Endothelial-Mesenchymal Transition and Possible Role of Cytokines in Streptozotocin-Induced Diabetic Heart

Abstract

Background: Although endothelial mesenchymal transition (EndMT) has been characterized as a basic process in embryogenesis, EndMT is the mechanism that accelerates the development of cardiovascular diseases, including heart failure, aging, and complications of diabetes or hypertension as well. Endothelial cells lose their distinct markers and take on a mesenchymal phenotype during EndMT, expressing distinct products. Methods: In this study, type 1 Diabetes mellitus (T1DM) was induced in rats with streptozotocin (STZ) by intraperitoneal injection at a 60 mg/kg dose. Diabetic rats were randomly divided into two groups, namely, control and diabetic rats, for 4 weeks. Heart, aorta, and plasma samples were collected at the end of 4 weeks. Sequentially, biochemical parameters, cytokines, reactive oxygen species (ROS), protein expression of EndMT markers (Chemokine C-X-C motif ligand-1 (CXCL-1), vimentin, citrullinated histone H3 (H3Cit), α-smooth muscle actin (α-SMA), and transforming growth factor beta (TGF-β) and versican), components of the extracellular matrix (matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase-1(TIMP-1), and discoidin domain tyrosine kinase receptor 2 (DDR-2)) were detected by ELISA or Western blot, respectively. Results: Cytokines and ROS were increased in diabetic hearts, which induced partial EndMT. Among EndMT markers, histone citrullination, α-SMA, and CXCL-1 were increased; vimentin was decreased in DM. The endothelial marker endothelin-1 was significantly higher in the aortas of DM rats. Interestingly, TGF-β showed a significant decrease in the diabetic heart, plasma, and aorta. Additionally, MMP-2/TIMP-1 levels also decreased in DM. Conclusions: To sum up, the identification of molecules and regulatory pathways involved in EndMT provided novel therapeutic approaches for cardiac pathophysiological conditions.

Keywords: Diabetic Heart; EndMT; STZ.

Figure 1

Cytokine expression in heart. *, p < 0.05; **, p < 0.01; NS, non-significant.

Figure 2

Peroxynitrite and MPO activity expression in heart. **, p < 0.01; NS, non-significant.

Figure 3

iNOS, eNOS, and nNOS expression in heart. *, p < 0.05; NS, non-significant.

Figure 4

MMP-2, MMP-2 zymography, DDR-2, and TIMP-1 expression in heart. *, p < 0.05; **, p < 0.01; *** p < 0.001.

Figure 5

CXCL-1, Vimentin, H3Cit, α-SMA and versican expression in heart. *, p < 0.05; **, p < 0.01; NS, non-significant.

Figure 6

TGF-β expression in heart, plasma, and aorta. *, p < 0.05; **** p < 0.0001.

Figure 7

Endothelin-1 expression in heart and aorta. *, p < 0.05; NS, non-significant.

Figure 8

Brief summary of EndMT pathway in the diabetic heart of T1DM. Dotted box, another assumed pathway results in fibrosis.

Figure 9

Scheme showing possible EndMT in the heart by STZ-induced T1DM. (A) In STZ DM rats, increased peroxynitrite truncates MMP-2, rendering it inactive. (B,C) MMP-2 facilitates the hydrolysis of TGF-β, causing activated TGF-β to induce the downstream pathway. In STZ DM, decreased MMP-2 suppressed TGF-β transduction. (D) Vimentin is one biomarker in mesenchymal cells during EndMT that is activated by TGF-β; a decrease in TGF-β causes a corresponding drop in vimentin. (E) On the other side, elevated TNF-α led to an increase in CXCL1. DDR2, a different biomarker during EndMT, was aberrantly elevated in the interim. Certain papers about in situ carcinoma suggest that diabetic heart disease may also have elevated DDR2. (F) In mesenchymal cells, higher α-SMA is a classical phenomenon during EndMT. (G) CXCL1 from endothelial cells is likely to attract immune cells, particularly neutrophils. In the diabetic heart, neutrophils may close as a result of an inflammatory signal being drawn to them. (H) CXCL-1 may induce citrullination in neutrophils.