Selección de Equipos para el Sistema de CCTV IP

We assessed the binding and internalisation of Dil-labelled Ac-LDL in HMCL treated with PMA and Ang II by FACS analysis. The parameters used to evaluate data were the percentage of scavenger receptor positive cells and the mean fluorescence intensity (MFI) of DiI labelled cells. The results showed that PMA and Ang II increased the percentage of Dil-labelled positive cells in a time dependent manner (Fig.3.7A). We also investigated if PMA and Ang II affected the density of scavenger receptor on HMCL. The data showed

that PMA and Ang II increased MFI in a time dependent manner (Fig.3.7B), indicating an increased intracellular level of DiI labelled Ac-LDL in HMCL. This suggests that PMA and Ang II increases scavenger receptor density on HMCL The specificity of the analysis was confirmed by showing that excess amounts of unlabelled Ac-LDL inhibited the uptake of DiI-Ac-LDL (Fig.3.8). These results suggest that the enhanced uptake of Ac-LDL result fi'om an increase in both the numbers of scavenger receptor positive cells and scavenger receptor density in HMCL.

3.3.2.2. Visualisation of Ac-LDL uptake and lipid droplets

Staining of HMCL with Oil Red O before (Fig. 3.9 A) and after PMA (Fig. 3.9B) & Ang II (Fig.3.9C) stimulation showed that both PMA & Ang II increased the number of intracellular Oil Red O stained lipid droplets. The stable cell line HMCL-Scr showed a stronger Oil Red O staining (Fig 3.9D) which confirmed that scavenger receptor had functional relevance in mesangial cells cultured in the appropriate environment. Visual inspection of Dil-Ac-LDL labelled cells was performed in both HMCL and primary cultures of HMC by fluorescence microscopy. HMCL and primary cultures of HMC were treated by 16 nmol/l of PMA for 72 hours, and then treated with colchicine which arrests mitotic cells in the metaphase by blocking the spindle apparatus for the chromosome separation. The synchronised HMCL and primary cultures of HMC were used to evaluate Dil-Ac-LDL uptake. Result showed that the majority of cells displayed high fluorescence after stimulation by PMA. However, some PMA treated cells were low - or non- fluorescent even after synchronisation with colchicine (Fig.3.9.E&F). This data suggests that not all cells in this population express scavenger receptor to the same degree when stimulated by PMA, and that the non-homogeneous behaviour of HMCL with

suspension of the cells during certain phases of the cycle. It is also excluded that heterogeneity results from different functional specificity of the HMCL cell line because the same heterogeneity also observed in the primary culture of HMC.

3.3.2.3. Expression of scavenger receptor mRNA

RT-PCR followed by Southern blotting showed that HMCL had a low mRNA level for scavenger receptor under normal tissue culture conditions. The stable cell line HMCL-Scr that was transfected by scavenger receptor cDNA showed a high level of scavenger receptor mRNA (Fig.3.2). However, HMCL expressed an inducible form of scavenger receptor when stimulated by PMA and Ang H. PMA (16 nmol/l) induced scavenger receptor expression in a time dependent manner. Ang H (1 pmol/1) also induced scavenger receptor expression (Figs.B.lOA &B). Both PMA at 1.6 nmol/l to 160 nmol/l and Ang II at 10 to 1000 nmol/l induced scavenger receptor mRNA expression in HMCL in a dose responsive manner (Figs.3.1 lA &B).

3.3.2.4. Activity of scavenger receptor promoter

To investigate whether enhanced scavenger receptor mRNA resulted from increased gene transcription in HMCL, we analysed the scavenger receptor promoter activity. The reporter gene (pGLBSCR) containing the full scavenger receptor promoter was transfected into HMCL. The transfected HMCL were stimulated by different concentrations of PMA and Ang II at various times, then promoter activity was measured. The results showed that PMA and Ang II induced scavenger receptor promoter activity in a time and dose dependent manner, which were consistent with the induction of scavenger receptor mRNA (Figs.3.12 A & B ).

3.3.2.5. The effect of Antiotensin H on HMCL proliferation

Mitogenesis assays involving ^H-thymidine incorporation revealed that Angiotensin II at 1000 nmol/l significantly increased HMCL proliferation (Fig.3.13.), suggesting that Ang II at high concentration may have a proliferation-dependent infiuence on scavenger receptor induction.

3.3.2.6. The Effect of various signal transduction pathway inhibitors on scavenger receptor promoter activity

To determine the role of signal transduction pathways in the regulation of scavenger receptor gene expression in PMA or Ang II -stimulated HMCL, we evaluated the effects of various signal transduction inhibitors on scavenger receptor transcription using the luciferase system. At non-cytotoxic concentrations of inhibitors, we observed that the promoter activity of scavenger receptor in PMA or Ang II treated HMCL in the presence of PKC inhibitor (calphostin C) or calmodulin inhibitor (W-7) was significantly lower than in the absence o f calphostin C or W-7, suggesting that both PKC and calmodulin pathways were involved in scavenger receptor upregulation. Additionally, a serine/threonine kinase inhibitor (staurosporine) significantly inhibited scavenger receptor promoter activity induced by PMA, suggesting that serine/threonine kinase pathway also involved in upregulation of scavenger receptor induced by PMA. (Table 3.1).

3.3.2.7. Functional analysis of the scavenger receptor promoter

The molecular mechanism of gene transcription was further investigated using several reporter gene constructs described in Fig.3.4. Functional analysis showed that the minimal prolactin promoter exhibited very little activity either before or after PMA and

motifs were introduced upstream of the minimal prolactin promoter, the promoter could respond to PMA stimulation in HMCL. Three copies of the AP-1/ets motifs increased basal activity by two to three folds in response to PMA stimulation. This suggested that AP-l/ets motifs are specific response elements to PMA stimulation (Fig.3.14). Mutation of either the AP-1 or the ets motifs decreased its ability to respond to PMA, suggesting that both AP-1 and ets motifs are necessary response elements for gene transcription induced by PMA (Fig.3.14). The reporter gene containing 3 copies of AP-l/ets responsive elements had a very small response to Ang II stimulation, suggesting that AP-l/ets motifs are not specific response elements for Ang II (Fig.3.14).

3.3.3. Inflammatory cytokines TNF-a and IL-ip induced scavenger receptor