Juno Deguchi

Inflammation in Atherosclerosis: Visualizing Matrix Metalloproteinase Action in Macrophages In Vivo

Background— Matrix metalloproteinases (MMPs) in inflamed atherosclerotic plaques may contribute to extracellular matrix remodeling and the onset of acute thrombotic complications. Methods and Results— To test the hypothesis that optical molecular imaging with the use of an activatable near-infrared fluorescence (NIRF) probe can detect enzymatic action of MMP in atherosclerotic plaques, we used a NIRF substrate for gelatinases (MMP-2/gelatinase-A and MMP-9/gelatinase-B) in apolipoprotein E–deficient (apoE−/−) mice that consumed a high-cholesterol diet for 12 weeks and age-matched apoE+/+ mice as control. The aortas of apoE−/− mice at 24 hours after probe yielded intense NIRF signals, as detected by NIRF reflectance ex vivo, compared with negligible signals in aortas of apoE+/+ mice with/without probe administration or atherosclerotic apoE−/− aortas without probe. Gelatinase inhibitor treatment abolished NIRF signals in apoE−/− mouse aortas ex vivo. Sites of gelatinase activity visualized by NIRF colocalized with macrophage accumulation, immunoreactive MMP-2 and MMP-9, and gelatinolytic activity detected by in situ zymography. Furthermore, fluorescence molecular tomography indicated in vivo that atherosclerotic aortas of apoE−/− mice produced NIRF signals for gelatinase action, whereas aortas of apoE+/+ mice injected with the probe or apoE−/− aortas with no probe exhibited negligible NIRF signals. Conclusions— These results suggest the feasibility of noninvasively imaging the enzymatic action of MMPs in vivo, an approach that may gauge inflammatory foci in atherosclerosis, assess cardiovascular risk, and evaluate the effects of therapeutic interventions.

Anastomotic aneurysms after surgical treatment of Takayasu's arteritis: A 40-year experience☆☆☆★

PURPOSE To evaluate the clinical characteristics of anastomotic aneurysms that develop in surgically treated patients with Takayasus arteritis. METHODS Among 103 patients with Takayasus arteritis treated surgically over 40 years, 91 patients with 259 anastomoses (allowing for exclusion of 12 operative deaths) participated in follow-up study from 1 month to 37.3 years with a mean value +/- SEM of 17.3 +/- 1.1 years with a follow-up completion rate of 93% at 30 years. The clinical characteristics of anastomotic aneurysms were clarified, and the influences of several factors (sites of anastomoses, occlusive or aneurysmal disease, suture material, preoperative systemic inflammation, and administration of corticosteroids) on formation of anastomotic aneurysms were analyzed by means of life-table method and Cox regression analysis. RESULTS Twenty-two uninfected anastomotic aneurysms were found among 14 patients (22 of 259 anastomoses, 8.5%). The interval between the previous operation and diagnosis varied from 1.6 to 30 years with a mean value +/- SEM of 9.8 +/- 1.8 years. The cumulative incidence of anastomotic aneurysm at 20 years was 12.0%. Systemic inflammation or steroid administration had little influence on formation of anastomotic aneurysm. Instead, anastomotic aneurysm tended to occur after operations for aneurysmal lesions. CONCLUSIONS Anastomotic aneurysm can occur anytime after operations for Takayasus arteritis. The development of anastomotic aneurysm is not influenced by any factor specific to this disease except the presence of an aneurysmal lesion.

Stimulated Activation of Platelet-Derived Growth Factor Receptor In Vivo in Balloon-Injured Arteries A Link Between Angiotensin II and Intimal Thickening

