Lysophosphatidic acid enhances stromal cell-directed angiogenesis

Abstract

Ischemic diseases such as peripheral vascular disease (PVD) affect more than 15% of the general population and in severe cases result in ulcers, necrosis, and limb loss. While the therapeutic delivery of growth factors to promote angiogenesis has been widely investigated, large-scale implementation is limited by strategies to effectively deliver costly recombinant proteins. Multipotent adipose-derived stromal cells (ASC) and progenitor cells from other tissue compartments secrete bioactive concentrations of angiogenic molecules, making cell-based strategies for in situ delivery of angiogenic cytokines an exciting alternative to the use of recombinant proteins. Here, we show that the phospholipid lysophosphatidic acid (LPA) synergistically improves the proangiogenic effects of ASC in ischemia. We found that LPA upregulates angiogenic growth factor production by ASC under two- and three-dimensional in vitro models of serum deprivation and hypoxia (SD/H), and that these factors significantly enhance endothelial cell migration. The concurrent delivery of LPA and ASC in fibrin gels significantly improves vascularization in a murine critical hindlimb ischemia model compared to LPA or ASC alone, thus exhibiting the translational potential of this method. Furthermore, these results are achieved using an inexpensive lipid molecule, which is orders-of-magnitude less costly than recombinant growth factors that are under investigation for similar use. Our results demonstrate a novel strategy for enhancing cell-based strategies for therapeutic angiogenesis, with significant applications for treating ischemic diseases.

Figure 1. LPA receptor expression in human ASC is dependent on oxygen microenvironment.

(a) LPAR1 expression is unchanged by SD/H, but (b) LPAR2 expression is significantly higher in SD/H. LPAR3 expression is undetectable in either condition, but SD/H also increases expression of (c) LPAR4 and (d) LPAR5 (n = 4). **p < 0.01 vs. control, ***p < 0.001 vs. control.

Figure 2. LPA enhances the proangiogenic effects of ASC under ischemia in vitro.

Expression of (a) VEGF and (b) FGF2 are upregulated by SD/H and further enhanced with the addition of 25 μM LPA. In both cases, the addition of Ki16425 abrogates this effect (n = 4). Data represents combined gene expression from three unique donors. *p < 0.05 vs. control, **p < 0.01 vs. control, ***p < 0.001 vs. control.

Figure 3. ASC treated with LPA promote endothelial cell migration.

(a) Medium conditioned by ASC in the presence of LPA under SD/H is significantly more chemoattractive to ECFC than medium from non-treated or inhibitor-treated ASC. The addition of LPA or Ki16425 to unconditioned medium had no effect on ECFC migration (n = 5). (b) LPA promotes VEGF secretion from ASC entrapped in 3D fibrin gels under SD/H compared to cells treated with no LPA or Ki16425. (n = 6). *p < 0.05 vs. control, **p < 0.01 vs. control, ***p < 0.001 vs. control (c) LPA also increases production of other angiogenic and inflammatory cytokines. Data are presented as average subtracted background fluorescence intensity normalized to positive controls.

Figure 4. Co-delivery of LPA with ASC in fibrin gels significantly improves angiogenesis in a murine model of critical limb ischemia.

Representative H&E stained sections from the quadriceps of (a) normal hindlimbs and (b) ischemic limbs treated with fibrin gels containing 25 μM LPA, (c) ASC, or (d) LPA and ASC show the presence of more large vessels in limbs treated with cells and LPA together. Similarly, CD31 staining reveals the formation of larger intramuscular blood vessels in limbs receiving (h) both LPA and ASC, while (e) normal tissue and defects receiving (f) LPA and (g) ASC alone have fewer and smaller vessels. (i) Blood vessel quantification from H&E stained sections confirms these results (n=8). Scale bars represent 100 μm; arrows indicative of vessels with defined lumens and erythrocytes. *p < 0.05 vs. other groups. (j) Hindlimbs were visually assessed for severity of toe and foot necrosis in each treatment group.