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Pagel, Judith-Irina Carola (2014): Functional characterization of the transcription factor early growth response 1 (Egr1) in arteriogenesis. Dissertation, LMU München: Medizinische Fakultät



The number of patients suffering from obstructive arterial disease is still increasing. Stimulation of a patient’s collateralization (arteriogenesis), though an auspicious therapeutic approach, is still not part of current therapy regimes. Further studies on the molecular level are needed to understand the genetic regulation in this process. The transcription factor early growth response 1 (Egr1) was shown to partic-ipate in leukocyte recruitment and cell proliferation in vitro. This work contributes to the acquisition of new insights into its mode of action in vivo. Using a model of peripheral arteriogenesis, Egr1 was found significantly upregulated in growing col-laterals of wild-type mice (WT), both on mRNA (2.24fold) and protein level (2.3fold). Egr1 stained positive in EC and vSMCs of collaterals as well as in nerves. In LDI measurements conducted over the period of 21 days evidenced a delayed perfusion recovery after femoral artery ligation in Egr1-/- mice compared to WT mice (day7: 0.46±0.05 in Egr1-/- vs. WT (0.73±0.04), day 14: 0.65±0.02 in Egr1-/- vs. 0.88±0.04 in WT and day 21: 0.79 ±0.03 in Egr1-/- vs. 0.96±0.02 in WT). Under baseline conditions, Egr1-/- showed increased levels of monocytes (521.89±52.9 cells/µl vs. 326.56±21.6 cells/µl in WT) and granulocytes (811.79±79.96 cells/µl vs. WT 559.88±34.57 cells/µl) in the circulation but reduced levels in adductor muscles (18.14±2.73 cells/µl vs. 51.22±4.38 cells/µl in WT) as evidenced by FACS analyses. After femoral artery ligation, more macrophages were detected in the perivascular space of collateral arteries in Egr1-/- (8.10±0.99 per vessel) vs. WT (6.12±0.45 per vessel) mice. The mRNA of leukocyte recruitment mediators monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1) and urokinase plasminogen activator (uPA) were found upregulated in both groups. Whereas other Egr family members (Egr2-4) did not show an upregulation in WT collateral arteries, they were found significantly upregulated in Egr1-/- mice suggesting a mechanism of counter-balancing Egr1 deficiency. A closer look at cell cycle regulators revealed that cyclin E and cdc20 were found upregulated in WT as well as in Egr1-/- mice. However, cyclin D1 was hardly detectable under Egr1 deficiency conferring Egr1 an unique role for cyclin D1 transcription. vSMC phenotype switch is a critical step towards vSMC proliferation and therefore arteriogenesis. In this context, the downregu-lation of alpha smooth muscle actin (αSM-actin) and of the transcriptional repressor, splicing factor-1 (SF-1) has been shown to be critical in vitro. During arteriogenesis, SF-1 has been found downregulat-ed in collaterals of WT mice but was 1.64fold upregulated in Egr1-/-. Similar was true for αSM-actin. Whereas in WT mice αSM-actin is downregulated at 12h after ligation Egr1 deficient mice evidenced an upregulation of αSM-actin. The strong upregulation of the nonselective proliferation marker ki67 in WT mice was not detectable under Egr1 deficiency evidencing furthermore a delay in vascular cell proliferation. Conclusion: Compensation for deficiency of Egr1 function in leukocyte recruitment can be mediated by other transcription factors; however, Egr1 is indispensable for effective vascular cell cycle progression and phenotype switch in arteriogenesis.