Immortalized Mesenchymal Stromal Cells Overexpressing Alpha-1 Antitrypsin Protect Acinar Cells from Apoptotic and Ferroptotic Cell Death.

Chronic pancreatitis (CP) is a progressive inflammatory disorder that impairs endocrine and exocrine function. Our previous work suggests that mesenchymal stem/stromal cells (MSCs) and MSCs overexpressing alpha-1 antitrypsin (AAT-MSCs) could be therapeutic tools for CP treatment in mouse models. However, primary MSCs have a predisposition to undergo senescence during culture expansion which limits their therapeutic applications. Here we generated and characterized immortalized human MSCs (iMSCs) and AAT-MSCs (iAAT-MSCs) and tested their protective effect on 2,4,6-Trinitrobenzenesulfonic acid (TNBS) -induced acinar cell death in an in vitro cell culture system. Primary MSCs were immortalized by transduction with simian virus 40 large T antigen (SV40LT), and the resulting iMSC and iAAT-MSC lines were analyzed for proliferation, senescence, phenotype, and multi-differentiation potential. Subsequently, the impact of these cells on TNBS-induced cell death was measured and compared. Both apoptosis and ferroptosis pathways were investigated by assessing changes of critical factors before and after cell treatment. Coculture of iMSCs and iAAT-MSCs with acinar cell lines inhibited early apoptosis induced by TNBS, reduced ER stress, and reversed TNBS-induced protein reduction at tight junctions. Additionally, iMSCs and iAAT-MSCs exerted such protection by regulating mitochondrial respiration, ATP content, and ROS production in TNBS-induced acinar cells. Furthermore, iMSCs and iAAT-MSCs ameliorated ferroptosis by regulating the ferritin heavy chain 1 (FTH1)/protein disulfide isomerase (PDI)/glutathione peroxide 4 (GPX4) signaling pathways and by modulating ROS function and iron generation in acinar cells. These findings identified ferroptosis as one of the mechanisms that leads to TNBS-induced cell death and offer mechanistic insights relevant to using stem cell therapy for the treatment of CP.

Treatment of atherosclerosis through transplantation of endothelial progenitor cells overexpressing dimethylarginine dimethylaminohydrolase (DDAH) in rabbits.

BACKGROUND: Endothelial dysfunction is a key event in the development of vascular diseases, including atherosclerosis. Endothelial progenitor cells (EPCs) play an important role in vascular repair. Decreased dimethylarginine dimethylaminohydrolase (DDAH) activity is observed in several pathological conditions, and it is associated with an increased risk of vascular disease. We hypothesized that bone marrow-derived EPCs and combination therapy with DDAH2-EPCs could reduce plaque size and ameliorate endothelial dysfunction in an atherosclerosis rabbit model. METHOD: Four groups of rabbits (n = 8 per group) were subjected to a hyperlipidemic diet for a month. After establishing the atherosclerosis model, rabbits received 4 × 106 EPC, EPCs expressing DDAH2, through femoral vein injection, or saline (the control group with basic food and the untreated group). One month after transplantation, plaque thickness, endothelial function, oxidative stress, and inflammatory mRNAs, DDAH, and eNOS function were assessed. RESULTS: DDAH2-EPCs transplantation (p < 0.05) and EPCs transplantation (p < 0.05) were both associated with a reduction in plaque size compared to the control saline injection. The antiproliferative and antiatherogenic effects of EPCs were further enhanced by the overexpression of DDAH2 (p < 0.05, DDAH2-EPCs vs. EPCs). Furthermore, DDAH2-EPCs transplantation significantly increased endothelium integrity compared to the EPCs transplantation. CONCLUSION: Transplantation of EPCs overexpressing DDAH2 may enhance the repair of injured endothelium by reducing inflammation and restoring endothelial function. Therefore, pCMV6-mediated DDAH2 gene-transfected EPCs are a potentially valuable tool for the treatment of atherosclerosis.

Comparative Analysis of the Rabbit Endothelial Progenitor Cells from Bone Marrow and Peripheral Blood Treated with Selenium Nanoparticles.

