Michiya Ohno

Expression of bcl-2 Protein, an Inhibitor of Apoptosis, and Bax, an Accelerator of Apoptosis, in Ventricular Myocytes of Human Hearts With Myocardial Infarction

BACKGROUND In general, myocyte death in myocardial infarctions (MIs) is attributed to necrosis, but recently the involvement of apoptosis has been suggested. The ratio of bcl-2 protein, an inhibitor of apoptosis, to Bax protein, an inducer of apoptosis, determines survival or death after an apoptotic stimulus. We speculated that bcl-2 or Bax expression is induced by ischemia and that it may be related to myocyte death in human hearts. METHODS AND RESULTS We studied immunohistochemically 37 autopsied human hearts (acute MI, n = 15; old MI, n = 12; normal hearts as a control, n = 10) with the use of bcl-2 and Bax antibodies. There were no myocytes with positive bcl-2 immunoreactivity in the controls or hearts with old MI. However, myocytes with positive bcl-2 immunoreactivity were seen in 9 of 15 hearts (60%) with acute MI, in that it was localized only in salvaged areas surrounding the infarcted tissues. Myocytes with slightly positive Bax immunoreactivity were observed in the control hearts. In the salvaged myocytes surrounding the infarcted tissues, Bax was overexpressed in 2 of 15 hearts (13%) with acute MI but in 10 of 12 hearts (83%) with old MI. CONCLUSIONS bcl-2 protein is induced in salvaged myocytes at the acute stage of infarction, but Bax protein is overexpressed at the old stage. The expression of bcl-2 and the overexpression of Bax may play an important pathophysiological role in the protection or acceleration of the apoptosis of human myocytes after ischemia and/or reperfusion.

“Apoptotic” Myocytes in Infarct Area in Rabbit Hearts May Be Oncotic Myocytes With DNA Fragmentation Analysis by Immunogold Electron Microscopy Combined With In Situ Nick End-Labeling

BACKGROUND Modes of cell death have been defined morphologically as apoptosis and oncosis. Infarcted myocytes have been reported to show apoptosis, as revealed by DNA fragmentation by DNA ladder and by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) at the light microscopic level. We investigated whether TUNEL-positive infarcted myocytes have apoptotic or oncotic ultrastructures by using electron microscopic TUNEL, which can simultaneously observe the ultrastructure and DNA fragmentation of the same myocytes. METHODS AND RESULTS Thirty rabbits were divided into 5 groups (n=6 each) that were subjected to a sham operation or to 30-minute ischemia followed by 0-minute, 30-minute, 2-hour, or 4-hour reperfusion of a coronary artery. In the 2- and 4-hour reperfusion groups only, DNA electrophoresis showed a ladder pattern, and the light microscopic TUNEL finding was positive in the nuclei of myocytes localized in the infarcted area (6+/-2% and 11+/-3%, respectively). Electron microscopic TUNEL showed that nuclei with a significant accumulation of immunogold particles (indicating an electronic microscopic TUNEL-positive result) were observed only in the infarcted myocytes with irreversibly oncotic ultrastructures that were found in the hearts of the 2- and 4-hour reperfusion groups (41+/-3% and 83+/-4%, respectively). Irreversibly oncotic myocytes (indicated by swelling, inhomogeneously clumped chromatin in nuclei, dense bodies in mitochondria, and/or ruptured plasma membranes) were also seen in the 0- and 30-minute reperfusion groups, which did not exhibit TUNEL-positive myocytes. There was no evidence of apoptotic ultrastructures in the myocytes. CONCLUSIONS DNA fragmentation occurs in the myocytes that had already shown irreversibly oncotic, but not apoptotic, ultrastructures with ischemia and/or reperfusion. Therefore, DNA fragmentation itself does not always mean apoptosis, and so-called apoptotic infarcted myocytes may belong to a category of cell death other than apoptosis.

Role of Apoptosis in the Disappearance of Infiltrated and Proliferated Interstitial Cells After Myocardial Infarction

