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Supplementary Data from COMP-Ang1 Potentiates the Antitumor Activity of 5-Fluorouracil by Improving Tissue Perfusion in Murine Lewis Lung Carcinoma

Capsanthin is the main carotenoid compound in red paprika (Capsicum annuum L.). However, little is known about the beneficial effects of capsanthin in nonalcoholic fatty liver disease (NAFLD). In t...

Breast cancer is the most common cancer and the leading cause of cancer-related mortality among females worldwide. Triple-negative breast cancer (TNBC) accounts for about 10–15% of all breast cancers and is usually more aggressive and has a poorer prognosis. Sericite has been known to have antitumor and immune-stimulatory effects. Although the chemopreventive potential of sericite has been demonstrated in other cancers, its molecular pathways in TNBC still require investigation. Thus, in the present study, the antitumor mechanism of sericite against MDA-MB231 breast cancer cells was examined in vitro and in an in vivo xenograft mouse model. Sericite treatment reduced cell proliferation and cell proliferation marker proliferating cell nuclear antigen (PCNA) in MDA-MB231 cells. It also decreased the total cell number and arrested cells in the G0/G1 phase of the cell cycle with an increase in the phosphorylation of P53 and upregulation of cell cycle regulatory proteins P21 and P16. In addition, sericite treatment also induced apoptosis signaling, which was evident by the upregulation of apoptotic protein markers cleaved caspases 3 and 9. A reduction in reactive oxygen species (ROS), NADPH oxidase 4 (NOX4), p22phox, and heat shock proteins (HSPs) was also observed. Similar results were obtained in vivo with significantly reduced tumor volume in sericite-administered mice. Collectively, these findings suggest that sericite has antitumor potential based on its property to induce cell cycle arrest and apoptotic cell death and therefore could serve as a potential therapeutic agent and crucial candidate in anticancer drug development for TNBC.

Triple-negative breast cancer (TNBC) is characterized with hormone receptor negative and frequently exhibits inherently aggressive clinical behavior, resulting in poor prognoses. TNBCs are highly heterogenic, leading to obstacles in identifying new therapeutic targets and performing targeted therapy. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1), a multifunctional protein, is mainly involved in DNA repair and redox regulation; it is upregulated in various cancers and is extracellularly secreted because of hyperacetylation. We previously reported that hyperacetylation induced APE1/Ref-1 release causes apoptosis in TNBCs. In the current study, we aimed to assess the therapeutic efficacy of combination treatment involving recombinant human APE1/Ref-1 (rhAPE1/Ref-1) plus acetylsalicylic acid (ASA) in TNBC xenograft mice. ASA co-treatment was used to induce and lasting acetylation of rhAPE1/Ref-1. For in vivo analysis, we constructed an orthotopic xenograft in vivo model of TNBC by using MDA-MB-231 cells. We performed combination therapy involving intravenous injection of purified rhAPE1/Ref-1 (1 mg/kg) and oral administration of ASA (20 mg/kg) thrice a week for 6 weeks in TNBC xenograft mice. This rhAPE1/Ref-1 plus ASA combination therapy inhibited tumor formation and growth, without damaging liver or kidney tissue, and decreased plasma levels of the cancer marker CEA and breast cancer-specific markers CA27-29 and CA15-3 in the TNBC xenograft mice. The TNBC group, which underwent combination therapy, had more apoptotic cells and greater inflammatory cell infiltration in tumor tissues than the vehicle group did. Additionally, in tumor tissue of TNBC with combination therapy exhibits increased apoptosis following upregulation of Bax, cleaved-caspase-3, and cleaved-PARP. Thus, we found that rhAPE1/Ref-1 plus ASA combination therapy enhanced antitumor effects in TNBCs via inflammatory cell infiltration and apoptosis signaling pathways. Our findings indicate the therapeutic potential of rhAPE1/Ref-1 plus ASA combination therapy for TNBC. Citation Format: Yu Ran Lee, Hee Kyoung Joo, Eun-Ok Lee, Sungmin Kim, Hao Jin, Yeon Hee Choi, Eun Ju Choi, Byeong Hwa Jeon. Recombinant apurinic/apyrimidinic endonuclease-1/redox factor-1 plus acetylsalicylic acid combination therapy inhibits tumorigenesis in an orthotopic xenograft murine model of triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6124.

Arterial thrombosis and its associated diseases are considered to constitute a major healthcare problem. Arterial thrombosis, defined as blood clot formation in an artery that interrupts blood circulation, is associated with many cardiovascular diseases. Oxidative stress is one of many important factors that aggravates the pathophysiological process of arterial thrombosis. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1) has a multifunctional role in cells that includes the regulation of oxidative stress and anti-inflammatory function. The aim of this study was to investigate the therapeutic effect of adenovirus-mediated Ref-1 overexpression on arterial thrombosis induced by 60% FeCl3 solution in rats. Blood flow was measured to detect the time to occlusion, thrombus formation was detected by hematoxylin and eosin staining, reactive oxygen species (ROS) levels were detected by high-performance liquid chromatography, and the expression of tissue factor and other proteins was detected by Western blot. FeCl3 aggravated thrombus formation in carotid arteries and reduced the time to artery occlusion. Ref-1 significantly delayed arterial obstruction via the inhibition of thrombus formation, especially by downregulating tissue factor expression through the Akt-GSK3β-NF-κB signaling pathway. Ref-1 also reduced the expression of vascular inflammation markers ICAM-1 and VCAM-1, and reduced the level of ROS that contributed to thrombus formation. The results showed that adenovirus-mediated Ref-1 overexpression reduced thrombus formation in the rat carotid artery. In summary, Ref-1 overexpression had anti-thrombotic effects in a carotid artery thrombosis model and could be a target for the treatment of arterial thrombosis.

