A Closer Look At Tissue And Cell Regeneration In CLI
Given the severe complications associated with critical limb ischemia (CLI), this author offers a revealing look at the research on tissue regeneration and stem cell therapy, and potential therapeutic applications in the future.
Critical limb ischemia (CLI) is an end-stage disease that is provoked by progressive obstruction of the peripheral arteries. It is often associated with long segments of involvement at multiple sites with distal vessel disease. The ischemic insult results in rest pain, which progresses to skin breakdown and gangrene unless ischemia is reversed immediately.
Treatment includes risk factor modification, medical treatment for the vascular symptoms, prevention of systemic complications and revascularization measures. The goals of therapy include restoration of straight line, pulsatile blood flow to relieve ischemia, achieve wound healing, relieve rest pain and avoid major amputation.
However, despite aggressive treatment, many patients with CLI either die or eventually have to undergo a major amputation. Surgical or endovascular (angioplasty and stenting) revascularization approaches are usually not possible due to the diffuse involvement and poor distal vessel reformation. Furthermore, comorbid conditions may adversely affect the outcome of surgical procedures.
Despite the technical advances in interventional and surgical revascularization procedures, a substantial number of patients with peripheral arterial occlusive disease (PAOD) and CLI remain. When it comes to these patients, amputation may be the only option.
Assessing The Research On Tissue Regeneration
Tissue regeneration via the use of stem/progenitor cells has been recognized as a maintenance or recovery system for many organs in adults. The isolation of endothelial progenitor cells (EPCs) derived from the peripheral blood was one of the amazing discoveries for the recognition of “neovessel formation” in adults that occurs as physiological and pathological responses.
These findings that EPCs are home to sites of neovascularization and differentiate into endothelial cells in situ are consistent with the notion of “vasculogenesis.”
Vasculogenesis is a critical paradigm that has been well described for embryonic neovascularization and was proposed recently for adults in whom a reservoir of stem or progenitor cells contributes to vascular organogenesis. On the basis of the regenerative potency, these stem cells/ progenitor cells are expected to be a key factor in therapeutic applications for ischemic diseases.1
The induction of therapeutic angiogenesis by stem cell implantation could provide therapeutic benefits and may help in limb salvage in patients with CLI. One can achieve angiogenesis (collateral formation) either through growth factors or gene encoding for these proteins. Asahara, et al., and Prockop showed that marrow stromal cells secrete many angiogenic cytokines and also have characteristics of stem cells for mesenchymal tissues. 2,3
Several investigators have reported beneficial effects of injecting bone marrow derived stem cells for those with coronary artery disease. 4-13
Using animal models with hind limb ischemia, Iba, et al., injected peripheral blood mononuclear cells (MNCs) or polymorphonuclear leukocytes along with platelets into ischemic limbs via intramuscular injection. Follow-up imaging revealed significant collateral vessel formation. 13,14
Al-Khaldi, et al., investigated the effect of autologous marrow stromal cells on neovascularization and blood flow in rat models with CLI. They detected an increase in vascular density, collateralization and increased blood flow in the ischemic limb. 15 Healing of skin ulcers has occurred in animal models with diabetes following stem cell injection. 16
Yamamoto, et al., evaluated the EPC content in different stem cell sources for the efficacy of therapeutic angiogenesis in limb ischemia in four patients. They quantified the mRNA expression of EPC specific molecules in autologous bone marrow derived or peripheral blood-derived MNCs, and then injected cells intramuscularly in the affected limb for each patient. Researchers observed that transplantation of autologous marrow MNCs increased circulating EPCs in patients and improved ischemic symptoms. 17
Recently, Tateishi-Yuyama, et al., reported that the autologous transplantation of bone marrow mononuclear cells was safe and effective for the achievement of therapeutic angiogenesis in patients with ischemic limbs. The authors also found that injections of bone marrow mononuclear cells could significantly improve the clinical status of these patients. 18
Various researchers have used both bone marrow derived and peripheral blood stem cells with CD34+ endothelial progenitor cells in small studies with good results. (See “A Pertinent Overview Of Studies On The Use Of Stem Cells For CLI” on page 60.)
