Low-flow intussusception and metastable VEGFR2 signaling launch angiogenesis in ischemic muscle.

Efforts to promote sprouting angiogenesis in skeletal muscles of individuals with peripheral artery disease have not been clinically successful. We discovered that, contrary to the prevailing view, angiogenesis following ischemic muscle injury in mice was not driven by endothelial sprouting. Instead, real-time imaging revealed the emergence of wide-caliber, primordial conduits with ultralow flow that rapidly transformed into a hierarchical neocirculation by transluminal bridging and intussusception. This process was accelerated by inhibiting vascular endothelial growth factor receptor-2 (VEGFR2). We probed this response by developing the first live-cell model of transluminal endothelial bridging using microfluidics. Endothelial cells subjected to ultralow shear stress could reposition inside the flowing lumen as pillars. Moreover, the low-flow lumen proved to be a privileged location for endothelial cells with reduced VEGFR2 signaling capacity, as VEGFR2 mechanosignals were boosted. These findings redefine regenerative angiogenesis in muscle as an intussusceptive process and uncover a basis for its launch.

Membrane Lipid-KIR2.x Channel Interactions Enable Hemodynamic Sensing in Cerebral Arteries.

Objective- Inward rectifying K+ (KIR) channels are present in cerebral arterial smooth muscle and endothelial cells, a tandem arrangement suggestive of a dynamic yet undiscovered role for this channel. This study defined whether distinct pools of cerebral arterial KIR channels were uniquely modulated by membrane lipids and hemodynamic stimuli. Approach and Results- A Ba2+-sensitive KIR current was isolated in smooth muscle and endothelial cells of rat cerebral arteries; molecular analyses subsequently confirmed KIR2.1/KIR2.2 mRNA and protein expression in both cells. Patch-clamp electrophysiology next demonstrated that each population of KIR channels was sensitive to key membrane lipids and hemodynamic stimuli. In this regard, endothelial KIR was sensitive to phosphatidylinositol 4,5-bisphosphate content, with depletion impairing the ability of laminar shear stress to activate this channel pool. In contrast, smooth muscle KIR was sensitive to membrane cholesterol content, with sequestration blocking the ability of pressure to inhibit channel activity. The idea that membrane lipids help confer shear stress and pressure sensitivity of KIR channels was confirmed in intact arteries using myography. Virtual models integrating structural/electrical observations reconceptualized KIR as a dynamic regulator of membrane potential working in concert with other currents to set basal tone across a range of shear stresses and intravascular pressures. Conclusions- The data show for the first time that specific membrane lipid-KIR interactions enable unique channel populations to sense hemodynamic stimuli and drive vasomotor responses to set basal perfusion in the cerebral circulation.

Study of the effect of stenosis severity and non-Newtonian viscosity on multidirectional wall shear stress and flow disturbances in the carotid artery using particle image velocimetry.

The development of atherosclerosis at the carotid bifurcation is impacted by local variations in wall shear stress (WSS) magnitude and direction, as well as flow complexity within the vessel. In this study, stereoscopic particle image velocimetry (PIV) was used to investigate multidirectional WSS and disturbed flow for idealized models of the carotid bifurcation with varying eccentric stenosis of the internal carotid artery (ICA) and both Newtonian (N-fluid) and non-Newtonian (nN-fluid) blood analogues. Turbulence intensity (TI) was reduced with the nN-fluid compared to N-fluid for mild to moderate stenosis, and comparable for more severely stenosed (70%) models. Differences in maximum TI due to viscosity model ranged from 0.02 m/s to 0.06 m/s compared to much larger differences due to geometry of up to 0.29 m/s between mild and severe stenosis. The level of time-averaged WSS (TAWSS) increased with stenosis severity from 5 Pa to 32 Pa, and nN-fluid led to higher WSS on average than N-fluid counterparts. Regions of elevated oscillatory shear index (OSI) demarcated recirculation regions, and mean OSI in the ICA branch was reduced for nN-fluid models by 9-19% compared to N-fluid. Transverse WSS (transWSS) increased with WSS magnitude and again was higher in nN-fluid models. Surface area exposure to shear metrics indicated that a Newtonian viscosity assumption predicted larger regions of low and oscillatory WSS, while predicting reduced regions of high transWSS, in comparison to the more physiological shear thinning fluid.

Investigation of Crossbeam Multi-receiver Configurations for Accurate 3-D Vector Doppler Velocity Estimation.

