•Aneurysmal hemodynamics are helpful in treatment planning and prediction.•In vitro models can validate patient-specific computational simulations of aneurysms.•Dimensionally-accurate, ...optically-transparent phantoms can be done by 3D printing.•Such models may inform optimal patient-specific neurointerventional strategies.
Perianeurysmal hemodynamics play a vital role in the initiation, growth and rupture of intracranial aneurysms. In vitro investigations of aneurysmal hemodynamics are helpful to visualize and measure blood flow, and aiding surgical planning approaches. Improving in vitro model creation can improve the feasibility and accuracy of hemodynamic investigations and surgical planning, improving clinical value. In this study, in vitro models were created from three-dimensional rotational angiography (3DRA) of six patients harboring intracranial aneurysms using a multi-step process involving 3D printing, index of refraction matching and silicone casting that renders the models transparent for flow visualization. Each model was treated with the same commercially-available, patient-specific, endovascular devices (coils and/or stents). All models were scanned by synchrotron X-ray microtomography to obtain high-resolution imaging of the vessel lumen, aneurysmal sac and endovascular devices. Dimensional accuracy was compared by quantifying the differences between the microtomographic reconstructions of the fabricated phantoms and the original 3DRA obtained during patient treatment. True-scale in vitro flow phantoms were successfully created for all six patients. Optical transparency was verified by using an index of refraction matched working fluid that replicated the mechanical behavior of blood. Synchrotron imaging of vessel lumen, aneurysmal sac and endovascular devices was successfully obtained, and dimensional errors were found to be O(100 μm). The creation of dimensionally-accurate, optically-transparent flow phantoms of patient-specific intracranial aneurysms is feasible using 3D printing technology. Such models may enable in vitro investigations of aneurysmal hemodynamics to aid in treatment planning and outcome prediction to devise optimal patient-specific neurointerventional strategies.
We developed a duplex ultrasound simulator for training and assessment of scanning skills. We used the simulator to test examiner performance in the measurement of flow velocities in dialysis access ...fistulas. Test cases were created from 3-D ultrasound scans of two dialysis access fistulas by reconstructing 3-D blood vessel models and simulating blood flow velocity fields within the lumens. The simulator displays a 2-D B-mode or color Doppler image corresponding to transducer position on a mannequin; a spectral waveform is generated according to Doppler sample volume location and system settings. Examiner performance was assessed by comparing the measured peak systolic velocity (PSV) with the true PSV provided by the computational flow model. The PSV measured by four expert examiners deviated from the true value by 7.8 ± 6.1%. The results indicate the ability of the simulator to objectively assess an examiner's measurement accuracy in complex vascular targets.
Objective:
We developed a duplex ultrasound simulator and used it to assess accuracy of volume flow measurements in dialysis access fistula (DAF) models.
Methods:
The simulator consists of a ...mannequin, computer, and mock transducer. Each case is built from a patient’s B-mode images that are used to create a 3-dimensional surface model of the DAF. Computational fluid dynamics is used to determine blood flow velocities based on model vessel geometry. The simulator displays real-time B-mode and color-flow images, and Doppler spectral waveforms are generated according to user-defined settings. Accuracy was assessed by scanning each case and measuring volume flow in the inflow artery and outflow vein for comparison with true volume flow values.
Results:
Four examiners made 96 volume flow measurements on four DAF models. Measured volume flow deviated from the true value by 35 ± 36%. Mean absolute deviation from true volume flow was lower for arteries than veins (22 ± 19%, N = 48 vs. 58 ± 33%, N = 48, p < 0.0001). This finding is attributed to eccentricity of outflow veins which resulted in underestimating true cross-sectional area. Regression analysis indicated that error in measuring cross-sectional area was a predictor of error in volume flow measurement (β = 0.948, p < 0.001). Volume flow error was reduced from 35 ± 36% to 9 ± 8% (p < 0.000001) by calculating vessel area as an ellipse.
Conclusions:
Duplex volume flow measurements are based on a circular vessel shape. DAF inflow arteries are circular, but outflow veins can be elliptical. Simulation-based analysis showed that error in measuring volume flow is mainly due to assumption of a circular vessel.
Objective:
Duplex ultrasound scanning with B-mode imaging and both color Doppler and Doppler spectral waveforms is relied upon for diagnosis of vascular pathology and selection of patients for ...further evaluation and treatment. In most duplex ultrasound applications, classification of disease severity is based primarily on alterations in blood flow velocities, particularly the peak systolic velocity (PSV) obtained from Doppler spectral waveforms. We developed a duplex ultrasound simulator for training and assessment of scanning skills.
Methods:
Duplex ultrasound cases were prepared from 2-dimensional (2D) images of normal and stenotic carotid arteries by reconstructing the common carotid, internal carotid, and external carotid arteries in 3 dimensions and computationally simulating blood flow velocity fields within the lumen. The simulator displays a 2D B-mode image corresponding to transducer position on a mannequin, overlaid by color coding of velocity data. A spectral waveform is generated according to examiner-defined settings (depth and size of the Doppler sample volume, beam steering, Doppler beam angle, and pulse repetition frequency or scale). The accuracy of the simulator was assessed by comparing the PSV measured from the spectral waveforms with the true PSV which was derived from the computational flow model based on the size and location of the sample volume within the artery.
Results:
Three expert examiners made a total of 36 carotid artery PSV measurements based on the simulated cases. The PSV measured by the examiners deviated from true PSV by 8% ± 5% (N = 36). The deviation in PSV did not differ significantly between artery segments, normal and stenotic arteries, or examiners.
