Human Internal Thoracic Artery Endothelial Cells

MMC-induced Genotoxic Stress in HCAEC and HITAEC

Mitomycin C (MMC), an alkylating chemotherapy drug, causes DNA crosslinking, leading to genotoxic stress, endothelial dysfunction, and potentially atherosclerosis. Endogenous and exogenous agents, including MMC, can induce such DNA damage, which triggers inflammation and promotes atherosclerosis development via endothelial dysfunction.

Sinitsk et al. explored the effects of MMC-induced DNA damage on gene expression in human coronary artery endothelial cells (HCAEC) and human internal thoracic endothelial cells (HITAEC) by exposed to 500 ng/mL MMC in vitro. After 6 hours of MMC treatment and 24 hours in MMC-free media, the CBMN assay results for HCAEC and HITAEC are shown in Fig. 1. MMC-treated cells exhibited significant increases in genotoxic stress markers: (i) binucleated (BN) cells with micronuclei (MNi) from chromosome fragments or missegregated chromosomes, (ii) nucleoplasmic bridges (NPBs) from dicentric chromosome misrepair or telomere fusion, and (iii) nuclear buds (NBUDs) indicating DNA and protein complex repair (Fig. 2). MMC led to a 3-fold and 2-fold rise in BN cells with MN and NBUDs in HCAEC and HITAEC, respectively, and a 4-fold and 3-fold increase in BN cells with NPBs. Baseline cytogenetic markers varied between cell types, with HITAEC showing higher initial levels, notably in BN cells with NPBs, similar to MMC-treated HCAEC levels (Fig. 1). Despite these differences, the fold-change in cytogenetic markers due to MMC was similar for both endothelial cell types. These results confirm that alkylating mutagen MMC inflicts cytogenetic damage in primary human endothelial cells.

Fig. 1. Frequency of BN cells with MN (A), NPBs (B) and NBUDs (C) in HCAEC and HITAEC in response to 500 ng/mL MMC (Sinitsky M Y, Kutikhin A G, et al., 2020).

Fig. 2. Morphology of undamaged BN endothelial cell (A), BN cell with MNi (B), BN cell with NPB (C) and BN cell with NBUD (D) at ×1000 magnification (Sinitsky M Y, Kutikhin A G, et al., 2020).

Co-Culture of Primary Human Coronary Artery and Internal Thoracic Artery Endothelial Cells Results in Mutually Beneficial Paracrine Interactions

Coronary artery bypass grafting (CABG) is a common surgical intervention for coronary artery disease. Despite the superior long-term patency of internal thoracic artery (ITA) grafts over saphenous vein (SV) conduits due to better resistance to atherosclerosis and other complications, SVs remain commonly used. Shishkova's team explored the underlying reasons for ITA grafts' superior performance by investigating the synergistic paracrine interactions between coronary artery endothelial cells (HCAECs) and internal thoracic artery endothelial cells (HITAECs).

To examine if HITAECs provide beneficial paracrine effects to HCAECs unlike HSaVECs, and to identify distinct patterns in their interactions, they used a co-culture model with HCAECs at the dish bottom and conduit ECs (HITAECs or HSaVECs) on a translucent polycarbonate insert. RT-qPCR revealed reduced pro-inflammatory and EndoMT transcripts in HCAECs with HITAECs compared to HSaVECs (Fig. 3A). Specifically, IL6 and CXCL8 were downregulated after 24 and 48 hours, while genes VCAM1, ICAM1, SELE, SNAI1, and SNAI2 showed varied expression (Fig. 3A). Western blotting confirmed eNOS increase and consistent Snail/Slug reduction in HCAECs with HITAECs post 48 hours (Fig. 3B and C). Despite an unexpected VCAM1 rise, it remained lower than in monocultures. Upregulation of arterial specification transcription factor HES1 suggested HITAECs aid arterial differentiation in HCAECs, as opposed to HSaVECs which disrupt it (Fig. 3B and C). Both CD31 and VE-cadherin levels stayed unchanged, indicating endothelial identity changes occur beyond observed time frames.

Fig. 3. Profiling of key endothelial molecules in human coronary artery endothelial cells (HCAECs) co-cultured with either human internal thoracic artery endothelial cells (HITAECs) (HIT) or human saphenous vein endothelial cells (HSaVECs) (HSVs) for 6, 24, or 48 h (Shishkova D, Markova V, et al., 2020).