What The Early Findings From One Recent Study Suggest
Researchers at Sir Ganga Ram Hospital (SGRH) in New Delhi, India conducted a pilot study which was aimed at assessing the “safety, feasibility and efficacy of transplantation of granulocyte colony stimulating factor (G-CSF)–mobilized peripheral blood mononuclear cells (PBMNCs) for the treatment of Buerger’s disease with CLI.”
The study involved patients who were diagnosed with Buerger’s disease (based on digital subtraction angiography (DSA) findings) of the lower limbs with pedal ulcers. Patients were randomized to either the transplant group or the control group. All patients received conventional care for their ulcers.
In the transplant group, the patients received treatment with 600 µg/day of recombinant human G-CSF via subcutaneous injection for five days to mobilize stem/progenitor cells. These patients also received a subcutaneous injection of 5,000 U of dalteparin every 12 hours to avoid the possible risks of embolism due to a G-CSF–induced increase of circulating blood cells.
Using flow cytometry with anti-CD34 antibody staining, researchers assessed the proportion of peripheral blood CD34+ cells in peripheral blood leukocytes after G-CSF treatment. The study authors took measurements every day between the third and fifth days of treatment. When researchers saw an increase in the ratio to >0.10, the G-CSF treatment stopped and researchers harvested peripheral blood stem cells on the next day.
Using a blood cell separator with a concentration to 1 x 108 mononuclear cells/mL, researchers collected a 30 to 50 mL suspension of blood circulating PBMNCs from patients treated with G-CSF. Three hours later, each diseased lower limb received an intramuscular injection (40 sites, 1 to 1.5 cm deep) into the calf under regional anesthesia.
The study authors prospectively collected clinical data, medication and safety laboratory data, and performed follow-up visits for six months. Researchers assessed lower limb blood perfusion via the ankle-brachial index (ABI), TcPO2 and laser Doppler.
The study authors also employed DSA to assess patients one week before and six months after treatment. Researchers assessed the angiographic scores for the formation of new collateral vessels as +0 (no collateral development), +1 (slight), +2 (moderate) and +3 (rich). 18
At the end of the six-month follow-up, the main manifestations, including lower limb pain and ulcers, significantly improved for patients in the transplant group. Their laser Doppler blood perfusion of lower limbs, mean ABI and TcPO2 (peri-ulcer) values increased significantly. Most of the patients in the transplant group demonstrated complete wound healing after cell transplantation in comparison to the control group. Some of the patients in the transplantation group with persistently non-healing ulcers ended up with below knee amputations after six months.
Researchers observed no adverse effects, specifically due to cell transplantation, and only minor toe amputation(s) occurred in the patients who underwent transplants. In contrast, an equal number of control patients had to undergo a lower limb amputation within six months.
Analysis via DSA revealed a significant formation of new vessels after cell transplantation. At the end of the visit, the number of ischemic limbs with rich new collateral vessels (+3) in the transplant patients was statistically significantly higher in comparison to the control patients.
During a six-month follow-up period, researchers observed no side effects specifically due to transplantation after analysis of ECG or dynamic ECG, ultrasound cardiogram, liver and kidney function testing, blood and urine testing, etc.
Key Points For Consideration On The Mechanism Of Action
Previous studies have shown that EPCs can be isolated from the peripheral blood of adult humans, mice and rabbits. Researchers have also found that G-CSF can mobilize peripheral blood CD34+, CD133+ and KDR+ cells with the capacity to differentiate into EPCs that are further able to incorporate into newly forming blood vessels in pathological and non-pathological conditions. 30-34
It has been established that G-CSF mobilization is a viable approach to collect stem/progenitor cells from peripheral blood for autologous transplantation for many diseases such as leukemia and solid cancer. 35
The objective in the aforementioned pilot study was to achieve therapeutic angiogenesis using similar techniques. We observed that pedal wounds in the transplanted patients significantly improved after autologous transplantation of PBMNCs and objective parameters such as ABI, angiographic scores and laser Doppler perfusion scores improved. In a six-month follow-up period, we did not detect angina pectoris, pulmonary embolism and other similar complications due to G-CSF mobilization and adverse effects specifically as a result of transplantation. 36,37
These results indicate that the autologous transplantation of mobilized PBMNCs is an effective and safe therapeutic approach for patients with CLI and Buerger’s disease. For the patients, the development of this potential treatment option would be significant as they may not be able to have conventional surgical bypasses or benefit from endovascular options for revacularization.
