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TME5C exerts cytoprotective and angiogenic functions
and PAI-1, which are recognized markers of endothelial cell damage and coagulopathy in these mice. All of these markers steeply increased in PBS-treated BMT mice at the 7th day after BMT. Levels of these markers were signifi- cantly lower in BMT recipients treated with TME5C than in those treated with PBS (Figure 7L).
Discussion
Our previous study found that the cytoprotective effects of TM were preserved in TME5.24,26 TME5 consists of three loops: A, B, and C (Figure 1A). The C-loop in the C-terminal subdomain is formed by a stretch of amino acids between the fifth and sixth cysteine residues, and its amino acid sequence is longer than those of the other two loops.27 The C loop contains a short tri-stranded b- sheet structure,33 and is more similar to EGF when com- pared to the A or B loops.27 The study herein found that TME5C, but not A or B, exerted pro-angiogenetic and cytoprotective effects in a GPR15-dependent manner both in vitro and in vivo. Strikingly, TME5C ameliorated HSCT-associated SOS in a murine model.
We previously showed that TME5 did not produce APC,24 but retained some binding capacity towards thrombin (Figure 6). The present study found that TME5C lost the ability to interact with thrombin, as TME5C did not affect PT or APTT (Figure 6). The ratio of concentration of TME5 that affect cytoprotection (30 nM) to coagulation (5000 nM) was approximately 1:166. The concentration of TME5C that produced cytoprotection was 500 nM. However, even the 166-fold higher concen- tration of TME5C (83 mM) did not prolong APTT (data not shown). Thus, the use of TME5C may be safe for BMT recipients as well as SOS patients who are at risk of bleeding due to low platelet counts and/or coagulopathy.
We have recently identified GPR15 as a binding partner of TME5 by performing a pull-down assay with mem- brane protein isolated from HUVECs followed by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis.25 We found that the cytoprotective and pro-angiogenic effects of TME5 were mediated by GPR15, as neither cytoprotection nor angiogenesis is noted in vascular endothelial cells isolated from Gpr15 KO mice after exposure to TME5.25 The study herein found that TME5C exerts cytoprotective and pro- angiogenic effects in vascular ECs isolated from wild-type C57BL/6 mice, but not in ECs isolated from Gpr15 KO mice, suggesting that TME5C also produces favorable effects in ECs via GPR15.
GPR15 is also expressed on T lymphocytes and is required for infection of HIV as a co-receptor.34 Accumulating evidence suggests the involvement of GPR15 in the regulation of inflammation; GPR15 expressed on murine TH1 and TH17 effector cells is
implicated in the development of colitis.35 However, the expression of GPR15 on regulatory T cells is associated with the accumulation of these cells in the intestines and the alleviation of colitis.36 GPR15 is also required for the homing of dendritic epidermal T cells into epidermal tis- sues.34 Intriguingly, TME5 inhibits mixed lymphocyte reactions in vitro in association with a decrease in the pro- duction of inflammatory cytokines such as interleukin-6 and tumor necrosis factor α.37 The anti-inflammatory effect of TME5 is also mediated by GPR15, as TME5 was not able to inhibit the mixed lymphocyte reaction when we used lymphocytes isolated from Gpr15 KO mice.37 Of note, the use of TME5 significantly alleviated graft-versus- host disease (GvHD) in a murine model.37 TME5 also res- cues mice from LPS-induced sepsis,26 further confirming the anti-inflammatory function of TME5C.
Liver sinusoidal ECs are not only the supporting cells of quiescent hepatocytes, but also the source of vasculariza- tion during liver regeneration.38 Hepatocytes undergo apoptosis in parallel with a decrease in vascularization when sinusoidal ECs are damaged.31 EC insults cause a hypercoagulable state through the upregulation of tissue factor and downregulation of TM on ECs, leading to the formation of fibrin clots in the microvasculature.31 As a consequence, the fibrinolytic system is activated to form various FDP fragments. Of note, PAI-1, an important antifibrinolytic regulator mainly produced by ECs, impedes fibrinolysis by inhibiting tissue plasminogen activator and augments thrombus formation.39 Interestingly, the levels of PAI-1 are elevated in SOS patients but not in patients with GvHD and other liver injuries, suggesting that PAI-1 might be a marker capable of discriminating SOS from other BMT-related complica- tions.40 Increases in plasma levels of TM, FDP, and PAI-1 were noted in the murine SOS model, and they were sig- nificantly counteracted by the use of TME5C (Figure 7L), indicating cytoprotective roles of TME5C in vivo.
The optimum effective concentration of TME5C in our experiment (500 nM) was higher than that of TME5 (30 nM). TME5 was produced by the yeast Pichia, while TME5C is a chemically synthesized peptide, therefore we cannot precisely compare the potency of these two com- pounds. However, it is possible that the C-loop alone is not enough to fully produce the pro-proliferative and proangiogenic effect of TM.
Taken together, TME5C, which does not affect the coagulation system, exerts pro-angiogenetic and cytopro- tective activities both in vitro and in vivo. Hence, the use of TME5C may be a promising strategy to prevent and/or treat HSCT-related complications, including SOS.
Funding
This study was supported by Asahi Kasei Pharma (Tokyo, Japan), Uehara Memorial Foundation, and KAKENHI (26461406).
References
1. Richardson PG, Ho VT, Cutler C, et al. Hepatic veno-occlusive disease after hematopoietic stem cell transplantation: novel insights to pathogenesis, current sta- tus of treatment, and future directions. Biol
Blood Marrow Transplant. 2013;19(1
Suppl):S88-90.
2. Mohty M, Malard F, Abecassis M, et al.
Sinusoidal obstruction syndrome/veno- occlusive disease: current situation and per- spectives-a position statement from the European Society for Blood and Marrow
Transplantation (EBMT). Bone Marrow
Transplant. 2015;50(6):781-789.
3. Senzolo M, Germani G, Cholongitas E, et al.
Veno occlusive disease: update on clinical management. World J Gastroenterol. 2007;13(29):3918-3924.
4. Palomo M, Diaz-Ricart M, Carbo C, et al.
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