A Novel Selectable Islet 1 Positive Progenitor Cell Reprogrammed to Expandable and Functional Smooth Muscle Cells.

Disorders affecting smooth muscle structure/function may require technologies that can generate large scale, differentiated and contractile smooth muscle cells (SMC) suitable for cell therapy. To date no clonal precursor population that provides large numbers of differentiated SMC in culture has been identified in a rodent. Identification of such cells may also enhance insight into progenitor cell fate decisions and the relationship between smooth muscle precursors and disease states that implicate differentiated SMC. In this study, we used classic clonal expansion techniques to identify novel self-renewing Islet 1 (Isl-1) positive primitive progenitor cells (PPC) within rat bone marrow that exhibited canonical stem cell markers and preferential differentiation towards a smooth muscle-like fate. We subsequently used molecular tagging to select Isl-1 positive clonal populations from expanded and de novo marrow cell populations. We refer to these previously undescribed cells as the PPC given its stem cell marker profile, and robust self-renewal capacity. PPC could be directly converted into induced smooth muscle cells (iSMC) using single transcription factor (Kruppel-like factor 4) knockdown or transactivator (myocardin) overexpression in contrast to three control cells (HEK 293, endothelial cells and mesenchymal stem cells) where such induction was not possible. iSMC exhibited immuno- and cytoskeletal-phenotype, calcium signaling profile and contractile responses similar to bona fide SMC. Passaged iSMC could be expanded to a scale sufficient for large scale tissue replacement. PPC and reprogramed iSMC so derived may offer future opportunities to investigate molecular, structure/function and cell-based replacement therapy approaches to diverse cardiovascular, respiratory, gastrointestinal, and genitourinary diseases that have as their basis smooth muscle cell functional aberrancy or numerical loss. Stem Cells 2016;34:1354-1368.

Bone Marrow-Derived Mesenchymal Stem Cells Have Innate Procoagulant Activity and Cause Microvascular Obstruction Following Intracoronary Delivery: Amelioration by Antithrombin Therapy.

Mesenchymal stem cells (MSCs) are currently under investigation as tools to preserve cardiac structure and function following acute myocardial infarction (AMI). However, concerns have emerged regarding safety of acute intracoronary (IC) MSC delivery. This study aimed to characterize innate prothrombotic activity of MSC and identify means of its mitigation toward safe and efficacious therapeutic IC MSC delivery post-AMI. Expression of the initiator of the coagulation cascade tissue factor (TF) on MSC was detected and quantified by immunofluorescence, FACS, and immunoblotting. MSC-derived TF antigen was catalytically active and capable of supporting thrombin generation in vitro. Addition of MSCs to whole citrated blood enhanced platelet thrombus deposition on collagen at arterial shear, an effect abolished by heparin coadministration. In a porcine AMI model, IC infusion of 25 × 10(6) MSC during reperfusion was associated with a decrease in coronary flow reserve but not when coadministered with an antithrombin agent (heparin). Heparin reduced MSC-associated thrombosis incorporating platelets and VWF within the microvasculature. Heparin-assisted therapeutic MSC delivery also reduced apoptosis in the infarct border zone at 24 hours, significantly improved infarct size, left ventricular (LV) ejection fraction, LV volumes, wall motion, and attenuated histologic evidence of scar formation at 6 weeks post-AMI. Heparin alone or heparin-assisted fibroblast control cell delivery had no such effect. Procoagulant TF activity of therapeutic MSCs is associated with reductions in myocardial perfusion when delivered IC may be successfully managed by heparin coadministration. This study highlights an important mechanistic insight into safety concerns associated with therapeutic IC MSC delivery for AMI.

Use of a Real Time PCR Assay to Assess the Effect of Pulsed Light Inactivation on Bacterial Cell Membranes and Associated Cell Viability.

Research into more rapid and effective means of disinfecting water has become necessary due to the recognition that not all pathogenic species are being removed by chemical means. There is an extent of research highlighting the benefits of pulsed light for the disinfection of water. This study aims to determine the ability of a real time polymerase chain reaction assay to evaluate microbial inactivation of pulsed light treated cells. Findings show that pulsed light is a more rapid means of inactivating test species than standard UV lamp systems. A linear relationship between cell number and polymerase chain reaction amplification was obtained. A difference in threshold value (Ct) of approximately 4 (p ≤ 0.05) was obtained for DNA amplification following the addition of the dye for pulsed ultrviolet (PUV)-treated Bacillus cells. Membrane protein leakage proved an effective means of determining membrane damage for both Bacillus and E. coli test species following PUV treatment. This membrane damage was not evident for cells exposed to low pressure ultraviolet (LPUV). Findings describe suggest that PUV treatment induced a viable but nonculturable state in treated cells.

