Effect of Rivaroxaban and Clopidogrel Combination Therapy on In-Stent Responses After Everolimus-Eluting Stent Implantation in a Porcine Coronary Model.

AIM: According to recent clinical trials, a combination of direct oral anticoagulants with antiplatelet drugs is often recommended for atrial fibrillation patients who receive drug-eluting stents (DESs). Although the optimal combination comprises direct factor Xa inhibitors and a P2Y12 receptor antagonist (or aspirin), their influence on vascular responses to DESs remains unclear. METHODS: Pigs were given either aspirin and clopidogrel (dual antiplatelet therapy [DAPT] group), aspirin and rivaroxaban (AR group), or clopidogrel and rivaroxaban (CR group), followed by everolimus-eluting stent (Promus Element) implantation into the coronary artery. Stented coronary arteries were evaluated via intravascular optical coherence tomography (OCT) and histological analysis at 1 and 3 months. RESULTS: OCT revealed lower neointimal thickness in the DAPT group and comparable thickness among all groups at 1 and 3 months, respectively. Histological analyses revealed comparable neointimal area among all groups and the smallest neointimal area in the CR group at 1 and 3 months, respectively. In the DAPT and AR groups, the neointima continued to grow from 1 to 3 months. A shortened time course for neointima growth was observed in the CR group, with rapid growth within a month (maintained for 3 months). A higher incidence of in-stent thrombi was observed in the AR group at 1 month; no thrombi were found in either group at 3 months. More smooth muscle cells with contractile features were found in the CR group at both 1 and 3 months. CONCLUSIONS: Our results proved the noninferiority of the combination of rivaroxaban with an antiplatelet drug, particularly the dual therapy using rivaroxaban and clopidogrel, compared to DAPT after DES implantation.

Defective autophagy in vascular smooth muscle cells enhances the healing of abdominal aortic aneurysm.

Autophagy is an evolutionarily conserved cellular catabolic process essential for cell homeostasis, and thus its failure is associated with several diseases. While autophagy has been reported to play a role in vascular smooth muscle cells (SMCs) in vascular disorders, its precise role in the pathogenesis of abdominal aortic aneurysm (AAA) has not yet been elucidated. In this study, we investigated the role of SMC autophagy in AAA formation. As a mouse model of AAA, we used control apolipoprotein E-deficient (apoeKO) mice and Atg7cKO (SMC-specific Atg7-deficient mice):apoeKO mice administered angiotensin II for 4 weeks. Intriguingly, Kaplan-Meier curves showed that the survival rates of Atg7cKO:apoeKO mice were significantly higher than those of apoeKO mice. The hematoma area in AAA of Atg7cKO:apoeKO mice was smaller than in apoeKO mice despite the lack of a significant difference in AAA incidence between the two groups. Furthermore, the amount of granulomatous tissues was significantly larger and the collagen-positive area within AAA was significantly larger in Atg7cKO:apoeKO mice than in apoeKO mice. In accordance with these findings, SMCs cultured from Atg7cKO mice showed increased expression of collagens, independent of angiotensin II action. Taken together, our data suggest that defective autophagy in SMCs elicits AAA healing that may underlie the better survival rate under dyslipidemia and angiotensin II infusion.

Defective autophagy in vascular smooth muscle cells enhances atherosclerotic plaque instability.