An accurate estimation of low blood velocities whose Doppler shifts span the wall filter cutoff, such as near the wall in recirculation or disturbed flow regions, is important for accurate mapping of velocities to achieve improved estimations of wall shear stress and turbulence, which are known risk factors for atherosclerosis and stroke. This paper presents the comparative benefit of increasing the number of receiver beams above three for an improved estimation of low 3-D velocities. The 3-D crossbeam vector Doppler ultrasound configurations were studied in terms of the number of receiver beams, interbeam angle, and beam selection method (criterion for discriminating between tissue and blood Doppler signals) for a range of velocity orientations, which may prove useful in the design of a future 2-D array for vascular imaging. For maximum velocity resolution, a shallow gradient of low flow velocities up to 5 cm/s was generated across a large-diameter (2.46 cm) straight vessel. Data were acquired using a linear array rotated around the central transmit beam axis to generate three- to eight-receiver (3R-8R) configurations;the rotation of each configuration relative to the flow axis was used to mimic a broad range of velocity vector orientations. Accuracy and precision for ≥5 receivers were consistently better over all velocity orientations and for all selection methods. For a velocity magnitude of 2 cm/s, the best accuracy and precision in both magnitude and direction (~21% ± 13%, <1° ± 9°, respectively) were seen with a 5R configuration using a weighted least-squares selection method. Asymmetry in the 5R configuration led to an improved accuracy and precision compared with that in symmetrical 6R and 8R configurations. The results demonstrated relatively little to no benefit from more than five receiver beams.

Spread-Spectrum Beamforming and Clutter Filtering for Plane-Wave Color Doppler Imaging.

Plane-wave imaging is desirable for its ability to achieve high frame rates, allowing the capture of fast dynamic events and continuous Doppler data. In most implementations of plane-wave imaging, multiple low-resolution images from different plane wave tilt angles are compounded to form a single high-resolution image, thereby reducing the frame rate. Compounding improves the lateral beam profile in the high-resolution image, but it also acts as a low-pass filter in slow time that causes attenuation and aliasing of signals with high Doppler shifts. This paper introduces a spread-spectrum color Doppler imaging method that produces high-resolution images without the use of compounding, thereby eliminating the tradeoff between beam quality, maximum unaliased Doppler frequency, and frame rate. The method uses a long, random sequence of transmit angles rather than a linear sweep of plane wave directions. The random angle sequence randomizes the phase of off-focus (clutter) signals, thereby spreading the clutter power in the Doppler spectrum, while keeping the spectrum of the in-focus signal intact. The ensemble of randomly tilted low-resolution frames also acts as the Doppler ensemble, so it can be much longer than a conventional linear sweep, thereby improving beam formation while also making the slow-time Doppler sampling frequency equal to the pulse repetition frequency. Experiments performed using a carotid artery phantom with constant flow demonstrate that the spread-spectrum method more accurately measures the parabolic flow profile of the vessel and outperforms conventional plane-wave Doppler in both contrast resolution and estimation of high flow velocities. The spread-spectrum method is expected to be valuable for Doppler applications that require measurement of high velocities at high frame rates.

In vitro shear stress measurements using particle image velocimetry in a family of carotid artery models: effect of stenosis severity, plaque eccentricity, and ulceration.

Atherosclerotic disease, and the subsequent complications of thrombosis and plaque rupture, has been associated with local shear stress. In the diseased carotid artery, local variations in shear stress are induced by various geometrical features of the stenotic plaque. Greater stenosis severity, plaque eccentricity (symmetry) and plaque ulceration have been associated with increased risk of cerebrovascular events based on clinical trial studies. Using particle image velocimetry, the levels and patterns of shear stress (derived from both laminar and turbulent phases) were studied for a family of eight matched-geometry models incorporating independently varied plaque features - i.e. stenosis severity up to 70%, one of two forms of plaque eccentricity, and the presence of plaque ulceration). The level of laminar (ensemble-averaged) shear stress increased with increasing stenosis severity resulting in 2-16 Pa for free shear stress (FSS) and approximately double (4-36 Pa) for wall shear stress (WSS). Independent of stenosis severity, marked differences were found in the distribution and extent of shear stress between the concentric and eccentric plaque formations. The maximum WSS, found at the apex of the stenosis, decayed significantly steeper along the outer wall of an eccentric model compared to the concentric counterpart, with a 70% eccentric stenosis having 249% steeper decay coinciding with the large outer-wall recirculation zone. The presence of ulceration (in a 50% eccentric plaque) resulted in both elevated FSS and WSS levels that were sustained longer (∼20 ms) through the systolic phase compared to the non-ulcerated counterpart model, among other notable differences. Reynolds (turbulent) shear stress, elevated around the point of distal jet detachment, became prominent during the systolic deceleration phase and was widely distributed over the large recirculation zone in the eccentric stenoses.

