Distinguishing the Cellular Transport of Folic Acid Conjugated Nano-Drugs among Different Cell Lines by Using Force Tracing Technique.

Folic acid (FA) is a ligand that has been renowned for its strong binding to FA receptor (FR), and the robustness of the specific interaction has led to the generation of multitudinous tumor-targeted nano-drug delivery systems. However, selecting the appropriate FA targeted nano-drugs according to types of cancerous cells to achieve a high effect is critical. Understanding of how the drug is transported through the cell membrane and is delivered intracellularly is very important in screening ideal targeted nano-drugs for cancerous changes in different organs. Herein, by using a force tracing technique based on atomic force microscopy (AFM), the dynamic process of FA-polyamidoamine-Doxorubicin (FA-PAMAM-DOX) entry into different tumor cells (HeLa and A549) and normal cells (Vero) was monitored in real time. The cell membrane transport efficacy of FA-PAMAM-DOX in tumor cells with an FR high overexpression level (HeLa) and FR low overexpression level (A549) is analyzed, which is significantly higher than that in normal cells (Vero), especially for HeLa cells. Subsequently, the intracellular delivery efficiency of FA-PAMAM-DOX in different cell lines was measured by using fluorescence imaging and AFM-based nanoindentation techniques. This report will help to discover the cellular transport mechanism of nano-drugs and screen out optimal therapeutic nano-drugs for different types of tumors.

Investigating the trans-membrane transport of HAIYPRH peptide-decorated nano-drugs.

Transferrin (Tf) has been effectively used to promote the cellular uptake of HAIYPRH (T7) peptide-conjugated nano-drugs. In this study, the enhancing effect of Tf on T7-decorated nano-drug transport was investigated using force tracing and nano-indentation techniques at a single-particle/cell level. Furthermore, the results were confirmed by ensemble fluorescence imaging.

Erastin induces ferroptosis via ferroportin-mediated iron accumulation in endometriosis.

STUDY QUESTION: Could erastin activate ferroptosis to regress endometriotic lesions? SUMMARY ANSWER: Erastin could induce ferroptosis to regress endometriotic lesions in endometriosis. WHAT IS KNOWN ALREADY: Ectopic endometrial stromal cells (EESCs) are in an iron overloading microenvironment and tend to be more sensitive to oxidative damage. The feature of erastin-induced ferroptosis is iron-dependent accumulation of lethal lipid reactive oxygen species (ROS). STUDY DESIGN, SIZE, DURATION: Eleven patients without endometriosis and 21 patients with endometriosis were recruited in this study. Primary normal and ectopic endometrial stromal cells were isolated, cultured and subjected to various treatments. The in vivo study involved 10 C57BL/6 female mice to establish the model of endometriosis. PARTICIPANTS/MATERIALS, SETTING, METHODS: The markers of ferroptosis were assessed by cell viability, lipid peroxidation level and morphological changes. The cell viability was measured by colorimetric method, lipid peroxidation levels were measured by flow cytometry, and morphological changes were observed by transmission electron microscopy. Immunohistochemistry and western blot were used to detect ferroportin (FPN) expression. Prussian blue staining and immunofluorescent microscopy of catalytic ferrous iron were semi-quantified the levels of iron. Adenovirus-mediated overexpression and siRNA-mediated knockdown were used to investigate the role of FPN on erastin-induced ferroptosis in EESCs. MAIN RESULTS AND THE ROLE OF CHANCE: EESCs were more susceptible to erastin treatment, compared to normal endometrial stromal cells (NESCs) (P<0.05). Treatment of cultured EESCs with erastin dramatically increased the total ROS level (P<0.05, versus control), lipid ROS level (P<0.05, versus NESCs) and intracellular iron level (P<0.05, versus NESCs). The cytotoxicity of erastin could be attenuated by iron chelator, deferoxamine (DFO), and ferroptosis inhibitors, ferrostatin-1 and liproxstatin-1, (P<0.05, versus erastin) in EESCs. In EESCs with erastin treatment, shorter and condensed mitochondria were observed by electron microscopy. These findings together suggest that erastin is capable to induce EESC death by ferroptosis. However, the influence of erastin on NESCs was slight. The process of erastin-induced ferroptosis in EESCs accompanied iron accumulation and decreased FPN expression. The overexpression of FPN ablated erastin-induced ferroptosis in EESCs. In addition, knockdown of FPN accelerated erastin-induced ferroptosis in EESCs. In a mouse model of endometriosis, we found ectopic lesions were regressed after erastin administration. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: This study was mainly conducted in primary human endometrial stromal cells. Therefore, the function of FPN in vivo need to be further investigated. WIDER IMPLICATIONS OF THE FINDINGS: Our findings reveal that erastin may serve as a potential therapeutic treatment for endometriosis. STUDY FUNDING/COMPETING INTEREST(S): This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. The authors declare no conflict of interest.

