Wasp controls oriented migration of endothelial cells to achieve functional vascular patterning

Endothelial cell migration and proliferation are essential for the establishment of a hierarchical organization of blood vessels and optimal distribution of blood. However, how these cellular processes are quantitatively coordinated to drive vascular network morphogenesis remains unknown. Here, using the zebrafish vasculature as a model system, we demonstrate that the balanced distribution of endothelial cells as well as the resulting regularity of vessel caliber, is a result of cell migration from veins towards arteries and cell proliferation in veins. We identify the Wiskott-Aldrich Syndrome protein (WASp) as an important molecular regulator of this process and show that loss of coordinated migration from veins to arteries upon wasb depletion results in aberrant vessel morphology and the formation of persistent arteriovenous shunts. We demonstrate that WASp achieves its function through the coordination of junctional actin assembly and PECAM1 recruitment and provide evidence that this is conserved in human. Overall, we demonstrate that functional vascular patterning in the zebrafish trunk is established through differential cell migration regulated by junctional actin, and that interruption of differential migration may represent a pathomechanism in vascular malformations.

Quantitative Three-dimensional High Definition Microvessel Imaging for characterization breast masses v1

Breast cancer, a major cause of morbidity and mortality in women, is highly dependent on angiogenesis for its growth and distant metastasis. Furthermore, new blood vessels in malignant tumors are structurally abnormal and different from benign. Imaging techniques that provide information regarding tumor microvasculature structures could aid cancer detection. This protocol is to advance the development and evaluate the performance of a quantitative 3D microvessel imaging technique to provide quantitative information of morphological features of tumor microvessel as new biomarker for differentiation of malignant and benign breast masses. Our team has developed a new 2D contrast-free ultrasound (US) microvessel imaging technique based on novel processing procedures for revealing and enhancing submillimeter size tumor microvessels and complemented this technique with novel quantification tools to quantify vessel morphology. The results of quantitative 2D-HDMI for differentiation of malignant and benign breast masses are promising; however, the 2D imaging method overlooks some important 3-dimensional (3D) morphological features, such as the connectivity of the blood microvessels, leading to under- or overestimation of these parameters. Here, we propose to advance the development of a new quantitative 3D High-Definition Microvascular Imaging (q3D-HDMI) to provide complementary diagnostic information to the conventional US. The new technique, q3D-HDMI, uses high frame rate ultrasound imaging and is based on novel processing and quantification procedures for 3D imaging to reveal and quantify microvessel morphology in tumor volume. In this protocol we evaluate the diagnostic performance of q3D-HDMI for characterization of breast masses in a population of pre-biopsy patients; correlate the results with pathology as the gold standard.We also compare q3D-HDMI and q2D-HDMI in differentiating malignant from benign breast lesions

The Effects of 5-Fluorouracil/Leucovorin Chemotherapy on Cognitive Function in Male Mice

5-Fluorouracil (5-Fu) and leucovorin (LV) are often given in combination to treat colorectal cancer. 5-Fu/LV prevents cell proliferation by inhibiting thymidylate synthase, which catalyzes the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate. While 5-Fu has been shown to cause cognitive impairment, the synergistic effect of 5-Fu with LV has not been fully explored. The present investigation was designed to assess how the combination of 5-Fu and LV affect cognition in a murine model. Six-month-old male mice were used in this study; 15 mice received saline injections and 15 mice received 5-Fu/LV injections. One month after treatment, the elevated plus maze, Y-maze, and Morris water maze behavioral tasks were performed. Brains were then extracted, cryosectioned, and stained for CD68 to assay microglial activation and with tomato lectin to assay the vasculature. All animals were able to locate the visible and hidden platform locations in the water maze. However, a significant impairment in spatial memory retention was observed in the probe trial after the first day of hidden-platform training (first probe trial) in animals that received 5-Fu/LV, but these animals showed spatial memory retention by day 5. There were no significant increases in inflammation as measured by CD68, but 5-Fu/LV treatment did modulate blood vessel morphology. Tandem mass tag proteomics analysis identified 6,049 proteins, 7 of which were differentially expressed with a p-value of <0.05 and a fold change of >1.5. The present data demonstrate that 5-Fu/LV increases anxiety and significantly impairs spatial memory retention.