Turbulence intensity measurements using particle image velocimetry in diseased carotid artery models: effect of stenosis severity, plaque eccentricity, and ulceration.

Clinical decision-making for the treatment of patients with diseased carotid artery is mainly based on the severity of the stenosis. However, stenosis severity alone is not a sensitive indicator, and other local factors for the assessment of stroke risk are required. Flow disturbance is of particular interest due to its proven association with increased thromboembolic activities. The objective of this study was to investigate the level of turbulence intensity (TI) with regards to certain geometrical features of the plaque - namely stenosis severity, eccentricity, and ulceration. A family of eight carotid-artery bifurcation models was examined using particle image velocimetry. Results showed a marked difference in turbulence intensity among these models; increasing degree of stenosis severity resulted in increased turbulence intensity, going from 0.12 m/s for mild stenosis to 0.37 m/s for severe stenosis (with concentric geometry). Moreover, independent of stenosis severity, eccentricity led to further elevations in turbulence intensity, increasing TI by 0.05-0.10 m/s over the counterpart concentric plaque. The presence of ulceration (in a 50% eccentric plaque) produced a larger portion of moderate turbulence intensity (~0.10 m/s) compared to the non-ulcerated model, more proximal to the bifurcation apex in the post-stenotic recirculation zone. The effect of plaque eccentricity and ulceration in enhancing the downstream turbulence has potential clinical implications for a more sensitive assessment of stroke risk beyond stenosis severity alone.

Transitional flow analysis in the carotid artery bifurcation by proper orthogonal decomposition and particle image velocimetry.

Blood flow instabilities in the carotid artery bifurcation have been highly correlated to clot formation and mobilization resulting in ischemic stroke. In this work, PIV-measured flow velocities in normal and stenosed carotid artery bifurcation models were analyzed by means of proper orthogonal decomposition (POD). Through POD analysis, transition to more complex flow was visualized and quantified for increasing stenosis severity. While no evidence of transitional flow was seen in the normal model, the 50%-stenosed model started to show characteristics of transitional flow, which became highly evident in the 70% model, with greatest manifestation during the systolic phase of the cardiac cycle. By means of a model comparison, we demonstrate two quantitative measures of the flow complexity through the power-law decay slope of the energy spectrum and the global entropy. The more complex flow in the 70%-stenosed model showed a flatter slope of energy decay (-0.91 compared to -1.34 for 50% stenosis) and higher entropy values (0.26 compared to 0.17). Finally, the minimum temporal resolution required for POD analysis of carotid artery flow was found to be 100 Hz when determined through a more typical energy-mode convergence test, as compared to 400 Hz based on global entropy values.

Evaluation of distal turbulence intensity for the detection of both plaque ulceration and stenosis grade in the carotid bifurcation using clinical Doppler ultrasound.

OBJECTIVES: To determine the interrelationship of stenosis grade and ulceration with distal turbulence intensity (TI) in the carotid bifurcation measured using conventional clinical Doppler ultrasound (DUS) in vitro, in order to establish the feasibility of TI as a diagnostic parameter for plaque ulceration. METHODS: DUS TI was evaluated in a matched set of ulcerated and smooth-walled carotid bifurcation phantoms with various stenosis severities (30, 50, 60 and 70 %), where the ulcerated models incorporated a type 3 ulceration. RESULTS: Post-stenotic TI was significantly elevated owing to ulceration in the mild and moderate stenoses (P < 0.001). TI increased with stenosis severity in both the ulcerated and non-ulcerated series, with a statistically significant effect of increasing stenosis severity (P < 0.001). Whereas TI in the mild and non-ulcerated moderate stenoses was less than 20.4 ± 1.3 cm s(-1), TI in the ulcerated moderate and severe models was higher than 25.6 ± 1.3 cm s(-1), indicating a potential diagnostic threshold. CONCLUSION: We report a two-curve relationship of stenosis grade and ulceration to distal TI measured using clinical DUS in vitro. Clinical DUS measurement of distal TI may be a diagnostic approach to detecting ulceration in the mild and moderately stenosed carotid artery. KEY POINTS: • Patients with carotid artery plaque ulcerations are at higher risk of stroke. • Clinical Doppler ultrasound is routinely used to detect carotid artery stenosis. • Doppler ultrasound turbulence intensity can detect ulceration in realistic flow models. • Turbulence intensity also increases with stenosis severity independent of ulceration. • Doppler ultrasound should help in assessing both stenosis severity and ulceration.

