The Antiangiogenic Activity of rPAI-123 Inhibits Vasa Vasorum and Growth of Atherosclerotic Plaque
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-123, that has significant antiangiogenic activity. Female LDLR−/−ApoB-48–deficient mice fed Paigen’s diet without cholate for 20 weeks received rPAI-123 treatment (n=21) 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 (n=16) and mice fed Paigen’s diet until the onset of treatment (n=14). The rPAI-123 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-123 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-123–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-123. In conclusion, rPAI-123 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−/−ApoB-48–deficient mice.
Atherosclerosis is a chronic disease of large and medium size arteries1,2 and is the most frequent cause of coronary, peripheral, and carotid artery disease. Neoangiogenesis associated with more advanced stages of human atherosclerosis is found in plaque3 and the vasa vasorum,4 the microvasculature in the adventitial layer of large arteries that provides arterial blood supply to the arterial wall.5 The presence and extent of vasa vasorum correlate with atherosclerotic lesion size and lumen diameter in hypercholesterolemic animal models.5–9 Vasa vasorum are considered to be the conduit for nutrient supplies to atherosclerotic plaque. Studies have demonstrated that inhibition of neovascularization in the vasa vasorum is associated with reduced plaque progression.9,10 At the same time, there is little information about the origin of plaque vasculature and the role of vasa vasorum in plaque growth, nor is there conclusive evidence that angiogenesis in vasa vasorum promotes plaque development.11
Proteases that degrade the extracellular matrix play an important role in angiogenesis and plaque remodeling. The plasminogen activator (PA) system contributes to both processes through the proteolytic activity of plasmin. Plasmin activity is tightly regulated by plasminogen, PAs, and PA inhibitor (PAI)-1. Activation of PAI-1 inhibitory function requires a conformational change to expose the reactive center loop containing a binding site for PA.12 The PA binding interaction with PAI-1 prevents PAs from cleaving plasminogen, thereby limiting plasmin levels and its role in extracellular matrix proteolysis.13
The role of PAI in plaque progression is poorly understood and controversial. It is implicated in promoting plaque progression in the carotid artery,14,15 enhancing thrombosis,15 and increasing neointima formation in atherosclerosis-prone mice.16 It has also been demonstrated that PAI-1 is atheroprotective in ApoE−/− mice, whereas the loss of PAI-1 promotes plaque progression in advanced stages of atherosclerosis by increasing matrix deposition.17 Still others have shown that PAI-1 has no effect on atherosclerosis progression in ApoE−/− and LDLR−/− mice.18
The role of PAI-1 in angiogenesis is also controversial. PAI-1 modulates the functions of plasmin, urokinase receptor,19 and αvβ3,20 which suggests that PAI-1 is antiangiogenic, and there is evidence to support this.21,22 However, there is also evidence that PAI-1 is proangiogenic.21
PAI-1 is cleaved by matrix metalloproteinase-3 and plasmin to produce a truncated PAI-1 protein with a molecular mass less than 30 kDa.23 We hypothesized that cleaved PAI-1 has a function and produced truncated PAI-1 proteins (rPAI-1) that lack a reactive center loop at the carboxyl terminus. In the absence of the reactive center loop, the rPAI-1 proteins could not bind and inactivate PAs, thus making them “inactive” PAI-1 proteins. Partial removal of the heparan sulfate binding domain at the amino terminus produced an rPAI-1 protein, rPAI-123, with significant antiangiogenic activity.24,25 It inhibits fibroblast growth factor (FGF)-2–stimulated migration, tube formation, and proliferation,26 vascular endothelial growth factor (VEGF)-stimulated migration in aortic endothelial cells,25 and VEGF- and FGF-2–stimulated tubulogenesis in embryonic chick aortic rings.24,26 The goal of this study was to determine whether rPAI-123 inhibits angiogenic vasa vasorum that corresponds with reduced plaque growth in mice on an atherogenic diet.
Materials and Methods
Female B6; 129S-Apobtm2SgyLdlrtm1Her/J mice (LDLR−/−/ApoB-48–deficient) (The Jackson Laboratory) were used for all experiments (see Method 1 in the online data supplement, available at http://circres.ahajournals.org).
