Exosomes from Adipose-Derived Mesenchymal Stem Cells Protect Against Cyclophosphamide-Induced Cardiotoxicity in Rats.

A certain dosage of cyclophosphamide (CYP) in clinical applications contributes to severe cardiotoxicity. Herein, this study explored the impact of adipose-derived mesenchymal stem cell (AdMSC)-exosomes (Exos) on CYP-induced cardiotoxicity.AdMSCs and AdMSCs-Exos were isolated and identified. CYP was utilized for developing a cardiotoxicity rat model, after which blood was collected and then the serum contents of cardiac injury-related indexes (creatine kinase-MB, lactate dehydrogenase, aspartate aminotransferase, and alkaline phosphatase) were detected with enzyme-linked immunosorbent assay kits. Oxidative stress (OS)-related indicators were measured with the corresponding kits. Myocardial pathological changes and collagen fibrosis were tested with hematoxylin-eosin and Masson staining, and apoptosis-related and autophagy-related proteins in rat cardiac tissues with immunohistochemistry and Western blot assays, respectively.AdMSCs and AdMSCs-Exos were successfully isolated. AdMSCs-Exos could target rat hearts. AdMSCs-Exos improved cardiac function and diminished the content of the cardiac injury-related indexes in CYP rats. In addition, AdMSCs-Exos reduced CYP-induced cardiac fibrosis, OS, apoptosis, and autophagy in rats.AdMSCs-Exos alleviated CYP-induced cardiotoxicity in rats via the repression of OS, apoptosis, and autophagy.

Clinical Comparative Study of Intravitreal Injection of Triamcinolone Acetonide and Aflibercept in the Treatment of Diabetic Retinopathy Cystoid Macular Edema.

Objective To compare the curative effect of intravitreal injection of triamcinolone acetonide and aflibercept on diabetic retinopathy (DR) cystoid macular edema. Methods A total of 102 patients with DR cystoid macular edema admitted to the hospital were enrolled between July 2018 and July 2021. According to random number table method, they were divided into the control group (intravitreal injection of triamcinolone acetonide) and the observation group (intravitreal injection of aflibercept), 51 cases in each group. All were followed up for half a year. The clinical curative effect, visual acuity, central subfield macular thickness (CSMT), macular volume, scores of quality of life, and levels of cytokines in aqueous humor (vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), human angiopoietin-like protein 4 (ANGPTL4)] at different time points (before and at 6 months after surgery) were compared between the two groups. The times of drugs injection and occurrence of adverse reactions in both groups were statistically analyzed. Results The total effective rate in observation group was higher than that in the control group (96.08% vs 82.35%) (P < 0.05). After 6 months of treatment, visual acuity was improved, and CSMT and macular volume were decreased in both groups. Also, the above changes were more significant in the observation group than those in the control group (P < 0.05). After 6 months of treatment, levels of cytokines in aqueous humor were decreased in both groups. The levels of VEGF, MCP-1, and ANGPTL4 in observation group were lower than those in the control group (P < 0.05). After 6 months of treatment, quality of life scores in observation group were higher than those in the control group (P < 0.05). In the follow-up period, average times of drugs injection in the observation group were more than those in the control group, and the incidence of adverse reactions was lower than that in control group (5.88% vs 21.57%) (P < 0.05). Conclusion The curative effect of intravitreal injection of both triamcinolone acetonide and aflibercept is good on DR cystoid macular edema. The curative effect of aflibercept is better, which can improve visual acuity and quality of life, and regulate cytokines in aqueous humor, with high safety. However, aflibercept has a high price, and further research is needed to determine whether its price can be matched with clinical benefits. In clinic, medication plan should be selected according to the actual situation.

Inhibition of miR-322-5p Protects Cardiac Myoblast Cells Against Hypoxia-Induced Apoptosis and Injury Through Regulating CIAPIN1.