In addition to producing a classical excitatory postsynaptic current via activation of synaptic NMDA receptors (NMDARs), glutamate in the brain also induces a tonic NMDAR current (INMDA) via activation of extrasynaptic NMDARs (eNMDARs). However, since Mg2+blocks NMDARs in nondepolarized neurons, the potential contribution of eNMDARs to the overall neuronal excitatory/inhibitory (E/I) balance remains unknown. Here, we demonstrate that chronic (7 d) salt loading (SL) recruited NR2D subunit-containing NMDARs to generate an Mg2+-resistant tonicINMDAin nondepolarized [Vh(holding potential) −70 mV] vasopressin (VP; but not oxytocin) supraoptic nucleus (SON) neurons in male rodents. Conversely, in euhydrated (EU) and 3 d SL mice, Mg2+-resistant tonicINMDAwas not observed. Pharmacological and genetic intervention of NR2D subunits blocked the Mg2+-resistant tonicINMDAin VP neurons under SL conditions, while an NR2B antagonist unveiled Mg2+-sensitive tonicINMDAbut not Mg2+-resistant tonicINMDA. In the EU group VP neurons, an Mg2+-resistant tonicINMDAwas not generated by increased ambient glutamate or treatment with coagonists (e.g.,d-serine and glycine). Chronic SL significantly increased NR2D expression but not NR2B expression in the SON relative to the EU group or after 3 d under SL conditions. Finally, Mg2+-resistant tonicINMDAselectively upregulated neuronal excitability in VP neurons under SL conditions, independent of ionotropic GABAergic input. Our results indicate that the activation of NR2D-containing NMDARs constitutes a novel mechanism that generates an Mg2+-resistant tonicINMDAin nondepolarized VP neurons, thus causing an E/I balance shift in VP neurons to compensate for the hormonal demands imposed by a chronic osmotic challenge.SIGNIFICANCE STATEMENTThe hypothalamic supraoptic nucleus (SON) consists of two different types of magnocellular neurosecretory cells (MNCs) that synthesize and release the following two peptide hormones: vasopressin (VP), which is necessary for regulation of fluid homeostasis; and oxytocin (OT), which plays a major role in lactation and parturition. NMDA receptors (NMDARs) play important roles in shaping neuronal firing patterns and hormone release from the SON MNCs in response to various physiological challenges. Our results show that prolonged (7 d) salt loading generated a Mg2+-resistant tonic NMDA current mediated by NR2D subunit-containing receptors, which efficiently activated nondepolarized VP (but not OT) neurons. Our findings support the hypothesis that NR2D subunit-containing NMDARs play an important adaptive role in adult brain in response to a sustained osmotic challenge.

Non-alcoholic fatty liver disease (NAFLD) is associated with hepatic metabolism dysfunction. However, the mechanistic role of miR204 in the development of NAFLD is unknown. We investigate the functional significance of miR204 in the evolution of NAFLD. IDH2 KO mice feed a normal diet (ND) or HFD increased body weight, epididymal fat-pad weight, lipid droplet in liver, blood parameter and inflammation compared to WT mice fed a ND or HFD. Moreover, the expression of miR204 is increased in mice with IDH2 deficiency. Increased miR204 by IDH2 deficiency regulates carnitine palmitoyltransferase 1a (cpt1a) synthesis, which inhibits fatty acid β-oxidation. Inhibition of miR204 prevents the disassembly of two fatty acid-related genes by activating CPT1a expression, which decreases lipid droplet in liver, inflammatory cytokines, epididymal fat pad weight, blood parameters. Increased miR204 by IDH2 deficiency promotes the pathogenesis of HFD-induced NAFLD by regulating hepatic fatty acid metabolism and inflammation.

Vascular endothelial cell senescence is an important cause of cardiac-related diseases. Mitochondrial reactive oxygen species (mtROS) have been implicated in cellular senescence and multiple cardiovascular disorders. CR6 interacting factor 1 (CRIF1) deficiency has been shown to increase mtROS via the inhibition of mitochondrial oxidative phosphorylation; however, the mechanisms by which mtROS regulates vascular endothelial senescence have not been thoroughly explored. The goal of this study was to investigate the effects of CRIF1 deficiency on endothelial senescence and to elucidate the underlying mechanisms. CRIF1 deficiency was shown to increase the activity of senescence-associated β-galactosidase along with increased expression of phosphorylated p53, p21, and p16 proteins. Cell cycle arrested in the G0/G1 phase were identified in CRIF1-deficient cells using the flow cytometry. Furthermore, CRIF1 deficiency was also shown to increase cellular senescence by reducing the expression of Sirtuin 3 (SIRT3) via ubiquitin-mediated degradation of transcription factors PGC1α and NRF2. Downregulation of CRIF1 also attenuated the function of mitochondrial antioxidant enzymes including manganese superoxide dismutase (MnSOD), Foxo3a, nicotinamide-adenine dinucleotide phosphate, and glutathione via the suppression of SIRT3. Interestingly, overexpression of SIRT3 in CRIF1-deficient endothelial cells not only reduced mtROS levels by elevating expression of the antioxidant enzyme MnSOD but also decreased the expression of cell senescence markers. Taken together, these results suggest that CRIF1 deficiency induces vascular endothelial cell senescence via ubiquitin-mediated degradation of the transcription coactivators PGC1α and NRF2, resulting in decreased expression of SIRT3.

Inhibition of mitochondrial protein CR6 interacting factor 1 (CRIF1) disturbs mitochondrial function, depolarizes membrane potential, and increases reactive oxygen species (ROS) levels in endothelial cells. Impaired mitochondrial function accompanied by oxidative damage is a major contributor to the initiation of mitophagy. We hypothesized that CRIF1 deficiency-induced harmful effects may promote mitophagy, and explored the mechanism underlying this effect in human umbilical vein endothelial cells (HUVECs). Our results showed that CRIF1 downregulation not only induced the mitophagy-related markers LC3 (LC3-II/Ⅰ), PTEN-induced putative kinase 1 (PINK1) and parkin, but also stimulated redox enzyme p66shc expression. Scavenging mitochondrial ROS markedly blunted the CRIF1 deficiency-induced increase in p66shc expression. In addition, knockdown of p66shc inhibited the CRIF1 deletion-triggered mitochondrial ROS increase, membrane potential depolarization, and mitochondrial fusion. The restoration of mitochondrial dysfunction by p66shc downregulation also decreased CRIF1 deficiency-induced mitophagy, by elevating the levels of LC3-II/Ⅰ, PINK1 and parkin. These findings suggest that CRIF1 deficiency induces mitophagy via p66shc-regulated ROS in endothelial cells.