Vitamin B6 inhibits macrophage activation to prevent lipopolysaccharide-induced acute pneumonia in mice.

Macrophage activation participates in the pathogenesis of pulmonary inflammation. As a coenzyme, vitamin B6 (VitB6) is mainly involved in the metabolism of amino acids, nucleic acids, glycogen and lipids. We have previously reported that activation of AMP-activated protein kinase (AMPK) produces anti-inflammatory effects both in vitro and in vivo. Whether VitB6 via AMPK activation prevents pulmonary inflammation remains unknown. The model of acute pneumonia was induced by injecting mice with lipopolysaccharide (LPS). The inflammation was determined by measuring the levels of interleukin-1 beta (IL-1ß), IL-6 and tumour necrosis factor alpha (TNF-α) using real time PCR, ELISA and immunohistochemistry. Exposure of cultured primary macrophages to VitB6 increased AMP-activated protein kinase (AMPK) Thr172 phosphorylation in a time/dose-dependent manner, which was inhibited by compound C. VitB6 downregulated the inflammatory gene expressions including IL-1ß, IL-6 and TNF-α in macrophages challenged with LPS. These effects of VitB6 were mirrored by AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR). However, VitB6 was unable to inhibit LPS-induced macrophage activation if AMPK was in deficient through siRNA-mediated approaches. Further, the anti-inflammatory effects produced by VitB6 or AICAR in LPS-treated macrophages were abolished in DOK3 gene knockout (DOK3-/- ) macrophages, but were enhanced in macrophages if DOK3 was overexpressed. In vivo studies indicated that administration of VitB6 remarkably inhibited LPS-induced both systemic inflammation and acute pneumonia in wild-type mice, but not in DOK3-/- mice. VitB6 prevents LPS-induced acute pulmonary inflammation in mice via the inhibition of macrophage activation.

CCAAT/enhancer-binding protein ß overexpression alleviates myocardial remodelling by regulating angiotensin-converting enzyme-2 expression in diabetes.

Diabetic cardiomyopathy, a major cardiac complication, contributes to heart remodelling and heart failure. Our previous study discovered that CCAAT/enhancer-binding protein ß (C/EBPß), a transcription factor that belongs to a family of basic leucine zipper transcription factors, interacts with the angiotensin-converting enzyme 2 (ACE2) promoter sequence in other disease models. Here, we aimed to determine the role of C/EBPß in diabetes and whether ACE2 expression is regulated by C/EBPß. A type 1 diabetic mouse model was generated by an intraperitoneal injection of streptozotocin. Diabetic mice were injected with a lentivirus expressing either C/EBPß or sh-C/EBPß or treated with valsartan after 12 weeks to observe the effects of C/EBPß. In vitro, cardiac fibroblasts and cardiomyocytes were treated with high glucose (HG) to investigate the anti-fibrosis, anti-apoptosis and regulatory mechanisms of C/EBPß. C/EBPß expression was down-regulated in diabetic mice and HG-induced cardiac neonatal cells. C/EBPß overexpression significantly attenuated collagen deposition and cardiomyocyte apoptosis by up-regulating ACE2 expression. The molecular mechanism involved the binding of C/EBPß to the ACE2 promoter sequence. Although valsartan, a classic angiotensin receptor blocker, relieved diabetic complications, the up-regulation of ACE2 expression by C/EBPß overexpression may exert greater beneficial effects on patients with diabetic cardiomyopathy.

Vitamin B6 Alleviates Lipopolysaccharide-induced Myocardial Injury by Ferroptosis and Apoptosis Regulation.

