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. 2018 Mar 27;18(7):1084-1093.
doi: 10.1039/c8lc00130h.

Flow dynamics control endothelial permeability in a microfluidic vessel bifurcation model

Ehsan Akbari  1 , Griffin B Spychalski, Kaushik K Rangharajan, Shaurya Prakash, Jonathan W Song
Affiliations

Affiliation

  • 1 Department of Mechanical and Aerospace Engineering, The Ohio State University, Scott Laboratory, 201 W. 19th Ave, Columbus, OH 43210, USA. song.1069@osu.edu prakash.31@osu.edu.

Flow dynamics control endothelial permeability in a microfluidic vessel bifurcation model

Ehsan Akbari et al. Lab Chip. .
. 2018 Mar 27;18(7):1084-1093.
doi: 10.1039/c8lc00130h.

Authors

Ehsan Akbari  1 , Griffin B Spychalski, Kaushik K Rangharajan, Shaurya Prakash, Jonathan W Song

Affiliation

  • 1 Department of Mechanical and Aerospace Engineering, The Ohio State University, Scott Laboratory, 201 W. 19th Ave, Columbus, OH 43210, USA. song.1069@osu.edu prakash.31@osu.edu.

Abstract

Endothelial barrier function is known to be regulated by a number of molecular mechanisms; however, the role of biomechanical signals associated with blood flow is comparatively less explored. Biomimetic microfluidic models comprised of vessel analogues that are lined with endothelial cells (ECs) have been developed to help answer several fundamental questions in endothelial mechanobiology. However, previously described microfluidic models have been primarily restricted to single straight or two parallel vessel analogues, which do not model the bifurcating vessel networks typically present in physiology. Therefore, the effects of hemodynamic stresses that arise due to bifurcating vessel geometries on ECs are not well understood. Here, we introduce and characterize a microfluidic model that mimics both the flow conditions and the endothelial/extracellular matrix (ECM) architecture of bifurcating blood vessels to systematically monitor changes in endothelial permeability mediated by the local flow dynamics at specific locations along the bifurcating vessel structure. We show that bifurcated fluid flow (BFF) that arises only at the base of a vessel bifurcation and is characterized by stagnation pressure of ∼38 dyn cm-2 and approximately zero shear stress induces significant decrease in EC permeability compared to the static control condition in a nitric oxide (NO)-dependent manner. Similarly, intravascular laminar shear stress (LSS) (3 dyn cm-2) oriented tangential to ECs located downstream of the vessel bifurcation also causes a significant decrease in permeability compared to the static control condition via the NO pathway. In contrast, co-application of transvascular flow (TVF) (∼1 μm s-1) with BFF and LSS rescues vessel permeability to the level of the static control condition, which suggests that TVF has a competing role against the stabilization effects of BFF and LSS. These findings introduce BFF at the base of vessel bifurcations as an important regulator of vessel permeability and suggest a mechanism by which local flow dynamics control vascular function in vivo.

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Conflict of interest statement

Conflicts of interest

There are no conflicts to declare.

Figures

Fig. 1

Fig. 1

Microfluidic bifurcating vessel model for…

Fig. 1

Microfluidic bifurcating vessel model for endothelial hydraulic conductivity measurements. (A) The photograph of…

Fig. 1
Microfluidic bifurcating vessel model for endothelial hydraulic conductivity measurements. (A) The photograph of the device stained with green dye in the perfusion channels and orange dye in the extracellular matrix (ECM) compartment. (B) Schematic of the microfluidic platform depicting the microchannels seeded with human umbilical vascular endothelial cells (HUVECs, green) branching around the central ECM (orange) compartment. (C) Zoomed-in view of the bifurcation region (shown by black box in A) depicting the apertures at the bifurcation point (BP) and branched vessel (BV), which enable interaction between the HUVECs and supporting ECM. The laminar in-flow stagnates at the BP, generating bifurcated fluid flow (BFF, black dashed line) and symmetrically branches around the central ECM region (black solid lines), generating two downstream regions of laminar shear stress (LSS) in BV. (D) Elevating the reservoir hydrostatic pressure produces a controlled pressure difference between the intravascular pressure (IVP) and interstitial fluid pressure (IFP), inducing transvascular flow (TVF) from both the BP and BV (white arrows). (E) Representative phase contrast image of the bifurcation region fully seeded with HUVECs adjacent to the polymerized ECM matrix. Scale bar is 500 μm. (F) Representative confocal reflectance image of the aperture at BP depicting the confluent HUVEC monolayer with well-defined junctions adjacent to the supporting fibrous ECM. The contact angle at each aperture facilitates the confinement of the ECM collagenous solution and formation of the ECM–HUVEC interface. The white dotted lines depict the PDMS microposts. Scale bar is 100 μm. (G) Representative fluorescence images of acellular versus cellular (or HUVEC-lined) microchannels with FITC conjugated dextran (10 kDa). Under a similar inlet pressure, the HUVEC monolayer at each of the apertures uniformly suppresses the level of transendothelial fluid flow, thereby confirming effective HUVEC barrier function. Scale bars are 500 μm.
Fig. 2