Autophagy is considered as an evolutionarily conserved cellular catabolic process. Defective autophagy has been implicated in various human diseases, including cardiovascular diseases. Recently, we and others demonstrated that defective autophagy in vascular smooth muscle cells (SMCs) promotes the progression of atherosclerosis. In this study, we investigated the role of autophagy in SMCs on plaque instability in vivo. We generated mice with a defect atg7in which is an essential gene for autophagy, in SMCs by crossing Atg7f/f mice with transgelin (Tagln) Cre+/0 mice (Atg7cKO). Then, Atg7cKO and apolipoprotein E (apoe)-deficient (apoeKO) mice were crossed to generate Atg7cKO:apoeKO mice. To generate a mouse model of plaque instability, we conducted to form a tandem stenosis in the carotid artery of Atg7cKO:apoeKO mice and their controls (apoeKO mice) at the age of 10 weeks. At 5 weeks after surgery, the percentage of cross-sectional stenosis area in the operated common carotid artery of Atg7cKO:apoeKO mice was significantly higher than that in apoeKO mice. In addition, thrombus, which was not observed in apoeKO mice, was frequently found in Atg7cKO:apoeKO mice. Furthermore, the number of Berlin blue staining-positive areas, which indicated intraplaque hemorrhage, was significantly higher in Atg7cKO:apoeKO mice than in control apoeKO mice. Taken together, our data suggest that defective autophagy in SMCs enhances plaque instability and the risk of plaque rupture.

Defective autophagy in vascular smooth muscle cells enhances cell death and atherosclerosis.

Macroautophagy/autophagy is considered as an evolutionarily conserved cellular catabolic process. In this study, we aimed to elucidate the role of autophagy in vascular smooth muscle cells (SMCs) on atherosclerosis. SMCs cultured from mice with SMC-specific deletion of the essential autophagy gene atg7 (Atg7cKO) showed reduced serum-induced cell growth, increased cell death, and decreased cell proliferation rate. Furthermore, 7-ketocholestrerol enhanced apoptosis and the expression of CCL2 (chemokine [C-C motif] ligand 2) with the activation of TRP53, the mouse ortholog of human and rat TP53, in SMCs from Atg7cKO mice. In addition, Atg7cKO mice crossed with Apoe (apolipoprotein E)-deficient mice (apoeKO; Atg7cKO:apoeKO) showed reduced medial cellularity and increased TUNEL-positive cells in the descending aorta at 10 weeks of age. Intriguingly, Atg7cKO: apoeKO mice fed a Western diet containing 1.25% cholesterol for 14 weeks showed a reduced survival rate. Autopsy of the mice demonstrated the presence of aortic rupture. Analysis of the descending aorta in Atg7cKO:apoeKO mice showed increased plaque area, increased TUNEL-positive area, decreased SMC-positive area, accumulation of macrophages in the media, and adventitia and perivascular tissue, increased CCL2 expression in SMCs in the vascular wall, medial disruption, and aneurysm formation. In conclusion, our data suggest that defective autophagy in SMCs enhances atherosclerotic changes with outward arterial remodeling.

Inhibition of plaque progression and promotion of plaque stability by glucagon-like peptide-1 receptor agonist: Serial in vivo findings from iMap-IVUS in Watanabe heritable hyperlipidemic rabbits.

BACKGROUND AND AIMS: Glucagon-like peptide-1 (GLP-1) is thought to inhibit development of aortic atherosclerosis and plaque formation. However, whether GLP-1 stabilizes fully developed atherosclerotic plaque or alters the complicated plaque composition remains unclarified. METHODS: Ten Watanabe heritable hyperlipidemic (WHHL) rabbits were divided into GLP-1 receptor agonist treatment group and control group. After confirmation of atherosclerotic plaques in brachiocephalic arteries by iMap intravascular ultrasound (iMAP-IVUS), GLP-1 receptor agonist lixisenatide was administered to WHHL rabbits at 30 nmoL/kg/day for 12 weeks by osmotic pump. An equal volume of normal saline was administered in a control group. After evaluation by iMAP-IVUS at 12 weeks, brachiocephalic arteries were harvested for pathological histological analysis. RESULTS: iMAP-IVUS analysis revealed larger fibrotic plaque components and smaller necrotic and calcified plaque components in the GLP-1 group than in the control group; %fibrotic area: 66.30 ± 2.06% vs. 75.14 ± 2.62%, p < 0.01, %necrotic area: 23.25 ± 1.87% vs. 16.17 ± 2.27%, p = 0.02, %calcified area: 2.15 ± 0.24% vs. 1.00 ± 0.18%, p < 0.01), indicating that GLP-1 receptor agonist might modify plaque composition and increase plaque stability. Histological analysis confirmed that GLP-1 receptor agonist treatment improved smooth muscle cell (SMC)-rich plaque with increased fibrotic content. Furthermore, plaque macrophage infiltration and calcification were significantly reduced by GLP-1 receptor agonist treatment; %SMC area: 6.93 ± 0.31% vs. 8.14 ± 0.48%, p = 0.02; %macrophage area: 9.11 ± 0.80% vs. 6.19 ± 0.85%, p < 0.01; %fibrotic area: 54.75 ± 1.63% vs. 69.60 ± 2.12%, p = 0.02; %calcified area: 3.25 ± 0.67% vs. 0.75 ± 0.15%, p = 0.02). CONCLUSIONS: GLP-1 receptor agonist inhibited plaque progression and promoted plaque stabilization by inhibiting plaque growth and modifying plaque composition.

