October 2009
Volume 50, Issue 10
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Retinal Cell Biology  |   October 2009
Plasminogen Activator Inhibitor-1 (PAI-1) Facilitates Retinal Angiogenesis in a Model of Oxygen-Induced Retinopathy
Author Affiliations
  • Anupam Basu
    From the Department of Cell Biology and Physiology and the
  • Gina Menicucci
    From the Department of Cell Biology and Physiology and the
  • Joann Maestas
    From the Department of Cell Biology and Physiology and the
  • Arup Das
    Division of Ophthalmology, Department of Surgery, University of New Mexico School of Medicine, Albuquerque, New Mexico; and
    New Mexico Veterans Administration Health Care System, Albuquerque, New Mexico.
  • Paul McGuire
    From the Department of Cell Biology and Physiology and the
Investigative Ophthalmology & Visual Science October 2009, Vol.50, 4974-4981. doi:10.1167/iovs.09-3619
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      Anupam Basu, Gina Menicucci, Joann Maestas, Arup Das, Paul McGuire; Plasminogen Activator Inhibitor-1 (PAI-1) Facilitates Retinal Angiogenesis in a Model of Oxygen-Induced Retinopathy. Invest. Ophthalmol. Vis. Sci. 2009;50(10):4974-4981. doi: 10.1167/iovs.09-3619.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

purpose. Angiogenesis, or the formation of new retinal blood vessels, is a key feature of many proliferative retinal diseases including diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. The aim of the present study was to investigate the role of the serine proteinase inhibitor plasminogen activator inhibitor -1 (PAI-1) in facilitating retinal angiogenesis.

methods. The temporal expression of PAI-1 was examined by real-time PCR, Western blot analysis, and immunohistochemistry in retinal tissues from mice with oxygen-induced retinopathy. The requirement for PAI-1 in facilitating the retinal angiogenic response in this model was examined by quantitating the angiogenic response with wild-type and PAI-1 null mice. The mechanism by which PAI-1 mediates angiogenesis was further investigated with isolated human retinal vascular endothelial cells.

results. PAI-1 expression was upregulated in the retinas of mice with oxygen-induced retinopathy, which coincided with a significant increase in the expression of vitronectin in the retina of the experimental mice. There was significant reduction in the angiogenic response of PAI-1−/− mice compared with wild-type mice. PAI-1 promotes endothelial cell migration in vitro and facilitates the migration of cells on a vitronectin substrate by regulating αv integrin cell surface expression.

conclusions. These observations suggest a role for PAI-1 during retinal angiogenesis and point to a potential new therapeutic target in the prevention or treatment of retinal neovascularization seen in many ocular diseases.

