Circulating Endothelial Progenitor Cells Are Preserved in Female Mice Exposed to Ambient Fine Particulate Matter Independent of Estrogen.

Males have a higher risk for cardiovascular diseases (CVDs) than females. Ambient fine particulate matter (PM) exposure increases CVD risk with increased reactive oxygen species (ROS) production and oxidative stress. Endothelial progenitor cells (EPCs) are important to vascular structure and function and can contribute to the development of CVDs. The aims of the present study were to determine if sex differences exist in the effect of PM exposure on circulating EPCs in mice and, if so, whether oxidative stress plays a role. Male and female C57BL/6 mice (8-10 weeks old) were exposed to PM or a vehicle control for six weeks. ELISA analysis showed that PM exposure substantially increased the serum levels of IL-6 and IL-1ß in both males and females, but the concentrations were significantly higher in males. PM exposure only increased the serum levels of TNF-α in males. Flow cytometry analysis demonstrated that ROS production was significantly increased by PM treatment in males but not in females. Similarly, the level of circulating EPCs (CD34+/CD133+ and Sca-1+/Flk-1+) was significantly decreased by PM treatment in males but not in females. Antioxidants N-acetylcysteine (NAC) effectively prevented PM exposure-induced ROS and inflammatory cytokine production and restored circulating EPC levels in male mice. In sharp contrast, circulating EPC levels remained unchanged in female mice with PM exposure, an effect that was not altered by ovariectomy. In conclusion, PM exposure selectively decreased the circulating EPC population in male mice via increased oxidative stress without a significant impact on circulating EPCs in females independent of estrogen.

CARD9 promotes autophagy in cardiomyocytes in myocardial ischemia/reperfusion injury via interacting with Rubicon directly.

Autophagy in cardiomyocyte is involved in myocardial ischemia/reperfusion (M-I/R) injury. Caspase recruitment domain-containing protein 9 (CARD9) plays a critical role in cardiovascular diseases (CVDs) such as hypertension and cardiac fibrosis. However, its role in autophagy following M-I/R injury is yet to be fully elucidated. Here, we found that CARD9 expression increased in M-I/R mouse hearts, and in H9c2 or neonatal rat ventricular myocytes (NRVMs) in response to hypoxia/reoxygenation (H/R) or H2O2. CARD9-/- mice exhibited a significant cardiac dysfunction following M-I/R injury (30 min of left ascending coronary (LAD) ischemia and 12 h of reperfusion) compared to wild-type (WT) mice. CARD9 deletion impaired autophagy during M-I/R in vivo and in vitro, evidenced by decrease of microtubule-associated protein 1 light chain 3 (LC3) lipidation and p62 accumulation. Conversely, CARD9 overexpression increased autophagic flux as indicated by enhanced expression of LC3 II/LC3 I and a reduction in p62. The protective effect of CARD9 on cardiomyocytes against H/R-induced oxidative stress was abolished by treatment with autophagy inhibitors, 3-methyladenine (3-MA) or Bafilomycin A1(BafA1). CARD9 interacted with RUN domain Beclin-1-interacting cysteine-rich-containing (Rubicon), a negative regulator of autophagy, and enhanced UV-irradiation-resistance-associated gene (UVRAG)-Beclin1-phosphatidylinositol 3-kinase catalytic subunit type 3 (PI3KC3) interaction and UVRAG-Vps16-mediated Rab7 activation to promote autophagosome formation, maturation, and endocytosis. Ablation of Rubicon by siRNA effectively prevented the detrimental effect of CARD9 knockdown on cardiomyocytes. These results suggest that CARD9 has protective effects on the myocardium against M-I/R injury by activating autophagy and restoring autophagic flux in vivo and in vitro.

Binge Alcohol Is More Injurious to Liver in Female than in Male Rats: Histopathological, Pharmacologic, and Epigenetic Profiles.

