The extracellular matrix is a novel attribute of endothelial progenitors and of hypoxic mature endothelial cells

Abstract

Extracellular matrix (ECM) production is critical to preserve the function and integrity of mature blood vessels. Toward the engineering of blood vessels, studies have centered on ECM production by supporting cells, whereas few studies implicate endothelial cells (ECs) with ECM synthesis. Here, we elucidate variations between cultured human arterial, venous, and progenitor ECs with respect to ECM deposition assembly, composition, and response to biomolecular and physiological factors. Our studies reveal that progenitor ECs, endothelial colony-forming cells (ECFCs), deposit collagen IV, fibronectin, and laminin that assemble to an organized weblike structure, as confirmed by decellularized cultures. Mature ECs only express these ECM proteins intracellularly. ECFC-derived ECM is abrogated in response to TGFβ signaling inhibition and actin cytoskeleton disruption. Hypoxic (1%) and physiological (5%) O(2) tension stimulate ECM deposition from mature ECs. Interestingly, deposition of collagen I is observed only under 5% O(2) tension. ECM production from all ECs is found to be regulated by hypoxia-inducible factors 1α and 2α but differentially in the different cell lines. Collectively, we suggest that ECM deposition and assembly by ECs is dependent on maturation stage and oxygen supply and that these findings can be harnessed to advance engineered vascular therapeutics.

Figure 1.

Comparison of ECFCs, HUAECs, and HUVECs. A) i) Quantitative RT-PCR analysis of EC markers. ii) Flow cytometry analysis of EC markers (shade indicates isotype control). iii) Mean fluorescent intensity of EC marker expression normalized to isotype control. Graphed values represent mean ± %CV fluorescent intensity. B, C) Quantitative RT-PCR analysis of arterial markers (B) and venous markers (C). **P <0.01.

Figure 2.

Expression of ECM proteins. ECM protein expression after 1 d (A) and 4 d (B). ECM proteins, F-actin (Ph, phalloidin), and nuclei (DAPI) are in green, red, and blue, respectively. Inset: high-magnification view of boxed area. Scale bar =50 μm.

Figure 3.

Decellularized ECFC matrices. A, B) Collagen IV (A) and fibronectin (B) production by ECFCs after 4 d. Red outline delineates magnified right panel. Scale bars = 100 μm (left panels); 500 μm (right panels). C, D) Confocal microscopy images of fibronectin (green) and collagen IV (red) matrix produced by ECFCs after 4 d. White arrowheads indicate an area in the ECM made up of only fibronectin.

Figure 4.

TGFβ and cytoskeleton effects in ECFC-derived ECM. Effect of TGFβ inhibition (A) and Rho kinase inhibition (B) on ECM production by ECFCs. Scale bar = 100 μm.

Figure 5.

Regulation of ECM production by oxygen tension. Effect of 1% O₂ (A) and 5% O₂ (B) on ECM protein expression from ECFCs, HUAECs, and HUVECs. Ph, phalloidin. Scale bar = 100 μm. C) Quantification of percentage area coverage of ECM deposition from tested conditions. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

HIF pathways in 1% O₂ conditions. Collagen IV and fibronectin expression under 1% O₂ conditions in ECs transfected with siRNA of luciferase, HIF1α, or HIF2α. Scale bar = 100 μm.

Figure 7.

HIF pathways in 5% O₂ conditions. Collagen IV and fibronectin expression under 5% O₂ conditions in ECs transfected with siRNA of luciferase, HIF1α, or HIF2α. Scale bar = 100 μm.

Figure 8.

HIF pathways in collagen I production under 5% O₂ conditions. Collagen I expression under 5% O₂ conditions in cells transfected with siRNA of luciferase, HIF1α, or HIF2α. Scale bar = 100 μm.

Figure 9.

HIF regulation of ECM. Schematic depicting HIF pathways in ECFCs and mature ECs that regulate ECM production under varying oxygen tension.