Hypoxia augments outgrowth endothelial cell (OEC) sprouting and directed migration in response to sphingosine-1-phosphate (S1P)

Abstract

Therapeutic angiogenesis provides a promising approach to treat ischemic cardiovascular diseases through the delivery of proangiogenic cells and/or molecules. Outgrowth endothelial cells (OECs) are vascular progenitor cells that are especially suited for therapeutic strategies given their ease of noninvasive isolation from umbilical cord or adult peripheral blood and their potent ability to enhance tissue neovascularization. These cells are recruited to sites of vascular injury or tissue ischemia and directly incorporate within native vascular endothelium to participate in neovessel formation. A better understanding of how OEC activity may be boosted under hypoxia with external stimulation by proangiogenic molecules remains a challenge to improving their therapeutic potential. While vascular endothelial growth factor (VEGF) is widely established as a critical factor for initiating angiogenesis, sphingosine-1-phosphate (S1P), a bioactive lysophospholipid, has recently gained great enthusiasm as a potential mediator in neovascularization strategies. This study tests the hypothesis that hypoxia and the presence of VEGF impact the angiogenic response of OECs to S1P stimulation in vitro. We found that hypoxia altered the dynamically regulated S1P receptor 1 (S1PR1) expression on OECs in the presence of S1P (1.0 μM) and/or VEGF (1.3 nM). The combined stimuli of S1P and VEGF together promoted OEC angiogenic activity as assessed by proliferation, wound healing, 3D sprouting, and directed migration under both normoxia and hypoxia. Hypoxia substantially augmented the response to S1P alone, resulting in ~6.5-fold and ~25-fold increases in sprouting and directed migration, respectively. Overall, this report highlights the importance of establishing hypoxic conditions in vitro when studying ischemia-related angiogenic strategies employing vascular progenitor cells.

Fig 1. S1PR1 expression by OECs is dynamically altered by microenvironment.

OEC expression of S1PR1 under normoxia and hypoxia was qualitatively confirmed by immunocytochemistry (A). The percentage of cells expressing S1PR1 on the cell surface, as confirmed with flow cytometry, was enhanced under hypoxia as compared to the normoxic control (B). Combined stimuli of S1P and VEGF resulted in greater S1PR1 mRNA production after 24h in normoxia (C). Combined stimuli also resulted in a greater proportion of S1PR1+ OECs under both normoxia and hypoxia (D and E). Scale bars represent 40 μm. Data are mean ± SD (n = 4) and normalized to the average untreated control values for either normoxia or hypoxia (C, E; indicated by dashed line) accordingly. An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively and N.S. displays no statistically significant difference between conditions.

Fig 2. The combination of S1P and VEGF induced greater OEC proliferation than either individual stimulus under both normoxia and hypoxia.

Data are mean ± SD (n = 6) and normalized to the initial cell seeding number (indicated by dashed line). An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively.

Fig 3. Combination therapy enhanced S1P-induced motility under both normoxia and hypoxia.

OEC motility was assessed in response to 1.0 μM S1P and/or 50 ng/mL VEGF for 12h under normoxia and hypoxia (A). S1P and VEGF combined resulted in accelerated wound closure compared to either stimuli alone under both normoxia and hypoxia (B). Data are mean ± SD (n = 5) and normalized to the average untreated control values for either normoxia or hypoxia (indicated by the dashed line) accordingly. An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively and N.S. displays no statistically significant difference between conditions.

Fig 4. Hypoxia augmented the angiogenic response of OECs to S1P stimulation in terms of 3D sprouting formation.

S1P and/or VEGF induced OEC sprouting in a fibrin gel after 1 day of culture in normoxia and hypoxia (A). S1P alone or in combination with VEGF led to significantly more sprouts per bead than VEGF alone under hypoxia (B). Scale bars represent 100 μm and 200 μm (DIC inlay). Data are mean ± SD (n = 4) and normalized to the average untreated control values for either normoxia or hypoxia (indicated by the dashed line) accordingly. An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively and N.S. displays no statistically significant difference between conditions.

Fig 5. Hypoxic culture led to enhanced directed migration towards a gradient of S1P in vitro.

A modified transwell assay was used to quantify directed migration/3D matrix invasion of OECs towards S1P in a fibrin gel (A). Gradients of S1P and VEGF were established by sustained delivery from an alginate hydrogel (B). While combined release of S1P and VEGF resulted in greater migration after 48h in both normoxia and hypoxia, S1P alone was equally sufficient under hypoxia (C). Data are mean ± SD (n = 4). An asterisk indicates statistically significant differences (P<0.05) between conditions in normoxia or hypoxia respectively and N.S. displays no statistically significant difference between conditions.