Development of Human-Like Advanced Coronary Plaques in Low-Density Lipoprotein Receptor Knockout Pigs and Justification for Statin Treatment Before Formation of Atherosclerotic Plaques.

BACKGROUND: Although clinical trials have proved that statin can be used prophylactically against cardiovascular events, the direct effects of statin on plaque development are not well understood. We generated low-density lipoprotein receptor knockout (LDLR(-/-)) pigs to study the effects of early statin administration on development of atherosclerotic plaques, especially advanced plaques. METHODS AND RESULTS: LDLR(-/-) pigs were generated by targeted deletion of exon 4 of the LDLR gene. Given a standard chow diet, LDLR(-/-) pigs showed atherosclerotic lesions starting at 6 months of age. When 3-month-old LDLR(-/-) pigs were fed a high-cholesterol, high-fat (HCHF) diet for 4 months (HCHF group), human-like advanced coronary plaques developed. We also fed 3-month-old LDLR(-/-) pigs an HCHF diet with pitavastatin for 4 months (Statin Prophylaxis Group). Although serum cholesterol concentrations did not differ significantly between the 2 groups, intravascular ultrasound revealed 52% reduced plaque volume in statin-treated pigs. Pathological examination revealed most lesions (87%) in the statin prophylaxis group were early-stage lesions, versus 45% in the HCHF diet group (P<0.01). Thin-cap fibroatheroma characterized 40% of the plaques in the HCHF diet group versus 8% in the statin prophylaxis group (P<0.01), intraplaque hemorrhage characterized 11% versus 1% (P<0.01), and calcification characterized 22% versus 1% (P<0.01). CONCLUSIONS: Results of our large animal experiment support statin prophylaxis before the occurrence of atherosclerosis. Early statin treatment appears to retard development of coronary artery atherosclerosis and ensure lesion stability. In addition, the LDLR(-/-) pigs we developed represent a large animal model of human-like advanced coronary plaque suitable for translational research.

Stabilization of atherosclerotic plaque by pitavastatin in Watanabe heritable hyperlipidemic rabbits: A serial tissue-characterizing intravascular ultrasound study.

BACKGROUND: To examine the effects of pitavastatin on atherosclerotic plaque in Watanabe heritable hyperlipidemic (WHHL) rabbits using serial in vivo tissue-characterizing intravascular ultrasound. METHODS: A total of 11 WHHL rabbits of 10-12 weeks of age were divided into two groups, control and pitavastatin-administered groups. A total of 29 atherosclerotic plaque segments from control group and 43 plaque segments from the pitavastatin group were serially imaged by 40MHz intravascular ultrasound in vivo with a tissue characterization software (iMAP™, Boston Scientific, Natick, MA, USA) at the baseline and the follow-up (16th week). RESULTS: The level of low-density lipoprotein cholesterol was significantly decreased in pitavastatin group. During the follow-up period, plaque area was significantly increased in the control group, whereas it was not significantly changed in the pitavastatin group. The fibrotic, necrotic, and necrotic plus lipidic areas were significantly increased in the control group, while no significant change was revealed for tissue profile in pitavastatin group. The change in the percent areas of fibrotic and lipidic plus necrotic tissues were significantly different between the two groups especially in the superficial half portion of plaque. CONCLUSIONS: These data indicate that pitavastatin could attenuate atherosclerotic plaque formation and that it could stabilize the plaque in WHHL rabbits. Considering the fact that these were observed even with a high follow-up level of cholesterol, these data might come from the pleiotropic effects of pitavastatin.