Downregulation of CR6 interacting factor 1 (CRIF1) has been reported to induce mitochondrial dysfunction, resulting in reduced activity of endothelial nitric oxide synthase (eNOS) and NO production in endothelial cells. Tetrahydrobiopterin (BH4) is an important cofactor in regulating the balance between NO (eNOS coupling) and superoxide production (eNOS uncoupling). However, whether the decreased eNOS and NO production in CRIF1-deficient cells is associated with relative BH4 deficiency-induced eNOS uncoupling remains completely unknown. Our results showed that CRIF1 deficiency increased eNOS uncoupling and depleted levels of total biopterin and BH4 by reducing the enzymes of BH4 biosynthesis (GCH-1, PTS, SPR, and DHFR) in vivo and vitro, respectively. Supplementation of CRIF1-deficient cells with BH4 significantly increased the recovery of Akt and eNOS phosphorylation and NO synthesis. In addition, scavenging ROS with MitoTEMPO treatment replenished BH4 levels by elevating levels of GCH-1, PTS, and SPR, but with no effect on the level of DHFR. Downregulation of DHFR synthesis regulators p16 or p21 in CRIF1-deficient cells partially recovered the DHFR expression. In summary, CRIF1 deficiency inhibited BH4 biosynthesis and exacerbated eNOS uncoupling. This resulted in reduced NO production and increased oxidative stress, which contributes to endothelial dysfunction and is involved in the pathogenesis of cardiovascular diseases.

Syndecan-2 (SDC2), a cell-surface heparin sulfate proteoglycan of the glycocalyx, is mainly expressed in endothelial cells. Although oxidative stress and inflammatory mediators have been shown to mediate dysfunction of the glycocalyx, little is known about their role in vascular endothelial cells. In this study, we aimed to identify the mechanism that regulates SDC2 expression in isocitrate dehydrogenase 2 (IDH2)-deficient endothelial cells, and to investigate the effect of ulinastatin (UTI) on this mechanism. We showed that knockdown of IDH2 induced SDC2 expression in human umbilical vein endothelial cells (HUVECs). Matrix metalloproteinase 7 (MMP7) influences SDC2 expression. When IDH2 was downregulated, MMP7 expression was increased, as was TGF-β signaling, which regulates MMP7. Inhibition of MMP7 activity using MMP inhibitor II significantly reduced SDC2, suggesting that IDH2 mediated SDC2 expression via MMP7. Moreover, expression of SDC2 and MMP7, as well as TGF-β signaling, increased in response to IDH2 deficiency, and treatment with UTI reversed this increase. Similarly, the increase in SDC2, MMP7, and TGF-β signaling in the aorta of IDH2 knockout mice was reversed by UTI treatment. These findings suggest that IDH2 deficiency induces SDC2 expression via TGF-β and MMP7 signaling in endothelial cells.

Tonic extrasynaptic GABAA receptor (GABAA R) activation is under the tight control of tonic GABA release from astrocytes to maintain the brain's excitation/inhibition (E/I) balance; any slight E/I balance disturbance can cause serious pathological conditions including epileptic seizures. However, the pathophysiological role of tonic GABA release from astrocytes has not been tested in epileptic seizures. Here, we report that pharmacological or genetic intervention of the GABA-permeable Bestrophin-1 (Best1) channel prevented the generation of tonic GABA inhibition, disinhibiting CA1 pyramidal neuronal firing and augmenting seizure susceptibility in kainic acid (KA)-induced epileptic mice. Astrocyte-specific Best1 over-expression in KA-injected Best1 knockout mice fully restored the generation of tonic GABA inhibition and effectively suppressed seizure susceptibility. We demonstrate for the first time that tonic GABA from reactive astrocytes strongly contributes to the compensatory shift of E/I balance in epileptic hippocampi, serving as a good therapeutic target against altered E/I balance in epileptic seizures.

There is growing evidence that apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) regulates inflammatory responses. Rheumatoid arthritis (RA) is an autoimmune disease, which is characterized with synovitis and joint destruction. Therefore, this study was planned to investigate the relationship between APE1/Ref-1 and RA. Serum and synovial fluid (SF) were collected from 46 patients with RA, 45 patients with osteoarthritis (OA), and 30 healthy control (HC) patients. The concentration of APE1/Ref-1 in serum or SF was measured using the sandwich enzyme-linked immunosorbent assay (ELISA). The disease activity in RA patients was measured using the 28-joint disease activity score (DAS28). The serum APE1/Ref-1 levels in RA patients were significantly increased compared to HC and OA patients (0.44 ± 0.39 ng/mL for RA group vs. 0.19 ± 0.14 ng/mL for HC group, p &lt, 0.05 and vs. 0.19 ± 0.11 ng/mL for OA group, p &lt, 0.05). Likewise, the APE1/Ref-1 levels of SF in RA patients were also significantly increased compared to OA patients (0.68 ± 0.30 ng/mL for RA group vs. 0.31 ± 0.12 ng/mL for OA group, p &lt, 0.001). The APE1/Ref-1 concentration in SF of RA patients was positively correlated with DAS28. Thus, APE1/Ref-1 may reflect the joint inflammation and be associated with disease activity in RA.

