Improved assay system for acidic peptide: N-glycanase (aPNGase) activity in plant extracts.

Plant acidic peptide: N-glycanase (aPNGase) release N-glycans from glycopeptides during the degradation process of glycoproteins in developing or growing plants. We have previously developed a new method to detect the aPNGase activity in crude extracts, which is prerequisite for the construction of aPNGase knockout or overexpression lines. However, this method has the disadvantage of requiring de-sialylation treatment and a lectin chromatography. In this study, therefore, we improved the simple and accurate method for detecting aPNGase activity using anion-exchange HPLC requiring neither the desialylation treatment nor the lectin affinity chromatography.

Direct evidence of cytosolic PNGase activity in Arabidopsis thaliana: in vitro assay system for plant cPNGase activity.

Cytosolic peptide:N-glycanase (cPNGase), which occurs ubiquitously in eukaryotic cells, is involved in the de-N-glycosylation of misfolded glycoproteins in the protein quality control system. In this study, we aimed to provide direct evidence of plant cPNGase activity against a denatured glycoprotein using a crude extract prepared from a mutant line of Arabidopsis thaliana lacking 2 acidic PNGase genes.

Novel assay system for acidic Peptide:N-glycanase (aPNGase) activity in crude plant extract.

Acidic peptide:N-glycanase (aPNGase) plays a pivotal role in plant glycoprotein turnover. For the construction of aPNGase-knockout or -overexpressing plants, a new method to detect the activity in crude plant extracts is required because endogenous peptidases present in the extract hamper enzyme assays using fluorescence-labeled N-glycopeptides as a substrate. In this study, we developed a new method for measuring aPNGase activity in crude extracts from plant materials.

Knee joint synovial hemangioma treated with arthroscopic resection without hemarthrosis: A case report.

INTRODUCTION: Intra-articular synovial hemangioma of the knee is a relatively rare benign tumor that if left undiagnosed and treated may be followed by degenerative cartilaginous changes and osteoarthritis. However, the non-specific symptoms of synovial hemangiomas limit its early diagnosis. We report our encounter with synovial hemangioma of the knee in which the diagnosis was based on a > 20-year history of chronic pain without joint swelling or hematoma. PRESENTATION OF CASE: A 34-year-old man with a localized vascular malformation on the upper edge of the left patella presented with pain and a restricted range of motion. CT and MRI revealed a tumorous lesion at this location. Upon excision of the lesion, the patient's symptoms disappeared, with no recurrence at the 1-year follow-up. DISCUSSION: Accurate diagnosis and appropriate early treatment are necessary for synovial hemangiomas to forestall articular cartilage degeneration due to recurrent intra-articular hemorrhages. CONCLUSION: Synovial hemangioma should be considered when a patient presents with recurrent knee pain, even in the absence of swelling or episodes of joint effusion.

Mobilized bone marrow progenitor cells serve as donors of cytoprotective genes for cardiac repair.

We proposed here that mobilized progenitor cells (MPCs) from the bone marrow are special cell types which carry cytoprotective proteins for cardiac repair following ischemia. Myocardial ischemia was induced by ligation of the left anterior descending coronary artery (LAD) in mice. Progenitor cells in peripheral blood were analyzed by fluorescence-activated cell sorting (FACS). The expression of cytoprotective genes was assayed by ELISA, RT-PCR, and/or real-time PCR. G-CSF was markedly up-regulated in the ischemic myocardium. A good correlation was observed between serum G-CSF and progenitor cells in circulation following LAD ligation. MPCs overexpressed cardiac transcription factor, GATA-4, and anti-apoptotic factor, Bcl-2, besides expression of the surface markers of bone marrow stem cells (BMSCs). Transplantation of cultured MPCs into the ischemic border area significantly improved cardiac function by reducing infarction size. More importantly, MPCs significantly protected cardiomyocytes against apoptosis when co-cultured with cardiomyocytes. The cardiac protection by MPCs was blocked by Bcl-2 neutralizing antibody and GATA-4 siRNA. In contrast, transfection of BMSCs with GATA-4 provided increased protection of myocytes against apoptosis. It is concluded that MPCs are highly cytoprotective and carry protective genes responsible for cardiac repair.

Bone marrow stem cells prevent left ventricular remodeling of ischemic heart through paracrine signaling.