Ocular angiogenesis, in response to tissue ischemia, is a leading cause of vision loss in numerous clinical conditions such as diabetic retinopathy, retinal vein occlusion, and retinopathy of prematurity. 1 Angiogenesis is a process in which new blood vessels are formed by the sprouting of endothelial cells from preexisting vessels. The process is multistep and involves the activation of endothelial cells, the breakdown of the capillary basal lamina, the migration and proliferation of endothelial cells, and the maturation of new capillary tubes. 2 3 Recent work suggests that in some circumstances the formation of new retinal vessels may also involve bone marrow–derived progenitor cells that contribute to the process. 4 5 6 7  
The activation of capillary endothelial cells is brought about by the activity of specific growth factors expressed in the retina in response to ischemic or hypoxic conditions. These growth factors include angiopoietin, VEGF, hepatocyte growth factor, and others. 8 9 10 Endothelial activation involves the upregulation of specific proteinases that mediate extracellular matrix remodeling and cell migration. We have previously reported increased levels of the serine proteinase urokinase plasminogen activator (uPA) in human diabetic neovascular membranes and in the retinas of mice with oxygen-induced retinopathy. 11 12  
Urokinase binds to a receptor (uPAR) on the cell surface, where it can efficiently convert plasminogen to plasmin. Through the activation of plasminogen, uPA is also capable of initiating a proteolytic cascade resulting in the activation of members of the matrix metalloproteinase (MMP) family that together with plasmin degrade components of the extracellular matrix, activate and release growth factors stored in the extracellular matrix, and modify the presentation of proteins at the cell surface. The interaction of uPA with uPAR results in conformational changes within the uPAR protein, leading to interactions of uPAR with other cell surface proteins. These interactions can lead to changes in gene expression, signaling events, and cell-matrix adhesion. 13 Inhibition of urokinase action or loss of its cell surface receptor uPAR can result in significant decreases in pathologic retinal angiogenesis. 12  
The activity of uPA and the surface expression of the uPA/uPAR complex is tightly regulated by plasminogen activator inhibitor type 1 (PAI-1). 14 15 PAI-1 is a member of the serine proteinase inhibitor (serpin) superfamily and is thought to play a role in the regulation of angiogenesis. 16 17 18 19 20 21 22 23 PAI-1 is a 50-kDa glycoprotein found in low concentrations in the blood and is expressed in response to cytokines and growth factors. The stability and half-life of PAI-1 is increased by interaction with and binding to the extracellular matrix protein vitronectin. 24 The aim of the present study was to investigate the role of the serine proteinase inhibitor PAI-1 in facilitating new retinal vessel formation in a model of oxygen-induced retinopathy. 
Materials and Methods
Animal Models
Wild-type C57BL/6J and genetically modified PAI-1−/− mice were bred at the University of New Mexico Animal Research Facility. The homozygous PAI-1−/− mice (B6.129S2-Serpine1 tm1Mlg /J) were originally obtained from The Jackson Laboratory (Bar Harbor, ME). All experiments were consistent with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Oxygen-Induced Retinopathy
Retinal angiogenesis was induced in wild-type and PAI-1−/− mice with use of the oxygen-induced retinopathy (OIR) model described by Smith et al. 25 Briefly, 7-day-old pups were placed with their nursing mothers in a chamber (ProOx model 110; BioSpherix, Redfield, NY) at 75% O2 until postnatal day (P) (P12). Mice were then placed in room air until P17, during which time angiogenesis occurred at the junction of the vascularized and nonvascularized regions of the retina. By P17, retinal neovascularization was achieved in 100% of the experimental mice. Age-matched mice, housed only in room air, served as controls. 
Quantitation of Vaso-obliteration and Retinal Neovascularization
To characterize the response of wild-type and PAI-1 deficient mice to the OIR protocol, endothelial cells were stained in retinal whole mounts from untreated P7 and oxygen-treated P12, P15, and P17 animals. The eyes were fixed for 2 hours with 2% paraformaldehyde in PBS, and the retinas were removed. The retinas were incubated in cold 70% ethanol followed by 1% triton X-100 and finally incubated overnight in PBS containing 5 μg/mL FITC-labeled isolectin (GSA-IB4; Invitrogen, Carlsbad, CA). The density of vessels in the retina as a percentage of total retinal area was quantitated from stained whole mounts using analytical software (MetaMorph; MDS Analytical Technologies, Concord, ON, Canada). For quantitation of retinal angiogenesis, eyes from control and experimental animals were fixed overnight in 10% neutral-buffered formalin and embedded in paraffin. Serial sections (6 μm) parallel to the optic nerve were mounted on gelatin-coated slides. After deparaffinization, sections were treated with 0.0125% trypsin at 37°C for 10 minutes. Sections were blocked with 10% normal goat serum and were stained for type IV collagen (Rockland Inc., Gilbertsville, PA) to label the vessels. The sections were mounted with medium (Vectashield; Vector Laboratories, Burlingame, CA) containing diamidinophenylindole (DAPI) to stain the nuclei. Nuclei associated with type IV collagen–positive vessels on the vitreous side of the inner limiting membrane of the retina were counted in every third section with the use of an inverted fluorescence microscope (Zeiss, Thornwood, NY). 
Cell Culture
Human retinal microvascular endothelial cells (ACBRI-181) were obtained from Cell Systems (Kirkland, WA) and were grown on fibronectin-coated cell culture dishes in MCDB-131 supplemented with 10% FBS, 10 ng/mL epidermal growth factor, 1 μg/mL hydrocortisone, 0.2 mg/mL medium (EndoGro; Millipore, Billerica, MA), and 0.09 mg/mL heparin (VEC Technologies, Rensselaer, NY). 
Quantitative Real-Time PCR
Retinas were collected from the oxygen-treated and room air control mice on P12, P15, and P17. Total mRNA was extracted using reagent (Trizol; Invitrogen), and first-strand cDNA was synthesized with 2 μg total RNA (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems, Foster City, CA). The following assays (TaqMan; Applied Biosystems) were used for real-time RT-PCR: PAI-1, Mm00435860 and 18s RNA Hs99999901. Amplification was performed using an Applied Biosystems (7500 Fast) system. Data were derived using the comparative Ct method for duplicate reactions. 26  
Western Blot Analysis
Retinal extracts were analyzed for PAI-1 and vitronectin proteins by Western blot analysis. Retinas were collected from control and oxygen-treated mice on P15 and P17 and were extracted with 1 × SDS PAGE sample buffer in a boiling water bath, and total protein was quantitated using the protein assay method (Micro BCA; Pierce, Rockford, IL). Equal amounts of total protein were separated on a 10% gel and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% milk in 1 × TBST and were incubated with anti–PAI-1 (BD Biosciences, San Jose, CA) or anti–vitronectin (AbCam, Cambridge, MA) antibody at 4°C overnight. After washing, the membranes were incubated with HRP-conjugated secondary antibody and were developed with enhanced chemiluminescence reagents. 
Immunostaining
Eyes from experimental and control mice were collected on P15 and P17 and were fixed overnight with fixative (HistoChoice; Electron Microscopy Sciences, Hatfield, PA) for frozen sections or 4% paraformaldehyde for paraffin embedding. The sections were blocked with 10% normal goat serum for 30 minutes and were incubated with anti–vitronectin antibody. Sections were washed with 1 × TBST and incubated with a fluorescence-labeled secondary antibody for 30 minutes. After a final wash, the sections were coverslipped with mounting medium (Vectashield; Vector Laboratories) and examined using fluorescence microscopy. For whole mount staining of neovascular tufts, eyes from P17 control and experimental animals were removed and fixed with 2% paraformaldehyde in PBS for 2 hours at 4°C. The cornea was cut approximately 2 mm anterior to the limbus and removed. The lens was removed from the eye, and any remaining hyaloid vessels were teased from the globe. The resultant eyecups were incubated for 30 minutes at room temperature with 5 mg/mL isolectin (GS-IB4; Invitrogen) conjugated to Alexa Fluor 488 (Invitrogen) and anti–PAI-1 antibody (AbCam). The eyes were washed extensively with PBS and incubated with an Alexa Fluor 568–labeled secondary antibody (Invitrogen) and were washed again. The retina was removed, flatmounted on glass slides, and imaged. 
Cell Migration Assay
Cell migration assays were performed using cell culture inserts (Falcon; BD Biosciences, San Jose, CA) containing a membrane with 8.0-μm pores. Human retinal microvascular endothelial cells (2 × 104 cells) were plated onto membranes coated with vitronectin or type I collagen in serum-free MCDB-131 media. The lower chamber contained serum-free medium with or without 40 ng/mL VEGF added as a chemoattractant. Some wells contained anti–PAI-1 antibody (BD Biosciences) at 6.25 μg/mL, 12.5 μg/mL, or 37.5 μg/mL. The cells were allowed to migrate for 18 hours, and the cells on the upper surface of the membrane were removed with a cotton swab. The cells that migrated to the lower chamber and attached to the lower surface of the membrane were fixed with 100% methanol for 5 minutes and rinsed with PBS. The membranes were excised and mounted on glass slides with mounting medium (Vectashield; Vector Laboratories) containing DAPI to stain the cell nucleus. The number of migrated cells was determined at 10 × magnification in four peripheral fields and one central field. The number of migrated cells was averaged from five fields per membrane. 
Flow Cytometry
Human retinal microvascular endothelial cells were plated at a subconfluent density (approximately 70%) onto vitronectin-coated substrates and grown in liquid medium (MCDB-131; Gibco, Grand Island, NY) in the presence of VEGF (40 ng/mL). Anti–PAI-1 antibody (12.5 μg/mL) or normal mouse IgG was added for 16 hours, and the cells were processed for immunostaining and flow cytometry. The cells were detached from the culture plate using EDTA for 30 minutes at 4°C and incubated with anti–αv/CD51 antibody (R&D Systems Inc., Minneapolis, MN) for 30 minutes. Cells were washed and incubated with an Alexa Fluor 568–conjugated secondary antibody for 30 minutes and washed again. After resuspension in 1 × PBS, the cells were analyzed (FACSCalibur system and Cell Quest software; Becton Dickinson, Franklin Lakes, NJ). 
Statistical Analysis
All statistical analysis was performed with commercial software (GraphPad Prism; GraphPad Software, Inc., San Diego, CA). Data were analyzed by Student’s t-test or by one-way ANOVA followed by Bonferroni multiple comparison test. 
Results
PAI-1 in the Retinas of Mice with Oxygen-Induced Retinopathy
The retinas of P7 wild-type mice, at the beginning of high oxygen exposure, have a well-developed vascular bed, as seen in GSA lectin-labeled wholemounts (Fig. 1A) . After 5 days of oxygen exposure, the central portion of the retina has undergone vaso-obliteration (Fig. 1B)that is followed over the next 5 days by the formation of new vessels at the interface of the vascularized and nonvascularized portions of the retina in 100% of the animals. These new vessels grow abnormally crossing the inner limiting membrane and invade the vitreous space. 25  
