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. 2017 May;96(21):e6978.
doi: 10.1097/MD.0000000000006978.

Carbon monoxide-releasing molecule-2 suppresses thrombomodulin and endothelial protein C receptor expression of human umbilical vein endothelial cells induced by lipopolysaccharide in vitro

Xianglin Meng  1 , Dongsheng Fei, Mingming Liu, Songlin Yang, Ning Song, Lei Jiang, Kai Kang, Chuanchuan Nan, Yunpeng Luo, Shangha Pan, Mingyan Zhao
Affiliations

Carbon monoxide-releasing molecule-2 suppresses thrombomodulin and endothelial protein C receptor expression of human umbilical vein endothelial cells induced by lipopolysaccharide in vitro

Xianglin Meng et al. Medicine (Baltimore). 2017 May.

Abstract

Objective: The aim of this study was to observe the counter-effect of carbon monoxide-releasing molecule-2 (CORM-2) on lipopolysaccharide (LPS)-suppressed thrombomodulin (TM) and endothelial protein C receptor (EPCR) expressions from human umbilical vein endothelial cell (HUVEC), and to reveal its mechanisms.

Methods: HUVECs were divided into 5 treatment groups, wherein reagents were added simultaneously. TM and EPCR proteins of the cells and the culture medium levels of soluble TM, soluble EPCR, and matrix metalloproteinase-2 (MMP-2) were detected after administration, whereas mRNA levels of TM and EPCR, as well as nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activity among groups, were also evaluated.

Results: No significant difference was observed in any indicator between CORM-2 and sham groups. Addition of LPS produced drastic increase in MMP-2 expression, NF-κB activity, shedding of TM and EPCR (into the culture medium), as well as remarkable decrease in both mRNA and protein expressions of TM and EPCR, and cell viability. LPS + CORM-2 treatment significantly reduced the increase in MMP-2, NF-κB activity, and TM/EPCR shedding, whereas maintained both mRNA and protein levels of TM and EPCR, and preserved cell viability.

Conclusions: CORM-2 protects HUVEC from LPS-induced injury, by way of suppressing NF-κB activity, which downregulates TM and EPCR mRNAs. It also decreases MMP-2 expression and prevents the shedding of TM and EPCR from the surface of endothelial cells, so as to preserve their protective effect.

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Conflict of interest statement

The authors report no conflicts of interest.

Figures

Figure 1
Figure 1
Cell viability determined by CCK-8 assay. LPS decreased cell viability in all groups compared with sham group. LPS, LPS + CORM, and LPS + iCORM groups had the most severe decrease. LPS + CORM group produced a lower cell viability than sham group (P < .05), but remained significantly higher than LPS and iCORM groups (P < .05). ∗P < .05, compared to sham group; +P < .05, compared to LPS group; #P < .05, compared to CORM group; &P < .05, compared to LPS + CORM group. CCK-8 = cell counting kit-8, CORM = carbon monoxide-releasing molecule, iCORM = inactive carbon monoxide-releasing molecule, LPS = lipopolysaccharides.
Figure 2
Figure 2
(A–C) sEPCR (A), sTM (B), MMP-2 (C) levels in cell culture medium. Compared with sham group, after addition of LPS, sEPCR, sTM, MMP-2 levels significantly increased in all other groups (P < .05), among which LPS and LPS + iCORM groups produced highest increase, whereas their increase in LPS + CORM group was significantly suppressed (P < .05). CORM and SHAM groups had no significant difference. ∗P < .05, compared to sham group; +P < .05, compared to LPS group; #P < .05, compared to CORM group; &P < 0.05, compared to LPS + CORM group. CORM = carbon monoxide releasing molecule, iCORM = inactive carbon monoxide releasing molecule, LPS = lipopolysaccharides, MMP-2 = matrix metalloproteinase-2, sEPCR = soluble endothelial protein C receptor, sTM = soluble thrombomodulin.
Figure 3
Figure 3
(A–D) Epcr and EPCR protein levels were analyzed by RT-PCR and Western blot in HUVECs stimulated with LPS for 8 hours. (A) Epcr (315 bp) and Gapdh (576 bp) were analyzed by PCR; ratio of Epcr/Gapdh (Gapdh as internal control) was compared (B). Semiquantity of EPCR protein level also was examined and quantified by Western blot (C and D). 1. Sham group, 2. LPS group, 3. CORM-2 group, 4. CORM-2 + LPS group, 5. iCORM-2 + LPS group, Marker: DL2000, from bottom to top: 100, 250, 500, 750, 1000, 2000; (B,D) semiquantitative analyses of mRNA. ∗P < .05, compared to sham group; +P < .05, compared to LPS group; #P < .05, compared to CORM group; &P < .05, compared to LPS + CORM group. CORM = carbon monoxide-releasing molecule, EPCR = endothelial protein C receptor, HUVECs = human umbilical vein endothelial cells, iCORM = inactive carbon monoxide releasing molecule, LPS = lipopolysaccharides, RT-PCR = reverse transcription polymerase chain reaction.
Figure 4
Figure 4
(A–D) Tm and TM protein levels were analyzed quantified by RT-PCR and Western blot in HUVECs stimulated with LPS for 8 hours. (A) Tm (227 bp) and Gapdh (576 bp) were analyzed by PCR; ratio of Epcr/Gapdh (Gapdh as internal control) was compared (B). 1. Sham group, 2. LPS group, 3. CORM-2 group, 4. CORM-2 + LPS group, 5. iCORM-2 + LPS group, Marker:DL2000, from bottom to top: 100, 250, 500, 750, 1000, 2000; B,D semi-quantitative analyses of mRNA. ∗P < .05, compared to sham group; +P < .05, compared to LPS group; #P < .05, compared to CORM group; &P < .05, compared to LPS + CORM group. Evaluation of tissue TM expression in the LPS-induced and CORM-2 pretreated groups by (C) Western blotting and (D) semiquantitative analyses of Western blots. ∗P < .05, compared to sham group; +P < .05, compared to LPS group; #P < .05, compared to CORM group; &P < .05, compared to LPS + CORM group. CORM = carbon monoxide-releasing molecule, iCORM = inactive carbon monoxide releasing molecule, HUVECs = human umbilical vein endothelial cells, LPS = lipopolysaccharides, RT-PCR = reverse transcription polymerase chain reaction, TM = thrombomodulin.
Figure 5
Figure 5
(A and B). NF-κB activity among groups 8 hours into LPS induction by EMSA. 1. Sham group, 2. LPS group, 3. CORM-2 group, 4. CORM-2 + LPS group, 5. iCORM-2 + LPS group, 6. Cold competition group, 7. Positive control, 8. Negative control. ∗P < 0.05, compared to sham group; +P < .05, compared to LPS group; #P < 0.05, compared to CORM group; &P < .05, compared to LPS + CORM group. CORM-2 = carbon monoxide-releasing molecule-2, EMSA = electrophoretic mobility shift assay, iCORM-2 = inactive carbon monoxide releasing molecule-2, LPS = lipopolysaccharides.
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