In this study, we hypothesized that bone marrow stem cells (BMSCs) protect ischemic myocardium through paracrine effects that can be further augmented with preconditioning. In in vitro experiments, cell survival factors such as Akt and eNOS were significantly increased in BMSCs following anoxia. In the second series of experiments following coronary ligation in mice, left ventricles were randomly injected with the following: DMEM (G-1), BMSCs (G-2), and preconditioned BMSCs (G-3). Four days after myocardial infarction, BMSCs were observed within injured myocardium in G-2 and G-3. Apoptotic cardiomyocytes within periinfarct area were significantly reduced in G-3. Four weeks after myocardial infarction, smaller left ventricular (LV) dimension and increased LV ejection fraction were observed in G-3. Infarct area was significantly reduced in G-3. However, GFP+ cardiomyocytes were observed in low numbers within periinfarct area in G-2 and G-3. In conclusion, BMSCs secreted cell survival factors under ischemia, and they prevented apoptosis in cardiomyocytes adjacent to the infarcted area. Preconditioning of BMSCs enhanced their survival and ability to attenuate LV remodeling, which was attributable, in part, to paracrine effects.

Progressed Multivessel Spontaneous Coronary Artery Dissection That Naturally Healed in a Male Patient with Non-ST Segment Elevation Myocardial Infarction.

Spontaneous coronary artery dissection (SCAD) is a rare condition that may have a serious outcome because of acute coronary syndrome. The condition especially affects young women. We evaluated a middle-aged male patient with a non-ST segment elevation myocardial infarction caused by multivessel SCAD. The SCAD had occurred in the distal right coronary artery (RCA), the mid left anterior descending artery (LAD), and the distal LAD at the same time. His culprit lesion was in the distal RCA, but the SCAD had progressed more proximally within the RCA 12 days later with no clinical symptoms. We treated the mid LAD with implantation of a drug-eluting stent on admission and the SCAD had not progressed 12 days later. Moreover, the SCAD in the distal RCA and distal LAD healed spontaneously 12 days later. He had no recurrent attack, and all SCAD lesions of the RCA and LAD had completely healed 6 months later. Given that SCAD appears in various forms over the clinical course, a strategy of intervention needs careful consideration.

A honeycomb-like structure in the left anterior descending coronary artery treated using a scoring device and drug-eluting stent implantation: a case report.

BACKGROUND: A honeycomb-like structure in the coronary artery is rarely diagnosed by intracoronary ultrasound or optical coherence tomography. Further, its structural mechanisms and response to interventional therapy remain unknown. CASE PRESENTATION: A 59-year-old Japanese man was referred to our hospital because of acute decompensated heart failure with rapid atrial fibrillation. After receiving anticoagulant therapy, a coronary angiogram revealed a braid-like appearance and an intracoronary ultrasound image confirmed a honeycomb-like structure in the mid left anterior descending coronary artery. We inserted two guide wires into different partitions. Although a balloon angioplasty with a scoring device could not completely fenestrate these partitions, a stent implant was able to completely compress the structure easily. CONCLUSIONS: The honeycomb-like structure of the left anterior descending coronary artery in our patient was suspected to be because of recanalization of a cardiogenic embolism. This structure may have been composed of relatively hard tissues, but was easily compressed by a stent implantation.

Elevated troponin T levels and lesion characteristics in non-ST-elevation acute coronary syndromes.

BACKGROUND: Elevated troponin T levels in non-ST-elevation acute coronary syndromes (NSTE-ACS) have been shown to predict an adverse outcome. Furthermore, it has been reported that troponin T could help improve the effectiveness of such new antithrombotic drugs as platelet GPIIb/IIIa antagonists and low-molecular-weight heparins. We hypothesized that such elevated troponin T levels in NSTE-ACS indicate the presence of thrombus at culprit lesions, and this hypothesis was verified through the use of coronary angioscopy. METHODS AND RESULTS: We studied 57 consecutive patients with NSTE-ACS who underwent preinterventional angioscopy. Before catheterization, we obtained blood samples to determine troponin positivity, and the patients were then classified as either troponin-positive or troponin-negative groups (diagnostic threshold, 0.1 ng/mL). Using angioscopy at the culprit lesions, we examined the presence of coronary thrombus, yellow plaque, and complex plaque. Moreover, we compared the preinterventional angiographic parameters (thrombus and complexity of the culprit lesion, and TIMI flow) between the two groups. Twenty-two patients were troponin-positive and 35 patients were troponin-negative. Univariate analyses indicated that the TIMI flow and the incidence of coronary thrombus detected with angioscopy correlate with the elevated troponin T levels. A multivariate logistic regression analysis showed the presence of coronary thrombus detected with angioscopy to be the only independent factor associated with elevated troponin T levels in patients with NSTE-ACS (odds ratio, 22.1; 95% CI, 2.59 to 188.42; P=0.0046). CONCLUSIONS: Using angioscopy, the elevated troponin T levels in NSTE-ACS were confirmed to be strongly associated with the presence of coronary thrombus.