Binge alcohol consumption is a health problem, but differences between the sexes remain poorly defined. We have examined the in vivo effects of three acute, repeat binge alcohol administration on the liver in male and female rats. Sprague-Dawley rats were gavaged with alcohol (5 g/kg body weight) three times at 12-hour intervals. Blood and liver tissues were collected 4 hours after the last binge ethanol. Subsequently, several variables were analyzed. Compared with male rats, females had higher levels of blood alcohol, alanine aminotransferase, and triglycerides. Liver histology showed increased lipid vesicles that were larger in females. Protein levels of liver cytochrome P4502E1 were higher in the liver of females than in the liver of males after binge. Hepatic phospho-extracellular signal-regulated kinase 1/2 and phosph-p38 mitogen-activated protein kinase levels were lower in females compared with males after binge alcohol, but no differences were found in the phospho-C-jun N-terminal kinase levels. Peroxisome proliferator-activated receptor γ-coactivator 1α and cyclic AMP response element binding (CREB) protein levels increased more in female than in male livers; however, increases in phospho-CREB levels were lower in females. Remarkably, c-fos was reduced substantially in the livers of females, but no differences in c-myc protein were found. Binge ethanol caused elevation in acetylated (H3AcK9) and phosphoacetylated (H3AcK9PS10) histone H3 in both sexes but without any difference. Binge alcohol caused differential alterations in the levels of various species of phosphatidylethanol and a larger increase in the diacylglycerol kinase-α protein levels in the liver of female rats compared with male rats. These data demonstrate, for the first time, similarities and differences in the sex-specific responses to repeat binge alcohol leading to an increased susceptibility of female rats to have liver injury in vivo. SIGNIFICANCE STATEMENT: This study examines the molecular responses of male and female rat livers to acute binge alcohol in vivo and demonstrates significant differences in the susceptibility between sexes.

Nucleolin protects macrophages from oxLDL-induced foam cell formation through up-regulating ABCA1 expression.

Our recent studies have indicated that nucleolin, as a multifunctional RNA-binding protein, exerts protective effects in the myocardial cells and endothelial cells under the condition of oxidative stress. However, the function of nucleolin and its potential mechanism in macrophage-derived foam cell formation remain largely unexplored. ApoE-/- mice were fed with a high-fat diet (HFD) for 10-24 weeks. Protein expression was measured by western blotting or immunofluorescence, and gene expression at the mRNA level was detected by qRT-PCR. The level of lipid in macrophages was examined by Oil Red O staining, high-performance liquid chromatography (HPLC) and NBD-cholesterol. Actinomycin D (Act D) was used to determine the stability of ABCA1 mRNA in macrophages. The interaction of nucleolin with ABCA1 mRNA was assessed using co-immunoprecipitation (co-IP). The aortas advanced plaques demonstrated significantly lower levels of nucleolin protein compared with early plaques in ApoE-/- mice, in which the macrophage foam cells occupied main body. Nucleolin expression at the mRNA and protein levels in RAW264.7 macrophages was significantly reduced by oxidized low-density lipoprotein (oxLDL) in a dose- and time-dependent manner. Furthermore, nucleolin overexpression markedly attenuated lipid accumulation in oxLDL-challenged macrophages through increasing cholesterol efflux. In addition, nucleolin overexpression significantly increased the expression of ATP-binding cassette transporter A1 (ABCA1) at the mRNA and protein levels without affecting expressions of scavenger receptors (SR)-A, SR-B1, CD36 and ATP-binding cassette transporter G1 (ABCG1) at the mRNA level. Moreover, nucleolin overexpression increased the stability of ABCA1 mRNA in macrophages, whereas nucleolin ablation abrogated the oxLDL-induced up-regulation of ABCA1. The up-regulation of ABCA1 by nucleolin resulted from its protein-RNA interaction. Our data suggested that nucleolin inhibited foam cell formation through enhancing stability of ABCA1 mRNA and subsequently increasing cholesterol efflux.

Sustained Release of a Peptide-Based Matrix Metalloproteinase-2 Inhibitor to Attenuate Adverse Cardiac Remodeling and Improve Cardiac Function Following Myocardial Infarction.

