Expression and Function of Ephrin-B1 and Its Cognate Receptor EphB2 in Human Abdominal Aortic Aneurysm.

We examined the expression of ephrin-B1 and its cognate receptor EphB2, key regulators of angiogenesis and embryogenesis, in human abdominal aortic aneurysm (AAA) and analyzed their functional roles in cell migration. From 10 patients (9 males and 1 female; age, 68.5 ± 2.4) who underwent vascular surgery for AAA, we obtained AAA and adjacent control tissues. Using real-time RT-PCR, we analyzed expression of ephrin-B1 and EphB2. We also histologically localized these molecules in AAA tissues. Finally, effects of ephrin-B1 and EphB2 on inflammatory cell chemotaxis were examined by cell migration assay. Expression levels of ephrin-B1 (0.410 ± 0.046 versus 1.198 ± 0.252, P = 0.027) and EphB2 (0.764 ± 0.212 versus 1.272 ± 0.137, P = 0.594) were higher in AAA than normal control. Both ephrin-B1 and EphB2 were expressed in macrophages, T lymphocytes, and endothelial cells within AAA. In chemotaxis assay, ephrin-B1 and EphB2 inhibited mononuclear-cell chemotaxis induced by stromal derived factor-1 down to 54.7 ± 12.7% (P = 0.01) and 50.7 ± 13.1% (P = 0.01), respectively. These data suggest that ephrin-B1 and EphB2 might be functional in human adult inflammatory cells and involved in the pathogenesis of AAA, although specific roles of these molecules should further be sought.

Expression and function of ephrin-B1 and its cognate receptor EphB2 in human atherosclerosis: from an aspect of chemotaxis.

Although several cytokines and chemokines have been demonstrated to play pivotal roles in the pathophysiological conditions of atherosclerosis, few findings exist regarding the expression and function of cytokine-modulating molecules such as ephrin-Bs and their cognate receptors, EphBs, in human atherosclerosis. Therefore, in the present study, we screened novel genes modulating atherogenesis by cDNA array and quantitatively determined them by real-time RT (reverse transcription)-PCR in human carotid atherosclerotic plaques. Ephrin-B1 and EphB2, key regulators of embryogenesis, were significantly up-regulated in plaques compared with those in adjacent control tissues [ephrin-B1, 0.638+/-0.106 compared with 0.831+/-0.152, or 130% (P<0.05); EphB2, 1.296+/-0.281 compared with 2.233+/-0.506, or 172% (P<0.05)]. Immunohistological analysis demonstrated that both ephrin-B1 and EphB2 were expressed in macrophages and T-lymphocytes in plaques as well as in monocytes, T-lymphocytes and arterial endothelial cells isolated from healthy adults. Interestingly, the extracellular domains of ephrin-B1 and EphB2, the expression of which were both enhanced in stimulated THP-1 cells, significantly inhibited spontaneous (22.5 and 27.6% respectively; P<0.01) and MCP-1 (monocyte chemoattractant protein-1)-dependent (29.7 and 22.6% respectively; P<0.01) migration of monocytes. In conclusion, these results demonstrate that ephrin-B1 and EphB2 are overexpressed in atherosclerotic tissue and might locally regulate cell migration, possibly through modulating cytokine-related chemotaxic activity; however, the functional role of these molecules in atherogenesis should be investigated further.

Altered expression balance of matrix metalloproteinases and their inhibitors in human carotid plaque disruption: results of quantitative tissue analysis using real-time RT-PCR method.

