Hiroaki Sasaki

Regulation of cardiomyocyte apoptosis by redox-sensitive transcription factors

Reperfusion of ischemic myocardium results in apoptotic cell death and DNA fragmentation. Several transcription factors are known to regulate the apoptotic cell death. This study sought to examine the regulation of cardiomyocyte apoptosis by these transcription factors. Isolated working rat hearts were divided into six groups: control, 15 min ischemia, 60 min ischemia, 15 min ischemia followed by 2 h reperfusion, ischemic stress adaptation by subjecting the hearts to four cyclic episodes to 5 min ischemia, each followed by 10 min of reperfusion, and adaptation followed by 15 min ischemia and 2 h reperfusion. Redox‐regulated transcription factors, NFκB and AP‐1 and the expression of two anti‐ and pro‐apoptotic genes, Bcl‐2 and p53 were determined. The results demonstrated NFκB and AP‐1 progressively and steadily increased as a function of the duration of ischemia. In the adapted heart, NFκB binding remained high while AP‐1 binding was lowered to almost baseline value. The anti‐oxidant gene, Bcl‐2 was downregulated in the ischemic/reperfused heart, but upregulated in the preconditioned myocardium. Significant induction of the expression of p53 occurred after ischemia and reperfusion. Apoptotic cells were barely detected in the adapted myocardium which was subjected to the same ischemia/reperfusion protocol. The results demonstrate for the first time differential regulation of cardiomyocyte apoptosis by pro‐ and anti‐apoptotic transcription factors and genes as a function of different durations of ischemia and reperfusion.

Apoptotic cell death during ischemia/reperfusion and its attenuation by antioxidant therapy

Apoptosis is a form of programmed cell death that can be induced in susceptible cells by a wide variety of normal physiological stimuli as well as by deleterious environmental conditions and cytotoxic agents. The common inducers of apoptosis include oxygen free radicals/oxidative stress and Ca(2+) which are also implicated in the pathogenesis of myocardial ischemic reperfusion injury. To examine how free radicals are directly involved in apoptosis, rats were divided into three groups. The first group of rat hearts were perfused for 15 min with KHB buffer, the second group of rat hearts were perfused with superoxide dismutase plus catalase, and the hearts were subjected to 30 min of ischemia and 120 min of reperfusion. The third group of rat hearts, served as control which were subjected to 165 min of perfusion with KHB buffer (where n=6 rats in each group). At the end of each experiment, hearts were saved (at -70 degrees C) and analysed for apoptosis, DNA laddening and MDA production. During the reperfusion continuous cardiac pressure measurements were recorded in real time with a data acquisition and analysis system (CORDAT II, Triton Technologies). Direct measurements of heart rate, developed pressure and the first derivative of the developed pressure were recorded before the intervention and during the reperfusion. Coronary flow was measured by timed collection of coronary effluent. The results of our study revealed apoptotic cells after 120 min of reperfusion as demonstrated by the intense fluorescence of the immunostained digoxigeninlabeled genomic DNA when observed under fluroscence microscopy. Evaluation of DNA fragmentation showed increased ladders of DNA bands in the same reperfused hearts representing integer multiples of the internucleosomal DNA, about 180 bp. The presence of apoptotic cells and DNA fragmentation in the myocardium were abolished by preperfusing the hearts in the presence of SOD and catalase, which also reduced the oxidative stress as evidenced by the MDA production. In concert, myocardial function was significantly better when compared with the ischemic control. Taken together, these results clearly demonstrate that oxidative stress developed in the ischemic reperfused myocardium induces apoptosis and free radical scavengers can play a crucial role in apoptotic cell death associated with ischemia/reperfusion.

Early effects of hypoxia/reoxygenation on VEGF, Ang-1, Ang-2 and their receptors in the rat myocardium: Implications for myocardial angiogenesis

Tissue hypoxia has been identified as being a particularly important stimulus for triggering angiogenesis. Here we report early effects of hypoxia/reoxygenation (H/R) on the protein expression profiles and localization patterns of the VEGF and Angiopoietin-Tie systems in adult rat myocardium. Western blot as well as immunohistochemical analyses were performed on hearts obtained from rats exposed to various durations of in vivo systemic hypoxemic hypoxia followed by 24 h reoxygenation. The relative time course of protein expression in response to increasing durations of hypoxia, as indicated from our experiments, seems to suggest the involvement of the VEGF system and the Ang-Tie system in early angiogenesis. An apparent relationship between the expression profiles of Flk-1 and Ang-2 was observed. The most significant and interesting relationship which came to light was the surprisingly coincident yet opposite temporal trends between Ang-1 and Ang-2 protein levels. In the 1 h hypoxia group, there was significant induction of Ang-2 expression (31.3% compared to its baseline control) in contrast to relatively mild Ang-1 expression (23.8% compared to its baseline control). Thereafter Ang-1 displayed a progressive increase in expression, parallel to a progressive decrease in Ang-2 expression, becoming most pronounced in the 4 h hypoxia group (Ang-1, 50% and Ang-2, 12.6% compared to respective baseline control values). This suggests that despite their being antagonists at the receptor level, regulation of Ang-1 and Ang-2 protein levels in response to hypoxia runs much deeper and seems to indicate modulatory control at the transcriptional and/or translational level. Two additional groups of rats were sacrificed 7 days after 4 h hypoxia + 24 h reoxygenation, or after a 28 h period of time-matched normoxia. Left ventricular tissue sections were used to determine capillary density (CD) by using anti-CD31 immunohistochemistry and computer-assisted morphometry. CD was significantly increased in the 4 h hypoxia group compared to control (1814 ± 56 vs. 1642 ± 43 counts/mm2) confirming that modulation of angiogenic factors and their receptors by H/R is capable of stimulating capillary proliferation in the myocardium. Our study presents the first evidence for the Ang-Tie systems involvement in early stages of myocardial angiogenesis along with the VEGF-Flk-1-Flt-1 system. The stimulation of myocardial angiogenesis by H/R may constitute a potential basis for a possible more long-lived adaptive response to stress afforded by preconditioning stimuli.