Following myocardial infarction (MI), degradation of extracellular matrix (ECM) by upregulated matrix metalloproteinases (MMPs) especially MMP-2 decreases tissue mechanical properties, leading to cardiac function deterioration. Attenuation of cardiac ECM degradation at the early stage of MI has the potential to preserve tissue mechanical properties, resulting in cardiac function increase. Yet the strategy for efficiently preventing cardiac ECM degradation remains to be established. Current preclinical approaches have shown limited efficacy because of low drug dosage allocated to the heart tissue, dose-limiting side effects, and cardiac fibrosis. To address these limitations, we have developed a MMP-2 inhibitor delivery system that can be specifically delivered into infarcted hearts at early stage of MI to efficiently prevent MMP-2-mediated ECM degradation. The system was based on an injectable, degradable, fast gelation, and thermosensitive hydrogel, and a MMP-2 specific inhibitor, peptide CTTHWGFTLC (CTT). The use of fast gelation hydrogel allowed to completely retain CTT in the heart tissue. The system was able to release low molecular weight CTT over 4 weeks possibly due to the strong hydrogen bonding between the hydrogel and CTT. The release kinetics was modulated by amount of CTT loaded into the hydrogel, and using chondroitin sulfate and heparin that can interact with CTT and the hydrogel. Both glycosaminoglycans augmented CTT release, while heparin more greatly accelerated the release. After it was injected into the infarcted hearts for 4 weeks, the released CTT efficiently prevented cardiac ECM degradation as it not only increased tissue thickness but also preserved collagen composition similar to that in the normal heart tissue. In addition, the delivery system significantly improved cardiac function. Importantly, the delivery system did not induce cardiac fibrosis. These results demonstrate that the developed MMP-2 inhibitor delivery system has potential to efficiently reduce adverse myocardial remodeling and improve cardiac function.

Causal effects from inflammatory bowel disease on liver function and disease: a two-sample Mendelian randomization study.

Background: Accumulating evidence has shown that patients with inflammatory bowel disease (IBD) have liver function abnormalities and are susceptible to liver diseases. However, the existence of a causal relationship between IBD and liver function or disease remains unclear. Methods: A two-sample Mendelian randomization (MR) analysis was performed using genetic associations from publicly available genome-wide association studies (GWAS). These associations encompass ulcerative colitis (UC), Crohn's disease (CD), liver function traits, and liver disease phenotypes. The liver function traits comprised hepatic biochemistries, percent liver fat, and liver iron content from the UK Biobank. Furthermore, the liver disease phenotypes included cholelithiasis, non-alcoholic fatty liver disease (NAFLD), primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC) in cohorts of European ancestry. The primary estimation used the inverse-variance weighted method, with GWAS of C-reactive protein (CRP) in the UK Biobank serving as a positive control outcome. Results: Genetically predicted UC is causally associated with decreased levels of albumin (ALB) and liver iron content, while genetically predicted CD is causally associated with increased levels of alkaline phosphatase (ALP). Moreover, genetically predicted UC or CD increases the risk of PSC, and CD increases the risk of PBC. Neither UC nor CD causally increases the risk of cholelithiasis and NAFLD. Conclusion: UC affects the levels of ALB and liver iron content, while CD affects the levels of ALP. Both UC and CD increase the risk of PSC, and CD increases the risk of PBC.

CARD9 deficiency improves the recovery of limb ischemia in mice with ambient fine particulate matter exposure.

Background: Exposure to fine particulate matter (PM) is a significant risk for cardiovascular diseases largely due to increased reactive oxygen species (ROS) production and inflammation. Caspase recruitment domain (CARD)9 is critically involved in innate immunity and inflammation. The present study was designed to test the hypothesis that CARD9 signaling is critically involved in PM exposure-induced oxidative stress and impaired recovery of limb ischemia. Methods and results: Critical limb ischemia (CLI) was created in male wildtype C57BL/6 and age matched CARD9 deficient mice with or without PM (average diameter 2.8 µm) exposure. Mice received intranasal PM exposure for 1 month prior to creation of CLI and continued for the duration of the experiment. Blood flow and mechanical function were evaluated in vivo at baseline and days 3, 7, 14, and 21 post CLI. PM exposure significantly increased ROS production, macrophage infiltration, and CARD9 protein expression in ischemic limbs of C57BL/6 mice in association with decreased recovery of blood flow and mechanical function. CARD9 deficiency effectively prevented PM exposure-induced ROS production and macrophage infiltration and preserved the recovery of ischemic limb with increased capillary density. CARD9 deficiency also significantly attenuated PM exposure-induced increase of circulating CD11b+/F4/80+ macrophages. Conclusion: The data indicate that CARD9 signaling plays an important role in PM exposure-induced ROS production and impaired limb recovery following ischemia in mice.

