Abstract-Plaque vascularity has been implicated in its growth and stability. However, there is a paucity of information regarding the origin of plaque vasculature and the role of vasa vasorum in plaque growth. To inhibit growth of vasa vasorum in atherogenic mice and assess its effect on plaque growth, we used a truncated plasminogen activator inhibitor (PAI)-1 protein, rPAI-1 23 , that has significant antiangiogenic activity. Female LDLR Ϫ/Ϫ ApoB-48 -deficient mice fed Paigen's diet without cholate for 20 weeks received rPAI-1 23 treatment (nϭ21) for the last 6 weeks. Plaque size and vasa vasorum density were compared to 2 controls: mice fed Paigen's diet and treated with saline for the last 6 weeks (nϭ16) and mice fed Paigen's diet until the onset of treatment (nϭ14). The rPAI-1 23 treatment significantly reduced plaque area and plaque cholesterol in the descending aorta and plaque area in the innominate artery. Measurements of reconstructed confocal microscopy images of vasa vasorum demonstrate that rPAI-1 23 treatment decreased vasa vasorum area and length, which was supported by microCT images. Confocal images provide evidence for vascularized plaque in the saline-treated group but not in rPAI-1 23 -treated mice. The increased vessel density in saline-treated mice is attributable, in part, to upregulated fibroblast growth factor-2 expression, which is inhibited by rPAI-1 23 . In conclusion, rPAI-1 23 inhibits growth of vasa vasorum, as well as vessels within the adjacent plaque and vessel wall, through inhibition of fibroblast growth factor-2, leading to reduced plaque growth in atherogenic female LDLR Ϫ/Ϫ ApoB-48 -deficient mice.

Interleukin-1 beta (IL-1beta) is a central mediator of inflammation and connective tissue destruction in rheumatoid arthritis. IL-1beta activates articular chondrocytes to produce matrix metalloproteinase-1 (MMP-1), an enzyme capable of dismantling the collagen scaffold of articular cartilage. To define the transcription factors and signaling intermediates that activate MMP-1 transcription in chondrocytes, we performed transient transfection of MMP-1 promoter constructs followed by reporter assays. These studies identified an IL-1beta-responsive region of the human MMP-1 promoter that contains a consensus CCAAT enhancer-binding protein (C/EBP) binding site. Deletion of this site reduced overall transcriptional activity of the MMP-1 promoter, as well as decreased fold induction by IL-1beta. IL-1beta stimulation of chondrocytes increased binding of C/EBP-beta to the MMP-1 C/EBP site. Extracellular signal regulated kinase (ERK) pathway-dependent phosphorylation of C/EBP-beta on threonine 235 activates this transcription factor. Here we show that IL-1beta stimulation of chondrocytes induced phosphorylation of C/EBP-beta on threonine 235, and that the ERK pathway inhibitor PD98059 reduced this phosphorylation. We further show that PD98059 reduces IL-1beta-induced MMP-1 mRNA expression in chondrocytes. Moreover, inhibition of the ERK pathway by expression of dominant-negative forms of ERK1 and ERK2 impaired the ability of IL-1beta to transactivate the MMP-1 promoter. Our findings demonstrate a novel role for C/EBP-beta in IL-1beta-induced connective tissue disease and define a new nuclear target for the ERK pathway in MMP-1 gene activation.

Objective. Systemic sclerosis (SSc; scleroderma) is a systemic connective tissue disease with an extensive vascular component that includes aberrant microvasculature and impaired wound healing. The aim of this study was to investigate the presence of antiangiogenic factors in patients with SSc.Methods. Plasma samples were obtained from 30 patients with SSc and from 10 control patients without SSc. The samples were analyzed for the ability of plasma to affect endothelial cell migration and vascular structure formation and for the presence of antiangiogenic activity.Results. Exposure of normal human microvascular dermal endothelial cells to plasma from patients with SSc resulted in decreased cell migration (mean ؎ SEM 52 ؎ 5%) and tube formation (34 ؎ 6%) compared with that in plasma from control patients (P < 0.001 for both). SSc plasma contained 2.9-fold more plasminogen kringle 1-3 fragments (angiostatin) than that in control plasma. The addition of angiostatin to control plasma resulted in inhibition of endothelial cell migration and proliferation similar to that observed in SSc plasma. In vitro studies demonstrated that granzyme B and other proteases contained in T cell granule content cleave plasminogen and plasmin into angiostatin fragments.Conclusion. Plasminogen conformation in patients with SSc enables granzyme B and granule content protease to limit the proangiogenic effects of plasmin and increase the levels of antiangiogenic angiostatin. This increase in angiostatin production may account for some of the vascular defects observed in patients with SSc.

