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Aortic adventitial angiogenesis and lymphangiogenesis promote intimal inflammation and hyperplasia
Cardiovascular Pathology 18 (2009) 269 – 278
Original Article
Aortic adventitial angiogenesis and lymphangiogenesis promote intimal inflammation and hyperplasia Xinsheng Xu a,b,1 , Huixia Lu a,1 , Huili Lin a,c , Xiaolu Li a,d , Mei Ni a , Huiwen Sun a , Changjiang Li a , Hong Jiang a , Fuhai Li a , Mei Zhang a , Yuxia Zhao a,⁎, Yun Zhang a,⁎ a
The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu Hospital, Jinan, Shandong, 250012, China b Department of Cardiology, Dongying People's Hospital, Dongying, Shandong, China c Department of Cardiology, the Second Affiliated Hospital of Fujian Medical University, Fujian, China d Department of Emergency, Qianfoshan Hospital, Jinan, Shandong, China Received 15 January 2008; received in revised form 18 June 2008; accepted 18 July 2008
Abstract Introduction: Adventitial inflammation is known to influence neointimal formation and vascular remodeling. The present study was aimed to clarify the relationship between neointima hyperplasia and adventitial angiogenesis and lymphangiogenesis after balloon-induced aortic endothelial injury. Methods: Seventy male Wistar rats were randomly divided into six interventional groups and one control group. The intimal area/medial area ratio (I/M ratio), the adventitial macrophage index, and the number of adventitial microvessels (Ad-MV) and lymphatic vessels (Ad-LV) in the aorta were measured, and the mRNA expressions of VEGF-A, VEGFR-1, VEGF-C, VEGFR-3, PDGFB, and PDGFR-β in the aortic wall were quantified by real-time RT-PCR. Results: Compared with the control group, the I/M ratio, macrophage index, Ad-MV, Ad-LV, and the mRNA expressions of VEGF-A, VEGFR-1, VEGF-C, VEGFR-3, PDGF-B, and PDGFR-β in interventional groups increased significantly after balloon-induced injury. I/M ratio showed significant correlations with Ad-MV and Ad-LV after balloon intervention. Multiple linear regression analysis indicated that Ad-MV and Ad-LV were independent factors of intimal hyperplasia. Conclusion: Adventitial angiogenesis and lymphangiogenesis are induced by intimal inflammation after balloon injury, and these neogenetic vessels in turn promote intimal inflammation and hyperplasia probably via delivery and activation of inflammatory cells. © 2009 Elsevier Inc. All rights reserved. Keywords: Angiogenesis; Lymphangiogenesis; Intimal injury; Inflammation
1. Introduction It is well known that lymphatic vessels, which commonly accompany blood vessels in tissues, drain extravasated This study was supported by the National 973 Basic Research Program of China (No.2005CB523301), the National High-tech Research and Development Program of China (No.2006AA02A406), the Program of Introducing Talents of Discipline to Universities (No.B07035), and grants from the National Natural Science Foundation of China (Nos. 30570747, 30670873, 30772810, 30700301). ⁎ Corresponding authors. Shandong University Qilu Hospital, Jinan, No. 107, Wen Hua Xi Road, Jinan, Shandong, 250012, PR China. E-mail address: [email protected] (Y. Zhang). 1 The first two authors contributed to this work equally. 1054-8807/08/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2008.07.004
bloodless fluid, protein, and inflammatory cells from the tissues and take an active part in inflammatory diseases [1,2]. Recent studies have reported that lymphatic vessels were enlarged in chronic inflammatory skin diseases and that rejected kidney transplants contained abundant lymphatic vessels [3,4]. Lately, it was found that lymphangiogenesis prevented mucosal edema and these neogenetic lymphatic vessels persisted in chronic airway inflammation [5]. Although the presence of adventitial inflammatory infiltration in close proximity to intimal atherosclerotic plaques has been recognized for more than four decades [6], the clinical relevance of this finding was not clear until recently when inflammatory infiltration was proved to be present in adventitia after coronary balloon injury [7]. Inflammatory
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cells like macrophages can secret cytokines and growth factors to promote angiogenesis and lymphangiogenesis [8,9]. However, the relationship between adventitial lymphangiogenesis and intimal hyperplasia is still unknown. In the present study, we tested the hypothesis that adventitial angiogenesis and lymphangiogenesis are initially induced by intimal inflammation after endothelial injury and these neogenetic vessels in turn promote intimal inflammation and neointimal hyperplasia by transportation and activation of inflammatory cells. 2. Materials and methods 2.1. Animal model Seventy male Wistar rats weighing 450 to 500 g were obtained from the Animal Center of Shandong University and divided randomly into seven groups with 10 rats in each group. Group 1 to Group 6 rats underwent balloon-induced aortic endothelial injury, while Group 7 rats received only sham operation and served as a control group. Rats were housed under conditions of constant room temperature (22°C) and a 12-h dark/12-h light cycle, and fed a normal diet. All rats underwent anesthesia with intraperitoneal injection of pentobarbital (30 mg kg−1) and the left external carotid artery was exposed to introduce a 2-F embolectomy catheter (Baxter Healthcare Corp., Irvine, CA, USA) into the distal abdominal aorta. The balloon catheter was then inflated with 50 μl of saline and went back and forth in the abdominal aorta three times to induce endothelial injury in Group 1 to Group 6 rats [10–13]. Sham operation was performed in Group 7 rats that underwent the same catheterization procedure but without balloon inflation. Group 1 to Group 6 rats were euthanized by intraperitoneal injection of a lethal dose of phenobarbitone on Days 1, 3, 7, 14, 28, and 90 after the procedure, respectively, and Group 7 rats were euthanized on Day 1 after sham operation. After the abdominal aorta was harvested, aortic segments were snap frozen in liquid nitrogen and stored at −80°C for quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, or fixed with 4% phosphate-buffered formaldehyde for 24 h for immunohistochemical and morphometric analysis. All animal procedures in the present study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and the Guide for the Care and Use of Laboratory Animals published by the Chinese National Institutes of Health. 2.2. Morphometric analysis Aortic segments were embedded in paraffin and cut into 6μm-thick sections which were stained with hematoxylin and eosin, and analyzed using a computer-assisted morphometric analysis system (Image-Pro Plus 5) with particular attention to the intimal and the medial thickness. Vascular area within the external elastic lamina (EELA) and the internal elastic
lamina (IELA) as well as the lumen area (LA) was measured. The I/M ratio was calculated as: I/M=(IELA−LA)/(EELA −IELA). All parameters were measured from three sections selected from the proximal, the middle, and the distal portion of the aortic segment, and the values averaged. 2.3. Antibodies The primary antibodies used were as follows: goat polyclonal antibody against human CD34 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) to identify the blood endothelial cells; rabbit polyclonal antibody against human LYVE-1 (1:100; Santa Cruz Biotechnology) to tag lymphatic endothelial cells; rabbit polyclonal antibody against human CD68 (1:400; Santa Cruz Biotechnology) to mark macrophages; mouse monoclonal anti-α-SMA antibody (1:2000; ab7817, Santa Cruz Biotechnolgy) to label vascular smooth muscle cells (VSMCs); rabbit polyclonal antibody against mouse VEGF-A (1:50; Santa Cruz Biotechnology); rabbit polyclonal antibody against human VEGF-C (1:50; Santa Cruz Biotechnology), and rabbit polyclonal antibody against PDGF-BB (1:100; ab15499, Abcam, Cambridge, UK). Second antibodies against IgG in rabbits, mice, and goats were obtained from Beijing Zhongshan Biotechnology Co., Ltd. (ZSBIO, Beijing Zhongshan Golden Bridge Technology, China). 2.4. Immunohistochemistry Sections were deparaffinized and incubated with 5% goat serum or 5% BSA for 20 min to minimize the nonspecific binding to the primary antibody and incubated with the primary antibodies overnight at 4°C in a moisture chamber. The sections were then incubated with the appropriate secondary antibody for 30 min at room temperature. To inhibit any endogenous peroxidase activity, the sections were incubated with 0.3% H2O2 in absolute methanol for 30 min. A peroxidase substrate solution containing 0.02% H2O2 and 0.1% 3,3′-diaminobenzidine tetrahydrochloride (ZSBIO) in PBS was applied to display the reaction product with a brown color, and the sections were then counterstained with hematoxylin. Incubation with PBS instead of the primary antibody was used as a negative control. 2.5. Quantification of adventitial angiogenesis and lymphangiogenesis The total number of microvessels (CD34+) (Fig. 1A) or lymph vessels (LYVE-1+) (Fig. 1B) in the whole adventitia was counted under a light microscope at a high power magnification (×400) [14–17]. Vascular and lymph vessels were identified as morphologically circumferential brown products formed by one or more stained endothelial cells with at least one counterstained nucleus. In this case, a single brown dot was not counted. The number of adventitial microvessels (Ad-MV) and adventitial lymphatic vessels
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Fig. 1. Adventitial microvessels and lymph vessels identified by immunohistochemical staining at a high power magnification (×400). The microvessels exhibited as CD34 positive (A, arrows), while lymph vessels exhibited as LYVE-1 positive (B, arrow heads). Scale bar, 50 μm.
