Critical limb ischemia (CLI) is a severe form of peripheral arterial disease (PAD) associated with high morbidity and mortality. It remains a significant threat to both life and limb of patients at risk. Treatment goals for CLI include relief of claudication, ulcer healing, avoidance of amputation, and improvement in patient's quality of life. Currently, treatment options for CLI patients include risk factor modification, endovascular strategies, open surgery, and amputation. However, despite the availability of treatment options, many patients are poor candidates for either endovascular or surgical approach due to their comorbidities. The era of gene therapy and cellular based modalities, or so-called therapeutic angiogenesis, emerged to fill in the gap of treatment options for these patients. Studies on therapeutic angiogenesis involve a complex process of determining the appropriate growth factors, dosing regimen, delivery, and route. While the existing data from multiple studies are conflicting and equivocal, some studies independently suggest clinical benefit, thus there is still reason to be hopeful.

This review summarizes the current findings of the emerging modalities for therapeutic angiogenesis as the search for the best treatment option continues.

EPIDEMIOLOGY

The prevalence of PAD based on several epidemiologic studies ranges from 3 to 10%.¹ This figure is based mostly on asymptomatic patients with PAD. Their prevalence can only be estimated by non-invasive measurements in the general population wherein ankle-brachial pressure index (ABI) is the most commonly used test. A resting ABI ≤0.90 is the hemodynamic definition of PAD caused by a significant peripheral arterial stenosis.¹ On the other hand, CLI is a subgroup of patients with an advanced stage of PAD. Patients with CLI have chronic ischemic rest pain, ischemic skin lesions, ulcers or gangrene.² Every year, an estimated 500 to 1,000 new cases per million will be diagnosed with CLI in North America. In the United States alone, the incidence is 12% in the adult population, which is an estimated 8 to 10 million in 2012.³ At the age of 70, nearly 20% of adults will be diagnosed with CLI.³

The prognosis of CLI is grim. The utmost significance of CLI in patient prognostication is exemplified by its classification as a coronary heart disease (CHD) equivalent, which carries a 20% risk of coronary event in 10 years. Left untreated, in 1 year, one-fourth will die, one-third will have undergone amputation of one or both legs, and the rest will be alive with both limbs. In 5 years, more than half of the patients with CLI will be dead.³ Furthermore, quality of life indices of patients with CLI were compared to those of terminal cancer patients and were noted to be similar.⁴

As with the clinical aspect of the disease, the health economic impact of CLI is considerable. A study conducted in 1990 analyzed the cost of the total clinical care of patients with CLI and was estimated to be $43,000 per patient per year.⁵ The costs not only included the hospital admission and procedures performed, but a substantial amount was also attributed to the health care services provided after discharge. This includes rehabilitation facilities, home health care, and transportation. The mean cost of patients undergoing surgical bypass was noted to be a third higher than the endovascular treatment approach. Interestingly, the median cost of managing a patient after amputation is double the cost of a patient who underwent successfully limb salvage either by angioplasty or surgical bypass.⁶ As with other cardiovascular diseases, CLI not only has an astounding clinical burden, but also has a considerable financial impact on society.

Risk Factors and Patient Demographics

CLI is associated with similar risk factors as most cardiovascular and cerebrovascular diseases. Risk factor identification is integral since risk modification remains a cornerstone for management. Increasing age, male gender, African-American race, smoking, diabetes mellitus (DM), hypertension and dyslipidemia are the "classic" risk factors for CLI. The second Trans-Atlantic Inter-society Consensus Document on the Management of Peripheral Artery Disease (TASC II) guidelines have also identified the following additional risk factors for CLI: increased C-reactive protein (CRP), hyperviscosity and hypercoagulable states, hyperhomocysteinemia, and chronic renal insufficiency.^1,7

MANAGEMENT OPTIONS FOR CRITICAL LIMB ISCHEMIA

Risk Factor Modification and Medical Therapy

Aggressive risk factor modification is the initial therapy of both intermittent claudication (IC) and CLI. Lifelong treatment should include abstaining from cigarette smoking and adequate control of DM, dyslipidemia, and hypertension. The American College of Cardiology (ACC) and American Heart Association (AHA) also recommend daily exercise and a non-atherogenic diet in their joint guidelines.⁸ Notable Class I recommendations include blood pressure control, use of aspirin or clopidogrel, and statins. It is also important to note that DM is an independent risk factor for amputations in patients with CLI.^9,10 Therefore, hemoglobin AIC and regulation of blood glucose levels in diabetics are also of utmost importance.^1,7

However, in contrast to IC, treatment of CLI warrants a more aggressive approach. The natural history of CLI and its grim prognosis mandates a more aggressive strategy and treatment of underlying ischemia.⁸ In addition, the evidence of the effectiveness of therapies such as aspirin to prevent cardiovascular events is not sufficiently established and proven in CLI patients. In fact, in the PREVENT III (Prevention of Autogenous Vein Graft Failure in Coronary Artery Bypass Procedures) trial comparing other pharmacological therapies, the only class of drug that was associated with improved survival in CLI patients was statins.¹¹