ABSTRACT: Hypoxia leads to insufficient supply of blood and nutrients, which is major incentive for cardiomyocyte injury and apoptosis. Previous studies reported the regulation effects of microRNAs (miRNAs) in myocardial infarction, whereas function and molecular mechanisms of miR-322-5p were still unclear. Therefore, our study focused on the biological role of miR-322-5p in hypoxia-induced cardiac myoblast cells apoptosis and injury. The expression levels of miR-322-5p and cytokine-induced apoptosis inhibitor 1 (CIAPIN1) were measured by real-time quantitative polymerase chain reaction in cardiac myoblast cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazol-3-ium bromide (MTT), lactic dehydrogenase, and flow cytometry assays were performed to examine proliferation, injury, and apoptosis of cardiac myoblast cells, respectively. The protein expression levels were evaluated with western blot assay. The relationship between miR-322-5p and CIAPIN1 was confirmed by dual-luciferase reporter analysis. We found that miR-322-5p level was increased in cardiac myoblast cells exposed to hypoxia. In addition, miR-322-5p silencing could weaken injury and apoptosis in cardiac myoblast cells induced by hypoxia; meanwhile, inhibition of miR-322-5p activation of phosphatidylinositol-3 kinases (PI3K)/protein kinase B (AKT) signal pathway. Besides, CIAPIN1 was a target mRNA of miR-322-5p based on bioinformatics prediction. CIAPIN1 knockdown reversed the effects of miR-322-5p silencing on hypoxic cardiac myoblast cells. Suppression of miR-322-5p protected cardiac myoblast cells against hypoxia-induced injury and apoptosis through regulation of CIAPIN1 expression and PI3K/AKT signal pathway.

SIRT4 suppresses the PI3K/Akt/NF­κB signaling pathway and attenuates HUVEC injury induced by oxLDL.

Atherosclerosis is a chronic and progressive disease. Its morbidity and mortality rates have demonstrated an increase in recent years. The present study aimed to explore the role of sirtuin (SIRT) 4 in the development of atherosclerosis. Alterations in SIRT4 expression in response to oxidized low density lipoprotein (oxLDL) were quantified in human umbilical vein endothelial cells (HUVECs) using western blotting. Cell counting kit­8 and flow cytometry assays were used in order to explore the effects of SIRT4 on HUVEC proliferation and apoptosis. The effect of SIRT4 on the expression of inflammatory factors in HUVECs was analyzed using ELISA. The expression and phosphorylation of proteins in the phosphoinositide 3­kinase (PI3K)/protein kinase B (Akt)/nuclear factor (NF)­κB pathway were comparatively analyzed using western blotting. Nuclear translocation of p65 NF­κB was examined using immunofluorescence. The present study indicated that oxLDL treatment decreased the expression of SIRT4 in HUVECs in a dose­ and time­dependent manner. SIRT4 overexpression promoted oxLDL­induced HUVEC proliferation and inhibited cell apoptosis. Furthermore, SIRT4 overexpression suppressed the PI3K/Akt/NF­κB pathway by inhibiting PI3K phosphorylation and phosphorylated (p)­Akt, p­nuclear factor of kappa light polypeptide gene enhancer in B­cells inhibitor α and p­p65 NF­κB expression; blocking p65 NF­κB nuclear translocation and decreasing interleukin (IL)­1ß, IL­6, and tumor necrosis factor α expression in oxLDL­induced HUVECs. In conclusion, SIRT4 overexpression enhanced HUVEC survival, suppressed the PI3K/Akt/NF­κB signaling pathway and inhibited the expression of inflammatory cytokines in oxLDL­induced HUVECs.

Fenofibrate Ameliorates Oxidative Stress-Induced Retinal Microvascular Dysfunction in Diabetic Rats.

PURPOSE: The aim of this study is to investigate the effects of fenofibrate (FA) on microvascular dysfunction in the retina of diabetic rats, and to determine the underlying mechanism. METHODS: Streptozotocin (STZ)-induced diabetic rats and control rats were used in this study. A subgroup of STZ-induced diabetic rats was treated orally with vehicle or FA (100 mg/kg/day) for 24 weeks along with regular monitoring of body weight and serum parameters. At the end of the 24-week treatment, retinal vascular permeability was quantified by confocal microscopy using Evans blue as a tracer. Retinal capillary basement membrane thickness (BMT) was examined by transmission electron microscopy. The retinal reactive oxygen species (ROS) level was quantified by flow cytometry using a fluorescent probe. Levels of vascular endothelial growth factor (VEGF), nuclear facto (NF)-κB (p65), and thioredoxin interacting protein (TXNIP) were measured by Western blotting or enzyme-linked immunosorbent assay and real-time reverse transcription polymerase chain reaction. RESULTS: Retinal vascular permeability and BMT were significantly increased in diabetic rats compared to in non-diabetic control rats. Diabetes also increased the retinal ROS levels. These effects were associated with increased levels of VEGF, phosphorylation of p65(P-p65), and TXNIP. FA significantly ameliorated the retinal vascular permeability, alleviated retinal BMT, and reduced retinal ROS level. Consistent with these effects, FA also decreased VEGF and P-p65 expression and, at the same time, decreased TXNIP expression. CONCLUSIONS: FA prevents the retinal microvascular dysfunction induced by diabetes, likely by restoring VEGF and P-p65 levels, and possibly by reducing oxidative stress and TXNIP expression.