Impact of vascular morphology and plaque characteristics on computed tomography derived fractional flow reserve in early stage coronary artery disease

Background FFRCT gradually decreases from the proximal to the distal part of a vessel and reach the pathological threshold for significant ischemia even in the absence of obstructive coronary artery disease (CAD). The exact mechanisms of such gradual FFRCT decline remain unknown. Purpose The aims of this study are (1) to clarify the mechanisms of the gradual decline of computed tomography (CT) derived fractional flow reserve (FFRCT); and (2) to identify the predictive factors of an FFRCT decline below the pathological value of 0.80 in no apparent CAD vessels. Methods A total of 1058 outpatients with suspected CAD and who underwent CT angiography (CTA) with FFRCT analysis between January 2017 and December 2019 were evaluated. Among them, 150 consecutive patients who had both a CTA coupled to an FFRCT analysis and an invasive angiogram showing <25% coronary stenosis were included for analysis. Vessels were divided into two groups according to FFRCT at the distal vessel: FFRCT >0.80 (n=317) and FFRCT ≤0.80 (n=114). ΔFFRCT was defined as the magnitude of the change in FFRCT from the proximal to the distal vessel. Plaque characterization and vessel morphology measurements were performed semi-automatically. Vessel constituents were characterized based on Hounsfield units (HU) into lumen volume (<−50 HU), non-calcified plaque (NCP) (−50–150 HU), and calcified plaque (>150 HU). Results FFRCT decreased continuously from the proximal to distal across the three major vessels in both FFRCT>0.80 and FFRCT ≤0.80 groups (Figure 1). Compared to FFRCT>0.80 group, NCP volume was significantly higher in all three major vessels in FFRCT ≤0.80 group (210.2±83.6 mm3 vs. 140.9±139.3 mm3 for the RCA, p=0.01; 177.5±150.2 mm3 vs. 133.2±112.2 mm3 for the LAD, p=0.04; 127.6±91.5 mm3 vs. 58.7±57.7 mm3 for the LCX, p<0.01). Next, we investigated the vessel parameters that correlated with ΔFFRCT. ΔFFRCT was correlated with lumen volume in FFRCT>0.80 group (r=−0.24, p<0.0001), whereas ΔFFRCT was correlated with NCP volume in FFRCT ≤0.80 group (r=0.42, p<0.001) (Figure 2). An NCP volume above 44.8 mm3 was the strongest predictor of distal FFRCT of ≤0.80 (area under the curve 0.69, p<0.0001, sensitivity 95%, specificity 39%). Conclusions FFRCT is affected by vascular morphology and plaque characteristics even in the early stage of coronary artery disease. Our study highlights that subclinical coronary artery disease strongly influences FFRCT by effects unrelated to coronary stenosis. The presence of NCP is a major predictor of the gradual decrease of FFRCT toward pathological values. Anatomical findings as vessel morphology and plaque characteristics should be taken into consideration when interpreting numerical values of FFRCT to avoid unnecessary referrals for invasive coronary angiography or percutaneous coronary intervention. FUNDunding Acknowledgement Type of funding sources: None. Figure 1 Figure 2

No direct involvement of Phactr-1 in non-atherosclerotic arteriopathies: results from 3 different Phactr-1 transgenic knockout mice

Introduction Genome-wide association studies have revealed robust associations of common genetic polymorphisms in the Phactr1 locus (6p24) (encoding Phosphatase and Actin Regulator 1) with cervico-cerebral artery dissection (CCeAD), spontaneous coronary artery dissection (SCAD) and fibromuscular dysplasia (FMD). These common elements of genetic predisposition provide a proof of concept for important shared mechanisms at the molecular level between CCeAD, SCAD and FMD vascular diseases. Objective Deciphering genetic, molecular and physiological importance of PHACTR1. Method Using various tissue-specific Cre-driver mouse lines, Phactr1 was deleted either in endothelial cells (EC) using 2 tissue-specific Cre-driver (one of them was embryonic) and Smooth Muscle Cells (SMC) with a third tissue-specific Cre-driver to assess its role in the pathogenesis of CCeAD, SCAD or FMD. Results To test the efficacy of the Phactr1 deletion after cre-induction, we confirmed firstly, a decrease in Phactr1 transcription and Phactr1 expression in EC and SMC isolated from Phactr1iPDGFB and Phactr1iSMA mice. Irrespective to the tissue or the duration of the deletion, mice did not spontaneously display pathological phenotype or vascular impairment: mouse survival, growth, blood pressure, large vessel morphology or actin organization were not different in KO mice than their comparatives littermates. Challenging vascular function and repair either by angiotensin-II-induced hypertension or limb ischemia did not lead to vascular morphology or function impairment in Phactr1-deleted mice. Similarly, there was no more consequences of Phactr1 deletion during embryogenesis in ECs. Conclusion Loss of function of PHACTR-1 in the cell types mostly involved in vascular physiology does not appear to induce a pathological vascular phenotype. FUNDunding Acknowledgement Type of funding sources: None.

