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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online April 8, 2003 as doi:10.1096/fj.02-0800fje. |
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Max-Planck-Institute for Physiological and Clinical Research, Bad Nauheim, Germany
* Institute for Biochemistry, Justus-Liebig-University, Giessen, Germany; and
Center for Transgene technology and Gene therapy, Flanders Interuniversity Institute for Biotechnology, KU Leuven, Leuven, Belgium
2Correspondence: Max-Planck-Institute, Department of Exp. Cardiology, Benekestrasse 2, D-61231 Bad Nauheim, Germany. E-mail: e.deindl{at}kerckhoff.mpg.de
SPECIFIC AIMS
The plasminogen system is known to play an important role in angiogenesis as well as negative arterial remodeling processes like neointima formation and arteriosclerosis that require cell migration and proliferation. The topic of our study was to define the role of the plasminogen system in arteriogenesis, which has never been elucidated before. We therefore investigated the expression of fibrinolytic components during arteriogenesis in a rabbit and mouse hindlimb model of arteriogenesis and studied their function in mice deficient for urokinase plasminogen activator (uPA), uPA receptor (uPAR), or tissue PA (tPA).
PRINCIPAL FINDINGS
1. Arteriogenesis is associated with an increased expression of uPA in rabbit and mouse
Northern blot analysis revealed a strong up-regulation of uPA but not of uPAR or tPA during the early phase of arteriogenesis. In rabbit the uPA mRNA was up-regulated 
1.7-fold in the upper leg (m. quadriceps) 6 h after femoral artery occlusion, followed by increased uPA activity (2.5-fold compared with sham-operated animals) at 12 h. In the lower leg (m. gastrocnemius), where angiogenesis takes place and which we analyzed for comparison, we found a continuous up-regulation of the uPA mRNA with peaking levels (2.3-fold of sham-operated animals) at 12 h. For the same period we found a significant up-regulation of the uPAR and PAI-I (2.8- and 2.2-fold of sham-operated animals, respectively) in the m. gastrocnemius.
In wild-type (WT) mice, Northern blot results showed a significant up-regulation of uPA (2-fold) in the upper leg (m. adductor) at 12 h after femoral artery ligation but unchanged levels of uPAR. The tPA levels were, like in rabbit, at the detection limit of Northern blot. Western blot analysis evidenced a significant up-regulation of the uPA protein (3-fold of sham-operated animals) at 24 h of femoral artery ligation associated with increased activity levels, as shown by zymography. Protein levels of uPAR and tPA, however, as well as tPA activity remained unchanged.
2. Perfusion recovery upon femoral artery ligation is impaired in uPA-deficient mice but not in uPAR or tPA deficiency
Measuring the perfusion of the distal mouse hindlimbs using a laser Doppler imager, we found no significant differences in right-to-left blood perfusion ratio (ligated vs. nonligated distal hindlimb) between the uPA-/-, tPA-/-, uPAR-/-, and WT groups before femoral artery ligation. Immediately after ligation, the blood flow ratio dropped to between 0.16 ± 0.05 (tPA-/- group) and 0.21 ± 0.07 (WT group), with no significant differences between all groups of animals. At day 3, a trend to an impaired perfusion recovery was observed in uPA-/- vs. WT mice. It reached statistical significance at day 7 (P<0.02 for uPA-/- vs. WT, P<0.02 for uPA-/- vs. uPAR -/-, and P<0.05 for uPA-/- vs. tPA-/- group) and persisted up to the 14th postoperative day (P<0.01 for uPA-/- vs. WT, P<0.05 for uPA-/- vs. uPAR-/- and nonsignificantly for uPA-/- vs. tPA-/- group). At day 21, the differences were no longer significant, but the trend toward an impaired perfusion recovery was still apparent (Fig. 1
). For tPA-/- and uPAR-/- mice, our results on perfusion recovery showed no significance in comparison to WT.