Elevated plasma homocysteine levels can induce vascular endothelial dysfunction, however, the mechanisms regulating homocysteine metabolism in impaired endothelial cells are currently unclear. In this study, we deleted the essential mitoribosomal gene CR6 interacting factor 1 (CRIF1) in human umbilical vein endothelial cells (HUVECs) and mice to induce endothelial cell dysfunction, then, we monitored homocysteine accumulation. We found that CRIF1 downregulation caused significant increases in intracellular and plasma concentrations of homocysteine, which were associated with decreased levels of folate cycle intermediates such as 5-methyltetrahydrofolate (MTHF) and tetrahydrofolate (THF). Moreover, dihydrofolate reductase (DHFR), a key enzyme in folate-mediated metabolism, exhibited impaired activity and decreased protein expression in CRIF1 knockdown endothelial cells. Supplementation with folic acid did not restore DHFR expression levels or MTHF and homocysteine concentrations in endothelial cells with a CRIF1 deletion or DHFR knockdown. However, the overexpression of DHFR in CRIF1 knockdown endothelial cells resulted in decreased accumulation of homocysteine. Taken together, our findings suggest that CRIF1-deleted endothelial cells accumulated more homocysteine, compared with control cells, this was primarily mediated by the disruption of DHFR expression.

The vascular endothelium is an active tissue that plays a crucial role in the maintenance of vascular homeostasis [...]

Apurinic/apyrimidinic endonuclease-1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that can be secreted, and recently suggested as new biomarker for vascular inflammation. However, the endogenous hormones for APE1/Ref-1 secretion and its underlying mechanisms are not defined. Here, the effect of twelve endogenous hormones on APE1/Ref-1 secretion was screened in cultured vascular endothelial cells. The endogenous hormones that significantly increased APE1/Ref-1 secretion was 17β-estradiol (E2), 5?-dihydrotestosterone, progesterone, insulin, and insulin-like growth factor. The most potent hormone inducing APE1/Ref-1 secretion was E2, which in cultured endothelial cells, E2 for 24 h increased APE1/Ref-1 secretion level of 4.56 ± 1.16 ng/mL, compared to a basal secretion level of 0.09 ± 0.02 ng/mL. Among the estrogens, only E2 increased APE1/Ref-1 secretion, not estrone and estriol. Blood APE1/Ref-1 concentrations decreased in ovariectomized (OVX) mice but were significantly increased by the replacement of E2 (0.39 ± 0.09 ng/mL for OVX vs. 4.67 ± 0.53 ng/mL for OVX + E2). E2-induced APE1/Ref-1secretion was remarkably suppressed by the estrogen receptor (ER) blocker fulvestrant and intracellular Ca2+ chelator 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), suggesting E2-induced APE1/Ref-1 secretion was dependent on ER and intracellular calcium. E2-induced APE1/Ref-1 secretion was significantly inhibited by exosome inhibitor GW4869. Furthermore, APE1/Ref-1 level in CD63-positive exosome were increased by E2. Finally, fluorescence imaging data showed that APE1/Ref-1 co-localized with CD63-labled exosome in the cytoplasm of cells upon E2 treatment. Taken together, E2 was the most potent hormone for APE1/Ref-1 secretion, which appeared to occur through exosomes that were dependent on ER and intracellular Ca2+. Furthermore, hormonal effects should be considered when analyzing biomarkers for vascular inflammation.

Keloids are a type of aberrant skin scarring characterized by excessive accumulation of collagen and extracellular matrix (ECM), arising from uncontrolled wound healing responses. While typically non-pathogenic, keloids are occasionally regarded as a form of benign tumor. CR6-interacting factor 1 (CRIF1) is a well-known CR6/GADD45-interacting protein, that has both nuclear and mitochondrial functions, and also exerts regulatory effects on cell growth and apoptosis. In this study, cell proliferation, cell migration, collagen production and TGF-β signaling was compared between normal fibroblasts (NFs) and keloid fibroblasts (KFs). Subsequently, the effects of CRIF1 deficiency were investigated in both NFs and KFs. Cell proliferation, cell migration, collagen production and protein expressions of TGF-β, phosphorylation of Smad2 and Smad3 were all found to be higher in KFs compared to NFs. CRIF1 deficiency in NFs and KFs inhibited cell proliferation, migration, and collagen production. In addition, phosphorylation of Smad2 and Smad3, which are transcription factors of collagen, was decreased. In contrast, mRNA expression levels of Smad7 and SMURF2, two important inhibitory proteins of Smad2/3, were increased, suggesting that CRIF1 may regulate collagen production. CRIF1 deficiency decreases the proliferation and migration of KFs, thereby inhibiting their overgrowth via the transforming growth factor-β (TGF-β)/Smad pathway. CRIF1 may therefore represent a potential therapeutic target in keloid pathogenesis.

The CR6-interacting factor1 (CRIF1) mitochondrial protein is indispensable for peptide synthesis and oxidative phosphorylation. Cardiomyocyte-specific deletion of CRIF1 showed impaired mitochondrial function and cardiomyopathy. We developed an endothelial cell-specific CRIF1 deletion mouse to ascertain whether dysfunctional endothelial CRIF1 influences cardiac function and is mediated by the antioxidant protein sirtuin 1 (SIRT1). We also examined the effect of the potent SIRT1 activator SRT1720 on cardiac dysfunction. Mice with endothelial cell-specific CRIF1 deletion showed an increased heart-to-body weight ratio, increased lethality, and markedly reduced fractional shortening of the left ventricle, resulting in severe cardiac dysfunction. Moreover, endothelial cell-specific CRIF1 deletion resulted in mitochondrial dysfunction, reduced ATP levels, inflammation, and excessive oxidative stress in heart tissues, associated with decreased SIRT1 expression. Intraperitoneal injection of SRT1720 ameliorated cardiac dysfunction by activating endothelial nitric oxide synthase, reducing oxidative stress, and inhibiting inflammation. Furthermore, the decreased endothelial junction-associated protein zonula occludens-1 in CRIF1-deleted mice was significantly recovered after SRT1720 treatment. Our results suggest that endothelial CRIF1 plays an important role in maintaining cardiac function, and that SIRT1 induction could be a therapeutic strategy for endothelial dysfunction-induced cardiac dysfunction.

