Therapeutic angiogenesis using autologous adipose-derived regenerative cells in patients with critical limb ischaemia in Japan: a clinical pilot study

Abstract

Adipose-derived regenerative cell (ADRC) is a promising alternative source of autologous somatic stem cells for the repair of damaged tissue. This study aimed to assess the safety and feasibility of autologous ADRC implantation for therapeutic angiogenesis in patients with critical limb ischaemia (CLI). A clinical pilot study-Therapeutic Angiogenesis by Cell Transplantation using ADRCs (TACT-ADRC) study-was initiated in Japan. Adipose tissue was obtained by ordinary liposuction method. Isolated ADRCs were injected into the ischaemic limb. We performed TACT-ADRC procedure in five patients with CLI. At 6 months, no adverse events related to the TACT-ADRC were observed. No patients required major limb amputation, and ischaemic ulcers were partly or completely healed during the 6-month follow-up. In all cases, significant clinical improvements were seen in terms of rest pain and 6-min walking distance. Numbers of circulating CD34⁺ and CD133⁺ cells markers of progenitor cell persistently increased after ADRC implantation. The ratio of VEGF-A₁₆₅b (an anti-angiogenic isoform of VEGF) to total VEGF-A in plasma significantly decreased after ADRC implantation. In vitro experiments, cultured with ADRC-conditioned media (CM) resulted in increased total VEGF-A and decreased VEGF-A₁₆₅b in C2C12 cells, but not in macrophages. ADRC-CM also increased CD206⁺ cells expression and decreased TNF-α in macrophages. Autologous ADRC implantation was safe and effective in patients with CLI and could repair damaged tissue via its ability to promote angiogenesis and suppress tissue inflammation.

Figure 1

Clinical outcomes after ADRC implantation. Tissue blood perfusion indicated by ABI (a) and SPP (b) did not significantly change after ADRC implantation. Pain scale was evaluated as per the NRS (c) and ulcer size was calculated as the grand total of major axis length times the minor axis length (d) and showed significant improvement after 1 and 6 months of ADRC implantation; walking distance covered in 6 min (e) after ADRC implantation significantly increased 6 months after ADRC implantation. (f) The changes in blood perfusion levels from pre-surgery to a follow-up period of 6 months. Blood perfusion levels were assessed using a LDBF analyzer. (g) The levels of CRP both before and after ADRC implantation. The values shown are the means ± SEMs. * indicates p < 0.05 and ** indicates p < 0.01, compared to the pre-procedure. ADRC: adipose-derived regenerative cell, ABI: ankle brachial index, SPP: skin perfusion pressure, and NRS: numerical rating scale.

Figure 2

Limb salvage after ADRC implantation. Representative pictures before and after ADRC implantation (Case 1–5).

Figure 3

Circulating progenitor cells after ADRC implantation. FACS analysis indicating an increase in the numbers of circulating CD34⁺ (a, b and c) and CD133⁺ cells (d, e and f) in all the patients after ADRC implantation. FACS: fluorescence-activated cell sorting, ADRC: adipose-derived regenerative cell, MNCs: Mononuclear cells.

Figure 4

Serum VEGF isoforms levels after ADRC implantation. Serum levels of total VEGF-A did not alter significantly (a), but levels of VEGF-A₁₆₅b decreased significantly at 1 month after ADRC implantation (b); the ratio of VEGF-A₁₆₅b to total VEGF-A significantly decreased at 1 and 6 months after ADRC implantation (c). The values shown are the means ± SEMs. * indicates p < 0.05 and ** indicates p < 0.01, compared to the pre-procedure. VEGF: vascular endothelial growth factor.

Figure 5

Effects of ADRC on the expression of VEGF isoforms in macrophages. Effect of ADRC-CM on the expression of total VEGF-A (a), VEGF-A₁₆₅b (b), F4/80 (c), CD206⁺ cells (d), and TNF-α (e) in peritoneal macrophages from WT mice. Macrophages were pretreated with ADRC-CM or control medium for 24 h. mRNA expression of total VEGF-A, VEGF-A₁₆₅b, and TNF-a was measured by real-time PCR and expressed relative to Gapdh levels (n = 12 in each group). The levels of proteins VEGF-A, VEGF-A₁₆₅b, and TNF-a in the ADRC-CM treated cells were assessed by western blotting analysis (n = 4 in each group). The expression of F4/80 and CD206⁺ cells was analyzed by flow cytometric analysis (n = 4 in each group).

Figure 6

Effects of ADRC on the expression of VEGF isoforms in C2C12 cells. Effect of ADRC-CM on the expression of total VEGF-A (a), VEGF-A₁₆₅b (b), and TNF-α (c) in differentiated C2C12 mouse skeletal muscle cells. C2C12 cells were pretreated with ADRC-CM or control medium for 24 h. The mRNA expression of molecules was measured by real-time PCR and expressed relative to Gapdh levels (n = 8 in each group). The levels of proteins VEGF-A, VEGF-A₁₆₅b, and TNF-a in the ADRC-CM treated cells were assessed by western blotting analysis (n = 4 in each group). (d) Proposed scheme for the effects of ADRC therapy in the repair of damaged tissue. Results are presented as the mean ± SEM. * indicates p < 0.05 and ** indicates p < 0.01.

Figure 7

Therapeutic angiogenesis using ADRCs in patients with CLI. (a) Outline of protocol for the TACT-ADRC study. TACT: therapeutic angiogenesis by cell transplantation, ADRC: adipose-derived regenerative cell, CLI: critical limb ischaemia, ABI: ankle brachial index, SPP: skin perfusion pressure, MACE: major adverse cardiovascular event. (b) Flowchart of the TACT-ADRC procedure.