Conclusion:
To our knowledge, this is the first simulation of duplex ultrasound that can create and display real-time color Doppler images and Doppler spectral waveforms. The results demonstrate that an examiner can measure PSV from the spectral waveforms using the settings on the simulator with a mean absolute error in the velocity measurement of less than 10%. With the addition of cases with a range of pathologies, this duplex ultrasound simulator will be a useful tool for training health-care providers in vascular ultrasound applications and for assessing their skills in an objective and quantitative manner.
Many cancers harbor oncogenic mutations of KRAS. Effectors mediating cancer progression, invasion, and metastasis in KRAS-mutated cancers are only incompletely understood. Here we identify cancer ...cell-expressed murine TRAIL-R, whose main function ascribed so far has been the induction of apoptosis as a crucial mediator of KRAS-driven cancer progression, invasion, and metastasis and in vivo Rac-1 activation. Cancer cell-restricted genetic ablation of murine TRAIL-R in autochthonous KRAS-driven models of non-small-cell lung cancer (NSCLC) and pancreatic ductal adenocarcinoma (PDAC) reduces tumor growth, blunts metastasis, and prolongs survival by inhibiting cancer cell-autonomous migration, proliferation, and invasion. Consistent with this, high TRAIL-R2 expression correlates with invasion of human PDAC into lymph vessels and with shortened metastasis-free survival of KRAS-mutated colorectal cancer patients.
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•mTRAIL-R promotes KRAS-driven lung and pancreatic cancer growth and metastasis•Human TRAIL-R2 promotes tumor growth, migration, invasion, and metastasis•Endogenous mTRAIL-R constitutively activates Rac1 in vivo in tumors•TRAIL-R2 expression positively correlates with the onset of metastasis in patients
von Karstedt et al. show that mouse TRAIL-R and human TRAIL-R2, but not TRAIL-R1, are important for the progression, invasion, and metastasis of KRAS-mutant tumors through the regulation of Rac-1.
The Adenomatous Polyposis Coli (APC) gene is mutated in the majority of colorectal cancers (CRCs). Loss of APC leads to constitutively active WNT signaling, hyperproliferation, and tumorigenesis. ...Identification of pathways that facilitate tumorigenesis after APC loss is important for therapeutic development. Here, we show that RAC1 is a critical mediator of tumorigenesis after APC loss. We find that RAC1 is required for expansion of the LGR5 intestinal stem cell (ISC) signature, progenitor hyperproliferation, and transformation. Mechanistically, RAC1-driven ROS and NF-κB signaling mediate these processes. Together, these data highlight that ROS production and NF-κB activation triggered by RAC1 are critical events in CRC initiation.
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•RAC1 is required for tumorigenesis after APC loss•LGR5 ISC transformation requires RAC1•RAC1-driven ROS production and NF-κB activation are required for CRC initiation
This study demonstrates that RAC1-mediated ROS production and NF-KB activation is required for LGR5 stem cell expansion and colorectal cancer initiation after loss of the APC gene.
Tumors and associated stroma manifest mechanical properties that promote cancer. Mechanosensation of tissue stiffness activates the Rho/ROCK pathway to increase actomyosin-mediated cellular tension ...to re-establish force equilibrium. To determine how actomyosin tension affects tissue homeostasis and tumor development, we expressed conditionally active ROCK2 in mouse skin. ROCK activation elevated tissue stiffness via increased collagen. β-catenin, a key element of mechanotranscription pathways, was stabilized by ROCK activation leading to nuclear accumulation, transcriptional activation, and consequent hyperproliferation and skin thickening. Inhibiting actomyosin contractility by blocking LIMK or myosin ATPase attenuated these responses, as did FAK inhibition. Tumor number, growth, and progression were increased by ROCK activation, while ROCK blockade was inhibitory, implicating actomyosin-mediated cellular tension and consequent collagen deposition as significant tumor promoters.
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► Collagen deposition and tissue stiffness induced by cellular actomyosin contraction ► ROCK-induced actomyosin contractility stimulates β-catenin transcriptional activity ► ROCK-induced proliferation blocked by β-catenin deletion or actomyosin inhibition ► Actomyosin contraction-driven tissue stiffness fosters tumor growth and progression
FRET biosensors have proven very useful tools for studying the activation of specific signalling pathways in living cells. Most biosensors designed to date have been predicated on fluorescent protein ...pairs that were identified by, and for use in, intensity based measurements, however fluorescence lifetime provides a more reliable measurement of FRET. Both the technology and fluorescent proteins available for FRET have moved on dramatically in the last decade. Lifetime imaging systems have become increasingly accessible and user-friendly, and there is an entire field of biology dedicated to refining and adapting different characteristics of existing and novel fluorescent proteins. This growing pool of fluorescent proteins includes the long-lifetime green and cyan fluorescent proteins Clover and mTurquoise2, the red variant mRuby2, and the dark acceptor sREACh. Here, we have tested these donors and acceptors in appropriate combinations against the standard or recommended norms (EGFP and mTFP as donors, mCherry and either Ypet or Venus as acceptors) to determine if they could provide more reliable, reproducible and quantifiable FLIM-FRET data to improve on the dynamic range compared to other donors and breadth of application of biosensor technologies. These tests were performed for comparison on both a wide-field, frequency domain system and a multiphoton, TCSPC time domain FLIM system. Clover proved to be an excellent donor with extended dynamic range in combination with mCherry on both platforms, while mRuby2 showed a high degree of variability and poor FRET efficiencies in all cases. mTFP-Venus was the most consistent cyan-yellow pair between the two FLIM methodologies, but mTurquoise2 has better dynamic range and transfers energy consistently over time to the dark acceptor sRCh. Combination of mTFP-sRCh with Clover-mCherry would allow the simultaneous use of two FLIM-FRET biosensors within one sample by eliminating the crosstalk between the yellow acceptor and green donor emissions.
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