Fibrinogen increases the development of atherosclerosis. The fibrin-derived peptide Bβ15–42 prevents leukocyte accumulation, protects endothelial function, and reduces inflammatory osmosis [9, 16]. EFEa cleaves fibrinogen, and it has been suggested that, similar to Bβ15–42, EFEa has clinical value for reducing fibrinogen expression and has anti-tumor, anti-virus, and anti-inflammatory effects [12]. Our study focused on understanding the anti-atherosclerotic mechanisms of these two drugs.
We found no difference in LDL levels after treatment of rabbits with Bβ15–42 and EFEa. However, fibrinogen protein levels were reduced in aortic lesions after drug treatment. The EFEa effects are likely due to molecular antagonism like Bβ15–42, rather than direct effects on fibrinogen expression. Previous studies demonstrated that fibrinogen primarily accumulates in early atherosclerotic tissues and this deposition results in destruction of endothelial cells and progression of atherosclerotic lesions [4, 5]. Therefore, treatment with Bβ15–42 or EFEa reduces fibrinogen deposition and overall atherosclerotic burden.
Local inflammation and cell activation are important underlying mechanisms of atherosclerosis. Macrophage expression is significantly increased in atherosclerotic plaques, and is closely related to the severity of plaque formation, development, and rupture [17]. We used immunohistochemistry to label macrophages on the aorta and found that Ros treatment significantly reduced inflammatory cells, demonstrating that statins account for at least part of the anti-inflammatory effects in our model; this is consistent with the effect of Ros given by Tziakas et al. in a rabbit model of atherosclerosis [18]. In addition, these data suggest that EFEa also has anti-inflammatory effects as seen by a reduction in inflammatory cells. Bβ15–42 and EFEa treatment reduced aortic atherosclerotic lesion areas, demonstrating that both drugs have potent anti-atherosclerotic effects.
However, the specific mechanisms of action of these drugs remain largely unknown. The Zbharowski group first confirmed in vitro that the fibrin fragment N-terminal disulfide knot (NDSK)-II can promote migration of human peripheral blood mononuclear cells through HUVEC monolayers [19]. The effect of Bβ15–42 treatment on the amino-terminus of the NDSK-IIβ chain can be blocked by antibodies against recombinant VE-cadherin, CD11b, and the amino terminal sequence AαGPRP of the NDSK-IIα chain [19]. Therefore, we propose that the Bβ15–42/VE-cadherin pathway is involved in the leukocyte transendothelial cell monolayer as a "molecular bridge". We used real-time fluorescence quantitative PCR, protein immunoblot, and immunofluorescence to show VE-cadherin expression in HUVECs by successfully expressing VE-cadherin protein in HUVECs through a lentivirus vector (HUVEC-CDH5+). HUVEC-CDH5 + cells linked tightly to each other and provided a good model to simulate the state of vascular endothelium in the body.
To test fibrinogen- and fibrin-mediated effects on VE-cadherin expression, we exposed HUVEC-CDH5 + cells to varying lengths of time and concentrations of fibrinogen and fibrin. The high-dose fibrinogen treatment (6 mg/mL for 24 hours) reduced VE-cadherin protein expression. Consistent with our results, Lominadze et al. [20] demonstrated that high concentrations of fibrinogen (4 mg/mL) decreased VE-cadherin expression in cerebral vascular endothelial cells and led to cerebral vascular dysfunction. In MMP9 knockout mice, the use of ICAM-1 function-blocking antibodies can antagonize fibrinogen-induced reduction in VE-cadherin expression, suggesting that ICAM-1 is involved in the interaction between fibrinogen and endothelial cells. The reduction in the amount of VE-cadherin protein caused by fibrinogen injection in vivo is associated with MMP9 [21]. Our results suggest that the effect of fibrin on reducing VE-cadherin protein is even more pronounced. Low-dose fibrin (1 mg/mL) treatment significantly reduced VE-cadherin protein expression and high-dose fibrin (6 mg/mL) treatment reduced VE-cadherin after 12 hours of treatment compared with 24-hours by fibrinogen, and by a greater extent. Therefore, we speculated that in vivo, fibrin deposited in the blood vessel or fibrinogen that was exposed to the β15–42 sequence [22] could reduce VE-cadherin expression, destroy the integrity of the original endothelium, and accelerate atherosclerosis.
We used immunofluorescence to observe the distribution of VE-cadherin and F-actin in HUVEC-CDH5 + treated with high concentrations of fibrinogen or fibrin. In response to the high dose of fibrinogen and, to a greater extent, fibrin, the total amount of VE-cadherin was decreased and aggregated, the structure of F-actin was disordered, and co-localization of F-actin with VE-cadherin at the cell junction was reduced. Higher concentrations of fibrinogen (4mg/mL) can activate the ERK signaling pathway and increase the formation of endothelial cytoskeletal protein F-actin, thereby increasing intercellular permeability. In addition, the activation of the ERK pathway in endothelial cells with fibrinogen alters the relationship between ICAM-1 and integrin α5β1 [23]. Our results suggest that fibrin has a more pronounced effect on VE-cadherin redistribution and F-actin morphological changes than does fibrinogen.
The central E region of fibrin consists of α17–78, β15–122, and γ1–62. Its homologue NDSK-II (α17–51, β15–118, γ1–78) promotes transmigration of leukocytes through the endothelium in vitro experiments. To understand the role of migration through cell monolayers [19], we stimulated HUVEC-CDH5 + cells with fibrin and observed an increase in macrophage migration through endothelial cells; however, fibrinogen did not have this effect. These results indicate that the α17-, β15-terminal exposure of fibrin is necessary to promote inflammation and intracellular migration. Next, we used Bβ15–42 and EFEa to interfere with fibrin-induced macrophage migration. Both drugs showed an inhibitory effect on fibrin-induced cell migration through endothelial cell monolayers and prevented loss of VE-cadherin. Several experiments have shown that Bβ15–42 reduces ischemia reperfusion injury and protects the integrity of the vascular endothelium. The main mechanism may be that Bβ15–42 mimics the amino-terminal sequence of the fibrin β chain and binds competitively to VE-cadherin, thereby preventing leukocyte migration through the endothelial monolayer. Bβ15–42 induces release of Fyn, inhibits RhoA activity, inhibits rearrangement of skeletal proteins, maintains the continuity of VE-cadherin between cells, and reduces inflammatory cell infiltration [19, 24]. EFEa has trypsin-like activity and multiple hydrolytic sites for plasminogen and fibrin that can hydrolyze fibrinogen [25]. EFEa cleaves the fibrinogen alpha chain at Val(21)-Glu(22). Our results suggest that the Bβ15–42/VE-cadherin "molecular bridge" links the amino terminus of the alpha chain of leukocyte CD11c and CD18 [26] and that Bβ15–42 has the effect of antagonizing fibrin mediated pro-inflammatory cell migration.
Our results together with previous studies indicate that fibrinogen is closely related to the occurrence and development of atherosclerosis. The fibrin Bβ15–42/VE-cadherin pathway participates in the migration process of macrophages through endothelial cells. The mechanism may be related to the down regulation of VE-cadherin protein expression and the rearrangement of F-actin. Bβ15–42 treatment protects endothelial function, reduces the deposition of local fibrinogen, reduces inflammatory cell levels in the aorta, and is anti-atherosclerotic. Our data suggest that the inhibitory effect of EFE on atherosclerosis in rabbits is caused by the peptide effect of sheared products like Bβ15–42.