Browsing by Author "Van der Merwe, Elizabeth Lael"

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    Pancreatic peptide hormone expression in the RINm5F cell line
    (1994) Van der Merwe, Elizabeth Lael; Dr S.H. Kidson and Prof B.B. Rawdon
    Phenotypic heterogeneity is a striking feature of the pancreatic islet cell lines (RIN and MSL) derived from a rat islet cell tumour (Chick et al., 1977) which may therefore be suitable for the study of islet cell differentiation in vitro. In the present study, the RINm5F cell line (Oie ~-, 1983) was investigated to determine whether or not it would be suitable for this purpose. This study presents the results from work aimed at determining which hormone-producing phenotypes were expressed under routine and modified culture conditions. Insulin, detected by immunoperoxidase labelling at earlier passages, was not detectable in later passages of RINm5P:-MRC cultures. Immunoblots and radio-immunoassay performed on cell extracts and culture medium respectively confirmed that RINm5F cells contained proinsulin and that low levels of insulin and/ or proinsulin were secreted into the medium. These findings suggested that the insulin content of individual cells was at the lower limit of detection for the immunoperoxidase technique. To overcome the problem of detecting and quantifying the number of cells containing low levels of insulin by microscopy, a protocol was established for immunolabelling cell suspensions for analysis by flow cytometry. Flow cytometry confirmed that RINm5F cultures contained a small percentage (less than 2%) of cells stained for insulin and that a greater subpopulation of cells (18-38%) were positively stained for insulin after cultures had been exposed to 2mM sodium butyrate for three days. Although positively stained cells were observed by immunoperoxidase or immunofluorescence microscopy in sodium butyrate-treated cultures, staining was barely above that of background staining in peroxidasestained preparations and positively labelled cells could only be identified at high magnification in fluorescent-stained preparations. Thus flow cytometry successfully provided an alternative approach to accurately quantify insulin expression in RINm5F-MRC cells. Flow cytometry, immunoblotting and immunocytochemistry established that all glucagon and somatostatin-like immunoreactivity in RINm5F cultures was non-specific and was most likely caused by the binding of non-specific components present in rabbit serum. Ultrastructural studies confirmed that morphologically differentiated islet cell phenotypes were not present in RINm5F-MRC cultures. The finding that RINm5F-MRC cultures consisted mainly of agranular cells and that only a small subpopulation of cells stained for insulin, indicates that this cell line appears to be poorly differentiated. However, the increase in the number of cells containing insulin and the increased insulin secretion in response to sodium butyrate shows that at least some of these cells have the potential to differentiate. These results indicate that RINm5F-MRC cells are suitable for the study of /1-cell differentiation. It remains to be determined whether or not they are suitable for studying the differentiation of other pancreatic islet cell types.
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    The role of the arterial glycocalyx in sphingosine-1- phosphate induced cardioprotection in the isolated heart of the Wistar rat
    (2018) Araibi, Hala; Kelly-Laubscher, Roisin; Gwanyanya, Asfree; Van der Merwe, Elizabeth Lael
    Background: Ischemic heart diseases (IHD) are a leading cause of death among cardiovascular diseases. Unfortunately, the myocardial damage due to ischemia in IHD may be worsened by reperfusion therapy, a phenomenon called ischemic-reperfusion (I/R) injury. Coronary vascular damage is a key feature of I/R injury. Among the coronary vascular structures, the endothelial glycocalyx is a delicate polysaccharide and protein-rich layer that plays an important role in the regulation of vascular permeability, and is easily damaged during I/R. Sphingosine-1-phosphate (S1P) is a membrane phospholipid metabolite that has been shown to protect the heart against I/R. It has also been shown to regulate the synthesis of glycocalyx, but its effects on coronary endothelial glycocalyx damage and possible mechanism during I/R are unknown. Therefore, we hypothesized that S1P-induced cardioprotection is mediated by modulation of the glycocalyx during I/R in the isolated rat heart. Methods: Isolated male Wistar hearts were perfused on a Langendorff system with Krebs-Henseleit buffer via retrograde perfusion at constant temperature and pressure. The hearts were stabilized and pre-treated with S1P (10 nM for 7 minutes) before inducing 20 minutes of global ischemia, followed by 60 minutes reperfusion. Functional parameters were recorded throughout the protocol, including left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVEDP), heart rate (HR) and coronary flow (CF). Ventricular infarct size was measured by using triphenyltetrazolium chloride stain. Coronary net filtration rate (NFR) was calculated as a ratio of the amount of transudate to CF. Cardiac edema was assessed by calculating the heart wet/dry weight ratio and histologically quantifying size of the interstitial compartment. The shedding of the glycocalyx was estimated by measuring the release of the glycocalyx component syndecan-1 in the coronary effluent using enzyme-linked immunosorbent assay (ELISA) and determining relative syndecan-1 staining intensity between groups in immuno-stained wax sections of perfusion-fixed hearts. In addition, the histo-morphology of the myocardium was assessed using hematoxylin and eosin staining. Results: The cardiac performance was depressed after I/R, as was reflected by decreased LVDP (P=0.02 vs. control), and an increased LVEDP (P<0.0001 vs. control). I/R also significantly increased infarct size (P=0.04 vs. control). Treatment with S1P before I/R significantly decreased infarct size (P=0.01 vs. I/R), but did not improve the post-ischemic decrease in LVDP or stabilize the LVEDP, and had no effect on CF. I/R significantly increased release of syndecan-1 in the coronary effluent (P=0.0002 vs. control). Immunohistochemically-stained imaging also revealed syndecan-1 staining intensity was significantly decreased or absent in ischemic hearts (P≤0.001 vs. control). Pretreatment with S1P had neither effect on syndecan-1 level in the coronary effluent nor on the intensity of syndecan-1 signal in immuno-stained sections (P=n.s vs. I/R). Histological analysis of cardiac edema revealed an increase in the extracellular area in ischemic hearts compared to the control hearts (P≤0.001 vs. control), and S1P treatment decreased the extracellular area (P≤0.01 I/R+S1P vs. I/R). The NFR, and heart wet/dry ratio were not significantly different post-reperfusion between the groups and S1P had no effect on these parameters. Conclusion: This study showed that pretreatment with S1P protects the heart against I/R injury, as was indicated by the decreased infarct size, and decreased extracellular cardiac edema. S1P had no effect on hemodynamic performance or the shedding of syndecan-1. These results suggest that S1P-induced cardioprotection is not mediated by protection of the glycocalyx via stabilization of syndecan-1. However, it is possible that S1P may stabilize other minor glycocalyx components which were not measured in this study, such as heparan sulphate and hyaluronic acid. This is the first study that evaluated syndecan-1 in the cardiac effluent of the isolated heart of rats with global ischemia, and the study opens up prospects for further investigation of the role of the glycocalyx in other models of I/R injury, such as the more clinically-relevant regional ischemia disease model.
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