The role of nanotopographical extracellular matrix (ECM) cues on vascular endothelial cell (EC) organization and function is not well-understood despite the composition of nano- to micro-scale fibrillar ECMs within blood vessels. collagen films that induce parallel EC alignment prior to stimulation with disturbed flow resulting from spatial wall shear stress gradients. Using real time live-cell imaging we tracked the alignment migration trajectories proliferation and anti-inflammatory behavior Bryostatin 1 of ECs when they were cultured on parallel-aligned or randomly oriented nanofibrillar films. Intriguingly ECs cultured on aligned nanofibrillar films remained well-aligned and migrated predominantly along the direction of aligned nanofibrils despite exposure to shear stress orthogonal to the direction of the aligned nanofibrils. Furthermore in stark contrast to ECs cultured on randomly oriented films ECs on aligned nanofibrillar films exposed to disturbed flow had significantly reduced inflammation and proliferation while maintaining intact intercellular junctions. This work reveals fundamental insights into the importance of nanoscale ECM interactions in the maintenance of endothelial function. Importantly it provides new insight into Bryostatin 1 how ECs respond to opposing cues derived from nanotopography and mechanical shear force and has strong implications in the design of polymeric conduits and bioengineered tissues. studies randomly oriented or aligned nanofibrillar films were sterilized with 70% ethanol Bryostatin 1 and rehydrated with 1× PBS for 2 hours. 5×105 primary human dermal microvascular ECs (Lonza P7-10) were seeded onto Itgal the collagen film in EGM-2MV growth media (Lonza) at 37°C and 5% CO2 until they reached approximately 80% confluence. Disturbed flow system A disturbed flow system resulting from spatial wall shear stress gradients was previously characterized15 to recapitulate the pathologic flow profile seen at the bifurcation points of blood vessels (Figure 1a). A Nikon TE-2000 inverted microscope with a motorized stage and enclosed in a plexiglass chamber maintained at 37°C housed the cells and flow orifice. A nine-roller dampened peristaltic pump (Idex) was used to deliver cell culture media at a flow rate of 3 mL/min through 1.3 mm (inner diameter) tubing corresponding to a fluid velocity range of 0-75.3 mm/s. Media flowed downward from the flow orifice (0.7 mm inner diameter) at the conserved flow rate of 3mL/min onto EC-cultured collagen films corresponding to a fluid velocity range between 0-259.8 mm/s and producing a shear stress range of 0-25.1 dynes/cm2 on the cell monolayer (Figure 1b-c) which is within physiological range.40 Cells were exposed to disturbed flow for 24 hours. Phase contrast images were collected every 25 min using Fiji Bryostatin 1 software for 24 hours. All images were bandpass filtered in ImageJ to increase contrast Bryostatin 1 of cell boundaries. To assess shear gradients the cell monolayer was assigned 5 regions of interest defined by concentric rings (R1 R2 R3 R4 R5) each with a radius of 185 μm. The stagnation point directly underneath the flow orifice corresponded to the center of R1 where the cells experience zero shear stress. The magnitude of the shear stress increased radially outward from the jetting center with maximum shear stress peaking within R2 (Figure 1c). The shear stress decreases from R3 to R5. The impinging flow was modeled byaxisymmetric flow using the commercial finite-element analysis (FEA) package COMSOL Multiphysics 3.5a following our previous study.15 A flow rate of 3 ml/min is prescribed at the orifice inlet and a pressure Bryostatin 1 boundary condition is used at the outlet. A “no slip” boundary condition was assumed at the wall (where z=0 at the cell monolayer) such that the velocity of the fluid directly at the wall is zero. The wall shear stress τwas calculated as a function of the velocity gradient
which quantifies how quickly fluid velocity (u) changes along the z-direction and the fluid viscosity (μ):
Quantification of cellular alignment.