Real-time PCR showed that Cx37, Cx40, and Cx43 existed in both HAEC and HUVEC. However, the expression levels differed markedly (Figure ). Regarding HAEC, Cx43 transcripts were more than 10-fold abundant, compared to Cx37 or Cx40 (Cx43 vs. Cx37 or Cx40, both P <0.01), in contrast to HUVEC, in which Cx37 was the most abundant. In addition, the relative mRNA expression levels of individual connexins in HAEC were lower than those in HUVEC (all P <0.05, see Figure ). Consistently, immunoconfocal examination of the untreated HAEC showed that Cx43 was abundantly expressed at the cell borders, typical for gap junctions, but Cx37 and Cx40 were rarely seen (Figure ). Treatment with AGE-BSA for 24 hours at doses of 100 and 500 μg/ml lead to a marked reduction of Cx43 and Cx37 and Cx40 remained rarely seen (Figure ). Therefore, the following experiments in HAEC were focused on Cx43.
Figure 1 Analysis of the mRNA profile of Cx37, Cx40, and Cx43 in HAEC and HUVEC showed distinct expression patterns in each type of cells and in HAEC Cx43 was the most abundant transcripts. Real-time PCR showed the existence of Cx37, Cx40, and Cx43 transcripts. (more ...)
Figure 2 Expression of Cx37, Cx40, and Cx43 gap junctions in HAEC evaluated by immunoconfocal microscopy showed that in untreated cells Cx43 was abundantly expressed at the cell borders but Cx37 and Cx40 were rarely observed. AGE-BSA reduced Cx43 signals in a (more ...)
In cells treated with AGE-BSA at doses from 25 to 250 μg/ml for 24-48 hours, no changes of the cell density and morphology were observed (Figure ). However, cells became slightly retracted and reduced in density after treated with 500 μg/ml of AGE-BSA for more than 24 hours (Figure ). For Cx43, regardless of the 24 or 48 hours treatment, the levels of expression gradually decreased as the dose of AGE-BSA increased (Figure ). Western blotting also verified the dose-dependent effect of AGE-BSA on Cx43 expression (Figure ). After exposure to 500 μg/ml of AGE-BSA for 24 hours (Figure ) and 48 hours (Figure ), the relative expression levels of Cx43 protein were respectively reduced to 61.2 ± 7.3% and 43.9 ± 8.8% (both P <0.05, compared to the control groups). A control group of cells treated with dialyzed, long term stored BSA for 24 hours showed no changes of Cx43 proteins (Additional file 1
Figure S1). There was no difference in decreasing trends or patterns of Cx43 proteins extracted from AGE-BSA-treated cells with lysis buffers containing NP40 or SDS as well as no difference in Cx43 expression when the samples were detected using anti-Cx43 antibodies from various sources (Additional file 2
Figure 2S and Additional file 3
Figure 3S). The function of gap-junction communication was checked using the method of scrape-loading/dye transfer (Figure ). The areas of dye transfer were significantly reduced in cells treated with 500 μg/ml of AGE-BSA for 24 hours and 250 μg/ml of AGE-BSA for 48 hours (Figure ; both P <0.05). Moreover, because cells were not well contacted after exposure to 500 μg/ml of AGE-BSA for 48 hours, the inhibition of gap-junction communication was unable to quantify (Figure ). The effects of AGE-BSA on viability of cells after exposure for 24 hours were assessed using MTT assay. The relative viability of cells was 94.2 ± 0.2% (100 μg/ml AGE-BSA), 90.2 ± 1.4% (250 μg/ml AGE-BSA), and 81.6 ± 0.8% (500 μg/ml AGE-BSA), respectively (all P <0.05, compared to the control groups) (Figure ).
Figure 3 Reduced expression of endothelial Cx43 gap junctions by AGE-BSA, as evaluated by immunoconfocal microscopy. The levels of Cx43 decreased as the dose of AGE-BSA increased regardless of 24 or 48-hour treatment. Images A-F and G-L were respectively obtained (more ...)
Figure 4 Dose-dependent reduction of expression of Cx43 protein by AGE-BSA, as detected by Western blotting. Note that a dose-dependent reduction is seen in cells treated with AGE-BSA for both 24 (A) and 48 hours (B). For each dose of treatment, the relative level (more ...)
Figure 5 Reduction of gap-junction communication by AGE-BSA, as evaluated using scrape loading assay. A. Bottom images are phase-contrast micrographs of the upper fluorescent images. The concentration of AGE-BSA (in μg/ml) is indicated in the upper left (more ...)