BACKGROUND Growth factors such as platelet-derived growth factor (PDGF) have been postulated to be important mediators of neointimal formation in balloon-injured artery. Binding of growth factors to their receptors activates intrinsic receptor tyrosine kinase, resulting in tyrosine phosphorylation of receptors themselves and cellular substrate proteins. We investigated in vivo activities of growth factors by determining the extent of tyrosine phosphorylation of growth factor receptors and substrate proteins in injured artery. METHODS AND RESULTS Rat balloon-injured carotid artery was analyzed for phosphotyrosine content of PDGF alpha- and beta-receptors, epidermal growth factor (EGF) receptors, and insulin receptor substrate-1 (IRS-1) by immunoprecipitation and anti-phosphotyrosine Western blot. The development of intimal thickening after deendothelializing balloon catheterization of rat carotid artery was accompanied by transient twofold to threefold increases in the extent of tyrosyl phosphorylation of PDGF alpha- and beta-receptors but not EGF receptor or IRS-1. The AT1 angiotensin II (Ang II) receptor antagonist TCV-116 markedly inhibited both tyrosyl phosphorylation of PDGF alpha- and beta-receptors and intimal thickening. The AT1 antagonist reduced mRNA levels of both PDGF-A and -B chains in injured arteries. CONCLUSIONS The present study provides direct evidence for increased PDGF activities in injured artery in situ and the involvement of Ang II in stimulated activation of PDGF receptors. These results are consistent with the pathogenetic role for PDGF in intimal thickening.

Targeting endogenous platelet-derived growth factor B-chain by adenovirus-mediated gene transfer potently inhibits in vivo smooth muscle proliferation after arterial injury

Platelet-derived growth factor (PDGF), especially its B chain, has been implicated in the pathogenesis of vascular proliferative disorders such as atherosclerosis and restenosis after angioplasty. We constructed a replication-deficient recombinant adenovirus containing the gene encoding the extracellular region of PDGF β-receptor (PDGFXR) that binds PDGF-B chain and acts as its antagonist. The administration into balloon-injured rat carotid arteries of an adenovirus containing the Escherichia coli lacZ gene as a marker gene at 5 days after injury markedly facilitated efficacy of gene transfer, as compared with its administration immediately after injury. Adenovirus-mediated gene transfer of PDGFXR into injured arteries performed at 5 days resulted in a more than 50% reduction in the neointimal area of injured arteries at 14 days. In con- trast, the administration of control adenoviruses containing lacZ gene or containing no foreign gene was without suppressive effects on neointima formation. The inhibition of neointima formation by the expression of PDGFXR was accompanied by a reduction in bromodeoxyuridine-labeled cells and nearly complete inhibition of tyrosine phosphorylation of both α- and β-receptors for PDGF, but not of epidermal growth factor receptor, in injured arteries. This is the first report to indicate the usefulness of targeting a growth factor by expressing an extracellular binding region of a receptor using an adenovirus for the treatment of vascular proliferative disorders, and provide direct evidence that PDGF-B chain plays an essential role in neointimal formation.

Angiotensin II Stimulates Platelet-Derived Growth Factor-B Chain Expression in Newborn Rat Vascular Smooth Muscle Cells and Neointimal Cells Through Ras, Extracellular Signal–Regulated Protein Kinase, and c-Jun N-Terminal Protein Kinase Mechanisms

Platelet-derived growth factors (PDGFs) have been implicated in the pathogenesis of vascular proliferative disorders. Vascular smooth muscle cells (VSMCs) are one of the cell types that produce PDGF-B chain in proliferative lesions, although the mechanism of regulation of PDGF-B chain production in these cells is not well understood. In the present study, we demonstrate that angiotensin II (Ang II), which is also implicated in vascular stenosis after angioplasty and atherosclerosis, markedly stimulates PDGF-B chain mRNA expression in cultured newborn rat medial VSMCs and neointimal VSMCs via an AT(1), but not in adult rat VSMCs. In newborn rat VSMCs, Ang II activates extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase. The mitogen-activated protein/ERK (MEK) inhibitor PD98059, but not the p38 inhibitor SB203580, abrogates Ang II-induced PDGF-B mRNA expression. Transient transfection analysis using a PDGF-B promoter-luciferase gene reporter construct reveals that Ang II induces transcriptional activation of PDGF-B chain gene, which is abolished by the expression of a dominant negative form of either ERK or JNK, but not of p38. The expression of a dominant negative form of Ras abolishes the stimulatory effects of Ang II on ERK activity and PDGF-B mRNA expression. In adult rat VSMCs, Ang II activates ERK and JNK, but weakly induces Egr-1, a transcription factor implicated in PDGF-B chain gene expression, compared with newborn VSMCs. These data indicate that Ang II activates PDGF-B chain gene expression in VSMCs through mechanisms involving Ras-ERK and JNK.

