Increased expression of urokinase during atherosclerotic lesion development causes arterial constriction and lumen loss, and accelerates lesion growth

Abstract

Overexpression of urokinase plasminogen activator (uPA) in endothelial cells can decrease intravascular thrombosis. However, expression of uPA is increased in atherosclerotic human arteries, which suggests that uPA might accelerate atherogenesis. To investigate whether elevated uPA expression accelerates atherogenesis, we cloned a rabbit uPA cDNA and expressed it in carotid arteries of cholesterol-fed rabbits. uPA gene transfer increased artery-wall uPA activity for at least 1 week, with a return to baseline by 2 weeks. One week after gene transfer, uPA-transduced arteries were constricted, with significantly smaller lumens and thicker walls, but no difference in intimal or medial mass. Two weeks after gene transfer, uPA- and control-transduced arteries were morphologically indistinguishable. By 4 weeks, however, uPA-transduced arteries had 70% larger intimas than control-transduced arteries (P < 0.01) and smaller lumens (P < 0.05). Intimal lesions appeared to be of similar composition in uPA- and control-transduced arteries, except that degradation of elastic laminae was evident in uPA-transduced arteries. These data suggest that elevated uPA expression in atherosclerotic arteries contributes to intimal growth and constrictive remodeling leading to lumen loss. Antagonists of uPA activity might, therefore, be useful in limiting intimal growth and preventing constrictive remodeling. Overexpression of uPA in endothelial cells to prevent intravascular thrombosis must be reconsidered, because this intervention could worsen underlying vascular disease.

Fig 1.

Zymography of medium conditioned by 293 cells transduced with either AdrbtuPA or AdCMVNull. Overnight collections of serum-free medium were analyzed by casein plasminogen zymography. The two large lysis zones likely indicate presence of both high- and low-molecular-mass forms of uPA. The position of migration of molecular mass markers is indicated in kilodaltons.

Fig 2.

In vivo expression of rabbit uPA. (A) Dose-response study. RNA was extracted from arteries harvested 3 days after transduction with either AdCMVNull at 1 × 10⁸ pfu/ml (as a control and to detect endogenous uPA mRNA) or AdrbtuPA at 1 × 10⁸ to 4 × 10⁹ pfu/ml. Phosphorimager analysis of Northern blots was used to calculate the ratio of the signals of uPA transgene mRNA to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA from the same artery. The relative level of endogenous uPA to GAPDH mRNA in the AdCMVNull-transduced arteries was assigned a value of 1.0. (B) Time course of vector-driven uPA expression. Arteries were transduced with AdrbtuPA (4 × 10⁹ pfu/ml) and harvested 1–14 days later. The relative abundance of 2.4-kb uPA transgene mRNA was quantified as in A, except that uPA:GAPDH ratios are presented without normalization to AdCMVNull values. For both panels, data are presented as mean ± SEM of groups of four arteries. *, uPA transgene mRNA was undetectable.

Fig 3.

Plasminogen activator activity in transduced artery segments. (A) Dose–response study. Arteries were transduced with AdrbtuPA or AdCMVNull at the indicated concentrations (pfu/ml). Three days later, segments of transduced arteries were incubated ex vivo with plasminogen and the plasmin substrate S-2390. Plasmin generation is measured as the increase in OD₄₀₅ over time. Data points represent the mean OD from segments of four separate arteries in each group. For clarity, SEMs are shown at 60 min only. Curves for AdCMVNull arteries are extended toward actual data points measured at 180 min. (B–E) Time course of increased plasminogen activator activity. Arteries were transduced with AdrbtuPA or AdCMVNull (both at 4 × 10⁹ pfu/ml) and segments explanted and assayed for plasminogen activator activity as in A at 1, 3, 7, or 14 days after gene transfer. Data points are means of n = 4 arteries in each group; error bars indicate SEM.

Fig 4.

uPA activity in arterial extracts. Arteries were transduced with AdrbtuPA or AdCMVNull (both at 4 × 10⁹ pfu/ml). Three days later, artery segments were harvested, and proteins were extracted. Extracts were assayed with the uPA-specific chromogenic substrate S-2444. Data points represent individual arteries; bar heights indicate group medians.

Fig 5.

Morphometry of transduced arteries. Arteries harvested 4 weeks (A–C) or 1 week (D–F) after gene transfer. Arteries transduced with AdrbtuPA had larger intimas at 4 weeks (B vs. A) and were constricted with smaller lumens at 1 week (E vs. D). Absence of elastic laminae (arrows) and fragmentation of elastic laminae (arrowhead) were more common in AdrbtuPA arteries (C and F and Inset). Movat's pentachrome stain. (Original magnifications: A–C and F, ×40; D–E, ×5.)