Background
The processes of scar formation during myocardial remodeling after ischemia/reperfusion (I/R) are intricate and tightly regulated by the immune system and its effector cells. The fusion protein LEA29Y is a widely used immunosuppressive medication that inhibits T-cell co-stimulation, thereby limiting immune cell activation. A pig model with systemic LEA29Y expression has been established as a model for general immunosuppression, enabling investigation of I/R injury and remodeling under immunocompromised conditions. Investigations include an emphasis on collagen content as a representation of scar formation, as well as the quantification of immune cells.
Methods
Using a catheter-based intervention, 60 minutes of ischemia in the left anterior descending artery (LAD) was induced, followed by a permanent reperfusion, leading to an infarction in the left ventricle (LV). LEA29Y animals were sampled after 3, 9, 14 and 26 days and compared to controls. Ejection fraction was calculated from LV angiograms, ECG was recorded, infarct size was measured and complemented by histological stainings with HE, Sirius Red, IBA1, CD3-T-Cells and neutrophils. Histological analysis was performed on wholly scanned slides using “QuPath”, enabling a high-resolution global measurement of stained tissue areas in per-cent and micrometers square.
Results
LEA29Y and WT animals displayed similar infarct sizes. Compared to preischemic levels LV angiography showed a tendency of higher loss of ejection fraction in the transgenic pigs up to 14 days, but after 26 days the loss was less aggravated in LEA29Y (-18.96) than in WT (-27.34, p=0.0016). QuPath analysis of stained collagen areas detects scar formation over the course of the investigation period. It showed a peak of collagen on day 14 but tended to be mitigated in LEA29Y animals (d14: 18.4% LEA29Y LV vs. 20.75% WT LV; d26: 16.49% LEA29Y LV vs. 14.38% WT LV). Quantitative analysis of global IBA1 stained macrophages revealed initially significantly lower levels in transgenic animals compared to WT (1.637% LEA29Y LV vs. 2,741% WT LV, p=0.000751) followed by a rapid recovery, reaching levels comparable to WT animals after 3 days (7.465% LEA29Y LV vs. 7,545% WT LV). Infarct area comparison revealed initially higher macrophage levels in LEA pigs that rapidly declined, while WT animal levels peaked at day 9, leading to significantly higher levels in WTs at day 9 but similar levels of both genotypes at day 26 (d9: 9.362% LEA29Y LV vs. 20.49% WT LV, p= 0.008634; day 26: 7.913% LEA29Y LV vs. 7.978% WT LV). Quantitative measurements of stained CD3 T-cells in LEA-animals show significantly lower levels compared to WT at baseline (0.08127% LEA29Y LV vs. 0.1517% WT LV, p=0.0411). In infarcted areas, the numbers consistently remain lower and less dynamic in the immunocompromised animals through day 26 (d26: 0.3326% LEA29Y LV vs. 0.4537% WT LV).
Conclusion
Blocking of T-cell co-stimulation initially leads to a higher loss of LV function with significantly greater recovery after 26 days and earlier scar contraction. The immune response of LEA29Y animals is characterized by attenuated and rapidly decreasing macrophage levels together with a persistently lower T-cell infiltration. To further investigate the connection between these immune cells and neutrophils, additional immunohistochemical stainings are conducted.
