doi: 10.1161/01.cir.0000032914.33237.3b
(Circulation. 2002;106:I-259.)
© 2002 American Heart Association, Inc.
Aortic and Peripheral Vascular Surgery |
Expression of Peroxisome Proliferator-Activated Receptor-
in Vascular Smooth Muscle Cells Is Upregulated in Cystic Medial Degeneration of Annuloaortic Ectasia in Marfan Syndrome
From the Departments of Cardiology (Y.S., H.N., Y.A., K.U., A.S., H. Kasanuki), Cardiovascular Surgery (S.A., H. Kurosawa), and Pathology (T.N.), The Heart Institute of Japan, Tokyo Women’s Medical University, Tokyo, Japan.
Correspondence to Yasunari Sakomura, MD, PhD, Heart Institute of Japan, Department of Cardiology, Tokyo Women’s Medical University, 8-1, Kawada-cho, Shinjyuku-ku, Tokyo, 162-8666, Japan. E-mail msakomur{at}hij.twmu.ac.jp
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Abstract |
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Background Cystic medial degeneration (CMD) is a histological abnormality that is common in annuloaortic ectasia (AAE) and aortic dissection with Marfan syndrome. Apoptosis and loss of vascular smooth muscle cells (VSMCs) is one of the features of CMD, but little is known about its pathogenesis. Peroxisome proliferator-activated receptor-
(PPAR), a transcription factor of the nuclear receptor superfamily, has been reported to show antiproliferative effects on VSMCs as well as anti-inflammatory effects on macrophages. PPAR agonist has been recently reported to induce apoptosis of cultured VSMCs.
Methods We examined the histopathology of ascending aortas in AAE of Marfan patients (n=21) and control patients (n=6) at surgery. RT-PCR was performed to demonstrate expression of PPAR in CMD. Localization of PPAR was determined by double immunostaining using antibodies against PPAR and cell-specific markers (ie, SMCs, macrophages, and T lymphocytes).
Results PPAR expression was upregulated in AAE samples but minimal in control samples by RT-PCR (P=0.07). Immunoreactivity against PPAR in numerous nuclei of VSMCs was observed in CMD lesions. Severity of CMD correlated with positive immunoreactivity of PPAR in medial VSMCs (P=0.03). No inflammatory cells (ie, macrophages or T lymphocytes) were detected in CMD lesions.
Conclusion PPAR expression is upregulated in SMCs of CMD without any inflammatory response. Activated PPAR in VSMCs might be involved in the pathogenesis of CMD in Marfan’s aortas. Regulation of PPAR might lead to clinical implication in protection against progression of AAE.
Key Words: peroxisome proliferator-activated receptor- • vascular smooth muscle cell • Marfan syndrome
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Introduction |
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Cystic medial degeneration (CMD) is an important histological abnormality common in annuloaortic ectasia (AAE) with Marfan syndrome. The abnormality is also observed in congenital aortic disease,1 atherosclerosis, and aging.2 These aortic diseases develop as consequences of disruption of medial elastic layers in association with loss of vascular smooth muscle cells (VSMCs) and accumulation of proteoglycans.
Apoptosis and loss of VSMCs is one of the features of CMD,3,4 but little is known about its pathogenesis. It is widely accepted that apoptosis is a major mechanism for the control of cell number in developing and mature tissues under physiological and pathological conditions.5 Vascular remodeling in the process of angiogenesis and during vascular diseases is thought to involve apoptosis of VSMCs. Some reports have suggested that apoptosis may play an important role in the pathogenesis of aortic dilatation in patients with Marfan’s syndrome and in congenital aortic diseases.6
Peroxisome proliferator-activated receptor- (PPAR), a transcription factor of the nuclear receptor superfamily, has been reported to show antiproliferative7,8 and antimigratory effects on VSMCs9 as well as anti-inflammatory effects on macrophages (M
s).10 PPAR agonist has been recently reported to induce apoptosis of cultured VSMCs11 and attenuate the development of intimal hyperplasia in animal models of balloon catheter–induced vascular injury.12
In this study, we examined whether expression of PPAR in VSMCs is upregulated in CMD of AAE in Marfan syndrome. We demonstrate that aortic tissue samples obtained from Marfan patients express PPAR in VSMCs, which correlates with the degree of CMD. We discuss its implication in Marfan’s aorta.
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Methods |
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Subjects
We examined the histopathology of nondissecting ascending aortas in AAE of 21 Marfan patients compatible with the Gherent diagnostic criteria13 at Bentall’s operation (16 men and 5 women; age range, 21–42 years; mean age, 32.8±7.4) and in 6 control patients with aortic regurgitation and mild aortic dilatation at aortic valve and graft replacement (all of 6 men, 36–56 years; mean age, 48.8±11.0). All operations were done at Tokyo Women’s Medical University Hospital between 1995 and 2000. Range of maximum aortic diameter in Marfan patients was measured by chest computed tomography or magnetic resonance imaging between 45 and 90 mm (66.9±13.2 mm); in control patients, between 40 and 57 mm (51.3±7.9 mm). This study was approved by the institutional guidelines of Tokyo Women’s Medical University, and all patients gave oral informed consent.