(Ad-LV) from three sections was counted and the mean values derived. All counting was performed by two independent investigators and the values were averaged. 2.6. Real-time RT-PCR Total RNA was extracted from the aortic tissue by Trizol (Invitrogen, Carlsbad, CA, USA) following the protocols recommended by the manufacturers. Reverse transcriptasepolymerase chain reaction was performed as described previously [18]. In brief, 1 μg of RNA in 20 μl of volume was reversely transcribed with oligo (dT) primer using the M-MLV Reverse Transcriptase System (Promega, Madison, WI, USA). The relative quantification of target genes was determined using the LightCycler (Roche Applied Science, USA) following the manufacturer's protocol. Real-time quantitative PCR (real-time PCR) analysis for mRNA expressions in the aortic tissue was performed using a TaqMan probe method. The reaction contained 0.2 μl of TaKaRa Ex Taq HS polymerase (TaKaRa Biotechnology,
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Dalian, China), 2 μl of 10× buffer (Mg2+ plus), 2 μl dNTP mixture (1 mM, respectively), 1 μl of forward primer, 1 μl of reverse primer, 1 μl of probe, and 1 μl of cDNA in a 20-μl final volume. The conditions for real-time PCR were as follows: denaturation at 95°C for 30 s, and in the following 50 cycles, denaturation at 95°C for 0 s, annealing for 10 s, and extension at 72°C for 10 s. Primer and probe sequence (Sangon, Shanghai, China), PCR product size, annealing temperature, and GenBank Access Number for each primer are listed in Table 1. Target gene expressions were calculated using the 2ΔΔCt method and expressed as N-fold difference between experimental groups (Group 1 to Group 6) and control group (Group 7) after normalizing to reference gene expressions [19]. 2.7. Statistical analysis Data analysis was conducted using a statistical software package (SPSS 11.5, Chicago, IL, USA). All measurements were expressed as mean±S.D. One-way ANOVA was used to compare the differences among seven groups. The relationship between the number of Ad-MV and Ad-LV and the I/M ratio was tested using simple (Pearson's correlation) and multiple linear regression analysis. Pb.05 was considered statistically significant. 3. Results 3.1. Neointima hyperplasia after balloon injury The aortic intima appeared as a single cell layer, the IELA was intact, and the EELA was in close contact with the adventitia in the control group of rats (Fig. 2A). The
Table 1 Primers and probes in real-time PCR assays Gene (size)
Primers and probes
Annealing temperature
GenBank number
APDH (78 bp)
F 5′-ATG TAT CCG TTG TGG ATC TGA C-3′ R 5′-CCT GCT TCA CCA CCT TCT TG-3′ P 5′-TGC CGC CTG GAG AAA CCT GCC A-3′ F 5′-CGA CAG AAG GGG AGC AGA AAG-3′ R 5′-GCA CTC CAG GGC TTC ATC ATT-3′ P 5′-AGC AGC CCG CAC ACC GCA TTA GG-3′ F 5′-ACA GAA GAG GAT GAG GGT GTC T-3′ R 5′-TGA AGA GAG TTA GAA GGA GCC AAA-3′ P 5′-TGC CGA GCC ACC AAC CAG AAG GG-3′ F 5′-CAG TTT TTC AGT CCA TCA TTT-3′ R 5′-CAG TCC ATT CCC ACA GTA A-3′ P 5′-CTG CCT TGA AAA ACT GTT GCC AC-3′ F 5′-GTT TTG TGT CCC ACC CCT ACC-3′ R 5′-GCC CTC GTT GTC TGA GTT TGA-3′ P 5′-TGC CCC TTA CAG CCT CCC TAT CCA G-3′ F 5′-ACT AAG AGC GTG CGT CAG TTG-3′ R 5′-ATG CTC ACC CAG ACA AAG TAA GAA-3′ P 5′-AGA GTG AGG GAG CAA CGG CGG CA-3′ F 5′-CAA GAG TGA CAG AGA AGG CAA G-3′ R 5′-GCT GGC AGT TGA GAT GGC T-3′ P 5′-CCT CAG CGT CGT CCT CTC ATG CCC A-3′
58°C
BC059110
62°C
NM_031836
60°C
NM_019306
60°C
NM_053653
60°C
NM_053652
62°C
XM_343293
60°C
AY090783
VEGF-A (194 bp)
VEGFR-1 (175 bp)
VEGF-C (145 bp)
VEGFR-3 (76 bp)
PDGF-B (77 bp)
PDGFR-β (158 bp)
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Fig. 2. Aortic intima and adventitia in the control group and the interventional groups (hematoxylin and eosin, ×400). (A) The intima appeared as a single layer of endothelial cells in the control group. There were few microvessels in the adventitia. (B) The aortic intima was found entirely denudated on Day 1 after balloon injury. (C) Infiltration of inflammatory cells in the adventitia on Day 3 after balloon injury. (D) Neointima consisting of SMCs and macrophages was evident on Day 7 after balloon injury. Inflammatory cells surrounded Ad-MVs. (E) to (G) represent the pathological changes on Days 14, 28, and 90 after balloon injury, respectively. Intimal hyperplasia consisting of multiple layers of SMCs became prominent and persisted until Day 90 after balloon intervention. Neogenetic microvessels and lymphatic vessels were evident in the adventitia. Scale bar, 50 μm.