Hormone replacement therapy may cause various side effects, including enhancing the risk of cardiovascular disease (CVD) in postmenopausal women. Here, we investigated the effect of red clover and hop extract combination (RHEC) on estrogen receptor (ER) binding and endothelial function of human umbilical vein endothelial cells (HUVECs) to develop an herbal agent for reducing the risk of CVDs. In ER competitor assay, RHEC showed binding affinity toward ERα and ERβ with IC50 values of 5.92 µg/ml and 1.66 µg/ml, respectively. In HUVECs, RHEC significantly increased the cell viability and reduced the reactive oxygen species production against oxidative stress-induced damage. We also showed that RHEC increased the NO production through upregulating the endothelial nitric oxide synthase expression via ER activation in estrogen depleted condition. In particular, RHEC showed greater efficacy with increase in NO and decrease in endothelin-1 than red clover or hop treatment alone. Additionally, 0.3-0.5 mg/ml of RHEC-induced vasorelaxation of rat aortic rings precontracted by phenylephrine. PRACTICAL APPLICATIONS: Recently, a large interest has grown in the synergistic effects of phytochemicals for better therapies to treat various diseases. Red clover and hop are well-known edible plants which are widely used to help relieve postmenopausal symptoms including CVD. However, their combination has not been studied so far. For the first time, we demonstrated that RHEC, a new herbal combination comprising the extracts from red clover and hop, appeared to be effective in protection of endothelial function against oxidative stress and estrogen depletion. Therefore, RHEC could be a potent herbal agent for reducing the risk of endothelial damage.

Endothelial dysfunction promotes vascular inflammation by inducing the production of reactive oxygen species and adhesion molecules. Vascular inflammation plays a key role in the pathogenesis of vascular diseases and atherosclerotic disorders. However, whether there is an endogenous system that can participate in circulating immune surveillance or managing a balance in homeostasis is unclear. Apurinic/apyrimidinic endonuclease 1/redox factor-1 (henceforth referred to as APE1/Ref-1) is a multifunctional protein that can be secreted from cells. It functions as an apurinic/apyrimidinic endonuclease in the DNA base repair pathway and modulates redox status and several types of transcriptional factors, in addition to its anti-inflammatory activity. Recently, it was reported that the secretion of APE1/Ref-1 into the extracellular medium of cultured cells or its presence in the plasma can act as a serological biomarker for certain disorders. In this review, we summarize the possible biological functions of APE1/Ref-1 according to its subcellular localization or its extracellular secretions, as therapeutic targets for vascular inflammation and as a serologic biomarker.

Elevated expression of human enhancer filamentation 1 (HEF1; also known as NEDD9 or Cas-L) is an essential stimulus for the metastatic process of various solid tumors. This process requires HEF1 localization to focal adhesions (FAs). Although the association of HEF1 with FAs is considered to play a role in cancer cell migration, the mechanism targeting HEF1 to FAs remains unclear. Moreover, up-regulation of Polo-like kinase 1 (Plk1) positively correlates with human cancer metastasis, yet how Plk1 deregulation promotes metastasis remains elusive. Here, we report that casein kinase 1δ (CK1δ) phosphorylates HEF1 at Ser-780 and Thr-804 and that these phosphorylation events promote a physical interaction between Plk1 and HEF1. We found that this interaction is critical for HEF1 translocation to FAs and for inducing migration of HeLa cells. Plk1-docking phosphoepitopes were mapped/confirmed in HEF1 by various methods, including X-ray crystallography, and mutated for functional analysis in HeLa cells. In summary, our results reveal the role of a phosphorylation-dependent HEF1–Plk1 complex in HEF1 translocation to FAs to induce cell migration. Our findings provide critical mechanistic insights into the HEF1–Plk1 complex–dependent localization of HEF1 to FAs underlying the metastatic process and may therefore contribute to the development of new cancer therapies.

Background and Objectives: Propofol-based total intravenous anesthesia (TIVA) is presumed to have more favorable effects on the prognosis of patients with cancer compared with volatile inhaled anesthesia (VIA). We hypothesized that these anesthetics target plasma apurinic apyrimidinic endonuclease/redox effector factor-1 (APE1/Ref-1) as a possible mechanism of action. Materials and Methods: The plasma APE1/Ref-1 level was evaluated three times during surgery for cancer, i.e., before anesthesia, immediately after cancer resection, and finally, in the recovery room. Blood (3 cc) was drawn from the radial artery catheter, and plasma APE1/Ref-1 levels were compared according to measurement time and between the two groups. Spearman’s Rho correlation analysis was performed to determine relationships among body mass index, American Society of Anesthesiologists classification, age, sex, cancer type, and tumor-node-metastasis (TNM) stage. A total of 166 patients (VIA: 129; TIVA: 37) were enrolled. Results: Plasma APE1/Ref-1 level increased significantly (p = 0.028) after cancer resection compared with before surgery, but no significant difference was observed between anesthetics (p = 0.134). The post-resection plasma APE1/Ref-1 level showed a positive correlation with the NM stages, but not the T stage. Conclusions: The plasma APE1/Ref-1 level increased during surgery with more severe lymph node invasion, but there were no significant differences according to the anesthetics used.

In this study, we fabricated a nerve guide conduit (NGC) with nerve growth factor (NGF) gradient along the longitudinal direction by rolling a porous polycaprolactone membrane with NGF concentration gradient. The NGF immobilized on the membrane was continuously released for up to 35 days, and the released amount of the NGF from the membrane gradually increased from the proximal to distal NGF ends, which may allow a neurotrophic factor gradient in the tubular NGC for a sufficient period. From the in vitro cell culture experiment, it was observed that the PC12 cells sense the NGF concentration gradient on the membrane for the cell proliferation and differentiation. From the in vivo animal experiment using a long gap (20 mm) sciatic nerve defect model of rats, the NGC with NGF concentration gradient allowed more rapid nerve regeneration through the NGC than the NGC itself and NGC immobilized with uniformly distributed NGF. The NGC with NGF concentration gradient seems to be a promising strategy for the peripheral nerve regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 52-64, 2018.

