Angiopoietin-like protein 2 promotes chondrogenic differentiation during bone growth as a cartilage matrix factor.

OBJECTIVE: Chondrocyte differentiation is crucial for long bone growth. Many cartilage extracellular matrix (ECM) proteins reportedly contribute to chondrocyte differentiation, indicating that mechanisms underlying chondrocyte differentiation are likely more complex than previously appreciated. Angiopoietin-like protein 2 (ANGPTL2) is a secreted factor normally abundantly produced in mesenchymal lineage cells such as adipocytes and fibroblasts, but its loss contributes to the pathogenesis of lifestyle- or aging-related diseases. However, the function of ANGPTL2 in chondrocytes, which are also differentiated from mesenchymal stem cells, remains unclear. Here, we investigate whether ANGPTL2 is expressed in or functions in chondrocytes. METHODS: First, we evaluated Angptl2 expression during chondrocyte differentiation using chondrogenic ATDC5 cells and wild-type epiphyseal cartilage of newborn mice. We next assessed ANGPTL2 function in chondrogenic differentiation and associated signaling using Angptl2 knockdown ATDC5 cells and Angptl2 knockout mice. RESULTS: ANGPTL2 is expressed in chondrocytes, particularly those located in resting and proliferative zones, and accumulates in ECM surrounding chondrocytes. Interestingly, long bone growth was retarded in Angptl2 knockout mice from neonatal to adult stages via attenuation of chondrocyte differentiation. Both in vivo and in vitro experiments show that changes in ANGPTL2 expression can also alter p38 mitogen-activated protein kinase (MAPK) activity mediated by integrin α5ß1. CONCLUSION: ANGPTL2 contributes to chondrocyte differentiation and subsequent endochondral ossification through α5ß1 integrin and p38 MAPK signaling during bone growth. Our findings provide insight into molecular mechanisms governing communication between chondrocytes and surrounding ECM components in bone growth activities.

Angiotensin converting enzyme as a genetic risk factor for coronary artery spasm. Implication in the pathogenesis of myocardial infarction.

It has been reported that individuals with the D allele of an insertion/deletion (I/D) polymorphism of the angiotensin converting enzyme (ACE) gene are at greater risk for myocardial infarction (MI), especially among subjects normally considered to be at low risk. However, little is known about the mechanism by which the ACE polymorphism affects the risk of MI. Coronary artery spasm (CAS) is considered to be one possible mechanism for developing MI. We therefore examined the ACE polymorphism relation to CAS to determine if this was the mechanism by which the DD genotype influences MI. We studied 150 angiographically assessed Japanese males, all more than 60 yr old. CASs were detected using intracoronary injection of ergonovine maleate. Subjects were divided into three groups: those with CAS (group 1), those without CAS, but with fixed organic stenosis (group 2); and those without CAS and no organic stenosis (group 3). DD subjects were significantly represented in group 1 when compared with groups 2 (P = 0.002) and 3 (P = 0.026). These results suggest that the DD genotype relates to the greater risk for MI in the patients with CAS.

Mobilization of endothelial progenitor cells in patients with acute myocardial infarction.

BACKGROUND: Endothelial progenitor cells (EPCs) circulate in adult peripheral blood (PB) and contribute to neovascularization. However, little is known regarding whether EPCs and their putative precursor, CD34-positive mononuclear cells (MNC(CD34+)), are mobilized into PB in acute ischemic events in humans. METHODS AND RESULTS: Flow cytometry revealed that circulating MNC(CD34+) counts significantly increased in patients with acute myocardial infarction (n=16), peaking on day 7 after onset, whereas they were unchanged in control subjects (n=8) who had no evidence of cardiac ischemia. During culture, PB-MNCs formed multiple cell clusters, and EPC-like attaching cells with endothelial cell lineage markers (CD31, vascular endothelial cadherin, and kinase insert domain receptor) sprouted from clusters. In patients with acute myocardial infarction, more cell clusters and EPCs developed from cultured PB-MNCs obtained on day 7 than those on day 1. Plasma levels of vascular endothelial growth factor significantly increased, peaking on day 7, and they positively correlated with circulating MNC(CD34+) counts (r=0.35, P=0.01). CONCLUSIONS: This is the first clinical demonstration showing that lineage-committed EPCs and MNC(CD34+), their putative precursors, are mobilized during an acute ischemic event in humans.