In comparison with the method of Tateishi-Yuyama, et al., which requires a large amount of marrow (500 mL), the autologous transplantation of G-CSF–mobilized PBMNCs reported here is a novel alternative for Buerger’s disease with CLI. 18
It has been suggested that G-CSF by itself causes some neovascularization and wound healing in people with ischemic diseases because of its ability to mobilize EPCs into peripheral blood. 38 The G-CSF augments the differentiation of marrow cells into endothelial cells of blood vessels. 39
There are at least two possibilities to explain why G-CSF mobilization plus the transplantation of PBMNCs into ischemic local muscles can result in an excellent therapeutic effectiveness.
The first is that the intramuscular injections of G-CSF–mobilized PBMNCs into ischemic legs directly bring a number of EPCs into ischemic foci, where the EPCs can initiate angiogenesis. The second is that a large number of transplanted PBMNCs can secrete in vivo in the injected sites several angiogenic factors to activate the EPCs nearby ischemic tissues to form new vessels and repair impaired vessels. 40 The two possibilities may coexist in this therapeutic approach.
In the aforementioned pilot study, 50 percent of the transplant patients demonstrated remarkable improvement in all the parameters. Therefore, one can conclude that GCSF mobilized PBMNCs constitute simple, safe and effective therapy for patients with Buerger’s disease and CLI.
Future studies are obviously needed to evaluate the precise efficacy of this therapy in a large number of patients and to determine the particularities of their mechanism of action in provoking therapeutic angiogenesis.
What About The Future Of Regenerative Medicine In CLI?
As evident from the current literature, the isolation of endothelial progenitor cells (EPCs) derived from peripheral blood, autologous adult bone marrow (BM) and most recently from the human umbilical cord blood is an epoch-making event for the recognition of neovessel formation.
In regard to stem cell therapy, Kim, et al., examined the use of umbilical cord blood-derived multipotent stem cells for Buerger’s disease and ischemic limb disease in an animal model. 41
In Kim’s study, four men with Buerger’s disease who had already received medical treatment and surgical therapies received transplanted human leukocyte, antigen-matched human umbilical cord blood (UCB)-derived mesenchymal stem cells (MSCs). After the stem cell transplantation, ischemic rest pain suddenly disappeared from their affected extremities. The necrotic skin lesions healed within four weeks. In the follow-up angiography, digital capillaries increased in number and size. In addition, vascular resistance in the affected extremities, in comparison with the preoperative examination, was markedly decreased due to improvement of the peripheral circulation.
Since an animal model of Buerger’s disease is absent, athymic mice with hind limb ischemia received transplants of human UCB-derived MSCs. Up to 60 percent of the hind limbs were salvaged in the animals with a ligated femoral artery. With in situ hybridization, researchers detected the human UCB-derived MSCs in the arterial walls of the ischemic hind limb in the treated group. These findings suggest that human UCB-derived MSC transplantation may be a new and useful therapeutic option for Buerger’s disease and similar ischemic diseases.
At our institution in India, we are about to commence a comparative study to assess the safety and efficacy of bone marrow derived MSCs and umbilical cord derived MSCs with the standard treatment in patients with ischemic limb disease.
Clarifying The Unanswered Questions
The current literature does not describe a definite number of cells that one should apply to obtain a certain benefit. Until now, no correlation between the applied cell number and clinical outcome is known. 42,43
To date, most studies of progenitor cell application do not elucidate which cell type is involved in the process of neovascularization. Other researchers have reported that the cell suspension used contained a heterogeneous population of circulating blood driven progenitor cells. 44,45 Approximately 50 percent of the cells showed endothelial characteristics as demonstrated by the expression of kinase insert domain (KDR) and Ve-cadherin.
Additionally, the cells expressed cell surface markers of stem/progenitor cells and the panleukocyte marker CD45, which is consistent with the literature. 46,47
Given the variant clinical nature of the many studies performed so far, little is known about the fate of the injected cells and one can only speculate about the amount of endothelial differentiation, cell fusion or migration into other tissue layers.
As there is a lack of control groups in some studies, the observed changes cannot be unambiguously attributed to the bone marrow driven or CPC administration. Other contributing factors might be spontaneous improvement, placebo effect and medical therapy.
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