intima of the abdominal aorta was found to be uniformly denudated on Day 1 after balloon injury (Fig. 2B). Hyperplastic intima consisting of multiple layers of VSMC and macrophages became evident on Day 7 after balloon intervention, and these changes reached a maximal level on Day 28 (Fig. 2D–F). The I/M ratio increased significantly on Day 28 compared with that on Day 7 after balloon injury (0.56±0.06 vs. 0.17±0.02, Pb.001). However, this ratio did not show any further increase on Day 90 compared with that on Day 28 after balloon intervention (0.55±0.06 vs. 0.56±0.06, PN.05; Fig. 3A). 3.2. Adventitial microvessels after balloon injury The value of Ad-MV began to increase significantly on Day 3 after balloon injury when compared with that in the control group (12.40±1.43 vs. 7.80±1.32, Pb.001), and this difference became even greater on Day 7 and Day 14 (19.80±2.74 vs. 7.80±1.32, Pb.001; and 24.20±2.15 vs. 7.80±1.32, Pb.001) after balloon intervention. However, the value of Ad-MV reached a plateau thereafter and did not show any further increase on Day 28 and Day 90 after balloon injury when compared with that on Day 14 (PN.05, Fig. 3C). 3.3. Adventitial lymphatic vessels after balloon injury Similar to the changes in Ad-LV, the value of Ad-LV began to rise on Day 3 (6.4±1.1 vs. 2.5±0.53, Pb.001) and continued to ascend on Day 7 (12.5±1.4 vs. 2.5±0.53, Pb.001) until a plateau was reached on Day 14 (12.8±1.0 vs. 2.5±0.53, Pb.001) when compared with that in the control group. Thereafter the value of Ad-LV remained at a high level on Day 28 (12.8±1.4 vs. 12.8±1.0, PN.05) and Day 90
(12.6±1.3 vs. 12.8±1.0, PN.05, Fig. 3D) after balloon intervention when compared with that on Day 14. 3.4. Macrophage distribution in the aortic wall Immunohistochemical staining using anti-macrophage antibody (CD68) revealed that CD68-positive cells were predominantly located in the adventitia near the external elastic membrane on Day 3 and scattered in the neointima and adventitia on Days 7 and 14 (Fig. 4). Thereafter, CD68positive cells were mainly present in the adventitia on Day 28. The percentage of macrophage number to total cell number in adventitia (macrophage index) was calculated [20]. The results demonstrated that the macrophage index increased on Day 3 (4.5±0.8% vs. 0.6±0.2%, Pb.001), peaked on Day 14 (12.5±1.3% vs. 0.6±0.2%, Pb.001), maintained this level on Day 28 (12.3±1.5% vs. 0.6±0.2%, Pb.001) when compared with that in the control group, and returned to the control level on Day 90 after balloon injury (Fig. 3B). 3.5. VEGF-A, VEGF-C, and PDGF-BB expressions in the aortic wall Immunohistochemical staining revealed that VEGF-Apositive expressions were barely visible, while VEGF-C and PDGF-BB expressions were not present in the control group. On the other hand, intensive VEGF-A expressions were evident in VSMCs and macrophages in the aortic wall on Days 3, 7, and 14 after balloon injury. Thereafter, the intensity of VEGF-A expressions diminished and only faint positive expressions were visible in these cells on Day 90 after balloon intervention (Fig. 5). VEGF-C-positive expressions were mainly present in the macrophages on Days 7 and
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Fig. 3. Dynamic changes in I/M ratio in (A), adventitial macrophage index in (B), the number of microvessels in (C), and the number of lymphatic vessels in (D). Cont: control group; D1 to D90: Days 1 to 90, respectively. ⁎⁎Pb.001 vs. D7 in (A), ⁎⁎Pb.001 vs. control group in (B) to (D).
14 in the neointima, appeared only in the macrophages in the aortic adventitia on Day 28, and became invisible on Day 90 after balloon injury (Fig. 6). PDGF-BB-positive expressions were mainly found in VSMCs and endothelial cells on Days 3 and 7, and went undetectable thereafter (Fig. 7).
3.6. mRNA expressions of VEGF-A and VEGFR-1 The mRNA expression of VEGF-A started to increase as early as on Day 1 after balloon injury, reached the peak level (approximately 14-fold of the control level) on Day 3,
Fig. 4. Immunohistochemical staining using anti-macrophage antibody (CD68) in the interventional groups revealed distribution of macrophages in the aortic wall (×400) on Days 1 (A), 3 (B), 7 (C), 14 (D), 28 (E), and 90 (F). CD68-positive cells were predominantly located in the adventitia near the external elastic membrane on Day 3, scattered in the neointima and adventitia on Days 7 and 14, and mainly appeared in the adventitia on Day 28. Scale bar, 50 μm.
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Fig. 5. Immunohistochemical staining using anti-VEGF-A antibody in interventional groups (×400). (A) VEGF-A expressed in VSMCs on Day 1 after balloon injury. (B–D) Intensive VEGF-A expressions were evident in VSMCs and macrophages in the aortic wall on Days 3, 7, and 14 after balloon injury. (E, F) Faint positive expressions of VEGF-A were visible in these cells on Day 90 after balloon intervention. Scale bar, 50 μm.
declined on Day 7, and reverted to the baseline level on Day 28 after balloon intervention (Fig. 8A). The pattern of mRNA expressions of VEGFR-1 was similar to that of VEGF-A (Fig. 8D). 3.7. mRNA expressions of VEGF-C and VEGFR-3 The mRNA expression level of VEGF-C began to ascend on Day 3, reached its plateau (approximately 12-fold of the control level) on Day 7, remained unchanged until Day 14, descended on Day 28, and returned to the baseline level on Day 90 after balloon intervention (Fig. 8B). The mRNA expression level of VEGFR-3 started to increase on Day 7 and peaked (approximately 13-fold of control group's level) on Day 14, decreased slightly on Day 28, and reverted to the baseline level on Day 90 after balloon injury (Fig. 8E).
3.8. mRNA expressions of PDGF-B and PDGFR-β The mRNA expression level of PDGF-B was up-regulated as early as on Day 1, peaked (approximately 16-fold of the control level) on Day 3, declined on Day 7, and resumed its baseline level on Day 14 after balloon injury (Fig. 8C). The mRNA expression of PDGFR-β also became up-regulated on Day 1, reached its summit (approximately 11-fold of the control level) on Day 7, decreased on Day 14, and returned to the baseline level on Day 28 after balloon intervention (Fig. 8F). 3.9. Relationship between adventitial angiogenesis and lymphangiogenesis, and neointima hyperplasia There were significant correlations between the macrophage index and the I/M ratio on Days 7, 14, and 28 (r=0.87,
Fig. 6. Immunohistochemical staining using anti-VEGF-C antibody in interventional groups (×400). (A, B) No positive expressions of VEGF-C in the aortic wall were found on Days 1 and 3 after balloon injury. (C, D) VEGF-C-positive expressions mainly appeared in the macrophages in the neointima on Days 7 and 14. (E) VEGF-C-positive expressions exhibited only in the macrophages in the aortic adventitia on Day 28. (F) Expressions of VEGF-C became invisible on Day 90 after balloon injury. Scale bar, 50 μm.
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Fig. 7. Immunohistochemical staining using anti-PDGF-BB antibody in the interventional groups (×400). (A) Negative expressions of PDGF-BB on Day 1 after balloon injury. (B, C) PDGF-BB-positive expressions were mainly found in VSMCs and endothelial cells on Days 3 and 7, and went undetectable on Days 14, 28, and 90 after balloon intervention (D–F). Scale bar, 50 μm.
P =.001; r=0.79, P=.006; and r=0.68, P=.03, respectively) with the highest correlation being found on Day 7 after balloon intervention. However, such a correlation became insignificant on Day 90 after balloon injury. Ad-MV showed significant correlations with I/M ratio on Days 14, 28, and 90 (r=0.68, P=.03; r=0.67, P=.032; and r=0.81, P=.004, respectively) with the highest correlation being found on Day 90 after balloon intervention. Similarly, significant correlations were found between Ad-LV and I/M
ratio on Days 14, 28, and 90 (r=0.78, P=.007; r=0.75, P=.013; and r=0.79, P=.006, respectively) with the highest correlation being found on Day 90 after balloon injury. In order to clarify whether Ad-MV and Ad-LV were independent factors of neointima hyperplasia, multiple regression analysis was performed using I/M ratio as a dependent variable and the result demonstrated that Ad-MV and Ad-LV were independent factors of neointima hyperplasia (R2=0.89, P=.000).
Fig. 8. mRNA expressions of VEGF-A (A), VEGF-C (B), PDGF-B (C), VEGFR-1 (D), VEGFR-3 (E), and PDGFR-β (F) after balloon injury. Cont: control group; D1–D90: Days 1 to 90, respectively. #Pb.05, ⁎Pb.01, ⁎⁎Pb.001 vs. control group.