Helicobacter pylori infection selectively attenuates endothelial function in male mice via exosomes-mediated ROS production.

Background: Substantial sex differences exist in atherosclerosis. Excessive reactive oxygen species (ROS) formation could lead to endothelial dysfunction which is critical to atherosclerosis development and progression. Helicobacter pylori (H. pylori) infection has been shown to attenuate endothelial function via exosomes-mediated ROS formation. We have demonstrated that H. pylori infection selectively increases atherosclerosis risk in males with unknown mechanism(s). The present study was to test the hypothesis that H. pylori infection impaired endothelial function selectively in male mice through exosome-mediated ROS formation. Methods and results: Age-matched male and female C57BL/6 mice were infected with CagA+ H. pylori to investigate sex differences in H. pylori infection-induced endothelial dysfunction. H. pylori infection attenuated acetylcholine (ACh)-induced endothelium-dependent aortic relaxation without changing nitroglycerine-induced endothelium-independent relaxation in male but not female mice, associated with increased ROS formation in aorta compared with controls, which could be reversed by N-acetylcysteine treatment. Treatment of cultured mouse brain microvascular endothelial cells with exosomes from H. pylori infected male, not female, mice significantly increased intracellular ROS production and impaired endothelial function with decreased migration, tube formation, and proliferation, which could be prevented with N-acetylcysteine treatment. Conclusions: H. pylori infection selectively impairs endothelial function in male mice due to exosome-mediated ROS formation.

CARD9 Signaling, Inflammation, and Diseases.

Caspase-recruitment domain 9 (CARD9) protein is expressed in many cells especially in immune cells, and is critically involved in the function of the innate and adaptive immune systems through extensive interactions between CARD9 and other signaling molecules including NF-κB and MAPK. CARD9-mediated signaling plays a central role in regulating inflammatory responses and oxidative stress through the productions of important cytokines and chemokines. Abnormalities of CARD9 and CARD9 signaling or CARD9 mutations or polymorphism are associated with a variety of pathological conditions including infections, inflammation, and autoimmune disorders. This review focuses on the function of CARD9 and CARD9-mediated signaling pathways, as well as interactions with other important signaling molecules in different cell types and the relations to specific disease conditions including inflammatory diseases, infections, tumorigenesis, and cardiovascular pathologies.

An Integrative Human Pan-Cancer Analysis of Cyclin-Dependent Kinase 1 (CDK1).

Cyclin-dependent kinase 1 (CDK1) is essential for cell division by regulating the G2/M phase and mitosis. CDK1 overexpression can also promote the development and progression of a variety of cancers. However, the significance of CDK1 in the formation, progression, and prognosis of human pan-cancer remains unclear. In the present study, we used The Cancer Genome Atlas database, Clinical Proteomic Tumor Analysis Consortium, Human Protein Atlas, Genotype-Tissue Expression, and other well-established databases to comprehensively examine CDK1 genetic alterations and gene/protein expression in various cancers and their relationships with the prognosis, immune reactivities, and clinical outcomes for 33 tumor types. Gene set enrichment analysis was also conducted to examine the potential mechanisms of CDK1 in tumorigenesis. The data showed that CDK1 mutation was frequently present in multiple tumors. CDK1 expression was significantly increased in various types of tumors as compared with normal tissues and was associated with poor overall and disease-free survival. In addition, CDK1 expression was significantly correlated with oncogenic genes, proteins, cellular components, myeloid-derived suppressor cell infiltration, ESTMATEScore, and signaling pathways associated with tumor development and progression and tumor microenvironments. These data indicate that CDK1 could serve as a promising biomarker for predicting tumor prognosis and a potential target for cancer treatment.