Far-infrared ray (FIR) therapy has been reported to exert beneficial effects on cardiovascular function by elevating endothelial nitric oxide synthesis (eNOS) activity and nitric oxide (NO) production. Tetrahydrobiopterin (BH4) is a key determinant of eNOS-dependent NO synthesis in vascular endothelial cells. However, whether BH4 synthesis is associated with the effects of FIR on eNOS/NO production has not yet been investigated. In this study, we investigated the effects of FIR on BH4-dependent eNOS/NO production and vascular function. We used FIR-emitting sericite boards as an experimental material and placed human umbilical vein endothelial cells (HUVECs) and Sprague–Dawley rats on the boards with or without FIR irradiation and then evaluated vascular relaxation by detecting NO generation, BH4 synthesis, and Akt/eNOS activation. Our results showed that FIR radiation significantly enhanced Akt/eNOS phosphorylation and NO production in human endothelial cells and aorta tissues. FIR can also induce BH4 storage by elevating levels of enzymes (e.g., guanosine triphosphate cyclohydrolase-1, 6-pyruvoyl tetrahydrobiopterin synthase, sepiapterin reductase, and dihydrofolate reductase), which ultimately results in NO production. These results indicate that FIR upregulated eNOS-dependent NO generation via BH4 synthesis and Akt phosphorylation, which contributes to the regulation of vascular function. This might develop potential clinical application of FIR to treat vascular diseases by augmenting the BH4/NO pathway.

Apurinic/apyrimidinic endonuclease/redox factor-1 (Ref-1), a multifunctional protein secreted from stimulated cells, has been identified as a new serological biomarker. Despite recent reports on the role of Ref-1 in inflammation, the biological function of secreted Ref-1 remains unknown, especially in vivo. This study aimed to evaluate the possible roles of secreted Ref-1 in lipopolysaccharide-induced systemic inflammation in vivo. We generated a secretory Ref-1 adenoviral vector system, AdPPT-LS-Ref-1, by conjugation of preprotrypsin leading sequence (PPT-LS) with full-length Ref-1 sequences. Expression of tumor necrosis factor-α (TNF-α)-induced vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells and lipopolysaccharide (LPS)-induced cyclooxygenase-2 in Raw264.7 cells was inhibited by secretory Ref-1, and this inhibitory effect was abrogated following neutralization of Ref-1 with anti-Ref-1 antibody. Plasma Ref-1 levels following administration of AdPPT-LS-Ref-1 (2 × 109 ifu, i.p.) for 24 h were substantially higher than those recorded following administration of Adβgal (84.6 ± 7.2 ng/ml vs. 4.4 ± 1.5 ng/ml). Treatment with LPS (10 mg/kg, i.v. for 6 h) markedly increased VCAM-1 expression, cathepsin or myeloperoxidase activity, which were significantly suppressed by treatment with AdPPT-LS-Ref-1. Furthermore, LPS-induced cytokines, such as TNF-α, interleukin (IL)-1β, IL-6, and monocyte chemoattractant protein 1, were significantly inhibited in AdPPT-LS-Ref-1-treated mice. However, LPS-induced myeloperoxidase activities were not suppressed by treatment with the redox mutant of secretory Ref-1, AdPPT-LS-Ref-1(C65A/C93A), or wild-type AdRef-1. Collectively, these results suggest that secreted Ref-1 has anti-inflammatory properties and that its redox cysteine residue is associated with the anti-inflammatory activity in vivo. Furthermore, our findings indicate that secretory Ref-1 may be useful as a therapeutic biomolecule against systemic inflammation.

Background/objectives Curcuma zedoaria R. (Zingiberaceae) has been used to treat headache, fever, and hypertension-related symptoms in Asian countries, including Korea, China, and Japan. We investigated whether dietary intake of a C. zedoaria extract (CzE) affected atherosclerosis in vivo. Materials/methods Apolipoprotein E-deficient (ApoE-/-) mice (n = 32) were fed a normal diet (ND), a high-cholesterol diet (HCD), an HCD containing CzE (100 mg/kg/day), or an HCD containing simvastatin (10 mg/kg/day) for 12 weeks. The anti-atherosclerotic effects were evaluated by observing changes in fatty streak lesions, immunohistochemical analysis, ex vivo fluorescence imaging, lipid profiles, and western blot analysis. Results The CzE-fed group showed a 41.6% reduction of atherosclerosis. Furthermore, CzE significantly reduced the levels of serum triglyceride, high-density lipoprotein, the chemokine (C-X3-C-motif) ligand 1, the adhesion molecules vascular cell adhesion molecule-1, intracellular adhesion molecule-1, and E-selectin; down-regulation of tumor necrosis factor-α, interleukin-6, high mobility group box-1, and cathepsin levels in the aortic sinuses and aortas of ApoE-/- mice were also observed. Conclusions The results suggest that the inclusion of a water extract of C. zedoaria in a HCD is closely correlated with reducing the risk of vascular inflammatory diseases in an ApoE mouse model.

Background: Paraplegia is a devastating complication following operations on the thoracoabdominal aorta. We investigated whether histidine-tryptophan-ketoglutarate (HTK) solution could reduce the extent of ischemia/reperfusion (IR) spinal cord injuries in a rat model using a direct delivery method. Methods: Twenty-four Sprague-Dawley male rats were randomly divided into four groups. The sham group (n=6) underwent a sham operation, the IR group (n=6) underwent only an aortic occlusion, the saline infusion group (saline group, n=6) underwent an aortic occlusion and direct infusion of cold saline into the occluded aortic segment, and the HTK infusion group (HTK group, n=6) underwent an aortic occlusion and direct infusion of cold HTK solution into the occluded aortic segment. An IR spinal cord injury was induced by transabdominal clamping of the aorta distally to the left renal artery and proximally to the aortic bifurcation for 60 minutes. A neurological evaluation of locomotor function was performed using the modified Tarlov score after 48 hours of reperfusion. The spinal cord was harvested for histopathological and immunohistochemical examinations. Results: The spinal cord IR model using direct drug delivery in rats was highly reproducible. The Tarlov score was 4.0 in the sham group, 1.17±0.75 in the IR group, 1.33±1.03 in the saline group, and 2.67±0.81 in the HTK group (p=0.04). The histopathological analysis of the HTK group showed reduced neuronal cell death. Conclusion: Direct infusion of cold HTK solution into the occluded aortic segment may reduce the extent of spinal cord injuries in an IR model in rats.