3-D flow characterization and shear stress in a stenosed carotid artery bifurcation model using stereoscopic PIV technique.

The carotid artery bifurcation is a common site of atherosclerosis which is a major leading cause of ischemic stroke. The impact of stenosis in the atherosclerotic carotid artery is to disturb the flow pattern and produce regions with high shear rate, turbulence, and recirculation, which are key hemodynamic factors associated with plaque rupture, clot formation, and embolism. In order to characterize the disturbed flow in the stenosed carotid artery, stereoscopic PIV measurements were performed in a transparent model with 50% stenosis under pulsatile flow conditions. Simulated ECG gating of the flowrate waveform provides external triggering required for volumetric reconstruction of the complex flow patterns. Based on the three-component velocity data in the lumen region, volumetric shear-stress patterns were derived.

Flow patterns in carotid bifurcation models using pulsed Doppler ultrasound: effect of concentric vs. eccentric stenosis on turbulence and recirculation.

Hemodynamics play a significant role in stroke risk, where thrombus formation may be accelerated in regions of slow or recirculating flow, high shear and increased turbulence. An in vitro investigation was performed with pulsed Doppler ultrasound (DUS) using the complete spectral data to investigate the three-dimensional (3-D) distribution of advanced parameters that may have potential for making a more specific in vivo diagnosis of carotid disease and stroke risk. The effect of stenosis symmetry and the potential of DUS spectral parameters for visualizing regions of recirculation or turbulence were explored. DUS was used to map pulsatile flow in four model geometries representing two different plaque symmetries (eccentricity) and two stenosis severities (mild, severe). Qualitative comparisons were made with flow patterns visualized using digital particle imaging. Color-encoded maps of DUS spectral parameters (mean velocity, spectral-broadening index and turbulence intensity) clearly distinguished regions of slow or recirculating flow and disturbed or turbulent flow. Distinctly different flow patterns resulted from stenoses of equal severity but different eccentricity. Noticeable differences were seen in both the size and location of recirculation zones and in the paths of high-velocity jets. Highly elevated levels of turbulence intensity were seen distal to severe stenosis. Results demonstrated the importance of plaque shape, which is typically not considered in standard diagnosis, in addition to stenosis severity.

Deriving a blood-mimicking fluid for particle image velocimetry in Sylgard-184 vascular models.

A new blood-mimicking fluid (BMF) has been developed for particle image velocimetry (PIV), which enables flow studies in vascular models (phantoms). A major difficulty in PIV that affects measurement accuracy is the refraction and distortion of light passing through the interface between the model and the fluid, due to the difference in refractive index (n) between the two materials. The problem can be eliminated by using a fluid with a refractive index matching that of the model. Such fluids are not commonly available, especially for vascular research where the fluid should also have a viscosity similar to human blood. In this work, a blood-mimicking fluid, composed of water (47.38% by weight), glycerol (36.94% by weight) and sodium iodide salt (15.68% by weight), was developed for compatibility with our silicone (Sylgard 184; n = 1.414) phantoms. The fluid exhibits a dynamic viscosity of 4.31+/-0.03 cP which lies within the range of human blood viscosity (4.4+/-0.6 cP). Both refractive index and viscosity were attained at 22.2+/-0.2 degrees C, which is a feasible room temperature, thus eliminating the need for a temperature-control system. The fluid will be used to study hemodynamics in vascular flow models fabricated from Sylgard 184.

Clinical Doppler ultrasound for the assessment of plaque ulceration in the stenosed carotid bifurcation by detection of distal turbulence intensity: a matched model study.