The level of PAI-1 mRNA was determined by real time RT-PCR during the oxygen-induced retinopathy process in these animals (Fig. 2A) . PAI-1 mRNA levels in P12 experimental retinas were significantly lower than that seen in the control retinas at this age. Because PAI-1 is expressed primarily by endothelial cells, this decrease in mRNA expression might have occurred simply as a result of the significant degree of vaso-obliteration and endothelial cell loss occurring in these animals at this time. Despite the difference in vascular density between control and experimental retinas, the level of PAI-1 mRNA was found to increase significantly in the experimental retinas compared with controls during the subsequent active stages of angiogenesis in this model (P15 and P17). The increased level of PAI-1 mRNA correlated with an increase in the level of protein, as determined by Western blot analysis (Fig. 2B)
PAI-1 protein in the experimental retina was localized by immunostaining and was found specifically associated with the neovascular tufts present on the surface of the retina (Fig. 3) . The PAI-1 staining appeared to be localized primarily to the endothelial cells of the new vessels. 
Vitronectin in Retinas during OIR-Induced Angiogenesis
PAI-1 has been shown to mediate the attachment of cells to vitronectin in the extracellular matrix. 22 A role for PAI-1 in mediating the angiogenic response in the OIR model would be supported if vitronectin were present in the retina, where it could provide a suitable migratory substrate for the newly forming vessels during the angiogenic process. Retinas were examined for vitronectin expression during the preangiogenic and angiogenic phases in the OIR model (P12 and P15, respectively) and were compared with control retinas of the same age. A significant increase in the amount of vitronectin was seen in the retinas of P15 OIR mice compared with retinas at the beginning of the angiogenic process (P12 OIR) and was significantly greater than that seen in the P15 control animals (Fig 4A)
In P15 experimental mice, vitronectin protein was localized by immunostaining to the vitreoretinal interface. Staining was localized to the region of the inner limiting membrane (ILM) and to some portions of the nerve fiber layer adjacent to the ILM (Fig. 4B)
The Loss of PAI-1 and Retinal Neovascularization in Mice with Oxygen-Induced Retinopathy
To determine a role for PAI-1 in retinal angiogenesis, wild-type and PAI-1−/− mice were subjected to the OIR protocol and were analyzed for the extent of retinal neovascularization, as described. 
Responses of mice with genetic loss of PAI-1 to the initial high oxygen treatment were similar to those of the wild-type animals. Retinas of P7 PAI-1−/− mice displayed a dense pattern of retinal vessels that was equally susceptible to vaso-obliteration, as demonstrated in the P12 PAI-1−/− OIR mice in comparison with the wild-type animals (Fig. 5) . The level of uPA expression in the PAI-1−/− retina was also found to be similar to that of the wild-type (data not shown). A role for the elevated levels of PAI-1 in mediating angiogenesis in the retina was confirmed because the genetic loss of PAI-1 resulted in a significant decrease in the extent of retinal angiogenesis (Fig. 6) . Retinas of PAI-1−/− mice had significantly decreased numbers of neovascular tufts on the vitreal side of the inner limiting membrane than did those of wild-type mice (average, 33 vs. 62 nuclei). This result demonstrates a 53% reduction in retinal angiogenesis when PAI-1 is absent. 
Blocking PAI-1 Inhibits the Migration of Retinal Endothelial Cells In Vitro
We next examined whether the inhibition of new vessel formation seen in the PAI-1−/− mice might be attributed to a migration defect in the endothelial cells. The migration of human retinal endothelial cells was examined in vitro, as described. Cells plated onto vitronectin- or collagen-coated migration inserts and stimulated with VEGF showed extensive migration after 18 hours. When a PAI-1–neutralizing antibody was added at the time of plating, the cells showed a significant reduction in the extent of migration, and this effect was dose dependent. In addition, the effect was found to be substrate dependent because the PAI-1 antibody had no effect when cells were migrating on a collagen substrate (Fig. 7)
One potential mechanism by which PAI-1 facilitates cell migration is the regulation of the surface expression of integrins. 27 We used flow cytometry to examine the surface expression of the αv integrin in human retinal endothelial cells growing on a vitronectin substrate in the presence and absence of anti–PAI-1 antibody. Cells exposed to the antibody for 16 hours demonstrated significantly more cell surface αv integrin than did cells in the absence of antibody (Fig. 8) . When the activity of PAI-1 was reduced with the PAI-1 antibody, 20.26% of the cells expressed αv integrin on the cell surface. When cells were grown in the absence of antibody, only 5.37% demonstrated surface αv integrin. These results suggest that PAI-1 activity can regulate the interaction of endothelial cells with a vitronectin substrate by facilitating the turnover of the αv integrin. 
Discussion
This study addresses the role of the proteinase inhibitor PAI-1 in the regulation of new vessel formation in a model of oxygen-induced retinal angiogenesis. The expression of PAI-1 is significantly elevated during the most active phases of new vessel growth in this model and localizes to the newly forming vessels. Animals lacking a functional PAI-1 gene exhibited a 53% reduction in the retinal neovascular response compared with wild-type animals. The level of the permissive migratory substrate vitronectin, to which PAI-1 has been shown to bind, was increased in the inner portion of the retinas of animals undergoing angiogenesis. Vitronectin forms a suitable substrate for the migration of isolated retinal endothelial cells in response to VEGF stimulation, and this migratory activity could be blocked with the use of an anti–PAI-1 antibody. Decreasing PAI-1 function appeared to alter cell migration by retaining higher levels of αv integrin on the cell surface that may lead to increased cell-substrate adhesion. 
PAI-1 is a 50-kDa protein whose primary function is the inhibition of plasminogen activator. The active form of PAI-1, with its short half-life, is generally unstable but can be stabilized by interaction with components of the extracellular matrix, including vitronectin. 24 28 Although PAI-1 binds to vitronectin, it has a significantly higher affinity for the serine proteinase uPA, which can be localized to the cell surface by binding to its receptor, uPAR. 29 30 The binding of PAI-1 to uPA at the cell surface leads to the internalization of the uPA/PAI-1/uPAR complex, followed by the degradation of uPA and PAI-1 and the recycling of uPAR back to the cell surface. 31 32  
We have used the oxygen-induced retinopathy model to examine the role of PAI-1 in retinal neovascularization. In this model, exposure of mice to 75% oxygen results in significant regression of the superficial vascular network in the central portion of the retina. A condition of relative hypoxia ensues, resulting in the upregulation of VEGF and other growth factors, followed by the formation of abnormal preretinal neovascular tufts at the interface of the vascularized and nonvascularized retina. These new vessels are formed by the process of angiogenesis, and their formation can be aided by the infiltration of bone marrow–derived myeloid progenitor cells that differentiate into microglia. 4 The hypoxia that develops in the retinal tissues of this model likely drives the expression of PAI-1 mRNA by the remaining endothelial cells and associated pericytes. In the present study we have demonstrated a significant increase in PAI-1 mRNA in the retinas during the active angiogenic period (P15-P17) and a corresponding increase in PAI-1 protein. The increase in PAI-1 is seen after an initial decrease in expression, most likely caused by the vaso-obliteration and the significant delay in development of the deep vascular plexus that occurs at this time in the model. 4 Other studies have reported that the PAI-1 gene is responsive to hypoxia, and, under these conditions, its activity is regulated by specific transcription factors, including HIF-1α. 33  
Previous studies have examined the expression and function of PAI-1 in diabetic retinopathy and suggest that PAI-1 may be relevant to the development and progression of this disorder. Elevated PAI-1 levels have been reported in the sera of patients with types 1 and 2 diabetes. 34 35 In addition, studies have shown increased expression of PAI-1 in the retinal microvessels of patients with nonproliferative diabetic retinopathy. 36 Transgenic mice overexpressing PAI-1 have significantly thickened basement membrane similar to that seen in diabetic animals and humans. 37  
A study using the rat model of retinal neovascularization found that treatment with high doses of recombinant PAI-1 could inhibit the formation of new vessels. 38 Decreased retinal angiogenesis was also observed in the present study in mice lacking PAI-1, which suggests that an optimal physiological level of PAI-1 is necessary to facilitate angiogenesis in the retina. Excessively high or excessively low levels of PAI-1 would thus lead to inhibition. This hypothesis is further supported in studies of choroidal angiogenesis, in which a dose-dependent modulation of choroidal neovascularization by PAI-1 was observed. 18 19 Mice lacking PAI-1 demonstrate a deficient angiogenic response after choroidal laser photocoagulation. The response could be reversed by injection of recombinant PAI-1 protein or by treatment with adenovirus expressing human PAI-1. If PAI-1 levels were increased above the normal physiological levels found in this model, the degree of neovascularization was again reduced. 
A role for PAI-1 in retinal angiogenesis is strengthened by the demonstrated presence of vitronectin in the region of the experimental retinas in which the new vessels will form. Although the cell types responsible for production of the vitronectin substrate are unknown, because of its location at the vitreoretinal interface, one can speculate that vitronectin may be produced by glial cells or ganglion cells. Astrocytes in the superficial layers of the retina have been shown to form a meshwork and to secrete VEGF that facilitates the outgrowth of retinal vessels during normal development of the retinal vasculature. 38 39 Alternatively, retinal ganglion cells may respond to the hypoxic environment by expression of vitronectin. These cells have been shown to express VEGF in an ROP model after the selective loss of astrocytes in the ischemic retina. 40  
In this study we have demonstrated that isolated retinal vascular endothelial cells rely on PAI-1 to migrate efficiently on a vitronectin substrate in vitro. PAI-1 could function by two independent or complementary mechanisms in this situation. PAI-1 may protect the matrix from excessive degradation by regulating the production of plasmin, thereby maintaining the migratory substrate. 41 In addition, PAI-1 may function by regulating cell-substrate interactions independent of its antiproteolytic activity. PAI-1 has been shown to demonstrate high-affinity binding to vitronectin in a region referred to as the somatomedin B domain. 42 43 Binding of PAI-1 in this region not only stabilizes the active form of PAI-1, it regulates the interaction of uPAR and integrin with vitronectin that would be expected to impact the adhesive and migratory behavior of cells. 44 45 46  
During cell migration, PAI-1 may be important for the normal detachment of cells from the matrix through the internalization and recycling of cell surface receptors. Because the αv integrin interacts with uPAR on the cell surface, the internalization of the uPA/uPAR/PAI-1 complex would be expected to decrease the overall density of αv receptors on the cell surface and to promote cell detachment and subsequent migration. 47 In this study we have demonstrated that a PAI-1 blocking antibody decreased the migration of retinal endothelial cells on vitronectin and increased the amount of αv integrin on the cell surface. These data would support the hypothesis that inhibition or loss of PAI-1 would decrease overall retinal angiogenesis by increasing cell adhesion to a vitronectin-containing substrate, and they identify PAI-1 as a potential therapeutic target for retinal neovascularization. 
 