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3. Association of leukocyte infiltration with arteriogenesis is strongly reduced in uPA-/- but not uPAR-/- or tPA-/- mice
Immunoconfocal analysis of the m. adductor from WT mice showed low levels of uPA in the wall of collateral arteries and high levels in interstitial cells, frequently adjacent to growing collateral arteries. We found a significant decrease in the density of activated macrophages and granulocytes in the m. adductor of uPA-/- compared with WT mice 3 days after femoral occlusion. However, we obtained no significant differences between uPAR-/- or tPA-/- and WT mice (Fig. 2
).
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CONCLUSIONS
In the present study, we investigated the expression of the fibrinolytic components during arteriogenesis in a rabbit and mouse model. Our results showed that arteriogenesis in its early phase is associated with increased uPA mRNA levels, followed by increased enzymatic uPA activity in rabbit and mouse. In agreement with published data, our data on angiogenesis derived from studies of the lower leg of rabbits show that this type of vessel growth is associated with increased mRNA levels of uPA, uPAR, and tPA. Our study is the first to highlight the differences between angiogenesis and arteriogenesis with respect to the components of the plasminogen activation system.
To define the role of the plasminogen system in arteriogenesis, we investigated collateral artery growth in mice with targeted disruption of the uPA-, the uPAR-, or the tPA gene. Laser Doppler analysis showed a pronounced reduction in perfusion recovery after femoral ligation in vivo in uPA-/- but not in uPAR-/- or tPA-/- mice, indicating an uPAR-independent role for uPA but not for tPA in arteriogenesis.
Although uPA binds to its specific receptor uPAR on macrophages, thereby enabling the cell to degrade interstitial tissue in the microenvironment, previous results on vascular wound healing have shown that uPA-dependent plasmin proteolysis during smooth muscle cell (SMC) migration is not dependent on uPAR. Since uPA and receptor-bound uPA have the property to catalyze plasminogen activation, uPA accumulation in the extracellular environment probably compensates for receptor deficiency. Even if uPAR binding may accelerate plasmin generation over a period of minutes or hours, this kinetic advantage is likely to be less significant for processes requiring days to weeks—like arteriogenesis.
uPA expression has been shown to coincide with the proliferative phase of vascular SMCs and to be implicated in SMC DNA biosynthesis, but the mitogenic effect of uPA can be brought about independent of uPAR. However, the role of uPA for SMC proliferation is controversial since uPA deficiency did not affect SMC proliferation in uPA-deficient mice.
Arteriogenesis in humans as well as in animal models is initiated mechanically. After the occurrence of hemodynamically relevant stenosis or femoral artery ligation, blood flow is redistributed to preexisting arteriolar connections that experience increased flow velocity and hence increased shear stress. This causes a marked activation of the endothelium with infiltration of monocytes that locally produce a cocktail of distinct cytokines and growth factors promoting collateral artery growth (Fig. 3
).
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Plasmin is a potent and specific chemoattractant for human peripheral monocytes. However, uPA also induces stimulation of cell chemotaxis, adhesion of monocytes and neutrophils, as well as expression of matrix metalloproteinases (MMPs) independent of its enzymatic activity. An up-regulation of MMP-2 and MMP-9 during arteriogenesis has been previously shown in our laboratory, indicating that a certain proteolytic cascade is required for controlled collateral vessel formation.
In the current study, we found that an infiltration of monocytes and granulocytes, as observed in WT mice after femoral occlusion, was strongly reduced in uPA-/- but not uPAR- or tPA-/- mice. These data not only confirm the importance of leukocytes for collateral artery growth, but also clearly demonstrate that the appearance of leukocytes in arteriogenesis is dependent on uPA. Our data also confirm previous studies with other experimental settings showing that leukocyte extravasation is not dependent on an uPA/uPAR interaction.
Not only does uPA play a prominent role in monocyte attraction, migration, and invasion; it may also induce cytokine gene expression and release in monocytes. Delay of blood flow recovery as observed in uPA-deficient mice after femoral ligation indicates that growth factors supplied by monocytes may also be produced by growing arterioles or the surrounding muscle tissue, yet less efficiently than by circulating blood cells.
Our study shows for the first time that arteriogenesis is promoted by an uPA-mediated infiltration of leukocytes that is not dependent on uPAR.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0800fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0800fje 
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