Lysophosphatidic Acid Enhances Stromal Cell-Directed Angiogenesis

Statement of Purpose: Ischemic diseases such as peripheral vascular disease (PVD) affect more than 15% of the general population and are characterized by loss of blood flow to the extremities. While the delivery of cells or growth factors to promote angiogenesis has shown promise, the high cost of producing and purifying proteins such as vascular endothelial growth factor (VEGF) limits large-scale implementation. Lysophosphatidic acid (LPA) is a platelet derived growth factor that has been reported to promote the secretion of angiogenic factors by human adipose-derived stromal cells (hASC) and is implicated in promoting angiogenesis in metastatic tumors. Additionally, hASC are readily available, have outstanding proliferative capacity, and can function as pericytes in vivo, thus making the concomitant delivery of LPA with hASC an attractive target for investigation. We hypothesized that LPA would increase the secretion of angiogenic cytokines by hASC under ischemic conditions, and that this combination would synergistically promote revascularization and recovery in a mouse critical hindlimb ischemia model.

Methods: hASC were cultured in FBS-free α-MEM supplemented with 0.1% (w/v) fatty-acid free BSA and incubated in hypoxia (1% O₂) for 24 h to simulate the serum deprivation and hypoxia (SD/H) of in vivo ischemia. To explore the effect of LPA on the transcriptional regulation of angiogenic factors, hASC were cultured in SD/H with 25 μM LPA. When used, 10 μM LPA_1/3 receptor inhibitor (Ki16425) was added to medium containing LPA. qPCR was performed using TaqMan primers for VEGF and basic fibroblast growth factor (bFGF). To determine the functional effects of LPA treatment, conditioned media from hASC incubated with 25 μM LPA for 24 h was removed and used to assay migration of endothelial colony forming cells (ECFC). To study the ability of LPA and hASC to promote angiogenesis in vivo, 1e6 hASC were entrapped in an 8 mm x 1 mm fibrin gel containing 25 μM LPA and placed over the defect site of a ligated and segmentally resected femoral artery in NOD SCID gamma (NSG) mice.

Results: After 24 h in SD/H, hASC cultured with 25 μM LPA exhibited significantly higher levels of VEGF and bFGF expression. These increases were abrogated by the addition of 10 μM Ki16425. Conditioned media from LPA-treated hASC was also significantly more chemoattractive to endothelial cells in a transwell migration assay, but the addition of Ki16425 negated this effect. Similarly, media from hASC embedded in fibrin gels with 25 μM LPA in SD/H in vitro showed higher levels of VEGF production as measured by ELISA. Importantly, ischemic hindlimbs treated with both ASC and LPA had significantly increased numbers of blood vessels and functional recovery after 2 weeks compared to ASC or LPA alone.

Conclusions:  These data demonstrate that LPA enhances the pro-angiogenic capacity of hASC under SD/H. This information will be valuable for optimizing cell-based treatments for PVD and ischemic disease in vivo.