Figure 6 Dose-dependent reduction in viability of cells treated with AGE-BSA, as detected by MTT assay. Note a dose-dependent reduction is seen in cells treated with AGE-BSA for 24 hours. For each dose of treatment, the relative level of the total amount of cells (more ...)
To explore whether alteration of Cx43 expression by AGE-BSA was regulated through MAPK cascades, three signal pathways, ERK, JNK, and p38 MAPK were separately blocked before addition of AGE-BSA. PD98059, an MEK1 inhibitor which blocks ERK pathway, significantly reversed the reduction of Cx43 proteins in cells treated with AGE-BSA for 24 hours (relative expression levels, without PD98059, 62.3 ± 4.8%; with PD98059, 84.1 ± 4.4%, p < 0.05 see Figure ). Similarly, SB203580, a p38 MAPK inhibitor, also significantly reversed the reductions (without SB203580, 60.5 ± 1.6%; with SB203580, 79.6 ± 2.9%, p < 0.05 see Figure ). The basal levels of Cx43 proteins were not affected after the addition of these inhibitors. In contrast, no such effects were seen in cells treated with SP600125, a JNK inhibitor (data not shown). Immunoconfocal microscopy also confirmed that AGE-BSA induced reduction of Cx43 was reversed by PD98059 or SB203580 to the levels comparable to the control group and the reversed Cx43 mainly located at cell borders (Figure ). The gap-junction communication was measured when cells were treated with 500 μg/ml of AGE-BSA for 24 hours in the presence of either PD98059 (40 μM) or SB203580 (15 μM). Whereas Cx43 proteins returned to approximately the control levels (Figure ), the areas of dye transfer remained close to those of cells treated with 500 μg/ml of AGE-BSA for 24 hours (all P <0.05, compared to the control groups; see Figure ). Taken together, our results indicated the reversed Cx43 by MEK1 or p38 MAPK inhibitor remained at the cell borders but exhibited impaired communication function.
Figure 7 PD98059 and SB203580 respectively blocked the down-regulating effect of AGE-BSA on Cx43 protein (as detected by Western blotting and immunoconfocal microscopy) but not the effect on the gap-junction communication (by scrape loading). Note the relative (more ...)
In addition, to understand the effect of AGE-BSA on protein synthesis at the transcription level, Cx43 transcripts was measured using semi-quantitative RT-PCR, which showed a dose-dependent decrease of the transcripts in cells treated with AGE-BSA for 24 hours (relative expression levels, 500 μg/ml of AGE-BSA, 57.1 ± 8.9%; p < 0.05 compared to the control group; see Figure ). This effect was more obvious after treatment for 48 hours (500 μg/ml of AGE-BSA, 38.3 ± 5.5%; p < 0.05; see Figure ). Moreover, the decrement of Cx43 transcripts at exposure of 500 μg/ml of AGE-BSA followed a time-dependent manner, as early as 6 hours, a significant decrease was noticed (77.5 ± 5.1%; p < 0.05; see Figure ). The down-regulation of Cx43 transcripts by AGE-BSA (500 μg/ml) seen at 24 hours was significantly reversed in cells pre-treated with PD98059 or SB203580 (without PD98059 or SB203580, 65.1 ± 2.1%; with PD98059, 95.1 ± 5.3%; with SB203580, 98.8 ± 9.4%, all p < 0.05; see Figure ). No affections on the basal levels of Cx43 transcripts were seen after the addition of these inhibitors. This indicated that the AGE-BSA-induced reduction in Cx43 transcription was mediated by ERK and p38 MAPK. Phosphorylation of ERK and p38 MAPK was activated dose-dependently in cells treated with AGE-BSA for 2 hours (Figure ) and 6 hours (Figure ), while no such change was seen in JNK. Taken together, these data indicated that the down-regulation of Cx43 expression was through ERK and p38 MAPK signaling pathways.
Figure 8 Reduced transcription of Cx43 by AGE-BSA and partial recovery of reduced Cx43 transcript by each of PD98059 and SB203580, as detected by semi-quantitative RT-PCR. Note that a dose-dependent reduction is seen in cells treated with AGE-BSA for both 24 (A) (more ...)
Figure 9 Activation of ERK and p38 MAPK but not JNK signaling pathways by AGE-BSA. Representative western blots of ERK, phosphorylated ERK, p38 MAPK, phosphorylated p38 MAPK, JNK, and phosphorylated JNK in cells treated with AGE-BSA for 2 (A) and 6 hours (B). (more ...)