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. Author manuscript; available in PMC: 2010 Feb 15.
Published in final edited form as: Circ Res. 2009 Jan 2;104(3):337–345. doi: 10.1161/CIRCRESAHA.108.184622

The Anti-Angiogenic Activity of rPAI-123 Inhibits Vasa Vasorum and Growth of Atherosclerotic Plaque: Drinane and Mollmark, rPAI-123 Inhibits Vasa Vasorum and Plaque Growth

Mary Drinane 1,2,, Jessica Mollmark 1,2,, Lyubomir Zagorchev 1,3, Karen Moodie 1,3, Baiming Sun 1,2, Amy Hall 1,3, Samantha Shipman 1,3, Peter Morganelli 5, Michael Simons 1,3,4, Mary Jo Mulligan-Kehoe 1,2,*
PMCID: PMC2737332  NIHMSID: NIHMS127178  PMID: 19122176

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. In this study we used a truncated PAI-1 protein, rPAI-123, that has significant anti-angiogenic activity, to inhibit growth of vasa vasorum in atherogenic mice and assessed its effect on plaque growth. Female LDLR−/− ApoB-48 deficient mice fed Paigen’s diet without cholate for 20 weeks received rPAI-1 23 treatment (n= 21) for the last six weeks. Plaque size and vasa vasorum density were compared to two controls, mice fed Paigen’s diet and treated with saline for the last six 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 due, in part, to upregulated FGF-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 FGF-2, leading to reduced plaque growth in atherogenic female LDLR−/− ApoB-48 deficient mice.

Keywords: atherosclerosis, angiogenesis, inhibitors, FGF-2

Atherosclerosis is a chronic disease of large and medium size arteries 1, 2 and is the most frequent cause of coronary, peripheral and carotid artery disease. Neo-angiogenesis associated with more advanced stages of human atherosclerosis is found in plaque 3 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 models5–9. 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 system contributes to both processes through the proteolytic activity of plasmin. Plasmin activity is tightly regulated by plasminogen, plasminogen activators (PA), and plasminogen activator inhibitor-1 (PAI-1). Activation of PAI-1 inhibitory function requires a conformational change to expose the reactive center loop (RCL) containing a binding site for PA12. 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, while 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, uPAR 19 and αvβ3 20, which suggests that PAI-1 is anti-angiogenic and there is evidence to support this21, 22. However, there is also evidence that PAI-1 is pro-angiogenic21.

PAI-1 is cleaved by MMP-3 and plasmin to produce a truncated PAI-1 protein with a molecular mass less than 30 kD23. We hypothesized that cleaved PAI-1 has a function and produced truncated PAI-1 proteins (rPAI-1) that lack a reactive center loop (RCL) at the carboxy terminus. In the absence of the RCL, the rPAI-1 proteins could not bind and inactivate plasminogen activators (PA), 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 anti-angiogenic activity 24, 25. It inhibits FGF-2 stimulated migration, tube formation and proliferation 26, 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 if rPAI-123 inhibits angiogenic vasa vasorum that corresponds with reduced plaque growth in mice on an atherogenic diet.

Materials and Methods

Animals

Female B6; 129S-Apobtm2Sgy Ldlrtm1Her/J mice (LDLR−/−/ApoB-48 deficient) (Jackson Laboratories) were used for all experiments (Online Method1).

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 diet 27 without cholate (PD)(Online Method 2) for a total of 20 weeks and 14 twelve-week-old female mice were fed PD for 14 weeks. Twenty one mice received intraperitoneal injections of rPAI-123 (5.4 μg/kg/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 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, 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 DA were placed in O.C.T. tissue embedding compound (Sakura Finetek USA, Inc.) in preparation for serial sectioning (Online Method 4).

Plaque cholesterol measurement

Mice were sacrificed, descending aortas were removed and weighed. Cholesterol was extracted, evaporated and rehydrated for measuring plaque cholesterol levels (Online Method 5).

