Primary cilia as biomechanical sensors in regulating endothelial function

Depending on the pattern of blood flow to which they are exposed and their proliferative status, vascular endothelial cells can present a primary cilium into the flow compartment of a blood vessel. The cilium modifies the response of endothelial cells to biomechanical forces. Shear stress, which is the drag force exerted by blood flow, is best studied in this respect. Here we review the structural composition of the endothelial cilia and the current status of knowledge about the relation between the presence of primary cilia on endothelial cells and the shear stress to which they are exposed.     

Accurate Blood Flow Measurements: Are Artificial Tracers Necessary?

Imaging-based blood flow measurement techniques, such as particle image velocimetry, have become an important tool in cardiovascular research. They provide quantitative information about blood flow, which benefits applications ranging from developmental biology to tumor perfusion studies. Studies using these methods can be classified based on whether they use artificial tracers or red blood cells to visualize the fluid motion. We here present the first direct comparison in vivo of both methods. For high magnification cases, the experiments using red blood cells strongly underestimate the flow (up to 50% in the present case), as compared to the tracer results. For medium magnification cases, the results from both methods are indistinguishable as they give the same underestimation of the real velocities (approximately 33%, based on in vitro reference measurements). These results suggest that flow characteristics reported in literature cannot be compared without a careful evaluation of the imaging characteristics. A method to predict the expected flow averaging behavior for a particular facility is presented.     

Vestibular Role of KCNQ4 and KCNQ5 K+ Channels Revealed by Mouse Models

The function of sensory hair cells of the cochlea and vestibular organs depends on an influx of K⁺ through apical mechanosensitive ion channels and its subsequent removal over their basolateral membrane. The KCNQ4 (K_v7.4) K⁺ channel, which is mutated in DFNA2 human hearing loss, is expressed in the basal membrane of cochlear outer hair cells where it may mediate K⁺ efflux. Like the related K⁺ channel KCNQ5 (K_v7.5), KCNQ4 is also found at calyx terminals ensheathing type I vestibular hair cells where it may be localized pre- or postsynaptically. Making use of Kcnq4^−/− mice lacking KCNQ4, as well as Kcnq4^dn/dn and Kcnq5^dn/dn mice expressing dominant negative channel mutants, we now show unambiguously that in adult mice both channels reside in postsynaptic calyx-forming neurons, but cannot be detected in the innervated hair cells. Accordingly, whole cell currents of vestibular hair cells did not differ between genotypes. Neither Kcnq4^−/−, Kcnq5^dn/dn nor Kcnq4^−/−/Kcnq5^dn/dn double mutant mice displayed circling behavior found with severe vestibular impairment. However, a milder form of vestibular dysfunction was apparent from altered vestibulo-ocular reflexes in Kcnq4^−/−/Kcnq5^dn/dn and Kcnq4^−/− mice. The larger impact of KCNQ4 may result from its preferential expression in central zones of maculae and cristae, which are innervated by phasic neurons that are more sensitive than the tonic neurons present predominantly in the surrounding peripheral zones where KCNQ5 is found. The impact of postsynaptic KCNQ4 on vestibular function may be related to K⁺ removal and modulation of synaptic transmission.     

Shear induced collateral artery growth modulated by endoglin but not by ALK1

Transforming growth factor-beta (TGF-β) stimulates both ischaemia induced angiogenesis and shear stress induced arteriogenesis by signalling through different receptors. How these receptors are involved in both these processes of blood flow recovery is not entirely clear. In this study the role of TGF-β receptors 1 and endoglin is assessed in neovascularization in mice. Unilateral femoral artery ligation was performed in mice heterozygous for either endoglin or ALK1 and in littermate controls. Compared with littermate controls, blood flow recovery, monitored by laser Doppler perfusion imaging, was significantly hampered by maximal 40% in endoglin heterozygous mice and by maximal 49% in ALK1 heterozygous mice. Collateral artery size was significantly reduced in endoglin heterozygous mice compared with controls but not in ALK1 heterozygous mice. Capillary density in ischaemic calf muscles was unaffected, but capillaries from endoglin and ALK1 heterozygous mice were significantly larger when compared with controls. To provide mechanistic evidence for the differential role of endoglin and ALK1 in shear induced or ischaemia induced neovascularization, murine endothelial cells were exposed to shear stress in vitro. This induced increased levels of endoglin mRNA but not ALK1. In this study it is demonstrated that both endoglin and ALK1 facilitate blood flow recovery. Importantly, endoglin contributes to both shear induced collateral artery growth and to ischaemia induced angiogenesis, whereas ALK1 is only involved in ischaemia induced angiogenesis.     

Quantification of Blood Flow and Topology in Developing Vascular Networks

Since fluid dynamics plays a critical role in vascular remodeling, quantification of the hemodynamics is crucial to gain more insight into this complex process. Better understanding of vascular development can improve prediction of the process, and may eventually even be used to influence the vascular structure. In this study, a methodology to quantify hemodynamics and network structure of developing vascular networks is described. The hemodynamic parameters and topology are derived from detailed local blood flow velocities, obtained by in vivo micro-PIV measurements. The use of such detailed flow measurements is shown to be essential, as blood vessels with a similar diameter can have a large variation in flow rate. Measurements are performed in the yolk sacs of seven chicken embryos at two developmental stages between HH 13+ and 17+. A large range of flow velocities (1 µm/s to 1 mm/s) is measured in blood vessels with diameters in the range of 25–500 µm. The quality of the data sets is investigated by verifying the flow balances in the branching points. This shows that the quality of the data sets of the seven embryos is comparable for all stages observed, and the data is suitable for further analysis with known accuracy. When comparing two subsequently characterized networks of the same embryo, vascular remodeling is observed in all seven networks. However, the character of remodeling in the seven embryos differs and can be non-intuitive, which confirms the necessity of quantification. To illustrate the potential of the data, we present a preliminary quantitative study of key network topology parameters and we compare these with theoretical design rules.     

In vivo micro particle image velocimetry measurements of blood-plasma in the embryonic avian heart

The measurement of blood-plasma velocity distributions with spatial and temporal resolution in vivo is inevitable for the determination of shear stress distributions in complex geometries at unsteady flow conditions like in the beating heart. A non-intrusive, whole-field velocity measurement technique is required that is capable of measuring instantaneous flow fields at sub-millimeter scales in highly unsteady flows. Micro particle image velocimetry (muPIV) meets these demands, but requires special consideration and methodologies in order to be utilized for in vivo studies in medical and biological research. We adapt muPIV to measure the blood-plasma velocity in the beating heart of a chicken embryo. In the current work, bio-inert, fluorescent liposomes with a nominal diameter of 400 nm are added to the flow as a tracer. Because of their small dimension and neutral buoyancy the liposomes closely follow the movement of the blood-plasma and allow the determination of the velocity gradient close to the wall. The measurements quantitatively resolve the velocity distribution in the developing ventricle and atrium of the embryo at nine different stages within the cardiac cycle. Up to 400 velocity vectors per measurement give detailed insight into the fluid dynamics of the primitive beating heart. A rapid peristaltic contraction accelerates the flow to peak velocities of 26 mm/s, with the velocity distribution showing a distinct asymmetrical profile in the highly curved section of the outflow tract. In relation to earlier published gene-expression experiments, the results underline the significance of fluid forces for embryonic cardiogenesis. In general, the measurements demonstrate that muPIV has the potential to develop into a general tool for instationary flow conditions in complex flow geometries encountered in cardiovascular research.