The assessment of flow disturbances due to carotid plaque ulceration may provide added diagnostic information to Doppler ultrasound (DUS) of the carotid stenosis, and indicate whether the associated hemodynamics are a potential thromboembolic source. We evaluated the effect of ulceration in a moderately stenosed carotid bifurcation on distal turbulence intensity (TI) measured using clinical DUS in matched anthropomorphic models. Several physiologically relevant ulcer geometries (hemispherical, mushroom-shaped, and ellipsoidal pointing distally and proximally) and sizes (2-mm, 3-mm and 4-mm diameter hemispheres) were investigated. An offline analysis was performed to determine several velocity-based parameters from ensemble-averaged spectral data, including TI. Significant elevations in TI were observed in the post-stenotic flow field of the stenosed carotid bifurcation by the inclusion of ulceration (P < 0.001) in a region two common carotid artery diameters distal to the site of ulceration during the systolic peak and the diastolic phase of the cardiac cycle. Both the size and shape of ulceration had a significant effect on TI in the distal region (P < 0.001). Due to the use of a clinical system, this method provides the means to evaluate for plaque ulcerations in patients with carotid atherosclerosis using DUS.

In vitro Doppler ultrasound investigation of turbulence intensity in pulsatile flow with simulated cardiac variability.

An in vitro investigation of turbulence intensity (TI) associated with a severe carotid stenosis in the presence of physiological cardiac variability is described. The objective of this investigation was to determine if fluctuations due to turbulence could be quantified with conventional Doppler ultrasound (DUS) in the presence of normal physiological cycle-to-cycle cardiac variability. An anthropomorphic model of a 70% stenosed carotid bifurcation was used in combination with a programmable flow pump to generate pulsatile flow with a mean flow rate of 6 mL/s. Utilizing the pump, we studied normal, nonrepetitive cycle-to-cycle cardiac variability (+/-3.9%) in flow, as well as waveform shapes with standard deviations equal to 0, 2 and 3 times the normal variation. Eighty cardiac cycles of Doppler data were acquired at two regions within the model, representing either laminar or turbulent flow; each measurement was repeated six times. Turbulence intensity values were found to be 11 times higher (p < 0.001), on average, in the turbulent region than in the laminar region, with a mean difference of 24 cm/s. Twenty cardiac cycles were required for confidence in TI values. In conclusion, these results indicate that it is possible to quantify TI in vitro, even in the presence of normal and exaggerated cycle-to-cycle cardiac variability.

Use of an ultrasound blood-mimicking fluid for Doppler investigations of turbulence in vitro.

Turbulence is an important factor in the assessment of stenotic disease and a possible causative mechanism for thromboembolism. Previous Doppler studies of turbulence have typically used whole-blood preparations or suspensions of erythrocytes. Recently, a water-glycerol based blood-mimicking fluid (BMF) has been developed for use in Doppler ultrasound studies. This fluid has desirable ultrasound properties but it has not previously been described during in vitro investigations of turbulence intensity. We report on investigations of grid-generated and constrained-jet turbulence in an in vitro test system. The BMF was found to generate significant levels of turbulence during steady flow at physiological flow rates, producing turbulent patterns in the distal region that were consistent with previous studies. Turbulence intensity increased significantly with flow rate (p < 0.005) for both the constrained jet and the constrained grid. Based on our observations, we conclude that a water-glycerol based BMF provides a suitable working fluid during in vitro investigations of turbulence using Doppler ultrasound.

Doppler ultrasound compatible plastic material for use in rigid flow models.

A technique for the rapid but accurate fabrication of multiple flow phantoms with variations in vascular geometry would be desirable in the investigation of carotid atherosclerosis. This study demonstrates the feasibility and efficacy of implementing numerically controlled direct-machining of vascular geometries into Doppler ultrasound (DUS)-compatible plastic for the easy fabrication of DUS flow phantoms. Candidate plastics were tested for longitudinal speed of sound (SoS) and acoustic attenuation at the diagnostic frequency of 5 MHz. Teflon was found to have the most appropriate SoS (1376 +/- 40 m s(-1) compared with 1540 m s(-1) in soft tissue) and thus was selected to construct a carotid bifurcation flow model with moderate eccentric stenosis. The vessel geometry was machined directly into Teflon using a numerically controlled milling technique. Geometric accuracy of the phantom lumen was verified using nondestructive micro-computed tomography. Although Teflon displayed a higher attenuation coefficient than other tested materials, Doppler data acquired in the Teflon flow model indicated that sufficient signal power was delivered throughout the depth of the vessel and provided comparable velocity profiles to that obtained in the tissue-mimicking phantom. Our results indicate that Teflon provides the best combination of machinability and DUS compatibility, making it an appropriate choice for the fabrication of rigid DUS flow models using a direct-machining method.

Acoustic speed and attenuation coefficient in sheep aorta measured at 5-9 MHz.