Recombinant rPAI-123 Protein Production and Purification
Recombinant, truncated PAI-1 protein rPAI-123 was produced, purified, and tested for bacterial, yeast, and endotoxin contaminants as previously described.24–26
Diet and Treatment
Thirty-seven 12-week-old, weight-matched female mice were fed Paigen’s atherogenic diet27 without cholate (PD) (online data supplement, Method 2) for a total of 20 weeks and fourteen 12-week-old female mice were fed PD for 14 weeks. Twenty-one mice received intraperitoneal injections of rPAI-123 (5.4 μg/kg per day) beginning at week 14 of PD, and 16 received saline treatment. The rPAI-123 dose was based on in vivo Matrigel plug assays in C57B6 mice (online data supplement, Method 3). Animal care and procedures were performed in accordance with the guidelines of the Animal Care and Use Committee and procedures outlined in the Guide for the Care and Use of Laboratory Animals (NIH publication No. 86-23, 1985). All procedures were approved by the Dartmouth College IACUC review committee.
Preparation of Tissue
At the end of each treatment period the animals were fasted overnight. The next day blood was drawn and mice were euthanized and perfused. The innominate artery, carotid arteries, aortic arch, and descending aorta (DA) to the iliac bifurcation were surgically removed. Innominate arteries and DAs were placed in OCT tissue embedding compound (Sakura Finetek USA Inc.) in preparation for serial sectioning (online data supplement, Method 4).
Plaque Cholesterol Measurement
Mice were euthanized, and DAs were removed and weighed. Cholesterol was extracted, evaporated, and rehydrated for measuring plaque cholesterol levels (online data supplement, Method 5).
Histological Measurements of Plaque Morphology
Alternating 5-micron frozen sections of innominate arteries from mice fed PD for 20 weeks and treated with either rPAI-123 (n=13) or saline (n=11), and mice fed PD for 14 weeks (n=8) were stained with hematoxylin/eosin (H&E), picro-Sirius red, and Masson’s trichrome (Dartmouth Clinical Pathology Laboratory). Vessel circumference and plaque and lumen area were measured using Regions of Interest imaging software (Philips Imaging). Macrophage content was examined by probing DA cross-sections for MOMA-2, a mouse macrophage and monocyte intracellular antigen (Abcam). Major histocompatibility class (MHC) II cells were detected with a rat I-A/I-E antibody (BD Pharmingen). Sections probed in the absence of the primary antibody served as negative controls (supplemental Figure III).
Detection of Apoptotic Cells
DNA strand breaks in apoptotic cells were detected with a TUNEL labeling and enzyme kit following the instructions of the manufacturer for cryopreserved tissue (Roche).
Confocal Microscopy and Vessel Reconstruction
Mice were perfused as described. DAs were removed, and adventitial vessels were probed for CD31 and imaged by confocal microscopy (online data supplement, Method 6).
Mice were perfused with fluorescein-labeled Lycopersicon esculentum lectin (tomato) (Vector Labs). Vessels in DA adventitia, wall, and plaque were analyzed for endothelial and smooth muscle cells by imaging lectin-bound endothelium and antibody-bound smooth muscle actin. Confocal Z-stack were collected with a ×63 objective (online data supplement, Method 7).
Probing for FGF-2
Mice were perfused with FITC-labeled lectin. DAs were permeabilized and then incubated with an antimouse FGF-2 antibody (Sigma). Amplification and detection of the binding reaction were as described.26 Confocal images were acquired at ×63, as described.
Quantitative Real-Time PCR of Growth Factor RNA
RNA was isolated and purified from the DA of atherogenic mice treated with rPAI-123 or saline for 6 weeks and age-matched chow-fed mice. RNA was reverse transcribed into cDNA and quantification of VEGF-A165, FGF-2, and β-actin gene expression was performed by the real-time PCR technique. Relative mRNA copy number was calculated by the 2ΔΔ method (online data supplement, Method 8).
Vessel Area and Length Measurements
Projection images of adventitial vessels from confocal microscopy Z-stack reconstruction were preformed with Volocity software and then transformed to black and white images using the thresholding tool in Volocity. Total vessel area and length were measured by MatLAB script. Vessel area is unskeletonized image and is the sum of white pixels in a field. Length is the same measurement on skeletonized image.
Reconstruction of Vessels in Plaque, Vessel Wall, and Adventitia
DA cross-sections probed for smooth muscle actin and lectin were imaged by confocal microscopy at ×63 resolution. Z-stacks consisting of 15 slices at physical resolution of 2.54 μm were acquired. Slices were aligned in a 3D volumetric image, and resolution was increased by trilinear interpolation. Reconstructed Z-stacks were manually segmented to represent colocalized probes in consecutive axial slices. Only blood vessels going all the way through the interpolated volumes were considered (online data supplement, Method 9).