Sodium ionophore converts growth manner of vascular smooth muscle cells from spontaneously hypertensive rats.

OBJECTIVES: Vasoconstrictor peptides such as endothelin (ET) cause hypertrophy of vascular smooth muscle cells (VSMC) in Wistar Kyoto rats (WKY) and hyperplasia in spontaneously hypertensive rats (SHR). They also induce an increase in Na+ concentration ([Na+]i) and activate protein kinase C (PKC) independently. Therefore, we tested the hypothesis that the increase in [Na+]i may be involved in the conversion of growth manner under activated PKC in SHR VSMC. METHODS AND RESULTS: 10(-7) M phorbol ester (TPA) increased the diameter and protein content of VSMC from both strains under 18% serum conditions. Further addition of 10(-6) M gramicidin (Na+ ionophore) converted TPA-induced hypertrophy to hyperplasia, which was due to the quick transition from S to G2/M phase, only in SHR VSMC. Western blot analysis showed that serum- and TPA-induced tyrosine phosphorylation of mitogen-activated protein (MAP) kinase was potentiated by 10(-6) M gramicidin in SHR. [Na+]i, which was measured by sodium-binding benzofuran isophthalate (SBFI), was increased about 35 mM by 10(-6) M gramicidin in both strains, but TPA did not affect basal [Na+]i and the gramicidin-induced increase in [Na+]i. CONCLUSIONS: We conclude that sodium ionophore may convert hypertrophy to hyperplasia synergistically with activated PKC in SHR VSMC, possibly by MAP kinase phosphorylation.

Isolation and characterization of human fibroblast tenascin. An extracellular matrix glycoprotein of interest for developmental studies.

We have developed a biochemical method for purifying human tenascin from cultured fibroblasts or the culture medium. The method is a series of biochemical procedures including gel filtration, gelatin gel affinity chromatography and ion-exchange high performance liquid chromatography. The final preparation was identified as tenascin from its immunological cross-reactivity to antibody against chicken tenascin, strong hemagglutination activity which has been reported to be one of the biological functions of chicken tenascin, and from the electron microscopic study demonstrating a six-armed structure. Gel chromatography showed that intact human tenascin has an apparent molecular weight of over one million. Analysis of the purified tenascin with SDS-PAGE under reducing conditions demonstrated that tenascin consists of two kinds of subunits (250K and 190K). We established rat x mouse heterohybridoma cell lines which produce tenascin-specific antibodies. One monoclonal antibody (RCB1) was selected for immunohistochemical study and partially characterized. RCB1 bound native tenascin but not reduced and alkylated tenascin. Immunohistochemistry of normal and neoplastic tissues demonstrated that RCB1 bound the connective tissues surrounding the cancer nests and various normal tissues including interstitium of renal distal tubule, periosteum, endosteum, smooth muscles of digestive tract and media of arteries and arterioles.

Hyperventilation as a specific test for diagnosis of coronary artery spasm.

