Changes in Shear Stress–Related Gene Expression After Experimentally Altered Venous Return in the Chicken Embryo

Abstract

Abstract-Hemodynamics play an important role in cardiovascular development, and changes in blood flow can cause congenital heart malformations. The endothelium and endocardium are subjected to mechanical forces, of which fluid shear stress is correlated to blood flow velocity. The shear stress responsive genes lung Krüppel-like factor (KLF2), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3) display specific expression patterns in vivo during chicken cardiovascular development. Nonoverlapping patterns of these genes were demonstrated in the endocardium at structural lumen constrictions that are subjected to high blood flow velocities. Previously, we described in chicken embryos a dynamic flow model (the venous clip) in which the venous return to the heart is altered and cardiac blood flow patterns are disturbed, causing the formation of congenital cardiac malformations. In the present study we test the hypothesis that disturbed blood flow can induce altered gene expression. In situ hybridizations indeed show a change in gene expression after venous clip. The level of expression of ET-1 in the heart is locally decreased, whereas KLF2 and NOS-3 are both upregulated. We conclude that venous obstruction results in altered expression patterns of KLF2, ET-1, and NOS-3, suggestive for increased cardiac shear stress. Key Words: cardiovascular physiology Ⅲ embryonic circulation Ⅲ endothelium Ⅲ gene expression Ⅲ shear stress H emodynamic forces generated by blood flow modulate the structure and function of both fetal and adult endothelial cells (reviewed by Gimbrone et al 1 ). In pathogenesis shear stress is important as atherosclerosis develops in low and unsteady shear stress areas. 2 During embryogenesis blood flow plays an important role in cardiac development. 3,4 We developed the chicken venous clip model 3 in which the right lateral vitelline vein is ligated. This results in immediate changes in blood-flow patterns through the heart, and eventually to cardiovascular malformations, including ventricular septal defects, semilunar valve anomalies, and several types of pharyngeal arch artery abnormalities. 5 Additionally, Stekelenburg-de Vos et al 6 have shown that up to 5 hours after venous clip the dorsal aortic mean and peak blood flow is decreased, demonstrating a change in hemodynamics. Shear stress is directly related to blood flow, therefore it is likely that this is also altered in the venous clip model and involved in the development of abnormalities in the cardiovascular system.Important genes encoding transcription factors and signaling molecules, eg, lung Krüppel-like factor (KLF2/LKLF), endothelin-1 (ET-1), and endothelial nitric oxide synthase (NOS-3/eNOS) are shear-dependent in their expression in vitro. 7-9 Previously, we suggested that these genes are also shear-related in vivo. 10 Endothelin-1 is a growth hormone and vasoconstrictor. NOS-3 catalyzes the conversion of L-arginine to L-citrullin, generating nitric oxide (NO). NO is involved in, eg, vasodilation. KLF2 is a member of t...

Primary cilia sensitize endothelial cells for fluid shear stress

“…We showed that already during embryonic development, patterning of KLF2 gene expression relates to areas of high shear stress (Groenendijk et al, 2004;Vennemann et al, 2006). Experimental changes in embryonic blood flow lead to changes in gene expression patterning of KLF2, ET1, and eNOS (Groenendijk et al, 2005), and lead to loss of proper cardiovascular function (Stekelenburg-de Vos et al, 2005Ursem et al, 2004) and the development of structural anomalies (Hogers et al, 1997(Hogers et al, , 1999. We have recently identified the inverse correlation between the expression of KLF2 and the presence of endothelial primary cilia in the embryonic heart (Van der Heiden et al, 2006).…”

“…This regulation apparently aims to level off shear stress, through activation or repression of vasoactive genes like endothelial nitric oxide synthase (eNOS, NOS3) or endothelin-1 (ET1), and modulate the activation state of the cells. In this respect, the zinc finger transcription factor Krü ppel-like factor-2 (KLF2/LKLF) plays a central role, in vitro (SenBanerjee et al, 2004), as well as in vivo in the adult (Lee et al, 2006;Dekker et al, 2005Dekker et al, , 2006 and embryonic vasculature (Groenendijk et al, 2004(Groenendijk et al, , 2005. KLF2 is expressed in areas of high and steady or pulsatile flow where it positively regulates eNOS and downregulates ET1, whereas prolonged oscillatory flow suppresses KLF2 expression (Wang et al, 2006).…”

Quantitative volumetric analysis of cardiac morphogenesis assessed through micro‐computed tomography

“…Photographic images, even with scales, do not have the capacity to measure depth, limiting their utility for three-dimensional (3D) quantification. The 3D serial reconstruction using histological sections has dramatically improved understanding of heart development with the ability to combine geometry and the expressions of cell and/or matrix proteins (Moorman et al, 2000;Groenendijk et al, 2005). However, accuracy of this process is extremely sensitive to the number of sections processed, limiting its use to research labs with specifically dedicated technicians and equipment.…”

Tgfβ/Alk5 signaling is required for shear stress induced klf2 expression in embryonic endothelial cells

“…In line with the necessity of adequate endothelial signaling, venous clipping eventually results in a spectrum of cardiac malformations, including including atrioventricular (AV) and outflow tract (OFT) anomalies (Hogers et al, 1997(Hogers et al, , 1999. Groenendijk et al (2005) described changes in expression patterns of KLF2, endothelin-1 (ET-1), and endothelial nitric oxide synthase-3 (eNOS/NOS-3) already within 3 hr after clipping, suggestive of an increase in cardiac shear stress (Groenendijk et al, 2005(Groenendijk et al, , 2007.On a cellular level, Tgfb is a multipotent cytokine that plays pivotal roles in the regulation of proliferation, differentiation, migration, and survival of EC (Lebrin et al, 2005). After secretion and activation, Tgfb ligands bind their specific type I and type II serine/threonine kinase receptors.…”