Effects of a high-fat diet on the electrical properties of porcine atria.

BACKGROUND: Because obesity is an important risk factor for atrial fibrillation (AF), we conducted an animal study to examine the effect of a high-fat diet (HFD) on atrial properties and AF inducibility. METHODS: Ten 8-week-old pigs (weight, 18-23 kg) were divided into two groups. For 18 weeks, five pigs were fed a HFD (HFD group) and five were fed a normal diet (control group). Maps of atrial activation and voltages during sinus rhythm were created for all pigs using the EnSite NavX system. Effective refractory period (ERP) and AF inducibility were also determined. When AF was induced, complex fractionated atrial electrogram (CFAE) mapping was performed. At 18 weeks, hearts were removed for comparing the results of histological analysis between the two groups. Body weight, lipid levels, hemodynamics, cardiac structures, and electrophysiological properties were also compared. RESULTS: Total cholesterol levels were significantly higher (347 [191-434] vs. 81 [67-88] mg/dL, P=0.0088), and left atrium pressure was higher (34.5 [25.6-39.5] vs. 24.5 [21.3-27.8] mmHg, P=0.0833) in the HFD group than in the control group, although body weight only increased marginally (89 [78-101] vs. 70 [66-91] kg, P=0.3472). ERPs of the pulmonary vein (PV) were shorter (P<0.05) and AF lasted longer in the HFD group than in the control group (80 [45-1350] vs. 22 [3-30] s, P=0.0212). Neither CFAE site distribution nor histopathological characteristics differed between the two groups. CONCLUSIONS: The shorter ERPs for the PV observed in response to the HFD increased vulnerability to AF, and these electrophysiological characteristics may underlie obesity-related AF.

Effect of endothelial cell proliferation on atherogenesis: a role of p21(Sdi/Cip/Waf1) in monocyte adhesion to endothelial cells.

OBJECTIVE: Uniform laminar shear stress (LS) and disturbed turbulent shear stress (DS) are thought to play opposite roles in preventing or inducing atherosclerosis. Endothelial cell (EC) growth and monocyte adhesion to ECs, an early event in atherosclerosis, are also oppositely regulated by LS and DS. However, how atherogenesis is affected by the regulation of growth by blood flow is unknown. Here we examined the role of p21(Sdi/Cip/Waf1) (p21), a growth inhibitor induced by LS, in monocyte adhesion to ECs. METHODS: p21 was overexpressed by transfecting a p21-expressing adenoviral vector into ECs. Factors linking EC growth, monocyte adhesion, and p21 were examined by microarray analysis, PCR and Western blotting. RESULTS: Compared with DS, in the presence or absence of TNFalpha, LS significantly inhibited EC growth and monocyte adhesion to ECs. Both EC proliferation and monocyte adhesion induced by DS were inhibited by p21-overexpression. LS suppressed the expression of thioredoxin-interacting protein (TXNIP). Thioredoxin (TRX) activity, which is suppressed by TXNIP, was therefore higher under LS than DS, as reported previously. p21-overexpression significantly suppressed the DS-induced TXNIP expression, and inhibited the expression of vascular cell adhesion molecule 1 and chemokine (C-C motif) ligand 5 (CCL5/RANTES), which stimulates leukocyte recruitment and is downregulated by ROS scavenging. CONCLUSION: p21 may function to prevent atherogenesis by regulating the redox balance, which leads to the inhibition of adhesion molecule and chemokine expression in ECs under LS.

Inflammatory pseudotumor (inflammatory myofibroblastic tumor) of the mitral valve of the heart.

