development of cardiac malformations : The involvement of the
endothelin-1 pathway
Groenendijk, B.C.W.
Citation
Groenendijk, B. C. W. (2006, March 23). Influence of blood flow on shear stress responsive
genes in the development of cardiac malformations : The involvement of the endothelin-1
pathway. Retrieved from https://hdl.handle.net/1887/4346
Version:
Corrected Publisher’s Version
Summary
149
Summary
The aim of this study was to determine the role of shear stress caused by blood flow on gene expression, and to investigate this effect on the development of cardiovascular malformations. The endothelin-1 (ET-1) pathway was in this respect investigated in more detail.
Chapter 1 provides information on blood flow and shear stress in chicken heart development, and maldevelopment after venous clip. Shear sensing, the effect of shear on gene expression, and three shear stress responsive genes: ET-1, Krüppel-like factor 2 (KLF2), and endothelial nitric oxide synthase (NOS-3), are introduced.
Chapter 2 shows the expression patterns of the three shear stress responsive genes, ET-1,
KLF2 and NOS-3 during normal chicken cardiovascular development. It was demonstrated
that, especially during later embryonic stages, KLF2 and NOS-3 were expressed in narrow regions of the cardiovascular system, i.e., the atrioventricular canal and the outflow tract, regions where shear stress is expected to be high. ET-1, on the other hand, was not present in these areas. This demonstrated that also in vivo NOS-3 is positively correlated with shear stress, whereas ET-1 is negatively correlated.
In Chapter 3 the shear distribution pattern in a normal stage HH14 chicken heart is provided, showing the overlap with KLF2 at HH18 (from Chapter 2) in the narrow regions and inner curvature. Furthermore, the gene patterns were investigated after disturbing the intracardiac flow patterns by venous clip. It was demonstrated that KLF2 and NOS-3 were increased in the heart after 3 hours of vitelline ligation, whereas ET-1 was down-regulated. In the dorsal aorta this was the opposite: KLF2 and NOS-3 were decreased and ET-1 up-regulated, which is in agreement with Doppler measurements on the dorsal aorta that show that blood flow is decreased for up to 5 hours after venous clip. The gene alterations after clip in predominantly the inner curvature of the heart suggest that shear stress is locally increased. Cardiovascular malformations may arise due to changes in gene expression upon alterations in shear, since abnormalities are formed by an impaired development of the regions where gene alterations were most conspicuous.
Since ET-1 is cardially down-regulated after clip and the Edn-1-/- mice show similar
was investigated in Chapter 4. At HH18 ET-1 and ET-1 receptor antagonists were infused in the extra-embryonic blood circulation. Ventricular diastolic filling characteristics were impaired at HH24, similarly to venous clip-induced alterations in ventricular function. In addition, small cardiovascular malformations were shown that were also encountered after venous clip. However, these functional and morphological impairments were less severe than after clip, where more genes are involved and are affected for a longer period of time. ET-1 is biologically active for only 2 hours, but in vitro experiments showed that ET-1 and its receptor antagonists were able to induce feedback loop mechanisms in cultured endothelial/muscle cells after 5 hours, suggesting that these mechanisms are involved in the impairments observed at later stages. The results of this study imply that components of the ET-1 pathway, but also other genes, are involved after venous ligation.
In Chapter 5 the direct effects of ET-1 and the ET-1 receptor antagonists on embryonic and vitelline hemodynamics were investigated with two different techniques. Embryonic hemodynamics were monitored with the Doppler ultrasound technique, and hemodynamics in the vitelline veins were measured by means of micro-Particle Image Velocimetry (μPIV). In the vitelline veins changes in hemodynamics were demonstrated, whereas with the embryonic Doppler measurements no alterations could be detected, concluding that the μPIV technique is much more sensitive than Doppler ultrasound. Furthermore, ET-1, endothelin-converting enzyme-1 (ECE-1) and endothelin-B receptor (ETB) mRNA were shown to be expressed in the vitelline vessels, whereas endothelin-A receptor (ETA) mRNA was absent, suggesting that ET-1 functions differently in the vitelline circulation than in the embryonic circulation, where all genes are expressed. It was postulated that the alterations in (embryonic and vitelline) hemodynamics influence gene expression and/or flow patterns in the vitelline circulation, which results in changes in the intracardiac flow patterns. In combination with the balance-shift of receptor types due to the feedback mechanisms in receptor mRNA, this will eventually lead to the impaired ventricular function and morphological malformations encountered at stages HH24 and HH35, that were demonstrated in Chapter 4.
Summary
151
ventricles, where KLF2 was not expressed. Under high shear stress circumstances primary cilia are disassembled, explaining the absense of cilia in these regions. The basal body of the cilium is connected to the cortical actin cytoskeleton, suggesting that the cytoskeleton functions as a central shear stress transducer. The monocilia themselves are postulated to be immotile primary cilia, which function as fluid shear stress sensors.