BACKGROUND: The balance between degradation and synthesis of extracellular matrix determines its content in atherosclerotic tissue. To examine the role of expression balance of matrix metalloproteinases (MMPs) to their inhibitors, tissue inhibitors of metalloproteinases (TIMPs) and tissue factor pathway inhibitor-2 (TFPI-2) in the development and disruption of atherosclerotic plaque, these gene expressions in human carotid plaque were quantitatively determined by real-time reverse transcription (RT)-polymerase chain reaction (PCR) method. METHODS: Total RNA for cDNA synthesis was extracted from tissues in 24 patients with carotid endarterectomy. The amounts of cDNAs for MMP-1, -2, -3 and -9, TFPI-2 and TIMP-1, -2 and -3 were determined by real-time RT-PCR method, and normalized with glutaraldehyde 3-dehydrogenase. RESULTS: In plaques, the expression MMP-1 (1.53+/-0.25, mean+/-S.E.M.), MMP-3 (1.99+/-0.59) and MMP-9 (2.00+/-0.51) was augmented compared to those in the adjacent control regions (0.60+/-0.16, 0.46+/-0.18 and 0.58+/-0.21, respectively, p<0.05). The expression of TFPI-2 was lower in plaques (0.32+/-0.08) than in controls (0.94+/-0.23, p<0.01). Although the expression of TIMP-1 was higher in plaques (1.28+/-0.23) than in controls (0.81+/-0.10, p<0.05), the indices of MMP-1/TIMP-1, MMP-3/TIMP-3 and MMP-9/TIMP-1 were still significantly higher in plaques. Interestingly, MMP-9 and the resulting MMP-9/TIMP-1 balance in plaques with disruption were significantly higher (3.36+/-1.52 and 1.66+/-0.12, n=11) than those in non-disrupted plaques (1.11+/-0.52 and 0.76+/-0.12, n=13, p<0.05). CONCLUSION: With the decreased expression of TFPI-2, upregulation of MMPs in atherosclerotic plaque was disproportional to that of TIMPs, suggesting that imbalanced degradation and synthesis of extracellular matrix persists in advanced lesions, particularly in plaques with disruption.

Sustained upregulation of inflammatory chemokine and its receptor in aneurysmal and occlusive atherosclerotic disease: results form tissue analysis with cDNA macroarray and real-time reverse transcriptional polymerase chain reaction methods.

BACKGROUND: Although cytokines are known to be pivotal in the development of atherosclerotic diseases, few data exist regarding their expressions in the established stages such as aneurysmal or occlusive lesions. Therefore, in the present study the gene expression levels of cytokine-related substances in abdominal aortic aneurysm (AAA) and carotid artery stenosis (CAS) were determined using cDNA macroarray and real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods. METHODS AND RESULTS: Tissue samples were obtained from 31 patients with AAA and 24 with CAS. The array-specific [33P]-labeled cDNA probe mixture synthesized from 2.5 microg total RNA with gene-specific primers was hybridized with nylon membranes containing 375 cDNA clones. Densitometric analysis confirmed differences in expression (>5-fold) for 97 of the cytokine-related gene products between AAA and adjacent control tissue. Among these, simultaneous upregulation was found in the expression of interleukin (IL)-8 (9-fold) and its receptor, CXCR-2 (11-fold). Thus, the expressions of IL-8 and CXCR-2 were further quantified by real-time RT-PCR. The expression of both the genes was significantly upregulated in both AAA and CAS compared with control regions as followed: IL-8=0.53+/-0.16 vs 0.11+/-0.04 (p<0.01); CXCR-2=2.04+/-0.75 vs 0.29+/-0.10 (p<0.01) in AAA, and IL-8=1.35 +/-0.25 vs 0.60+/-0.16; CXCR-2=2.00 +/-0.51 vs 0.58+/-0.21 (p<0.05) in CAS. Under these conditions, the gene expressions of monocyte chemotactic protein-1 and its receptor, CCR-2, were not significantly different in the control and diseased regions of both AAA and CAS. CONCLUSIONS: Sustained upregulation of IL-8 and CXCR-2 was observed in both AAA and CAS, suggesting the inflammatory process is still active in established dilated and occlusive atherosclerotic diseases. Whether upregulation of this system could be protective or not protective for disease development requires further study.

Application of real-time RT-PCR to quantifying gene expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human abdominal aortic aneurysm.

