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.
Purpose Wrist-worn accelerometer devices measure rest in free-living configurations. of women through the Healthy Women Research (n=145; age group 73.3±1.7y) wore an Actiwatch-2 on the nondominant wrist and an ActiGraph GT1M on the dominant hip for 7-consecutive times. Participants documented their leisure-time exercise inside a 7-day time diary and finished the past-year edition from the Modifiable Activity Questionnaire. Analyses were conducted for many wake intervals as well as for dynamic intervals when both products were worn separately. Outcomes Spearman rank-order correlation coefficients for total movement volume between Actiwatch-2 and ActiGraph GT1M were significant for wake periods (r=0.47 p<0.001) and to a lesser extent for active periods (r=0.26 p<0.01). However Actiwatch-2 did not rank participant physical activity levels similarly to self-reported leisure-time physical activity estimates (kappa≤0.05 p>0.05). Multilevel model analyses comparing temporally-matched activity measured via ActiGraph GT1M and Actiwatch-2 suggest that the two devices yielded similar levels of activity during wake periods (B=0.90 SE=0.008 p<0.001) as well as during active periods (B=0.81 SE=0.01 p<0.001). Conclusions A wrist-worn Actiwatch-2 may be useful for ranking total movement volume and for assessing the pattern of activity over a day in older women. Bryostatin Bryostatin 1 1 However our data does not support using a wrist-worn Actiwatch-2 device for measuring physical activity. acceleration over a user-specified time period (i.e. epoch) (9 17 One limitation of past data collection using ActiGraph GT1M and other similar waist-worn accelerometers however is that participants are typically instructed to remove the device when sleeping; therefore capturing movement only during waking hours within each 24-hour period. In contrast to the Bryostatin 1 standard placement of accelerometers on the waist to assess waking movement behavior the most accurate placement of accelerometer devices to measure sleep is on the wrist (7). These devices detect small movements at the wrist which are used to determine sleep-wake intervals. The Actiwatch make of sleep displays specifically has been utilized to directly measure sleep in free-living settings widely. Even though the Actiwatch displays identify accelerations in the vertical airplane like the ActiGraph GT1M the Actiwatch displays differs from ActiGraph GT1M for the reason that they possess a larger regularity response range (e.g. 0.35 Hz) and integrate data being a acceleration detected over each epoch (9). A significant benefit of the Actiwatch displays and other equivalent rest monitoring devices is certainly their modern/compact style (just like a wristwatch) making wearing these devices super easy Bryostatin 1 and unobtrusive. Furthermore the Actiwatch displays are typically put on for a complete 24-hour period producing them attractive being a potential one gadget that could catch both waking motion behavior and rest. However to your knowledge no research in adults possess looked into the validity of the wrist-worn Actiwatch gadget to measure waking motion behavior concurrently with rest within a free-living placing. The Healthy Females Research (HWS) (26) can be an on-going longitudinal research of healthy maturing and cardiovascular risk Rabbit Polyclonal to Caspase 1 (Cleaved-Asp210). getting conducted on the College or university of Pittsburgh. Individuals in the HWS had been between the age range of 42 and 50 years at enrollment (1983-1984) and also have been followed regularly since then. Provided the need for both waking motion behavior and rest for cardiovascular wellness in older females an ancillary research was put into the HWS process in the 2010-2011 follow-up go to including 7-consecutive times of sleep-wake monitoring in the individuals house environment. Notably individuals used both an ActiGraph GT1M on the waistline and an Actiwatch-2 on the wrist for 24-hours every day over the analysis week. Hence the HWS research provides a exclusive possibility to investigate the electricity of the wrist-worn Actiwatch-2 gadget to assess free-living waking motion behavior set alongside the ActiGraph GT1M in an example of older females. This understanding will be beneficial to support supplementary analyses of existing data choices using Actiwatch-2 or various other similar wrist-worn rest devices and you will be.