Novel Cell-Free Strategy for Therapeutic Angiogenesis: In Vitro Generated Conditioned Medium Can Replace Progenitor Cell Transplantation

BACKGROUND: Current evidence suggests that endothelial progenitor cells (EPC) contribute to ischemic tissue repair by both secretion of paracrine factors and incorporation into developing vessels. We tested the hypothesis that cell-free administration of paracrine factors secreted by cultured EPC may achieve an angiogenic effect equivalent to cell therapy. METHODOLOGY/PRINCIPAL FINDINGS: EPC-derived conditioned medium (EPC-CM) was obtained from culture expanded EPC subjected to 72 hours of hypoxia. In vitro, EPC-CM significantly inhibited apoptosis of mature endothelial cells and promoted angiogenesis in a rat aortic ring assay. The therapeutic potential of EPC-CM as compared to EPC transplantation was evaluated in a rat model of chronic hindlimb ischemia. Serial intramuscular injections of EPC-CM and EPC both significantly increased hindlimb blood flow assessed by laser Doppler (81.2+/-2.9% and 83.7+/-3.0% vs. 53.5+/-2.4% of normal, P<0.01) and improved muscle performance. A significantly increased capillary density (1.62+/-0.03 and 1.68+/-0.05/muscle fiber, P<0.05), enhanced vascular maturation (8.6+/-0.3 and 8.1+/-0.4/HPF, P<0.05) and muscle viability corroborated the findings of improved hindlimb perfusion and muscle function. Furthermore, EPC-CM transplantation stimulated the mobilization of bone marrow (BM)-derived EPC compared to control (678.7+/-44.1 vs. 340.0+/-29.1 CD34(+)/CD45(-) cells/1x10(5) mononuclear cells, P<0.05) and their recruitment to the ischemic muscles (5.9+/-0.7 vs. 2.6+/-0.4 CD34(+) cells/HPF, P<0.001) 3 days after the last injection. CONCLUSIONS/SIGNIFICANCE: Intramuscular injection of EPC-CM is as effective as cell transplantation for promoting tissue revascularization and functional recovery. Owing to the technical and practical limitations of cell therapy, cell free conditioned media may represent a potent alternative for therapeutic angiogenesis in ischemic cardiovascular diseases

Paracrine factors secreted by endothelial progenitor cells prevent oxidative stress-induced apoptosis of mature endothelial cells

Endothelial progenitor cells (EPC) play a fundamental role in tissue regeneration and vascular repair. Current research suggests that EPC are more resistant to oxidative stress as compared to differentiated endothelial cells. Here we hypothesized that EPC not only possess the ability to protect themselves against oxidative stress but also confer this protection upon differentiated endothelial cells by release of paracrine factors. To test this hypothesis, HUVEC incubated with conditioned medium obtained from early EPC cultures (EPC-CM) were exposed to H2O2 to assess the accumulation of intracellular ROS, extent of apoptosis and endothelial cell functionality. Under oxidative stress conditions HUVEC treated with EPC-CM exhibited substantially lower levels of intracellular oxidative stress (0.2+/-0.02 vs. 0.4+/-0.03 relative fluorescence units, p0.05), suggesting that soluble factors secreted by EPC, possibly via broad synergistic actions, exert strong cyto-protective properties on differentiated endothelium through modulation of intracellular antioxidant defensive mechanisms and pro-survival signals

Effect of EPC-CM and EPC transplantation on ischemic muscle neovascularization.

<p>(A) Representative images of healthy (non-operated) and ischemic hindlimb muscle of animals treated with EPC-CM, EPC or control medium stained with BS-1 lectin (FITC) to localize capillaries. (B) Quantitative analysis of capillary density expressed by the number of capillaries per muscle fiber. *, P<0.05. (C) NG2⁺ pericytes (white arrows) were identified (red fluorescence) by being adjacent to endothelial cells stained for von Willebrand Factor (green fluorescence). (D) Quantitative analysis of NG2⁺ cells per capillary in healthy and ischemic hindlimbs treated with EPC-CM, EPC and control medium. *, P<0.05.</p

Concentration of selected angiogenic growth factors in EPC-CM.

