Tnf-Alpha Alters Intra- and Extracellular Isomir Profiles of Endothelial Cells

Coronary artery disease (CAD) is a chronic inflammatory process that is associated with alterations in the intracellular expression and extracellular release of microRNAs (miRNAs) from different cell types, including endothelial cells (ECs), white blood cells, platelets and red blood cells. MiRNAs, in turn, are involved in modulating the expression of pro‐ and anti‐inflammatory genes that impact disease progression. It has been shown that variations in the circulating miRNA profile of CAD patients are associated with alterations in extracellular miRNA transport as well as a shift in the ratio between the consensus miRNA and isoforms of the miRNA (isomiRs). IsomiRs are relatively abundant variants of consensus miRNAs that differ by one to three nucleotides, but their biological function is not yet known. Previously, we demonstrated that TNF‐α alters the expression of select miRNAs in cultured human aortic endothelial cells (HAECs) and their release into microvesicles (MVs). MVs are major extracellular transport modalities for miRNAs. MV formation was altered by inhibitors that targeted pathways involved in MV release from ECs. Here, we hypothesized that TNF‐α and agents that target pathways involved in EC MV release would alter the abundance of select isomiRs in HAECs and in EC MVs. Using qRT‐PCR, the intracellular and MV abundance of seven isomiRs (isomiR‐10b, ‐ 30d,‐93, ‐143, ‐181a, ‐182, and ‐744) was quantitated in cultured HAECs treated with TNF‐α (100 ng/mL) plus or minus inhibitors of the caspase (Q‐VD‐OPH, 10 μM) and RhoA/ROCK pathways (Y‐27632, 10 μM). Overall, the intracellular abundance of isomiR‐10b, ‐143,‐181a, and ‐744 among all treatment groups were 10–10,000 fold higher in cells compared to their levels in EC MVs. In contrast, among all treatment groups the abundance of isomiR‐30d and ‐182 were 10–100 fold higher in EC MVs compared to their intracellular levels. TNF‐α treatment alone significantly increased the intracellular abundance of isomiR‐181a (P<0.05; 0.016) only. Treatment of HAECs with the caspase inhibitor alone increased intracellular levels (100–10,000‐fold)of isomiR‐10b, ‐93, and ‐181a compared to their levels in EC MVs while the ROCK inhibitor alone increased (100–10,000‐fold) intracellular levels of isomiR‐10band ‐143 versus their levels in EC MVs. In contrast, the ROCK inhibitor alone increased isomiR‐182 (100‐fold) in EC MVs compared to levels in HAECs. Co‐treatment of HAECs with TNF‐α and caspase inhibitor (P<0.05; 0.027) or TNF‐α and ROCK inhibitor (P<0.05; 0.017) significantly increased intracellular isomiR‐181a levels compared to isomiR‐181a levels in EC MVs. However, co‐treatment of HAECs with TNF‐α and ROCK inhibitor increased isomiR‐30dlevels in EC MVs compared to those in HAECs. Co‐treatment with TNF‐α and caspase inhibitor showed a similar trend for isomiR‐30d levels. These data provide insight into the relationship between the intracellular expression of isomiRs in ECs and their extracellular release in response to inflammation and modulation of pathways involved in MV release. These data also enhance our understanding of using MV‐encapsulated isomiR profiles as biomarkers for determining CAD severity.