We report a rare case of inflammatory pseudotumor/inflammatory myofibroblastic tumor (IPT/IMT) of the heart, involving the mitral valve. A 58-year-old woman presenting with dyspnea was immediately admitted to the hospital, and found to have congestive heart failure due to the invagination of a tumor-like mass of the mitral valve. This mass arose from and involved almost the entire posterior leaflet of the mitral valve and occupied almost the entire mitral valve orifice. The tumor was a yellowish-white well-circumscribed mass with a smooth surface. The excised mass was 3.0 x 2.3 x 1.8 cm, and consisted of abundant Sudan III-positive foam cells, histiocytes, lymphocytes, plasma cells, and loosely arrayed spindle cells, in vascular-rich fibrous tissue. Immunohistochemistry showed that the spindle cells were positive for vimentin and alpha-smooth muscle actin, and negative for desmin, CD34, and human muscle actin (HHF-35), suggesting they were myofibroblastic cells. The plasma cells and lymphocytes showed no monoclonality. There were few mitotic cells, and, except for the lymphocytes, few Ki-67-positive cells. The findings suggested IPT/IMT. The 39 cardiac IPT/IMT cases appearing in the English-language literature are discussed.

Laminar high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in endothelium. A quantitative analysis.

Remodeling of endothelial basement membrane is important in atherogenesis. Since little is known about the actual relationship between type IV collagen and matrix metalloprotease-2 (MMP-2) in endothelial cells (ECs) under shear stress by blood flow, we performed quantitative analysis for type IV collagen and MMP-2 in ECs under high shear stress. The mRNA of type IV collagen from ECs exposed to high shear stress (10 and 30 dyn/cm(2)) had a higher expression compared to ECs exposed to a static condition or low shear stress (3 dyn/cm(2)) (P < 0.01). (3)H-proline uptake analysis and fluorography revealed a remarkable increase of type IV collagen under high shear stress (P < 0.01). In contrast, zymography revealed that exposing to high shear stress, however similar positivity was leveled in the intracellular MMP-2 in the control and high shear stress-exposed ECs, reduced the secretion of MMP-2 in ECs. The results of Northern blotting, gelatin zymography and monitoring the intracellular trafficking of GFP-labeled MMP-2 revealed that MMP-2 secretion by ECs was completely suppressed by high shear stress, but the intracellular mRNA expression, protein synthesis, and transport of MMP-2 were not affected. In conclusion, we suggest that high shear stress up-regulates type IV collagen synthesis and down-regulates MMP-2 secretion in ECs, which plays an important role in remodeling of the endothelial basement membrane and may suppress atherogenesis.

Increased expression of peroxisome proliferator-activated receptor (PPAR)-alpha and PPAR-gamma in human atherosclerosis.

To clarify the mechanism of atherosclerosis development in humans, we studied the mRNA and protein expression of PPAR subtypes in various types of atherosclerotic lesions and their correlation with cell proliferation and macrophage invasion. Human aortas were obtained from 35 autopsied cases, and each sample was divided into halves. One half was used for the analysis of mRNA or protein expression with RT-PCR or Western blotting, respectively. The other was microscopically classified into atheromatous plaque (AP), fatty streak (FS), and diffuse intimal thickening (DIT), and was analyzed immunohistochemically. The mRNA levels of both PPARs increased significantly in atherosclerosis and tended to increase in proportion to the severity of the lesion, and the expression of PPAR-alpha correlated with that of PPAR-gamma in FS and AP. The PPAR-gamma protein increased in AP. Monocytes/macrophages, as well as endothelial and smooth muscle cells, expressed the PPAR-gamma protein in plaques. This expression in the DIT was noted mainly in macrophages but was not correlated with the density of macrophages, suggesting that only certain macrophages express the PPARs in DIT. Cell proliferation did not correlate with PPARs expression in any lesion type. These findings suggest that PPARs may be associated with atheromatous plaque formation, and that PPAR-gamma may be involved in the early stages of human atherosclerosis.

Appropriate doses of granulocyte-colony stimulating factor reduced atherosclerotic plaque formation and increased plaque stability in cholesterol-fed rabbits.