Histological measurements of plaque morphology

Alternating five 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 and eosin (H & E), picro-sirius red and Masson’s trichrome (Dartmouth clinical pathology laboratory). Vessel circumference, 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 histocompatability class II cells (MHC II) 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 (Online Figure III).

Detection of apoptotic cells

DNA strand breaks in apoptotic cells were detected with a TUNEL labeling and enzyme kit following the manufacturer’s instructions for cryopreserved tissue (Roche).

Confocal microscopy and vessel reconstruction

Mice were perfused as described. Descending aortas were removed, adventitial vessels were probed for CD-31 and imaged by confocal microscopy (Online 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 63x objective (Online method 7).

Probing for FGF-2

Mice were perfused with FITC-labeled lectin. Descending aortas were permeabilized, then incubated with an anti-mouse FGF-2 antibody (Sigma). Amplification and detection of the binding reaction were as described26. Confocal images were acquired at 63x, as described.

Quantitative realtime 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 Realtime PCR technique. Relative mRNA copy number was calculated by the 2ΔΔ method (Online Method 8).

Vessel area and length measurements

Projection images of adventitial vessels from confocal microscopy Z-stack reconstruction were preformed with Volocity software, 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 unskeltonized 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

Descending aorta cross sections probed for SMA and lectin were imaged by confocal microscopy at 63x resolution. Z-stacks consisting of 15 slices at physical resolution of 2.54 μm were acquired. Slices were aligned in a 3-D volumetric image and resolution was increased by tri-linear interpolation. Reconstructed Z-stacks were manually segmented to represent co-localized probes in consecutive axial slices. Only blood vessels going all the way through the interpolated volumes were considered (Online 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. Descending aortas with a wide adventitial margin were removed after polymerization was complete (Online Method 10). Three mice per group were imaged.

MicroCT imaging of the vasa vasorum

Descending aortas containing microfil were scanned with a GE eXplore Locus SP microCT scanner at 6.5 micron resolution. Three-dimensional volumetric images were reconstructed from acquired two dimensional projections without averaging, yielding a final voxel size of 6.5 microns (Online Method 11).

Statistical analysis

Statistical analysis was performed with a two-tailed indirect Student’s t-test, one way analysis of variance (ANOVA) with a post-hoc least significant difference (LSD) test with or without repeated measures or with a chi square test, as appropriate, using the SPSS 12.0.1 statistical software package.

Results

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 six 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 (Figures 1A–D).

Figure 1. Sudan IV stained lipid in the aortic arch and descending aorta.

Figure 1

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Twelve week old female mice were fed Paigen’s diet without cholate (PD) for varied time periods and received variable treatment in the final 6 weeks of diet. Descending aortas stained with Sudan IV were cut longitudinally for en face preparations. (A) PD for 14 weeks (n=14); (B) PD for 20 weeks, saline weeks 14–20 (n=16); (C) PD for 20 weeks, rPAI-123 weeks 14–20 (n=21). (D) Sudan IV stained plaque area relative to total area of the descending aorta.

(E) Plaque cholesterol measured in the descending aorta from fasted mice. White bars represent chow diet in age matched mice (n=6); grey bars are PD for 20 weeks, saline weeks 14–20 (n=6); black bars are PD for 20, rPAI-123 weeks 14–20 (n=6). Data shown as mean ± standard error of the mean and p values were determined by ANOVA. * p<0.05 vs. rPAI-123, **p<0.001 vs. rPAI-123.

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 vs. saline, 17.2±2.9 μg/ml, p<0.01), but remained 39% higher than chow fed mice (5.4±0.7, 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 if 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), while similarities were observed between rPAI-123 and the 14 week PD control (Online Figure I). Quantification of unskeletonized (Figures 2B and 2E) and skeletonized (Figures 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 1H) in rPAI-123 treated mice maintained on PD compared to the saline counterpart.

Figure 2. Reconstructed confocal images of adventitial vessels.