Figure 1.
 
Vascular density of retinal whole mounts from (A) P7 and (B) P12 OIR wild-type C57Bl6 mice. Retinas were fixed and stained with FITC-GSA lectin and photographed. Retinas from P12 mice after 5 days of high oxygen treatment display a significant loss of vessels in the central portion of the retina, which, over time, becomes revascularized by an angiogenic process.
Figure 1.
 
Vascular density of retinal whole mounts from (A) P7 and (B) P12 OIR wild-type C57Bl6 mice. Retinas were fixed and stained with FITC-GSA lectin and photographed. Retinas from P12 mice after 5 days of high oxygen treatment display a significant loss of vessels in the central portion of the retina, which, over time, becomes revascularized by an angiogenic process.
Figure 2.
 
(A) PAI-1 mRNA expression in control and experimental OIR mice normalized to endothelial cell density. The level of PAI-1 mRNA increases significantly in the retinas of OIR mice in comparison with control mice on P15 and P17 after an initial decrease on day 12. Values are mean ± SEM for n = 4 mice in each group. The values have been normalized to the density of endothelial cells by measuring the area of the retina occupied by endothelial cells in FITC-GSA–stained wholemounts. *Significantly different from controls (P12 control vs. P12 OIR, P = 0.0018; P15 control vs. P15 OIR, P = 0.0057; P17 control vs. P17 OIR, P = 0.0103). (B) Representative Western blot of PAI-1 protein in control and OIR retinas from P12 (lanes 1, 4), P15 (lanes 2, 5), and P17 (lanes 3, 6) animals.
Figure 2.
 