Nafamostat mesilate (NM), a synthetic serine protease inhibitor, has anticoagulant and anti-inflammatory properties. The intracellular mediator and external anti-inflammatory external signal in the vascular wall have been reported to protect endothelial cells, in part due to nitric oxide (NO) production. This study was designed to examine whether NM exhibit endothelium dependent vascular relaxation through Akt/endothelial nitric oxide synthase (eNOS) activation and generation of NO. NM enhanced Akt/eNOS phosphorylation and NO production in a dose- and time-dependent manner in human umbilical vein endothelial cells (HUVECs) and aorta tissues obtained from rats treated with various concentrations of NM. NM concomitantly decreased arginase activity, which could increase the available arginine substrate for NO production. Moreover, we investigated whether NM increased NO bioavailability and decreased aortic relaxation response to an eNOS inhibitor in the aorta. These results suggest that NM increases NO generation via the Akt/eNOS signaling pathway, leading to endothelium-dependent vascular relaxation. Therefore, the vasorelaxing action of NM may contribute to the regulation of cardiovascular function.

Nafamostat mesilate (NM), a serine protease inhibitor, has a broad range of clinical applications that include use as an anticoagulant during hemodialysis in cerebral hemorrhage patients, as a hemoperfusion anticoagulant for patients with intravascular coagulation, hemorrhagic lesions, and hemorrhagic tendencies, and for the improvement of acute pancreatitis. However, the effects of NM on acute cerebral ischemia have yet to be investigated. Thus, the present study utilized a rat model in which transient middle cerebral artery occlusion (MCAO) was used to induce ischemic injury to investigate the effects of NM on infarct volume and histological and biological changes. NM (1mg/kg) was intravenously administered prior to and after the MCAO procedure. Compared to control rats, the administration of NM significantly decreased infarct size and the extent of brain edema after the induction of focal ischemia via MCAO. Additionally, NM treatment attenuated MCAO-induced neuronal degeneration and activation of microglia and astrocytes. NM treatment also inhibited the MCAO-induced expression levels of glucose-regulated protein 78 (GRP78), CATT/EBP homologous protein (CHOP), and p-eukaryotic initiation factor 2α (eIF2α), which are endoplasmic reticulum (ER) stress markers, in the cerebral cortex. The present findings demonstrate that NM exerts neuroprotective effects in the brain following focal ischemia via, at least in part, the inhibition of ER stress.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is involved in DNA base repair and reducing activity. However, the role of APE1/Ref-1 in atherosclerosis is unclear. Herein, we investigated the role of APE1/Ref-1 in atherosclerotic apolipoprotein E (ApoE&minus, /&minus, ) mice fed with a Western-type diet. We found that serologic APE1/Ref-1 was strongly correlated with vascular inflammation in these mice. Neutrophil/lymphocyte ratio (NLR), endothelial cell/macrophage activation, and atherosclerotic plaque formation, reflected by atherosclerotic inflammation, were increased in the ApoE&minus, mice fed with a Western-type diet. APE1/Ref-1 expression was upregulated in aortic tissues of these mice, and was co-localized with cells positive for cluster of differentiation 31 (CD31) and galectin-3, suggesting endothelial cell/macrophage expression of APE1/Ref-1. Interestingly, APE1/Ref-1 plasma levels of ApoE&minus, mice fed with a Western-type diet were significantly increased compared with those of the mice fed with normal diet (15.76 &plusmn, 3.19 ng/mL vs. 3.51 &plusmn, 0.50 ng/mL, p &lt, 0.05), and were suppressed by atorvastatin administration. Correlation analysis showed high correlation between plasma APE1/Ref-1 levels and NLR, a marker of systemic inflammation. The cut-off value for APE1/Ref-1 for predicting atherosclerotic inflammation at 4.903 ng/mL showed sensitivity of 100% and specificity of 91%. We conclude that APE1/Ref-1 expression is upregulated in aortic endothelial cells/macrophages of atherosclerotic mice, and that plasma APE1/Ref-1 levels could predict atherosclerotic inflammation.

Cenobamate is a novel antiepileptic drug under investigation for use in patients with focal (partial-onset) seizures. To understand its potential molecular mechanism of action, the effects of cenobamate on GABAA-mediated currents and GABAA receptors in rodent hippocampal neurons were examined. Cenobamate potentiated GABA-induced currents (IGABA) in acutely isolated CA3 pyramidal cells in a concentration-dependent manner (EC50, 164 μM), which was not affected by flumazenil, a benzodiazepine receptor antagonist. Cenobamate enhanced tonic GABAA currents (Itonic), which is defined as a holding current shift by the GABAA receptor antagonist bicuculline (EC50, 36.63 μM). At therapeutically relevant concentrations, cenobamate induced minimal changes in the frequency, amplitudes, and decay time of spontaneous inhibitory postsynaptic currents in the CA1 neurons. Flumazenil failed to affect cenobamate-potentiated Itonic and Iphasic in CA1 neurons. Cenobamate showed positive allosteric modulation of GABA-induced IGABA mediated by GABAA receptors. This effect was similar for all tested hGABAA receptors containing six different alpha subunits (α1β2γ2 or α2-6β3γ2), with EC50 values ranging from 42 to 194 μM. Cenobamate did not displace the binding of flunitrazepam, a benzodiazepine derivative, or flumazenil to GABAA receptors. The results showed that cenobamate, a novel antiepileptic drug, acts as a positive allosteric modulator of high-affinity GABAA receptors, activated by GABA at a site independent of the benzodiazepine binding site and efficiently enhances Itonic inhibition in hippocampal neurons, which could be an underlying molecular mechanism stabilizing neural circuits of the epileptic hippocampus.

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 Ca 2+ /Ca 2+ /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 Ca 2+ ([Ca 2+ ]c) and decreased mitochondrial Ca 2+ ([Ca 2+ ]m) in microscopic and fluorescence‐activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca 2+ /Ca 2+ /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 Ca 2+ 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 [Ca 2+ ]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 Ca 2+ /Ca 2+ /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 Ca 2+ ‐dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.

