Motoo Kanoh

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.

Inhibition of Granulation Tissue Cell Apoptosis During the Subacute Stage of Myocardial Infarction Improves Cardiac Remodeling and Dysfunction at the Chronic Stage

Background Granulation tissue cells at the subacute stage of myocardial infarction (MI) are eliminated by apoptosis to finally make a scar at the chronic stage. We hypothesized that postinfarct inhibition of apoptosis might preserve myofibroblasts and endothelial cells in granulation and modulate chronic left ventricular (LV) remodeling and heart failure. Methods and Results A pancaspase inhibitor, Boc‐Asp‐fmk (BAF, 10 μmol/kg per day), or vehicle (control) was given to rats with experimental large MI. The treatment was started on the third day after MI and continued until 4‐week‐old MI. Two weeks later, the apoptosis of granulation tissue cells was significantly reduced and conversely, the cell population was greater in BAF. Twelve weeks later, BAF showed significantly greater survival rates (84% versus 42%) with significantly smaller LV cavity, lower LV end‐diastolic pressure and central venous pressure, and higher LV dP/dt, which indicated improvement of LV remodeling and dysfunction. A scar was established in old infarct of control subjects, but in BAF, the infarct wall was thicker because of greater old infarct area, which contained abundant myofibroblasts and vessels. Surprisingly, many of the &agr;‐smooth muscle actin‐positive myofibroblast‐like cells in BAF, making bundles and running parallel to the survived cardiomyocytes, were ultrastructurally mature smooth muscle cells with contractile phenotype. Cardiomyocyte apoptosis in the infarct area was equally rare in each group. Conclusions The postinfarct treatment with BAF improved LV remodeling and dysfunction through inhibition of granulation tissue cell apoptosis. These findings imply a new therapeutic strategy against postinfarct heart failure. (Circulation. 2003;108:104‐109.)

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.