Observation and Analysis of Coronary Microcirculation by Establishing Rat Myocardial Ischemia and In Vitro CMECs Hypoxia Model

Zheng JIA; Zhengjiang XING; Qian LIU; Yibin DU; Bing LI; Ying XIE; Yi ZHAO

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Zheng JIA, Zhengjiang XING, Qian LIU, Yibin DU, Bing LI, Ying XIE, Yi ZHAO. Observation and Analysis of Coronary Microcirculation by Establishing Rat Myocardial Ischemia and In Vitro CMECs Hypoxia Model[J]. Journal of Kunming Medical University.

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Observation and Analysis of Coronary Microcirculation by Establishing Rat Myocardial Ischemia and In Vitro CMECs Hypoxia Model

1.
Dept. of Cardiovascular Surgery
2.
Generae Medicine Ward，Dept. of Cardiovascular Medicine，Yan'an Hospital Affiliated to Kunming Medical University，Kunming Yunnan 650051
3.
Dept. of Geriatrics，Traditional Chinese Medicine Hospital of Yunnan Province，Kunming Yunnan 650051，China

Received Date: 2024-06-06
Available Online: 2024-11-09

Abstract

Abstract

Objective To establish in vivo rat ischemic myocardial injury and in vitro cardiac microvascular endothelial cell （CMEC） hypoxia models so as to investigate the structural and biological changes and probe the angiogenesis basis of coronary microcirculation. Methods In vivo rat myocardial ischemia model was established using the 1/3 ligation of the left anterior descending coronary artery and myocardial tissue structure and ultrastructure were detected using HE, Masson staining, and transmission electron microscopy, respectively. In vitro time-gradient hypoxia model of rat CMECs （hypoxia times set at 0 h, 4 h, 8 h, 12 h, 24 h, 48 h, 72 h） was established using a hypoxic incubator. An inverted phase-contrast microscope was used to observe the morphological and growth characteristics of CMECs. The proliferation rate was determined by CCK-8 method, and the survival rate was determined by counting method. The expression of inflammatory factors （IL-1β, IL-6, TNF-α） and angiogenic factors （VEGF, Ang-2） were detected using ELISA method. Results After 72 hours of coronary artery ligation, HE and MASS staining indicated the successful establishment of a rat model of myocardial ischemia and hypoxia. The transmission electron microscopy revealed the ischemic and hypoxic changes in the ultrastructure of cells. . CMECs exhibited the distinct morphological characteristics and adhered to the surface. With the prolonged hypoxia time, the proliferation rate significantly decreased after 48 h （P = 0.0426）, and the survival rate significantly decreased after 24 h （72.8%）. Long-term hypoxia led to significantly higher release levels of IL-1β （24～72 h, P = 0.0007, 0.0007, 0.001）, IL-6 （24～72 h, P = 0.0015, 0.0005, 0.0007）, and TNF-α （24～72 h, P = 0.0015, 0.0063, 0.0008 respectively） compared to short-term hypoxia IL-1β （4～12 h, P = 0.007, 0.0034, 0.0009 respectively）, IL-6 （4～12 h, P = 0.0026, 0.0013, 0.0045 respectively）, and TNF-α （12 h, P = 0.0087）. In addition, the expression of angiogenic factor VEGF began to increase 8 hours after hypoxia （P < 0.0001）, decreased at 12-24 hours （P < 0.0001 respectively）, and then increased rapidly （P < 0.01）; The expression of Ang-2 decreased from 4-12 hours （P < 0.05）, and gradually increased from 24 hours （P < 0.01）. Conclusions Myocardial tissues and CMECs exhibit the different biological changes at different ischemia-hypoxia time points with inflammatory reactions beginning in the early stages and angiogenesis reactions occurring in the late stages. These findings contribute to elucidating the key cellular and molecular mechanisms underlying ischemic myocardial injury.
- Coronary artery disease,
- Hypoxia,
- Cardia microvascular endothelial cells,
- Coronary microcirculation,
- Inflammation,
- Angiogenesis.

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References(18)

References

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