Adipose-derived stem cell-derived microvesicle-released miR-210 promoted proliferation, migration and invasion of endothelial cells by regulating RUNX3.

The potential mechanism of miRNA released from adipose-derived stem cell (ADSC)-derived micro vesicle (MV) onthe modulation of proliferation, migration and invasion of endothelial cells were explored. In this study, miR-210 level was detected by qT-PCR. Alix, VEGF and RUNX3 expressions were detected by Western blot. The proliferation, migration and invasion of human umbilical vein endothelial cells (HUVECs) were observed by MTT assay and Transwell assay. Luciferase reporter gene assay was conducted to validate the targeting activity of MVs-released miR-210 on RUNX3. We found hypoxia significantly increased the expression of MVs-released miR-210. MVs released from ADSCsin hypoxic group significantly promoted the proliferation, migration and invasion of HUVECs. Overexpression of miR-210 significantly upregulated VEGF expression, and promoted the proliferation, migration and invasion of HUVECs. Besides, RUNX3 was identified as the direct of miR-210 in HUVECs. Overexpression of miR-210 decreased RUNX3 expression and promoted the proliferation, migration and invasion of HUVECs, while overexpression of RUNX3 inhibited these promotion effects. In vivo experiment showed that MVs derived from ADSCs under hypoxia increased miR-210 level and capillary density, and inhibition of miR-210 decreased capillary density. We also found MVs downregulated RUNX3 expression, and inhibition of miR-210 upregulated RUNX3 expression. Therefore, miR-210 released from ADSCs-derived MVs promoted proliferation, migration and invasion of HUVECs by targeting RUNX3, which revealed one of the mechanisms of ADSCs-derived MVs on the promotion of proliferation, migration and invasion of HUVECs. ABBREVIATIONS: ADSC, adipose-derived stem cell; MV, micro vesicle; HUVECs, human umbilical vein endothelial cells; RUNX3, Runtrelatedtranscription factor-3.

Genistein attenuates monocrotaline-induced pulmonary arterial hypertension in rats by activating PI3K/Akt/eNOS signaling.

INTRODUCTION: Phytoestrogen genistein may be useful to treat pulmonary arterial hypertension (PAH). However, its mechanism is still not clear. The aim of the present study was to confirm the therapeutic effects of phytoestrogen genistein on PAH in monocrotaline-induced rat model and to explore its mechanism. MATERIALS AND METHODS: Sprague-Dawley male rats were randomly divided into 4 groups: control group (n=8), PAH group (n=8), genistein treament group with three different doses (n=8 in each dose group) and group of PI3K inhibitor LY294002. The rat model of PAH was induced by monocrotaline (MCT). The situation of survival of rats was observed. Pathological studies of lung and heart tissues were performed. Western-blot detection of P-Akt and P-eNOS expression levels in lung tissue was carried out. Nitrate reductase analysis was used to measure nitric oxide (NO) in lung tissue. RESULTS: Genistein treatment resulted in significant improvement in the speed of tricuspid regurgitation, diameter of pulmonary artery, mean pulmonary artery pressure and right ventricular hypertrophy index. Genistein treatment also resulted in significant improvement in the stenosis of pulmonary artery, proliferation of smooth muscle, right ventricular hypertrophy and myocardial hypertrophy. These therapeutic effects were more obvious with increasing dose of genistein. After genistein treatment, amelioration in survival rates of PAH rats was observed. PI3K inhibitor LY294002 could block these therapeutic effects. In rat lung tissue, P-Akt, P-eNOS and NO expressions were increased significantly in genistein treatment group when compared with PAH group (p<0.05, respectively). The increase in expression level of P-Akt, P-eNOS and NO was correlated with genistein dose. P-Akt, P-eNOS and NO expressions in lung tissue increased slightly in the PI3K inhibitor LY294002 group when compared with PAH group, but the difference was not statistically significant (p>0.05). CONCLUSIONS: We confirmed that genistein could relax pulmonary vascular resistance, reduce pulmonary artery pressure, improve right heart function and ameliorate survival rate in the rat model of PAH. Our study suggested that its mechanism was related with PI3K/Akt/eNOS signal pathway. Phytoestrogen genistein may become a new and effective drug for patients with PAH.

