Contrast-enhanced micro-CT imaging in murine carotid arteries: a new protocol for computing wall shear stress.

BACKGROUND: Wall shear stress (WSS) is involved in the pathophysiology of atherosclerosis. The correlation between WSS and atherosclerosis can be investigated over time using a WSS-manipulated atherosclerotic mouse model. To determine WSS in vivo, detailed 3D geometry of the vessel network is required. However, a protocol to reconstruct 3D murine vasculature using this animal model is lacking. In this project, we evaluated the adequacy of eXIA 160, a small animal contrast agent, for assessing murine vascular network on micro-CT. Also, a protocol was established for vessel geometry segmentation and WSS analysis. METHODS: A tapering cast was placed around the right common carotid artery (RCCA) of ApoE-/- mice (n = 8). Contrast-enhanced micro-CT was performed using eXIA 160. An innovative local threshold-based segmentation procedure was implemented to reconstruct 3D geometry of the RCCA. The reconstructed RCCA was compared to the vessel geometry using a global threshold-based segmentation method. Computational fluid dynamics was applied to compute the velocity field and WSS distribution along the RCCA. RESULTS: eXIA 160-enhanced micro-CT allowed clear visualization and assessment of the RCCA in all eight animals. No adverse biological effects were observed from the use of eXIA 160. Segmentation using local threshold values generated more accurate RCCA geometry than the global threshold-based approach. Mouse-specific velocity data and the RCCA geometry generated 3D WSS maps with high resolution, enabling quantitative analysis of WSS. In all animals, we observed low WSS upstream of the cast. Downstream of the cast, asymmetric WSS patterns were revealed with variation in size and location between animals. CONCLUSIONS: eXIA 160 provided good contrast to reconstruct 3D vessel geometry and determine WSS patterns in the RCCA of the atherosclerotic mouse model. We established a novel local threshold-based segmentation protocol for RCCA reconstruction and WSS computation. The observed differences between animals indicate the necessity to use mouse-specific data for WSS analysis. For our future work, our protocol makes it possible to study in vivo WSS longitudinally over a growing plaque.

Identification, rapid screening, docking mechanism and in vitro digestion stability of novel DPP-4 inhibitory peptides from wheat gluten with ginger protease.

To better understand the hypoglycemic potential of wheat gluten (WG), we screened dipeptidyl peptidase IV (DPP-4) inhibitory active peptides from WG hydrolysates. WG hydrolysates prepared by ginger protease were found to have the highest DPP-4 inhibitory activity among the five enzymatic hydrolysates, from which a 1-3 kDa fraction was isolated by ultrafiltration. Further characterization of the fraction with nano-HPLC-MS/MS revealed 1133 peptides. Among them, peptides with P'2 (the second position of the N-terminal) and P2 (the second position of the C-terminal) as proline residues (Pro) accounted for 12.44% and 43.69%, respectively. The peptides including Pro-Pro-Phe-Ser (PPFS), Ala-Pro-Phe-Gly-Leu (APFGL), and Pro-Pro-Phe-Trp (PPFW) exhibited the most potent DPP-4 inhibitory activity with IC50 values of 56.63, 79.45, and 199.82 µM, respectively. The high inhibitory activity of PPFS, APFGL, and PPFW could be mainly attributed to their interaction with the S2 pocket (Glu205 and Glu206) and the catalytic triad (Ser630 and His740) of DPP-4, which adopted competitive, mixed, and mixed inhibitory modes, respectively. After comparative analysis of PPFS, PPFW, and PPF, Ser was found to be more conducive to enhancing the DPP-4 inhibitory activity. Interestingly, peptides with P2 as Pro also exhibited good DPP-4 inhibitory activity. Meanwhile, DPP-4 inhibitory peptides from WG showed excellent stability, suggesting a potential application in type 2 diabetes (T2DM) therapy or in the food industry as functional components.

Secondary, somatic mutations might promote cyst formation in patients with autosomal dominant polycystic liver disease.