Myocardial infarction (MI) progresses from the acute death of myocytes and the infiltration of inflammatory cells into granulation, followed by scars. During the healing process, the myocardial interstitial cell population in the infarcted tissues increases markedly and then decreases. We postulated that apoptosis is responsible for this process. Twenty-four male Japanese white rabbits underwent a 30-minute occlusion of the left coronary artery followed by reperfusion for 2 days, 2 weeks, or 4 weeks (n=8 each). The histological features consisted of dead cardiomyocytes and marked leukocyte infiltration at 2 days after MI and granulation consisting of numerous alpha-smooth muscle actin-positive myofibroblasts, macrophage antigen-positive macrophages, and neovascularization at 2 weeks. At 4 weeks, the cellularity decreased markedly, and scars were evident. Interstitial cells with positive nick end labeling were significantly more frequent at the light microscopic level in the 2-day MI samples (5.3+/-3.6% in the center and 6.9+/-3.3% in the periphery of the infarct region) than in the 2-week (2.5+/-1.0%) and 4-week (0.5+/-0.5%) samples. DNA electrophoresis showed a clear ladder in tissues from the ischemic areas at 2 days after MI but not at 2 and 4 weeks after MI. Ultrastructurally, typical apoptotic figures, including apoptotic bodies and condensed nuclei without ruptured plasma membranes, were detected in leukocytes from all hearts with 2-day MI and in myofibroblasts, endothelial cells, and macrophages from all hearts with 2-week MI. In the electron microscopic in situ nick end labeling, immunogold particles intensely labeled the condensed chromatin of the typical apoptotic nuclei. These particles were also accumulated on nuclei of the interstitial cells showing homogeneous density but not definite condensation as typical apoptotic nuclei, suggesting an early stage of apoptosis. Thus, apoptosis plays an important role in the disappearance of both the infiltrated leukocytes and the proliferated interstitial cells after MI. This finding may have therapeutic implications for postinfarct ventricular remodeling through apoptosis handling during the healing stage of MI.

C-reactive protein, lipoprotein(a), homocysteine, and male sex contribute to carotid atherosclerosis in peritoneal dialysis patients

BACKGROUND In patients with end-stage renal disease, the morbidity and mortality of cardiovascular disease are substantially greater than in the general population. Advancement in understanding the pathogenesis of atherosclerotic vascular disease suggests a central role of inflammation in atherogenesis. However, clinical data evaluating the role of inflammation in atherogenesis are sparse in peritoneal dialysis (PD) patients. METHODS We measured serum C-reactive protein (CRP), intact parathyroid hormone, lipoprotein(a) [Lp(a)], interleukin-1 receptor antagonist (IL-1Ra), tumor necrosis factor soluble receptor (TNF-sR), fibrinogen, and plasma homocysteine (Hcy), as well as intima-media thickness (IMT) and number of atherosclerotic plaques (plaque score [PS]) in the carotid arteries by means of carotid B-mode ultrasonography in 59 PD patients (35 men, 24 women; mean age, 52.4 years; average dialysis period, 36 months). All patients had chronic glomerulonephritis. RESULTS Sixty-eight percent of PD patients had at least 1 plaque. Serum CRP level was greater than the upper limit of the normal range in 52.5% of patients. Compared with PD patients with normal CRP levels, concentrations of such proinflammatory cytokines as IL-1Ra and TNF-sR, Lp(a), and Hcy were increased in PD patients with elevated CRP levels. However, no differences in plasma fibrinogen and intact parathyroid hormone levels were found between PD patients with increased and normal CRP levels. In a multiple regression model, age, male sex, CRP level, and Lp(a) level were independent predictors of IMT. Similarly, male sex, CRP level, Lp(a) level, and Hcy level were independent correlates of PS. CONCLUSION This study suggests that Lp(a) and Hcy levels and male sex, and especially CRP level, have an important role in carotid atherosclerosis in PD patients.

Apoptosis and Overexpression of Bax Protein and bax mRNA in Smooth Muscle Cells Within Intimal Hyperplasia of Human Radial Arteries Analysis With Arteriovenous Fistulas Used for Hemodialysis

