ASAP: Super-Contrast Vasculature Imaging Using Coherence Analysis and High Frame-Rate Contrast Enhanced Ultrasound.

The very high frame rate afforded by ultrafast ultrasound, combined with microbubble contrast agents, opens new opportunities for imaging tissue microvasculature. However, new imaging paradigms are required to obtain superior image quality from the large amount of acquired data while allowing real-time implementation. In this paper, we report a technique-acoustic sub-aperture processing (ASAP)-capable of generating very high contrast/signal-to-noise ratio (SNR) images of macro-and microvessels, with similar computational complexity to classical power Doppler (PD) imaging. In ASAP, the received data are split into subgroups. The reconstructed data from each subgroup are temporally correlated over frames to generate the final image. As signals in subgroups are correlated but the noise is not, this substantially reduces the noise floor compared to PD. Using a clinical imaging probe, the method is shown to visualize vessels down to $200~\mu \text{m}$ with a SNR of 10 dB higher than PD and to resolve microvascular flow/perfusion information in rabbit kidneys noninvasively in vivo at multiple centimeter depths. With careful filter design, the technique also allows the estimation of flow direction and the separation of fast flow from tissue perfusion. ASAP can readily be implemented into hardware/firmware for real-time imaging and can be applied to contrast enhanced and potentially noncontrast imaging and 3-D imaging.

Ultrasound imaging velocimetry with interleaved images for improved pulsatile arterial flow measurements: a new correction method, experimental and in vivo validation.

Blood velocity measurements are important in physiological science and clinical diagnosis. Doppler ultrasound is the most commonly used method but can only measure one velocity component. Ultrasound imaging velocimetry (UIV) is a promising technique capable of measuring two velocity components; however, there is a limit on the maximum velocity that can be measured with conventional hardware which results from the way images are acquired by sweeping the ultrasound beam across the field of view. Interleaved UIV is an extension of UIV in which two image frames are acquired concurrently, allowing the effective interframe separation time to be reduced and therefore increasing the maximum velocity that can be measured. The sweeping of the ultrasound beam across the image results in a systematic error which must be corrected: in this work, we derived and implemented a new velocity correction method which accounts for acceleration of the scatterers. We then, for the first time, assessed the performance of interleaved UIV for measuring pulsatile arterial velocities by measuring flows in phantoms and in vivo and comparing the results with spectral Doppler ultrasound and transit-time flow probe data. The velocity and flow rate in the phantom agreed within 5-10% of peak velocity, and 2-9% of peak flow, respectively, and in vivo the velocity difference was 9% of peak velocity. The maximum velocity measured was 1.8 m s-1, the highest velocity reported with UIV. This will allow flows in diseased arteries to be investigated and so has the potential to increase diagnostic accuracy and enable new vascular research.

Flow Velocity Mapping Using Contrast Enhanced High-Frame-Rate Plane Wave Ultrasound and Image Tracking: Methods and Initial in Vitro and in Vivo Evaluation.

Ultrasound imaging is the most widely used method for visualising and quantifying blood flow in medical practice, but existing techniques have various limitations in terms of imaging sensitivity, field of view, flow angle dependence, and imaging depth. In this study, we developed an ultrasound imaging velocimetry approach capable of visualising and quantifying dynamic flow, by combining high-frame-rate plane wave ultrasound imaging, microbubble contrast agents, pulse inversion contrast imaging and speckle image tracking algorithms. The system was initially evaluated in vitro on both straight and carotid-mimicking vessels with steady and pulsatile flows and in vivo in the rabbit aorta. Colour and spectral Doppler measurements were also made. Initial flow mapping results were compared with theoretical prediction and reference Doppler measurements and indicate the potential of the new system as a highly sensitive, accurate, angle-independent and full field-of-view velocity mapping tool capable of tracking and quantifying fast and dynamic flows.

Mass transport properties of the rabbit aortic wall.

Uptake of circulating macromolecules by the arterial wall may be a critical step in atherogenesis. Here we investigate the age-related changes in patterns of uptake that occur in the rabbit. In immature aortas, uptake was elevated in a triangle downstream of branch ostia, a region prone to disease in immature rabbits and children. By 16-22 months, uptake was high lateral to ostia, as is lesion prevalence in mature rabbits and young adults. In older rabbits there was a more upstream pattern, similar to the disease distribution in older people. These variations were predominantly caused by the branches themselves, rather than reflecting larger patterns within which the branches happened to be situated (as may occur with patterns of haemodynamic wall shear stress). The narrow streaks of high uptake reported in some previous studies were shown to be post mortem artefacts. Finally, heparin (which interferes with the NO pathway) had no effect on the difference in uptake between regions upstream and downstream of branches in immature rabbits but reversed the difference in older rabbits, as does inhibiting NO synthesis directly. Nevertheless, examination of uptake all around the branch showed that changes occurred at both ages and that they were quite subtle, potentially explaining why inhibiting NO has only minor effects on lesion patterns in mature rabbits and contradicting the earlier conclusion that mechanotransduction pathways change with age. We suggest that recently-established changes in the patterns of haemodynamic forces themselves are more likely to account for the age-dependence of uptake patterns.