An Integrative Pan-Cancer Analysis of Kinesin Family Member C1 (KIFC1) in Human Tumors.

Kinesin family member C1 (KIFC1) is a minus-end-directed motor protein that is critically involved in microtubule crosslinking and spindle formation. KIFC1 is essential for supernumerary centrosomes, and it is associated with the initiation and progression of cancers. In the present study, we initially reviewed the The Cancer Genome Atlas database and observed that KIFC1 is abundantly expressed in most types of tumors. We then analyzed the gene alteration profiles, protein expressions, prognoses, and immune reactivities of KIFC1 in more than 10,000 samples from several well-established databases. In addition, we conducted a gene set enrichment analysis to investigate the potential mechanisms for the roles of KIFC1 in carcinogenesis. The pan-cancer analysis of KIFC1 demonstrates significant statistical correlations of the KIFC1 expression with the clinical prognoses, the oncogenic signature gene sets, the myeloid-derived suppressor cell infiltration, the ImmunoScore, the immune checkpoints, the microsatellite instabilities, and the tumor mutational burdens across multiple tumors. These data may provide important information on the understanding of the role and mechanisms of KIFC1 in carcinogenesis and immunotherapy, as well as on the clinical progression of a variety of cancers.

Tempol Preserves Endothelial Progenitor Cells in Male Mice with Ambient Fine Particulate Matter Exposure.

Ambient fine particulate matter (PM) exposure associates with an increased risk of cardiovascular diseases (CVDs). Major sex differences between males and females exist in epidemiology, pathophysiology, and outcome of CVDs. Endothelial progenitor cells (EPCs) play a vital role in the development and progression of CVDs. PM exposure-induced reduction of EPCs is observed in male, not female, mice with increased reactive oxygen species (ROS) production and oxidative stress. The lung is considered an important source of ROS in mice with PM exposure. The aim of the present study was to investigate the sex differences in pulmonary superoxide dismutase (SOD) expression and ROS production, and to test the effect of SOD mimic Tempol on the populations of EPCs in mice with PM exposure. Both male and female C57BL/6 mice (8-10 weeks) were exposed to intranasal PM or vehicle for 6 weeks. Flow cytometry analysis demonstrated that PM exposure significantly decreased the levels of EPCs (CD34+/CD133+) in both blood and bone marrow with increased ROS production in males, but not in females. ELISA analysis showed higher levels of serum IL-6 and IL-1ßin males than in females. Pulmonary expression of the antioxidant enzyme SOD1 was significantly decreased in males after PM exposure, but not in females. Administration of the SOD mimic Tempol in male mice with PM exposure attenuated the production of ROS and inflammatory cytokines, and preserved EPC levels. These data indicated that PM exposure-induced reduction of EPC population in male mice may be due to decreased expression of pulmonary SOD1 in male mice.

N-acetylcysteine attenuates atherosclerosis progression in aging LDL receptor deficient mice with preserved M2 macrophages and increased CD146.

BACKGROUND AND AIMS: Inflammation and reactive oxygen species (ROS) are important to the pathogenesis of atherosclerosis. The effect of antioxidants on atherosclerosis is inconsistent, and sometimes controversial. We aimed to test the hypothesis that attenuation of atherosclerosis by N-acetylcysteine (NAC) depends on NAC treatment timing and duration. METHODS: Male LDL receptor deficient (LDLR-/-) mice were fed a normal diet (ND) and divided into controls (on ND for 24 months), models 1-2 (at age of 9 months, starting NAC treatment for 3 or 6 months), and model 3 (at age of 18 months, starting NAC treatment for 6 months). To determine if hyperlipidemia compromises NAC treatment outcome, mice were fed a high fat diet (HFD) starting at age of 6 weeks and treated with NAC starting at 9 months of age for 6 months. RESULTS: NAC treatment for 6 months, not for 3 months, significantly attenuated atherosclerosis progression, but did not reverse atherosclerotic lesions, in aging LDLR-/- mice on ND. NAC had no effect on atherosclerotic lesions in mice on HFD. NAC treatment significantly decreased aortic ROS production, and the levels of inflammatory cytokines in serum and aorta of aging LDLR-/- mice with increased CD146 level. Bone marrow transplantation study with GFP-positive bone marrow cells showed that NAC treatment preserved M2 population and M2 polarization in the aorta of LDLR-/- mice. CONCLUSIONS: Early and adequate NAC treatment could effectively attenuate inflammation and atherosclerosis progression with preserved M2 population and increased CD146 level in aging LDLR-/- mice without extreme hyperlipidemia.