B-mode ultrasound (US) images from blood vessels in vivo differ significantly from vascular flow phantom images. Phantoms with acoustic properties more closely matched to those of in vivo arteries may give better images. A method was developed for measuring the speed and attenuation coefficient of US over the range 5 to 9 MHz in samples of sheep aorta using a pulse-echo technique. The times-of-flight method was used with envelope functions to identify the reference points. The method was tested with samples of tissue-mimicking material of known acoustic properties. The tissue samples were stored in Krebs physiologic buffer solution and measured over a range of temperatures. At 37 degrees C, the acoustic speed and attenuation coefficient as a function of frequency in MHz were 1600 +/- 50 ms(-1) and 1.5 +/- 4f(0.94 +/- 1.3) dB cm(-1), respectively.

Real-time numerical simulation of Doppler ultrasound in the presence of nonaxial flow.

Numerical simulations of Doppler ultrasound (DUS) relying on computational fluid dynamics (CFD) models of nonaxial flow have traditionally employed detailed (but computationally intensive) models of the DUS physics, or have sacrificed much of the physics in the interest of computational or conceptual simplicity. In this paper, we present a compromise between these extremes, with the objective of simulating the essential characteristics of DUS spectrograms in a real-time manner. Specifically, a precomputed pulsatile CFD velocity field is interrogated at some number, N, of discrete points distributed spatially within a sample volume of prescribed geometry and power distribution and temporally within a prescribed sampling window. Intrinsic spectral broadening is accounted for by convolving each of the point velocities with a semiempirical broadening function. Real-time performance is facilitated through the use of an efficient algorithm for interpolating the unstructured CFD data. A spherical sample volume with Gaussian power distribution, N = 1000 sampling points, and quadratic broadening function are shown to be adequate for simulating, at frame rates of 86 Hz on a 1.5 GHz desktop workstation, realistic-looking spectrograms at representative locations within a stenosed carotid bifurcation model. Via qualitative comparisons with matched in vitro data, these simulated spectrograms are shown to mimic the distinctive spectral envelopes, broadening and power characteristics associated with common carotid, stenotic jet and poststenotic recirculating flows. We conclude that the complex interaction between Doppler ultrasound and complicated clinically relevant blood flow dynamics can be simulated in real time via this relatively straightforward semiempirical approach.

Origins of the edge shadowing artefact in medical ultrasound imaging.

Ultrasound (US) B-mode images distal to smooth, rounded cavities, such as cysts, containing a fluid with a speed of sound mismatch to the surrounding tissue, often exhibit a "refractile" edge shadowing artefact. This usually appears as narrow, hypoechoic, shadow lines extending a significant distance distal to the lateral edges of the fluid cavity and parallel to the US beam. The true reasons for this artefact are likely to be complex and to vary from case to case, with many different explanations found in the literature. However, we present a simplified theoretical model for the phenomenon based on a pulsed, finite-beam solution of US scattering from circular fluid-filled cylinders that suggests that "edge" shadows can occur distal not only to edges but also to points where the incident beam intersects the cavity near to the critical angle. Both mechanisms support the view that edge shadows can arise from the combination of unusually high wavefront spreading and the speckle-generating nature of the surrounding tissue. In vitro data from a tissue-mimicking phantom confirms that the edge shadow structure depends on the sign of the speed of sound contrast between the cylinder fluid and the surrounding medium.

A thin-walled carotid vessel phantom for Doppler ultrasound flow studies.

A technique is discussed for producing a robust ultrasound (US)-compatible flow phantom that consists of a thin-walled silicone-elastomer vessel with a lumen of arbitrary geometry, embedded in an agar-based tissue-mimicking material (TMM). The TMM has an acoustic attenuation of 0.56 dB cm(-1) MHz(-1) at 5 MHz, with nearly linear frequency-dependence and acoustic velocity of 1539 +/- 4 m s(-1). The vessel-mimicking material (VMM) has an acoustic attenuation of 3.5 dB cm(-1) MHz(-1) with linear frequency-dependence and an acoustic velocity of 1020 +/- 20 m s(-1). Scattering particles, which are added to the VMM to increase echogenicity and add speckle texture, lead to higher attenuation, depending on particle concentration and frequency. The VMM is stable over time, with a Young's elastic modulus of 1.3 to 1.7 MPa for strains of up to 10%, which mimics human arteries under typical physiological conditions. The phantom is sealed to prevent TMM exposure to air or water, to avoid changes to the acoustic velocity.