BACKGROUND & AIMS: Heterozygous germline mutations in PRKCSH cause autosomal dominant polycystic liver disease (PCLD), but it is not clear how they lead to cyst formation. We investigated whether mutations in cyst epithelial cells and corresponding loss of the PRKCSH gene product (hepatocystin) contributed to cyst development. METHODS: Liver cyst material was collected through laparoscopic cyst fenestration from 8 patients with PCLD who had a heterozygous germline mutation in PRKCSH. Tissue sections from 71 cysts (2-14 per patient) were obtained for hepatocystin staining and mutation analysis. Cyst epithelium was acquired using laser microdissection; DNA was isolated and analyzed for loss of heterozygosity (LOH) and somatic mutations using restriction analysis and sequencing. Common single nucleotide polymorphisms (SNPs) in a 70-kilobase region surrounding the germline mutation were used to determine variations in the genomic region with LOH. RESULTS: The wild-type allele of PRKCSH was lost (LOH) in 76% of cysts (54/71). Hepatocystin was not detected in cyst epithelia with LOH, whereas heterozygous cysts still expressed hepatocystin. The variation observed in the LOH region analysis indicates that cysts develop independently. We also detected somatic mutations in PRKCSH in 17% (2/12) of the cysts without LOH. Trans-heterozygous mutations in SEC63 were not observed. CONCLUSIONS: Among patients with PCLD who have a heterozygous germline mutation in PRKCSH, we found secondary, somatic mutations (second hits) in more than 76% of the liver cyst epithelia. PCLD is recessive at the cellular level, and loss of functional PRKCSH is an important step in cystogenesis.

Heart rate reduction with ivabradine promotes shear stress-dependent anti-inflammatory mechanisms in arteries.

Blood flow generates wall shear stress (WSS) which alters endothelial cell (EC) function. Low WSS promotes vascular inflammation and atherosclerosis whereas high uniform WSS is protective. Ivabradine decreases heart rate leading to altered haemodynamics. Besides its cardio-protective effects, ivabradine protects arteries from inflammation and atherosclerosis via unknown mechanisms. We hypothesised that ivabradine protects arteries by increasing WSS to reduce vascular inflammation. Hypercholesterolaemic mice were treated with ivabradine for seven weeks in drinking water or remained untreated as a control. En face immunostaining demonstrated that treatment with ivabradine reduced the expression of pro-inflammatory VCAM-1 (p<0.01) and enhanced the expression of anti-inflammatory eNOS (p<0.01) at the inner curvature of the aorta. We concluded that ivabradine alters EC physiology indirectly via modulation of flow because treatment with ivabradine had no effect in ligated carotid arteries in vivo, and did not influence the basal or TNFα-induced expression of inflammatory (VCAM-1, MCP-1) or protective (eNOS, HMOX1, KLF2, KLF4) genes in cultured EC. We therefore considered whether ivabradine can alter WSS which is a regulator of EC inflammatory activation. Computational fluid dynamics demonstrated that ivabradine treatment reduced heart rate by 20 % and enhanced WSS in the aorta. In conclusion, ivabradine treatment altered haemodynamics in the murine aorta by increasing the magnitude of shear stress. This was accompanied by induction of eNOS and suppression of VCAM-1, whereas ivabradine did not alter EC that could not respond to flow. Thus ivabradine protects arteries by altering local mechanical conditions to trigger an anti-inflammatory response.

Animal models of surgically manipulated flow velocities to study shear stress-induced atherosclerosis.

Atherosclerosis is a chronic inflammatory disease of the arterial tree that develops at predisposed sites, coinciding with locations that are exposed to low or oscillating shear stress. Manipulating flow velocity, and concomitantly shear stress, has proven adequate to promote endothelial activation and subsequent plaque formation in animals. In this article, we will give an overview of the animal models that have been designed to study the causal relationship between shear stress and atherosclerosis by surgically manipulating blood flow velocity profiles. These surgically manipulated models include arteriovenous fistulas, vascular grafts, arterial ligation, and perivascular devices. We review these models of manipulated blood flow velocity from an engineering and biological perspective, focusing on the shear stress profiles they induce and the vascular pathology that is observed.