Changes in coronary plaque color and morphology by lipid-lowering therapy with atorvastatin: serial evaluation by coronary angioscopy.

OBJECTIVES: Changes in coronary plaque color and morphology by statin therapy were evaluated using coronary angioscopy. BACKGROUND: Coronary plaque stabilization by statin therapy has not been clarified in humans. METHODS: Thirty-one patients with coronary artery disease were divided into either the comparison group (n = 16) or the atorvastatin group (n = 15). Before treatment and 12 months after, the color and complexity of 145 coronary plaques were determined according to angioscopic findings. The yellow score of the plaque was defined as 0 (white), 1 (light yellow), 2 (yellow), or 3 (dark yellow), and its disrupted score was defined as 0 (smooth surface) or 1 (irregular surface) and as 0 (without thrombus) or 1 (with thrombus). In each patient, the mean yellow score and mean disrupted score were calculated. RESULTS: Mean low-density lipoprotein cholesterol (LDL-C) decreased by 45% in the atorvastatin group, whereas an increase of 9% was seen in the comparison group. The mean yellow score decreased from 2.03 to 1.13 in the atorvastatin group, whereas it increased from 1.67 to 1.99 in the comparison group. There was a good correlation between the change in the mean yellow score and the change in LDL-C levels (r = 0.81, p < 0.0001). The change in the mean yellow score and mean disrupted score differed significantly between the two groups (p = 0.002 and p = 0.03, respectively). CONCLUSIONS: This is the first report clarifying detailed changes in coronary plaque by statin in humans. This study indicated that lipid-lowering therapy changes plaque color and morphology and should then lead to coronary plaque stabilization.

Morphologic changes in infarct-related plaque after coronary stent placement: a serial angioscopy study.

OBJECTIVES: The aim of this study was to investigate the morphologic changes in infarct-related lesions after stenting in acute or recent myocardial infarction (MI) with coronary angioscopy. BACKGROUND: There is no information on the serial morphologic changes, which occur after stenting, and the time course of neointimal coverage of stents for disrupted unstable plaques. METHODS: Forty-three patients with MI within seven days of onset were examined. Angioscopy was serially performed for the infarct-related lesions at baseline (n = 43), after balloon angioplasty (n = 35), and after stenting following balloon angioplasty (n = 39) and at one (n = 36) and six months (n = 30) after stenting. RESULTS: At baseline, most of the lesions had complex morphology, yellow plaque color, and protruding thrombus (96%, 96%, and 74%, respectively). Although balloon angioplasty reduced the protruding thrombus, it remained in 37%, and an intimal flap was observed in 89% of the lesions. After stenting, the protruding thrombus and intimal flap disappeared, with an increased luminal size obtained in all lesions. At one-month follow-up, an irregular and yellow surface, along with a lining thrombus, was still observed, with partial neointimal stent coverage in most of the lesions. At six-month follow-up, the neointima was found to have sufficiently formed over the stent. The plaque shape and color were almost all classified as smooth (97%) and white (93%). CONCLUSIONS: These results suggest that a stent not only compressed and covered a disrupted plaque with a protruding thrombus and intimal flap, leading to a wide vessel lumen, but also helped to seal the unstable plaque through neointimal proliferation.

Extended follow-up by serial angioscopic observation for bare-metal stents in native coronary arteries: from healing response to atherosclerotic transformation of neointima.

