Extracellular matrix composition alters endothelial force transmission

Abstract

Extracellular matrix (ECM) composition is important in a host of pathophysiological processes such as angiogenesis, atherosclerosis, and diabetes, and during each of these processes ECM composition has been reported to change over time. However, the impact ECM composition has on the ability of endothelium to respond mechanically is currently unknown. Therefore, in this study, we seeded human umbilical vein endothelial cells (HUVECs) onto soft hydrogels coated with an ECM concentration of 0.1 mg/mL at the following collagen I (Col-I) and fibronectin (FN) ratios: 100% Col-I, 75% Col-I-25% FN, 50% Col-I-50% FN, 25% Col-I-75% FN, and 100% FN. We subsequently measured tractions, intercellular stresses, strain energy, cell morphology, and cell velocity. Our results revealed that tractions and strain energy are maximal at 50% Col-I-50% FN and minimal at 100% Col-I and 100% FN. Intercellular stress response was maximal on 50% Col-I-50% FN and minimal on 25% Col-I-75% FN. Cell area and cell circularity displayed a divergent relationship for different Col-I and FN ratios. We believe that these results will be of great importance to the cardiovascular field, biomedical field, and cell mechanics.NEW & NOTEWORTHY The endothelium constitutes the innermost layer of all blood vessels and plays an important role in vascular physiology and pathology. During certain vascular diseases, the extracellular matrix has been suggested to transition from a collagen-rich matrix to a fibronectin-rich matrix. In this study, we demonstrate the impact various collagen and fibronectin ratios have on endothelial biomechanical and morphological response.

Keywords: extracellular matrix; fibronectin; intercellular stresses; traction force microscopy; type 1 collagen.

Graphical abstract

Figure 1.

Cropped HUVEC monolayer phase-contrast images (within 651 × 651 µm²) for different Col-I and FN coating concentration ratios: Col-I 100% (A), Col-I 75% FN 25% (B), Col-I 50% FN 50% (C), Col-I 25% FN 75% (D), FN 100% (E). Col-I, collagen I; FN, fibronectin; HUVEC, human umbilical vein endothelial cell.

Figure 2.

RMS traction (within 651 × 651 µm² cropped section) distributions (Pa) for different Col-I and FN coating concentration ratios: Col-I 100% (A), Col-I 75% FN 25% (B), Col-I 50% FN 50% (C), Col-I 25% FN 75% (D), FN 100% (E), and average RMS tractions (Pa) for different Col-I and FN coating concentrations based on averages from five samples for each ratio (F). *Statistical significance (***P ≤ 1E-3). Error bars represent standard errors. Col-I, collagen I; FN, fibronectin; HUVEC, human umbilical vein endothelial cell; RMS, root mean square.

Figure 3.

Average strain energies (pJ) for different Col-I and FN coating concentrations based on averages from five samples for each ratio. *Statistical significance (***P ≤ 1E-3). Error bars represent standard errors. Col-I, collagen I; FN, fibronectin.

Figure 4.

Average normal stress (within 651 × 651 µm² cropped section) distributions (Pa) for different Col-I and FN coating concentration ratios: Col-I 100% (A), Col-I 75% FN 25% (B), Col-I 50% FN 50% (C), Col-I 25% FN 75% (D), FN 100% (E), and average normal stress (Pa) for different Col-I and FN coating concentrations based on averages from five samples for each ratio (F). *Statistical significance (***P ≤ 1E-3; no star P > 0.05). Error bars represent standard errors. Col-I, collagen I; FN, fibronectin; HUVEC, human umbilical vein endothelial cell.

Figure 5.

Maximum shear stress (within 651 × 651 µm² cropped section) distributions (Pa) for different Col-I and FN coating concentration ratios: Col-I 100% (A), Col-I 75% FN 25% (B), Col-I 50% FN 50% (C), Col-I 25% FN 75% (D), FN 100% (E), and average maximum shear stress (Pa) for different Col-I and FN coating concentrations based on averages from five samples for each ratio (F). *Statistical significance (**P ≤ 1E-2; ***P ≤ 1E-3). Error bars represent standard errors. Col-I, collagen I; FN, fibronectin; HUVEC, human umbilical vein endothelial cell.

Figure 6.

RMS velocity (within 651 × 651 µm² cropped section) distributions (µm/min) for different Col-I and FN coating concentration ratios: Col-I 100% (A), Col-I 75% FN 25% (B), Col-I 50% FN 50% (C), Col-I 25% FN 75% (D), FN 100% (E), and average RMS (Pa) for different Col-I and FN coating concentrations based on averages from five samples for each ratio (F). *Statistical significance (***P ≤ 1E-3). Error bars represent standard errors. Col-I, collagen I; FN, fibronectin; HUVEC, human umbilical vein endothelial cell; RMS, root mean square.

Figure 7.

HUVEC area (µm²) (within 651 × 651 µm² cropped section) for different Col-I and FN coating concentration ratios: Col-I 100% (A), Col-I 75% FN 25% (B), Col-I 50% FN 50% (C), Col-I 25% FN 75% (D), FN 100% (E), and average cell area (µm²) for different Col-I and FN coating concentrations based on averages of 1,057 to 1,460 cells from five samples for each concentration ratio (F). *Statistical significance (***P ≤ 1E-3). Error bars represent standard errors. Col-I, collagen I; FN, fibronectin; HUVEC, human umbilical vein endothelial cell.

Figure 8.

HUVEC circularity computed as (4 × Area × π)/(Perimeter²) with a value of 1 for a perfect circle (within 651 × 651 µm² cropped section) for different Col-I and FN coating concentration ratios: Col-I 100% (A), Col-I 75% FN 25% (B), Col-I 50% FN 50% (C), Col-I 25% FN 75% (D), FN 100% (E), and average cell area (µm²) for different Col-I and FN coating concentrations based on averages of 1,057 to 1,460 cells from five samples for each concentration ratio (F). *Statistical significance (*P ≤ 0.05; ***P ≤ 1E-3). Error bars represent standard errors. Col-I, collagen I; FN, fibronectin; HUVEC, human umbilical vein endothelial cell.