BACKGROUND: The relative expression levels of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs), key regulators in remodeling of extracellular matrix, are considered to play a pivotal role in the development of abdominal aortic aneurysm (AAA). However, few data exist regarding quantitative assessment of their expression in clinical settings. METHODS: In 22 patients with AAA who underwent graft replacement, tissue samples of the AAA and non-dilated aorta were obtained. Using a real-time RT-PCR method that enabled quantitative measurement of mRNA levels in small tissue samples, we determined gene expression levels of MMPs and TIMPs relative to that of glutaraldehyde 3-phosphate dehydrogenase in each sample. RESULTS: The expression levels of the MMP-1 and -3 genes were significantly augmented in AAA compared with non-dilated regions (4.48 +/- 2.01 versus 0.26 +/- 0.12, P < 0.01 and 1.89 +/- 1.00 versus 5.01 +/- 0.97, P < 0.05, respectively). Although genes for TIMP-1, -2 and -3 tended to be upregulated in AAA, relative expression levels of MMP-1 to TIMP-1, MMP-1 to TIMP-2, MMP-1 to TIMP-3, and MMP-3 to TIMP-2 were still higher in AAA than in non-dilated regions (1.12 +/- 0.63 versus 0.10 +/- 0.03, 4.13 +/- 1.12 versus 0.43 +/- 0.11, 1.61 +/- 0.59 versus 0.14 +/- 0.03, and 7.81 +/- 1.60 versus 2.56 +/- 0.76, respectively, P < 0.05). CONCLUSION: These results demonstrate that the present real-time RT-PCR method is reliable for the determination of mRNA levels in small samples of vascular tissue and that disproportional expression of both MMP-1 and MMP-3 relative to TIMPs relates pathologically to the evolution of AAA.

Plaque morphology at coronary sites with focal spasm in variant angina: study using intravascular ultrasound.

In the present study, the intravascular ultrasound (IVUS) morphologic appearance of coronary atherosclerotic plaque associated with focal spasm was prospectively studied in 45 patients with or without focal coronary spasm provoked by ergonovine or acetylcholine. The percent plaque area and plaque arc were determined from the IVUS images at the sites of spasm. Calcified lesion was defined as the presence of high-intensity echo with acoustic shadowing. Twenty-three patients had focal coronary spasm defined as angiographic narrowing >75% and IVUS demonstrated atherosclerotic plaque in these 23 sites. In the 22 patients without focal spasm, IVUS demonstrated 18 atherosclerotic lesions in 17 patients and the remaining 5 patients did not have significant lesions. There was no difference in the percent plaque area and plaque arc between plaque lesions with (47+/-10%, 298+/-71 degrees ) and without (39+/-15%, 249+/-83 degrees ) coronary spasm. Interestingly, calcified lesion was less frequently present at the sites with than at those without spasm (p<0.05). These results indicate that the presence of plaque without calcification is likely to be related to the occurrence of focal vasospasm, although the severity and distribution of the disease did not differ between each patient group.

Effect of disease eccentricity on compensatory remodeling of coronary arteries: evidence from intravascular ultrasound before interventions.

BACKGROUND: Compensatory remodeling occurs to maintain lumen area in human coronary vessels. However, few data exist regarding the relationship between vessel remodeling and plaque distribution. Therefore, we studied coronary sites with or without remodeling by intravascular ultrasound and correlated with disease distribution. METHODS AND RESULTS: A total of 90 coronary sites with significant stenosis (>50%) from 80 patients were examined before interventions. For identifying the vessel remodeling, external elastic membrane (EEM) area was measured at the stenotic sites and the adjacent proximal and distal sites. The reference EEM area was calculated by averaging proximal and distal EEM areas, and percent enlargement of the EEM area was calculated by the formula: [(stenosis EEM area-reference EEM area)/reference EEM area]x100. Plaque area was determined by reducing the lumen from EEM areas. The maximal (max) and minimal (min) distances from the center of the lumen to the EEM were also measured, and the disease eccentricity index was calculated by the formula: [(max-min)/max]. The lesion was defined as eccentric if the index was >0.5 and as concentric if