Oxidative stress due to hypoxia/reoxygenation induces angiogenic factor VEGF in adult rat myocardium: possible role of NFκB

INTRODUCTION Oxidative stress, as exerted by free radicals within biological systems, is known to exert numerous physiological and pathological effects on the cardiovascular system. Short-term exposure to environmental conditions such as low oxygen tension can cause such oxidative stress in vivo through inhalational hypoxia/reoxygenation. In this report the effects of different durations of hypoxia were investigated on myocardial protein expression of vascular endothelial growth factor (VEGF). a major angiogenic growth factor, and also explore the possible modulatory role of transcription factor NFkappaB on such expression. METHODS Forty eight male Sprague-Dawley rats (300 g b.w.) were randomly divided into four groups and subjected to either 1, 2 or 4 h of systemic normobaric hypoxemic hypoxia (10+/-0.4% O2) in an anesthesia chamber, or to 4 h of normoxia (ambient 20.9+/-0.4% O2) to time-match the maximal hypoxic duration. All rats were then kept under normoxic conditions. Rats were sacrificed and hearts harvested either after 2 h for later electrophoretic mobility gel shift assay for NFkappaB, or after 24 h for later Western blot analysis for VEGF. RESULTS Western blot analysis for VEGF revealed significantly elevated protein expression (2.4-fold compared to baseline control) in the I h group. This elevated level persisted in the 2 and 4 h groups as well. Two hours post-hypoxia gel shift assay for NFkappaB indicated significant nuclear translocation and DNA binding of this transcription factor in the 1 and 2 h groups, with moderate decrease in the 4 h group. CONCLUSION In vivo oxidative stress caused by systemic inhalational hypoxemic hypoxia increases cardiac VEGF protein expression and may trigger myocardial angiogenesis. The results suggest that NFkappaB modulates such an effect.

Differential Regulation of Apoptosis by Ischemia‐Reperfusion and Ischemic Adaptationa

Abstract: Ischemia and reperfusion injure the heart, as manifested by myocardial infarction, postischemic ventricular functional dysfunctions, arrhythmias, and cardiomyocyte apoptosis. Hearts can be adapted to ischemicreperfusion injury by subjecting them to non‐lethal cyclic episodes of shortterm ischemia and reperfusion. The adapted myocardium becomes resistant to subsequent lethal ischemic injury. Reactive oxygen species and oxidative stress play crucial roles in the pathophysiology of ischemic‐reperfusion injury. The adapted hearts, when subjected to subsequent ischemia and reperfusion, generate a reduced amount of oxygen free radicals compared to the nonadapted hearts. The number of cardiomyocytes undergoing apoptotic cell death is reduced in the adapted hearts subjected to ischemia and reperfusion. In concert, the adapted myocardium is associated with increased antioxidant gene Bcl‐2, increased binding activity of the nuclear transcription factor NFκB, and reduced binding activity of AP‐1 compared to nonadapted hearts. Yet when nonadapted hearts are subjected to ischemia and reperfusion, Bcl‐2 is downregulated while NFκB is moderately upregulated and AP‐1 is significantly upregulated.

Effects of Hypoxia/Reoxygenation on Angiogenic Factors and Their Tyrosine Kinase Receptors in the Rat Myocardium

The process of angiogenesis is initiated primarily as a consequence of hypoxic stimulation at the cellular and molecular level. Although several angiogenic growth factors have been identified, at present a detailed understanding of the interplay among inducing stimuli, growth factors, and their respective molecular targets remains to be evaluated. Here we report the effects of progressively increasing durations of moderate hypoxia on the protein expression profiles and tissue distribution patterns of the vascular endothelial growth factor system and the angiopoietin/Tie system in the adult rat myocardium. The relative temporal trends of expression of the various components of these two systems, as well as apparent relationships between Flk-1 and angiopoietin-2 and between Flt-1 and Tie-1, suggest a probable sequence of involvement during myocardial angiogenesis, as proposed in our model. Such relationships may potentially be utilized in formulating strategies for sequential gene therapy to achieve clinically relevant myocardial angiogenesis.

[34] A survival model for the study of myocardial angiogenesis

Publisher Summary The chapter presents a study on myocardial angiogenesis. Angiogenesis is known to be the bodys natural healing process in which new blood vessels grow in response to injury. In the myocardium, hypoxia usually occurs during ischemic episodes. Such ischemic hypoxia is often followed by reperfusion. It is during this subsequent reperfusion period that reoxygenation occurs and reactive oxygen intermediates are formed. For the study of angiogenesis in the infarcted myocardium, a rat survival model of chronic myocardial infarction surgery is developed, and the examination of the left ventricular response during pharmacological stress, testing with dobutamine as a measure of cardiac reserve is conducted. The experimental protocol described in the chapter demonstrates myocardial angiogenesis stimulated by nonlethal moderate hypoxic challenge by utilizing an animal model of chronic myocardial infarction progressing to heart failure. Such angiogenic effects of preconditioning were found to be dim to its ability to augment vascular endothelial growth factor (VEGF), protein expression, which presumably played a crucial role in reducing endothelial cell apoptosis, a proved determinant for congestive heart failure.