The influence of hypoxia on the differentiation capacities and immunosuppressive properties of clonal mouse mesenchymal stromal cell lines.

Multipotent mesenchymal stromal cells are multipotent cells capable of differentiating into different mesodermal cell types. Enigmatically, mesenchymal stromal cells present in the bone marrow support early lymphopoiesis yet can inhibit mature lymphocyte growth. Critical features of the bone marrow microenvironment, such as the level of oxygen, play an important role in mesenchymal stromal cell biology. Herein, we show that a panel of continuously growing mouse mesenchymal stromal cell lines, namely OP9, MS5, PA6, ST2 and B16-14, exhibit mesenchymal stromal cell characteristic phenotypes and respond physiologically to oxygen deprivation. Culturing freshly isolated bone marrow-derived mesenchymal stromal cells or cell lines at 5% O2 resulted in a dramatic increase in expression of hypoxia-inducible factor family members and of key genes involved in their differentiation. Phenotypically, their osteogenic and adipogenic differentiation capacity was generally improved in hypoxia, whereas their inhibitory effects on in vitro T-cell proliferation were preserved. Taken together, we conclude that these continuously growing mouse cell lines behave as canonical mesenchymal stromal cells and respond physiologically to hypoxia, thereby providing a potent tool for the study of different aspects of mesenchymal stromal cell biology.

Development of a combined in vitro cell culture--quantitative PCR assay for evaluating the disinfection performance of pulsed light for treating the waterborne enteroparasite Giardia lamblia.

Giardia lamblia is a flagellated protozoan parasite that is recognised as a frequent cause of water-borne disease in humans and animals. We report for the first time on the use of a combined in vitro HCT-8 cell culture-quantitative PCR assay for evaluating the efficacy of using pulsed UV light for treating G. lamblia parasites. Findings showed that current methods that are limited to using vital stains before and after cyst excystation are not appropriate for monitoring or evaluating cyst destruction post PUV-treatments. Use of the human ileocecal HCT-8 cell line was superior to that of the human colon Caco-2 cell line for in vitro culture and determining PUV sensitivity of treated cysts. G. lamblia cysts were also shown to be more resistant to PUV irradiation compared to treating similar numbers of Cryptosporidium parvum oocysts. These observations also show that the use of this HCT-8 cell culture assay may replace use of animal models for determining disinfection performances of PUV for treating both C. parvum and G. lamblia.

Hemodialysis induces p66(shc) gene expression in nondiabetic humans: correlations with oxidative stress and systemic inflammation.

OBJECTIVE: Oxidative stress and inflammation characterize hemodialysis (HD) and are associated with malnutrition, cardiovascular disease, and poor clinical outcome. p66(shc) stimulates oxidative stress and atherogenesis. The objective of the present study was to assess p66(shc) expression levels in HD and their associations with inflammatory and oxidative stress markers. DESIGN: p66(shc) messenger ribonucleic acid (mRNA) was compared with systemic oxidative stress and inflammation markers in control subjects and patients on HD before and after a single HD session in a cross-sectional analysis. SETTING: Outpatient hemodialysis unit. PATIENTS: The study included stable HD patients (n = 21, men/women: 18/3) who were on HD 3 times per week for a minimum of 8 weeks; age-matched control subjects (n = 22, men/women:17/5). MAIN OUTCOME MEASURE: mRNA levels of p66(shc), tumor necrosis factor α (TNF-α), and pentraxin 3 (PTX3), p66(shc) protein levels in white blood cells, lipid peroxidation (in the form of plasma thiobarbituric acid-reactive substance [TBARS]) and serum C-reactive protein. RESULTS: In patients on dialysis, of the p66(shc), TNF-α, and PTX3 mRNAs, p66(shc) protein levels were higher (P < .05) than in control subjects, as well as plasma TBARS and C-reactive protein (P < .05). p66(shc) mRNA directly correlated with TBARS (r = 0.69, P = .0005) and with TNF-α mRNA (r = 0.63, P = .003). These associations were confirmed in the whole study population (TBARS: r = 0.541, P = .0003; TNF-α: r = 0.581, P < .0001), whereas in the control group only the positive association between p66(shc) and TNF-α was detected. TNF-α was directly correlated with PTX3 both in HD patients (r = 0.72, P = .0005) and in the whole study group (r = 0.678, P < .0001). The dialysis session affected neither p66(shc) and TNF-α mRNA nor p66(shc) protein expression, whereas it further increased (P = .002) PTX3 mRNA. As compared with predialysis levels, TBARS were reduced (P < .05) after dialysis. In these conditions, p66(shc) remained directly correlated with TNF-α (r = 0.901, P < .0001). CONCLUSIONS: Increased p66(shc) gene expression correlates with TNF-α mRNA and with levels of markers of oxidative stress in HD. We suggest a novel link between HD-associated inflammation and p66(shc) gene expression contributing to systemic oxidative stress.