Recovery of Ischemic Limb and Femoral Artery Endothelial Function Are Preserved in Mice with Dextran Sodium Sulfate-Induced Chronic Colitis.

Inflammatory bowel disease (IBD) produces significant systemic inflammation and increases the risk of endothelial dysfunction and peripheral artery disease. Our recent study demonstrated that abdominal aortic endothelial cell function was impaired selectively in female mice with chronic colitis. This study aimed to test the hypothesis that experimental colitis leads to femoral artery endothelial cell dysfunction and impairs limb ischemia recovery in female mice. An experimental chronic colitis model was created in female C57BL/6 mice with dextran sodium sulfate (DSS) treatment. Unilateral hind limb ischemia was produced by femoral artery ligation. Limb blood perfusion, vascular density, tissue ROS levels, and plasma levels of proinflammatory cytokines were assessed. Femoral artery endothelium-dependent and -independent vasodilation of the contralateral limb were evaluated ex vivo using acetylcholine and nitroglycerin, respectively. As expected, the plasma levels of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and IL-17, were significantly increased in the DSS-induced colitis model. However, ROS levels in the ischemic muscle tissues were not significantly increased in colitis model as compared to the controls. There were no significant changes in endothelium-dependent or -independent vasodilation of the femoral artery between colitis model and the control. Recovery of function and blood flow in the ischemic limb and capillary density in the ischemic gastrocnemius muscle were preserved in the colitis model as compared with the control. The data demonstrated that DSS-induced chronic colitis had no significant impact on femoral artery endothelial function or ischemic limb recovery in female mice.

Abdominal Aortic Endothelial Dysfunction Occurs in Female Mice With Dextran Sodium Sulfate-Induced Chronic Colitis Independently of Reactive Oxygen Species Formation.

Background and Objective: Inflammatory bowel disease (IBD) produces significant local and systemic inflammation with increased reactive oxygen species (ROS) formation. IBD Patients are at an increased risk for developing endothelial dysfunction and cardiovascular diseases. The present study tested the hypothesis that IBD impairs aortic endothelial function via ROS formation and investigate potential sex-related differences. Methods and Results: Acute and chronic colitis models were induced in male and female C57BL/6 mice with dextran sodium sulfate (DSS) treatment. Aortic wall stiffness, endothelial function, and ROS levels, as well as serum levels of pro-inflammatory cytokines were evaluated. Acetylcholine (Ach)-induced endothelium-dependent relaxation of abdominal aorta without perivascular adipose tissue (PVAT) was significantly reduced in female mice, not males, with chronic colitis without a change in nitroglycerin-induced endothelium-independent relaxation. PVAT effectively preserved Ach-induced relaxation in abdominal aorta of female mice with chronic colitis. Aortic peak velocity, maximal intraluminal diameters, pulse wave velocity, distensibility and radial strain were preserved in mice with both acute and chronic colitis. Although pro-inflammatory cytokines levels were increased in mice with acute and chronic colitis, aortic ROS levels were not increased. Conclusion: The data demonstrate that abdominal aortic endothelial function was attenuated selectively in female mice with chronic colitis independent of ROS formation. Further, PVAT played an important role in preserving endothelial function in female mice with chronic colitis.

N-Acetylcysteine Enhances the Recovery of Ischemic Limb in Type-2 Diabetic Mice.