Infusion of Microfil Into Vessels
Mice were anesthetized, heparinized, and perfused. A silicone rubber compound, Microfil Blue, was infused through the aortic cannula. DAs with a wide adventitial margin were removed after polymerization was complete (online data supplement, Method 10). Three mice per group were imaged.
MicroCT Imaging of the Vasa Vasorum
DAs containing Microfil were scanned with a GE eXplore Locus SP microCT scanner at 6.5-μm resolution. Three-dimensional volumetric images were reconstructed from acquired 2D projections without averaging, yielding a final voxel size of 6.5 μm (online data supplement, Method 11).
Statistical analysis was performed with a 2-tailed indirect Student’s t test, 1-way ANOVA with a post hoc least significant difference test with or without repeated measures, or with a χ2 test, as appropriate, using the SPSS 12.0.1 statistical software package.
rPAI-123 Inhibits Plaque Size
The effects of rPAI-123 on plaque growth were studied by visualizing the extent of Sudan IV–stained plaque located between the top of the DA and the iliac bifurcation. Plaque area relative to total DA area was calculated. Measurements were taken in mice fed PD for 14 weeks or PD for 20 weeks and received either saline or rPAI-123 treatment for the last 6 weeks of the diet. Plaque area in rPAI-123–treated mice was reduced by 73% (P<0.001) when compared to saline-treated mice fed the same diet and 39% (P=0.02) less than mice fed PD for 14 weeks (Figure 1A through 1D).
Plaque cholesterol in rPAI-123–treated mice was significantly reduced by 49% when compared to control mice fed PD for 20 weeks (rPAI-123, 8.8±0.4 μg/mL; versus saline, 17.2±2.9 μg/mL; P<0.01) but remained 39% higher than chow fed mice (5.4±0.7 μg/mL; P<0.001) (Figure 1E). These experiments demonstrate that rPAI-123 treatment has a dramatic effect on reducing plaque area and plaque cholesterol levels.
rPAI-123 Inhibits Adventitial Vessel Growth
Whole mount DAs from each test group were probed for endothelial marker CD31 to determine whether rPAI-123 treatment had a corresponding effect on adventitial vasa vasorum. Confocal Z-stack microscopic images of adventitial vessels showed substantial structural and density differences between rPAI-123 (Figure 2D) and saline-treated mice fed PD for 20 weeks (Figure 2A), whereas similarities were observed between rPAI-123 and the 14-week PD control (supplemental Figure I). Quantification of unskeletonized (Figure 2B and 2E) and skeletonized (Figure 2C and 2F) reconstructed vessel images demonstrated a 37% (P=0.01) reduction in total vessel area (Figure 2G) and a 43% (P=0.004) reduction in vessel length (Figure 2H) in rPAI-123–treated mice maintained on PD compared to the saline counterpart.
MicroCT images of vasa vasorum were acquired to confirm the confocal microscopy results. Microfil, a microvascular contrast agent, was infused into the ascending aorta of mice fed PD for 14 weeks or 20 weeks with rPAI-123 or saline treatment for the final 6 weeks of diet. DAs were removed for ex vivo microCT scanning and 3D reconstruction of the scanned images. Reconstructed images of rPAI-123–treated mice showed an absence of second order vasa vasorum (Figure 3C) that were abundantly dense in the saline counterpart (Figure 3B, white arrows) and beginning to develop in the 14-week-diet group (Figure 3A). The microCT images were rotated to visualize plaque in the luminal side of the DA. Plaque was not detected in the lumen of rPAI-123–treated mice (Figure 3F), but the saline group had extensive plaque (Figure 3E) and the 14 week atherogenic control group had detectable plaque (Figure 3D).
rPAI-123 Inhibits Vessel Growth Into Plaque Area
Confocal microscopic Z-stack images of DA cross-sections were examined for vessels within the plaque and vessel wall of atherogenic mice treated with rPAI-123 or saline for 6 weeks. Vascular structures were probed for lectin and smooth muscle actin. Mice treated with saline had distinct vessels in the plaque (Figure 4C) and DA wall (Figure 4B), while vessels in the plaque and DA wall of atherogenic mice treated with rPAI-123 were absent (Figure 5B and 5C). Moreover, these vessels abutted highly vascularized adventitia (Figure 4A), which was absent in rPAI-123–treated mice (Figure 5A).