The hyperventilation test has been used as a clinical tool to induce coronary spasm. However, its diagnostic and prognostic values have not been fully elucidated. This study was designed to establish the sensitivity and specificity of the hyperventilation test and to clarify the characteristics of hyperventilation test-positive patients. We examined 206 patients in whom coronary spasm was documented by angiography (spasm group), and 183 patients without angina at rest in whom acetylcholine failed to induce spasm (nonspasm group). All patients performed vigorous hyperventilation for 6 minutes in the early morning. Of the spasm group patients, 127 showed positive responses to the test, including ST elevation (n = 111), ST depression (n = 15) and negative U wave (n = 1). None in the nonspasm group showed any ischemic electrocardiographic change. Thus, the sensitivity and specificity of this test for diagnosis of coronary spasm were 62% and 100%, respectively. In the spasm group, there were no significant differences between hyperventilation test-positive and test-negative patients in age, sex, the prevalence of hypertension, diabetes mellitus, obesity, smoking, and the number of diseased vessels. When clinical characteristics were compared, the proportions of the patients with high disease activity (> or =5 attacks a week), with severe arrhythmias (second- or third-degree atrioventricular block and/or ventricular tachycardia) during attacks, and with multivessel spasm were significantly higher in the hyperventilation test-positive patients than in the negative patients (69% vs 20%, p <0.0001; 31% vs 11%, p <0.005; and 58% vs 34%, p <0.01, respectively). These findings imply that hyperventilation is a highly specific test for the diagnosis of coronary artery spasm, and that hyperventilation test-positive patients are likely to have life-threatening arrhythmias during attacks and multivessel spasm.

Efficiency of recombination by Cre transient expression in embryonic stem cells: comparison of various promoters.

The Cre-loxP recombination system of bacteriophage P1 is frequently utilized in genetic manipulation in embryonic stem (ES) cells. The level of Cre expression is critical to induce loxP site-specific recombination in ES cells. To compare the efficiency of recombination, we constructed four Cre expression vectors driven by different promoters: cytomegarovirus/chicken beta-actin (CAG) promoter, human polypeptide chain elongation factor 1alpha (hEF-1alpha) promoter, mouse phosphoglycerate kinase-1 (mPGK) promoter, and polyoma enhancer/herpes simplex virus thymidine kinase (MC1) promoter. We introduced these Cre expression vectors by electroporation into three ES cell lines carrying a single copy of CAG-loxP-chloramphenicol acetyltransferase (CAT) gene-loxP-beta-galactosidase (beta-gal) gene construct. Since the Cre-mediated recombination leads to excision of the CAT gene, the efficiency of recombination can be monitored as beta-gal expression. No selection system was used in the experiments. The maximum recombination frequency was obtained when the CAG promoter was used, followed by the hEF-1alpha promoter, the mPGK promoter and the MC1 promoter in order. These results indicate that the efficiency of recombination in transient expression system correlates with the promoter activity of Cre expression vector. Thus, it is important to choose the promoter for effective recombination by Cre.

Hematopoiesis and angiogenesis.

Hematopoiesis is closely linked with angiogenesis, because they interact with each other and have common ancestors: hemangioblasts or hematogenic endothelial cells. The relationship is reasonable, because vascular and hematopoietic systems must develop together in order to establish the body's oxygen-delivery system during organogenesis. Hematopoietic stem cells have been shown to originate from the para-aortic splanchnopleural mesoderm region or aorta-gonads-mesonephros at successive stages. We discuss the molecular events in the differentiation of hematopoietic cells and endothelial cells. Transcription factors SCL/tal-1 and AML1; tyrosine kinase receptors Flk-1, Tie-2, Eph, and the sialomucins; CD34; thrombomucin; and AA4 play important biological roles in these lineages.

A clinical study on urinary kallikrein in patients with renal diseases.

The urinary kallikrein activity (KA) was measured to investigate its significance in the renal diseases by using tosyl-arginine methyl ester (TAME) as a substrate. The examinees were 94 patients with renal diseases and 25 normal persons. The daily urinary kallikrein excretion (KE, KE=KAxdaily urinary volume) is less in chronic glomerulonephritis and outstandingly less in chronic renal failure than in the normal controls. The KE also shows a positive correlation moderately to 15-min PSP excretion and relatively to creatinine clearance. KE is closely related to renal function and decreases with the degree of renal damage. KA has no relation to the concentration of urine protein, but it was parallel, in general, to the urokinase activity. In nephrotic syndrome, KA tends to show a negative correlation to the urinary alpha 1-antitrypsin. alpha 1-antitrypsin may have a function as an inhibitor to the urinary kallikrein.

Fatal angioedema associated with enalapril.

