The extracellular matrix (ECM) supports vascular integrity during embryonic development. Proteolytic degradation of ECM components is required for angiogenesis, but excessive ECM proteolysis causes blood vessel fragility and hemorrhage. Little is understood about how ECM proteolysis is transcriptionally regulated during embryonic vascular development. We now show that the NuRD ATP-dependent chromatin-remodeling complex promotes vascular integrity by preventing excessive ECM proteolysis in vivo. Mice lacking endothelial CHD4—a catalytic subunit of NuRD complexes—died at midgestation from vascular rupture. ECM components surrounding rupture-prone vessels in Chd4 mutants were significantly downregulated prior to embryonic lethality. Using qPCR arrays, we found two critical mediators of ECM stability misregulated in mutant endothelial cells: the urokinase-type plasminogen activator receptor (uPAR or Plaur) was upregulated, and thrombospondin-1 (Thbs1) was downregulated. Chromatin immunoprecipitation assays showed that CHD4-containing NuRD complexes directly bound the promoters of these genes in endothelial cells. uPAR and THBS1 respectively promote and inhibit activation of the potent ECM protease plasmin, and we detected increased plasmin activity around rupture-prone vessels in Chd4 mutants. We rescued ECM components and vascular rupture in Chd4 mutants by genetically reducing urokinase (uPA or Plau), which cooperates with uPAR to activate plasmin. Our findings provide a novel mechanism by which a chromatin-remodeling enzyme regulates ECM stability to maintain vascular integrity during embryonic development.
Blood vessels are surrounded by an extracellular matrix (ECM), which provides structural support and helps vessels withstand the biomechanical pressure of flowing blood. As blood vessels grow during embryonic development, the ECM is locally degraded to allow new vessels to sprout from existing ones. This localized ECM degradation must be tightly regulated, however, because excessive matrix breakdown leaves vessels susceptible to rupture and lethal hemorrhage. We now present a novel mechanism by which production of proteins that promote or repress ECM degradation is coordinated. We show that a chromatin-remodeling enzyme called CHD4 is critical for maintaining blood vessel integrity during embryonic development. CHD4 achieves this task by limiting production of proteins that degrade ECM and promoting production of proteins that protect ECM around developing blood vessels. A better understanding of how ECM degradation is regulated during development could have an impact on our ability to combat adult pathologies, such as aneurysms, which are associated with excessive ECM degradation and vascular fragility.
Oxidative stress plays a major role in diabetes. In vivo levels of membrane-bound radicals (MBRs) in a streptozotocin-induced diabetic mouse model were uniquely detected by combining molecular magnetic resonance imaging (mMRI) and immunotrapping techniques. An anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibody (Ab) covalently bound to an albumin (BSA)-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin MRI contrast agent (anti-DMPO probe), and mMRI, were used to detect in vivo levels of DMPO-MBR adducts in kidneys, livers, and lungs of diabetic mice, after DMPO administration. Magnetic resonance signal intensities, which increase in the presence of a Gd-based molecular probe, were significantly higher within the livers, kidneys, and lungs of diabetic animals administered the anti-DMPO probe compared with controls. Fluorescence images validated the location of the anti-DMPO probe in excised tissues via conjugation of streptavidin-Cy3, which targeted the probe biotin moiety, and immunohistochemistry was used to validate the presence of DMPO adducts in diabetic mouse livers. This is the first report of noninvasively imaging in vivo levels of MBRs within any disease model. This method can be specifically applied toward diabetes models for in vivo assessment of free radical levels, providing an avenue to more fully understand the role of free radicals in diabetes.