Vitamin B6 (VitB6) is a water-soluble vitamin and includes pyridoxine, pyridoxal, pyridoxamine, and their phosphorylated forms. In the current study, we demonstrated that VitB6 could improve lipopolysaccharide (LPS)-induced myocardial injury. We demonstrated that VitB6 can suppress LPS-induced oxidative stress and lipid peroxidation that lead to ferroptosis and apoptosis in vivo and in vitro. Moreover, we found that VitB6 can regulate the expression of iron regulatory proteins, maintaining intracellular iron homeostasis. To confirm that VitB6 could inhibit LPS-induced ferroptosis and apoptosis, we pretreated mice with ferrostatin-1 (Fer-1) and emricasan that efficiently mimicked VitB6 pharmacological effects. This improved the survival rate of mice challenged with a high LPS dose. In addition, VitB6 regulated the expression of LPS-induced apoptosis-related proteins and iron regulatory proteins. It mediated the expression of Nrf2, transcription factor NF-E2-related factor 2, which promoted the expression of antioxidant enzymes and restrained LPS-induced ferroptosis and apoptosis. Overall, our results indicated that VitB6 can be used on novel therapies to relieve LPS-induced myocardial injury.

S-Nitrosylation of Prostacyclin Synthase Instigates Nitrate Cross-Tolerance In Vivo.

Development of nitrate tolerance is a major drawback to nitrate therapy. Prostacyclin (PGI2) is a powerful vasodilator produced from prostaglandin (PGH2) by prostacyclin synthase (PGIS) in endothelial cells. This study aimed to determine the role of PGIS S-nitrosylation in nitrate tolerance induced by nitroglycerin (GTN). In endothelial cells, GTN increased PGIS S-nitrosylation and disturbed PGH2 metabolism, which were normalized by mutants of PGIS cysteine 231/441 to alanine (C231/441A). Clearance of nitric oxide by carboxy-PTIO or inhibition of S-nitrosylation by N-acetyl-cysteine decreased GTN-induced PGIS S-nitrosylation. Enforced expression of mutated PGIS with C231/441A markedly abolished GTN-induced PGIS S-nitrosylation and nitrate cross-tolerance in Apoe-/- mice. Inhibition of cyclooxygenase 1 by aspirin, supplementation of PGI2 by beraprost, and inhibition of PGIS S-nitrosylation by N-acetyl-cysteine improved GTN-induced nitrate cross-tolerance in rats. In patients, increased PGIS S-nitrosylation was associated with nitrate tolerance. In conclusion, GTN induces nitrate cross-tolerance through PGIS S-nitrosylation at cysteine 231/441.

AMPKα inactivation destabilizes atherosclerotic plaque in streptozotocin-induced diabetic mice through AP-2α/miRNA-124 axis.

Diabetes mellitus is one of risk factors of cardiovascular diseases including atherosclerosis. Whether and how diabetes promotes the formation of unstable atherosclerotic plaque is not fully understood. Here, we show that streptozotocin-induced type 1 diabetes reduced collagen synthesis, leading to the formation of unstable atherosclerotic plaque induced by collar placement around carotid in apolipoprotein E knockout (Apoe-/-) mice. These detrimental effects of hyperglycemia on plaque stability were reversed by metformin in vivo without altering the levels of blood glucose and lipids. Mechanistically, we found that high glucose reduced the phosphorylated level of AMP-activated protein kinase alpha (AMPKα) and the transcriptional activity of activator protein 2 alpha (AP-2α), increased the expression of miR-124 expression, and downregulated prolyl-4-hydroxylase alpha 1 (P4Hα1) protein expression and collagen biosynthesis in cultured vascular smooth muscle cells. Importantly, these in vitro effects produced by high glucose were abolished by AMPKα pharmacological activation or adenovirus-mediated AMPKα overexpression. Further, adenovirus-mediated AMPKα gain of function remitted the process of diabetes-induced plaque destabilization in Apoe-/- mice injected with streptozotocin. Administration of metformin enhanced pAP-2α level, reduced miR-124 expression, and increased P4Hα1 and collagens in carotid atherosclerotic plaque in diabetic Apoe-/- mice. We conclude that streptozotocin-induced toxic diabetes promotes the formation of unstable atherosclerotic plaques based on the vulnerability index in Apoe-/- mice, which is related to the inactivation of AMPKα/AP-2α/miRNA-124/P4Hα1 axis. Clinically, targeting AMPKα/AP-2α/miRNA-124/P4Hα1 signaling should be considered to increase the plaque stability in patients with atherosclerosis. KEY MESSAGES: Hyperglycemia reduced collagen synthesis, leading to the formation of unstable atherosclerotic plaque induced by collar placement around carotid in apolipoprotein E knockout mice. Hyperglycemia destabilizes atherosclerotic plaque in vivo through an AMPKα/AP-2α/miRNA-124/P4Hα1-dependent collagen synthesis. Metformin functions as a stabilizer of atherosclerotic plaque to reduce acute coronary accent.