Rationale The antiangiogenic activity of rPAI-123, a truncated plasminogen activator inhibitor-1 (PAI-1) protein, induces vasa vasorum collapse and significantly reduces plaque area and plaque cholesterol in hypercholesterolemic low-density lipoprotein receptor–deficient/apolipoprotein B48–deficient mice. Objective The objective of this study was to examine rPAI-123–stimulated mechanisms that cause vasa vasorum collapse. Methods and Results The rPAI-123 protein opposed PAI-1 antiproteolytic function by stimulating a 1.6-fold increase in plasmin activity compared with the saline-treated counterpart. The increased proteolytic activity corresponded to increased activity of matrix metalloproteinase-3 and degradation of fibrin(ogen), nidogen, and perlecan in the adventitia of descending aortas. PAI-1 activity was reduced by 48% in response to rPAI-123; however, PAI-1 protein expression levels were similar in the rPAI-123– and saline-treated hypercholesterolemic mice. Coimmunoprecipitation assays demonstrated a novel PAI-1–plasminogen complex in protein from the descending aorta of rPAI-123– and saline-treated mice, but complexed PAI-1 was 1.6-fold greater in rPAI-123–treated mice. Biochemical analyses demonstrated that rPAI-123 and PAI-1 binding interactions with plasminogen increased plasmin activity and reduced PAI-1 antiproteolytic activity. Conclusions We conclude that rPAI-123 causes regression or collapse of adventitial vasa vasorum in hypercholesterolemic mice by stimulating an increase in plasmin activity. The rPAI-123–enhanced plasmin activity was achieved through a novel mechanism by which rPAI-123 and PAI-1 bound plasminogen in a cooperative manner to increase plasmin activity and reduce PAI-1 activity.

Objective Vasa vasorum are angiogenic in advanced stages of human atherosclerosis and hypercholesterolemic mouse models. Fibroblast growth factor-2 (FGF-2) is the predominant angiogenic growth factor in the adventitia and plaque of hypercholesterolemic low-density lipoprotein receptor–deficient/apolipoprotein B100/100 mice (DKO). FGF-2 seems to play a role in the formation of a distinct vasa vasorum network. This study examined the vasa vasorum structure and its relationship to FGF-2. Methods and Results DKO mice treated with saline, antiangiogenic recombinant plasminogen activator inhibitor-123 (rPAI-123), or soluble FGF receptor 1 were perfused with fluorescein-labeled Lycopersicon esculentum lectin. Confocal images of FGF-2–probed descending aorta adventitia show that angiogenic vasa vasorum form a plexus-like network in saline-treated DKO similar to the FGF-2 pattern of distribution. Mice treated with rPAI-123 and soluble FGF receptor 1 lack a plexus; FGF-2 and vasa vasorum density and area are significantly reduced. A perlecan/FGF-2 complex is critical for plexus stability. Excess plasmin produced in rPAI-123-treated DKO mice degrades perlecan and destabilizes the plexus. Plasmin activity and plaque size measured in DKO and DKO/plasminogen activator inhibitor-1−/− mice demonstrate that elevated plasmin activity contributes to reduced plaque size. Conclusion An FGF-2/perlecan complex is required for vasa vasorum plexus stability. Elevated plasmin activity plays a significant inhibitory role in vasa vasorum plexus and plaque development.

Many angiogenesis inhibitors are breakdown products of endogenous extracellular matrix proteins. Plasmin and matrix metalloproteinase-3 generate breakdown products of matrixbound plasminogen activator inhibitor-1 (PAI-1). We produced a truncated form of PAI-1, rPAI-1 23 , that possesses significant anti-angiogenic activity and stimulates high levels of apoptosis in quiescent arterial endothelial cells. Quiescent endothelial cells are less susceptible to apoptosis than angiogenic endothelial cells. The present study was designed to determine the mechanism of the rPAI-1 23 effects in bovine aortic endothelial cells. Apoptosis was measured in annexin V and caspase 3 assays. Expression of death and survival signaling molecules were examined by Western blot and kinase activity. The vascular endothelium is normally maintained in a differentiated, quiescent state. Pro-angiogenic factors destabilize the quiescent endothelium into migratory, proliferative endothelial cells that are attenuated by anti-angiogenic factors. The pro-and anti-angiogenic molecules have cell survival and death functions that are tightly controlled to maintain a balance (1-4).Many negative regulators of angiogenesis are cleavage products of an existing cellular protein that is not inhibitory in its normal intact conformation (5-7). Induction of endothelial cell apoptosis is one characteristic common to these inhibitors (8). Endothelial cells are more susceptible to apoptosis when they are activated (angiogenic) as compared with quiescent (9, 10). Survival of the latter is dependent upon angiogenic growth factors in the local environment, which can be blocked by antiangiogenic factors. If growth factor functions are blocked, endothelial cells are removed by apoptosis (4, 9, 11).Apoptosis can be induced by diverse stimuli that initiate specific signal transduction pathways. The apoptosis pathways are regulated by pro-and anti-apoptotic molecules that are controlled in part by kinases (12) and proteasomal degradation (13). The c-Jun NH 2 -terminal kinase (JNK) 2 signaling pathway is activated by cellular stress, and its role in apoptosis versus survival remains unclear (14). The role of JNK in cellular proliferation, apoptosis, differentiation, and motility is dependent upon the activity and stability of JNK isoforms and their associated substrates (15-18). The cellular function associated with JNK-substrate complexes is tightly regulated by proteasomal activity (19,20).Plasminogen activator inhibitor-1 (PAI-1) has been shown to have both pro-and anti-angiogenic activity (21-24). It has been suggested that PAI-1 pro-angiogenic versus anti-angiogenic activity is based on the relative amounts of the inhibitor that are in active versus inactive conformations (22,25). Proteolytic molecules plasmin (26) and matrix metalloproteinase 3 (27) cleave and inactivate PAI-1. Potential functions of cleaved PAI-1 have not been studied. We made truncated PAI-1 cDNAs and produced truncated PAI-1 proteins (rPAI-1) to investigate potential PAI-1 functions in the absen...