(A) PAI-1 mRNA expression in control and experimental OIR mice normalized to endothelial cell density. The level of PAI-1 mRNA increases significantly in the retinas of OIR mice in comparison with control mice on P15 and P17 after an initial decrease on day 12. Values are mean ± SEM for n = 4 mice in each group. The values have been normalized to the density of endothelial cells by measuring the area of the retina occupied by endothelial cells in FITC-GSA–stained wholemounts. *Significantly different from controls (P12 control vs. P12 OIR, P = 0.0018; P15 control vs. P15 OIR, P = 0.0057; P17 control vs. P17 OIR, P = 0.0103). (B) Representative Western blot of PAI-1 protein in control and OIR retinas from P12 (lanes 1, 4), P15 (lanes 2, 5), and P17 (lanes 3, 6) animals.
Figure 3.
 
Representative images of PAI-1 localization in P17 OIR retinal wholemounts. Wholemount double stained with FITC-GSA lectin to identify endothelial cells in neovascular tufts on the surface of the retina (A) and the anti-PAI-1 antibody (B). Overlay demonstrating colocalization of PAI-1 and endothelial cells (C). Scale bar, 33 μm.
Figure 3.
 
Representative images of PAI-1 localization in P17 OIR retinal wholemounts. Wholemount double stained with FITC-GSA lectin to identify endothelial cells in neovascular tufts on the surface of the retina (A) and the anti-PAI-1 antibody (B). Overlay demonstrating colocalization of PAI-1 and endothelial cells (C). Scale bar, 33 μm.
Figure 4.
 
(A) Western blot analysis of vitronectin protein in P12 and P15 wild-type control and OIR retinas. Duplicate samples of P12 and P15 retinal extracts are shown. Relative band intensity indicates a 1.7-fold increase in vitronectin levels from P12 to P15 in the control retina compared with a fourfold increase in the OIR samples. (B) Representative images of vitronectin immunostaining in P15 wild-type OIR retina. Vitronectin is localized to the inner limiting membrane and extends down into the nerve fiber layer (arrows). (C) Representative section from P15 wild-type OIR retina; no primary antibody control. Scale bar, 100 μm.
Figure 4.
 
(A) Western blot analysis of vitronectin protein in P12 and P15 wild-type control and OIR retinas. Duplicate samples of P12 and P15 retinal extracts are shown. Relative band intensity indicates a 1.7-fold increase in vitronectin levels from P12 to P15 in the control retina compared with a fourfold increase in the OIR samples. (B) Representative images of vitronectin immunostaining in P15 wild-type OIR retina. Vitronectin is localized to the inner limiting membrane and extends down into the nerve fiber layer (arrows). (C) Representative section from P15 wild-type OIR retina; no primary antibody control. Scale bar, 100 μm.
Figure 5.
 
Vascular density in retinal wholemounts from (A) P7 and (B) P12 OIR PAI-1−/− mice. Retinas were fixed and stained with FITC-GSA lectin and photographed. Retinas from P12 mice after 5 days of high oxygen treatment display a significant loss of vessels in the central portion of the retina, which, over time, becomes revascularized by an angiogenic process. This response is similar to that seen in the wild-type mice (compare with Fig. 1 ).
Figure 5.
 
Vascular density in retinal wholemounts from (A) P7 and (B) P12 OIR PAI-1−/− mice. Retinas were fixed and stained with FITC-GSA lectin and photographed. Retinas from P12 mice after 5 days of high oxygen treatment display a significant loss of vessels in the central portion of the retina, which, over time, becomes revascularized by an angiogenic process. This response is similar to that seen in the wild-type mice (compare with Fig. 1 ).
Figure 6.
 
Quantitation of retinal neovascularization in P17 wild-type C57Bl6 and PAI-1−/− mice. Neovascularization was quantitated by counting the number of vascular nuclei present on the vitreous side of the inner limiting membrane, as described in Materials and Methods. A 53% reduction in the angiogenic response is seen in PAI-1−/− mice compared with wild-type animals. Data are the mean ± SEM for n = 4 animals per group. *P = 0.0001, significantly less than wild-type animals.
Figure 6.
 
Quantitation of retinal neovascularization in P17 wild-type C57Bl6 and PAI-1−/− mice. Neovascularization was quantitated by counting the number of vascular nuclei present on the vitreous side of the inner limiting membrane, as described in Materials and Methods. A 53% reduction in the angiogenic response is seen in PAI-1−/− mice compared with wild-type animals. Data are the mean ± SEM for n = 4 animals per group. *P = 0.0001, significantly less than wild-type animals.
Figure 7.
 
Role of PAI-1 in the migration of human retinal endothelial cells. Cell migration was examined using a modified Boyden chamber assay. Representative images of migratory cells on either vitronectin-coated (A, B) or type I collagen-coated (C, D) inserts. Cells were stimulated to migrate to the underside of the filter with VEGF in the absence (A, C) or presence (B, D) of anti–PAI-1 antibody. (E) Quantification of cell migration. Values are the mean ± SEM of n = 3 wells for each condition. Differences among means were tested by ANOVA and were corrected with the Bonferroni posttest. *P < 0.05, significant. No antibody versus 6.25 mg/mL anti–PAI-1, P < 0.001; no antibody versus 12.5 mg/mL anti–PAI-1, P < 0.001; no antibody versus 37.5 mg/mL anti–PAI-1, P < 0.001. Scale bar, 100 μm.
Figure 7.
 
Role of PAI-1 in the migration of human retinal endothelial cells. Cell migration was examined using a modified Boyden chamber assay. Representative images of migratory cells on either vitronectin-coated (A, B) or type I collagen-coated (C, D) inserts. Cells were stimulated to migrate to the underside of the filter with VEGF in the absence (A, C) or presence (B, D) of anti–PAI-1 antibody. (E) Quantification of cell migration. Values are the mean ± SEM of n = 3 wells for each condition. Differences among means were tested by ANOVA and were corrected with the Bonferroni posttest. *P < 0.05, significant. No antibody versus 6.25 mg/mL anti–PAI-1, P < 0.001; no antibody versus 12.5 mg/mL anti–PAI-1, P < 0.001; no antibody versus 37.5 mg/mL anti–PAI-1, P < 0.001. Scale bar, 100 μm.
Figure 8.
 
Effect of PAI-1 on the surface localization of αv integrin assessed by flow cytometry. Representative histograms of human retinal endothelial cells grown in the (A) absence or (B) presence of anti–PAI-1 neutralizing antibody and stained for αv integrin. Gating is indicated by horizontal lines, and the percentage of positively stained cells is noted. (C) Cells plus anti–PAI-1 antibody, nonspecific staining (secondary antibody only).
Figure 8.
 
