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.

Improved Vascular Gene Transfer With a Helper-Dependent Adenoviral Vector

“…The endogenous uPA mRNA and transgene uPA mRNA are different sizes and can therefore be discriminated by Northern analysis. 22 In 11 of 12 FG-AduPA arteries, uPA transgene mRNA was less abundant than endogenous uPA mRNA. In contrast, in all HD-AduPA arteries, uPA transgene mRNA was more abundant than endogenous uPA mRNA.…”

“…We constructed a HD-Ad vector that expresses rabbit urokinase plasminogen activator (HD-AduPA; Figure 1) by ligation of the uPA expression cassette from the FG-Ad AdrbtuPA 22 (hereafter called FG-AduPA) into the SwaI site of the HD-Ad backbone plasmid pC4HSU, digestion of the product with PmeI, transfection into 293Cre4 cells, and infection of the cells with "H14," a helper virus that is a null, first-generation, E1-negative adenovirus. 23 HD-AduPA was expanded by serial passage and purified by CsCl ultracentrifugation.…”

“…uPA is also potentially a therapeutic gene, 25 although not under all circumstances. 22 Preparations of HD-AduPA, FG-AduPA, and H14 were characterized by spectrophotometry to measure vector particle (part) concentration. 26 E1A-positive virus was quantified by real-time polymerase chain reaction: primers, AATGGCCGCCAGTCTTTG and AAATGGCTAGGAGGTG-GAAGATT; probe, TCAGCCAGTACCTCTTCGATCAGCTGGT.…”

Improved Animal Models for Testing Gene Therapy for Atherosclerosis

“…FGAdNull is a first-generation E1/E3-deleted vector with an ''empty'' expression cassette (Falkenberg et al, 2002). HDAdNull is a third-generation (also known as a ''helper-dependent'') vector that lacks all viral genes and contains the same empty expression cassette as FGAdNull (Flynn et al, 2010).…”

“…This animal model is useful for testing short-term vascular-walltargeted gene therapy because the carotid arteries are easily accessible with only a minor surgical protocol (expensive catheters are not required); lesions develop quickly and reproducibly; and the side branches of the common carotid can be ligated without morbid consequences, facilitating vector dwell time and gene delivery. Moreover, vector infusion is inherent in the model: to test whether a transgene will prevent early atherosclerosis, one needs to only insert the transgene into an FGAd vector and compare lesions in arteries treated with the new vector to lesions in arteries treated with FGAdNull (Schneider et al, 2000;Falkenberg et al, 2002).…”