A 37-year-old female with a history of hypertension for 5 years was brought to the emergency room with swelling of the tongue and neck after the second dose of enalapril. After administration of hydrocortisone by her physician, she went to the emergency room. Her dyspnea and dysarthria were relieved. However, she experienced recurrence of the symptoms followed by respiratory arrest. She suffered severe anoxic brain damage and died three days later. Although angioedema is a rare occurrence with the use of enalapril, it is potentially life threatening.

The effect of radix Salviae Miltiorrhizae Composita on peroxidation of low density lipoprotein due to copper dichloride.

It is well known that plasma lipoprotein, particularly oxidized LDL, plays an important role in the pathogenesis of atherosclerosis and atherosclerotic diseases. We used oxidized LDL generated by incubating LDL from healthy persons with copper dichloride as a model to investigate the antioxidate property of Radix Salviae Miltiorrhizae Composita (RSMC). On photos, the spot X1 and the spot X2 were clearly found in the control group after the dialysis into copper dichloride for 24 and 48 hours, but they could not found in the RSMC group. The analysis of the constituents of lipids in LDL (by charring method) showed that after dialysis the percentages of the spot X1 and the spot X2 in the RSMC group were significantly lower than those in the control group (P < 0.01). The results suggest that RSMC plays a potential role in antioxidation of lipids or LDL.

The effects of radix Salviae miltiorrhizae on lipid accumulation of peroxidized low density lipoprotein in mouse peritoneal macrophages--lipid analysis and morphological studies.

Mouse peritoneal macrophages were incubated in DMEM with pox-LDL and Rradix Salviae Miltiorrhizae (RSM) to investigate the effects of RSM on the internalization of peroxidized low density lipoprotein (pox-LDL) by using lipid analysis and electron microscopy. Lipid peroxide (LPO) concentrations were increased slightly in the medium after incubation of macrophages with normal LDL (n-LDL), while decreased significantly in the media after incubation of macrophages with pox-LDL. In the three groups with pox-LDL, it could be found that there was a dose-dependent decrease of concentrations of LPO and total cholesterol (TCH) in the two RSM groups, and the decrease in the two RSM groups was much greater than in the group without RSM. RSM accelerated a more decrease of LPO than cholesterol contents in the media containing pox-LDL. The ultrastructural studies also showed that RSM induced the accumulation of lipid droplets in the cytoplasm of mouse peritoneal macrophages. The results suggested that RSM could accelerate the phagocytosis and degradation of pox-LDL by macrophages.

[A case of Schönlein-Henoch purpura with myocardial complications].

A rare case of Schönlein-Henoch purpura with myocardial complications was reported. A 64-year-old man was admitted to our hospital for further examination of hematuria, associated with arthralgia, proteinuria, and previous episodes of anasarca and orthopnea. The examinations revealed moderate renal dysfunction due to mesangial proliferative glomerulonephritis secondary to Schönlein-Henoch purpura. In addition, the patient proved to have cardiac dilatation and dysfunction of the left ventricle. The histological examination of the right ventricular subendocardium sampled by myocardial biopsy showed myocardial damage, suggesting the invasion of microvasculitis in Schönlein-Henoch purpura to the peripheral coronary vessels. These findings indicate that myocardial damage induced in the patient may be one of the complications caused by Schönlein-Henoch purpura.

[Are the first repetitive reentrant VPCs a trigger or an initiation of reentrant circuit for VPCs?: Evaluation by Holter ECG in patients with reentrant sustained ventricular tachycardia].

To evaluate whether the first repetitive reentrant VPCs are a trigger or an initiation of a reentrant circuit for VPCs, we studied Holter ECGs in 13 patients with sustained ventricular tachycardia, the morphology of reentrant VPCs, the relationship between the coupling interval (N-V) and the first ventricular cycle length (V-V), and the relationship of the coupling interval (N-V) between single VPC and repetitive VPCs. In 7 patients who showed episodes of repetitive VPCs more than twice on one recording of Holter ECG, most of VPCs in the first and second beats were the same in shape (1195 out of 1466 episodes, 82%). No obvious relationship between the coupling interval (N-V) and the first ventricular cycle length (V-V) was found in 5 patients, whereas a weak inverse relationship (r = 0.32) was found in one patient, and a weak positive relationship (r = 0.38) was found in another patient. In addition, the coupling interval in repetitive VPCs was longer than that in single VPCs in 4 out of 7 patients. These results imply that, in most cases, the first VPC is the expression of initiation of a reentrant circuit for repetitive reentrant VPCs developing spontaneously.