Blood vessels in plaque, adventitia, and vessel wall were visualized by aligning confocal Z-stacks images in a 3D volumetric image and increasing resolution by trilinear interpolation. Reconstructed Z-stacks were manually segmented to represent the detected colocalized probes in consecutive axial slices. Contours of blood vessels going all the way through the interpolated volumes were modeled and stacked in 3D to provide volumetric surface representation. The reconstructed images show the presence of fully formed vessels in the DA wall, plaque and adventitia of saline-treated mice (Figure 4D through 4F) and their absence in the rPAI-123–treated mice (Figure 5D through 5F). The collective data from this series of experiments clearly demonstrate that rPAI-123 has a profound inhibitory effect on second order vasa vasorum. The absence of these vessels corresponds to a lack of vessels invading the plaque and to dramatically reduced plaque area (supplemental Figure II, C and D), which is the opposite of what was found in control mice that were not treated with the angiogenesis inhibitor (supplemental Figure II, A and B).
Adventitial and Intraplaque FGF-2 Expression Levels
The adventitia was probed for the angiogenic growth factors FGF-228 and VEGF-A16511 to determine whether they were influential in expansion of the vessels observed in Figures 2A, 3B, and 4⇑⇑A. VEGF-A165 was not detected, but FGF-2 was expressed abundantly along vascular structures in the adventitia of saline-treated atherogenic mice (Figure 6A) and to a significantly lesser degree in rPAI-123–treated atherogenic mice (Figure 6B). Vessels in the saline-treated group have a lumen and follow the well-defined pattern of FGF-2-bound vascular structures. Lectin-probed vessels in rPAI-123–treated mice do not have a lumen. Although they follow the FGF-2-bound vascular structures, the pattern of the structures is disordered and in many instances disrupted. Similarly, FGF-2 was detected in plaque from saline-treated mice (Figure 6C) and was associated with vessels, whereas FGF-2 and lectin were barely detectable in rPAI-123-treated mice (Figure 6D) (rPAI-123, 0.25±0.25 vessels per field at ×65 magnification; versus saline, 7±0.9 vessels per field at ×65 magnification).
Quantitative PCR showed no significant reduction in FGF-2 mRNA copy number in rPAI-123–treated mice compared to age-matched chow-fed mice. However, it was reduced 8-fold compared to the saline counterpart (P=0.003). Quantitative analysis of VEGF-A165 mRNA levels showed no significant differences among the 3 groups. These data suggest that FGF-2 has a significant role in stimulating angiogenesis in the vasa vasorum and contributes to plaque growth. Furthermore, it shows that rPAI-123 blocks FGF-2-stimulated angiogenesis by regulating its transcription.
Innominate Artery Morphological Changes in Response to rPAI-123
The relationship between plaque growth and vessel remodeling was addressed by using morphological analyses of innominate artery cross-sections. Differences in vessel circumference, plaque and luminal area, and plaque composition at a site more proximal to the origin of diseased vessels were examined. Measurements of innominate artery circumferences in H&E-stained sections did not show a difference between rPAI-123–treated (Figure 7C) and saline-treated (Figure 7B) mice fed PD for 20 weeks (rPAI-123, 2.62±0.11 mm; saline, 2.46±0.18 mm; P=NS) (Figure 7D). However, the average circumference length of both groups was significantly greater than the 14-week PD group (1.85±0.06 mm; P<0.001) (Figures 7A and 6⇑D). A comparison with age-matched chow-fed mice determined that the average circumference of 14-week PD mice was 18% larger than its chow-diet control (1.52±0.02 mm; P<0.001) (Figure 7D). Similarly, the 20-week PD groups had an average innominate artery circumference that was 20% larger than age-matched chow controls (1.98±0.18; P=0.01). The combined data indicate that larger vessel circumferences in 20-week atherogenic diet groups are attributable to outward remodeling and age-related vessel size. The data also demonstrate that vessel wall outward remodeling began to occur by 14 weeks of atherogenic diet.