Effect of PAI-1 on the surface localization of αv integrin assessed by flow cytometry. Representative histograms of human retinal endothelial cells grown in the (A) absence or (B) presence of anti–PAI-1 neutralizing antibody and stained for αv integrin. Gating is indicated by horizontal lines, and the percentage of positively stained cells is noted. (C) Cells plus anti–PAI-1 antibody, nonspecific staining (secondary antibody only).
DasA, McGuirePG. Retinal and choroidal angiogenesis: pathophysiology and strategies for inhibition. Prog Retinal Eye Res. 2003;22:721–748. [CrossRef]
CarmelietP, JainRK. Angiogenesis in cancer and other diseases. Nature. 2000;407:249–257. [CrossRef] [PubMed]
CarmelietP. Mechanisms of angiogenesis and arteriogenesis. Nat Med. 2000;6:389–395. [CrossRef] [PubMed]
RitterMR, BaninE, MorenoSK, AguilarE, DorrellMI, FriedlanderM. Myeloid progenitors differentiate into microglia and promote vascular repair in a model of ischemic retinopathy. J Clin Invest. 2006;116:3266–3276. [CrossRef] [PubMed]
FriedlanderM, DorrellMI, RitterMR, et al. Progenitor cells and retinal angiogenesis. Angiogenesis. 2007;10:89–101. [CrossRef] [PubMed]
GrantMB, Stratford MayW, CaballeroS, et al. Adult hematopoietic stem cells provide functional hemangioblast activity during retinal neovascularization. Nat Med. 2002;8:607–612. [CrossRef] [PubMed]
SenguptaN, CaballeroS, MamesRN, et al. The role of adult bone marrow-derived stem cells in choroidal neovascularization. Invest Ophthalmol Vis Sci. 2003;44:4908–4913. [CrossRef] [PubMed]
AielloLP, AveryRL, ArriggPG, et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N Engl J Med. 1994;331:1480–1487. [CrossRef] [PubMed]
DasA, FanslowW, CerrettiD, WarrenE, TalaricoN, McGuirePG. Angiopoietin/Tek interactions regulate MMP-9 expression and retinal neovascularization. Lab Invest. 2003;83:1637–1645. [CrossRef] [PubMed]
ColomboES, MenicucciG, McGuirePG, DasA. Hepatocyte growth factor/scatter factor promotes retinal angiogenesis through increased urokinase expression. Invest Ophthalmol Vis Sci. 2007;48:1793–1800. [CrossRef] [PubMed]
DasA, McGuirePG, EriqatC, et al. Human diabetic neovascular membranes contain high levels of urokinase and metalloproteinase enzymes. Invest Ophthalmol Vis Sci. 1999;40:809–813. [PubMed]
McGuirePG, JonesTR, TalaricoN, WarrenE, DasA. The urokinase/urokinase receptor system in retinal neovascularization: inhibition by Å6 suggests a new therapeutic target. Invest Ophthalmol Vis Sci. 2003;44:2736–2742. [CrossRef] [PubMed]
BinderBR, MihalyJ, PragerGW. uPAR-uPA-PAI-1 interactions and signaling: a vascular biologist’s view. Thromb Haemost. 2007;97:336–342. [PubMed]
HerzJ, ClouthierDE, HammerRE. LDL receptor-related protein internalizes and degrades uPA/PAI-1 complexes and is essential for embryo implantation. Cell. 1992;71:411–421. [CrossRef] [PubMed]
ZhangJC, SakthivelR, KnissD, GrahamCH, StricklandDK, McCraeKR. The low density lipoprotein receptor-related protein/alpha2- macroglobulin receptor regulates cell surface plasminogen activator activity on human trophoblast cells. J Biol Chem. 1998;273:32273–32280. [CrossRef] [PubMed]
BalsaraRD, CastellinoFJ, PloplisVA. A novel function of plasminogen activator inhibitor-1 in modulation of the AKT pathway in wild-type and plasminogen activator inhibitor-1 deficient endothelial cells. J Biol Chem. 2006;281:22527–22536. [CrossRef] [PubMed]
McMahonGA, PetitclercE, StefanssonS, et al. Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis. J Biol Chem. 2001;276:33964–33968. [CrossRef] [PubMed]
LambertV, MunautC, CarmelietP, et al. Dose-dependent modulation of choroidal neovascularization by plasminogen activator inhibitor type I: implications for clinical trials. Invest Ophthalmol Vis Sci. 2003;44:2791–2797. [CrossRef] [PubMed]
LambertV, MunautC, NoëlA, et al. Influence of plasminogen activator inhibitor type 1 on choroidal neovascularization. FASEB J. 2001;15:1021–1027. [CrossRef] [PubMed]
EitzmanDT, KraussJC, ShenT, CuiJ, GinsburgD. Lack of plasminogen activator inhibitor-1 effect in a transgenic mouse model of metastatic melanoma. Blood. 1996;87:4718–4722. [PubMed]
AlmholtK, NielsenBS, FrandsenTL, BrunnerN, DanoK, JohnsenM. Metastasis of transgenic breast cancer in plasminogen activator inhibitor-1 gene-deficient mice. Oncogene. 2003;22:4389–4397. [CrossRef] [PubMed]
StefanssonS, PetitclercE, WongMK, McMahonGA, BrooksPC, LawrenceDA. Inhibition of angiogenesis in vivo by plasminogen activator inhibitor-1. J Biol Chem. 2001;276:8135–8141. [CrossRef] [PubMed]
MaudJ, MaillardC, LecomteJ, et al. Tumoral and choroidal vascularization: differential cellular mechanisms involving plasminogen activator inhibitor type 1. Am J Pathol. 2007;171:1369–1380. [CrossRef] [PubMed]
ZhouA, HuntingtonJA, PannuNS, CarrellRW, ReadRJ. How vitronectin binds PAI-1 to modulate fibrinolysis and cell migration. Nat Struct Biol. 2003;10:541–544. [CrossRef] [PubMed]
SmithLEH, WesolowskiE, McLellanA, et al. Oxygen-induced retinopathy in the mouse. Invest Ophthalmol Vis Sci. 1994;35:101–111. [PubMed]
LivakKJ, SchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method. Methods. 2001;25:402–408. [CrossRef] [PubMed]
CzekayRP, AertgeertsK, CurridenSA, LoskutoffDJ. Plasminogen activator inhibitor-1 detaches cells from extracellular matrices by inactivating integrins. J Cell Biol. 2003;160:781–791. [CrossRef] [PubMed]
HekmanCM, LoskutoffDJ. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem. 1985;260:11581–11587. [PubMed]
NielsenLS, KellermanGM, BehrendtN, PiconeR, DanoK, BlasiF. A 55,000–60,000 Mr receptor protein for urokinase-type plasminogen activator: identification in human tumor cell lines and partial purification. J Biol Chem. 1988;263:2358–2363. [PubMed]
DanielA, Selvi PalaniappanL, StefanssonS, et al. Characterization of the binding of different conformational forms of plasminogen activator inhibitor-1 to vitronectin: implications for the regulation of pericellular proteolysis. J Biol Chem. 1997;272:7676–7680. [CrossRef] [PubMed]
EstreicherA, MulhauserJ, CarpentierJL, OrciL, VassalliJD. The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes. J Cell Biol. 1990;111:783–792. [CrossRef] [PubMed]
NykjaerA, ConeseM, ChristensenEI, et al. Recycling of the urokinase receptor upon internalization of the uPA: serpine complexes. EMBO J. 1997;16:2610–2620. [CrossRef] [PubMed]
LiaoH, HymanMC, LawrenceDA, PinskyDJ. Molecular regulation of the PAI-1 gene by hypoxia: contributions of Egr-1, HIF-1α and C/EBPα. FASEB J. 2007;21:935–949. [CrossRef] [PubMed]
GramJ, JespersenJ. Induction and possible role of fibrinolysis in diabetes mellitus. Semin Thromb Hemost. 1991;17:412–416. [CrossRef] [PubMed]
SmallM, KluftC, MacCuishAC, LoweGD. Tissue plasminogen activation inhibition in diabetes mellitus. Diabetes Care. 1989;12:655–658. [CrossRef] [PubMed]
GrantMB, SpoerriPE, PlayerDW, et al. Plasminogen activator inhibitor (PAI-1) overexpression in retinal microvessels of PAI-1 transgenic mice. Invest Ophthalmol Vis Sci. 2000;41:2296–2302. [PubMed]
GrantMB, EllisEA, CaballeroS, MamesRN. Plasminogen activator inhibitor-1 overexpression in nonproliferative diabetic retinopathy. Exp Eye Res. 1996;63:233–244. [CrossRef] [PubMed]
PennJS, RajaratnamVS. Inhibition of retinal neovascularization by intravetreal injection of human rPAI-1 in a rat model of retinopathy of prematurity. Invest Ophthalmol Vis Sci. 2003;44:5423–5429. [CrossRef] [PubMed]
FruttigerM. Development of the mouse retinal vasculature: angiogenesis vs. vasculogenesis. Invest Ophthalmol Vis Sci. 2002;43:522–527. [PubMed]
StoneJ, Chan-LingT, Pe'erJ, ItinA, GnessinH, KeshetE. Roles of vascular endothelial growth factor and astrocyte degeneration in the genesis of retinopathy of prematurity. Invest Ophthalmol Vis Sci. 1996;37:290–299. [PubMed]
BajouK, MassonV, GerardRD, et al. The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin: implications for antiangiogenic strategies. J Cell Biol. 2001;152:777–784. [CrossRef] [PubMed]
DengG, RoyleG, WangS, CrainK, LoskutoffDJ. Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin. J Biol Chem. 1996;271:12716–12723. [CrossRef] [PubMed]
LijnenHR. Pleiotropic functions of plasminogen activator inhibitor-1. J Thromb Haemost. 2004;3:35–45.
OkumuraY, KamikuboY, CurridenSA, et al. Kinetic analysis of the interaction between vitronectin and the urokinase receptor. J Biol Chem. 2002;277:9395–9404. [CrossRef] [PubMed]
StefanssonS, LawrenceDA. The serpin PAI-1 inhibits cell migration by blocking integrin αvβ3 binding to vitronectin. Nature. 1996;383:441–443. [CrossRef] [PubMed]
PepperMS. Role of the matrix metalloproteinase and plasminogen activator-plasmin systems in angiogenesis. Arterioscler Thromb Vasc Biol. 2001;21:1104–1117. [CrossRef] [PubMed]
CarrieroMV, Del VecchioS, CapozzoliM, et al. Urokinase receptor interacts with αvβ5 vitronectin receptor, promoting urokinase-dependent cell migration in breast cancer. Cancer Res. 1999;59:5307–5314. [PubMed]
Figure 1.
 