[A case of myocardial infarction due to the left main trunk lesion, in which percutaneous coronary recanalization and emergency coronary-aorto bypass graft, and subsequent percutaneous coronary angioplasty were effective not only from a viewpoint of survival but from comeback to social life].

A case of acute myocardial infarction due to the lesion in the left main coronary artery was reported. A 50-year male was referred to our department for suspected acute myocardial infarction. Physical examination on admission revealed slight cyanosis with cold sweating due to severe chest pain. Pulse was irregular and heart rate was 78 beats/min. Blood pressure was 100/80 mmHg. A series of electrocardiograms (ECG) and laboratory data provided the diagnosis of wide-ranged anterolateral infarction in the left ventricle. Emergency coronary angiograms taken without delay showed a subtotal occlusion (99% stenosis) of the left main coronary trunk (LMT) before the initiation of intracoronary thrombolysis (PTCR). Following the intracoronary infusion of urokinase of 1,200,000 units, symptoms and ECG changes transiently improved but worsened later, and LMT stenotic lesion and delayed filling of myocardium were similar with before PTCR. Emergency coronary-aorto bypass graft (CABG) was undertaken without a significant delay to both the left anterior descending artery (LAD) and left circumflex coronary artery (LCX). With these treatments, the patient could survive despite the wide area of infarction due to LMT lesion. Coronary angiograms performed 37 days after the CABG showed that the graft to LAD was completely occluded and the LCX graft was patent with partial stenosis. Treadmill test at this time induced an anginal episode with ischemic ECG changes on moderate exercise, indicating the presence of significant area of ischemic myocardium. For salvage of the ischemic myocardium, percutaneous transluminal coronary angioplasty (PTCA) was successfully performed for the LMT stenosis, resulting in no episode of angina nor ischemic ECG changes during exercise loading.(ABSTRACT TRUNCATED AT 250 WORDS)

[Evaluation of myocardial oxygen extraction dynamics in syndrome X by continuous measurement of coronary sinus oxygen saturation and its relation to clinical features].

We investigated the relation of myocardial oxygen extraction dynamics to pathophysiology and clinical features in syndrome X. In patients with syndrome X who underwent cardiac catheterization, coronary sinus oxygen saturation (n = 21) during rapid atrial pacing loading was continuously measured using a fiberoptic catheter system, and global and regional left ventricular function (n = 14) was evaluated before and immediately after pacing loading. Results were as follows: 1) In 5 of 21 patients with syndrome X, coronary sinus oxygen saturation during pacing loading fell less than 5% below the baseline without any impairments of global and regional left ventricular function. 2) In 16 patients with syndrome X, coronary sinus oxygen saturation during the pacing loading continuously fell over 5% below the baseline accompanied by impairment of both global and regional left ventricular function. The decrease in regional wall motion of the left ventricle was mainly observed in the apical area. These findings imply that changes in myocardial oxygen extraction dynamics in syndrome X during rapid atrial pacing may show the extent of a patchy area where myocardial oxygen demand-supply imbalance occurs due to coronary microcirculatory disturbances.

[Coronary hemodynamics in a patient developing chest pain in the middle age in congenital coronary-pulmonary fistula].

