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.

Characterizing Human Pluripotent-Stem-Cell-Derived Vascular Cells for Tissue Engineering Applications

“…Vascular derivatives, including both EVCs and derived ECs, obtained under low-oxygen differentiation conditions demonstrated a greater propensity to produce ECM proteins collagen IV, fibronectin, and laminin. In our previous studies, we also document a correlation between hypoxic conditions and ECM production [26,30]; hypoxic exposure promoted ECM deposition from mature ECs and yielded a more fibrous ECM ultrastructure from cocultures of fibroblasts and breast cancer cells. In both our previous studies and current study, we could not observe collagen I deposition from any types of ECs tested in atmospheric conditions; however, 5% O 2 conditions stimulated collagen I production by ECs harvested from human umbilical vein or artery [26].…”

“…To derive continuous EVCs, the differentiation took place under 5% O 2 conditions for the entire 12 days, as previously described [25]. To observe ECM production capacity, the differentiation media were replaced with endothelial growth medium (EGM2) supplemented with 10% FBS for 48 h [26].…”

“…Cells were prepared for immunofluorescence as previously described [26]. Briefly, cells were fixed with 3.7% formaldehyde; blocked in 1% bovine serum albumin; treated with 0.1% Triton-X (Sigma); incubated with the mouse antihuman collagen I (Abcam), mouse anti-human collagen IV (Santa Cruz Biotechnology), rabbit anti-human fibronectin (Santa Cruz Biotechnology), rabbit anti-human laminin (Abcam), or mouse anti-human Ki67 (Invitrogen), followed by the either anti-mouse FITC (Sigma) or anti-rabbit Alexa Fluor 488 conjugate (Molecular Probes) and Phalloidin-546 (Invitrogen); and counterstained with DAPI (Roche Diagnostics) to visualize cell nuclei.…”

“…Two-step reverse transcription-polymerase chain reaction (RT-PCR) was performed on the same cDNA amount for all samples as previously described in accordance with Applied Biosystems manufacturer instructions [26]. Collagen I, collagen IV, fibronectin, laminin, VEGF, Ang1, Ang2, VEcad, ephrinB2, EphB4, Nrp1, Nrp2, and GAPDH (endogenous control) primer sets (Applied Biosystems) were assessed using the comparative computerized tomography method (Applied Biosystems) to calculate the amplification differences between samples.…”

“…Flow cytometry was performed as previously described [26]. Briefly, cells were incubated with PE-VEcad or PEIgG.…”