Components of the plasminogen activation system promote engraftment of porous polyethylene biomaterial via common and distinct effects

Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial

Components of the Plasminogen Activation System Promote Engraftment of Porous Polyethylene Biomaterial via Common and Distinct Effects

Rapid fibrovascularization is a prerequisite for successful biomaterial engraftment. In addition to their well-known roles in fibrinolysis, urokinase-type plasminogen activator (uPA) and tissue plasminogen activator (tPA) or their inhibitor plasminogen activator inhibitor-1 (PAI-1) have recently been implicated as individual mediators in non-fibrinolytic processes, including cell adhesion, migration, and proliferation. Since these events are critical for fibrovascularization of biomaterial, we hypothesized that the components of the plasminogen activation system contribute to biomaterial engraftment. Employing in vivo and ex vivo microscopy techniques, vessel and collagen network formation within porous polyethylene (PPE) implants engrafted into dorsal skinfold chambers were found to be significantly impaired in uPA-, tPA-, or PAI-1-deficient mice. Consequently, the force required for mechanical disintegration of the implants out of the host tissue was significantly lower in the mutant mice than in wild-type controls. Conversely, surface coating with recombinant uPA, tPA, non-catalytic uPA, or PAI-1, but not with non-catalytic tPA, accelerated implant vascularization in wild-type mice. Thus, uPA, tPA, and PAI-1 contribute to the fibrovascularization of PPE implants through common and distinct effects. As clinical perspective, surface coating with recombinant uPA, tPA, or PAI-1 might provide a novel strategy for accelerating the vascularization of this biomaterial

Neutrophils promote venular thrombosis by shaping the rheological environment for platelet aggregation

In advanced inflammatory disease, microvascular thrombosis leads to the interruption of blood supply and provokes ischemic tissue injury. Recently, intravascularly adherent leukocytes have been reported to shape the blood flow in their immediate vascular environment. Whether these rheological effects are relevant for microvascular thrombogenesis remains elusive. Employing multi-channel in vivo microscopy, analyses in microfluidic devices, and computational modeling, we identified a previously unanticipated role of leukocytes for microvascular clot formation in inflamed tissue. For this purpose, neutrophils adhere at distinct sites in the microvasculature where these immune cells effectively promote thrombosis by shaping the rheological environment for platelet aggregation. In contrast to larger (lower-shear) vessels, this process in high-shear microvessels does not require fibrin generation or extracellular trap formation, but involves GPIb alpha-vWF and CD40-CD40L-dependent platelet interactions. Conversely, interference with these cellular interactions substantially compromises microvascular clotting. Thus, leukocytes shape the rheological environment in the inflamed venular microvasculature for platelet aggregation thereby effectively promoting the formation of blood clots. Targeting this specific crosstalk between the immune system and the hemostatic system might be instrumental for the prevention and treatment of microvascular thromboembolic pathologies, which are inaccessible to invasive revascularization strategies

Effect of surface coating of implants with DFP-uPA or NE-tPA on vascularization of PPE biomaterial.

<p>Vascularization of PPE implants, which were coated with DFP-uPA or NE-tPA embedded in Matrigel, was analyzed by <i>in vivo</i> fluorescence microscopy in WT mice. Panels show results for the relative increase in absolute (<b>A</b>) and functional (<b>B</b>) vessel density within the implant as compared to Matrigel-coated control implants (mean ± SEM for n = 5 – 6).</p

Effect of surface coating of implants with recombinant uPA, tPA, or PAI-1 on vascularization of PPE biomaterial.

<p>Vascularization of PPE implants, which were coated with recombinant uPA, tPA, or PAI-1 embedded in Matrigel, was analyzed by <i>in vivo</i> fluorescence microscopy in WT mice. Panels show results for the relative increase in absolute (<b>A</b>) and functional (<b>B</b>) vessel density within the implant as well as for the increase in the number of branches (<b>C</b>) and junctions (<b>D</b>) in the vessel network as compared to Matrigel-coated control implants (mean ± SEM for n = 5 – 6).</p

Role of endogenous uPA, tPA, and PAI-1 for leukocyte-endothelial cell interactions in PPE biomaterial.

<p>Interactions of fluorescence-labeled leukocytes and endothelial cells in newly formed microvessels within the PPE implants in WT as well as in uPA-, tPA-, or PAI-1-deficient mice were analyzed by <i>in vivo</i> fluorescence microscopy. Representative <i>in vivo</i> microscopy images of these processes in a WT mouse are shown (<b>A</b>; scale bar: 100 μm). Panels show results for the number of intravascularly rolling (<b>B</b>) and adherent (<b>C</b>) leukocytes (mean ± SEM for n = 6; * p < 0.05 vs. WT).</p

Effect of uPA, tPA, and PAI-1 on migration of endothelial cells.

<p>The effect of recombinant uPA, tPA, and PAI-1 on migration of endothelial cells was analyzed <i>in vitro</i>. Representative migration plots are shown (<b>A</b>). Panels show results for the forward migration index (FMI) of migrating endothelial cells upon exposure to recombinant uPA, tPA, or PAI-1 (<b>B</b>; mean ± SEM for n = 3; * p < 0.05 vs. +/-).</p

Role of endogenous uPA, tPA, and PAI-1 for vessel network formation in PPE biomaterial.

<p>A detailed analysis of the vessel network within the PPE implants in WT as well as in uPA-, tPA-, or PAI-1-deficient mice was performed by software-assisted skeletonizing of the vessel network. Representative images of this analysis in a WT mouse are shown (<b>A;</b> scale bar: 100 μm). Panels show results for the number of branches (<b>B</b>) and total junctions (<b>C</b>) as well as triple (<b>D</b>) and quadruple (<b>E</b>) junctions in the vessel network (mean ± SEM for n = 6; * p < 0.05 vs. WT).</p

Role of endogenous uPA, tPA, and PAI-1 for mechanical tissue integration of PPE biomaterial.

<p>Panel (<b>A</b>) shows results for the disintegration force (mean ± SEM for n = 6; * p < 0.05 vs. WT). The disintegration force required for mechanical removal of the implant out of the host tissue was determined in WT as well as in uPA-, tPA-, or PAI-1-deficient mice. Collagen deposition within the PPE biomaterial was analyzed <i>ex vivo</i> in implants from WT as well as from uPA-, tPA-, or PAI-1-deficient mice by second harmonic imaging microscopy. A representative image from a WT mouse is shown (<b>B</b>; scale bar: 100 μm; red dotted lines delineate pores of the implant material, in which collagen (white) is deposited). Panel (<b>C</b>) shows quantitative results for the second harmonic signal (mean ± SEM for n = 4; * p < 0.05 vs. WT).</p

Role of endogenous uPA, tPA, and PAI-1 for vascularization of PPE biomaterial.

<p>The distribution of uPA, tPA, and PAI-1 in engrafted PPE biomaterial was analyzed <i>ex vivo</i> in implants from WT mice by confocal microscopy (<b>A;</b> scale bar: 100 μm). Vascularization of PPE implants was analyzed in WT as well as in uPA-, tPA-, or PAI-1-deficient mice by <i>in vivo</i> fluorescence microscopy. Representative <i>in vivo</i> microscopy images of PPE biomaterial vascularization in a WT mouse are shown (<b>B</b>; scale bar: 100 μm). Panels show results for the absolute (<b>C</b>) and functional (<b>D</b>) vessel density within the implant (mean ± SEM for n = 6; * p < 0.05 vs. WT).</p