BACKGROUND: Selenium Nanoparticles (Se-NPs) are known for their antioxidant and anti-inflammatory activities, which are effective in preventing oxidative damage and improving physiological processes. OBJECTIVES: This study aimed at investigating the effects of biosynthesized Se-NPs on bone marrow-derived Endothelial Progenitor Cells (bone marrow-derived EPCs) and blood-derived endothelial progenitor cells (blood-derived EPCs) isolated from rabbits in vitro. METHODS: The cultured EPCs incubated with biosynthesized Se-NPs at the concentrations of 0.19, 0.38, 0.76, 1.71, 3.42, 7.03, 14.25, 28.50, 57, 114, and 228µg/ml for 48h. After screening the proliferative potential of the Se-NPs by the MTT assay, the best concentrations were selected for Real-Time quantitative Polymerase Chain Reaction (RT-qPCR). Real-time quantification of Vascular Cell Adhesion Molecule 1 (VCAM-1), lectin-like oxidized Low-Density Lipoprotein (LDL) receptor-1 (LOX-1), endothelial Nitric Oxide Synthase (eNOS), and Monocyte Chemoattractant Protein-1 (MCP-1) gene expressions were analyzed by normalizing with Glyceraldehyde- 3-Phosphate Dehydrogenase (GAPDH) as an endogenous reference gene. RESULTS: Blood-derived EPCs and bone marrow-derived EPCs showed morphological differences before treatment in vitro. Se-NPs treated EPCs indicated a significant dose-dependent proliferative activity (p<0.01). In general, the expression levels of VCAM-1, LOX-1, and MCP-1 mRNA were significantly decreased (p<0.01), whereas that of the eNOS expression was significantly increased at the concentrations of 7.3 and 14.25µg/ml (p<0.01). Although the expressions of MCP-1, LOX-1, and eNOS mRNA were decreased at certain concentrations of Se-NPs (p<0.01 and p<0.05, respectively) in the treated bone marrow-derived EPCs, no significant differences were observed in the VCAM-1 mRNA expression levels in bone marrow-derived EPCs compared with the control group (p>0.05). CONCLUSION: This was the first report to demonstrate the effects of Se-NPs on proliferative, anti-oxidative, and anti-inflammatory activities for bone marrow-derived EPCs and blood-derived EPCs. Our findings suggested that Se-NPs could be considered as an effective agent that may ameliorate vascular problems.

Diagnostic and theranostic microRNAs in the pathogenesis of atherosclerosis.

MicroRNAs (miRNAs) are a group of small single strand and noncoding RNAs that regulate several physiological and molecular signalling pathways. Alterations of miRNA expression profiles may be involved with pathophysiological processes underlying the development of atherosclerosis and cardiovascular diseases, including changes in the functions of the endothelial cells and vascular smooth muscle cells, such as cell proliferation, migration and inflammation, which are involved in angiogenesis, macrophage function and foam cell formation. Thus, miRNAs can be considered to have a crucial role in the progression, modulation and regulation of every stage of atherosclerosis. Such potential biomarkers will enable us to predict therapeutic response and prognosis of cardiovascular diseases and adopt effective preclinical and clinical treatment strategies. In the present review article, the current data regarding the role of miRNAs in atherosclerosis were summarized and the potential miRNAs as prognostic, diagnostic and theranostic biomarkers in preclinical and clinical studies were further discussed. The highlights of this review are expected to present opportunities for future research of clinical therapeutic approaches in vascular diseases resulting from atherosclerosis with an emphasis on miRNAs.

Important signals regulating coronary artery angiogenesis.

Angiogenesis is a complex process of budding, the formation of new blood vessels from pre-existing microvessels, via migration, proliferation and survival. Vascular angiogenesis factors include different classes of molecules that have a fundamental role in blood vessel formation. Numerous inducers of angiogenesis, such as the members of the vascular endothelial growth factor (VEGF) family, basic fibroblast growth factor (bFGF), angiopoietin (Ang), hepatocyte growth factor (HGF), and hypoxia inducible factor-1 (HIF-1), have an important role in angiogenesis. However, VEGF, platelet-derived growth factor (PDGF), and transforming growth factor ß (TGF-ß) expression appear to be important in intraplaque angiogenesis. Interaction and combined effects between growth factors is essential in endothelial cell migration, proliferation, differentiation, and endothelial cell-cell communication that ultimately lead to the microvessel formation. Since VEGF has a key role during angiogenesis; it may be considered as a good therapeutic target in the clinic. The essential function of several angiogenic factors involved in coronary angiogenesis and intraplaque angiogenesis in atherosclerosis are carefully considered along with the use of angiogenic factors in clinical practice.

Biogenesis of Selenium Nanoparticles Using Green Chemistry.

Selenium binds some enzymes such as glutathione peroxidase and thioredoxin reductase, which may be activated in biological infections and oxidative stress. Chemical and physical methods for synthesizing nanoparticles, apart from being expensive, have their own particular risks. However, nanoparticle synthesis through green chemistry is a safe procedure that different biological sources such as bacteria, fungi, yeasts, algae and plants can be the catalyst bed for processing. Synthesis of selenium nanoparticles (SeNPs) by macro/microorganisms causes variation in morphology and shape of the particles is due to diversity of reduction enzymes in organisms. Reducing enzymes of microorganisms by changing the status of redox convert metal ions (Se2-) to SeNPs without charge (Se0). Biological activity of SeNPs includes their protective role against DNA oxidation. Because of the biological and industrial properties, SeNPs have wide applications in the fields of medicine, microelectronic, agriculture and animal husbandry. SeNPs can show strong antimicrobial effects on the growth and proliferation of microorganisms in a dose-dependent manner. The objective of this review is to consider SeNPs applications to various organisms.

Determine exogenous human DDAH2 gene function in rabbit bone marrow-derived endothelial progenitor cells in vitro.