Critical limb ischemia (CLI) is a severe complication of diabetes mellitus that occurs without effective therapy. Excessive reactive oxygen species (ROS) production and oxidative stress play critical roles in the development of diabetic cardiovascular complications. N-acetylcysteine (NAC) reduces ischemia-induced ROS production. The present study aimed to investigate the effect of NAC on the recovery of ischemic limb in an experimental model of type-2 diabetes. TALLYHO/JngJ diabetic and SWR/J non-diabetic mice were used for developing a CLI model. For NAC treatment, mice received NAC (1 mg/mL) in their drinking water for 24 h before initiating CLI, and continuously for the duration of the experiment. Blood flow, mechanical function, histology, expression of antioxidant enzymes including superoxide dismutase (SOD)-1, SOD-3, glutathione peroxidase (Gpx)-1, catalase, and phosphorylated insulin receptor substrate (IRS)-1, Akt, and eNOS in ischemic limb were evaluated in vivo or ex vivo. Body weight, blood glucose, plasma advanced glycation end-products (AGEs), plasma insulin, insulin resistance index, and plasma TNF-a were also evaluated during the experiment. NAC treatment effectively attenuated ROS production with preserved expressions of SOD-1, Gpx-1, catalase, phosphorylated Akt, and eNOS, and enhanced the recovery of blood flow and function of the diabetic ischemic limb. NAC treatment also significantly decreased the levels of phosphorylated IRS-1 (Ser307) expression and plasma TNF-α in diabetic mice without significant changes in blood glucose and AGEs levels. In conclusion, NAC treatment enhanced the recovery of blood flow and mechanical function in ischemic limbs in T2D mice in association with improved tissue redox/inflammatory status and insulin resistance.

Curcumin nicotinate decreases serum LDL cholesterol through LDL receptor-mediated mechanism.

Curcumin nicotinate (Curtn) is a synthesized ester derivative of curcumin and niacin. Our previous study has shown that Curtn lowers serum low-density lipoprotein cholesterol (LDL-C) levels in apoE-/- mice and promotes LDL-C uptake into HepG2 cells in vitro. The present study was to test the hypothesis that Curtn decreases serum LDL-C levels through decreased expression of pro-protein convertase subtilisin/kexin type 9 (PCSK9) and subsequent increase in LDL receptor expression. Male Wistar rats on high-fat diet (HFD) were treated with Curtn or rosuvastatin. Curtn or rosuvastatin treatment significantly decreased serum levels of total cholesterol (TC) and LDL-C in rats on HFD with increased liver LDL receptor expression. LDL-C-lowering effect of Curtn was not observed in LDL receptor deficient (LDLR-/-) mice on HFD, while rosuvastatin still decreased serum lipid levels in LDLR-/- mice, indicating that the reduction of serum LDL-C levels by Curtn treatment was LDL receptor-dependent. Curtn treatment also significantly decreased the protein expression of PCSK9 in Wistar rats and LDLR-/- mice. In HepG2 cells with overexpression of human PCSK9, Curtn treatment significantly increased LDL-C uptakes into hepatocytes, and increased LDL receptor distribution on cell surface in association with decreased PCSK9 protein expression. RNAi-LDLR significantly attenuated the effect of Curtn on LDLR distribution on cell surface. These data indicates that Curtn would decrease serum LDL-C level at least partially through inhibition of PCSK9 expression, and subsequent increase in LDL receptor expression and distribution in hepatocytes, serving as a potential novel compound to treat hyperlipidemia.

CagA+ Helicobacter pylori, Not CagA- Helicobacter pylori, Infection Impairs Endothelial Function Through Exosomes-Mediated ROS Formation.