Curcumin Attenuates Retinal Vascular Leakage by Inhibiting Calcium/Calmodulin-Dependent Protein Kinase II Activity in Streptozotocin-Induced Diabetes.

BACKGROUND: Curcumin possesses many pharmacological properties including anti-inflammatory effects. Although prior studies indicate that curcumin has beneficial effects for diabetic retinopathy, the mechanism of action is not known. To address this issue, we investigated the effect of curcumin against diabetes-induced retinal vascular damage and its mechanism of action by using cultured retinal Müller cells stimulated with high glucose. METHODS: We studied the effects of curcumin in vivo in the retinas of rats rendered diabetic by streptozotocin and in vitro in Müller cells stimulated with high glucose. We administered curcumin, or KN93, an inhibitor of calcium/calmodulin dependent protein kinase II (CaMKII), or saline vehicle to experimental animals on a daily basis for 12 weeks. Primary cultures of rat Müller cells were incubated with normal glucose or high glucose, with or without curcumin, KN93, or pyrrolidine dithiocarbamate (PDTC), an inhibitor of the transcription protein nuclear factor κB (NF-κB). We examined mRNA and protein levels of vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS) and intercellular adhesion molecule-1 (ICAM-1) by real-time RT-PCR and Western blotting, respectively. Retinal levels of CaMKII and NF-κB were examined by Western blotting. Vascular leakage was evaluated using Evans blue. RESULTS: Curcumin and KN93 significantly inhibited the activation of CaMKII/NF-κB signaling induced by diabetes or elevated glucose, and subsequently decreased the expression of VEGF, iNOS and ICAM-1. These changes were associated with a decrease of diabetes-induced retinal vascular leakage. CONCLUSION: Curcumin protects the diabetic rat retina against early retinal vascular damage, by inhibition of CaMKII activity. Curcumin is currently used to treat a number of clinical conditions, and may prove beneficial for the management of diabetic retinopathy.

Endoscopic Transretracaruncular-Middle Meteas Tract for Insertion of a Porous Polyethylene-Coated Jones Tube.

PURPOSE: The purpose of this article is to describe a modified lacrimal bypass with a porous polyethylene-coated Jones tube. METHODS: A total of 180 patients (180 eyes) with a nonreconstructable lacrimal obstruction underwent lacrimal bypass with a porous polyethylene-coated Jones tube through a retrocaruncular-middle meatus tract approach with endoscopic assistance. All patients were followed up at least for 24 months. Success rate of lacrimal bypass was analyzed and complications were recorded. RESULTS: A total of 174 patients were finally included. Duration of surgery ranged from 28 to 47 minutes (mean 37.2 ±â€Š4.2 minutes). The mean duration of follow-up was 30.0 ±â€Š6.4 months (range 24-48 months). The mean tube length was 23.2 ±â€Š1.9 mm (range 20-28 mm). At the final review, complete success was achieved in 138 (79.3%) patients. Moderate success was achieved in 23 (13.2%) patients, and 13 (7.5%) patients failed. Of the 161 patients successfully treated, 24 patients underwent revision surgery to excise granulomas (15 patients) or adjust tube position (9 patients). The complications included granuloma proliferation around the openings of the tube (28 eyes), downward displacement of the tube (17 eyes), and ocular discomfort (15 eyes). The majority of downward tube migration occurred in patients who had a prior history of dacryocystorhinostomy. The treatment failed for 5 patients because of repeated granulomas covering the nasal tube openings, and the treatment failed for 8 patients because of downward displacement of the tube. CONCLUSIONS: Our procedure appears to be an effective method for closed insertion of a porous polyethylene-coated Jones.

