulin‐dependent kinase II. These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca2+ from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II‐null mice resulted in reduced p32 protein level. In the aortas of wild‐type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO‐dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca2+]m were increased in the aortas from apolipoprotein E (ApoE−/−) mice fed a high‐cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca2+/Ca2+/calmodulin‐dependent kinase II‐dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice. Conclusions These data demonstrate a novel function of arginase II in regulation of Ca2+‐dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.

Heme oxygenase-1 (HO-1) exerts beneficial effects, including angiogenesis and energy metabolism via the hypoxia-inducible factor-1α (HIF-1α) and peroxisome-proliferator-activating receptor-γ coactivator-1α (PGC-1α)/estrogen-related receptor α (ERRα) pathways, respectively, in astrocytes. However, evidence of cross-talk between both pathways in HO metabolite-mediated mitochondrial biogenesis has not been well elucidated. Here, we found that HIF-1α was upregulated in astrocytes after ischemic brain injury following exposure to the carbon monoxide (CO)-releasing compound CORM-2. Experiments with pharmacological inhibitors and target-specific siRNAs revealed that HIF-1α levels were highly correlated with increased PGC-1α and ERRα levels, which were linked to the HO metabolites CO- and bilirubin-induced activation of apical L-type Ca2+ channel and sequential Ca2+-dependent signal transduction. Moreover, HIF-1α was stabilized in a proline hydroxylase-dependent manner by transient induction of intracellular hypoxia via the PGC-1α/ERRα-induced increases in mitochondrial biogenesis and oxygen consumption. HIF-1α knockdown blocked HO-1 system-mediated transcriptional expression of ERRα, but not of PGC-1α, suggesting a possible involvement of HIF-1α in ERRα-mediated mitochondrial biogenesis. These data suggest that the HO-1-derived metabolites, CO and bilirubin, elevate astrocytic mitochondrial function via a HIF-1α/ERRα circuit coupled with L-type Ca2+ channel activation and PGC-1α-mediated oxygen consumption. This circuit may play an important role in repairing neurovascular function after focal ischemic brain injury by stimulating mitochondrial biogenesis.

Vascular branching morphogenesis is activated and maintained by several signaling pathways. Among them, vascular endothelial growth factor receptor 2 (VEGFR2) signaling is largely presented in arteries, and VEGFR3 signaling is in veins and capillaries. Recent reports have documented that Snail, a well-known epithelial-to-mesenchymal transition protein, is expressed in endothelial cells, where it regulates sprouting angiogenesis and embryonic vascular development. Here, we identified Snail as a regulator of VEGFR3 expression during capillary branching morphogenesis. Snail was dramatically upregulated in sprouting vessels in the developing retinal vasculature, including the leading-edged vessels and vertical sprouting vessels for capillary extension toward the deep retina. Results from in vitro functional studies demonstrate that Snail expression colocalized with VEGFR3 and upregulated VEGFR3 mRNA by directly binding to the VEGFR3 promoter via cooperating with early growth response protein-1. Snail knockdown in postnatal mice attenuated the formation of the deep capillary plexus, not only by impairing vertical sprouting vessels but also by downregulating VEGFR3 expression. Collectively, these data suggest that the Snail-VEGFR3 axis controls capillary extension, especially in vessels expressing VEGFR2 at low levels.

Stroke is the second leading cause of death. Experimental animal models of cerebral ischemia are widely used for researching mechanisms of ischemic damage and developing new drugs for the prevention and treatment of stroke. The present study aimed to comparatively investigate neuroprotective effects of aspirin (ASA), decursinol (DA) and new synthetic aspirin-decursinol adduct (ASA-DA) against transient focal and global cerebral ischemic damage. We found that treatment with 20 mg/kg, not 10 mg/kg, ASA-DA protected against ischemia-induced neuronal death after transient focal and global ischemic damage, and its neuroprotective effect was much better than that of ASA or DA alone. In addition, 20 mg/kg ASA-DA treatment reduced the ischemia-induced gliosis and maintained antioxidants levels in the corresponding injury regions. In brief, ASA-DA, a new synthetic drug, dramatically protected neurons from ischemic damage, and neuroprotective effects of ASA-DA may be closely related to the attenuation of ischemia-induced gliosis and maintenance of antioxidants.

Endothelial barrier integrity is essential for vascular homeostasis and increased vascular permeability and has been implicated in many pathological processes, including diabetic retinopathy. Here, we investigated the effect of Rk1, a ginsenoside extracted from sun ginseng, on regulation of endothelial barrier function. In human retinal endothelial cells, Rk1 strongly inhibited permeability induced by VEGF, advanced glycation end-product, thrombin, or histamine. Furthermore, Rk1 significantly reduced the vessel leakiness of retina in a diabetic mouse model. This anti-permeability activity of Rk1 is correlated with enhanced stability and positioning of tight junction proteins at the boundary between cells. Signaling experiments revealed that Rk1 induces phosphorylation of myosin light chain and cortactin, which are critical regulators for the formation of the cortical actin ring structure and endothelial barrier. These findings raise the possibility that ginsenoside Rk1 could be exploited as a novel prototype compound for the prevention of human diseases that are characterized by vascular leakage.

Aberrant activation of the Wnt pathway contributes to human cancer progression. Antagonists that interfere with Wnt ligand/receptor interactions can be useful in cancer treatments. In this study, we evaluated the therapeutic potential of a soluble Wnt receptor decoy in cancer gene therapy. We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells. In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes. Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition.

Background/Aims Inflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC), are chronic and recurring conditions with intricate pathogenesis, resulting in a lack of effective medications for therapy. The efficacy of CU104, a new blocker of endothelial dysfunction, in other IBD models with a CD-like phenotype has not been evaluated and its precise cellular or molecular targets in colitis remain unknown. Methods We evaluated the effectiveness of CU104 in treating dextran sodium sulfate (DSS)-induced colitis in IL-10 knockout (KO) mice, which spontaneously develope colitis. We also performed FITC-dextran permeability assay, TEER assay, NF-κB/IRF reporter assay, and gene expression profiling in Caco-2, HCT-116, and HT-29 cells. Results CU104 improved DSS-induced colitis in IL-10 KO mice. CU104 demonstrated potent suppressive effects on innate immune responses, dysfunction in vascular and intestinal barriers, and immune cell recruitment in these colitis models. Furthermore, our mechanistic studies revealed that CU104 inhibited the activation of the transcription factors nuclear factor kappa-light-chain-enhancer of activated B cells and interferon regulatory factor, as well as the ezrin/radixin/moesin (ERM) signaling pathway in vitro and in vivo. These components play critical roles in both maintaining the vascular and intestinal barriers and regulating immune cell recruitment during inflammation regulating actin dynamics via ERM pathway as well as inflammatory responses. Conclusion Collectively, our findings highlight potential therapeutic targets of CU104, a pseudo-sugar derivative of cholesterol that regulates actin dynamics as well as inflammatory signaling pathways, and present promising prospects as a novel therapeutic agent for treating CD along with UC. [ABSTRACT FROM AUTHOR]