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In this article a number of whole-field blood velocity measurement techniques are concisely reviewed. We primarily focus on optical measurement techniques for in vivo applications, such as laser Doppler velocimetry (including time varying speckle), laser speckle contrast imaging and particle image velocimetry (including particle tracking). We also briefly describe nuclear magnetic resonance and ultrasound particle image velocimetry, two techniques that do not rely on optical access, but that are of importance to in vivo whole-field blood velocity measurement. Typical applications for whole-field methods are perfusion monitoring, the investigation of instantaneous blood flow patterns, the derivation of endothelial shear stress distributions from velocity fields, and the measurement of blood volume flow rates. These applications require individual treatment in terms of spatial and temporal resolution and number of measured velocity components. The requirements further differ for the investigation of macro-, meso-, and microscale blood flows. In this review we describe and classify those requirements and present techniques that satisfy them.

The wall shear stress plays a key role in the interaction between blood flow and the surrounding tissue. To obtain quantitative information about this parameter, velocity measurements are required with sufficient spatial (and temporal) resolution. We present a methodology for the determination of the wall shear stress in vivo in the vitelline network of a chick embryo. Velocity data is obtained by microscopic particle image velocimetry using correlation ensemble averaging; the latter is used to increase the signal-to-noise ratio of the measurements. The temporal evolution of the pulsatile flow is reconstructed by sorting the image pairs based on a phase estimate. From these flow measurements, the wall shear stress can be derived either directly from the magnitude of the gradients or from fits to velocity profiles. Both methods give results that are in good agreement with each other, while the former method is significantly easier to implement. For more accurate studies, the full threedimensional velocity field may be required. It is demonstrated how this velocity field can be obtained by scanning the measurement volume.

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...

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