AIM: To evaluate the dose-response effects of granulocyte-colony stimulating factor (G-CSF) on atherosclerosis, we examined how G-CSF treatment at different concentrations affects atherosclerotic plaque formation in the aorta of cholesterol-fed rabbits. METHODS: Japanese White rabbits (n=8 each) fed on a 1.5% cholesterol diet were subcutaneously injected with G-CSF at 50 (GL), 100 (GM), or 300 microg/kg/day (GH) for five days, or with 3 cycles of G-CSF at 100 microg/kg/day at 3-week intervals (GM3), or human serum albumin (Control). The extent and composition of atherosclerosis was evaluated 14 weeks after cholesterol feeding. RESULTS: Although G-CSF treatment did not affect plasma lipid levels, the percentage of aortic surface involvement in the GM3 group was significantly decreased (p<0.05) compared with the Control group. Histological analysis revealed that the intima media ratio was also diminished in GM and GM3 groups. The extent of intimal smooth muscle cell accumulation was higher in GL and GM3 groups than in the Control group. TIMP-2 mRNA expression in the aortic tissue was increased by G-CSF treatment. CONCLUSIONS: Our results suggest that appropriate doses of G-CSF reduced atherosclerotic plaque formation and increased plaque stability in cholesterol-fed rabbits.

Low invasive angiogenic therapy for myocardial infarction by retrograde transplantation of mononuclear cells expressing the VEGF gene.

BACKGROUND: Although transplantation of mononuclear cells (MNCs) induces angiogenesis in myocardial infarction, transplantation requires a large amount of bone marrow or peripheral blood cells. We examined the effects of transplantation of peripheral MNCs expressing an exogenous vascular endothelial growth factor (VEGF) gene in a pig model of acute myocardial infarction (AMI). METHODS: MNCs were isolated from 20 ml peripheral blood from pigs and transfected with 10 microg of human VEGF165 plasmid (phVEGF). Myocardial infarction was induced by occlusion of the mid portion of the left anterior descending coronary artery (LAD) in anesthetized pigs. At 4 h after total occlusion, 5 x 10(6) VEGF-transfected MNCs were retrogradely transplanted into the pig via the coronary vein. Cardiac function, neovascularization and histology of the ischemic tissue were evaluated 4 weeks after transplantation. RESULTS: MNCs expressing hVEGF and infused via the coronary vein were efficiently delivered the heart in pigs with myocardial infarction. Transplantation of MNCs expressing hVEGF significantly increased left ventricular (LV) function, collateral vessels, and capillary density in heart from AMI model pigs. Transplantation of MNCs expressing hVEGF increased the wall thickness of the scar in the heart after AMI. CONCLUSIONS: Retrograde transplantation of peripheral blood MNCs expressing hVEGF efficiently induced angiogenesis and improved the impaired LV function in hearts of pigs with AMI. These findings indicate that angiogenic cells and gene therapy may be useful to treat ischemic heart disease.

Ubiquitin-positive foam cells are identified in the aortic and mitral valves with atherosclerotic involvement.

AIM: Our aim was to determine the roles of the ubiquitin (Ub)-proteasome system (UPS) in valvular diseases by immunohistochemically identifying Ub-positive cells in aortic and mitral valves and determining if Ub+cells were associated with the severity of valvular diseases. METHODS: We evaluated surgically removed aortic and mitral valves from 60 patients (mean age, 64.5 years) for thickening, fibrosis, foam cell infiltration, thrombus, and atheromatous plaques by using grading scores. U+cells were detected immunohistochemically. RESULTS: We found Ub+cells in 16 (26.7%) of the 60 patients. Eleven (28.2%) of the 39 aortic valves and 5 (23.8%) of the 21 mitral valves were Ub-positive. Ub was found with granular depositions in the cytoplasm of monocyte-derived foam cells that were CD68+. The aortic valvular thickness of the Ub+group was significantly greater than that of the Ub- group (3.9+/-1.6mm vs. 3.2+/-1.6mm, p<0.05). Foam cells and fibrosis were greater in the Ub+group (p<0.05), and calcifications were prominent in aortic valves. There was no difference in the number of apoptotic cells in Ub+ and Ub- groups. Ub+cells were present in the affected valves and ubiquitinated proteins were accumulated in macrophage-derived foam cells. CONCLUSIONS: Ub+ foam cells are present in valves that are vulnerable to valvular disease, and UPS may contribute to the development of atherosclerosis through the inflammatory process.

