Circulating microRNAs Associated With Reversible Cerebral Vasoconstriction Syndrome.

OBJECTIVE: The purpose of this study was to investigate the significance of circulating micro RNAs (miRNAs) in the pathogenesis of reversible cerebral vasoconstriction syndrome (RCVS). METHODS: We prospectively recruited 3 independent cohorts of patients with RCVS and age-matched and sex-matched controls in a single medical center. Next-generation small RNA sequencing followed by quantitative polymerase chain reaction (PCR) was used to identify and validate differentially expressed miRNAs, which was cross-validated in migraine patients in ictal stage or interictal stage. Computational analysis was used to predict the target genes of miRNAs, followed by in vitro functional analysis. RESULTS: We identified a panel of miRNAs including miR-130a-3p, miR-130b-3p, let-7a-5p, let-7b-5p, and let-7f-5p that well differentiated patients with RCVS from controls (area under the receiver operating characteristics curve [AUC] was 0.906, 0.890, and 0.867 in the 3 cohorts, respectively). The abundance of let-7a-5p, let-7b-5p, and let-7f-5p, but not miR-130a-3p nor miR-130b-3p, was significantly higher in patients with ictal migraine compared with that of controls and patients with interictal migraine. Target prediction and pathway enrichment analysis suggested that the transforming growth factor-ß signaling pathway and endothelin-1 responsible for vasomotor control might link these miRNAs to RCVS pathogenesis, which was confirmed in vitro by transfecting miRNAs mimics or incubating the patients' cerebrospinal fluid (CSF) in 3 different vascular endothelial cells. Moreover, miR-130a-3p was associated with imaging-proven disruption of the blood-brain barrier (BBB) in patients with RCVS and its overexpression led to reduced transendothelial electrical resistance (ie, increased permeability) in in vitro human BBB model. INTERPRETATION: We identified the circulating miRNA signatures associated with RCVS, which may be functionally linked to its headache, BBB integrity, and vasomotor function. ANN NEUROL 2021;89:459-473.

Nitric Oxide Mobilizes Intracellular Zn2+ via the GC/cGMP/PKG Signaling Pathway and Stimulates Adipocyte Differentiation.

Plasma and tissue zinc ion levels are associated with the development of obesity. Previous studies have suggested that zinc ions may regulate adipocyte metabolism and that nitric oxide (NO) plays a pivotal role in the regulation of adipocyte physiology. Our previous study showed that chronic NO deficiency causes a significant decrease in adipose tissue mass in rats. Studies also suggested that zinc ions play an important modulatory role in regulating NO function. This study aims to explore the role of zinc ions in NO-regulated adipocyte differentiation. We hypothesized that NO could increase intracellular Zn2+ level and then stimulate adipocyte differentiation. ZnCl2 and the NO donor, NONOate, were used to explore the effects of Zn2+ and NO on adipocyte differentiation. Regulatory mechanisms of NO on intracellular Zn2+ mobilization were determined by detection. Then, Zn2+-selective chelator TPEN was used to clarify the role of intracellular Zn2+ on NO-regulated adipocyte differentiation. Furthermore, the relationship between adipocyte size, Zn2+ level, and NOS expression in human subcutaneous fat tissue was elucidated. Results showed that both ZnCl2 and NO stimulated adipocyte differentiation in a dose-dependent manner. NO stimulated intracellular Zn2+ mobilization in adipocytes through the guanylate cyclase (GC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) pathway, and NO-stimulated adipocyte differentiation was Zn2+-dependent. In human subcutaneous adipose tissue, adipocyte size was negatively correlated with expression of eNOS. In conclusion, NO treatment stimulates intracellular Zn2+ mobilization through the GC/cGMP/PKG pathway, subsequently stimulating adipocyte differentiation.

Cysteine-Cysteine Motif Chemokine Receptor 5 Expression in Letrozole-Induced Polycystic Ovary Syndrome Mice.

Polycystic ovary syndrome (PCOS), which affects 5-10% of women of reproductive age, is associated with reproductive and metabolic disorders, such as chronic anovulation, infertility, insulin resistance, and type 2 diabetes. However, the mechanism of PCOS is still unknown. Therefore, this study used a letrozole-exposed mouse model in which mice were orally fed letrozole for 20 weeks to investigate the effects of letrozole on the severity of reproductive and metabolic consequences and the expression of cysteine-cysteine motif chemokine receptor 5 (CCR5) in letrozole-induced PCOS mice. The letrozole-treated mice showed a disrupted estrous cycle and were arrested in the diestrus phase. Letrozole treatment also increased plasma testosterone levels, decreased estradiol levels, and caused multicystic follicle formation. Furthermore, histological analysis of the perigonadal white adipose tissue (pgWAT) showed no significant difference in the size and number of adipocytes between the letrozole-treated mice and the control group. Further, the letrozole-treated mice demonstrated glucose intolerance and insulin resistance during oral glucose and insulin tolerance testing. Additionally, the expression of CCR5 and cysteine-cysteine motif ligand 5 (CCL5) were significantly higher in the pgWAT of the letrozole-treated mice compared with the control group. CCR5 and CCL5 were also significantly correlated with the homeostasis model assessment of insulin resistance (HOMA-IR). Finally, the mechanisms of insulin resistance in PCOS may be caused by an increase in serine phosphorylation and a decrease in Akt phosphorylation.

