Flow shear stress regulates endothelial barrier function and expression of angiogenic factors in a 3D microfluidic tumor vascular model

Abstract

Endothelial cells lining blood vessels are exposed to various hemodynamic forces associated with blood flow. These include fluid shear, the tangential force derived from the friction of blood flowing across the luminal cell surface, tensile stress due to deformation of the vessel wall by transvascular flow, and normal stress caused by the hydrodynamic pressure differential across the vessel wall. While it is well known that these fluid forces induce changes in endothelial morphology, cytoskeletal remodeling, and altered gene expression, the effect of flow on endothelial organization within the context of the tumor microenvironment is largely unknown. Using a previously established microfluidic tumor vascular model, the objective of this study was to investigate the effect of normal (4 dyn/cm(2)), low (1 dyn/cm(2)), and high (10 dyn/cm(2)) microvascular wall shear stress (WSS) on tumor-endothelial paracrine signaling associated with angiogenesis. It is hypothesized that high WSS will alter the endothelial phenotype such that vascular permeability and tumor-expressed angiogenic factors are reduced. Results demonstrate that endothelial permeability decreases as a function of increasing WSS, while co-culture with tumor cells increases permeability relative to mono-cultures. This response is likely due to shear stress-mediated endothelial cell alignment and tumor-VEGF-induced permeability. In addition, gene expression analysis revealed that high WSS (10 dyn/cm(2)) significantly down-regulates tumor-expressed MMP9, HIF1, VEGFA, ANG1, and ANG2, all of which are important factors implicated in tumor angiogenesis. This result was not observed in tumor mono-cultures or static conditioned media experiments, suggesting a flow-mediated paracrine signaling mechanism exists with surrounding tumor cells that elicits a change in expression of angiogenic factors. Findings from this work have significant implications regarding low blood velocities commonly seen in the tumor vasculature, suggesting high shear stress-regulation of angiogenic activity is lacking in many vessels, thereby driving tumor angiogenesis.

Keywords: Angiopoietin; Collagen Hydrogel; Permeability; Tissue Engineering; Tumorigenesis; VEGF.

Figure 1.

Schematic of microfluidic tumor vascular model for 3D culture of tumor and endothelial cells under flow conditions. Type I collagen seeded with MDA-MB-231 cells is injected into FEP tubing fit concentrically with a 22G needle and capped with PDMS sleeves. Endothelial cells are seeded along the luminal surface of the central microchannel, after which flow is introduced via insertion of syringe needles interfaced with tubing that leads to a syringe pump that controls the flow rate.

Figure 2.

VEGF protein secretion during 72 hr low flow preconditioning scheme. ELISA of perfused culture media from endothelial mono-culture relative to co-culture with tumor cells reveals a significant increase in total VEGF protein over time P < 0.05. Concentration (ng/ml) was multiplied by volume of perfusate at time of collection (5, 25, and 60 ml at t = 24, 48, and 72 hrs, respectively).

Figure 3.

High WSS down-regulates tumor-expressed angiogenic factors in the presence of an endothelium. Tumor mono-cultures (A) or co-cultures with endothelial cells (B) were cultured under the 72 hr precondition flow rate, after which the target WSS (τ_W = 1, 4, or 10 dyn/cm²) was introduced through the microchannel for a total 6 hrs. Total tumor mRNA was then isolated for gene expression analysis. Relative mRNA to GAPDH mRNA expressed as a fold induction ± standard deviation (n = 4) *P < 0.05. Scale bar 200 μm.

Figure 4.

Effective permeability coefficient, P_d, decreases as a function of increasing WSS and increases during co-culture with tumor cells. (A) P_d of 70 kDa Oregon Green-conjugated Dextran across the endothelialized microchannel decreases as τ_W increases for both mono-culture and co-culture with tumor cells, with a statistically significant reduction at τ_W = 10 dyn/cm² relative to the preconditioned endothelium *P < 0.05. P_d during co-culture was significantly increased for all τ_W relative to mono-cultures #P < 0.05. (B) Representative images of 70 kDa Oregon Green-conjugated Dextran diffusion across the endothelialized microchannel for the case of τ_W = 10 dyn/cm². Scale bar 200 μm.