Calcium entry mediates hyperglycemia-induced apoptosis through Ca(2+)/calmodulin-dependent kinase II in retinal capillary endothelial cells.

PURPOSE: Hyperglycemia-induced vascular cell apoptosis is a seminal early event in diabetic retinopathy. Prolonged hyperglycemia is known to increase intracellular cytosolic free calcium ([Ca(2+)]i) in retinal vascular endothelial cells (RECs), suggesting that [Ca(2+)]i is a critical trigger for microvascular degeneration. This study aims to elucidate Ca(2+)-dependent signaling mechanisms that mediate hyperglycemia-induced apoptosis in RECs. METHODS: A cultured macaque choroid-retinal endothelial cell line (RF/6A) was incubated in normal glucose (NG), NG plus the Ca(2+) entry blocker 2-aminoethoxydiphenyl borate (2-APB), high glucose (HG), or HG plus either 2-APB, the c-jun N-terminal kinase (JNK) inhibitor SP600125, or the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93. Changes in [Ca(2+)]i evoked by adenosine 5'-triphosphate (ATP) were measured in fluo-3/AM-loaded RF/6A cells by confocal microscopy. The mitochondrial membrane potential (ΔΨm) and apoptosis were assessed by flow cytometry. Expression levels of CaMKII, phosphorylated CaMKII (p-CaMKII), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), the death receptor (Fas), and cytochrome c were detected by western blotting analysis. RESULTS: Prolonged exposure to HG (96 h) potentiated ATP-evoked Ca(2+) entry as well as CaMKII phosphorylation and RF/6A cell apoptosis. Enhanced apoptosis was blocked by 2-APB and KN93. Furthermore, HG increased JNK phosphorylation and Fas expression, and both responses were partially blocked by 2-APB and KN93, while the JNK inhibitor SP600125 partially reduced HG-induced Fas expression. In addition, HG depolarized the ΔΨm and triggered the release of mitochondrial cytochrome c. These early signs of mitochondria-dependent apoptosis were partially reversed by 2-APB and KN93. CONCLUSIONS: HG-induced apoptosis in RF/6A cells depends on Ca(2+) entry and CaMKII activation, leading to the activation of both Fas-dependent and mitochondria-dependent apoptosis pathways. The CaMKII-JNK-Fas pathway is involved in HG-evoked apoptosis of RECs.

Calcium mediates high glucose-induced HIF-1α and VEGF expression in cultured rat retinal Müller cells through CaMKII-CREB pathway.

AIM: To investigate the effects of high glucose (HG) medium on expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in cultured rat retinal Müller cells and to determine the signaling pathways mediating the effects. METHODS: Primary cultures of retinal Müller cells were prepared from Sprague-Dawley rats, and incubated in a medium containg HG (30 mmol/L) in the presence of the membrane-permeable Ca(2+) chelator BAPTA-AM (10 µmol/L) or the CaMKII inhibitor KN93 (10 µmol/L). The levels of CaMKII, p-CaMKII, CREB, p-CREB, HIF-1α, and VEGF proteins were measured with Western blotting, while HIF-1á and VEGF mRNA levels were determined using real-time RT-PCR. RESULTS: The stimulation of retinal Müller cell with HG for 24 h remarkably increased the expression levels of HIF-1α and VEGF. These responses were significantly inhibited in the presence of BAPTA-AM or KN93. Both BAPTA-AM and KN93 also significantly inhibited HG-induced phosphorylation of CaMKII and CREB in the cultured retinal Müller cells. Transfection of the cultured retinal Müller cells with antisense CREB oligonucleotide (300 nmol/L) was similarly effective in blocking the HG-induced increase of HIF-1α and VEGF. CONCLUSION: HG-induced HIF-1α and VEGF expression in cultured rat retinal Müller cells depends on intracellular free Ca(2+) and activation of CaMKII-CREB pathway. The activation of CaMKII-CREB pathway by HG may be a possible mechanism underlying the pathogenesis of diabetic retinopathy.