The in vitro amplification of endothelial progenitor cells (EPCs) is an important method because of its role in gene transferring and regenerative medicine. In this study, we isolated rabbit bone marrow-derived EPCs to further manipulation and overexpression of dimethylarginine dimethylaminohydrolase (DDAH) in EPCs. Isolated EPCs were cultured, expanded in endothelial basal medium. Morphology of EPCs and expression levels of surface markers detected using immunocytochemistry staining and through the use of flow cytometery. Endothelial progenitor cells were transfected with plasmid vectors expressing human DDAH2 (DDAH2-EPCs). Three days after gene transfer, positive transfected-EPCs proliferation and DDAH activity were assayed. We observed colonies conformation and endothelium-like morphology gradually in the third week of culture. Characterization results revealed positive expression of EPC surface markers CD106, Flk-1, vWF, and CD34 using few identification techniques. Overexpression of DDAH2 increased citrulline production after 96 hours of transfection, 235.34 ± 0.69 vs 95.26 ± 5.76 ng/mL; P = .023. These results suggest that cell population with EPC characteristics can be simply isolated from rabbit bone marrow and successfully engineered to overexpress exogenous gene. In this study, we offer a feasible method to isolate and identify EPCs from bone marrow. In addition, an efficient transfection with a plasmid vector (without risk of interference) can be constructed a hybrid structure with EPC and DDAH2 gene to examine their function in vitro.

Biosynthesis of selenium nanoparticles using Enterococcus faecalis and evaluation of their antibacterial activities.

Microorganisms are capable of synthesizing metal nanoparticles, and specifically Enterococcus faecalis bacteria were tested for its ability to synthesize selenium nanoparticles (Se-NPs) from sodium selenite. The biosynthesized Se-NPs were spherical in shape with the size range of 29-195nm. Also, the TEM microscopy showed the accumulation of nano-structures as extracellular deposits. The ability of the bacteria to tolerate high levels of toxic selenite was studied by changing with different concentrations of sodium selenite (0.19mM-2.97mM). Also, the effect of Se-NPs was studied on the growth profile of number of pathogenic Gram-positive and -negative bacteria. High concentrations of sodium selenite in the medium led to the production of small amounts of selenium nanostructures by bacteria. In addition, Se-NPs can be used as an anti-staphylococcal element to effectively prevent and treat S. aureus infections.

Association of Serum hs-CRP Levels With the Presence of Obesity, Diabetes Mellitus, and Other Cardiovascular Risk Factors.

BACKGROUND: Diabetes mellitus remains one of the major health problems of the 21st century and is associated with comorbidities including obesity and metabolic abnormalities. The study was conducted to evaluate serum high-sensitivity C-reactive protein (hs-CRP) levels, as a marker of inflammation, in a large sample of Iranian population without a history of cardiovascular or inflammatory disease and cancer, and to relate this to fasting blood glucose (FBG) and the presence of diabetes mellitus. METHODS: The study consisted of 7,762 subjects divided into four groups-nonobese/nondiabetic, obese/nondiabetic, nonobese/diabetic and obese/diabetic-based on the BMI classification and their FBG. Anthropometric characteristics were measured and blood was collected for the evaluation of fasted lipid profile, FBG and serum hs-CRP levels. RESULTS: Several clinical and biochemical characteristics were significantly different among the four groups: FBG, P < 0.001; total cholesterol (TC), P < 0.001; and triglyceride (TG), P < 0.001. The subjects with a serum hs-CRP >3 mg/dl had higher TC (P < 0.001), low-density lipoprotein cholesterol (LDL-C, P < 0.001), TG (P < 0.001), fat percentage (P < 0.001), and systolic and diastolic blood pressure (P < 0.001) compared with subjects with a serum hs-CRP <3 mg/dl. Multivariate analysis showed FBG, LDL-C, and waist circumference (WC) associated with increased serum hs-CRP levels (P < 0.001). CONCLUSIONS: FBG, LDL-C, WC and gender are independently associated with serum hs-CRP concentrations.

Cytokine profiles in overweight and obese subjects and normal weight individuals matched for age and gender.

Background Obesity is associated with a state of systemic inflammation, mediated by adipose tissue-derived cytokines that may also have metabolic effects, including an effect on insulin resistance. The aim of this study was to compare the serum profile of pro- and anti-inflammatory cytokines in obese and non-obese subjects. Methods A total of 242 subjects who were either overweight or obese (body mass index [BMI] ≥ 25 kg/m2) and non-obese subjects (body mass index <25 kg/m2), were recruited in Mashhad in northeastern Iran. The concentrations of serum interleukin-1α, -1ß, -2, -4, -6, -8 and -10 (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8 and IL-10), were measured in all subjects, together with serum vascular endothelial growth factor, interferon-γ, epidermal growth factor, monocyte chemoattractant protein-1 and tumour necrosis factor-α. Results The groups differed significantly with respect to measures of adiposity and fasted lipid profile. Serum pro-inflammatory cytokines interferon-γ and interleukin-1α, and anti-inflammatory cytokines, interleukin-10, and epidermal growth factor were significantly different between obese and non-obese individuals, as was serum high-sensitivity C-reactive protein. Multivariate regression showed that waist circumference was significantly and independently related to serum monocyte chemoattractant protein-1concentrations ( P = 0.001). Conclusion Despite significant differences in several cytokines between the groups, only monocyte chemoattractant protein-1appeared to be independently related to a measure of adiposity in this population sample from Iran.