Chronic Hypoxia Activates the Akt and β-Catenin Pathways in Human Macrophages

Objective—Macrophage activation contributes importantly to the pathogenesis of inflammatory diseases including atherosclerosis. Macrophages exist chronically under moderate hypoxia (2% to 5% O2) in inflamed tissues such as atherosclerotic plaques. However, macrophage phenotypes in such environments remain incompletely understood. This study tested the hypothesis that chronic moderate hypoxia induces macrophage activation and explored the underlying mechanisms. Methods and Results—We cultured primary human macrophages derived from peripheral blood monocytes in moderate hypoxia (2% O2 tension) or normoxia (21% O2) for 10 days. Moderate hypoxia did not affect macrophage differentiation assessed via expression levels of scavenger receptor A. Chronic moderate hypoxia, but not normoxia, activated Akt and inactivated GSK-3&bgr;, a negative effector of Akt, thus allowing nuclear translocation of &bgr;-catenin. 2% O2 tension increased accumulation of hypoxia-inducible factors 1&agr; (HIF-1&agr;) transiently at 3 to 5 days. Hypoxia induced mRNA expression of the &bgr;-catenin-associated genes: MMP-7, CD44, and c-Myc. RNAi of TCF7L2, a cofactor of &bgr;-catenin, suppressed MMP-7 expression induced by hypoxia. Inhibition of Akt phosphorylation with LY294002 abolished hypoxia-induced GSK-3&bgr; inactivation, &bgr;-catenin activation, and MMP-7 expression. Macrophages under hypoxia were more resistant for oxLDL-induced apoptosis. Moreover, phospho-Akt colocalized with MMP-7 and CD44 expression in macrophages of human atherosclerotic plaques. Conclusions—Chronic moderate hypoxia induces macrophage activation via the Akt and &bgr;-catenin pathways, providing new insight into the pathogenesis of inflammatory diseases.

Successful treatment tailored to each splanchnic arterial lesion due to segmental arterial mediolysis (SAM): report of a case.

Segmental arterial mediolysis (SAM) is a rare condition characterized by splanchnic arterial catastrophe caused by mediolysis. We report a 59-year-old man with a ruptured splenic arterial aneurysm who was successfully treated by coil embolization. He underwent additional resection of large gastroepiploic and residual splenic aneurysms. Pathological examination showed mediolysis and tearing, compatible with SAM. Furthermore, he developed acute dissection of the superior mesenteric artery (SMA) one and a half years later, demonstrated by computed tomography. This report demonstrates that SAM is characterized by multiple lesions of the splanchnic arteries at different times, and requires treatment suited to the lesions, including careful long-term observation.

Family history of aortic aneurysm is an independent risk factor for more rapid growth of small abdominal aortic aneurysms in Japan

OBJECTIVE We aimed to investigate risk factors associated with more rapid growth of abdominal aortic aneurysms (AAA) <50 mm (small AAAs) in Japan. METHODS We retrospectively investigated the clinical data of 374 patients with small AAAs (maximum diameter, ≤50 mm) who were referred to The University of Tokyo Hospital, Tokyo Medical University Hospital, or Saitama Medical Center, between 1995 and 2008. RESULTS A total of 374 patients (321 men and 53 women) were followed up for a median of 66 months. The median diameter on initial examination was 40 mm, and the median growth rate of the AAAs was 2.1 mm/y. The growth rate of AAAs with an initial diameter ≥45 mm was significantly greater than those with an initial diameter <45 mm (3.3 mm/y vs 2.0 mm/y, respectively; P = .007). The growth rate of AAAs was significantly greater in patients with hypertension than in those without (2.3 mm/y vs 1.7 mm/y, respectively; P = .006) and in patients with a family history of aortic aneurysm than in those without (4.2 mm/y vs 2.0 mm/y, respectively; P = .009). Logistic regression analysis revealed that a large initial diameter and family history of aortic aneurysm were independent predictors of accelerated growth rate of small AAAs in Japan. CONCLUSIONS In the present study, a large initial diameter and family history of aortic aneurysm were independent risk factors for more rapid growth of small AAAs. Although few studies have reported similar findings thus far, family history of aortic aneurysm should be carefully considered during follow-up of patients with small AAAs.

