Yujiro Asada

C-Reactive Protein in Atherosclerotic Lesions: Its Origin and Pathophysiological Significance

C-reactive protein (CRP) is frequently deposited in the lesions of the arterial intima; however, the origin and pathological significance of CRP in these lesions are not completely understood. In this study, we measured CRP levels in the plasma of hypercholesterolemic rabbits and investigated CRP expression at both the mRNA and protein levels using rabbit and human atherosclerotic specimens. CRP levels were significantly elevated in both cholesterol-fed and Watanabe heritable hyperlipidemic rabbits, and CRP levels were clearly correlated with aortic atherosclerotic lesion size. Immunohistochemical staining coupled with Western blotting analysis revealed that CRP-immunoreactive proteins were found at all stages of atherosclerosis from the early to advanced lesions. CRP was present extracellularly and co-localized with apolipoprotein B but was rarely associated with the cytoplasm of macrophages and foam cells. Real-time reverse transcriptase-polymerase chain reaction analysis revealed that CRP mRNA in atherosclerotic lesions was barely detectable, and isolated macrophages did not express CRP mRNA, suggesting that CRP proteins found in the lesions were essentially derived from the circulation rather than synthesized de novo by vascular cells. These results suggest that there is a link between plasma CRP and the degree of atherosclerosis and that inhibition of plasma CRP may represent a therapeutic modality for the treatment of cardiovascular disease.

Adventitial Mast Cells Contribute to Pathogenesis in the Progression of Abdominal Aortic Aneurysm

Abdominal aortic aneurysm (AAA) is histologically characterized by medial degeneration and various degrees of chronic adventitial inflammation, although the mechanisms for progression of aneurysm are poorly understood. In the present study, we carried out histological study of AAA tissues of patients, and interventional animal and cell culture experiments to investigate a role of mast cells in the pathogenesis of AAA. The number of mast cells was found to increase in the outer media or adventitia of human AAA, showing a positive correlation between the cell number and the AAA diameter. Aneurysmal dilatation of the aorta was seen in the control (+/+) rats following periaortic application of calcium chloride (CaCl2) treatment but not in the mast cell–deficient mutant Ws/Ws rats. The AAA formation was accompanied by accumulation of mast cells, T lymphocytes and by activated matrix metalloproteinase 9, reduced elastin levels and augmented angiogenesis in the aortic tissue, but these changes were much less in the Ws/Ws rats than in the controls. Similarly, mast cells were accumulated and activated at the adventitia of aneurysmal aorta in the apolipoprotein E–deficient mice. The pharmacological intervention with the tranilast, an inhibitor of mast cell degranulation, attenuated AAA development in these rodent models. In the cell culture experiment, a mast cell directly augmented matrix metalloproteinase 9 activity produced by the monocyte/macrophage. Collectively, these data suggest that adventitial mast cells play a critical role in the progression of AAA.

Involvement of C-reactive protein obtained by directional coronary atherectomy in plaque instability and developing restenosis in patients with stable or unstable angina pectoris

We investigated whether positive immunohistochemical staining of C-reactive protein (CRP) in initial culprit lesions is related to coronary plaque instability and whether it could affect the outcome of directional coronary atherectomy (DCA). The plasma level of CRP is a reliable marker of the risk of coronary events and restenosis after percutaneous coronary intervention. However, the influence of tissue CRP in atheromatous plaque on plaque vulnerability and restenosis remains unknown. Samples of DCA obtained from 12 patients with stable angina pectoris and 15 patients with unstable angina pectoris were immunohistochemically stained with a monoclonal antibody against CRP. We performed follow-up coronary angiography on 22 of 27 patients to evaluate the presence of restenosis after DCA. Immunoreactivity to CRP was localized to macrophages, smooth muscle cells, and necrotic areas. The ratio of CRP positive cells to total cells was significantly higher in DCA samples from patients with unstable (17.9 +/- 2.0%) than with stable angina (11.0 +/- 2.5%) (p <0.05). Follow-up coronary angiography showed that 12 of 22 patients developed restenosis after DCA. The ratio was also significantly higher in DCA specimens from patients with restenosis (19.3 +/- 2.8%) compared with those without restenosis (11.0 +/- 2.0%) (p <0.05). In addition, the ratio significantly correlated with late luminal loss (r = 0.428, p <0.05) and loss index (r = 0.636, p = 0.0011) after DCA. Immunoreactivity to CRP in coronary atheromatous plaque increases in culprit lesions of unstable angina, and it affects restenosis after DCA. These findings suggest that CRP in atheromatous plaque plays an important role in the pathogenesis of unstable angina and restenosis after coronary intervention.

