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Forskare från Lund och Amman i Jordanien  har i ett samarbetsprojekt fått sina studier om kärlsjukdomar godkända och publicerade (idag). Ämnet tillhör inte min starka sida så jag går direkt till studien.

Transcription factor GATA6 promotes migration of human coronary artery smooth muscle cells in vitro

29 November 2022

Introduction

Vascular diseases are among the leading causes for morbidity and mortality worldwide. A large body of work has recognized that vascular smooth muscle cells (VSMCs) play a prominent role in these pathological processes. Unlike other terminally differentiated cells, VSMCs retain a remarkable capability to undergo phenotypic modulation. In response to changes in the surrounding environment, VSMCs can switch from a differentiated contractile phenotype to a more proliferative and migratory phenotype, often referred to as a synthetic phenotype. Although this is beneficial during various biological processes such as wound repair, phenotypic modulation can play a fundamental role in the development of various vascular diseases, including atherosclerosis, hypertension and restenosis following angioplasty.

SMC migration is a key process in the development of coronary artery disease. SMCs respond to a perceived injury to the vascular wall by migrating from the vascular media towards the lumen where they proliferate and form a neointima. Eventually, this effect together with an atherosclerotic process can limit blood flow and increase the risk of thrombosis. Although synthetic smooth muscle cells often have both increased proliferative and migratory capacity, these are separate biological processes that are regulated by distinct signaling pathways.

Despite extensive effort to characterize the transcriptional program that defines smooth muscle phenotype, the role of endogenous regulators that control smooth muscle specific gene expression are not fully understood. One of the transcription factors that appears to have a complex role in smooth muscle gene expression is GATA6, which is the predominantly expressed GATA factor in VSMCs. GATA6 belongs to a family of highly conserved zinc-finger transcription factors that regulates the expression of genes required for developmental processes and tissue-specific functions. 

Several studies have demonstrated a role for GATA6 in maintaining the differentiated state of VSMCs by regulating the expression of smooth muscle-specific genes including smooth muscle myosin heavy chain (MYH11) and smooth muscle alpha actin (ACTA2). Moreover, GATA6 has been shown to reduce smooth muscle proliferation and neointimal formation in vivo following balloon injury in mice. Consistent with these findings, rapamycin, a common stent drug preventing restenosis, mediates positive effects on SMC differentiation and prevents vascular disease by phosphorylation-mediated activation of GATA6. However, several studies have demonstrated that GATA6 can promote expression of genes associated with the synthetic function of SMCs. This effect of GATA6 may be caused by inhibitory or activating interaction with myocardin, a master regulator of SMC identity, depending on the target gene. Thus, GATA6 appears to play multifaceted roles in the regulation of smooth muscle phenotype and exploring the molecular action of GATA6 may have pivotal implications for our understanding of smooth muscle cell function and the underlying mechanisms of vascular disease.

The aim of this study was to further elucidate the importance of GATA6 for human vascular smooth muscle gene expression and cell migration. Surprisingly, our results suggest that GATA6 promotes cell migration of human coronary artery smooth muscle cells (HCASMCs). Furthermore, a microarray screen of GATA6-sensitive gene transcription identified several members of the transforming growth factor beta (TGF-β) signaling.

Material and Methods (urval)

Digital holographic cytometry

HoloMonitor M4, a label-free imaging technology from Phase Holographic Imaging (PHI, Lund, Sweden), was used to analyze single cell motility. Cell motility was quantified by recording a time-lapse sequence of migrating cells in standard culture conditions. 

Briefly, cells were seeded in 6-well culture plates (Sarstedt #83.3920.005) and transfected with GapmeRs as described previously. On day four following transfection a pipette tip was used to create a scratch wound to induce cell migration. 

The lid was replaced with PHI HoloLids^TM imaging covers, sterilized in 70% ethanol. 

The plate was placed onto a HoloMonitor microscope inside an incubator and set to automatically capture images at 20 min intervals for 36 h using the Hstudio software. 

For reproducibility, multiple fields and wells were analyzed. The number of cells that migrated to the wound area and their migration speed and distance was obtained by tracking individual cells over time.

 

GATA6 induces migration of human coronary artery smooth muscle cells

Consistent with these findings, loss of function studies using GapmeRs against GATA6 resulted in reduced cellular motility following downregulation of GATA6 (Figure 2C). This was further supported by live imaging and tracking of individual cell movement over time using digital holographic cytometry (Figure 2D). Both migration speed and distance were significantly reduced in cells treated with GATA6 GapmeRs. The live imaging also confirmed that wound healing in this assay is almost exclusively due to cell migration, and not cell proliferation. All together, these data suggest a role for GATA6 in regulating migration of HCASMCs.

FIGURE 2. GATA6 promotes migration of human vascular smooth muscle cells. Human coronary artery smooth muscle cells were transduced with Ad-CMV-GATA6/Ad-CMV-null or transfected with GapmeRs against GATA6. Cell migration was assessed using a (A) wound-healing assay, (B,C) transwell migration assay (24 h). (D) Digital holographic cytometry was used to measure migration of human coronary artery smooth muscle cells after GapmerRs transfection to downregulate GATA6. Photos were taken at the indicated time points. Number of migrating cells were analyzed after 24 h. Migration speed and distance after 19 h. Data are presented as mean ± SEM (n = 3–6). *p < 0.05, **p < 0.01, ***p < 0.001.

I slutordet skriver forskarna :

To our knowledge, only one earlier study has demonstrated a link between GATA6 and smooth muscle migration, which suggests that GATA6 knockdown leads to an increased VSMC migration. 

Our results support a complex role of GATA6 in the regulation of smooth muscle phenotype, involving both upregulation of specific contractile smooth muscle markers and increased smooth muscle cell migration.  

Cell migration is dependent on changes in the structure of the cytoskeleton, a process driven by dynamic treadmilling of actin filaments.

In conclusion, in this study we provide further insight into the functional role of GATA6 in vascular smooth muscle. We demonstrate that GATA6 promotes migration of HCASMCs, which is of clinical relevance as VSMC migration is a key step in the development and progression of vascular diseases. Finding ways to reduce GATA6-mediated migration may potentiate the positive effects of GATA6. For future work it will be important to elucidate the involvement of TGF-β superfamily in GATA6-induced cell migration and to determine if GATA6 or members of the TGF-β superfamily could be used to promote atherosclerotic plaque stability by stimulating SMCs to strengthen the fibrotic cap. A better understanding of the relationship between GATA6 and cell migration could provide a deeper insight into the molecular mechanisms through which GATA6 regulates smooth muscle phenotype and enable us to identify therapeutic strategies in vascular disease. "

Studien är delvis finansierad av bjässen Novo Nordisk. Minns detta PM om NN.

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..... the HoloMonitor® system (PHI [11]) are in situ microscopy systems based on holographic imaging placed within an incubator. It allows for cell counting directly within different volumes including 96 well plates for high throughputs and/or multiple simultaneous experiments.

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