Effects of left ventricular mass index on computed tomography derived fractional flow reserve in significant obstructive coronary artery disease

Background In significant obstructive coronary artery disease (SOCAD), a mismatched assessment of the severity of coronary artery stenosis may occur between invasive coronary angiography and computed tomography (CT) derived fractional flow reserve (FFRCT). The exact mechanisms of unexpected underestimation of FFRCT remain unknown. Purpose The aims of this study are (1) to clarify the mechanisms of underestimation on FFRCT; and (2) to identify the predictive factors of FFRCT underestimation above the value of 0.80 in SOCAD vessels. Methods A total of 1160 outpatients who underwent CT angiography (CTA) with FFRCT analysis for suspected coronary artery disease (CAD) between January 2017 and June 2020 were evaluated. Among them, 141 consecutive patients who had both CTA coupled to FFRCT analysis and invasive angiogram showing >75% coronary stenosis were included for analysis. Vessels were divided into two groups according to FFRCT at the distal vessel: FFRCT >0.80 (n=12) and FFRCT ≤0.80 (n=153). Vessel-related parameters, including vessel morphology (vessel length and lumen volume) and plaque components (non-calcified plaque volume and calcified plaque volume) and left ventricular (LV) myocardial-related parameters, including LV wall thickness at each site of the myocardium, and LV mass were evaluated semi-automatically. Results Vessel morphology and plaque components did not differ between FFRCT >0.80 and ≤0.80, whereas LV wall thickness (average; 10.7±2.7 vs. 8.4±1.6 mm, and maximal; 13.5±3.0 vs. 10.6±1.8 mm, all p value <0.001), LV mass (136.4±38.4 vs. 98.8±26.8 g, p<0.001), and LV mass index (73.8±22.6 vs. 51.8±12.2 g/m2, p<0.001) were significantly higher in FFRCT >0.80. Next, we investigated the parameters that correlated with FFRCT. Of all, vessel morphology and plaque components were not related to FFRCT, whereas maximal LV wall thickness, r=0.24, p=0.01; LV mass, r=0.19. p=0.04; and LV mass index, r=0.30, p=0.001) correlated with FFRCT. In the vessels showing FFRCT >0.80, only LV mass (r=0.84, p=0.005) and LV mass index (r=0.67, p=0.047) correlated with FFRCT. (Figure 1). LV mass index was the strongest predictor of a distal FFRCT of >0.80 with the area under curve (AUC) 0.81, 95% CI 0.62 – 1.00, P<0.0001 and an optimal cut-off value of 66.5 g/m2 sensitivity 77.8%, specificity 89.6% (Figure 2). Conclusions FFRCT is affected not by vessel-related parameters but LV myocardial-related parameters in SOCAD. The presence of an excessive LV mass is a major predictor of underestimation of FFRCT in SOCAD vessels. LV myocardial-related parameters should be considered when interpreting numerical values of FFRCT to avoid the possibility of overlooked SOCAD. FUNDunding Acknowledgement Type of funding sources: None. Figure 1 Figure 2

Genetic Regulation of Vessel Morphology in Populus

During secondary growth, forest trees can modify the anatomy of the wood produced by the vascular cambium in response to environmental conditions. Notably, the trees of the model angiosperm genus, Populus, reduce the risk of cavitation and hydraulic failure under water stress by producing water-conducting vessel elements with narrow lumens, which are more numerous and more interconnected with each other. Here, we determined the genetic architecture of vessel traits affecting hydraulic physiology and resilience to water stress. Vessel traits were measured for clonally replicated genotypes of a unique Populus deltoides x nigra population carrying genomically defined insertions and deletions that create gene dosage variation. We found significant phenotypic variation for all traits measured (mean vessel diameter, height-corrected mean vessel diameter, vessel frequency, height-corrected vessel frequency, vessel grouping index, and mean vessel circularity), and that all traits were under genetic control and showed moderate heritability values, ranging from 0.32 to 0.53. Whole-genome scans of correlations between gene dosage and phenotypic traits identified quantitative trait loci for tree height, mean vessel diameter, height-corrected mean vessel diameter, height-corrected vessel frequency, and vessel grouping index. Our results demonstrate that vessel traits affecting hydraulic physiology are under genetic control, and both pleiotropic and trait-specific quantitative trait loci are found for these traits.