Primary and secondary lymphatic valve development: molecular, functional and mechanical insights.

Fluid homeostasis in vertebrates critically relies on the lymphatic system forming a hierarchical network of lymphatic capillaries and collecting lymphatics, for the efficient drainage and transport of extravasated fluid back to the cardiovascular system. Blind-ended lymphatic capillaries employ specialized junctions and anchoring filaments to encourage a unidirectional flow of the interstitial fluid into the initial lymphatic vessels, whereas collecting lymphatics are responsible for the active propulsion of the lymph to the venous circulation via the combined action of lymphatic muscle cells and intraluminal valves. Here we describe recent findings on molecular and physical factors regulating the development and maturation of these two types of valves and examine their role in tissue-fluid homeostasis.

Flow control in our vessels: vascular valves make sure there is no way back.

The efficient transport of blood and lymph relies on competent intraluminal valves that ensure unidirectional fluid flow through the vessels. In the lymphatic vessels, lack of luminal valves causes reflux of lymph and can lead to lymphedema, while dysfunction of venous valves is associated with venous hypertension, varicose veins, and thrombosis that can lead to edema and ulcerations. Despite their clinical importance, the mechanisms that regulate valve formation are poorly understood and have only recently begun to be characterized. Here, we discuss new findings regarding the development of venous and lymphatic valves that indicate the involvement of common molecular mechanisms in regulating valve formation in different vascular beds.

Genes regulating lymphangiogenesis control venous valve formation and maintenance in mice.

Chronic venous disease and venous hypertension are common consequences of valve insufficiency, yet the molecular mechanisms regulating the formation and maintenance of venous valves have not been studied. Here, we provide what we believe to be the first description of venous valve morphogenesis and identify signaling pathways required for the process. The initial stages of valve development were found to involve induction of ephrin-B2, a key marker of arterial identity, by venous endothelial cells. Intriguingly, developing and mature venous valves also expressed a repertoire of proteins, including prospero-related homeobox 1 (Prox1), Vegfr3, and integrin-α9, previously characterized as specific and critical regulators of lymphangiogenesis. Using global and venous valve-selective knockout mice, we further demonstrate the requirement of ephrin-B2 and integrin-α9 signaling for the development and maintenance of venous valves. Our findings therefore identified molecular regulators of venous valve development and maintenance and highlighted the involvement of common morphogenetic processes and signaling pathways in controlling valve formation in veins and lymphatic vessels. Unexpectedly, we found that venous valve endothelial cells closely resemble lymphatic (valve) endothelia at the molecular level, suggesting plasticity in the ability of a terminally differentiated endothelial cell to take on a different phenotypic identity.

Integrin-alpha9 is required for fibronectin matrix assembly during lymphatic valve morphogenesis.

Dysfunction of lymphatic valves underlies human lymphedema, yet the process of valve morphogenesis is poorly understood. Here, we show that during embryogenesis, lymphatic valve leaflet formation is initiated by upregulation of integrin-alpha9 expression and deposition of its ligand fibronectin-EIIIA (FN-EIIIA) in the extracellular matrix. Endothelial cell-specific deletion of Itga9 (encoding integrin-alpha9) in mouse embryos results in the development of rudimentary valve leaflets characterized by disorganized FN matrix, short cusps, and retrograde lymphatic flow. Similar morphological and functional defects are observed in mice lacking the EIIIA domain of FN. Mechanistically, we demonstrate that in primary human lymphatic endothelial cells, the integrin-alpha9-EIIIA interaction directly regulates FN fibril assembly, which is essential for the formation of the extracellular matrix core of valve leaflets. Our findings reveal an important role for integrin-alpha9 signaling during lymphatic valve morphogenesis and implicate it as a candidate gene for primary lymphedema caused by valve defects.

Anterograde Jelly belly and Alk receptor tyrosine kinase signaling mediates retinal axon targeting in Drosophila.

Anaplastic lymphoma kinase (Alk) has been proposed to regulate neuronal development based on its expression pattern in vertebrates and invertebrates; however, its function in vivo is unknown. We demonstrate that Alk and its ligand Jelly belly (Jeb) play a central role as an anterograde signaling pathway mediating neuronal circuit assembly in the Drosophila visual system. Alk is expressed and required in target neurons in the optic lobe, whereas Jeb is primarily generated by photoreceptor axons and functions in the eye to control target selection of R1-R6 axons in the lamina and R8 axons in the medulla. Impaired Jeb/Alk function affects layer-specific expression of three cell-adhesion molecules, Dumbfounded/Kirre, Roughest/IrreC, and Flamingo, in the medulla. Moreover, loss of flamingo in target neurons causes some R8-axon targeting errors observed in Jeb and Alk mosaic animals. Together, these findings suggest that Jeb/Alk signaling helps R-cell axons to shape their environment for target recognition.