Volume 166, Issue 2, Pages 243-251 (February 2003) Enhanced bridging function and augmented monocyte adhesion by lipoprotein lipase N9: insights into increased risk of coronary artery disease in N9 carriers  Rachel M. Fisher, Ferdaous Benhizia, Renate Schreiber, Elena Makoveichuk, Wendy Putt, Maysoon Al-Haideri, Richard J. Deckelbaum, Gunilla Olivecrona, Steve E. Humphries, Philippa J. Talmud  Atherosclerosis  Volume 166, Issue 2, Pages 243-251 (February 2003) DOI: 10.1016/S0021-9150(02)00337-4

Fig. 1 Expression levels of LPL mRNA in D9 and N9 stable cell lines assessed by reverse transcription and competitive PCR. A constant amount of cDNA prepared by reverse transcription of RNA isolated from D9 and N9 cells was mixed with increasing amounts (0, 1, 2, 4, 6, 8 and 10 μl) of internal standard (13.5×106 copies per μl). PCR amplification with LPL specific primers gave products of 342 bp and 284 bp from target and competitor sequences, respectively. For both D9 and N9 cells, competition between internal standard and endogenous LPL cDNA is just visible when 2 μl of internal standard were mixed with the cDNA and competition is clearly visible with 4 μl. The ratios of LPL cDNA to internal control when 4 and 10 μl of internal standard were added were D9=3.76 and N9=2.99 (4 μl) and D9=2.90 and N9=2.04 (10 μl), respectively. Therefore, cDNA, and hence RNA, for LPL-D9 and LPL-N9 were present at similar levels. ‘L’ indicates size marker ladder. Atherosclerosis 2003 166, 243-251DOI: (10.1016/S0021-9150(02)00337-4)

Fig. 2 Time course of LPL-D9 (open circles) and LPL-N9 (filled circles) activity released in to the medium after incubation with 5 IU heparin per ml media at 4°C. Results were measured in quintuplicate, corrected for cell protein concentration and presented as mean±S.D. Atherosclerosis 2003 166, 243-251DOI: (10.1016/S0021-9150(02)00337-4)

Fig. 3 Binding (measured at 4°C) and internalisation (measured at 37°C) of 125I-LDL by D9 (open bars) and N9 (hatched bars) stably transfected cells compared with that of untransfected CHO K1 cells. Measurements were made in untreated cells and in cells treated with either heparin (100 IU/ml media) or heparinase (4 U/ml media). In all cells, the uniformly very low levels of LDL binding following heparin or heprainase pre-treatment explain why the ratio relative to untransfected CHO K1 cells may be less than one. Results were measured in duplicate or triplicate, corrected for cell protein concentration and presented as mean±S.D. The absolute values for binding and internalisation of LDL in untreated cells were 39.9±9.2 and 113.7±10.5 ng LDL per mg cell protein for untransfected CHO K1 cells, 46.7±12.0 and 88.5±3.1 ng LDL per mg cell protein for D9 cells, and 213.9±8.6 and 231.0±19.5 ng LDL per mg cell protein for N9 cells, respectively. It should be noted that these values were obtained from a different series of experiments to those presented in Fig. 4 and as such are not directly comparable. Atherosclerosis 2003 166, 243-251DOI: (10.1016/S0021-9150(02)00337-4)

Fig. 4 Binding (measured at 4°C) and internalisation (measured at 37°C) of native and oxidised (oxLDL) 125I-LDL by untransfected CHO K1 cells (crosshatched bars) and by D9 (open bars) and N9 (hatched bars) stably transfected cells corrected for cell protein concentration. In these experiments 125I-LDL internalisation was not determined in untransfected CHO K1 cells and hence only absolute data are presented (in contrast to Fig. 3 where values for D9 and N9 stable cell lines are expressed relative to untransfected cells). Measurements were performed in triplicate or quadruplicate and are presented as mean±S.D. Atherosclerosis 2003 166, 243-251DOI: (10.1016/S0021-9150(02)00337-4)

Fig. 5 Binding of THP-1 monocytes labelled with [3H]leucine to culture plates pre-coated with 5 or 10 μg/ml of purified dimeric LPL-D9 (open bars) or LPL-N9 (hatched bars). Data is corrected for non-specific binding (to un-coated plates) and all results are expressed relative to binding to plates coated with 5 μg/ml of LPL-D9. Values are presented as mean±S.D. for three independent experiments (for each experiment measurements were performed in quadruplicate or quintuplicate). Atherosclerosis 2003 166, 243-251DOI: (10.1016/S0021-9150(02)00337-4)