There is a type of arteriosclerosis with remodeling of middle-size arteries in which intimal hyperplasia of smooth muscle cells (SMCs) plays the main role, and there are few macrophages, T lymphocytes, and foam cells. It is unknown whether apoptosis and the expression of Bax, an inducer of apoptosis, are increased according to the progression of this type of human arteriosclerosis, which is different from so-called atherosclerosis. Bax heterodimerizes with Bcl-2, an inhibitor of apoptosis, and the ratio of Bax to Bcl-2 determines cellular apoptosis or survival. Thus, we investigated apoptosis and the expressions of Bax, bax mRNA, and Bcl-2 in human arteriovenous (AV) fistulas used for hemodialysis, a representative of arteriosclerosis of the aforementioned type. The material was 20 radial arteries obtained from 20 patients with chronic renal failure undergoing AV shunt surgery. SMCs, macrophages, and T lymphocytes were immunohistochemically identified at the light microscopic (LM) level. Apoptosis was detected by in situ terminal deoxynucleotidyl transferase (TdT)-mediated digoxigenin-dUTP nick end labeling (TUNEL) at both the LM and electron microscopic (EM) level. Cell proliferating activity was estimated by proliferating cell nuclear antigen (PCNA). Bax and Bcl-2 were detected by immunohistochemistry and Western blot analysis. Expression of bax mRNA was detected by in situ hybridization. LM TUNEL-positive cells in both the intima and media were significantly increased according to the percent stenosis of the vessels. EM analysis revealed that ultrastructures of apoptotic SMCs were seen in both synthetic and contractile phenotypes. Their frequency of occurrence in the intima and media were greater in those vessels with >50% stenosis than in those with <50% stenosis (5.2+/-0.7% versus 1.0+/-0.3% in the intima and 2. 1+/-0.5% versus 0.2+/-0.1% in the media). The proportion of apoptotic SMCs with ruptured plasma membranes was greater than that of apoptotic SMCs with intact membranes in the intima of the former (4.1+/-0.6% versus 1.1+/-0.1%). Only those SMCs with apoptotic ultrastructures had TUNEL-positive nuclei with moderate or marked accumulation of immunogold particles at the EM level. However, ultrastructures of oncosis (primary necrosis) were not observed. Immunohistochemical analyses showed significant positive correlations between percent stenosis of vessels and the percentage of either PCNA-positive intimal cells or Bax-positive areas in the intima and media. Bcl-2-positive cells were not observed in the intima but mainly in the outer media. The percentage of Bcl-2-positive medial cells was definitely decreased at an early stage after formation of the AV fistula but did not change according to the duration of hemodialysis or the progression of arteriosclerosis. Western blot analysis of Bax or Bcl-2 and in situ hybridization of bax mRNA confirmed the immunohistochemical data. Thus, regulation of cellularity in intimal hyperplasia of SMCs in human arteriosclerosis with remodeling is mediated by proliferation and apoptosis but not oncosis. The apoptosis is probably induced by an increase in the Bax to Bcl-2 ratio.

T-0162, a novel free radical scavenger, reduces myocardial infarct size in rabbits.

1. We investigated the effects of 1‐(3‐tert‐butyl‐2‐hydroxy‐ 5‐methoxyphenyl)‐3‐(3‐pyridylmethyl)urea hydrochloride (T‐0162), a novel low‐molecular weight free radical scavenger, on the generation of superoxide anions and hydroxyl radicals in vitro and in vivo and on myocardial infarct (MI) size in an in vivo model of MI in rabbits.

Losartan reduces proteinuria and preserves renal function in hypertensive patients with IgA nephropathy

AbstractBackground. The objectives of this study were to evaluate the effects of the angiotensin II receptor antagonist losartan on blood pressure (BP), proteinuria, and renal function in hypertensive patients with IgA nephropathy. Method. The study subjects comprised 18 patients with biopsy-proven IgA nephropathy with mild hypertension. Patients were classified into three groups (good/relatively good, relatively poor, poor) according to renal histologic findings and treated once a day with losartan 50 mg for 12 months. Changes in BP, proteinuria, renal function, and biochemical parameters were prospectively evaluated before and after the treatment. Results. BP began to fall after 1 month and proteinuria decreased significantly after 9 months of therapy. Glomerular filtration rate (GFR), renal plasma flow (RPF), and filtration fraction (FF) did not change throughout the observation period. There was no significant difference between the three different histologic groups in relation to the effects of losartan on BP, proteinuria, and renal function. We found that in patients with a proteinuria reduction rate of more than 50%, RPF increased and FF decreased significantly. Although it has been reported that losartan has a uric acid lowering effect, this study could not confirm such an effect. Conclusions. Losartan lowers blood pressure and decreases proteinuria in hypertensive patients with IgA nephropathy. These effects appear to be independent of renal histologic findings. This study suggests that the antiproteinuric effect of losartan is primarily mediated by changes in glomerular hemodynamics.

Renal congestion related to worsening renal function in patients with acute decompensated heart failure: Diuretic strategy for acute cardiorenal syndrome

Deterioration of renal function in patients with acute decompensated heart failure (ADHF) influences the prognosis, suggesting that ADHF should be managed as an acute cardiorenal syndrome. Close collaboration between cardiologists and nephrologists is frequently crucial for management of this condition. It is noteworthy that renal congestion promotes worsening renal function (WRF). High-dose loop diuretics can cause WRF, but are often necessary for treatment of congestion, which is the main symptom of ADHF. However, it is controversial whether WRF associated with diuretic therapy actually has a poor prognosis. In this review, we focus on the mechanism of renal congestion related to WRF in patients with ADHF and on the current status of WRF. We also review the use of loop diuretics to treat ADHF and chronic heart failure, as well as the current role of selective vasopressin-2 receptor antagonist therapy.