The effect of substrate modulus on the growth and function of matrix-embedded endothelial cells

Abstract

Endothelial cells (EC) are potent bioregulatory cells, modulating thrombosis, inflammation and control over mural smooth muscle cells and vascular health. The biochemical roles of EC are retained when cells are embedded within three-dimensional (3D) denatured collagen matrices. Though substrate mechanics have long been known to affect cellular morphology and function and 3D-EC systems are increasingly used as therapeutic modalities little is known about the effect of substrate mechanics on EC in these 3D systems. In this work, we examined the effect of isolated changes in modulus on EC growth and morphology, extracellular matrix gene expression, modulation of smooth muscle cell growth, and immunogenicity. EC growth, but not morphology was dependent on scaffold modulus. Increased scaffold modulus reduced secretion of smooth muscle cell growth inhibiting heparan sulfate proteoglycans (HSPGs), but had no effect on secreted growth factors, resulting in a loss of smooth muscle cell growth inhibition by EC on high modulus scaffolds. Expression of ICAM-1, VCAM-1 and induction of CD4(+) T cell proliferation was reduced by increased scaffold modulus, and correlated with changes in integrin α5 expression. Expression of several common ECM proteins by EC on stiffer substrates dropped, including collagen IV(α1), collagen IV(α5), fibronectin, HSPGs (perlecan and biglycan). In contrast, expression of elastin and TIMPs were increased. This work shows even modest changes in substrate modulus can have a significant impact on EC function in three-dimensional systems. The mechanism of these changes is not clear, but the data presented here within suggests a model wherein EC attempt to neutralize changes in environmental force balance by altering ECM and integrin expression, leading to changes in effects on downstream signaling and function.

Figure 1. Scaffold Modulus vs. Crosslinking/121C, 20mmHgTreatment

Scaffold stiffness was characterized by uniaxial compression, at a rate of 0.01mm/s, of 12mm scaffold disks in PBS using a Zwick mechanical tester. Young’s modulus was calculated as the slope of the linear region of the resulting stress strain curve. All error bars are ± SEM.

Figure 2

EC retain a normal, cobble-stone appearance on scaffolds of varying modulus. All cells show a normal immuno-fluorescent pattern of EC markers, including CD31 (green). Actin (red) stress fibers are visible on all scaffold types. Left: 50Pa scaffold Right: 1345Pa scaffold

Figure 3. Endothelial Cell Growth is Dependent on Scaffold Modulus

Top: Cell per scaffold at day 21 (confluence) vs. modulus. Cell number peaks at 508Pa and falls off at both lower and higher moduli. Error bars are mean cell number ± SEM. Bottom: EC growth rate, as measured by slope of growth curve between days 5–21. As with cell number, growth rate peaks at 508Pa and falls at lower and higher moduli. Error bars represent standard error of linear regression to cell number data.

Figure 4. Smooth Muscle Cell Growth Inhibition and Heparan Sulfate Proteoglycan Secretion Are Affected by Scaffold Modulus

Top) Inhibition of smooth muscle cell growth by EC is related to scaffold modulus. Bottom) Inhibition and HSPG secretion are linearly related. Correlation r² = 0.80, p < 0.001. All error bars are ± SEM.

Figure 5. Endothelial Cell Induced T-Cell Proliferation

Top: MFI (inversely related to T-cell proliferation) increases with scaffold modulus, indicating decreased proliferation at higher modulus. (left) MFI is linearly related to endothelial α₅ integrin expression, r² = 0.83. (right) Bottom: MFI is linearly related to endothelial ICAM-1 (r² = 0.81) (left) and VCAM-1 (r² = 0.95) (right) expression. All error bars are ± SEM.

Figure 6. Proposed Model for Mechanism of Modulus Effect on Endothelial Cell Function

Upper Left: In a natural environment, external forces on the cell are in balance and the cell carries out its normal inhibitory and stimulatory functions. Upper Right: When substrate modulus is changed, the balance is disrupted. Bottom: In order to restore balance of external forces, the cell alters ECM production and integrin expression, leading to changes in cell function.