Vascular endothelial growth factor receptor 2 (VEGFR2) is an important angiogenic marker over-expressed in gliomas. With the use of molecular magnetic resonance imaging (mMRI) differing levels of VEGFR2 can be characterized in vivo with in rodent gliomas varying in angiogenesis. VEGFR2 levels were assessed by intravenous administration of an anti-VEGFR2 probe (anti-VEGFR2-albumin-Gd (gadolinium)-DTPA (diethylene triamine penta acetic acid)-biotin) into C6 or RG2 glioma-bearing rats, and visualized with mMRI. A non-specific IgG was coupled to the albumin-Gd-DTPA-biotin construct as a contrast agent molecular weight control. VEGFR2 levels are heterogeneous in different regions of C6 gliomas, whereas VEGFR2 was more homogenous or evenly distributed in RG2 gliomas. RG2 gliomas have less VEGFR2 within tumor periphery and peri-necrotic (p<0.05) regions, but more VEGFR2 within tumor interior regions (p<0.01), compared to C6 gliomas. mMRI results were confirmed with fluorescence staining and mean fluorescence intensity (MFI) quantification of the anti-VEGFR2 probe in excised glioma and brain tissues, as well as detection of VEGFR2 in C6 and RG2 gliomas and corresponding contalateral brain tissues. Ex vivo VEGFR2 levels were found to be significantly higher in C6 gliomas compared to RG2 tumors (p<0.001), which corresponded with in vivo detection using the VEGFR2 probe. Immunohistochemistry staining for HIF-1α (hypoxia inducible factor 1α), which is associated with angiogenesis, indicated higher levels in RG2 (p<0.01) compared to C6 gliomas. The data suggests that C6 gliomas have angiogenesis which is associated more with large blood vessels in tumor periphery and peri-necrotic regions, and less microvascular angiogenesis within the tumor interior, compared to RG2 gliomas.
Molecular magnetic resonance imaging (mMRI); vascular endothelial growth factor receptor 2 (VEGFR2); C6 and RG2 rat gliomas; in vivo; fluorescence imaging
The lung is the site of entry for Bacillus anthracis in inhalation anthrax, the deadliest form of the disease. Bacillus anthracis produces virulence toxins required for disease. Alveolar macrophages were considered the primary target of the Bacillus anthracis virulence factor lethal toxin because lethal toxin inhibits mouse macrophages through cleavage of MEK signaling pathway components, but we have reported that human alveolar macrophages are not a target of lethal toxin. Our current results suggest that, unlike human alveolar macrophages, the cells lining the respiratory units of the lung, alveolar epithelial cells, are a target of lethal toxin in humans. Alveolar epithelial cells expressed lethal toxin receptor protein, bound the protective antigen component of lethal toxin, and were subject to lethal-toxin-induced cleavage of multiple MEKs. These findings suggest that human alveolar epithelial cells are a target of Bacillus anthracis lethal toxin. Further, no reduction in alveolar epithelial cell viability was observed, but lethal toxin caused actin rearrangement and impaired desmosome formation, consistent with impaired barrier function as well as reduced surfactant production. Therefore, by compromising epithelial barrier function, lethal toxin may play a role in the pathogenesis of inhalation anthrax by facilitating the dissemination of Bacillus anthracis from the lung in early disease and promoting edema in late stages of the illness.
We review our baboon models of E. coli sepsis that mimic, respectively, the shock/disseminated intravascular coagulation (DIC) and organ failure variants of severe sepsis, and analyze the pathophysiologic processes that are unique to each. The multistage, multifactorial characteristics of severe sepsis develop as a result of the initial insult, which -depending on its intensity- activates components of the intravascular compartment leading to overwhelming shock/DIC; or initiates a sequence of events involving both the intra- and extravascular (tissues) compartments that lead to organ failure. In the latter case, the disorder passes through two stages: an initial inflammatory/coagulopathic intravascular first stage triggered by E. coli, followed by an extravascular second stage, involving components unique to each organ and triggered by ischemia/reperfusion (oxidative stress and histone release).
