Hepatic overexpression of soluble full-length uPAR inhibits atherosclerotic lesion development and macrophage accumulation in LDLR^-/- mice.

<p>A, Immunostaining for the c-myc-tag revealed expression of soluble uPAR in the livers of mice after hydrodynamic transfection. Bar = 100 μm B. Oil red-O staining of aortic valve cryosections. Bar = 250 μm C. Atherosclerotic lesion size was assessed as % total aortic sinus lumen area occupied. D, Representative CD68 immunostaining and E. % CD68-positive lesion area. F, Representative anti-αSMC immunostaining and G. % anti-αSMA-positive lesion area. Bar = 250 μm, Mean±SEM, n = 6/8. *<i>P<</i>0.05, **<i>P<</i>0.01, ***<i>P<</i>0.001. αSMA: alpha smooth-muscle-actin, DAPI: 4′,6-Diamidin-2-phenylindol</p

Lipoprotein profiles.

<p>Mean±SEM, n = 10. n.s. = not significant, HDL: high-density lipoprotein, LDL: low-density lipoprotein.</p><p>The cholesterol rich diet induced hyperlipidemia in uPAR wild-type and knockout mice in the LDLR^-/- background. Within three weeks total and LDL-cholesterol were significantly increased along with triglycerides. No uPAR-dependent differences between the cohorts were observed. HDL-cholesterol increased along with the atherogenic lipoproteins and triglycerides.</p

Soluble uPAR reduces macrophage adhesion to TNFα activated murine endothelial cells.

<p>Wild-type macrophage adhesion to resting and activated endothelium is significantly reduced in the presence of soluble uPAR in vitro. Mean±SEM, n = 4/5. **<i>P<</i>0.01. TNFα: tumor necrosis factor-alpha, suPAR: soluble urokinase-type plasminogen activator receptor.</p

Guide-wire injury induced intimal hyperplasia.

<p>Mice were subjected to guide wire injury of the internal carotid artery after hypercholesterolemia had been induced. (A) Representative Micrographs of carotid artery lesions in the injured and the contralateral artery. GWI induced concentric lesions did not differ in (B) size or (C) lipid content. (D) Macrophage recruitment did occur, but (E) differences between uPAR-deficient and uPAR-wild-type lesions were not observed in this model. Additionally, no differences between uPAR^+/+ and uPAR^-/- animals were observable with respect to (F-G) VSMC content of the lesions. No relevant lesion formation was observed in the contralateral sham vessels. (H) Proliferating cells in atherosclerotic lesions are mainly non-VSMC. The amount of proliferating VSMC appears similar for the two genotypes. A, D, F: Bar = 250μm, H: Bar: 50μm, mean±SEM, n = 8. n.s.: not significant, αSMA: alpha smooth-muscle-actin, DAPI: 4′,6-Diamidin-2-phenylindol, GWI: guide-wire injury, L: left, R: right, PCNA: Proliferating Cell Nuclear Antigen.</p

Macrophage and VSMC accumulation is reduced in uPAR^-/-/LDLR^-/-.

<p>(A) Representative Mac-3 immunostaining and (B) %-Mac-3-positive lesion per lesion area. (C) Representative anti-SMC immunostaining and (D) % anti-SMC-positive lesion per lesion area. (E) PCNA/CD68 double staining excludes differences in macrophage proliferation in the lesions. (F) Macrophage apoptosis was not responsible for the differences in macrophage content as evidenced by cleaved caspase 3 staining. Mean±SEM, n = 8. *P<0.05, **P<0.01, ***P<0.001. A/C: Bar = 250 μm, E/F Bar = 100μm αSMA: alpha smooth-muscle-actin, PCNA: Proliferating-Cell-Nuclear-Antigen, cC3: cleaved caspase-3, DAPI: 4′,6-Diamidin-2-phenylindol.</p

Macrophage adhesion and VSMC migration are dependent on uPAR.

<p>Macrophages were subjected to a low shear adhesion assay on murine EC. A. Macrophage adhesion to resting and activated endothelium in vitro is reduced if macrophages are harvested from uPAR-deficient animals. B. The capability of uPAR^-/--VSMC to migrate into a scratch was assessed in vitro and revealed increase in the migrated distance. C. mRNA expression of VCAM but not ICAM in primary murine ECs is reduced in uPAR deficient animals (n = 6) compared to controls. Mean±SEM, n = 5–8. *<i>P<</i>0.05, **<i>P<</i>0.01. Bar = 250 μm. hpf: high-power fields, WT: wild-type, TNFα: tumor necrosis factor-alpha, VSMC: vascular smooth muscle cells.</p

Hepatic Overexpression of Soluble Urokinase Receptor (uPAR) Suppresses Diet-Induced Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient (LDLR^-/-) Mice