Histological Examination
Paraformaldehyde-fixed aortas of 21 AAE and 6 control patients were processed for light microscopy and immunohistochemistry. In addition to hematoxylin and eosin staining, Alcian-blue staining for detection of acidic mucopolysaccharides (MPS) deposition, Victoria-blue staining for evaluation of fragmented elastic layers, and Masson’s trichrome staining for fibrosis were performed and scored on a scale of 1 to 4 points according to the severity of each finding (grading for MPS deposition: 1, minimal; 2, mild; 3, 1 cystic lesion; and 4, more than 2 cystic lesions; grading for elastic layer fragmentation: 1, minimal; 2, mild; 3, less than 1/2 medial layer; and 4, more than 1/2 medial layer; grading for fibrosis: 1, minimal; 2, mild; 3, focal fibrosis in less than 1/2 medial layer, and 4, focal fibrosis more than 1/2 medial layer). The total score was summated as the CMD score to assess the degree of CMD.
Immunohistochemical Analysis
Nuclear localization of PPAR was determined by immunostaining with monoclonal antibody against PPAR (Santa Cruz Biotechnology, Inc) by using an LSAB kit (Dako Japan Co Ltd.). VSMCs were identified by immunoreactivity for mature form of smooth muscle myosin heavy chain antibody (SM1; donated by Dr Ryozo Nagai, Tokyo University, Tokyo). CD68 (clone KP-1, Dako Co.) and CD45RO (clone UCHL-1, Dako Co.) were immunostained for detection of Ms and T lymphocytes, respectively. The reaction was visualized with deaminobenzidine, and nuclei were counterstained with hematoxylin. To determine the proportion of PPAR-positive VSMCs for each sample, the total number of SM1-positive cells in the aortic media was counted for each section. Six fields per section were examined at X200 magnification, and measurements were performed independently by 2 investigators. Their observations were averaged. The investigators performing histological evaluation were blinded to the clinical data.
Reverse Transcription Polymerase Chain Reaction
Media of aortic tissues from 8 Marfan and 3 control patients were separated from both intima and adventitia to avoid contamination of endothelium and fatty tissue. Total RNA from these tissues was isolated by the single-step guanidinium thiocyanate-phenol-chloroform method by using RNAzol (TelTest, Friendswood, Tex). RT-PCR was performed to demonstrate expression of PPAR in CMD. Two micrograms of total RNA was reverse-transcribed into cDNA with 1U/mL of reverse transcriptase (Superscript, Gibco-BRL, Gaithersburg, Md) at 37 °C for 1 hour in standard buffer. The PCR reaction was carried out with Ready To Go PCR beading (Pharmacia Biotech). For the amplification of human PPAR cDNA, 2 primers (5'-AAC TGC GGG GAA ACT TGG GAG ATT CTC C-3' and 5'- AAT AAT AAG GTG GAG ATG CAG GCT CC-3') were used. The DNA fragments of human PPAR cDNA (351 bp) and ms16 (a housekeeping gene as internal control: 103 bp) were amplified with the oligonucleotide primers as described previously.14,15 The PCR products were run on 2.0% agarose gels.
Statistical Analysis
Analyses were performed with the SAS System 6.12 (SAS Institute Inc., Cary, NC). The data were presented as frequency or means± SEM. Chi-square test or Fisher’s exact probability test was applied for dichotomous data. Student’s t test or Welch’s t test was used for continuous data. The F test was used for homogeneity of variance testing.
Spearman correlation coefficients (2-tailed) were used to evaluate the correlation between the CMD score and the percentage of PPAR-positive VSMCs and between aortic diameter and relative PPAR mRNA expression. Two-tailed P values<0.05 were considered statistically significant. >
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Results |
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Light Microscopy and Immunohistochemistry in Marfan’s Aortas
Typical CMD lesions with more than grade 3 MPS deposition and more than 7 points on total CMD score were observed in aortic media in 15 of 21 patients (Figure 1A and B). Intimal thickening was minimal, and there were no fatty streaks in any ascending aortas in Marfan and control patients. The majority of cell type was VSMC in media. No inflammatory cells (ie, macrophages or T lymphocytes) or foam cells were detected in CMD lesions. A few mast cells and macrophages were seen in the adventitia (Figure 2A-D).
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Expression of PPAR in Marfan’s Aortas
RT-PCR showed that PPAR expression was upregulated in half of the AAE samples obtained from Marfan patients but minimal in 3 control samples (Figure 3A and B). Immunoreactivity against PPAR in numerous nuclei of medial VSMCs was observed in CMD lesions (Figure 1C). The percentage of PPAR-positive VSMCs was significantly higher in Marfan’s aortas than control aortas (21.3±2.7% versus 3.3±0.8%; P<0.001; Figure 4A).
