Project 8: The role of STAT proteins in controlling leukocyte migration into ischaemic areas in myocardial infarction

PI Göttingen: T. Meyer; PI London: A. Ivetic; PhD student: Sana M. Sheikh

Scientific background and preliminary results

Infiltration of the necrotic zone by leucocytes and macrophages is a histological hallmark in myocardial infarction resulting from the acute occlusion of a coronary artery. In this collaborative project, we will assess the effects of cytokine-driven STAT (signal transducer and activator of transcription) transcription factors in myocardial infarction using a mouse line with a STAT1 knock-in mutation in the amino-terminal domain, which results in a loss of STAT1 cooperative DNA binding and a global defect in interferon-γ (IFNγ) signalling.

In a model of myocardial infarction performed by ligation of the left anterior descending coronary artery, we found that transgenic male mice expressing the IFNγ-irresponsive N-terminal STAT1 point mutant displayed higher survival rates than their wild-type littermates and, in line, female mice were protected from adverse cardiac remodelling, as shown by echocardiographic assessment. Furthermore, the pool of total STAT1 was significantly reduced in the infarcted areas of the knock-in animals as compared to the mice expressing wild-type STAT1.

These findings highlight the role of cooperative DNA binding and tetramer formation of STAT1 in orchestrating complex immunological processes in the heart. In preliminary experiments, we observed that, in the damaged myocardium of the STAT1 knock-in mice, the ratio of tyrosine-phosphorylation of STAT1 to its homologue phospho-STAT3 was decreased as compared to myocardial samples from wild-type mice, suggesting that the balance between the activation state of both members of the STAT protein family mediates the remodelling process after myocardial infarction.

Hypotheses and aims

Based on these preliminary data, we aimed at studying the impact of STAT1 and its interaction partner STAT3 on the course of myocardial infarction. In particular, we will examine the migration of circulating innate immune cells, such as monocytes, into the damaged, ischaemic heart tissue. Since leukocyte transendothelial migration is a fundamental process in the inflammatory response during myocardial infarction, we therefore plan to investigate the role of STAT1 and STAT3 in monocyte protrusive behaviour during transendothelial migration.

We have recently shown that ezrin binds preferentially to L-selectin both in resting cells and during early leukocyte transendothelial migration (TEM) and, furthermore, that the knockdown of ezrin, but not moesin, impairs the recruitment of monocytes to activated endothelial monolayers under flow. Since the two ERM proteins ezrin and moesin play mutually exclusive roles in modulating monocyte TEM, we want to test the hypothesis that the expression level and the cytokine-induced tyrosine-phosphorylation state of STAT1 and STAT3 are modulating the contribution of ezrin to TEM. Another hypothesis to be tested in this study is that the down-regulation of STAT3 expression is associated with increased recruitment of monocytes to activated endothelial monolayers under flow.

Using this experimental approach, we hope to gain a deeper insight into the role of the two STAT proteins in controlling the migration of circulating innate immune cells towards the damaged, ischaemic tissue during myocardial infarction.

Work programme

Gene expression profiles in mice with normal and defective STAT1 signalling in unaffected myocardium and ischaemic areas of the heart

In a first set of experiments, we want to follow the expression of STAT1-regulated target genes on a global level. RNA samples will be obtained from damaged heart tissue in the myocardial infarction-operated STAT1-knockin mice and compared to their wild-type littermates. RNA isolates obtained from heart samples of sham-operated mice expressing either mutant or wild-type STAT1 will serve as controls. For selected STAT1 target genes, these data will be confirmed by means of real-time PCR. In particular, we want to identity markers of stem cell-ness, which may differ in their expression with regard to the STAT1 phenotype. In addition, we will test the expression of the Adam17 and other genes. The transcriptome data will be correlated with the clinical data from the hemodynamic assessment of the left ventricle by means of echocardiography and with the histopathological results with regard to the cellular infiltration by STAT1-expressing leukocytes and monocytes. 

Transendothelial migration of monocytes in STAT1- and STAT3-depleted cells

In a second set of experiments, we will use shRNA lentiviral clones to modulate the expression level of the two STAT proteins in monocyte-like THP-1 cells and neutrophil-like HL-60 cells. Parallel plate flow chamber assays will be used for live-cell imaging and performed using a flow chamber of 35 mm in diameter. In these experiments, we will study their influence of STAT proteins on the recruitment of monocyte-like cells to activated endothelial monolayers under constant flow conditions. All perfusion experiments will be performed at 1.5 dyn/cm2 assisted by a syringe pump. Before each perfusion assay, exogenously transduced human umbilical vein endothelial cell (HUVECs) will be either left untreated or stimulated for 16 hours with 50 ng/ml of recombinant interferon-ɣ. THP-1 cells will be perfused at a density of 0.5×106 cells per ml, and primary CD14+ monocytes will be perfused at a density of 1×106 cells per ml. All flow assays will be conducted using an inverted epifluorescence microscope, housed in an environmental chamber and maintained at a stable temperature of 37°C. Tracking of transmigrating cells will be performed as described in the literature.

Studying functional interactions between STAT1 and STAT3 proteins in the myocardium

In order to study the functional interaction between STAT1 and STAT3, which as heterodimers may signal differently as compared to the homodimers, we are planning to simultaneously stimulate cardiomyocytes isolated from the transgenic mouse line, homozygous knock-in and wild-type animals, with the two cytokines IFNɣ and IL-6. In control experiments, isolated cardiomyocytes will be treated by with the same concentration of either cytokine. In these co-stimulated experiments, we will assess the tyrosine-phosphorylation level of STAT1 and STAT3 by means of Western blotting. In addition, immunocytochemical experiments will be performed to study the extent of nuclear accumulation of STAT1 and STAT3 in the cytokine-treated cells. Fluorescence microscopy of these cells will provide information on the process of STAT1-STAT3 interaction, since STAT1/STAT3 heterodimers may differ in their kinetics from the corresponding STAT1/STAT1 and STAT3/STAT3 homodimers. Immunoblotting will be performed to analyse the total protein and phosphorylation state of either STAT protein in response to the different cytokine stimulation conditions.

In summary, the experimental approach outlined in this proposal will lead us to a better understanding of the molecular basis of the distinct functions of STAT proteins in myocardial infarction. In addition, we want to identify new STAT1-driven candidate genes important for tissue remodelling in the ischaemic myocardium. Results from these experiments may help to identify novel pharmacological interventions in the prevention of heart failure following severe myocardial damage due to the acute occlusion of an epicardial coronary artery.

Prof. Dr. mult. Thomas Meyer
Psychosomatic medicine and psychotherapy
+49 (0)551-39 4881

Research interests: STAT1 signalling

Dr. Aleksandar Ivetic
Speaker British Heart Foundation Centre of Research Excellence
Cardiovascular Division, KCL

Research interests: Mechanisms of inflammation

Sana M. Sheikh
PhD student 3rd cohort

RP 8.3: The role of STAT proteins in controlling leukocyte migration into ischaemic areas in myocardial infarction