Pravastatin activates activator protein 2 alpha to augment the angiotensin II-induced abdominal aortic aneurysms.

We have previously reported that activation of AMP-activated kinase alpha 2 (AMPKα2) by nicotine or angiotensin II (AngII) instigates formation of abdominal aortic aneurysms (AAA) in Apoe-/- mice. Statins, used to treat hyperlipidemia widely, activate AMPK in vascular cells. We sought to examine the effects of pravastatin on AAA formation and uncover the molecular mechanism. The AAA model was induced by AngII and evaluated by incidence, elastin degradation, and maximal abdominal aortic diameter in Apoe-/- mice. The phosphorylated levels of AMPKα2 and activator protein 2 alpha (AP-2α) were examined in cultured vascular smooth muscle cells (VSMCs) or in mice. We observed that pravastatin (50 mg/kg/day, 8 weeks) remarkably increased the AngII-induced AAA incidence in mice. In VSMCs, pravastatin increased the levels of pAMPK, pAP-2α, and MMP2 in both basal and AngII-stressed conditions, which were abolished by tempol and compound C. Pravastatin-upregulated MMP2 was abrogated by AMPKα2 or AP-2α siRNA. Lentivirus-mediated gene silence of AMPKα2 or AP-2α abolished pravastatin-worsened AAA formations in AngII-infused Apoe-/- mice. Clinical investigations demonstrated that both AMPKα2 and AP-2α phosphorylations were increased in AAA patients or human subjects taking pravastatin. In conclusion, pravastatin promotes AAA formation through AMPKα2-dependent AP-2α activations.

Lovastatin upregulates microRNA-29b to reduce oxidative stress in rats with multiple cardiovascular risk factors.

AIMS: Proteasome-linked oxidative stress is believed to cause endothelial dysfunction, an early event in cardiovascular diseases (CVD). Statin, as HMG-CoA reductase inhibitor, prevents endothelial dysfunction in CVD. However, the molecular mechanism of statin-mediated normalization of endothelial function is not completely elucidated. METHODS AND RESULTS: Lovastatin time/dose-dependently increased miR-29b expression and decreased proteasome activity in cultured human umbilical vein endothelial cells (HUVECs). Anti-miR-29b or overexpression of PA200 abolished lovastatin-induced inhibition of proteasome activity in HUVECs. In contrast, pre-miR-29b or PA200 siRNA mimics these effects of lovastatin on proteasome activity. Lovastatin inhibited oxidative stress induced by multiple oxidants including ox-LDL, H2O2, TNFα, homocysteine thiolactone (HTL), and high glucose (HG), which were reversed by inhibition of miR-29b in HUVECs. Ex vivo analysis indicated that lovastatin normalized the acetylcholine-induced endothelium-dependent relaxation and the redox status in isolated rat aortic arteries exposure to multiple cardiovascular risk factors. In vivo analysis revealed that administration of lovastatin remarkably suppressed oxidative stress and prevented endothelial dysfunction in rats with hyperglycemia, dyslipidemia, and hyperhomocysteinemia, as well as increased miR-29b expressions, reduced PA200 protein levels, and suppression of proteasome activity in aortic tissues. CONCLUSION: Upregulation of miR-29b expression is a common mechanism contributing to endothelial dysfunction induced by multiple cardiovascular risk factors through PA200-dependent proteasome-mediated oxidative stress, which is prevented by lovastatin.