Adenovirus-Mediated Local Expression of Human Tissue Factor Pathway Inhibitor Eliminates Shear Stress–Induced Recurrent Thrombosis in the Injured Carotid Artery of the Rabbit

The main cause of acute coronary syndrome may be recurrent thrombosis, which is initiated by the activation of the extrinsic coagulation pathway. Tissue factor (TF) pathway inhibitor (TFPI) efficiently inhibits an early step in this pathway by the formation of a complex with factor VIIa, TF, and factor Xa. We determined whether local TFPI gene transfer can inhibit thrombosis in an injured artery without inducing systemic side effects. Balloon-injured rabbit carotid arteries were infected with an adenoviral vector that expressed either human TFPI (AdCATFPI) or bacterial beta-galactosidase (AdCALacZ). Two to 6 days after gene transfer, thrombosis was induced by the production of constant stenosis of the artery, and blood flow was measured continuously with an electromagnetic flow probe. A cyclic flow variation, which is thought to reflect the recurrent formation and dislodgment of mural thrombi, was observed in all AdCALacZ-infected arteries as well as in saline-infused arteries. In contrast, no cyclic flow variation was detectable in AdCATFPI-transfected arteries, even in the presence of epinephrine (1 microg. kg-1. min-1 infusion). Prothrombin time, activated partial thromboplastin time, and the ex vivo platelet aggregation induced by either adenosine diphosphate or collagen were unaltered in AdCATFPI-infected rabbits. We found that in vivo TFPI gene transfer into an injured artery completely inhibits the recurrent thrombosis induced by shear stress even in the presence of catecholamine, without affecting systemic coagulation status. Adenovirus-mediated local expression of TFPI may have the potential for the treatment of human thrombosis.

Proportion of fibrin and platelets differs in thrombi on ruptured and eroded coronary atherosclerotic plaques in humans

Objective: To determine the proportion of platelets and fibrin in coronary thrombi. Design: Immunohistochemical and morphometric means to examine the coronary arteries of 31 patients who died of acute myocardial infarction. Results: Fresh thrombi were detected in the feeding arteries of infarction areas in 23 cases (74%) and were associated with plaque rupture in 18 (78%) and plaque erosion in 5 (22%). An immunohistochemical study showed that the thrombi consisted of a mixture of fibrin and platelets as well as some other types of blood cells. The fibrin and platelet positive areas in the thrombi associated with plaque rupture accounted for 74 (19)% and 35 (20)% (p < 0.01) and those associated with erosion accounted for 51 (6)% and 70 (21)%, respectively, of the total areas. Areas of positive immunoreactivity for tissue factor and C reactive protein were also significantly greater in ruptured than in eroded plaques. Conclusion: These results indicate that the proportions of fibrin and of platelets differ in coronary thrombi on ruptured and eroded plaques. Higher proportions of tissue factor and C reactive protein contribute more significantly to thrombus formation on plaque rupture than on plaque erosion.

Adrenomedullin Administration Immediately After Myocardial Infarction Ameliorates Progression of Heart Failure in Rats

Background—Adrenomedullin (AM) is expressed in cardiac tissue, and plasma AM levels increase in patients with acute myocardial infarction (MI). This study was performed to determine whether AM administration immediately after acute MI inhibits progression of heart failure in rats. Methods and Results—Rats were infused with 1.0 &mgr;g/h IP AM or saline over 7 days immediately after MI inducted by left coronary ligation and were examined 9 weeks after MI. Compared with the saline infusion, AM infusion significantly improved survival (59% versus 81%; P<0.05) and body weight gain (32%; P<0.01) and reduced heart weight (−28%; P<0.01), lung weight (−26%; P<0.01), left ventricular (LV) end-diastolic pressure (11.4±2.0 versus 4.0±0.6 mm Hg, mean± SEM; P<0.01), collagen volume fraction of noninfarcted LV (−39%; P<0.05), and plasma levels of endogenous rat AM (−38%; P<0.05) without affecting infarct size. To investigate the mechanism of AM actions, another series of MI rats infused with AM were killed on day 7. AM infusion had no effect on organ weights and hemodynamic parameters on day 7 of MI but significantly reduced urinary excretion of isoprostane (−61%; P<0.01) and noninfarcted LV mRNA levels of ACE (−31%; P<0.05) and p22-phox (−30%; P<0.05). Conclusions—AM administration during the early period of MI improved the survival and ameliorated progression of LV remodeling and heart failure. This beneficial effect was accompanied by reductions in oxidative stress and ACE mRNA expression in noninfarcted LV in the AM infusion period.

Reciprocal Contribution of Pentraxin 3 and C-Reactive Protein to Obesity and Metabolic Syndrome

Pentraxin 3 (PTX3) is an acute‐phase protein that shares structural homology with C‐reactive protein (CRP). PTX3 is produced in macrophages, endothelial cells, and adipocytes in response to inflammatory stimuli, whereas hepatocytes are the main source of CRP. Because obesity and metabolic syndrome (MetS) are considered chronic inflammatory states, PTX3 might be involved in the pathogenesis of obesity and MetS as well as CRP. Levels of CRP correlated positively with body weight, BMI, waist circumference (WC), fasting plasma glucose and interleukin (IL)‐6, and negatively with high‐density lipoprotein cholesterol and adiponectin in healthy males. In contrast, PTX3 correlated positively with adiponectin, and negatively with body weight, BMI, WC, and triglyceride. Plasma CRP significantly increased, whereas plasma PTX3 significantly decreased with increasing BMI. Plasma CRP and PTX3 levels were significantly higher and lower, respectively, in individuals who had more than one MetS component compared with those who had none. In conclusion, PTX3 and CRP antagonistically participate in the development of obesity or MetS.