Figure 2

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Mice were fed PD for 20 weeks and treated with (A–C) saline (n=7) or (D–F) rPAI-123 (n= 8). Descending aortas were removed and probed for CD31. Confocal Z-stack images of adventitial vessels from A and D were reconstructed with Volocity V.3.7 software. (B and E) Reconstructed projection images of CD31-probed adventitial vessels, preformed with Volocity software, were (C and F) transformed to black and white images using the thresholding tool in Photoshop. MatLAB script calculated (G) the vessel area from the unskeletonized image and (H) vessel length from the skeletonized image. Black bars: PD for 20 weeks, saline weeks 14–20; striped bars: PD for 20 weeks, rPAI-123 from weeks 14–20.

Data shown as mean ± standard error of the mean and p values were determined by student’s t-test. * p<0.05 vs. control.

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. Descending aortas were removed for ex vivo microCT scanning and 3-D 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).

Figure 3. MicroCT images of second order vasa vasorum.

Figure 3

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Mice fed PD for either 14 or 20 weeks were infused with microfil. Descending aortas were scanned ex vivo at 6.5 microns and three-dimensional volumetric images of vasa vasorum were reconstructed from mice receiving: (A) PD for 14 weeks, no treatment (n=3); (B) PD for 20 weeks, saline weeks 14–20 (n=3); (C) PD for 20, rPAI-123 weeks 14–20 (n=3). Reconstructed images were rotated to obtain luminal images from mice receiving: (D) PD for 14 weeks, no treatment; (E) PD for 20 weeks, saline weeks 14–20; (F) PD for 20 weeks, rPAI-123 weeks 14–20. White arrows, second order vasa vasorum. Red arrows, plaque.

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 six weeks. Vascular structures were probed for lectin and SMA. Vessels in the plaque and DA wall of atherogenic mice treated with rPAI-123 were absent (Figures 5B and 5C), while mice treated with saline had distinct vessels in the plaque (Figure 4C) and DA wall (Figure 4B). Moreover, these vessels abutted highly vascularized adventitia (Figure 4A), which was absent in rPAI-123 treated mice (Figure 5A).

Figure 5. Vessels in the descending aorta adventitia, wall and plaque of rPAI-123 treated mice.

Figure 5

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Descending aorta cross sections probed for smooth muscle actin (green) and lectin (red) were imaged by confocal microscopy at 63x resolution. Z-stacks at physical resolution of 2.54 μm were acquired: (A) adventitia, (B) vessel wall, (C) plaque. To visualize blood vessels in the (D) adventitia, (E) vessel wall and (F) plaque, Z-stacks were reconstructed as described for Figure 4. N=five descending aortas per group.

Figure 4. Vessels in the descending aorta adventitia, vessel wall and plaque in saline treated mice.

Figure 4

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Descending aorta cross sections probed for smooth muscle actin (green) and lectin (red) were imaged by confocal microscopy at 63x resolution. Z-stacks at physical resolution of 2.54 μm were acquired: (A) adventitia, (B) vessel wall, (C) plaque. To visualize blood vessels in the (D) adventitia, (E) vessel wall and (F) plaque the reconstructed Z-stacks were manually segmented to represent co-localized probes in consecutive axial slices. The obtained contours were modeled and stacked in 3-D for volumetric surface representation. Arrows point to blood vessels. N=five descending aortas per group.

Blood vessels in plaque, adventitia, and vessel wall were visualized by aligning confocal Z-stacks images in a 3-D volumetric image and increasing resolution by trilinear interpolation. Reconstructed Z-stacks were manually segmented to represent the detected co-localized probes in consecutive axial slices. Contours of blood vessels going all the way through the interpolated volumes were modeled and stacked in 3-D 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 (Figures 4D- 4F) and their absence in the rPAI-123 treated mice (Figures 5D–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 (Online Figures II C and II D), which is opposite of what is found in control mice that were not treated with the angiogenesis inhibitor (Online Figures II A and II B).

