The role of urokinase plasminogen activator and plasmin activator inhibitor-1 on vein wall remodeling in experimental deep vein thrombosis

Abstract

Objective: Deep vein thrombosis (DVT) resolution instigates an inflammatory response, resulting in vessel wall damage and scarring. Urokinase-plasminogen activator (uPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1), are integral components of the fibrinolytic system, essential for venous thrombosis (VT) resolution. This study determined the vein wall response when exposed to increased and decreased plasmin activity.

Methods: A mouse inferior vena cava (IVC) ligation model in uPA -/- or PAI-1 -/- and their genetic wild types (B6/SvEv and C57/BL6, respectively) was used to create stasis thrombi, with tissue harvest at either 8 or 21 days. Tissue analysis included gene expression of vascular smooth muscle cells (alpha smooth muscle actin [αSMA], SM22) and endothelial marker (CD31), by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, matrix metalloproteinase (MMP)-2 and -9 activity by zymography, and vein wall collagen by picro-Sirius red histologic analysis. A P < .05 was considered significant.

Results: Thrombi were significantly larger in both 8-day and 21-day uPA -/- as compared with wild type (WT) and were significantly smaller in both 8-day and 21-day PAI-1 -/- as compared with WT. Correspondingly, 8-day plasmin levels were reduced in half in uPA -/- and increased three-fold in PAI-1 -/- when compared with respective WT thrombi (P < .05; n = 5-6). The endothelial marker CD31 was elevated two-fold in PAI-1 -/- mice at 8 days, but reduced 2.5-fold at 21 days in uPA -/- as compared with WT (P = .02; n = 5-6), suggesting less endothelial preservation. Vein wall vascular smooth muscle cell (VSMC) gene expression showed that 8-day and 21-day PAI-1 -/- mice had 2.3- and 3.8-fold more SM22 and 1.8- and 2.3-fold more αSMA expression than respective WT (P < .05; n = 5-7), as well as 1.8-fold increased αSMA (+) cells (P ≤ .05; n = 3-5). No significant difference in MMP-2 or -9 activity was found in the PAI-1 -/- mice compared with WT, while 5.4-fold more MMP-9 was present in 21-day WT than 21-day uPA -/- (P = .03; n = 5). Lastly, collagen was ∼two-fold greater at 8 days in PAI-1 -/- IVC as compared with WT (P = .03; n = 6) with no differences observed in uPA -/- mice.

Conclusions: In stasis DVT, plasmin activity is critical for thrombus resolution. Divergent vein wall responses occur with gain or loss of plasmin activity, and despite smaller VT, greater vein wall fibrosis was associated with lack of PAI-1.

Figure 1

A) Thrombus size was larger in uPA −/− as compared with WT. B) Plasmin activity was decreased in uPA−/− at 8d as compared with WT; C) Despite larger thrombi, the fibrinogen content was lower, as was TAT at 8d in uPA −/− mice compared with WT. D) At 8 and 21d, VT were smaller in the PAI-1 −/− mice as compared with WT. E) Thrombus size was smaller in the PAI-1−/− as compared with WT. F) Plasmin activity was increased in PAI-1−/− at 8d as compared with WT. * = P < .05.

Figure 2

Hematoxylin and Eosin stained IVC sections of 21d WT (A) and uPA −/− (B) examples. Note large and relative acellularity of the uPA −/− thrombus. The 21d WT (C) and PAI-1 −/− (D) are shown with similar thrombus morphology and cellularity.

Figure 3

Gene expression of the endothelial marker CD31 was reduced in 21d uPA−/− compared with WT (A). Conversely, CD31 gene expression was increased at 8d in PAI-1 −/− (B) as compared with WT. Vein wall intimal thickness was reduced in 8d uPA−/− vein wall as compared with WT (C). Mild neointimal thickening is present in 8d WT vein walls(D), but little in the uPA −/− vein wall (E). Note again of marked cellularity of 8d WT thrombi, but few present in uPA −/− thrombi.

Figure 4

Vein wall procollagen I (A) and III (B) gene expression was elevated in 8d PAI-I −/− as compared with WT. By Picosirius red analysis, collagen content was elevated at 8d in PAI-1 −/− compared with WT, and a trend at 21d while noo difference in collagen content was found in uPA −/− at 8 or 21d (C). Birefringence images show a thin vein wall collagen in WT (D), but greater amount in PAI-1 −/− vein wall (E). P < .05. T = thrombus; W = wall.

Figure 5

VSMC gene marker showed increased SM22 (A) and αSMA (B) expression in PAI-1 −/− vein wall compared to WT at 8 and 21d. Medial αSMA + cells were increased in PAI-1 −/− at 8d as compared with WT (C). Immunohistology showed the mononuclear medial location of these positive cells in WT (D) and PAI-1 −/− (E) IVC sections. *P < .05; arrows denote (+) cells.