Isocitrate dehydrogenase 2 (IDH2) is an essential enzyme in the mitochondrial antioxidant system, which produces nicotinamide adenine dinucleotide phosphate, and thereby defends against oxidative stress. We have shown that IDH2 downregulation results in mitochondrial dysfunction and reactive oxygen species (ROS) generation in mouse endothelial cells. The redox enzyme p66shc is a key factor in regulating the level of ROS in endothelial cells. In this study, we hypothesized that IDH2 knockdown-induced mitochondrial dysfunction stimulates endothelial inflammation, which might be regulated by p66shc-mediated oxidative stress. Our results showed that IDH2 downregulation led to mitochondrial dysfunction by decreasing the expression of mitochondrial oxidative phosphorylation complexes I, II, and IV, reducing oxygen consumption, and depolarizing mitochondrial membrane potential in human umbilical vein endothelial cells (HUVECs). The dysfunction not only increased mitochondrial ROS levels but also activated p66shc expression in HUVECs and IDH2 knockout mice. IDH2 deficiency increased intercellular adhesion molecule (ICAM)-1 expression and mRNA levels of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α, and interleukin [IL]-1β) in HUVECs. The mRNA expression of ICAM-1 in endothelial cells and plasma levels of TNF-α and IL-1β were also markedly elevated in IDH2 knockout mice. However, p66shc knockdown rescued IDH2 deficiency-induced mitochondrial ROS levels, monocyte adhesion, ICAM-1, TNF-α, and IL-1β expression in HUVECs. These findings suggest that IDH2 deficiency induced endothelial inflammation via p66shc-mediated mitochondrial oxidative stress.

Anthocyanins, the most prevalent flavonoids in red/purple fruits and vegetables, are known to improve immune responses and reduce chronic disease risks. In this study, the anti-inflammatory activities of an anthocyanin-rich extract from red Chinese cabbage (ArCC) were shown based on its inhibitory effects in cultured endothelial cells and hyperlipidemic apolipoprotein E-deficient mice. ArCC treatment suppressed monocyte adhesion to tumor necrosis factor-α-stimulated endothelial cells. This was validated by ArCC’s ability to downregulate the expression and transcription of endothelial adhesion molecules, determined by immunoblot and luciferase promoter assays, respectively. The regulation of adhesion molecules was accompanied by transcriptional inhibition of nuclear factor-κB, which restricted cytoplasmic localization as shown by immunocytochemistry. Administration of ArCC (150 or 300 mg/kg/day) inhibited aortic inflammation in hyperlipidemic apolipoprotein E-deficient mice, as shown by in vivo imaging. Immunohistochemistry and plasma analysis showed that the aortas from these mice exhibited markedly lower leukocyte infiltration, reduced plaque formation, and lower concentrations of blood inflammatory cytokines than those observed in the control mice. The results suggest that the consumption of anthocyanin-rich red Chinese cabbage is closely correlated with lowering the risk of vascular inflammatory diseases.

Triple-negative breast cancer (TNBC) represents a relatively small proportion of all BCs but a relatively large proportion of BC-related death. Thus, more effective therapeutic strategies are needed for the management of TNBC. We demonstrated that the stimulation of apoptosis by the binding of secreted acetylated-apurinic apyrimidinic endonuclease 1/redox factor-1 (Ac-APE1/Ref-1) to the receptor for advanced glycation end products (RAGE) was essential for TNBC cell death in response to hyperacetylation. The aim of the present study was to assess the potential therapeutic efficacy of secretory Ac-APE1/Ref-1 in orthotopic TNBC xenografts in vivo. We found that hyperacetylation in xenografts caused secretion of Ac-APE1/Ref-1 into the blood, where the factor bound directly to RAGE in hyperacetylated tumor tissues. Hyperacetylation in the TNBC xenografts induced strong inhibition of tumor growth and development, leading to apoptotic cell death, accompanied by increased RAGE expression and generation of reactive oxygen species. Tissues exhibited markedly higher counts of apoptotic bodies, a reduced proliferation index, and reduced neovascularization compared with control tumors. Ac-APE1/Ref-1-stimulated apoptosis was markedly reduced in RAGE-knockdown tumors compared with RAGE-overexpressing tumors, even in the presence of hyperacetylation. The function of secreted Ac-APE1/Ref-1 was confirmed in other hyperacetylated TNBCs xenografts using BT-549 and MDA-MB-468 cells, demonstrating its relevance as an anti-cancer molecule.

Aims CR6 interacting factor 1 (CRIF1) deficiency impairs mitochondrial oxidative phosphorylation complexes, contributing to increased mitochondrial and cellular reactive oxygen species (ROS) production. CRIF1 downregulation has also been revealed to decrease sirtuin 1 (SIRT1) expression and impair vascular function. Inhibition of SIRT1 disturbs oxidative energy metabolism and stimulates nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB)-induced inflammation. Therefore, we hypothesized that both CRIF1 deficiency-induced mitochondrial ROS production and SIRT1 reduction play stimulatory roles in vascular inflammation. Methods and results Plasma levels and mRNA expression of proinflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) were markedly elevated in endothelium-specific CRIF1-knockout mice and CRIF1-silenced endothelial cells, respectively. Moreover, CRIF1 deficiency-induced vascular adhesion molecule-1 (VCAM-1) expression was consistently attenuated by the antioxidant N-acetyl-cysteine and NF-κB inhibitor (BAY11). We next showed that siRNA-mediated CRIF1 downregulation markedly activated NF-κB. SIRT1 overexpression not only rescued CRIF1 deficiency-induced NF-κB activation but also decreased inflammatory cytokines (TNF-α, IL-1β, and IL-6) and VCAM-1 expression levels in endothelial cells. Conclusions These results strongly suggest that CRIF1 deficiency promotes endothelial cell inflammation by increasing VCAM-1 expression, elevating inflammatory cytokines levels, and activating the transcription factor NF-κB, all of which were inhibited by SIRT1 overexpression.