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4. Discussion A major finding of the present study is that aortic neointimal hyperplasia following balloon-induced endothelial injury was accompanied not only by significant adventitial angiogenesis but also by extensive proliferation of Ad-LVs, and both Ad-MV and Ad-LV had good correlation with and were independent factors of neointimal hyperplasia. Adventitial microvessels and lymphatic vessels increased significantly and maintained a high density for at least 90 days after balloon injury. Inflammatory cells such as macrophages increased significantly in the whole vascular wall after balloon intervention but existed for a longer time in the adventitia than in the intima. VEGF-A, VEGF-C, and PDGF-BB were expressed in inflammation-related cells including VSMCs, macrophages, and fibroblasts, and their mRNA expression levels were up-regulated after balloon intervention. All these findings indicated an important role played by adventitial angiogenesis and lymphangiogenesis in the pathogenesis of neointimal hyperplasia induced by endothelial injury. To our knowledge, this is the first study in the literature to report the causal relationship between adventitia lymphangiogenesis and neointima hyperplasia after intimal injury. It is always important to establish an optimal animal model in basic research. Although mice have been widely used in animal studies, the thickness of the aortic wall in mice is too thin to allow a detailed histopathological observation in the adventitial layer. The lack of appropriate antibodies to mark the endothelial cells of the lymphatic vessels in rabbits precluded the use of this type of animals. Therefore, we finally established an animal model in rats to investigate the relationship between adventitial angiogenesis and lymphangiogenesis and neointima hyperplasia. Although the lymphatic system has been recognized for several decades, only recently was LYVE-1 discovered as a specific lymphatic endothelium marker [21]. Several studies have revealed that the vascular expression pattern displayed by the antibody against LYVE-1 and antibody against CD34, a specific blood vessel marker, was mutually exclusive [14–17]. Thus, LYVE-1 and CD34 were expressed only in lymphatic vessels and blood vessels, respectively. As a result, LYVE-1 has been adopted in clinical studies to detect lymphatic vessel distribution [22]. For these reasons, LYVE-1 and CD34 antibodies were used in the present study to identify lymphatic vessels and blood vessels, respectively. Angiogenesis is regulated by a number of growth factors and their corresponding receptors. In arterial injury models, the expression levels of growth factors such as VEGF-A and PDGF-BB and their corresponding receptors have been found to be up-regulated [23–25]. The inflated balloon going through the aorta can remove most of the endothelial cells and injure the VSMCs located in the innermost layers of the media. As a result, acute inflammation may happen in the aortic wall [26,27], in which endothelial cells, VSMCs,
macrophages, and T cells are activated and start to express growth factors. In the present study, VEGF-A were initially expressed by VSMCs in the tunica media, fibroblasts in the adventitia, and macrophages in the media and adventitia early after balloon intervention. Thereafter, considerable VEGF-A-expressing macrophages were present in the neointima and adventitia. These VEGF-A-positive cells can potentially stimulate angiogenesis and lymphangiogenesis through VEGFR-1 and VEGFR-2 receptors, respectively [28]. VEGF-C has been demonstrated to play a critical role in lymphangiogenesis via activation of VEGFR-3, which is expressed mainly by lymphatic endothelial cells in normal adult tissues [29]. VEGFR-3 signaling is important for the development of the embryonic lymphatic system, lymphatic regeneration in the adult, and tumor lymphangiogenesis [30]. In the present study, the mRNA expression levels of VEGF-C and VEGFR-3 increased significantly, thereby promoting active lymphangiogenesis. Macrophages and fibroblasts with VEGF-C-positive staining were found to be present early in the neointima, and most of these positive staining macrophages moved to the adventitia later after endothelial injury, which suggests that macrophages play an important role in adventitial lymphangiogenesis. One of the major effects of PDGF-BB in vivo is to stimulate the migration of SMC from the media into the intima with a modest effect on SMC proliferation [31]. PDGF-BB can also promote angiogenesis and lymphangiogenesis in two ways [32,33]: First, PDGF-BB can generate functional new blood vessels in vivo by a potent mitogenic and chemotactic effect on VSMCs, leading to maturation and stabilization of neogenetic unstable vessels [34,35]. Second, PDGF-BB may be a survival factor for newly formed lymphatic vessels since PDGF-BB activates the Akt kinase, which promotes antiapoptotic signaling. In the present study, high mRNA expression of PDGF-B and intensive positive staining of PDGF-BB in VSMCs indicate that PDGF-BB is indeed an important promoter of adventitial angiogenesis and lymphangiogenesis. In the current study, VEGF-A-expressing macrophages were found to aggregate in the aortic intima at the early stage, and then they infiltrated into the area of neogenetic microvessels and lymphatic vessels in the adventitia, which suggests that adventitial angiogenesis and lymphangiogenesis after balloon injury may be in fact induced by inflammatory cells originating from intimal inflammation. It has been reported that mononuclear cells are a source of VEGF-C [36]. In this study, VEGF-C-positive expressions were mainly found in macrophages and fibroblast cells located mainly in the area of neogenetic microvessels and lymphatic vessels in the adventitia. It has been suggested that macrophages play a dual role in lymphangiogenesis induced by inflammation by secreting lymphangiogenic growth factor VEGF-C, which stimulates the growth of existing lymphatic endothelial cells, and by trans-differentiating to
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lymphatic endothelial cells, which incorporate into the lymphatic endothelium [37]. A growing body of evidence has lent support to the view that atherosclerosis is a chronic inflammatory disease [38,39]. Endothelial injury, which may cause intimal inflammation, has been regarded as an early feature of atherosclerosis. Our results clearly demonstrated that the I/ M ratio had the highest correlation with the macrophage index in the early phase of balloon injury, while it had the highest correlation with the number of Ad-MVs in the late phase of balloon injury. These results reveal that accumulation of VSMCs in the intima is largely dependent on the stimulation of growth factors released by inflammatory cells in the early phase, while adventitial neovascularization is responsible for the promotion of intimal hyperplasia in the late phase [40]. Adventitial microvessels promote neointimal hyperplasia by delivering fibroblasts and circulating smooth muscle progenitor cells as well as inflammatory cells and cytokines into the aortic wall [41]. The number of Ad-LVs had a high and independent correlation with the I/M ratio, but the mechanisms involved may be quite different from those underlying the relationship between angiogenesis and intimal hyperplasia. Lymphatic vessels have been confirmed to play an important role in the afferent phase of the immune response, while blood vessels act in the efferent phase of the immune response by delivering inflammatory cells and factors [14]. Our reasoning is that Ad-LVs may drain local inflammatory cells and cytokines to the lymphatic nodes and lymphoid tissues where inflammatory cells can be sensitized and activated. On the other hand, blood vessels may deliver sensitized inflammatory cells and cytokines to the inflammatory site of the vascular wall. Therefore, both blood and lymphatic vessels constitute a complete circle of immune response, by which the inflammatory cells and cytokines are effectively delivered and their effects magnified. Under certain circumstances, however, this situation may lead to a vicious cycle of inflammation such as in atherosclerosis, resulting in perpetuating intimal hyperplasia and vascular remodeling. There were several limitations in our study. First, although intimal hyperplasia induced by balloon injury is similar to the early lesion of atherosclerosis, it is different from other animal models of atherosclerosis induced by a high cholesterol diet. The latter method was not used in this study simply because it is very difficult, if not impossible, to induce dyslipidemia-related atherosclerosis in rats. Second, the impact of stimulation or inhibition of angiogenesis and/or lymphangiogenesis on intimal hyperplasia is not clear despite the fact that angiogenesis and lymphangiogenesis have a close spatial and temporal correlation with intimal inflammation and hyperplasia as revealed in the present study. Therefore, further therapeutic interventions are required to explore the causal relationship between neointima hyperplasia and adventitial angiogenesis and lymphangiogenesis..