Recently reports of congenital coronary-pulmonary fistula have been increasing with the wide-spread use of coronary angiography. However, the cause of the angina sometimes seen as a chief complaint in coronary fistula has not been well demonstrated although it has been suggested that coronary steal phenomenon accounts for it. This report documented coronary hemodynamics in a patient who came to develop anterior chest pain in the middle age owing to congenital coronary-pulmonary fistula, measuring coronary flow before and after the fistula-closure operation. A 35-year-old woman suffered from a sudden onset of severe anterior chest pain in April, 1986. She was referred to our hospital on suspicion of ruptured aneurysm of Valsalva. Auscultation disclosed continuous murmur at 3 LSB, but no evidence of ruptured aneurysm of Valsalva was detected by echocardiography nor aortography. Coronary angiography showed both left and right coronary fistula into the stem of pulmonary artery and otherwise normal angiogram. Great cardiac vein flow (GCVF) measured with regional thermodilution method was 25 ml/min at rest (70 bpm) and 30 ml/min during rapid atrial pacing (150 bpm) before the operation, and 30 ml/min (78 bpm) and 58 ml/min (150 bpm) after the operation, respectively. Before the surgery, anterior coronary resistance (CRant) was higher than that in normal subjects at rest and remained almost steady during atrial pacing. After the surgery, CRant was still higher at rest but remarkably reduced during pacing of 150 bpm. These findings suggest that the gradual increase in peripheral coronary resistance for a long time may lead to the inducement of coronary steal in the middle-later age in patients with coronary fistula.

[A case of left atrial ball thrombus observed in a patient with combined valvular heart disease, accompanying anomalous coronary venous run].

A 53-year woman, who had been under observation for combined valvular heart disease, was admitted to our hospital for further examination of embolic episodes to brain and kidney. Echocardiographic examination showed the evidence of free-moving ball thrombus in the left atrium, and emergent cardiac catheterization following the echocardiography confirmed the diagnosis of it as well as mitral stenosis, aortic regurgitation with stenosis, and tricuspid regurgitation. In addition, coronary angiography disclosed the anomalous coronary venous run. With these findings, the cause of embolic episodes was found to be due to the thrombus in the left atrium. In the surgery performed a ball thrombus of 40 x 35 x 36 mm and a mural thrombus of 15 x 35 x 20 mm in size in the left atrium were detected and removed, and both mitral and aortic valves were replaced to artificial ones. She had a good hospital course after the surgery and discharged without any complication. In this report, we discussed a case of left atrial thrombus observed in a combined valvular heart disease, with 29 literatures reported in our country.

Angiopoietin-like protein 2 sensitively responds to weight reduction induced by lifestyle intervention on overweight Japanese men.

OBJECTIVE: Overexpression of Angiopoietin-like protein 2 (Angptl2) in obese adipose tissues promotes adipose tissue inflammation and its-related metabolic abnormalities. In a comparative study with adiponectin, we investigated whether alterations in serum Angptl2 concentrations reflect the effect of lifestyle intervention on weight loss and improved metabolic parameters in overweight subjects. METHODS: A total of 154 Japanese men (age, 40.9±5.1 years; body mass index, 26.9±3.6 kg m(-2); abdominal circumference, 94.1±8.9 cm) underwent a 3-month lifestyle intervention and underwent follow-up for 3 months thereafter. RESULTS: Decreased serum Angptl2 levels, but not increased serum adiponectin levels, were immediately apparent at the end of 3-month lifestyle intervention. Angptl2 levels continued to decrease for 3 months in parallel with body weight loss and improvement in metabolic indicators. In subjects showing 6% weight reduction, markedly reduced Angptl2 levels were detected at the end of 3-month intervention, whereas increased adiponectin levels were detected 3 months after the end of intervention. Multivariate analysis revealed changes in serum Angptl2 levels associated with changes in triglycerides (TGs), aspartate aminotransferase and alanine aminotransferase. In contrast, changes in serum adiponectin levels were associated with altered high-density lipoprotein cholesterol (HDL-C) and fasting plasma glucose levels. CONCLUSION: A 3-month lifestyle intervention promoted weight reduction and improved glucose and lipid metabolism, an effect maintained 3 months later. Notably, our findings indicate that decreased Angptl2 levels are a good indicator of reduced visceral fat and metabolic improvement at early stages of lifestyle intervention. Thus, Angptl2 reflects adiposity and might be a key protein to regulate inflammation and TG metabolism, whereas adiponectin levels could reflect improved glucose and HDL-C metabolism.