BACKGROUND: Although coronary angiograms after bare-metal stent (BMS) implantation show late luminal narrowing beyond 4 years, the detailed changes inside the BMS have not yet been fully elucidated. METHODS AND RESULTS: Serial angiographic and angioscopic examinations were performed immediately (baseline), 6 to 12 months (first follow-up), and >or=4 years (second follow-up) after stenting without target lesion revascularization in 26 segments of 26 patients who received BMS deployment for their native coronary arteries. Angioscopic observation showed atherosclerotic yellow plaque crushed out by stent struts in 22 patients (85%) and mural thrombus in 21 patients (81%) at baseline. At first follow-up, white neointimal hyperplasia was almost completely buried inside the struts, and both yellow plaque and thrombus had decreased in comparison with baseline (12% and 4%, respectively; P<0.001). The frequencies of yellow plaque and thrombus increased from the first to second follow-ups (58% and 31%, respectively; P<0.05). All of the yellow plaques in the second follow-up were located not exterior to the struts but protruding from the vessel wall into the lumen. Late luminal narrowing, defined as an increasing of percent diameter stenosis between the first and second follow-ups, was greater in segments with yellow plaque than in those without yellow plaque (18.4+/-17.3% versus 3.6+/-4.2%, respectively; P=0.011). CONCLUSIONS: This angiographic and angioscopic study suggests that white neointima of the BMS may often change into yellow plaque over an extended period of time, and atherosclerotic progression inside the BMS may contribute to late luminal narrowing.

Multiple yellow plaques assessed by angioscopy with quantitative colorimetry in patients with myocardial infarction.

BACKGROUND: Multiple angioscopic yellow plaques are associated with diffuse atherosclerotic plaque, and may be prevalent in patients with myocardial infarction (MI), so in the present study the yellow plaques in the coronary arteries of patients with MI was evaluated using quantitative colorimetry, and compared with those of patients with stable angina (SA). METHODS AND RESULTS: In the recorded angioscopic images of 3 coronary vessels in 29 patients (15 patients with MI, 14 with SA), yellow plaques were determined as visually yellow regions with b* value >0 (yellow color intensity) measured by the quantitative colorimetric method. A total of 90 yellow plaques were identified (b* =19.35+/-8.3, 3.05-45.35). Yellow plaques were significantly more prevalent in 14 (93%) of 15 culprit lesions of MI as compared with 8 (57%) of 14 of SA (p=0.03). In non-culprit segments, yellow plaques were similarly prevalent in 13 (87%) patients with MI and 11 (79%) with SA (p=0.65). Overall, multiple (> or =2) yellow plaques were prevalent in 13 (87%) patients with MI, similar to the 10 (71%) with SA (p=0.38). The number of yellow plaques was significantly higher in patients with MI (3.8+/-1.9) than in those with SA (2.4+/-1.6, p=0.03). CONCLUSION: The present study suggests that patients with MI tend to have diffuse atherosclerotic plaque in their coronary arteries.

In vitro and in vivo effects of bone marrow stem cells on cardiac structure and function.

It is hypothesized that the protection of bone marrow stem cells (BMSCs) on ischemic myocardium might be related to the anti-apoptotic effect via paracrine mechanisms. In this study, a wide array of cytokines including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), stromal cell-derived factor-1 (SDF-1) and insulin growth factor-1 (IGF-1) were detected in the BMSCs cultured medium by ELISA. Myocyte apoptosis was assayed by DNA fragmentation and annexin-V staining. Myocardial infarction model was produced by ligation of mouse left anterior descending coronary artery (LAD). Before LAD ligation, mice were myoablated by irradiation and transplanted with bone marrow cells from transgenic mice expressing green fluorescent protein (GFP). After LAD ligation, animals were administered stem cell factor (SCF, 200 mug/day/kg, i.p.) or saline for 6 days. Animals were sacrificed at 4 weeks after SCF treatment. Apoptotic cardiomyocytes were analyzed by TUNEL. Myocardial function was analyzed by echocardiography and pressure-volume system. Bcl-2 protein was analyzed by Western blotting. Our results showed that cultured BMSCs released VEGF, bFGF, SDF-1 and IGF-1. Hypoxia-induced cell apoptosis was diminished in cardiomyocytes co-cultured with BMSCs. Smaller LV dimension and increased LV ejection fraction were seen in SCF-treated animals. SCF significantly reduced cardiomyocytes apoptosis within peri-infarct area and increased up-regulation expression of Bcl-2 in ischemic area. Moreover, conditioned medium from cultured BMSCs also induced up-regulation of Bcl-2 protein in cardiomyocytes. It is concluded that paracrine mediators secreted by BMSCs might be involved in early repair of ischemic heart by preventing cardiomyocytes apoptosis and improving cardiac function.

Impact of histological plaque characteristics on intravascular ultrasound parameters at culprit lesions in coronary artery disease.