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Original Article
Aortic adventitial angiogenesis and lymphangiogenesis promote intimal inflammation and hyperplasia Xinsheng Xu a,b,1 , Huixia Lu a,1 , Huili Lin a,c , Xiaolu Li a,d , Mei Ni a , Huiwen Sun a , Changjiang Li a , Hong Jiang a , Fuhai Li a , Mei Zhang a , Yuxia Zhao a,⁎, Yun Zhang a,⁎ a
The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Shandong University Qilu Hospital, Jinan, Shandong, 250012, China b Department of Cardiology, Dongying People's Hospital, Dongying, Shandong, China c Department of Cardiology, the Second Affiliated Hospital of Fujian Medical University, Fujian, China d Department of Emergency, Qianfoshan Hospital, Jinan, Shandong, China Received 15 January 2008; received in revised form 18 June 2008; accepted 18 July 2008
Abstract Introduction: Adventitial inflammation is known to influence neointimal formation and vascular remodeling. The present study was aimed to clarify the relationship between neointima hyperplasia and adventitial angiogenesis and lymphangiogenesis after balloon-induced aortic endothelial injury. Methods: Seventy male Wistar rats were randomly divided into six interventional groups and one control group. The intimal area/medial area ratio (I/M ratio), the adventitial macrophage index, and the number of adventitial microvessels (Ad-MV) and lymphatic vessels (Ad-LV) in the aorta were measured, and the mRNA expressions of VEGF-A, VEGFR-1, VEGF-C, VEGFR-3, PDGFB, and PDGFR-β in the aortic wall were quantified by real-time RT-PCR. Results: Compared with the control group, the I/M ratio, macrophage index, Ad-MV, Ad-LV, and the mRNA expressions of VEGF-A, VEGFR-1, VEGF-C, VEGFR-3, PDGF-B, and PDGFR-β in interventional groups increased significantly after balloon-induced injury. I/M ratio showed significant correlations with Ad-MV and Ad-LV after balloon intervention. Multiple linear regression analysis indicated that Ad-MV and Ad-LV were independent factors of intimal hyperplasia. Conclusion: Adventitial angiogenesis and lymphangiogenesis are induced by intimal inflammation after balloon injury, and these neogenetic vessels in turn promote intimal inflammation and hyperplasia probably via delivery and activation of inflammatory cells. © 2009 Elsevier Inc. All rights reserved. Keywords: Angiogenesis; Lymphangiogenesis; Intimal injury; Inflammation
1. Introduction It is well known that lymphatic vessels, which commonly accompany blood vessels in tissues, drain extravasated This study was supported by the National 973 Basic Research Program of China (No.2005CB523301), the National High-tech Research and Development Program of China (No.2006AA02A406), the Program of Introducing Talents of Discipline to Universities (No.B07035), and grants from the National Natural Science Foundation of China (Nos. 30570747, 30670873, 30772810, 30700301). ⁎ Corresponding authors. Shandong University Qilu Hospital, Jinan, No. 107, Wen Hua Xi Road, Jinan, Shandong, 250012, PR China. E-mail address: [email protected] (Y. Zhang). 1 The first two authors contributed to this work equally. 1054-8807/08/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.carpath.2008.07.004
bloodless fluid, protein, and inflammatory cells from the tissues and take an active part in inflammatory diseases [1,2]. Recent studies have reported that lymphatic vessels were enlarged in chronic inflammatory skin diseases and that rejected kidney transplants contained abundant lymphatic vessels [3,4]. Lately, it was found that lymphangiogenesis prevented mucosal edema and these neogenetic lymphatic vessels persisted in chronic airway inflammation [5]. Although the presence of adventitial inflammatory infiltration in close proximity to intimal atherosclerotic plaques has been recognized for more than four decades [6], the clinical relevance of this finding was not clear until recently when inflammatory infiltration was proved to be present in adventitia after coronary balloon injury [7]. Inflammatory
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cells like macrophages can secret cytokines and growth factors to promote angiogenesis and lymphangiogenesis [8,9]. However, the relationship between adventitial lymphangiogenesis and intimal hyperplasia is still unknown. In the present study, we tested the hypothesis that adventitial angiogenesis and lymphangiogenesis are initially induced by intimal inflammation after endothelial injury and these neogenetic vessels in turn promote intimal inflammation and neointimal hyperplasia by transportation and activation of inflammatory cells. 2. Materials and methods 2.1. Animal model Seventy male Wistar rats weighing 450 to 500 g were obtained from the Animal Center of Shandong University and divided randomly into seven groups with 10 rats in each group. Group 1 to Group 6 rats underwent balloon-induced aortic endothelial injury, while Group 7 rats received only sham operation and served as a control group. Rats were housed under conditions of constant room temperature (22°C) and a 12-h dark/12-h light cycle, and fed a normal diet. All rats underwent anesthesia with intraperitoneal injection of pentobarbital (30 mg kg−1) and the left external carotid artery was exposed to introduce a 2-F embolectomy catheter (Baxter Healthcare Corp., Irvine, CA, USA) into the distal abdominal aorta. The balloon catheter was then inflated with 50 μl of saline and went back and forth in the abdominal aorta three times to induce endothelial injury in Group 1 to Group 6 rats [10–13]. Sham operation was performed in Group 7 rats that underwent the same catheterization procedure but without balloon inflation. Group 1 to Group 6 rats were euthanized by intraperitoneal injection of a lethal dose of phenobarbitone on Days 1, 3, 7, 14, 28, and 90 after the procedure, respectively, and Group 7 rats were euthanized on Day 1 after sham operation. After the abdominal aorta was harvested, aortic segments were snap frozen in liquid nitrogen and stored at −80°C for quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, or fixed with 4% phosphate-buffered formaldehyde for 24 h for immunohistochemical and morphometric analysis. All animal procedures in the present study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and the Guide for the Care and Use of Laboratory Animals published by the Chinese National Institutes of Health. 2.2. Morphometric analysis Aortic segments were embedded in paraffin and cut into 6μm-thick sections which were stained with hematoxylin and eosin, and analyzed using a computer-assisted morphometric analysis system (Image-Pro Plus 5) with particular attention to the intimal and the medial thickness. Vascular area within the external elastic lamina (EELA) and the internal elastic
lamina (IELA) as well as the lumen area (LA) was measured. The I/M ratio was calculated as: I/M=(IELA−LA)/(EELA −IELA). All parameters were measured from three sections selected from the proximal, the middle, and the distal portion of the aortic segment, and the values averaged. 2.3. Antibodies The primary antibodies used were as follows: goat polyclonal antibody against human CD34 (1:200; Santa Cruz Biotechnology, Santa Cruz, CA) to identify the blood endothelial cells; rabbit polyclonal antibody against human LYVE-1 (1:100; Santa Cruz Biotechnology) to tag lymphatic endothelial cells; rabbit polyclonal antibody against human CD68 (1:400; Santa Cruz Biotechnology) to mark macrophages; mouse monoclonal anti-α-SMA antibody (1:2000; ab7817, Santa Cruz Biotechnolgy) to label vascular smooth muscle cells (VSMCs); rabbit polyclonal antibody against mouse VEGF-A (1:50; Santa Cruz Biotechnology); rabbit polyclonal antibody against human VEGF-C (1:50; Santa Cruz Biotechnology), and rabbit polyclonal antibody against PDGF-BB (1:100; ab15499, Abcam, Cambridge, UK). Second antibodies against IgG in rabbits, mice, and goats were obtained from Beijing Zhongshan Biotechnology Co., Ltd. (ZSBIO, Beijing Zhongshan Golden Bridge Technology, China). 2.4. Immunohistochemistry Sections were deparaffinized and incubated with 5% goat serum or 5% BSA for 20 min to minimize the nonspecific binding to the primary antibody and incubated with the primary antibodies overnight at 4°C in a moisture chamber. The sections were then incubated with the appropriate secondary antibody for 30 min at room temperature. To inhibit any endogenous peroxidase activity, the sections were incubated with 0.3% H2O2 in absolute methanol for 30 min. A peroxidase substrate solution containing 0.02% H2O2 and 0.1% 3,3′-diaminobenzidine tetrahydrochloride (ZSBIO) in PBS was applied to display the reaction product with a brown color, and the sections were then counterstained with hematoxylin. Incubation with PBS instead of the primary antibody was used as a negative control. 2.5. Quantification of adventitial angiogenesis and lymphangiogenesis The total number of microvessels (CD34+) (Fig. 1A) or lymph vessels (LYVE-1+) (Fig. 1B) in the whole adventitia was counted under a light microscope at a high power magnification (×400) [14–17]. Vascular and lymph vessels were identified as morphologically circumferential brown products formed by one or more stained endothelial cells with at least one counterstained nucleus. In this case, a single brown dot was not counted. The number of adventitial microvessels (Ad-MV) and adventitial lymphatic vessels
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Fig. 1. Adventitial microvessels and lymph vessels identified by immunohistochemical staining at a high power magnification (×400). The microvessels exhibited as CD34 positive (A, arrows), while lymph vessels exhibited as LYVE-1 positive (B, arrow heads). Scale bar, 50 μm.
