Immortalized Human Coronary Artery Endothelial Cells

Single Irradiation Induces Changes in the Gene Expression of Atheroprotective Cx37 and Cx40 and Proatherogenic Cx43

Radiotherapy has been linked to increased cardiovascular disease (CVD) risk, notably atherosclerosis, due to endothelial dysfunction post-ionizing radiation. Connexin (Cx) proteins in endothelial cells, forming gap junctions and hemichannels, play a crucial role in mediating these effects. Ramadan's team exposed immortalized human coronary artery endothelial cells (TICAE) and immortalized human dermal microvascular endothelial cells (TIME) to X-rays to analyze Cx expression and channel functions.

TICAE and TIME cells were exposed to X-ray doses (0.1, 0.5, and 5 Gy) to study changes in Cx gene expression over time (6h to 14d). In TICAE cells, Cx37 expression decreased from 24h to 14d, significant at 0.5 and 5 Gy. At 0.1 Gy, it increased at 6h but decreased by 7d (Fig. 1a). For TIME cells, 5 Gy reduced Cx37 expression at 24h and 14d but increased it at 48h. Doses of 0.1 and 0.5 Gy increased expression at 6h and 48h, respectively (Fig. 1a). Cx40 expression in TICAE cells decreased from 6h to 7d at 5 Gy, while 0.1 Gy increased it at 6h and 48h (Fig. 1b). In TIME cells, Cx40 increased early at 6h for 0.1 Gy and at 48h for higher doses, with a dose-dependent decrease from 72h to 14d at 5 Gy (Fig. 1b). Cx43 expression largely increased at 0.5 and 5 Gy. In TICAE, 5 Gy caused increases from 6h to 7d, while at 0.5 Gy, it normalized faster (Fig. 1c). In TIME cells, 5 Gy significantly increased Cx43 at 24h, 72h, and 14d, whereas lower doses showed no major changes.

Fig. 1. The effect of single and fractionated irradiation on Cx37, Cx40 and Cx43 gene expression (Ramadan R, Vromans E, et al., 2019).

TAT-Gap19 Protects Against Radiation-Induced Cell Death

Radiotherapy in thoracic cancer patients elevates cardiovascular disease risk, notably atherosclerosis, through IR-induced endothelial dysfunction. Ramadan's team examined the effects of X-ray irradiation on human coronary/microvascular endothelial cells, focusing on Cx43 hemichannels.

To study IR-induced cell death, Annexin V and Caspase 3/7 activities were monitored in immortalized human coronary artery endothelial cells (TICAE) and immortalized human dermal microvascular endothelial cells (TIME) up to 100h post-IR using Incucyte imaging, with hemichannel inhibition assessed using TAT-Gap19. In TICAE cells, a significant increase in Caspase 3/7 activity and Annexin V was observed at 5 Gy dose from 4h to 100h post-IR (Fig. 2A and B). TAT-Gap19 reduced these activities at 5 Gy dose from 12h onward, and at lower doses from 16h onward for Caspase 3/7 and starting from 4-8h for Annexin V (Fig. 2A and B). In TIME cells, no significant Caspase 3/7 changes were noted at 0.1 and 5 Gy (Fig. 2D), but Annexin V increased significantly at 5 Gy from 4h onward (Fig. 2C), reduced by TAT-Gap19 during 4-48h. TAT-Gap19 also reduced Annexin V transiently at 0 Gy (12-24h; Fig. 2C). To further validate cell death in TIME cells, dextran fluorescein staining was assessed at 6 and 72h post-IR. Increased staining was observed at 5 Gy at 6h and at both 0.1 and 5 Gy at 72h (Fig. 2E and F), inhibited by TAT-Gap19. A minor increase with TAT-Gap19 was noted at 6h for control and 0.1 Gy (Fig. 2E). They also evaluated the effect of TAT peptide alone (100 μm) on cell death in TICAE and TIME cells. Results showed no significant effects on TICAE cells and only slightly increased Annexin V in TIME cells between 24-30h (Fig. 3).

Fig. 2. Radiation-induced cell death as assessed by Caspase 3/7 activity, Annexin V and 10 kDa dextran fluorescein staining, and effect of TAT-Gap19 (Ramadan R, Vromans E, et al., 2020).

Fig. 3. Caspase 3/7 activity was assessed in TICAE cell and Annexin V was assessed in TICAE and TIME cells from 4 h until 100 h of 100 µl of TAT peptide exposure (Ramadan R, Vromans E, et al., 2020).