Visfatin Promotes Monocyte Adhesion by Upregulating ICAM-1 and VCAM-1 Expression in Endothelial Cells via Activation of p38-PI3K-Akt Signaling and Subsequent ROS Production and IKK/NF-κB Activation.

BACKGROUND/AIMS: Visfatin is known to act as a mediator in several metabolic disorders, such as obesity, diabetes, and cardiovascular diseases. This study aimed to investigate the effect of visfatin on the adhesion of THP-1 monocytes to human vascular endothelial cells and the underlying mechanism. METHODS: Monocytes adhesion to endothelial cells was determined by using fluorescence-labeled monocytes. ICAM-1 and VCAM-1 expression in endothelial cells were measured by western blotting. Production of reactive oxygen species (ROS) was measured by using a fluorescent dye. The amounts of nuclear factor-kappa B (NF-κB) and phosphorylation of inhibitory factor of NF-κB (IκB) were determined by using western blot analysis. The translocation of NF-κB from the cytoplasm to the nucleus was determined by using immunofluorescence. RESULTS: Here we showed that visfatin significantly caused the upregulation of intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in endothelial cells, as well as enhanced monocyte adhesion to endothelial cells. Moreover, we found that inhibition of PI3K, Akt, and p38 MAPK activation significantly prevented visfatin-enhanced expression of ICAM-1 and VCAM-1 and monocyte adhesion to endothelial cells. Visfatin enhanced ROS production and IKK/NF-кB activation and then led to upregulation of ICAM-1 and VCAM-1 and enhanced monocyte adhesion to endothelial cells. These effects were also p38/PI3K/Akt-dependent. CONCLUSION: These results demonstrated that visfatin promoted monocyte-endothelial cell adhesion by increasing ICAM-1 and VCAM-1 expression via the activation of p38/PI3K/Akt signaling and downstream ROS production and IKK/NF-кB activation.

Cysteine-cysteine Chemokine Receptor Type 5 Plays a Critical Role in Exercise Performance by Regulating Mitochondrial Content in Skeletal Muscle.

Cysteine-cysteine chemokine receptor type 5 (CCR5) is thought to play an important role in the trafficking of lymphoid cells but has recently also been associated with AMPK signaling pathways that are implicated in energy metabolism in skeletal muscle. We hypothesized that genetic deletions of CCR5 would alter mitochondria content and exercise performance in mice. CCR5-/- and wild-type mice with the same genetic background were subjected to endurance exercise and grip strength tests. The soleus muscle was stained with immunofluorescence for myosin heavy chain 7 (MYH7) and succinate dehydrogenase (SDH) analysis as well as the expression of genes associated with muscle atrophy and mitochondrial oxidative phosphorylation were measured using qPCR. Although there were no differences in the weight of the soleus muscle between the CCR5-/- group and the wild-type mice, the CCR5-/- mice showed the following muscular dysfunctions: (i) decreased MYH7 percentage and cross-section area, (ii) higher myostatin and atrogin-1 mRNA levels, (iii) dropped expression of mitochondrial DNA-encoded electron respiratory chain genes (cytochrome B, cytochrome c oxidase subunit III, and ATP synthase subunit 6) as well as mitochondrial generation genes (PPARγ and PGC-1α), and (iv) lower SDH activity and exercise performance when compared with wild-type mice. In addition, genes associated with mitochondrial biogenesis (PGC-1α, PPARγ, and MFN2) and mitochondrial complex (ND4 and Cytb) were upregulated when the skeletal muscle cell line C2C12 was exposed to cysteine-cysteine chemokine ligand 4 (a ligand of CCR5) in vitro. These findings suggested that attenuation of endurance exercise performance is related to the loss of mitochondrial content and lower SDH activity of soleus muscle in CCR5 knockout mice. The present study provides evidence indicating that the chemokine receptor CCR5 might modulate the skeletal muscle metabolic energy system during exercise.