(Ad-LV) from three sections was counted and the mean values derived. All counting was performed by two independent investigators and the values were averaged. 2.6. Real-time RT-PCR Total RNA was extracted from the aortic tissue by Trizol (Invitrogen, Carlsbad, CA, USA) following the protocols recommended by the manufacturers. Reverse transcriptasepolymerase chain reaction was performed as described previously [18]. In brief, 1 μg of RNA in 20 μl of volume was reversely transcribed with oligo (dT) primer using the M-MLV Reverse Transcriptase System (Promega, Madison, WI, USA). The relative quantification of target genes was determined using the LightCycler (Roche Applied Science, USA) following the manufacturer's protocol. Real-time quantitative PCR (real-time PCR) analysis for mRNA expressions in the aortic tissue was performed using a TaqMan probe method. The reaction contained 0.2 μl of TaKaRa Ex Taq HS polymerase (TaKaRa Biotechnology,
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Dalian, China), 2 μl of 10× buffer (Mg2+ plus), 2 μl dNTP mixture (1 mM, respectively), 1 μl of forward primer, 1 μl of reverse primer, 1 μl of probe, and 1 μl of cDNA in a 20-μl final volume. The conditions for real-time PCR were as follows: denaturation at 95°C for 30 s, and in the following 50 cycles, denaturation at 95°C for 0 s, annealing for 10 s, and extension at 72°C for 10 s. Primer and probe sequence (Sangon, Shanghai, China), PCR product size, annealing temperature, and GenBank Access Number for each primer are listed in Table 1. Target gene expressions were calculated using the 2ΔΔCt method and expressed as N-fold difference between experimental groups (Group 1 to Group 6) and control group (Group 7) after normalizing to reference gene expressions [19]. 2.7. Statistical analysis Data analysis was conducted using a statistical software package (SPSS 11.5, Chicago, IL, USA). All measurements were expressed as mean±S.D. One-way ANOVA was used to compare the differences among seven groups. The relationship between the number of Ad-MV and Ad-LV and the I/M ratio was tested using simple (Pearson's correlation) and multiple linear regression analysis. Pb.05 was considered statistically significant. 3. Results 3.1. Neointima hyperplasia after balloon injury The aortic intima appeared as a single cell layer, the IELA was intact, and the EELA was in close contact with the adventitia in the control group of rats (Fig. 2A). The
Table 1 Primers and probes in real-time PCR assays Gene (size)
Primers and probes
Annealing temperature
GenBank number
APDH (78 bp)
F 5′-ATG TAT CCG TTG TGG ATC TGA C-3′ R 5′-CCT GCT TCA CCA CCT TCT TG-3′ P 5′-TGC CGC CTG GAG AAA CCT GCC A-3′ F 5′-CGA CAG AAG GGG AGC AGA AAG-3′ R 5′-GCA CTC CAG GGC TTC ATC ATT-3′ P 5′-AGC AGC CCG CAC ACC GCA TTA GG-3′ F 5′-ACA GAA GAG GAT GAG GGT GTC T-3′ R 5′-TGA AGA GAG TTA GAA GGA GCC AAA-3′ P 5′-TGC CGA GCC ACC AAC CAG AAG GG-3′ F 5′-CAG TTT TTC AGT CCA TCA TTT-3′ R 5′-CAG TCC ATT CCC ACA GTA A-3′ P 5′-CTG CCT TGA AAA ACT GTT GCC AC-3′ F 5′-GTT TTG TGT CCC ACC CCT ACC-3′ R 5′-GCC CTC GTT GTC TGA GTT TGA-3′ P 5′-TGC CCC TTA CAG CCT CCC TAT CCA G-3′ F 5′-ACT AAG AGC GTG CGT CAG TTG-3′ R 5′-ATG CTC ACC CAG ACA AAG TAA GAA-3′ P 5′-AGA GTG AGG GAG CAA CGG CGG CA-3′ F 5′-CAA GAG TGA CAG AGA AGG CAA G-3′ R 5′-GCT GGC AGT TGA GAT GGC T-3′ P 5′-CCT CAG CGT CGT CCT CTC ATG CCC A-3′
58°C
BC059110
62°C
NM_031836
60°C
NM_019306
60°C
NM_053653
60°C
NM_053652
62°C
XM_343293
60°C
AY090783
VEGF-A (194 bp)
VEGFR-1 (175 bp)
VEGF-C (145 bp)
VEGFR-3 (76 bp)
PDGF-B (77 bp)
PDGFR-β (158 bp)
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Fig. 2. Aortic intima and adventitia in the control group and the interventional groups (hematoxylin and eosin, ×400). (A) The intima appeared as a single layer of endothelial cells in the control group. There were few microvessels in the adventitia. (B) The aortic intima was found entirely denudated on Day 1 after balloon injury. (C) Infiltration of inflammatory cells in the adventitia on Day 3 after balloon injury. (D) Neointima consisting of SMCs and macrophages was evident on Day 7 after balloon injury. Inflammatory cells surrounded Ad-MVs. (E) to (G) represent the pathological changes on Days 14, 28, and 90 after balloon injury, respectively. Intimal hyperplasia consisting of multiple layers of SMCs became prominent and persisted until Day 90 after balloon intervention. Neogenetic microvessels and lymphatic vessels were evident in the adventitia. Scale bar, 50 μm.