Inhibitory effects of fenofibrate on apoptosis and cell proliferation in human endothelial cells in high glucose.

Fenofibrate has beneficial effects on the progression and clinical emergence of atherosclerosis in normoglycemic and in diabetic patients. Given the involvement of endothelium in these processes, we speculated that fenofibrate may influence endothelial cell apoptosis and proliferation, regulators of endothelium integrity. Fenofibrate effects on apoptosis and proliferation were studied in human umbilical vein endothelial cells under normal (5.5 mmol/l, NG) and high (22 mmol/l, HG) glucose with or without fenofibrate (50 micromol/l). Apoptosis was evaluated by annexin V, by poly(ADP-ribose) polymerase protein cleavage, and cyclooxygenase-2 (COX-2), Bax/Bcl-2, and p53 protein levels; proliferation was assessed by determining cell cycle phase distribution and the amounts of the cell cycle regulators E2F1, cyclin D1, E1, and A and the levels of the hyper-phosphorylated form of the retinoblastoma protein (ppRb). HG resulted in increased (p<0.05) apoptosis rate associated with COX-2 protein overexpression, without modification of Bax/Bcl2 ratio and p53 levels. Fenofibrate decreased apoptosis and normalized increased COX-2 expression in HG (p<0.05). Both in HG and NG, fenofibrate dramatically reduced cell proliferation (p<0.05) through a G1/G0 block mediated by the reduction in ppRb and the decrease in E2F1, cyclin E1, A, and D1 protein expression, with a mechanism that, for cyclin E1, occurred at the posttranscriptional level. In conclusion, our data show that fenofibrate reduces apoptosis caused by HG but severely interferes with endothelial cell proliferation both in NG and HG. The resulting effect may influence endothelium integrity in vivo and may impact the outcome of acute complications of atherosclerosis in diabetes.

Vascular sources of oxidative stress: implications for uremia-related cardiovascular disease.

Chronic oxidative stress that characterizes uremia has potentially devastating effects on the vasculature and has been advocated in the pathogenesis of accelerated atherosclerosis in this disease. Recent advances have been made in our understanding of the molecular mechanisms that regulate expression and activity of key enzymes of vascular oxidative stress (eg, nicotinamide adenine dinucleotide phosphate [NAD{P}H] oxidase) and that dissect their interactions with signalling pathways of inflammation. The finding that NAD(P)H oxidase is upregulated in experimental uremia has important consequences from a physiologic and a therapeutic standpoint. In addition, identification of novel proteins involved in systemic oxidative stress has shed some new light on the pathogenesis of vascular disease. p66(shc) is a cytoplasmic protein that is expressed in a wide range of cell types. Initially believed to be involved in signalling pathways that regulate cell growth and oxidative stress, it has now been shown to play a pivotal role in promoting endothelial dysfunction and atherosclerosis. Although a specific role in uremia-related vascular disease has not yet been shown, available data in humans suggest involvement of p66(shc) in clinical conditions associated with increased oxidative stress.

Mesenchymal stem cell survival in the infarcted heart is enhanced by lentivirus vector-mediated heat shock protein 27 expression.

Mesenchymal stem cell (MSC) therapy offers the potential to promote recovery after myocardial infarction (MI). However, therapeutic efficacy may be limited by poor survival and retention of transplanted cells. A combination of gene and cell therapy has the capacity to prevent donor cell death and augment the reparative and regenerative effects of cell transfer. The present study investigates the effect of exogenous heat shock protein 27 (Hsp27) expression in MSCs in an in vitro model of ischemia and in an in vivo rat MI model and aims to determine if this could enhance the therapeutic benefit associated with cell delivery. Hsp27 overexpression by lentivirus vector modification resulted in increased MSC survival in vitro and in vivo. Furthermore, decreased apoptosis in the infarcted tissue and improved cardiac function was observed in the Hsp27 group, enhancing the therapeutic effect of MSCs. Together, these data demonstrate that ex vivo genetic modification-specifically Hsp27 overexpression-offers the possibility of enhancing the efficacy of MSC therapy in MI.