Though a myriad of overlapping cellular and molecular components are involved, it is the context in which these components are brought into play that determine whether shock/DIC or organ failure predominate. For example, inflammatory and thrombotic responses amplified by thrombin in the first case while similar responses are amplified by complement activation products in the second. Rather than blocking specific mediators, we found that attenuation of the thrombin and complement amplification pathways can effectively reverse the shock/DIC and organ failure exhibited by the LD100 and LD50
E. coli models of severe sepsis, respectively. Translation of these concepts to successful intervention in the respective baboon models of E. coli sepsis and the application to their clinical counterparts is described.
sepsis; E. coli; staging; animal model; multiple organ failure; complement
Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.
Infection frequently elicits a coagulation response. Endotoxin triggers the formation of tissue factor initiating coagulation, down regulates anticoagulant mechanisms including the protein C pathway and heparin-like proteoglycans and up regulates plasminogen activator inhibitor. The overall physiological result of this is to promote coagulation through enhancing initiation, suppressing negative regulation and impairing fibrin removal. The response to infection also leads to tissue destruction. Nucleosomes and histones released from the injured cells trigger further inflammation, protection from the pathogen but further tissue injury leading to multi-organ failure. Such a complex response to infection presumably arises due to the role of coagulation in the control and clearance of the infectious agent.
Histones; innate immunity; natural anticoagulants; nucleosomes; protein C; thrombosis
Angiogenesis is essential to tumor progression and a precise evaluation of angiogenesis is important for tumor early diagnosis and treatment. The quantitative and dynamic in vivo assessment of tumor angiogenesis can be achieved by molecular magnetic resonance imaging (mMRI). Vascular endothelial growth factor (VEGF) and VEGF receptors (VEGFRs) are the main regulatory system in angiogenesis and have been used as hot targets for radionuclide-based molecular imaging. However, little research has been accomplished in targeting VEGF/VEGFRs by mMRI. In our study, we aimed to assess the expression of VEGFR2 in C6 gliomas by using a specific molecular probe with mMRI. The differential uptake of the probe conjugated to anti-VEGFR2 monoclonal antibody, shown by varied increases in T1 signal intensity during a two-hour period, demonstrated the heterogeneous expression of VEGFR2 in different tumor regions. Microscopic fluorescence imaging, obtained for the biotin group in the probe with streptavidin-Cy3, along with staining for cellular VEGFR2 levels, laminin and CD45, confirmed the differential distribution of the probe which targeted VEGFR2 on endothelial cells. The angiogenesis process was also assessed using MR angiography (MRA), which quantified tumor blood volume and provided a macroscopic view and a dynamic change of the correlation between tumor vasculature and VEGFR2 expression. Together these results suggest mMRI can be very useful in assessing and characterizing the expression of specific angiogenic markers in vivo and help evaluate angiogenesis associated with tumor progression.
VEGFR2; molecular MRI (mMRI); biotin-Gd-DTPA-albumin-anti-VEGFR2 probe; angiogenesis; C6 rat glioma
Basal and inducible expression of human P-selectin in transgenic mice differs from that of murine P-selectin, resulting in distinct functions.
Leukocytes roll on P-selectin after its mobilization from secretory granules to the surfaces of platelets and endothelial cells. Tumor necrosis factor (TNF), IL-1β, and lipopolysaccharide increase synthesis of P-selectin in murine but not in human endothelial cells. To explore the physiological significance of this difference in gene regulation, we made transgenic mice bearing the human Selp gene and crossed them with mice lacking murine P-selectin (Selp−/−). The transgenic mice constitutively expressed human P-selectin in platelets, endothelial cells, and macrophages. P-selectin mediated comparable neutrophil migration into the inflamed peritoneum of transgenic and wild-type (WT) mice. Leukocytes rolled similarly on human or murine P-selectin on activated murine platelets and in venules of the cremaster muscle subjected to trauma. However, TNF increased murine P-selectin in venules, slowing rolling and increasing adhesion, whereas it decreased human P-selectin, accelerating rolling and decreasing adhesion. Both P- and E-selectin mediated basal rolling in the skin of WT mice, but E-selectin dominated rolling in transgenic mice. During contact hypersensitivity, murine P-selectin messenger (m) RNA was up-regulated and P-selectin was essential for leukocyte recruitment. However, human P-selectin mRNA was down-regulated and P-selectin contributed much less to leukocyte recruitment. These findings reveal functionally significant differences in basal and inducible expression of human and murine P-selectin in vivo.