Prior intravascular ultrasound (IVUS) studies have demonstrated that a positive remodeling pattern of a culprit lesion is observed more frequently in acute coronary syndrome (ACS) than stable angina (SA). However, the relationship between the plaque morphology detected by IVUS and the histological type of atherosclerotic plaque has not been well defined. This is a prospective study on 37 consecutive patients who underwent directional coronary atherectomy. The 37 patients were divided into 2 groups; 21 patients with SA and 16 with ACS. Vessel and plaque cross sectional area were measured at the culprit lesion and the remodeling index (RI) was calculated by IVUS. The plaque tissue was assessed for the presence of inflammatory cells and lipids, and the presence of each was scored as 0 (absent), 1 (sparse), 2 (dense), or 3 (predominant). The RI of the patients with ACS was higher than that of SA. Inflammatory cells were present to a greater extent in patients with ACS. Inflammatory cells and lipids were significantly correlated with the RI (Inflammatory cell score grade > or = 2 patients; 1.14 +/- 0.13 versus grade 0 patients; 0.87 +/- 0.24, and grade 1 patients; 0.93 +/- 0.17, P < 0.01 and lipid score grade > or = 2 patients; 1.13 +/- 0.17 versus grade 0 patients; 0.85 +/- 0.18, P < 0.001 and grade 1 patients; 0.95 +/- 0.19, P < 0.05). The results clearly indicate that the evaluation of vessel morphology by vascular imaging is an important indicator of plaque instability.

[Left ventricular peak systolic pressure/end-systolic volume ratio change after dobutamine infusion for predicting left ventricular contractile reserve: comparison with Emax].

OBJECTIVES: Genuine left ventricular contractile function is difficult to assess in the clinical setting. Left ventricular peak systolic pressure/end-systolic volume (Pps/Ves) ratio may be misleading because this index takes no account of the left ventricular end-systolic point and V0 intercept in the pressure-volume relation geographic curve. End-systolic pressure-volume relation and maximum chamber elastance derived from left ventricular pressure-volume loops can provide reliable estimates of contractile function. However, the feasibility of this technique for clinical purposes is limited, because it requires instantaneous measurement of left ventricular pressure and volume. This study assessed the feasibility of using Pps/Ves ratio for predicting the left ventricular contractile reserve by direct comparison with maximum elastance (Emax) derived from left ventricular pressure-volume loops. METHODS: Studies were undertaken in 18 consecutive patients aged 60 +/- 9 years who underwent cardiac catheterization. On-line instantaneous left ventricular volume was derived from the acoustic quantification method by transthoracic echocardiography. Pps was determined by pressure manometer tipped wire and Ves was measured automatically from acoustic quantification software in an ultrasound system. Pps/Ves was compared with Emax derived from each simultaneous pressure-volume loop during inferior vena caval occlusion before and after dobutamine infusion. Emax was determined as the slope of end-systolic points for each loop with the use of an automated iterative linear regression technique. Left ventricular contractile reserve was assessed by evaluating its functional response to 10 micrograms/kg/min of dobutamine infusion. RESULTS: Pps/Ves showed significant correlation with Emax in all patients (r = 0.70, p < 0.0001). However, scattered distribution of V0 value differences were noted. Contractile reserve (Pps/Ves) showed strong correlation with contractile reserve (Emax) despite V0 value differences (r = 0.927, p < 0.0001). CONCLUSIONS: Pps/Ves change after dobutamine infusion may minimize individual V0 distribution. This simple index could be used to evaluate left ventricular systolic performance without requiring the left ventricular pressure-volume relationship and volume unloading maneuver.

Unique single coronary artery with acute myocardial infarction: observation of the culprit lesion by intravascular ultrasound and coronary angioscopy.

We report an acute myocardial infarction in a patient with a single coronary artery. The right coronary artery arose from the middle portion in the left anterior descending artery through the transverse branch. This type of single coronary artery has not been previously reported. Moreover, this is the first report in which the culprit lesion in a patient with a single coronary artery was observed by intravascular ultrasound and coronary angioscopy. The patient underwent successful coronary stent deployment.

Visualized plaque debris as a cause of distal embolization after percutaneous coronary intervention in patient with unstable angina.

Procedural complications of percutaneous transluminal coronary angioplasty for unstable angina are higher than for stable angina. We report a case in which coronary angioscopy proved the dislodgment of a large plaque fragment after Cutting Balloon angioplasty and confirmed our suspicion that plaque fragmentation can cause distal embolization.