intima of the abdominal aorta was found to be uniformly denudated on Day 1 after balloon injury (Fig. 2B). Hyperplastic intima consisting of multiple layers of VSMC and macrophages became evident on Day 7 after balloon intervention, and these changes reached a maximal level on Day 28 (Fig. 2D–F). The I/M ratio increased significantly on Day 28 compared with that on Day 7 after balloon injury (0.56±0.06 vs. 0.17±0.02, Pb.001). However, this ratio did not show any further increase on Day 90 compared with that on Day 28 after balloon intervention (0.55±0.06 vs. 0.56±0.06, PN.05; Fig. 3A). 3.2. Adventitial microvessels after balloon injury The value of Ad-MV began to increase significantly on Day 3 after balloon injury when compared with that in the control group (12.40±1.43 vs. 7.80±1.32, Pb.001), and this difference became even greater on Day 7 and Day 14 (19.80±2.74 vs. 7.80±1.32, Pb.001; and 24.20±2.15 vs. 7.80±1.32, Pb.001) after balloon intervention. However, the value of Ad-MV reached a plateau thereafter and did not show any further increase on Day 28 and Day 90 after balloon injury when compared with that on Day 14 (PN.05, Fig. 3C). 3.3. Adventitial lymphatic vessels after balloon injury Similar to the changes in Ad-LV, the value of Ad-LV began to rise on Day 3 (6.4±1.1 vs. 2.5±0.53, Pb.001) and continued to ascend on Day 7 (12.5±1.4 vs. 2.5±0.53, Pb.001) until a plateau was reached on Day 14 (12.8±1.0 vs. 2.5±0.53, Pb.001) when compared with that in the control group. Thereafter the value of Ad-LV remained at a high level on Day 28 (12.8±1.4 vs. 12.8±1.0, PN.05) and Day 90
(12.6±1.3 vs. 12.8±1.0, PN.05, Fig. 3D) after balloon intervention when compared with that on Day 14. 3.4. Macrophage distribution in the aortic wall Immunohistochemical staining using anti-macrophage antibody (CD68) revealed that CD68-positive cells were predominantly located in the adventitia near the external elastic membrane on Day 3 and scattered in the neointima and adventitia on Days 7 and 14 (Fig. 4). Thereafter, CD68positive cells were mainly present in the adventitia on Day 28. The percentage of macrophage number to total cell number in adventitia (macrophage index) was calculated [20]. The results demonstrated that the macrophage index increased on Day 3 (4.5±0.8% vs. 0.6±0.2%, Pb.001), peaked on Day 14 (12.5±1.3% vs. 0.6±0.2%, Pb.001), maintained this level on Day 28 (12.3±1.5% vs. 0.6±0.2%, Pb.001) when compared with that in the control group, and returned to the control level on Day 90 after balloon injury (Fig. 3B). 3.5. VEGF-A, VEGF-C, and PDGF-BB expressions in the aortic wall Immunohistochemical staining revealed that VEGF-Apositive expressions were barely visible, while VEGF-C and PDGF-BB expressions were not present in the control group. On the other hand, intensive VEGF-A expressions were evident in VSMCs and macrophages in the aortic wall on Days 3, 7, and 14 after balloon injury. Thereafter, the intensity of VEGF-A expressions diminished and only faint positive expressions were visible in these cells on Day 90 after balloon intervention (Fig. 5). VEGF-C-positive expressions were mainly present in the macrophages on Days 7 and
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Fig. 3. Dynamic changes in I/M ratio in (A), adventitial macrophage index in (B), the number of microvessels in (C), and the number of lymphatic vessels in (D). Cont: control group; D1 to D90: Days 1 to 90, respectively. ⁎⁎Pb.001 vs. D7 in (A), ⁎⁎Pb.001 vs. control group in (B) to (D).
14 in the neointima, appeared only in the macrophages in the aortic adventitia on Day 28, and became invisible on Day 90 after balloon injury (Fig. 6). PDGF-BB-positive expressions were mainly found in VSMCs and endothelial cells on Days 3 and 7, and went undetectable thereafter (Fig. 7).
3.6. mRNA expressions of VEGF-A and VEGFR-1 The mRNA expression of VEGF-A started to increase as early as on Day 1 after balloon injury, reached the peak level (approximately 14-fold of the control level) on Day 3,
Fig. 4. Immunohistochemical staining using anti-macrophage antibody (CD68) in the interventional groups revealed distribution of macrophages in the aortic wall (×400) on Days 1 (A), 3 (B), 7 (C), 14 (D), 28 (E), and 90 (F). CD68-positive cells were predominantly located in the adventitia near the external elastic membrane on Day 3, scattered in the neointima and adventitia on Days 7 and 14, and mainly appeared in the adventitia on Day 28. Scale bar, 50 μm.
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Fig. 5. Immunohistochemical staining using anti-VEGF-A antibody in interventional groups (×400). (A) VEGF-A expressed in VSMCs on Day 1 after balloon injury. (B–D) Intensive VEGF-A expressions were evident in VSMCs and macrophages in the aortic wall on Days 3, 7, and 14 after balloon injury. (E, F) Faint positive expressions of VEGF-A were visible in these cells on Day 90 after balloon intervention. Scale bar, 50 μm.
declined on Day 7, and reverted to the baseline level on Day 28 after balloon intervention (Fig. 8A). The pattern of mRNA expressions of VEGFR-1 was similar to that of VEGF-A (Fig. 8D). 3.7. mRNA expressions of VEGF-C and VEGFR-3 The mRNA expression level of VEGF-C began to ascend on Day 3, reached its plateau (approximately 12-fold of the control level) on Day 7, remained unchanged until Day 14, descended on Day 28, and returned to the baseline level on Day 90 after balloon intervention (Fig. 8B). The mRNA expression level of VEGFR-3 started to increase on Day 7 and peaked (approximately 13-fold of control group's level) on Day 14, decreased slightly on Day 28, and reverted to the baseline level on Day 90 after balloon injury (Fig. 8E).
3.8. mRNA expressions of PDGF-B and PDGFR-β The mRNA expression level of PDGF-B was up-regulated as early as on Day 1, peaked (approximately 16-fold of the control level) on Day 3, declined on Day 7, and resumed its baseline level on Day 14 after balloon injury (Fig. 8C). The mRNA expression of PDGFR-β also became up-regulated on Day 1, reached its summit (approximately 11-fold of the control level) on Day 7, decreased on Day 14, and returned to the baseline level on Day 28 after balloon intervention (Fig. 8F). 3.9. Relationship between adventitial angiogenesis and lymphangiogenesis, and neointima hyperplasia There were significant correlations between the macrophage index and the I/M ratio on Days 7, 14, and 28 (r=0.87,
Fig. 6. Immunohistochemical staining using anti-VEGF-C antibody in interventional groups (×400). (A, B) No positive expressions of VEGF-C in the aortic wall were found on Days 1 and 3 after balloon injury. (C, D) VEGF-C-positive expressions mainly appeared in the macrophages in the neointima on Days 7 and 14. (E) VEGF-C-positive expressions exhibited only in the macrophages in the aortic adventitia on Day 28. (F) Expressions of VEGF-C became invisible on Day 90 after balloon injury. Scale bar, 50 μm.
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Fig. 7. Immunohistochemical staining using anti-PDGF-BB antibody in the interventional groups (×400). (A) Negative expressions of PDGF-BB on Day 1 after balloon injury. (B, C) PDGF-BB-positive expressions were mainly found in VSMCs and endothelial cells on Days 3 and 7, and went undetectable on Days 14, 28, and 90 after balloon intervention (D–F). Scale bar, 50 μm.
P =.001; r=0.79, P=.006; and r=0.68, P=.03, respectively) with the highest correlation being found on Day 7 after balloon intervention. However, such a correlation became insignificant on Day 90 after balloon injury. Ad-MV showed significant correlations with I/M ratio on Days 14, 28, and 90 (r=0.68, P=.03; r=0.67, P=.032; and r=0.81, P=.004, respectively) with the highest correlation being found on Day 90 after balloon intervention. Similarly, significant correlations were found between Ad-LV and I/M
ratio on Days 14, 28, and 90 (r=0.78, P=.007; r=0.75, P=.013; and r=0.79, P=.006, respectively) with the highest correlation being found on Day 90 after balloon injury. In order to clarify whether Ad-MV and Ad-LV were independent factors of neointima hyperplasia, multiple regression analysis was performed using I/M ratio as a dependent variable and the result demonstrated that Ad-MV and Ad-LV were independent factors of neointima hyperplasia (R2=0.89, P=.000).
Fig. 8. mRNA expressions of VEGF-A (A), VEGF-C (B), PDGF-B (C), VEGFR-1 (D), VEGFR-3 (E), and PDGFR-β (F) after balloon injury. Cont: control group; D1–D90: Days 1 to 90, respectively. #Pb.05, ⁎Pb.01, ⁎⁎Pb.001 vs. control group.