Cell exposed tissue factor (TF) is generally in a low procoagulant (“cryptic”) state, and requires an activation step (decryption) to exhibit its full procoagulant potential. Recent data suggest that TF decryption may be regulated by the redox environment through the oxidoreductase activity of protein disulfide isomerase (PDI). In this article we review PDI contribution to different models of TF decryption, namely the disulfide switch model and the phosphatidylserine dynamics, and hypothesize on PDI contribution to TF self-association and association with lipid domains. Experimental evidence debate the disulfide switch model of TF decryption and its regulation by PDI. More recently we showed that PDI oxidoreductase activity regulates the phosphatidylserine equilibrium at the plasma membrane. Interestingly, PDI reductase activity could maintain TF in the reduced monomeric form, while also maintaining low exposure of PS, both states correlated with low procoagulant function. In contrast, PDI inhibition or oxidants may promote the adverse effects with a net increase in coagulation. The relative contribution of disulfide isomerization and PS exposure needs to be further analyzed to understand the redox control of TF procoagulant function. For the moment however TF regulation remains cryptic.
tissue factor; decryption; protein disulfide isomerase; phosphatidylserine
The levels of Met, a tyrosine kinase receptor for the hepatocyte growth factor or scatter factor, are elevated during tissue regeneration, and can be used to assess tissue regeneration associated with engineered tissue grafts. This study involved the development and assessment of a novel magnetic resonance imaging (MRI) molecular probe for the in vivo detection of Met in an experimental rodent (rat) model of disease (C6 glioma). The implication of using these probes in tissue engineering is discussed. The molecular targeting agent we used in our study incorporated a magnetite-based dextran-coated nanoparticle backbone covalently bound to an anti-Met antibody. We used molecular MRI with an anti-Met probe to detect in vivo Met levels as a molecular marker for gliomas. Tumor regions were compared to normal tissue, and found to significantly (p < 0.05) decrease MR signal intensity and T2 relaxation in tumors. Nonimmune nonspecific normal rat IgG coupled to the dextran-coated nanoparticles was used as a control. Met levels in tumor tissues were confirmed in Western blots. Based on our results, in vivo evaluation of tissue regeneration using molecular MRI is possible in tissue engineering applications.
In tissue engineering it is often necessary to assess angiogenesis associated with engineered tissue grafts. The levels of vascular endothelial growth factor receptor 2 (VEGF-R2) is elevated during angiogenesis. The goal of this study was to develop and assess a novel magnetic resonance imaging (MRI) molecular probe for the in vivo detection of VEGF-R2 in an experimental rodent model of disease. The possible use of the probe in tissue engineering applications is discussed. The molecular targeting agent we used in our study incorporated a magnetite-based dextran-coated nanoparticle backbone covalently bound to an anti-VEGF-R2 antibody. We used molecular MRI with an anti-VEGF-R2 probe to detect in vivo VEGF-R2 levels as a molecular marker for gliomas (primary brain tumors). Tumor regions were compared with normal tissue. Nonimmune nonspecific normal rat immunoglobulin G coupled to the dextran-coated nanoparticles was used as a control. Prussian blue staining for iron-based nanoprobes was used to confirm the specificity of the probe for VEGF-R2 in glioma tissue. VEGF-R2 levels in tumor tissues were also confirmed in western blots and via immunohistochemistry. Based on our results, in vivo evaluation of tissue angiogenesis using molecular MRI is possible in tissue engineering applications.