Dedifferentiated fat cells convert to cardiomyocyte phenotype and repair infarcted cardiac tissue in rats.

Adipose tissue-derived stem cells have been demonstrated to differentiate into cardiomyocytes and vascular endothelial cells. Here we investigate whether mature adipocyte-derived dedifferentiated fat (DFAT) cells can differentiate to cardiomyocytes in vitro and in vivo by establishing DFAT cell lines via ceiling culture of mature adipocytes. DFAT cells were obtained by dedifferentiation of mature adipocytes from GFP-transgenic rats. We evaluated the differentiating ability of DFAT cells into cardiomyocytes by detection of the cardiac phenotype markers in immunocytochemical and RT-PCR analyses in vitro. We also examined effects of the transplantation of DFAT cells into the infarcted heart of rats on cardiomyocytes regeneration and angiogenesis. DFAT cells expressed cardiac phenotype markers when cocultured with cardiomyocytes and also when grown in MethoCult medium in the absence of cardiomyocytes, indicating that DFAT cells have the potential to differentiate to cardiomyocyte lineage. In a rat acute myocardial infarction model, transplanted DFAT cells were efficiently accumulated in infarcted myocardium and expressed cardiac sarcomeric actin at 8 weeks after the cell transplantation. The transplantation of DFAT cells significantly (p<0.05) increased capillary density in the infarcted area when compared with hearts from saline-injected control rats. We demonstrated that DFAT cells have the ability to differentiate to cardiomyocyte-like cells in vitro and in vivo. In addition, transplantation of DFAT cells led to neovascuralization in rats with myocardial infarction. We propose that DFAT cells represent a promising candidate cell source for cardiomyocyte regeneration in severe ischemic heart disease.

Association of serum oxidized lipoprotein(a) concentration with coronary artery disease: potential role of oxidized lipoprotein(a) in the vasucular wall.

AIM: A new antibody reacted with an epitope in Lp(a) that has undergone oxidation treatment, but is not present in native Lp(a), was developed. Thus, we determined serum oxidized Lp(a) concentration in healthy volunteers, and coronary artery disease (CAD), diabetes mellitus (DM), and hypertensive patients. METHODS: We measured serum levels of oxidized Lp(a), Lp(a), LDL-cholesterol and HDL-cholesterol in 122 consecutive patients who underwent routine coronary angiography and had significant coronary artery stenosis (>75%), and 164 age-matched healthy volunteers. Moreover, serum native Lp(a), oxidized Lp(a) concentration, and pulse wave velocity (PWV) were determined in 181 hypertensive patients. RESULTS: Oxidized Lp(a) level in CAD patients with DM was significantly higher than in healthy volunteers (p<0.01). Moreover, serum oxidized Lp(a) concentration showed a significant positive correlation with pulse wave velocity, an index of arteriosclerosis (r=0.431, p<0.01). Of importance, the deposition of oxidized Lp(a) was readily detected in calcified areas of coronary arteries in patients with myocardial infarction. CONCLUSION: The present study demonstrated that oxidized Lp(a) may be a new risk factor for coronary artery disease. As the deposition of oxidized Lp(a) was detected in calcified areas of coronary arteries, oxidized Lp(a) might be implicated in endothelial dysfunction.

[Immunohistochemical study of nonspecific interstitial pneumonia and ubiquitin-positive pneumocytes in elderly patients].