Introduction: Neovascularized areas in atherosclerosis are primarily supplied by angiogenic vasa vasorum. The vasa vasorum density increases during plaque progression, but the role of angiogenesis in plaque growth is uncertain. A truncated PAI‐1 protein, rPAI‐123, has significant anti‐angiogenic activity. We hypothesized that rPAI‐123 inhibition of angiogenic vasa vasorum in atherogenic mice would reduce plaque progression. Methods: Female LDLR−/−/ApoB‐100 mice were fed a high fat diet (HFD) for 14 weeks prior to initiating 6 weeks of rPAI‐123 (n= 16) or saline (n=11) treatment with continued HFD. Control animals received chow diet and saline treatment. Lipid content, vasa vasorum density, vessel circumference, plaque and lumen areas were measured. Results: rPAI‐123 reduced lipid area by 60% and 30% (p<0.001) in the descending aorta and aortic root, respectively as compared to HFD saline treatment. Measurements of reconstructed vessels in the plaque area show that rPAI‐123 decreased vessel area and length by 43 and 37% (p = 0.01), respectively. Carotid artery circumferences in rPAI‐123 and saline HFD groups were similar. However, rPAI‐123 decreased plaque area by 67% (p<0.001) and increased lumen area by 74% (p<0.001). Conclusions: rPAI‐123 inhibits angiogenic vasa vasorum, reduces plaque growth and promotes plaque regression in atherogenic female LDLR−/−/ApoB‐100 mice.

Objective— Vasa vasorum are angiogenic in more advanced stages of human atherosclerosis and in mouse models of atherosclerosis. Fibroblast growth factor-2 (FGF-2) is the predominant angiogenic growth factor in the adventitia and plaque of hypercholesterolemic LDLR -/- ApoB100 + mice (DKO). Our previous studies suggest that FGF-2 plays an important role in guiding angiogenic vasa vasorum into a distinct plexus-like network. This study further investigated the role of FGF-2 in plexus development and the ability of an angiogenesis inhibitor, rPAI-1 23 , to inhibit the plexus. Methods and Results— DKO mice treated with saline, anti-angiogenic rPAI-1 23 or adeno-soluble FGFR1 (sFGFR1) were perfused with fluorescein-labeled Lycopersicon esculentum lectin. Descending aortas were probed for FGF-2 and whole mounts were imaged by confocal microscopy. Angiogenic vasa vasorum are abundant and form a plexus-like network in saline treated DKO that is similar to the FGF-2 pattern of distribution. Mice treated with rPAI-1 23 and sFGFR1 lack a plexus; FGF-2 and vasa vasorum density and area are significantly reduced. Examination of potential FGF-2 binding partners indicates that a perlecan/FGF-2 complex is critical for plexus development. Excess plasmin produced in rPAI-1 23 treated DKO/PAI-1 -/- mice degrades perlecan, disperses FGF-2 causing collapse of the plexus and destabilization of the vascular tree. Conclusion— Vasa vasorum develop an angiogenic plexus essential for maintaining a vascular tree during the atherosclerotic disease process. A perlecan/FGF-2 complex is required for structural organization and stability of the plexus. Disruption of the complex by rPAI-1 23 results in loss of the plexus causing the vasa vasorum to collapse. We conclude that a novel rPAI-1 23 stimulated pathway for regulating plasmin activity is the mechanism for inhibiting the expanded vasa vasorum in atherosclerotic DKO mice.