Hyper–inflammatory responses can lead to a variety of diseases including sepsis1. We now report that extracellular histones released in response to inflammatory challenge contribute to endothelial dysfunction, organ failure and death during sepsis. They can be targeted pharmacologically by antibody to histone or by activated protein C (APC). Antibody to histone reduced the mortality of mice in lipopolysaccharide (LPS), tumor necrosis factor (TNF) or cecal ligation and puncture models of sepsis. Extracellular histones are cytotoxic toward endothelium in vitro and are lethal in mice. In vivo, histone administration resulted in neutrophil margination, vacuolated endothelium, intra–alveolar hemorrhage and macro and microvascular thrombosis. Histone was detected in the circulation of baboons challenged with E. coli and the increase in histone levels accompanied the onset of renal dysfunction. APC cleaves histones and reduces their cytotoxicity. Co–infusion of APC with E. coli in baboons or histones in mice prevented lethality. Blockade of protein C activation exacerbated sublethal LPS challenge into lethality which was reversed by antibody to histone. We conclude that extracellular histones are potential molecular targets for therapeutics for sepsis and other inflammatory diseases.
Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1–derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn–/– mice). EHC T-syn–/– mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn–/– mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn–/– lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn–/– defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn–/– mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn–/– pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.
Apoptosis of vascular cells is considered to be a major determinant of atherosclerotic plaque vulnerability and potential rupture. Plasmin can be generated in atherosclerotic plaques and recent in vitro data suggest that plasminogen activation may trigger vascular smooth muscle cell (VSMC) apoptosis.
To determine whether plasminogen activation may induce aortic VSMC apoptosis ex vivo and in vivo.
Methods and results
Mice with single or combined deficiencies of ApoE and PAI-1 were used. Ex vivo incubation of plasminogen (1.3 μM) with isolated aortic tunica media from PAI-1-deficient mice induced plasminogen activation and VSMC apoptosis, which was inhibited by α2-antiplasmin. In vivo, levels of plasmin, active caspase 3 and VSMC apoptotic index were significantly higher in atherosclerotic aortas from mice with combined ApoE−/− and PAI-1−/− deficiencies than in those from littermates with single ApoE deficiency. A parallel decrease in VSMC density was also observed.
These data strongly suggest that, in vivo, plasminogen activation may contribute to VSMC apoptosis in atherosclerotic plaques.
Animals; Antiplasmin; pharmacology; Aorta; drug effects; metabolism; pathology; Apolipoproteins E; genetics; Apoptosis; Atherosclerosis; metabolism; pathology; Disease Models; Animal; Mice; Mice; Inbred C57BL; Mice; Knockout; Muscle; Smooth; Vascular; drug effects; metabolism; pathology; Plasmin; metabolism; Plasminogen; metabolism; Plasminogen Activator Inhibitor 1; genetics; Tunica Media; drug effects; metabolism; pathology; apoptosis; atherosclerosis; genetically altered mice; plasminogen; vascular smooth muscle cell
We recently reported that junctional adhesion molecule (JAM)-C plays a role in leukocyte transendothelial migration. Here, the role of JAM-C in vascular permeability was investigated in vitro and in vivo. As opposed to macrovascular endothelial cells that constitutively expressed JAM-C in cell–cell contacts, in quiescent microvascular endothelial cells, JAM-C localized mainly intracellularly, and was recruited to junctions upon short-term stimulation with vascular endothelial growth factor (VEGF) or histamine. Strikingly, disruption of JAM-C function decreased basal permeability and prevented the VEGF- and histamine-induced increases in human dermal microvascular endothelial cell permeability in vitro and skin permeability in mice. Permeability increases are essential in angiogenesis, and JAM-C blockade reduced hyperpermeability and neovascularization in hypoxia-induced retinal angiogenesis in mice. The underlying mechanisms of the JAM-C–mediated increase in endothelial permeability were studied. JAM-C was essential for the regulation of endothelial actomyosin, as revealed by decreased F-actin, reduced myosin light chain phosphorylation, and actin stress fiber formation due to JAM-C knockdown. Moreover, the loss of JAM-C expression resulted in stabilization of VE-cadherin–mediated interendothelial adhesion in a manner dependent on the small GTPase Rap1. Together, through modulation of endothelial contractility and VE-cadherin–mediated adhesion, JAM-C helps to regulate vascular permeability and pathologic angiogenesis.