Endothelin-1 induces lipolysis through activation of the GC/cGMP/Ca2+/ERK/CaMKIII pathway in 3T3-L1 adipocytes.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide produced and secreted mainly by endothelial cells. Recent studies indicate that ET-1 can regulate lipid metabolism, which may increase the risk of insulin resistance. Our previous studies revealed that ET-1 induced lipolysis in adipocytes, but the underlying mechanisms were unclear. 3T3-L1 adipocytes were used to investigate the effect of ET-1 on lipolysis and the underlying mechanisms. Glycerol levels in the incubation medium and hormone-sensitive lipase (HSL) phosphorylation were used as indices for lipolysis. ET-1 significantly increased HSL phosphorylation and lipolysis, which were completely inhibited by ERK inhibitor (PD98059) and guanylyl cyclase (GC) inhibitor (LY83583). LY83583 reduced ET-1-induced ERK phosphorylation. A Ca2+-free medium and PLC inhibitor caused significant decreases in ET-1-induced lipolysis as well as ERK and HSL phosphorylation, and IP3 receptor activator (D-IP3) increased lipolysis. ET-1 increased cGMP production, which was not affected by depletion of extracellular Ca2+. On the other hand, LY83583 diminished the ET-1-induced Ca2+ influx. Transient receptor potential vanilloid-1 (TRPV-1) antagonist and shRNA partially inhibited ET-1-induced lipolysis. ET-1-induced lipolysis was completely suppressed by CaMKIII inhibitor (NH-125). These results indicate that ET-1 stimulates extracellular Ca2+ entry and activates the intracellular PLC/IP3/Ca2+ pathway through a cGMP-dependent pathway. The increased cytosolic Ca2+ that results from ET-1 treatment stimulates ERK and HSL phosphorylation, which subsequently induces lipolysis. ET-1 induces HSL phosphorylation and lipolysis via the GC/cGMP/Ca2+/ERK/CaMKIII signaling pathway in 3T3-L1 adipocytes.

Glucosamine Interferes With Myelopoiesis and Enhances the Immunosuppressive Activity of Myeloid-Derived Suppressor Cells.

Glucosamine (GlcN) is the most widely consumed dietary supplement and exhibits anti-inflammatory effects. However, the influence of GlcN on immune cell generation and function is largely unclear. In this study, GlcN was delivered into mice to examine its biological function in hematopoiesis. We found that GlcN promoted the production of immature myeloid cells, known as myeloid-derived suppressor cells (MDSCs), both in vivo and in vitro. Additionally, GlcN upregulated the expression of glucose transporter 1 in hematopoietic stem and progenitor cells (HSPCs), influenced HSPC functions, and downregulated key genes involved in myelopoiesis. Furthermore, GlcN increased the expression of arginase 1 and inducible nitric oxide synthase to produce high levels of reactive oxygen species, which was regulated by the STAT3 and ERK1/2 pathways, to increase the immunosuppressive ability of MDSCs. We revealed a novel role for GlcN in myelopoiesis and MDSC activity involving a potential link between GlcN and immune system, as well as the new therapeutic benefit.

Curcumin Attenuates Adipogenesis by Inducing Preadipocyte Apoptosis and Inhibiting Adipocyte Differentiation.

Patients with metabolic syndrome are at an increased risk of developing type 2 diabetes and cardiovascular diseases. The principal risk factor for development of metabolic syndrome is obesity, defined as a state of pathological hyperplasia or/and hypertrophy of adipose tissue. The number of mature adipocytes is determined by adipocyte differentiation from preadipocytes. The purpose of the present study is to investigate the effects of curcumin on adipogenesis and the underlying mechanism. To examine cell toxicity of curcumin, 3T3-L1 preadipocytes were treated with 0-50 µM curcumin for 24, 48, or 72 h, then cell viability was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The effect of curcumin on the cell cycle was determined by flow cytometry. Curcumin-induced cell apoptosis was determined by the TUNEL assay and curcumin-induced caspase activation was measured by immunoblotting. The effect of curcumin on adipocyte differentiation was determined by measuring mitotic clonal expansion (MCE), expression of adipogenic transcription factors, and lipid accumulation. Results showed the viability of preadipocytes was significantly decreased by treatment with 30 µM curcumin, a concentration that caused apoptosis in preadipocytes, as assessed by the TUNEL assay, and caused activation of caspases 8, 9, and 3. A non-cytotoxic dose of curcumin (15 µM) inhibited MCE, downregulated the expression of PPARγ and C/EBPα, prevented differentiation medium-induced ß-catenin downregulation, and decreased the lipid accumulation in 3T3-L1 adipocytes. In conclusion, our data show that curcumin can induce preadipocyte apoptosis and inhibit adipocyte differentiation, leading to suppression of adipogenesis.

Adipocytes play an etiological role in the podocytopathy of high-fat diet-fed rats.

