Project description:Blood flow promotes emergence of definitive hematopoietic stem cells (HSCs) in the developing embryo, yet the signals generated by hemodynamic forces that influence hematopoietic potential remain poorly defined. In transplantation assays of hematopoietic reconstitution, we find that fluid shear stress endows long-term multilineage engraftment potential upon early hematopoietic tissues at E9.5 not previously described to harbor HSCs. Effects on hematopoiesis appear to be mediated in part by prostaglandin E2 (PGE2) and the cyclic AMP-protein kinase A (cAMP-PKA) signaling axis. Studies of Ncx1 cardiac mutants corroborate that blood flow is required for sufficient COX2 levels and phosphorylation of CREB. Further implicating PGE2 in mediating the effects of shear stress, we find that E10.5 and E11.5 AGM treated transiently with the synthetic analog dmPGE2 engraft more robustly and contribute to greater lymphoid reconstitution. These data provide a mechanism by which biomechanical forces induced by blood flow modulate hematopoietic potential. - AGM from C57BL/6J embryos at 10.5 days gestation (E10.5) were isolated by microdissection from uteri of pregnant dams, following by gentle dissociation by Accutase with agitation at room temperature for 20 minutes. Single cell suspension was seeded on microfluidic IBIDI VI^0.4 6-channel slides (0.8 to 1x10^7 cells per channel) and permitted to attach for 8 hours. Fluid movement was then applied to each channel using a Harvard Apparatus PHD ULTRA programmable syringe pump for manangement of M5300 Myelocult medium. Cells were exposed either to static/low flow (0.0001 dyne/cm^2) or wall shear stress (WSS) of 5 dyne/cm^2 for 6 hours or 36 hours. In addition, some cells were treated with 10 uM indomethacin (indo) to inhibit COX2 activity and PGE2 synthesis. Upon collection of cells with RLT lysis buffer (QIAGEN RNeasy kit), six channels of identical treatment were pooled to comprise a single sample. 24 samples total are included in this study. 12 samples were collected after 6 hours and 12 after 36 hours. In detail, samples included at 6 hours: 3 static, 3 static with indo, 3 WSS, 3 WSS with indo; and at 36 hours: 3 static, 3 static with indo, 3 WSS, 3 WSS with indo. Sample labels begin with the timepoint collected and end with the replicate number, i.e., 06WSS1 for the 6 hour collection of the first replicate of the WSS sample.

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: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.

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Flow dependent gene expression in the rat aorta under physiological conditions

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