Background: Helicobacter pylori (H. pylori) infection increases the risk for atherosclerosis, and ROS are critical to endothelial dysfunction and atherosclerosis. CagA is a major H. pylori virulence factor associated with atherosclerosis. The present study aimed to test the hypothesis that CagA+ H. pylori effectively colonizes gastric mucosa, and CagA+ H. pylori, but not CagA- H. pylori, infection impairs endothelial function through exosomes-mediated ROS formation. Methods: C57BL/6 were used to determine the colonization ability of CagA+ H. pylori and CagA- H. pylori. ROS production, endothelial function of thoracic aorta and atherosclerosis were measured in CagA+ H. pylori and CagA- H. pylori infected mice. Exosomes from CagA+ H. pylori and CagA- H. pylori or without H. pylori infected mouse serum or GES-1 were isolated and co-cultured with bEND.3 and HUVECs to determine how CagA+ H. pylori infection impairs endothelial function. Further, GW4869 was used to determine if CagA+ H. pylori infection could lead to endothelial dysfunction and atherosclerosis through an exosomes-mediated mechanism. Results: CagA+ H. pylori colonized gastric mucosa more effectively than CagA- H. pylori in mice. CagA+ H. pylori, not CagA- H. pylori, infection significantly increased aortic ROS production, decreased ACh-induced aortic relaxation, and enhanced early atherosclerosis formation, which were prevented with N-acetylcysteine treatment. Treatment with CagA-containing exosomes significantly increased intracellular ROS production in endothelial cells and impaired their function. Inhibition of exosomes secretion with GW4869 effectively prevented excessive aortic ROS production, endothelial dysfunction, and atherosclerosis in mice with CagA+ H. pylori infection. Conclusion: These data suggest that CagA+ H. pylori effectively colonizes gastric mucosa, impairs endothelial function, and enhances atherosclerosis via exosomes-mediated ROS formation in mice.

High-fat diet selectively decreases bone marrow lin- /CD117+ cell population in aging mice through increased ROS production.

Bone marrow (BM) stem cells (BMSCs) are an important source for cell therapy. The outcome of cell therapy could be ultimately associated with the number and function of donor BMSCs. The present study was to evaluate the effect of long-term high-fat diet (HFD) on the population of BMSCs and the role of reactive oxygen species (ROS) in aging mice. Forty-week-old male C57BL/6 mice were fed with HFD for 3 months with regular diet as control. Experiments were repeated when ROS production was reduced in mice treated with N-acetylcysteine (NAC) or using mice overexpressing antioxidant enzyme network (AON) of superoxide dismutase (SOD)1, SOD3, and glutathione peroxidase. BM and blood cells were analyzed with flowcytometry for lineage negative (lin- ) and Sca-1+ , or lin- /CD117+ , or lin- /CD133+ cells. Lin- /CD117+ cell population was significantly decreased with increased intracellular ROS and apoptosis and decreased proliferation in BM, not in blood, in HFD-treated mice without change for Sca-1+ or CD133+ cell populations in BM or blood. NAC treatment or AON overexpression effectively prevented HFD-induced intracellular ROS production and reduction of BM lin- /CD117+ population. These data suggested that long-term HFD selectively decreased BM lin- /CD117+ cell population in aging mice through increased ROS production.

Bioinspired One Cell Culture Isolates Highly Tumorigenic and Metastatic Cancer Stem Cells Capable of Multilineage Differentiation.

Cancer stem cells (CSCs) are rare cancer cells that are postulated to be responsible for cancer relapse and metastasis. However, CSCs are difficult to isolate and poorly understood. Here, a bioinspired approach for label-free isolation and culture of CSCs, by microencapsulating one cancer cell in the nanoliter-scale hydrogel core of each prehatching embryo-like core-shell microcapsule, is reported. Only a small percentage of the individually microencapsulated cancer cells can proliferate into a cell colony. Gene and protein expression analyses indicate high stemness of the cells in the colonies. Importantly, the colony cells are capable of cross-tissue multilineage (e.g., endothelial, cardiac, neural, and osteogenic) differentiation, which is not observed for "CSCs" isolated using other contemporary approaches. Further studies demonstrate the colony cells are highly tumorigenic, metastatic, and drug resistant. These data show the colony cells obtained with the bioinspired one-cell-culture approach are truly CSCs. Significantly, multiple pathways are identified to upregulate in the CSCs and enrichment of genes related to the pathways is correlated with significantly decreased survival of breast cancer patients. Collectively, this study may provide a valuable method for isolating and culturing CSCs, to facilitate the understanding of cancer biology and etiology and the development of effective CSC-targeted cancer therapies.