Project description:Intracranial aneurysms tend to form at bifurcation apices, where flow impingement causes high frictional force (or wall shear stress, WSS) and flow acceleration and deceleration that create positive and negative streamwise gradients in WSS (WSSG), respectively. In vivo, intracranial aneurysms initiate under high WSS and positive WSSG. Little is known about the responses of endothelial cells (ECs) to either positive or negative WSSG under high WSS conditions. We used cDNA microarrays to profile EC gene expression exposed to positive WSSG vs. negative WSSG for 24 hours in a flow chamber with converging and diverging channels, respectively. WSS varied between 3.5 and 28.4 Pa in each gradient channel. GO and biological pathway analysis indicated that positive WSSG favored proliferation, apoptosis, and extracellular matrix processing while decreasing expression of pro-inflammatory genes. A subset of characteristic genes was validated using qPCR: Genes for ADAMTS1, CKAP2 and NCEH1 had higher expression under positive WSSG compared to negative WSSG while TAGLN, THBS1, VCAM1, CCL2, and CSF2 had lower expression. To determine if these patterns of expression are also exhibited in vivo, we tested whether the extracellular matrix related protein ADAMTS1 and proliferation were modulated by positive WSSG during intracranial aneurysm initiation. An aneurysm was induced at the basiliar terminus in rabbits by bilateral carotid ligation. WSSG at the bifurcation was determined by computational fluid dynamic simulations from 3D angiography and mapped on immunofluorescence staining for ADAMTS1 and the proliferation marker, Ki-67. Endothelial ADAMTS1 protein and Ki-67 were significantly higher in regions with positive WSSG compared to adjacent sites where WSSG was negative. Our results indicate that WSSG can elicit distinct gene expression profiles in ECs. Increased matrix processing and high levels of proliferation under positive WSSG could contribute to intracranial aneurysm initiation by causing transient gaps in the endothelium or disrupting EC signals to smooth muscle cells. Time-matched bovine aortic endothelial cells were exposed to positive wall shear stress gradient, negative wall shear stress gradient, and two no gradient samples: uniform WSS of 3.5 Pa and high WSS of 28.4 Pa for 24 hrs in an in vitro flow loop system. RNA was extracted and hybridized on Affymetrix microarrays. There were 12 samples in total, four flow conditions with three replicates each.

Project description:The opportunistic pathogen, Staphylococcus aureus, encounters a wide variety of fluid shear levels within the human host, which may play a key role in dictating whether this organism adopts a commensal interaction with the host or transitions to cause disease. Using rotating-wall vessel bioreactors to create a physiologically-relevant, low fluid shear environment, S. aureus was evaluated for cellular responses that could impact its colonization and virulence. S. aureus cells grown in a low fluid shear environment initiated a novel attachment-independent biofilm phenotype and were completely encased in extracellular polymeric substances. Compared to controls, low-shear cultured cells displayed slower growth and repressed virulence characteristics, including decreased carotenoid production, increased susceptibility to oxidative stress, and reduced survival in whole blood. Transcriptional whole genome microarray profiling suggested alterations in metabolic pathways. Further genetic expression analysis revealed the down-regulation of the RNA chaperone Hfq, which parallels low fluid shear responses of certain Gram negative organisms. This is the first study to report an Hfq association to fluid shear in a Gram positive organism, suggesting an evolutionarily conserved response to fluid shear among structurally diverse prokaryotes. Collectively, our results suggest S. aureus responds to a low fluid shear environment by initiating a biofilm/colonization phenotype with diminished virulence characteristics, which could lead to insight into key factors influencing the divergence between infection and colonization during initial host pathogen interaction. Genetic expression profiles of Staphylococcus aureus cultured under low fluid shear conditions was compared to control cultures of S. aureus which was cultured in identical hardware in an orientation disrupting the low fluid shear effect. Samples from the same date of culture were compared (control 21:low 21 and control 30: low 30). S. aureus was cultured for 20 hours in either the low fluid shear or control orientated rotating wall vessel (RWV) bioreactor at which point the cells were removed and RNA extracted. At 20 hours, both cultures were in the same stage of growth (stationary phase) and at this point phenotypic differences between control and low fluid shear cultures were noted.

Project description:Background: Intracranial aneurysms (IAs) result from abnormal enlargement of the arterial lumen. IAs are mostly quiescent and asymptomatic, but their rupture leads to severe brain damage or death. As the evolution of IAs is hard to predict and intricates medical decision, it is essential to improve our understanding of their pathophysiology. Wall shear stress (WSS) is proposed to influence IA growth and rupture. In this study, we investigated the effects of low and supra-high aneurysmal WSS on endothelial cells (ECs). Methods: Porcine arterial ECs were exposed for 48 h to defined levels of shear stress (2, 30, or 80 dyne/cm2) using an Ibidi flow apparatus. Immunostaining for CD31 or γ-cytoplasmic actin was performed to outline cell borders or to determine cell architecture. Geometry measurements (cell orientation, area, circularity and aspect ratio) were performed on confocal microscopy images. mRNA was extracted for RNAseq analysis. Results: ECs exposed to low or supra-high aneurysmal WSS were more circular and had a lower aspect ratio than cells exposed to physiological flow. Furthermore, they lost the alignment in the direction of flow observed under physiological conditions. The effects of low WSS on differential gene expression were stronger than those of supra-high WSS. Gene set enrichment analysis highlighted that extracellular matrix proteins, cytoskeletal proteins and more particularly the actin protein family were among the protein classes the most affected by shear stress. Interestingly, most genes showed an opposite regulation under both types of aneurysmal WSS. Immunostainings for γ-cytoplasmic actin suggested a different organization of this cytoskeletal protein between ECs exposed to physiological and both types of aneurysmal WSS. Conclusion: Under both aneurysmal low and supra-high WSS the typical arterial EC morphology molds to a more spherical shape. Whereas low WSS down-regulates the expression of cytoskeletal-related proteins and up-regulates extracellular matrix proteins, supra-high WSS induces opposite changes in gene expression of these protein classes. The differential regulation in EC gene expression observed under various WSS translate into a different organization of the ECs’ architecture. This adaptation of ECs to different aneurysmal WSS conditions may affect vascular remodeling in IAs.

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Effects of aneurysmal low and supra-high wall shear stress on endothelial gene expression

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