Fig. 2

Quantification of transendothelial volumetric flux…

Fig. 2

Quantification of transendothelial volumetric flux ( J V ). (A) The representative epifluorescence…

Fig. 2
Quantification of transendothelial volumetric flux (JV). (A) The representative epifluorescence images of the cellular microfluidic platform perfused with 10 μM FITC- Dextran. A user-defined region of interest was used to analyze the rate of convective transport for the FITC-dextran across the endothelial monolayer at each aperture (dashed red line). Scale bars, 500 μm. (B) The rate of increase in normalized average fluorescence intensity within the defined Eulerian control volume multiplied by the volume of the analysis region () equates the level of transendothelial flux (JV) multiplied by the monolayer area (S). The R2 value of the linear fit was 0.98.
Fig. 3

Fig. 3

Bifurcated fluid flow (BFF) and…

Fig. 3

Bifurcated fluid flow (BFF) and laminar shear stress (LSS) elicit a time-dependent effect…

Fig. 3
Bifurcated fluid flow (BFF) and laminar shear stress (LSS) elicit a time-dependent effect on endothelial hydraulic conductivity at the bifurcation point (BP) and branched vessel (BV) apertures, respectively. (A) Schematic of the microfluidic platform, depicting the experimental outline of: (i) static control, (ii) perfused microfluidic device generating BFF (black dashed line) and LSS (black solid lines) under equilibrated intravascular pressure (IVP) and interstitial fluid pressure (IFP), which results in minimal transvascular flow (TVF), and (iii) perfused microfluidic platform under elevated IVP, which results in luminal to abluminal TVF (white solid lines). (B) Representative images of LP measurement after treatment with each experimental test condition at (i) BP and (ii) BV. The white dotted lines represent the PDMS microposts. The red dotted lines represent the semipermeable HUVEC monolayer at each aperture. Scale bars are 100 μm. (C) Quantitative effects of 38 dyn cm−2 BFF and ~1 μm s−1 TVF on endothelial hydraulic conductivity after 1 hour and 6 hours of treatment, compared to static control condition. (D) Quantitative effects of 3 dyn cm−2 LSS and ~1 μm s−1 TVF on endothelial hydraulic conductivity after 1 hour and 6 hours of treatment, compared to static control condition *, p < 0.05 **, p < 0.01, ***, p < 0.001.
Fig. 4

Fig. 4

The stabilizing effect of bifurcated…

Fig. 4

The stabilizing effect of bifurcated fluid flow (BFF) at the bifurcation point (BP)…

Fig. 4
The stabilizing effect of bifurcated fluid flow (BFF) at the bifurcation point (BP) and laminar shear stress (LSS) at the branched vessel (BV) is nitric oxide (NO) dependent. (A) Schematics of the experimental conditions: (i) static control, (ii) perfused microdevice in the absence of transvascular flow (TVF), and (iii) perfused microdevice in the absence of TVF and addition of the NO inhibitor L-NMMA (200 μM) to the reservoir media. (B) Representative fluorescent images of LP measurements at the: (i) BP and (ii) BV that demonstrate the effects of L-NMMA on endothelial permeability. Scale bars are 100 μm. (C) Quantitative effects of blocking NO release with L-NMMA on BFF and LSS mediated decreases in LP at the BP and BV respectively. In addition, application of L-NMMA under static conditions did not cause a significant change in LP **, p < 0.01, ***, p < 0.001.
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