Measurements of innominate artery luminal area in rPAI-123–treated mice were 2.14-fold greater than saline-treated mice (rPAI-123, 0.3±0.04 mm2; versus saline, 0.14±0.04 mm2; P<0.001) and 3.2-fold more than the 14-week PD group (0.094±0.01; P<0.001). Conversely, plaque area in rPAI-123–treated mice was reduced 64% when compared to saline treatment (rPAI-123, 0.09±0.01 mm2; versus saline, 0.25± 0.02 mm2; P=0.01) and 25% less than 14-week PD mice (0.12±0.02) (Figure 7E). We conclude that both groups fed PD for 20 weeks experienced the same degree of outward remodeling in the innominate artery. However, the effects of rPAI-123 over a 6-week treatment period significantly reduced plaque area, which in combination with the enlarged circumference dramatically increased luminal area.
A comparison of average cell number in innominate artery plaques relative to plaque area determined that rPAI-123–treated mice had 1.9-fold more cells/mm2 than saline-treated mice and 1.7-fold more than 14-week PD controls (rPAI-123, 11.16±1.6; saline, 5.6±0.87 [rPAI-123 vs saline, P=0.01]; 14-week PD, 6.4±1.1 [rPAI-123 vs 14-week PD, P=0.05]). Measurements of collagen area as a percentage of total plaque area show that rPAI-123–treated plaques had 1.7-fold more collagen/mm2 than either the saline or 14-week PD control (rPAI-123, 72%; saline, 42%; 14-week PD, 43%; P=0.01).
Similarly, cell number/mm2 of H&E-stained DA plaques was 1.58-fold greater in rPAI-123–treated mice (rPAI-123, 11.5±0.6; versus saline, 8.5±1.0; P=0.003), whereas collagen degradation was significantly reduced in these mice compared to the saline counterpart (59%; P=0.002). Analysis of plaque composition showed the presence of macrophages and MHCII cells in rPAI-123 and saline-treated mice (Figure 8A, 8B, 8D, and 8E). MHCII cells were relatively few in number (rPAI-123, 19±5vs.saline,26±5, P=NS) (Figure 8A and 8D), whereas macrophages were abundant in both treatment groups (Figure 8B and 8E). The significant difference between the 2 groups was the excess of apoptotic cells in macrophage-rich and/or collagen degraded areas in plaques of saline-treated mice (Figure 8C), which are absent in plaques responding to rPAI-123 treatment (Figure 8F) (rPAI-123, 3±1; versus saline, 18±1.5 TUNEL-positive degraded areas >2 mm2 at ×100 magnification; P<0.001). Increased apoptosis appears to explain the presence of large acellular areas in saline-treated plaques, a characteristic indicative of advanced plaque progression.29
In this study, we found that inhibition of adventitial angiogenesis by a novel antiangiogenic protein, rPAI-123, dramatically reduced the number and size of second order vasa vasorum in atherogenic female LDLR−/−ApoB-48–deficient mice. Inhibition of angiogenesis led to a highly statistically significant reduction in plaque growth and plaque cholesterol levels. These results strongly suggest that adventitial angiogenesis and vasa vasorum play a key role in plaque growth and atherosclerotic arterial remodeling and that targeting this process can induce regression of atherosclerotic lesions.
We demonstrate that increased vasa vasorum density corresponds with adjacent vessels in significantly larger plaques accompanied by vessels in the DA wall. This evidence is supported by the antiangiogenic activity of rPAI-123 that significantly inhibits vasa vasorum expansion to the second order to result in an absence of vessels invading the plaque and reduced plaque area.
Plaque growth is a complex and poorly understood process. A previous study9 suggested that inhibition of plaque vasculature may reduce plaque progression, but the relationship between plaque vasculature and arterial wall vasculature remains undefined. A number of studies suggest that increased second order vasa vasorum is associated with increased atherosclerotic lesion size in humans and hypercholesterolemic animal models.8,9,30,31 The primary function of these vessels is thought to be transport of nutrients to the vessel wall. Increased microvessel density in the adventitia and plaque in humans is associated with plaque instability, hemorrhage, and rupture, all life-threatening events that can accompany atherosclerosis.32,33
Confocal Z-stack and microCT images were reconstructed to show that rPAI-123 inhibits adventitial vessel density in LDLR−/−ApoB-48–deficient atherogenic mice such that the vascular architecture no longer resembles the saline control. Vessels observed in reconstructed confocal images of rPAI-123–treated mice are short and discontinuous and in many cases do not appear to have a lumen.