Intratracheal gene transfer of adrenomedullin using polyplex nanomicelles attenuates monocrotaline-induced pulmonary hypertension in rats.

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive PAH and right ventricular failure. Despite recent advances in therapeutic approaches using prostanoids, endothelin antagonists, and so on, PAH remains a challenging condition. To develop a novel therapeutic approach, we have established a nonviral gene delivery system of poly(ethylene glycol) (PEG)-based block catiomers, which form a polyplex nanomicelle with a nanoscaled core-shell structure in the presence of DNA. The polyplex nanomicelle from PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-P[Asp(DET)]), having ethylenediamine units at the side chain, showed ~100-fold increase in luciferase transgene expression activity in mouse lung via intratracheal administration with a minimal toxicity compared with the polyplex from linear poly(ethylenimine) (LPEI). The transfection activity was highest on day 3 after administration and remained detectable until day 14. PEG-b-P[Asp(DET)] polyplex nanomicelles were formulated with a therapeutic plasmid bearing the human adrenomedullin (AM) gene and intratracheally administered to rats with monocrotaline-induced pulmonary hypertension. The right ventricular pressure significantly decreased 3 days after administration as confirmed by a notable increase of pulmonary human AM mRNA levels. Intratracheal administration of PEG-b-P[Asp-(DET)] polyplex nanomicelles showed remarkable therapeutic efficacy with PAH animal models without compromising biocompatibility.

Pressure-independent effects of pharmacological stimulation of soluble guanylate cyclase on fibrosis in pressure-overloaded rat heart

Cardiac fibrosis is a hallmark of cardiovascular remodeling associated with hypertension. The purpose of this study was to explore the effect and mechanism of soluble guanylate cyclase (sGC) stimulator BAY 41–2272, leading to intracellular cyclic guanosine monophosphate (cGMP) elevation, on the remodeling process induced by pressure overload. Seven-week-old male Wistar rats with hypertension induced by suprarenal aortic constriction (AC) were treated orally with 2 mg kg−1 day−1 of BAY 41–2272 for 14 days. BAY 41–2272 had no effects on blood pressure, but decreased AC-induced collagen accumulation in the left ventricle (LV), inhibiting the number of myofibroblasts and gene expressions of transforming growth factor-β1 and type 1 collagen. In addition, the antifibrotic action of BAY 41–2272 was accompanied by reducing AC-induced angiotensin-converting enzyme (ACE) mRNA and its enzymatic activity, and angiotensin II concentration in LV. In cultured cardiac fibroblasts, BAY 41–2272 inhibited ACE synthesis and myofibroblast transformation, accompanied by elevating the intracellular cGMP concentration. These results suggest that sGC stimulator BAY 41–2272 might be effective to reduce fibrosis in hypertensive heart disease by attenuating angiotensin II generation through myofibroblast transformation.

Co-localization of von Willebrand factor with platelet thrombi, tissue factor and platelets with fibrin, and consistent presence of inflammatory cells in coronary thrombi obtained by an aspiration device from patients with acute myocardial infarction.

Summary.  Background: Detailed histochemical analysis of coronary thrombi obtained freshly from acute phase of myocardial infarction patients may provide information necessary to understand the mechanism of coronary occlusive thrombus formation. Methods and Results: Coronary thrombi causing myocardial infarction were obtained from 10 consecutive patients of myocardial infarction in the acute phase, using a newly developed aspiration catheter. All the fixed specimens of coronary thrombi, by hematoxylin and eosin staining, were found to contain three major constituents, namely, platelets, densely packed fibrin and inflammatory cells, including polymorphonuclear and mononuclear cells, although their distribution in each specimen is totally heterogeneous. Immunohistochemical staining revealed the prominent presence of von Willebrand factor (VWF) at the sites of platelet accumulation, presence of tissue factor and platelets at the sites of deposition of fibrin fibrils. It also revealed the presence of CD16‐, CD45‐ and CD34‐positive cells, yet the functional roles of these cells have still to be elucidated. There are weak positive correlation between the number of inflammatory cells involved in the unit area of coronary thrombi specimen and the time of collection of the specimens after the onset of chest pain. Conclusions: In spite of various limitations, our results contain information suggesting the possible role of VWF in platelet‐thrombus formation, possible important role played by tissue factor and activated platelets in the formation of fibrin fibrils, and the positive relationship between inflammatory cells migration and the formation of occlusive thrombi in human coronary arteries.