Vascular density of retinal whole mounts from (A) P7 and (B) P12 OIR wild-type C57Bl6 mice. Retinas were fixed and stained with FITC-GSA lectin and photographed. Retinas from P12 mice after 5 days of high oxygen treatment display a significant loss of vessels in the central portion of the retina, which, over time, becomes revascularized by an angiogenic process.
Figure 1.
 
Vascular density of retinal whole mounts from (A) P7 and (B) P12 OIR wild-type C57Bl6 mice. Retinas were fixed and stained with FITC-GSA lectin and photographed. Retinas from P12 mice after 5 days of high oxygen treatment display a significant loss of vessels in the central portion of the retina, which, over time, becomes revascularized by an angiogenic process.
Figure 2.
 
(A) PAI-1 mRNA expression in control and experimental OIR mice normalized to endothelial cell density. The level of PAI-1 mRNA increases significantly in the retinas of OIR mice in comparison with control mice on P15 and P17 after an initial decrease on day 12. Values are mean ± SEM for n = 4 mice in each group. The values have been normalized to the density of endothelial cells by measuring the area of the retina occupied by endothelial cells in FITC-GSA–stained wholemounts. *Significantly different from controls (P12 control vs. P12 OIR, P = 0.0018; P15 control vs. P15 OIR, P = 0.0057; P17 control vs. P17 OIR, P = 0.0103). (B) Representative Western blot of PAI-1 protein in control and OIR retinas from P12 (lanes 1, 4), P15 (lanes 2, 5), and P17 (lanes 3, 6) animals.
Figure 2.
 
(A) PAI-1 mRNA expression in control and experimental OIR mice normalized to endothelial cell density. The level of PAI-1 mRNA increases significantly in the retinas of OIR mice in comparison with control mice on P15 and P17 after an initial decrease on day 12. Values are mean ± SEM for n = 4 mice in each group. The values have been normalized to the density of endothelial cells by measuring the area of the retina occupied by endothelial cells in FITC-GSA–stained wholemounts. *Significantly different from controls (P12 control vs. P12 OIR, P = 0.0018; P15 control vs. P15 OIR, P = 0.0057; P17 control vs. P17 OIR, P = 0.0103). (B) Representative Western blot of PAI-1 protein in control and OIR retinas from P12 (lanes 1, 4), P15 (lanes 2, 5), and P17 (lanes 3, 6) animals.
Figure 3.
 