Obesity is a risk factor that promotes progressive kidney disease. Studies have shown that an adipocytokine imbalance contributes to impaired renal function in humans and animals, but the underlying interplay between adipocytokines and renal injury remains to be elucidated. We aimed to investigate the mechanisms linking obesity to chronic kidney disease. We assessed renal function in high-fat (HF) diet-fed and normal diet-fed rats, and the effects of preadipocyte- and adipocyte-conditioned medium on cultured podocytes. HF diet-fed and normal diet-fed Sprague Dawley rats were used to analyze the changes in plasma BUN, creatinine, urine protein and renal histology. Additionally, podocytes were incubated with preadipocyte- or adipocyte-conditioned medium to investigate the effects on podocyte morphology and protein expression. In the HF diet group, 24 h urinary protein excretion (357.5 ± 64.2 mg/day vs 115.9 ± 12.4 mg/day, P < 0.05) and the urine protein/creatinine ratio were significantly higher (1.76 ± 0.22 vs 1.09 ± 0.15, P < 0.05), increased kidney weight (3.54 ± 0.04 g vs 3.38 ± 0.04 g, P < 0.05) and the glomerular volume and podocyte effacement increased by electron microscopy. Increased renal expression of desmin and decreased renal expression of CD2AP and nephrin were also seen in the HF diet group (P < 0.05). Furthermore, we found that adipocyte-conditioned medium-treated podocytes showed increased desmin expression and decreased CD2AP and nephrin expression compared with that in preadipocyte-conditioned medium-treated controls (P < 0.05). These findings show that adipocyte-derived factor(s) can modulate renal function. Adipocyte-derived factors play an important role in obesity-related podocytopathy.

Induced pluripotent stem cells alleviate lung injury from mesenteric ischemia-reperfusion.

BACKGROUND: Mesenteric ischemia-reperfusion (I/R) injury is a serious pathophysiologic process that can trigger the development of multiorgan dysfunction. Acute lung injury is a major cause of death among mesenteric I/R patients, as current treatments remain inadequate. Stem cell-based therapies are considered novel strategies for treating several devastating and incurable diseases. This study examined whether induced pluripotent stem cells (iPSCs) lacking c-myc (i.e., induced using only the three genes oct4, sox2, and klf4) can protect against acute lung injury in a mesenteric I/R mouse model. METHODS: C57BL/6 mice were randomly divided into the following groups: sham/no treatment, vehicle treatment with phosphate-buffered saline, treatment with iPSCs, and treatment with iPSC-conditioned medium. The mice were subjected to mesenteric ischemia for 45 minutes followed by reperfusion for 24 hours. After I/R, the lungs and the ileum of the mice were harvested. Lung injury was evaluated by histology, immunohistochemistry, and analyses of the levels of inflammatory cytokines, cleaved caspase 3, and 4-hydroxynonenal. RESULTS: The intravenously delivered iPSCs engrafted to the lungs and the ileum in response to mesenteric I/R injury. Compared with the phosphate-buffered saline-treated group, the iPSC-treated group displayed a decreased intensity of acute lung injury 24 hours after mesenteric I/R. iPSC transplantation significantly reduced the expression of proinflammatory cytokines, oxidative stress markers, and apoptotic factors in injured lung tissue and remarkably enhanced endogenous alveolar cell proliferation. iPSC-conditioned medium treatment exerted a partial effect compared with iPSC treatment. CONCLUSION: When considering the anti-inflammatory, antioxidant, and antiapoptotic properties of iPSCs, the transplantation of iPSCs may represent an effective treatment option for mesenteric I/R-induced acute lung injury.

Long tract of untranslated CAG repeats is deleterious in transgenic mice.

The most frequent trinucleotide repeat found in human disorders is the CAG sequence. Expansion of CAG repeats is mostly found in coding regions and is thought to cause diseases through a protein mechanism. Recently, expanded CAG repeats were shown to induce toxicity at the RNA level in Drosophila and C. elegans. These findings raise the possibility that CAG repeats may trigger RNA-mediated pathogenesis in mammals. Here, we demonstrate that transgenic mice expressing EGFP transcripts with long CAG repeats in the 3' untranslated region develop pathogenic features. Expression of the transgene was directed to the muscle in order to compare the resulting phenotype to that caused by the CUG expansion, as occurs in myotonic dystrophy. Transgenic mice expressing 200, but not those expressing 0 or 23 CAG repeats, showed alterations in muscle morphology, histochemistry and electrophysiology, as well as abnormal behavioral phenotypes. Expression of the expanded CAG repeats in testes resulted in reduced fertility due to defective sperm motility. The production of EGFP protein was significantly reduced by the 200 CAG repeats, and no polyglutamine-containing product was detected, which argues against a protein mechanism. Moreover, nuclear RNA foci were detected for the long CAG repeats. These data support the notion that expanded CAG repeat RNA can cause deleterious effects in mammals. They also suggest the possible involvement of an RNA mechanism in human diseases with long CAG repeats.