<p>Selected cytokine levels were measured in the conditioned media from culture expanded EPC incubated in hypoxic or normoxic condition for 72 hours.</p

EPC-CM promotes progenitor cells homing to the ischemic tissue.

<p>Representative fluoresence pictures of CD34⁺ immunostaining in ischemic hindlimb tissue 3 days (left panel, iDAY10) and 4 weeks after treatment (right panel, iDAY35). The number of CD34⁺ cells on iDAY10 was significantly higher in EPC-CM treated limbs (A) as compared to control treated animals (B) with no evidence for focal recruitment, as CD34⁺ cells were found to similar extent in different anatomic regions (C). In comparison, 4 weeks after treatment (iDAY35), tissue sections from show decreased numbers of CD34⁺ cells in EPC-CM treated limbs (D) equivalent to numbers found in control (E). Quantitative analysis is depicted reflects the temporary recruitment of CD34⁺ cells to the ischemic limbs in EPC-CM treated animals (F). *, P<0.001.</p

EPC-CM and EPC transplantation improve blood perfusion in the ischemic hindlimb.

<p>(A) Representative images of hindlimb blood flow measured by laser Doppler immediately after intramuscular injection of EPC-CM, EPC or control medium (iDAY1, 4 weeks after occlusion of the femoral artery) and the end of the experiment (5 weeks after treatment, iDAY35). (B) Quantitative analysis of blood flow expressed as perfusion ratio of the ischemic to the contralateral (non-operated) hindlimb over the observation period (iDAY1: day of EPC-CM or EPC injection; iDAY7; iDAY14; iDAY21; iDAY28 and iDAY35: 1, 2, 3, 4 and 5 weeks after injection, respectively). *, EPC-CM vs. Control, P<0.01; †, EPC vs. Control, P<0.01.</p

Effect of EPC-CM and EPC transplantation on ischemic muscle function and activity.

<p>(A) Muscle function was tested by swimming exercise and expressed as the ratio of ischemic to healthy hindlimb stroke numbers in animals treated with EPC-CM, EPC, control medium or non-operated animals. Swimming activity was monitored for 3 minutes at 1 minute intervals. Rats treated with control medium were not able to complete the exercise due to obvious exhaustion with drowning. *, EPC-CM vs. Control, P<0.05; †, EPC vs. Control, P<0.05; ‡ Non-operated vs. EPC-CM and EPC, P<0.05. (B) Muscle mitochondrial activity in animals treated with EPC-CM, EPC or control medium was assessed by MTT reduction in the healthy and ischemic hindlimbs. The activity index is indicated as the ratio ischemic to healthy MTT values per gram of dry tissue. *, P<0.05.</p

EPC-CM stimulates the mobilization of bone marrow-derived EPC.

<p>Representative FACS analysis charts of CD34⁺/CD45⁻ cells isolated from bone marrow (A and B) and peripheral blood (D and E) of EPC-CM and control media treated animals 3 days after the last intramuscular injection. Quantitative analyses show significantly increased numbers of CD34⁺/CD45⁻ progenitor cells in the BM (C), and the peripheral blood (F) of EPC-CM treated animals. *, P<0.05.</p

Pro-survival properties of EPC-CM.

<p>Serum starved HUVEC were incubated in EPC-CM or control medium for 24 hrs and analyzed for cell survival and extent of apoptosis. (A) The number of viable cells was assessed by CyQuant® NF and expressed relative to control. (B) Apoptosis was measured by the level of caspase −3/7 activity by Apo-ONE® and expressed relative to control. *, P<0.001.</p

Design of <i>in vivo</i> experiments.

<p>Two <i>in vivo</i> experimental settings were designed to address the effect of the treatment modalities on tissue regeneration and neovascularization (Exp. Set 1) as well as progenitor cells mobilization and recruitment (Exp. Set 2). In both settings, rats were treated by 3 separate intramuscular injections within 7 days (iDAY1- iDAY7), 4 weeks after inducing ischemia as indicated by the white arrowheads (∇). Black arrowheads (▾) indicate blood flow measurements by Laser-Doppler of the hindlimb. T indicates tissue harvest and immunohistochemistry analysis.</p