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

Abstract

Endothelial cells (EC) translate biomechanical forces into functional and phenotypic responses that play important roles in cardiac development. Specifically, EC in areas of high shear stress, i.e., in the cardiac outflow tract and atrioventricular canal, are characterized by high expression of Krü ppel-like factor 2 (Klf2) and by transforming growth factor-beta (Tgfb)-driven endothelial-to-mesenchymal transition. Extraembryonic venous obstruction (venous clip model) results in congenital heart malformations, and venous clip-induced alterations in shear stress-related gene expression are suggestive for an increase in cardiac shear stress. Here, we study the effects of shear stress on Klf2 expression and Tgfb-associated signaling in embryonic EC in vivo using the venous clip model and in vitro by subjecting cultured EC to fluid flow. Cellular responses were assessed by analysis of Klf2, Tgfb ligands, and their downstream signaling targets. Results show that, in embryonic EC, shear stress activates Tgfb/Alk5 signaling and that induction of Klf2 is an Alk5 dependent process. Developmental Dynamics 240: [1670][1671][1672][1673][1674][1675][1676][1677][1678][1679][1680] 2011. V C 2011 Wiley-Liss, Inc.Key words: shear stress; cardiac cushions; endothelium; Klf2; Tgfb; Alk5 Accepted 14 April 2011 INTRODUCTIONBlood flow is a decisive factor guiding proper cardiovascular development and preventing vascular pathology. Biomechanical forces acting upon the vessel wall include flow-induced shear stress and pressure related cyclic stretch. Shear stress modulates endothelial structure and function and results in an accurate regulation of endothelial gene expression in vitro (Dekker et al., 2002) and in vivo (Groenendijk et al., 2004;Dekker et al., 2005). Several animal models show that congenital malformations can result from disturbed blood flow (Hogers et al., 1997;Sedmera et al., 1999;Tobita et al., 2002;Hove et al., 2003;Dealmeida et al., 2007;Butcher et al., 2007). The large and rapid changes in volume and geometry of cardiac compartments during development are translated into local changes in shear stress and concomitant gene expression patterns of, e.g., Krü ppel like factor 2 (KLF2) (Groenendijk et al., 2004).KLF2 is a shear responsive transcription factor essential in establishing and maintaining endothelial function by regulating the expression of many genes (Dekker et al., 2002(Dekker et al., , 2006SenBanerjee et al., 2004;Boon and Horrevoets, 2009). Exposure of adult endothelium to high and pulsatile shear stress causes an increase in KLF2 expression (Dekker et al., 2002;Wang et al., 2006), which induces a quiescent and atheroprotective phenotype, in part through inhibition of transforming growth factor-beta (TGFb) signaling (Boon et al., 2007). The increase of KLF2 protein levels under shear stress is mediated by two mechanisms, i.e., activation of the KLF2 promoter and stabilization of KLF2 mRNA (Dekker et al., 2002;van Thienen et al., 2006). Myocyte enhancer binding factor 2C (Mef2C) is one of the transcriptiona...

Endothelial colony-forming cells show a mature transcriptional response to shear stress

“…In the present study, KLF2 expression levels were induced under 0.5 and 2.5 Pa shear stress in HUVEC, HUAEC, and ECFC. The relative induction of KLF2 in HUVEC under 0.5 Pa shear stress was approximately factor 5, comparable to the previously reported values in these cells and adult human aortic EC (Egorova et al 2011b). The absolute KLF2 mRNA levels under low and high flow did not differ among the three cell types, indicating their universal capacity to upregulate KLF2 in response to shear stress.…”

“…ECFC, HUVEC, and HUAEC were seeded on fixed 1% (w/v) gelatin-coated coverslips and grown to confluency. Cells were plated in a parallel plate flow chamber and exposed to laminar shear stress in a recirculation flow system for 5 h at 37°C and 5% CO 2 to create a steady, laminar flow, essentially as described before Egorova et al 2011b). Shear stress levels of 0.5 (5 dyn/cm 2 ) and 2.5 Pa (25 dyn/cm 2 ) were used to compare the response of ECFC (n=4), HUVEC (n= 4), and HUAEC (n=4) to low and high flow rates, typically found in the venous and arterial parts of the vasculature, respectively.…”

SLUG Is Expressed in Endothelial Cells Lacking Primary Cilia to Promote Cellular Calcification

“…According to previous reports, 43 Tg737 orpk/orpk cells exhibited a higher basal phosphorylation of SMAD2. To investigate whether TGF-β signaling is responsible for the mineralizing phenotype of Tg737 orpk/orpk cells, we cultured these cells in the Human umbilical vein ECs isolated from 8 different donors were immunostained with acetylated α-TUBULIN to detect primary cilia (red channel, white arrows point to primary cilia), and nuclei were visualized with DAPI (blue channel).…”

Vasa Vasorum in Normal and Diseased Arteries

“…96 Events leading to endothelial TGF-β signaling activation are less clear. A number of processes have been implicated, including shear stress, [97][98][99] stimulation with endothelin-1, 90 Notch, 100 and caveolin deficiency, 101 among others. One of the recently identified mechanisms regulating TGF-β signaling in the endothelium involves fibroblast growth factor (FGF)-mediated suppression of TGF receptor expression.…”