Fibrinogen induces alterations of endothelial cell tight junction proteins

Abstract

We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin-associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell-substrate impedance system, 8-well chambered, or in 12-well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen-activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti-ICAM-1 antibody or BQ788 (endothelin type B receptor blocker), endothelin-1, endothelin-1 with BQ788, or medium alone for 24 h. Fg induced a dose-dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT-PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden-1 (ZO-1), and zona occluden-2 (ZO-2) in ECs. Fg-induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti-ICAM-1 antibody. While BQ788 inhibited endothelin-1-induced decrease in TEER, it did not affect Fg-induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO-1, and ZO-2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM-1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs.

A Role for Vascular Deficiency in Retinal Pathology in a Mouse Model of Ataxia-Telangiectasia

“…37,[62][63][64][65][66] Furthermore, increased levels of fibrinogen have been shown to downregulate endothelial tight junction proteins. 67 Both of these processes lead to widening of interendothelial junctions and increased vascular leakage in the paracellular route, as demonstrated by fibrinogen and hemosiderin labeling in this study. Fibrinogen can also induce vasoconstriction through the production of endothelin-1.…”

“…67 We demonstrated here that occludin is significantly reduced in the retinas of Atm Ϫ/Ϫ mice at 2 months of age. In parallel, we found indications for interstitial accumulation of blood by hemosiderin staining.…”

Pro-inflammatory effect of fibrinogen and FDP on vascular smooth muscle cells by IL-6, TNF-α and iNOS

“…According to the previous reports (Ge et al, 1991;Kuhns et al, 2001;Patibandla et al, 2009) and the results of our preliminary experiment, the significant mRNA and protein expressions of IL-6, TNF-α and iNOS in VSMCs were observed after stimulating VSMCs with 1.25 μM fibrinogen for 3 h or with 1/60 FDP (about equal to 0.5 μM fibrinogen) for 1 h. The approximately maximal protein expressions of IL-6, TNF-α and iNOS in VSMCs were achieved following stimulation of VSMCs with 10 μM fibrinogen for 12 h or with 1/20 FDP (about equal to 1.5 μM fibrinogen) for 4 h. This seems to mean that smaller fibrinogen fragments produce greater pro-inflammatory activity. This result is also in agreement with the finding that the effect of plasmin on fibrinogen, leading to the formation of their degradation products, will amplify the destructive effects initiated by an increased content of fibrinogen (Lominadze et al, 2010).…”

“…The existing evidence show that fibrinogen is an independent predictor for cardiovascular death and atherosclerotic burden (Espinola-Klein et al, 2007). An elevated fibrinogen level participates in the formation of atherosclerosis through activating platelet aggregation, increasing plasma viscosity, injuring endothelial cells and stimulating migration and proliferation of vascular smooth muscle cells (VSMCs) (Koenig, 2003;Patibandla et al, 2009). A number of studies report that fibrinogen has the ability to stimulate production of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β, IL-8, and ET-1) in endothelial cells (Lominadze et al, 2010;Sen et al, 2009), macrophages (Szaba and Smiley, 2002), monocytes (Robson et al, 1994), and neutrophils (Kuhns et al, 2001), which are mediated mainly via the integrin receptor (Ly et al, 2003).…”

Hyperglycemic Arterial Disturbed Flow Niche as an In Vitro Model of Atherosclerosis

“…Density of bands was quantified by optical scanning and analysis using Gel-Pro Analyzer software (Media Cybernetics, Silver Spring, MD). 31 Results are normalized for protein loading. Each experiment was performed in triplicate on two separate occasions.…”