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Severity of CMD, Aortic Dilatation, and PPAR Expression
PPAR expression estimated by RT-PCR showed association with maximum diameter of the ascending aorta (P=0.07; Figure 3A and B). The percentage of PPAR-positive medial SMCs correlated with the severity of CMD (P=0.03) and with maximum aortic diameter (P=0.06; Figure 4B and C).
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Discussion |
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Previous studies have demonstrated evidence that PPAR
might influence macrophage-dependent events in the development of atherosclerosis associated with inflammation.16,17 PPAR was expressed not only in normal human monocytes but also in foam cells and atherosclerotic plaques. Natural and synthetic PPAR ligands were demonstrated to inhibit expression of proinflammatory genes and to have an anti-atherogenic effect in the vessel wall.16 Several reports have shown that PPAR is expressed in cultured VSMCs18,8 and in rat neointima after balloon injury.19 This study provides the first documentation of the expression of PPAR in aneurysms of Marfan’s aorta with pathological CMD. Upregulation of PPAR expression is associated with severity of CMD and with increase of aortic diameter, suggesting that PPAR expression in medial VSMCs might be based on pathogenesis of CMD and disease progression of Marfan’s aorta. Moreover, immunohistochemical analysis of cell components revealed that there was no inflammatory cell in CMD lesion. PPAR expression in the CMD of Marfan’s aorta was not associated with inflammation, which is unique pathology.
What is the pathophysiological role of PPAR in aortic aneurysm with Marfan syndrome? Dedifferentiation of VSMCs is an important phenotypic change during the progression of athrosclerosis, restenosis,20 and aneurysm formation.21,22 Bunton et al have previously reported that fibrillin-1-deficient mouse, a mouse model of Marfan syndrome, showed phenotypic alteration of medial VSMCs preceding elastolysis.23 Segura et al have shown expression of matrix methalloproteinases in VSMCs in thoracic aortic aneurysms with Marfan syndrome that causes fragmentation of medial elastic layers.24 Elastolysis induced by matrix metalloproteinases is one important feature in pathology of Marfan patients. These investigators suggest phenotypic change of medial VSMCs to the activated phenotype might have a pivotal role in aorta of Marfan syndrome. Upregulation of PPAR in VSMCs might thus be involved in the pathogenesis of CMD via the activated phenotype of medial SMCs in Marfan’s aortas. PPAR agonists such as troglitazone and pioglitazone inhibit proliferation and matrix metalloproteinase expression in cultured VSMCs.8 PPAR agonists also inhibit intimal VSMC growth in the balloon-injury animal model7,25 Although the pathophysiological significance of PPAR expression in VSMCs of Marfan’s aorta without any inflammatory response is unclear, we speculate that PPAR may act to counterbalance activation and proliferation of VSMCs.
We have previously reported acceleration of VSMC apoptosis in Marfan’s aorta3 and that this might be involved in the progression of pathogenesis in aortic dilatation. Aizawa et al have shown that the PPAR agonist pioglitazone induces apoptosis in cultured VSMCs in a nitric oxide-dependent manner by cytokine activation, and reduces intimal hyperplasia in a rat balloon-injured model by enhancing VSMC apoptosis.12 Although evidence for a direct causal relationship between PPAR and its ligand was not demonstrated, pioglitazone was a potent inducer of apoptosis in vascular lesions. In aortic aneurysm with Marfan syndrome, VSMC apoptosis may play a substantial role in progression of aortic disease and formation of CMD via PPAR activation.
One question that remains is whether PPAR is a primary or secondary component of CMD formation in Marfan syndrome. CMD is also observed in congenital aortic disease,1 atherosclerosis, and aging.2 We examined aortas in the size-matched non-Marfan’s AAE patients, ie, coarctation of aorta and atherosclerosis at graft operation. We could find CMD in these patients. PPAR expression could be demonstrated in these patients by immunohistochemical and RT-PCR methods (data not shown). So we suppose that the expression of PPAR is associated with aneurysm formation with CMD because of various causes and not unique phenomena in Marfan’s syndrome. Further studies are required to characterize the importance of PPAR within CMD. If this is clarified, regulation of PPAR might lead to the clinical implication of protection against progression in AAE with Marfan’s syndrome.
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Conclusion |
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Expression of PPAR
was upregulated in VSMCs of Marfan’s aortas with CMD and associated with severity of CMD and increasing aortic diameter. Our results suggest that PPAR expression might reflect pathogenesis of CMD and disease progression of Marfan’s aorta without any inflammatory response. |
Acknowledgments |
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This study was supported in part by an open research grant from the Japan Research Promotion Society for Cardiovascular Diseases. We thank Toru Suzuki for reviewing the manuscript, Hiroaki Nagao for his excellent technical assistance, and Keiko Komatsu for her skillful technical help in immunohistochemistry.
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References |
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