<div><p>Objective</p><p>Atherosclerosis, a chronic inflammatory disease, arises from metabolic disorders and is driven by inappropriate recruitment and proliferation of monocytes / macrophages and vascular smooth-muscle-cells. The receptor for the urokinase-type plasminogen activator (uPAR, Plaur) regulates the proteolytic activation of plasminogen. It is also a coactivator of integrins and facilitates leukocyte-endothelial interactions and vascular smooth-muscle-cell migration. The role of uPAR in atherogenesis remains elusive.</p><p>Methods and Results</p><p>We generated C57Bl6/J low-density lipoprotein receptor (LDL) and uPAR double knockout (uPAR^-/-/LDLR^-/-) mice to test the role of uPAR in two distinct atherosclerosis models. In LDLR^-/- mice, hepatic overexpression following hydrodynamic transfection of soluble uPAR that competes with endogenous membrane-bound uPAR was performed as an interventional strategy. Aortic root atherosclerotic lesions induced by feeding a high-fat diet were smaller and comprised less macrophages and vascular smooth-muscle-cells in double knockout mice and animals overexpressing soluble uPAR when compared to controls. In contrast, lesion size, lipid-, macrophage-, and vascular smooth muscle cell content of guide-wire-induced intima lesions in the carotid artery were not affected by uPAR deficiency. Adhesion of uPAR^-/--macrophages to TNFα-stimulated endothelial cells was decreased <i>in vitro</i> accompanied by reduced VCAM-1 expression on primary endothelial cells. Hepatic overexpression of soluble full-length murine uPAR in LDLR^-/- mice led to a reduction of diet-induced atherosclerotic lesion formation and monocyte recruitment into plaques. <i>Ex vivo</i> incubation with soluble uPAR protein also inhibited adhesion of macrophages to TNFα-stimulated endothelial cells <i>in vitro</i>.</p><p>Conclusion</p><p>uPAR-deficiency as well as competitive soluble uPAR reduced diet-promoted but not guide-wire induced atherosclerotic lesions in mice by preventing monocyte recruitment and vascular smooth-muscle-cell infiltration. Soluble uPAR may represent a therapeutic tool for the modulation of hyperlipidemia-associated atherosclerotic lesion formation.</p></div

Signaling cascades of HLA I Ab-dependent VCAM-1 up-regulation in HUVECs.

<p><b>(A, B)</b> HUVECs were incubated with HLA I Ab W6/32 or isotype control (10 μg/ml) for 18 h after pre-treatment for 30 min with, <b>(A)</b> the PI3K/Akt pathway inhibitors wortmannin (1 μM) and LY294002 (4 μM), or with, <b>(B)</b> the NF-κB pathway inhibitors MG132 (100 nM) and Bay 11–7085 (10 μM). Cells were lysed and subjected to RT-PCR analysis. The fold induction of mRNA levels is indicated. Data are mean ± SEM from three independent experiments. * p≤ 0.05, significant differences treatment versus control; ** p≤ 0.05, W6/32 versus W6/32 plus inhibitor. Wort, wortmannin; LY, LY294002; Bay, Bay 11–7085.</p

CORMs decrease HLA class I Ab-induced VCAM-1 expression in HUVECs.

<p>Confluent HUVECs were incubated with W6/32 or isotype control (10 μg/ml) for 18 h in the presence or absence of CORM-2 (25 μM), iCORM-2 (25 μM), CORM-3 (75 μM) or iCORM-3 (75 μM), as indicated. Cells were lysed and subjected to RT-PCR analysis. The fold induction of mRNA levels is indicated. Data are represented as mean ± SEM from three independent experiments. * p≤ 0.05, significant differences CORM-2/3 versus W6/32; ** p≤ 0.05, iCORM-2/3 versus CORM2/3.</p

Pharmacological inhibition and siRNA-mediated knockdown of HO-1 reduce HLA I Ab-induced VCAM-1 expression in HUVECs.

<p><b>(A)</b> HUVECs were incubated with HLA I Ab W6/32 alone (10 μg/ml) and for 18 h in the presence of CoPPIX (5 μM) or ZnPPIX (5 μM), as indicated. Cells were lysed and subjected to RT-PCR analysis. The fold induction of VCAM-1 mRNA levels is shown. <b>(B-D)</b> HUVECs were transfected with siRNA for HO-1 or scrambled control siRNA. <b>(B)</b> mRNA expression was determined by RT-PCR analysis and relative levels of HO-1 mRNA are shown. <b>(C)</b> Protein expression was determined by Western blot analysis and probed sequentially with Abs against HO-1 and GAPDH. A representative of three independent experiments is shown. <b>(D)</b> Transfected HUVECs were treated with TNFα (15 ng/ml) or W6/32 for 18 h. Cells were lysed and subjected to RT-PCR analysis. The fold induction of mRNA levels is indicated. Bar graphs represent mean ± SEM from three independent experiments. * p≤ 0.05, significant differences treatment versus control; ** p≤ 0.05, W6/32 versus W6/32 plus CoPPIX/ ZnPPIX. Con, control.</p