Adventitial and intraplaque FGF-2 expression levels

The adventitia was probed for angiogenic growth factors, fibroblast growth factor-2 (FGF-2)28 and vascular endothelial growth factor-A165 (VEGF-A165)11, to determine if they were influential in expansion of the vessels observed in Figures 2A, 3B and 4A. 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, while FGF-2 and lectin were barely detectable in rPAI-123-treated mice (Figure 6D) (rPAI-123, 0.25±0.25 vs. saline, 7±0.9 vessels per field at 65x magnification).

Figure 6. Adventitial fibroblast growth factor-2 (FGF-2) expression.

Figure 6

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Mice fed PD for 20 weeks were treated with saline (A and C) or rPAI-123 (B and D). Mice were perfused, descending aortas with a wide margin were removed for confocal imaging of (A and B) the adventitia or (C and D) cross sections probed for lectin (red) and FGF-2 (white). Confocal Z-stacks were acquired at 63x magnification (n=6 per group). Arrows point to merged FGF-2 and lectin vessels.

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 three 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 employing 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 (Figure 7C) and saline (Figure 7B) treated mice fed PD for 20 weeks (rPAI-123,2.62 ±0.11 and 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) (Figure 7A, 6D). 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 due 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.

Figure 7. Morphology measurements of the innominate artery.

Figure 7

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Innominate arteries, from mice fed PD for 14 or 20 weeks with variable treatment in the weeks 14–20, were stained with H&E. Diet and treatment groups are: (A) PD for 14 weeks (n=8); (B) PD for 20 weeks, saline weeks 14–20 (n=11); (C) PD for 20 weeks, rPAI-123 weeks 14–20 (n=13). Regions of Interest software was used to obtain measurements of (D) vessel circumference and (E) plaque area and lumen area. White bars, normal chow diet; grey bars, PD for 14 weeks; black bars, PD for 20 weeks, saline weeks 14–20; striped bars, PD for 20 weeks, rPAI-123 weeks 14–20.

Data shown as mean ± standard error of the mean and p values were determined by ANOVA. * p<0.05 vs. control, **p<0.001 vs. control.

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 vs. 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 vs. 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 six 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 salinetreated mice and 1.7-fold more than 14 week PD controls (rPAI-123, 11.16±1.6 vs. saline, 5.6±0.87, p= 0.01; rPAI-123 vs. 14 week PD, 6.4±1.1, p=0.05). Measurements of collagen area as a percent 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% vs. saline, 42% and rPAI-123 vs. 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 vs. saline, 8.5±1.0, p= 0.003) while 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 (Figures 8A, B, D, E). MHCII cells were relatively few in number (rPAI-123, 19±5vs.saline,26±5, p=NS)(Figures 8A and D) while macrophages were abundant in both treatment groups (Figures 8B and E). The significant difference between the two 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 vs. saline, 18±1.5 TUNEL positive degraded areas > 2 mm2 at 100x 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.

Figure 8. Characterization of descending aorta plaque composition.

Figure 8

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Sections of descending aorta plaque from mice fed PD for 20 weeks and treated with saline (A–C) or rPAI-123 (D–F) were probed for: (A and D) I-A/I-E; (B and E) MOMA-2; (C and F) TUNEL. Magnification is 100x and inserts are 40x. N= 5 mice per group.

Discussion

In this study we found that inhibition of adventitial angiogenesis by a novel anti-angiogenic 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 anti-angiogenic 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 study 9 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 atherosclerosis32, 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, while 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 3-D 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 functions26.

We examined plaque growth in relationship to outward remodeling in the innominateartery, 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 due 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 MHCII cells in both treatment groups that presumably enter the plaque via the vasa vasorum prior to 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 anti-angiogenic 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/dy 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/dy for 16 weeks achieved 57% reduction in DA plaque area10. 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/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.

Acknowledgments

Sources of Funding

This study was supported by National Institutes of Health grants HL69948 (MJM-K) and HL53793 (MS).

Footnotes

Disclosures: None

References

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