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4. Discussion A major finding of the present study is that aortic neointimal hyperplasia following balloon-induced endothelial injury was accompanied not only by significant adventitial angiogenesis but also by extensive proliferation of Ad-LVs, and both Ad-MV and Ad-LV had good correlation with and were independent factors of neointimal hyperplasia. Adventitial microvessels and lymphatic vessels increased significantly and maintained a high density for at least 90 days after balloon injury. Inflammatory cells such as macrophages increased significantly in the whole vascular wall after balloon intervention but existed for a longer time in the adventitia than in the intima. VEGF-A, VEGF-C, and PDGF-BB were expressed in inflammation-related cells including VSMCs, macrophages, and fibroblasts, and their mRNA expression levels were up-regulated after balloon intervention. All these findings indicated an important role played by adventitial angiogenesis and lymphangiogenesis in the pathogenesis of neointimal hyperplasia induced by endothelial injury. To our knowledge, this is the first study in the literature to report the causal relationship between adventitia lymphangiogenesis and neointima hyperplasia after intimal injury. It is always important to establish an optimal animal model in basic research. Although mice have been widely used in animal studies, the thickness of the aortic wall in mice is too thin to allow a detailed histopathological observation in the adventitial layer. The lack of appropriate antibodies to mark the endothelial cells of the lymphatic vessels in rabbits precluded the use of this type of animals. Therefore, we finally established an animal model in rats to investigate the relationship between adventitial angiogenesis and lymphangiogenesis and neointima hyperplasia. Although the lymphatic system has been recognized for several decades, only recently was LYVE-1 discovered as a specific lymphatic endothelium marker [21]. Several studies have revealed that the vascular expression pattern displayed by the antibody against LYVE-1 and antibody against CD34, a specific blood vessel marker, was mutually exclusive [14–17]. Thus, LYVE-1 and CD34 were expressed only in lymphatic vessels and blood vessels, respectively. As a result, LYVE-1 has been adopted in clinical studies to detect lymphatic vessel distribution [22]. For these reasons, LYVE-1 and CD34 antibodies were used in the present study to identify lymphatic vessels and blood vessels, respectively. Angiogenesis is regulated by a number of growth factors and their corresponding receptors. In arterial injury models, the expression levels of growth factors such as VEGF-A and PDGF-BB and their corresponding receptors have been found to be up-regulated [23–25]. The inflated balloon going through the aorta can remove most of the endothelial cells and injure the VSMCs located in the innermost layers of the media. As a result, acute inflammation may happen in the aortic wall [26,27], in which endothelial cells, VSMCs,
macrophages, and T cells are activated and start to express growth factors. In the present study, VEGF-A were initially expressed by VSMCs in the tunica media, fibroblasts in the adventitia, and macrophages in the media and adventitia early after balloon intervention. Thereafter, considerable VEGF-A-expressing macrophages were present in the neointima and adventitia. These VEGF-A-positive cells can potentially stimulate angiogenesis and lymphangiogenesis through VEGFR-1 and VEGFR-2 receptors, respectively [28]. VEGF-C has been demonstrated to play a critical role in lymphangiogenesis via activation of VEGFR-3, which is expressed mainly by lymphatic endothelial cells in normal adult tissues [29]. VEGFR-3 signaling is important for the development of the embryonic lymphatic system, lymphatic regeneration in the adult, and tumor lymphangiogenesis [30]. In the present study, the mRNA expression levels of VEGF-C and VEGFR-3 increased significantly, thereby promoting active lymphangiogenesis. Macrophages and fibroblasts with VEGF-C-positive staining were found to be present early in the neointima, and most of these positive staining macrophages moved to the adventitia later after endothelial injury, which suggests that macrophages play an important role in adventitial lymphangiogenesis. One of the major effects of PDGF-BB in vivo is to stimulate the migration of SMC from the media into the intima with a modest effect on SMC proliferation [31]. PDGF-BB can also promote angiogenesis and lymphangiogenesis in two ways [32,33]: First, PDGF-BB can generate functional new blood vessels in vivo by a potent mitogenic and chemotactic effect on VSMCs, leading to maturation and stabilization of neogenetic unstable vessels [34,35]. Second, PDGF-BB may be a survival factor for newly formed lymphatic vessels since PDGF-BB activates the Akt kinase, which promotes antiapoptotic signaling. In the present study, high mRNA expression of PDGF-B and intensive positive staining of PDGF-BB in VSMCs indicate that PDGF-BB is indeed an important promoter of adventitial angiogenesis and lymphangiogenesis. In the current study, VEGF-A-expressing macrophages were found to aggregate in the aortic intima at the early stage, and then they infiltrated into the area of neogenetic microvessels and lymphatic vessels in the adventitia, which suggests that adventitial angiogenesis and lymphangiogenesis after balloon injury may be in fact induced by inflammatory cells originating from intimal inflammation. It has been reported that mononuclear cells are a source of VEGF-C [36]. In this study, VEGF-C-positive expressions were mainly found in macrophages and fibroblast cells located mainly in the area of neogenetic microvessels and lymphatic vessels in the adventitia. It has been suggested that macrophages play a dual role in lymphangiogenesis induced by inflammation by secreting lymphangiogenic growth factor VEGF-C, which stimulates the growth of existing lymphatic endothelial cells, and by trans-differentiating to
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lymphatic endothelial cells, which incorporate into the lymphatic endothelium [37]. A growing body of evidence has lent support to the view that atherosclerosis is a chronic inflammatory disease [38,39]. Endothelial injury, which may cause intimal inflammation, has been regarded as an early feature of atherosclerosis. Our results clearly demonstrated that the I/ M ratio had the highest correlation with the macrophage index in the early phase of balloon injury, while it had the highest correlation with the number of Ad-MVs in the late phase of balloon injury. These results reveal that accumulation of VSMCs in the intima is largely dependent on the stimulation of growth factors released by inflammatory cells in the early phase, while adventitial neovascularization is responsible for the promotion of intimal hyperplasia in the late phase [40]. Adventitial microvessels promote neointimal hyperplasia by delivering fibroblasts and circulating smooth muscle progenitor cells as well as inflammatory cells and cytokines into the aortic wall [41]. The number of Ad-LVs had a high and independent correlation with the I/M ratio, but the mechanisms involved may be quite different from those underlying the relationship between angiogenesis and intimal hyperplasia. Lymphatic vessels have been confirmed to play an important role in the afferent phase of the immune response, while blood vessels act in the efferent phase of the immune response by delivering inflammatory cells and factors [14]. Our reasoning is that Ad-LVs may drain local inflammatory cells and cytokines to the lymphatic nodes and lymphoid tissues where inflammatory cells can be sensitized and activated. On the other hand, blood vessels may deliver sensitized inflammatory cells and cytokines to the inflammatory site of the vascular wall. Therefore, both blood and lymphatic vessels constitute a complete circle of immune response, by which the inflammatory cells and cytokines are effectively delivered and their effects magnified. Under certain circumstances, however, this situation may lead to a vicious cycle of inflammation such as in atherosclerosis, resulting in perpetuating intimal hyperplasia and vascular remodeling. There were several limitations in our study. First, although intimal hyperplasia induced by balloon injury is similar to the early lesion of atherosclerosis, it is different from other animal models of atherosclerosis induced by a high cholesterol diet. The latter method was not used in this study simply because it is very difficult, if not impossible, to induce dyslipidemia-related atherosclerosis in rats. Second, the impact of stimulation or inhibition of angiogenesis and/or lymphangiogenesis on intimal hyperplasia is not clear despite the fact that angiogenesis and lymphangiogenesis have a close spatial and temporal correlation with intimal inflammation and hyperplasia as revealed in the present study. Therefore, further therapeutic interventions are required to explore the causal relationship between neointima hyperplasia and adventitial angiogenesis and lymphangiogenesis..
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