Surgical Treatment and Long-term Outcome of Renovascular Hypertension in Children and Adolescents

OBJECTIVES This article describes the long-term outcome of surgical treatment in children with renovascular hypertension (RVH) over a 40-year period. DESIGN Retrospective study. MATERIALS AND METHODS Twenty-five consecutive patients, aged 5-21 years, underwent renal artery (RA) repair from 1967 to 1995. The disease consisted of fibromuscular dysplasia in 17 patients, Takayasus arteritis in 7 and neurofibromatosis type 1 in one patient. RESULTS Twenty-nine RAs were repaired. Primary procedures included aortorenal bypass (ARB) with prosthesis in 10 RAs, autologous vein in five or internal iliac artery in four as conduits, direct reimplantation (DR) in four and nephrectomy in two RAs. Immediate graft failure occurred in three patients despite no peri-operative deaths. After a mean follow-up of 24.4 years, seven patients required secondary nephrectomy. Autologous ARB or DR showed better RA patency and fewer chances for secondary nephrectomy than prosthetic ARB. Hypertension was cured or improved in 21 patients. The overall cumulative survival rate at 20 years was 84%. All five deaths, observed a mean of 12.6 years after the initial operation, were attributed to cardiovascular events. CONCLUSIONS Surgical treatment, especially autologous ARB or DR, seems to provide durable results for paediatric RVH. Long-term observation and control of hypertension is mandatory.

Tyrosine phosphorylation of platelet derived growth factor β receptors in coronary artery lesions: implications for vascular remodelling after directional coronary atherectomy and unstable angina pectoris

Background Growth factors such as platelet derived growth factor (PDGF) have been postulated to be important mediators of neointimal proliferation observed in atherosclerotic plaques and restenotic lesions following coronary interventions. Binding of PDGF to its receptor results in intrinsic receptor tyrosine kinase activation and subsequent cellular migration, proliferation, and vascular contraction. Aims To investigate whether the concentration of PDGF β receptor tyrosine phosphorylation obtained from directional coronary atherectomy (DCA) samples correlate with atherosclerotic plaque burden, the ability of diseased vessels to remodel, coronary risk factors, and clinical events. Methods DCA samples from 59 patients and 15 non-atherosclerotic left internal thoracic arteries (LITA) were analysed for PDGF β receptor tyrosine phosphorylation content by receptor immunoprecipitation and antiphosphotyrosine western blot. The amount of PDGF β receptor phosphorylation was analysed in relation to angiographic follow up data and clinical variables. Results PDGF β receptor tyrosine phosphorylation in the 59 DCA samples was greater than in the 15 non-atherosclerotic LITA (mean (SD) 0.84 (0.67) v 0.17 (0.08) over a control standard, p < 0.0001). As evaluated by stepwise regression analysis, incorporation of both PDGF β receptor tyrosine phosphorylation and immediate gain correlated strongly (adjustedr 2 = 0.579) with late loss, although PDGF β receptor tyramine phosphorylation alone correlated poorly with late loss. Multivariate regression analysis of coronary risk factors and clinical events revealed unstable angina as the most significant correlate of PDGF β receptor tyrosine phosphorylation (F value 20.009, p < 0.0001). Conclusions PDGF β receptor tyrosine phosphorylation in atherosclerotic lesions is increased compared with non-atherosclerotic arterial tissues. The association of PDGF β receptor tyrosine phosphorylation with immediate gain strongly correlates with vascular remodelling. PDGF β receptor tyrosine phosphorylation correlates with unstable angina pectoris.