Representative images of PAI-1 localization in P17 OIR retinal wholemounts. Wholemount double stained with FITC-GSA lectin to identify endothelial cells in neovascular tufts on the surface of the retina (A) and the anti-PAI-1 antibody (B). Overlay demonstrating colocalization of PAI-1 and endothelial cells (C). Scale bar, 33 μm.
Figure 3.
 
Representative images of PAI-1 localization in P17 OIR retinal wholemounts. Wholemount double stained with FITC-GSA lectin to identify endothelial cells in neovascular tufts on the surface of the retina (A) and the anti-PAI-1 antibody (B). Overlay demonstrating colocalization of PAI-1 and endothelial cells (C). Scale bar, 33 μm.
Figure 4.
 
(A) Western blot analysis of vitronectin protein in P12 and P15 wild-type control and OIR retinas. Duplicate samples of P12 and P15 retinal extracts are shown. Relative band intensity indicates a 1.7-fold increase in vitronectin levels from P12 to P15 in the control retina compared with a fourfold increase in the OIR samples. (B) Representative images of vitronectin immunostaining in P15 wild-type OIR retina. Vitronectin is localized to the inner limiting membrane and extends down into the nerve fiber layer (arrows). (C) Representative section from P15 wild-type OIR retina; no primary antibody control. Scale bar, 100 μm.
Figure 4.
 
(A) Western blot analysis of vitronectin protein in P12 and P15 wild-type control and OIR retinas. Duplicate samples of P12 and P15 retinal extracts are shown. Relative band intensity indicates a 1.7-fold increase in vitronectin levels from P12 to P15 in the control retina compared with a fourfold increase in the OIR samples. (B) Representative images of vitronectin immunostaining in P15 wild-type OIR retina. Vitronectin is localized to the inner limiting membrane and extends down into the nerve fiber layer (arrows). (C) Representative section from P15 wild-type OIR retina; no primary antibody control. Scale bar, 100 μm.
Figure 5.
 
Vascular density in retinal wholemounts from (A) P7 and (B) P12 OIR PAI-1−/− mice. Retinas were fixed and stained with FITC-GSA lectin and photographed. Retinas from P12 mice after 5 days of high oxygen treatment display a significant loss of vessels in the central portion of the retina, which, over time, becomes revascularized by an angiogenic process. This response is similar to that seen in the wild-type mice (compare with Fig. 1 ).
Figure 5.
 
Vascular density in retinal wholemounts from (A) P7 and (B) P12 OIR PAI-1−/− mice. Retinas were fixed and stained with FITC-GSA lectin and photographed. Retinas from P12 mice after 5 days of high oxygen treatment display a significant loss of vessels in the central portion of the retina, which, over time, becomes revascularized by an angiogenic process. This response is similar to that seen in the wild-type mice (compare with Fig. 1 ).
Figure 6.
 
Quantitation of retinal neovascularization in P17 wild-type C57Bl6 and PAI-1−/− mice. Neovascularization was quantitated by counting the number of vascular nuclei present on the vitreous side of the inner limiting membrane, as described in Materials and Methods. A 53% reduction in the angiogenic response is seen in PAI-1−/− mice compared with wild-type animals. Data are the mean ± SEM for n = 4 animals per group. *P = 0.0001, significantly less than wild-type animals.
Figure 6.
 
Quantitation of retinal neovascularization in P17 wild-type C57Bl6 and PAI-1−/− mice. Neovascularization was quantitated by counting the number of vascular nuclei present on the vitreous side of the inner limiting membrane, as described in Materials and Methods. A 53% reduction in the angiogenic response is seen in PAI-1−/− mice compared with wild-type animals. Data are the mean ± SEM for n = 4 animals per group. *P = 0.0001, significantly less than wild-type animals.
Figure 7.
 
Role of PAI-1 in the migration of human retinal endothelial cells. Cell migration was examined using a modified Boyden chamber assay. Representative images of migratory cells on either vitronectin-coated (A, B) or type I collagen-coated (C, D) inserts. Cells were stimulated to migrate to the underside of the filter with VEGF in the absence (A, C) or presence (B, D) of anti–PAI-1 antibody. (E) Quantification of cell migration. Values are the mean ± SEM of n = 3 wells for each condition. Differences among means were tested by ANOVA and were corrected with the Bonferroni posttest. *P < 0.05, significant. No antibody versus 6.25 mg/mL anti–PAI-1, P < 0.001; no antibody versus 12.5 mg/mL anti–PAI-1, P < 0.001; no antibody versus 37.5 mg/mL anti–PAI-1, P < 0.001. Scale bar, 100 μm.
Figure 7.
 
Role of PAI-1 in the migration of human retinal endothelial cells. Cell migration was examined using a modified Boyden chamber assay. Representative images of migratory cells on either vitronectin-coated (A, B) or type I collagen-coated (C, D) inserts. Cells were stimulated to migrate to the underside of the filter with VEGF in the absence (A, C) or presence (B, D) of anti–PAI-1 antibody. (E) Quantification of cell migration. Values are the mean ± SEM of n = 3 wells for each condition. Differences among means were tested by ANOVA and were corrected with the Bonferroni posttest. *P < 0.05, significant. No antibody versus 6.25 mg/mL anti–PAI-1, P < 0.001; no antibody versus 12.5 mg/mL anti–PAI-1, P < 0.001; no antibody versus 37.5 mg/mL anti–PAI-1, P < 0.001. Scale bar, 100 μm.
Figure 8.
 
Effect of PAI-1 on the surface localization of αv integrin assessed by flow cytometry. Representative histograms of human retinal endothelial cells grown in the (A) absence or (B) presence of anti–PAI-1 neutralizing antibody and stained for αv integrin. Gating is indicated by horizontal lines, and the percentage of positively stained cells is noted. (C) Cells plus anti–PAI-1 antibody, nonspecific staining (secondary antibody only).
Figure 8.
 
Effect of PAI-1 on the surface localization of αv integrin assessed by flow cytometry. Representative histograms of human retinal endothelial cells grown in the (A) absence or (B) presence of anti–PAI-1 neutralizing antibody and stained for αv integrin. Gating is indicated by horizontal lines, and the percentage of positively stained cells is noted. (C) Cells plus anti–PAI-1 antibody, nonspecific staining (secondary antibody only).
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