AIM: The Ubiquitin (Ub)-proteasome system maintains cellular homeostasis through proteolysis, and Ub appears in the damaged cells of many organs. Nonspecific interstitial pneumonia (NSIP)in elderly patients was studied to clarify the relationship between Ub-positive cells, cellular damage, and resistance to therapy. METHODS: Specimens of surgical lung biopsy with the NSIP pattern (NSIP/P) from 13 patients (mean age, 68 years old) were examined histologically and immunohistochemically. Pneumocytes examined under high-power microscopy were counted for eosinophilic inclusion bodies and Ub-positive cells. NSIP/P was histologically evaluated and cases were scored for erosion and intraluminal granulation tissue subtypes (polypoid, mural, or occluded) as lung injury markers. NSIP/P was subdivided into cellular NSIP and fibrosing NSIP according to the proportions of interstitial inflammation and fibrosis. RESULTS: 1) Six of 13 patients with NSIP/P had Ub-positive cells (Ub+ group), and all inclusions identified by light-microscope were positive for Ub. A greater number of Ub+ pneumocytes were found compared with the inclusions by light-microscope, and Ub immunostaining was useful for the detection of the inclusions. 2) Granulation tissue scores in the Ub+ group were significantly greater than in the Ub- group (p<0.05); there was no difference in granulation tissue subtypes between the groups. 3) Granulation tissue scores in fibrosing NSIP/P (including each subtype) were significantly greater than in cellular NSIP/P. 4) After a follow-up period, there was no correlation between Ub+ group and NSIP therapy resistance or between the granulation tissue subtypes and therapy resistance. CONCLUSION: Some elderly patients with NSIP had inclusions, and these inclusions were Ub+. Pneumocyte injury might occur via the Ub-proteasome system pathway in elderly patients with NSIP/P, although there was no correlation between Ub+ pneumocytes and therapy resistance.

Presence of alpha-smooth muscle actin-positive endothelial cells in the luminal surface of adult aorta.

Alpha-smooth muscle actin-positive endothelial cells have not been found in adult aortic endothelium except valve leaflets. Here, using en face immunostaining method, we identified alpha-smooth muscle actin-positive endothelial cells in the luminal surface of rat, mouse and human thoracic aortas. These cells express both endothelial markers and definite smooth muscle cell markers and were only occasionally observed in thoracic aorta of wild type mice and rats. Their density did not increase with aging. Given that alpha-smooth muscle actin-positive endothelial cells express low level of vascular endothelial-cadherin that is important for the maintenance of cell contact, these cells were frequently detected in the thoracic aorta of 5-week-old apolipoprotein-E deficient mice. In 20- to 24-week-old apolipoprotein-E deficient mice, marked accumulation of alpha-smooth muscle actin-positive endothelial cells was observed especially in the luminal surface of atheromatous plaques. Our findings indicate the existence of alpha-smooth muscle actin-positive endothelial cells in adult aortic endothelium and the possible association with progression of atherosclerosis.

Atherogenic dyslipidemia and altered hepatic gene expression in SHRSP.Z-Leprfa/IzmDmcr rats.

We investigated lipid and lipoprotein abnormalities in SHRSP fatty rats as a new animal model of metabolic syndrome. We examined differentially expressed genes in the liver, one of the major tissues contributing to lipid metabolism. Using gel filtration high performance liquid chromatography, increased cholesterol concentrations of small particle size low-density lipoprotein (LDL) fractions were observed in SHRSP fatty rats, whereas the Zucker Fatty strain did not show a similar elevation of cholesterol content. Existence of apolipoprotein B in these fractions was confirmed by Western blotting. The small particle size of the LDL fractions was significantly decreased by a 4-week fenofibrate treatment. Microarray analysis identified seventeen genes that were significantly upregulated and ten that were significantly decreased in liver tissues of SHRSP fatty rats compared with levels in SHRSP rats. Stearoyl-coenzyme A desaturase 1, fatty acid synthase, ATP citrate lyase, and sterol regulatory element binding factor 1 genes were among the upregulated genes. These findings suggest that SHRSP fatty rats carry small dense LDL like particles which is a common lipid abnormality in the metabolic syndrome. Three of ten genes upregulated in liver tissues of SHRSP fatty rats play a role in this metabolic abnormality and are a therapeutic target of this metabolic syndrome.