Bacterial invasion during sepsis induces disregulated systemic responses that could lead to fatal lung failure. The purpose of this study was to relate the temporal dynamics of gene expression to the pathophysiological changes in the lung during the first and second stages of E. coli sepsis in baboons.
Using human oligonucleotide microarrays, we have explored the temporal changes of gene expression in the lung of baboons challenged with sublethal doses of E. coli. Temporal expression pattern and biological significance of the differentially expressed genes were explored using clustering and pathway analysis software. Expression of selected genes was validated by real-time PCR. Cytokine levels in tissue and plasma were assayed by multiplex ELISA. Changes in lung ultrastructure were visualized by electron microscopy. We found that genes involved in primary inflammation, innate immune response, and apoptosis peaked at 2 hrs. Inflammatory and immune response genes that function in the stimulation of monocytes, natural killer and T-cells, and in the modulation of cell adhesion peaked at 8 hrs, while genes involved in wound healing and functional recovery were upregulated at 24 hrs.
The analysis of gene expression modulation in response to sepsis provides the baseline information that is crucial for the understanding of the pathophysiology of systemic inflammation and may facilitate the development of future approaches for sepsis therapy.
The core 1 β1-3-galactosyltransferase (T-synthase) transfers Gal from UDP-Gal to GalNAcα1-Ser/Thr (Tn antigen) to form the core 1 O-glycan Galβ1-3GalNAcα1-Ser/Thr (T antigen). The T antigen is a precursor for extended and branched O-glycans of largely unknown function. We found that wild-type mice expressed the NeuAcα2-3Galβ1-3GalNAcα1-Ser/Thr primarily in endothelial, hematopoietic, and epithelial cells during development. Gene-targeted mice lacking T-synthase instead expressed the nonsialylated Tn antigen in these cells and developed brain hemorrhage that was uniformly fatal by embryonic day 14. T-synthase–deficient brains formed a chaotic microvascular network with distorted capillary lumens and defective association of endothelial cells with pericytes and extracellular matrix. These data reveal an unexpected requirement for core 1–derived O-glycans during angiogenesis.
T-synthase; endothelial cell; galactosyltransferase; mucin; development
It has been proposed that the urokinase receptor (u-PAR) is essential for the various biological roles of urokinase-type plasminogen activator (u-PA) in vivo, and that smooth muscle cells require u-PA for migration during arterial neointima formation. The present study was undertaken to evaluate the role of u-PAR during this process in mice with targeted disruption of the u-PAR gene (u-PAR−/−). Surprisingly, u-PAR deficiency did not affect arterial neointima formation, neointimal cell accumulation, or migration of smooth muscle cells. Indeed, topographic analysis of arterial wound healing after electric injury revealed that u-PAR−/− smooth muscle cells, originating from the uninjured borders, migrated over a similar distance and at a similar rate into the necrotic center of the wound as wild-type (u-PAR+/+) smooth muscle cells. In addition, u-PAR deficiency did not impair migration of wounded cultured smooth muscle cells in vitro. There were no genotypic differences in reendothelialization of the vascular wound. The minimal role of u-PAR in smooth muscle cell migration was not because of absent expression, since wild-type smooth muscle cells expressed u-PAR mRNA and functional receptor in vitro and in vivo. Pericellular plasmin proteolysis, evaluated by degradation of 125I-labeled fibrin and activation of zymogen matrix metalloproteinases, was similar for u-PAR−/− and u-PAR+/+ cells. Immunoelectron microscopy of injured arteries in vivo revealed that u-PA was bound on the cell surface of u-PAR+/+ cells, whereas it was present in the pericellular space around u-PAR−/− cells. Taken together, these results suggest that binding of u-PA to u-PAR is not required to provide sufficient pericellular u-PA–mediated plasmin proteolysis to allow cellular migration into a vascular wound.