MicroCT images of 14- and 20-week saline-treated atherogenic mice clearly show that second order vasa vasorum are associated with neighboring luminal plaque, whereas the absence of second order vasa vasorum in rPAI-123–treated mice is accompanied by undetectable plaque. These data demonstrate a distinct association between rPAI-123 induced reduction of vasa vasorum density and plaque growth. The association is validated by reconstructed 3D confocal images, which show the presence of fully formed vessels in the DA wall, plaque, and adventitia of saline-treated mice and their absence in rPAI-123–treated mice. These collective data provide evidence to support the concept that angiogenic vasa vasorum supply the plaque nutritional requirements for enhanced growth.
The high levels of FGF-2 expression detected in adventitial vascular structures of saline-treated mice appear to stimulate tubulogenesis and provide patterning guidance to vessels with a lumen. The significantly diminished FGF-2 levels in rPAI-123–treated mice are accompanied by short discontinuous vessels that lack a lumen. The association of FGF-2 with intraplaque vessels in saline-treated mice and its absence in rPAI-123–treated mice suggests that FGF-2 expression contributes to plaque growth. These conclusions are further validated by the highly significant loss of FGF-2 gene expression in response to rPAI-123 treatment, which inhibited plaque growth and progression. These data are consistent with our in vitro studies, which demonstrate rPAI-123 inhibition of FGF-2 angiogenic functions.26
We examined plaque growth in relationship to outward remodeling in the innominate artery, a smaller artery located in an area where blood flow would be more impaired by plaque growth. Treatment with rPAI-123 or saline was initiated after mice were fed PD for 14 weeks; therefore, both treatment groups would have plaque content similar to 14-week control mice at the onset of treatment. Plaque area in the DA and innominate arteries of rPAI-123–treated mice was reduced 73% and 64%, respectively, when compared to 20-week saline control mice and comparable to or less than the 14 week saline control. The results suggest that rPAI-123 inhibits plaque progression and promotes plaque regression.
Further analysis of the innominate artery indicates that the circumferences are expanded in all mice fed PD. However, the outward remodeling is more extensive in rPAI-123 and saline treatment groups that were fed PD for 20 weeks when compared to mice fed PD for 14 weeks. Expansion of the innominate circumference in rPAI-123–treated mice could partially explain their larger lumen area when compared to 14-week PD controls. However, the differences in lumen area between rPAI-123 and saline-treated mice fed PD for 20 weeks are clearly attributable to reduced plaque in response to rPAI-123. The collective data indicate that rPAI-123 inhibitory effects do not immediately prevent progression of the disease process but reduce plaque size over the course of treatment. This is supported by the presence of macrophages and MHC II cells in both treatment groups that presumably enter the plaque via the vasa vasorum before the onset of treatment. These data, combined with reduced plaque cholesterol levels, further suggest that rPAI-123 promotes plaque regression.
The vasa vasorum has been implicated as the origin of plaque vasculature and the promoter of plaque growth; however, there has not been any conclusive evidence that angiogenesis in the vasa vasorum promotes plaque development. Others have shown that cleavage products of extracellular matrix proteins with antiangiogenic activity can also reduce vasa vasorum and plaque growth, but they have not demonstrated the existence of plaque vessels.9,10 Moulton et al demonstrated that angiostatin, a plasminogen cleavage product, reduces plaque progression.9 Seventy-five days of angiostatin treatment at a concentration of 20 mg/kg per day reduced plaque area in the DA by 36%.9 Similarly, ApoE−/− mice treated with endostatin, a cleavage product of collagen XVIII, at a dose of 20 mg/kg per day for 16 weeks achieved 57% reduction in DA plaque area.10 The rPAI-123 protein, a cleavage product of PAI-1, reduced plaque area by 62% in LDLR−/−ApoB-48–deficient mice within 6 weeks of treatment at a dose of 5.4 μg/kg per day.
This study supports our in vitro and ex vivo studies, which show that rPAI-123 is a potent inhibitor of arterial endothelial cell tubulogenesis, migration, and proliferation.24–26 Taken together, the data suggest that rPAI-123 could have significant therapeutic potential in the treatment of atherosclerosis.
Sources of Funding
This study was supported by NIH grants HL69948 (to M.J.M.-K.) and HL53793 (to M.S.).
↵*Both authors contributed equally to this work.
Original received July 31, 2008; revision received November 17, 2008; accepted December 18, 2008.
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