Pharmacological Treatment Options

Among the conservative or pharmacological options in CLI, vasodilator therapy is a physiological and logical approach. By dilating peripheral arteries and arterioles in the limb, flow and subsequently skeletal muscle perfusion may improve. Prostaglandin E-1 (PGE-1), iloprost (synthetic prostacyclin analogue), and ciprostene (epoprostenol analogue) have been studied in multiple placebo-controlled trials for patients with CLI who are not candidates for revascularization procedures.⁸ However, in the eight short-term trials of parenteral administration of PGE-1, the results were inconsistent in controlling pain and ulcer healing. In 1999, the Ischemia Cronica degli Arti Inferiori (ICAI) study group conducted a randomized, controlled, open-label trial with PGE-1 on 1,560 patients with CLI, the largest study on vasodilator therapy in CLI. The CLI patients were randomized to have intravenous infusion of 60 mcg of PGE-1 (n=771) or no PGE-1 (n=789) daily up to 28 days. The initial results were encouraging as the combined outcome of death, major amputation, persistence of CLI, or major adverse cardiovascular event (MACE) were lower in the PGE-1 group (RR, 0.87 [95% CI, 0.81 to 0.93]; P <0.001).¹² However, compared to the post-hospital discharge, the 6-month follow up results were not as impressive. The ICAI group concluded that treatment with prostaglandin provides clinical benefit in CLI only in the short term period.¹² As of the latest guideline, parenteral administration of PGE-1 or iloprost for 7-28 days is only a class IIB recommendation and may be considered to reduce ischemic pain and facilitate ulcer healing in patients with CLI.⁸

A non-pharmacologic approach of relieving symptoms of CLI is by spinal cord stimulation (SCS). Several studies have evaluated the efficacy of SCS with the aim of providing relief from ischemic rest pain, delaying or avoiding surgery, and improving the quality of life of patients. However, similar to vasodilator studies, five randomized clinical trials demonstrated conflicting results. A meta-analysis in 2009 demonstrated that there is insufficient evidence for the higher efficacy of SCS compared to optimal or best medical treatment alone with regards to mortality and limb survival.^13,14

Endovascular and Surgical Therapies

Patients who do not undergo revascularization, whether endovascular or surgical bypass, may ultimately require amputation. Surgical bypass was historically the gold standard for revascularization in patients with CLI. However, the Bypass versus Angioplasty in Severe Ischemia of the Leg (BASIL) trial showed that either approach has similar outcomes but surgery was more expensive than angioplasty in the first year.¹⁵ The same study group in 2010 demonstrated that balloon angioplasty patients have a higher early failure rate compared to bypass surgery and that most patients with initial balloon angioplasty ultimately required surgery.¹⁶ Failure rate in balloon angioplasty can be explained by restenosis caused by neointimal hyperplasia. Since the BASIL trial, trials on endovascular therapies using drug eluting stents and drug eluting balloons (DEB) attempted to address the concern for neointimal hyperplasia in peripheral artery interventions. In the Local Taxane with Short Exposure for Reduction of Restenosis in Distal Arteries (THUNDER) trial, 154 patients with femoropopliteal artery stenosis were treated either with balloon catheters coated with paclitaxel, uncoated balloons with paclitaxel dissolved in the contrast medium or uncoated balloons without paclitaxel. The study concluded that the use of paclitaxel-coated balloon catheters when compared to uncoated balloons significantly lowered the incidence of restenosis (using late lumen loss and rate of angiographic stenosis as endpoints) at 6 months. At 6, 12, and 24 months, the rate of target-lesion revascularization was also significantly reduced.¹⁷ From 2008 to 2013, including the THUNDER trial, there were six randomized trials that were published (total n=535) that compared DEB to uncoated balloons in the treatment of chronic, symptomatic femoropopliteal disease.^17-22 All six trials used late lumen loss as one of the primary endpoint and showed comparable and statistically significant reductions in late lumen loss with the use of DEB compared to controls.

Currently, either endovascular therapy or open vascular surgery is an acceptable management of infrainguinal PAD and CLI. However, there is a disagreement on which option is more successful for CLI patients. The data available supporting endovascular therapies on CLI patients are mostly limited to nonrandomized trials focusing on a single device. Investigators will attempt to address these concerns in the Best Endovascular versus Best Surgical Therapy in Patients with CLI (BEST-CLI) trial. With its unique and pragmatic design, the trial will allow the site investigators to use almost any device or technique that they believe to be appropriate for their respective patients. Acknowledging that endovascular therapy in PAD is a rapidly evolving field, the trial will also review and use novel techniques or devices as they emerge into the market. Since the fall of 2014, the trial has been enrolling patients with a target of 2,100 patients with CLI over the next four years. They will be randomized into receiving the "best" available endovascular therapy and "best" available open vascular surgery with a primary endpoint of major adverse limb event-free survival.²³ This trial will hopefully resolve the existing disagreement on the optimal management of CLI patients.

Invasive treatment with either percutaneous intervention or surgery with the aim of revascularization is the best therapeutic option in CLI patients; however, many patients are poor candidates for either procedure because of concomitant diseases or unfavorable anatomy. This subgroup of patients is often classified as "no-option" patients. Despite the available treatment options, oftentimes the only remaining route is palliative.

HISTORY OF ANGIOGENESIS

Angiogenesis is defined as the process of forming new blood vessels from pre-existing blood vessels. Its history can be traced as far back in the late 19th century. Rudolf Virchow along with other German pathologists observed that human tumors are highly vascularized.²⁴ It was not until 1939 when Sandison, Ide, and their colleagues postulated the presence of tumor derived vascular growth factors after they implanted a tumor in a rabbit's ear and observed the development of new blood vessels. After isolating a protein that is mitogenic only to endothelial cells, Ferrara and colleagues named the protein vascular endothelial growth factor (VEGF) in 1989.²⁵ Several studies on growth factors have evolved since then and the race for the use of these newly discovered proteins started. In 1992, while studying glioblastoma multiforme, Keshet and Plate observed that the most ischemic portions of the tumor had the highest VEGF expression.²⁶ This vital hypothesis was the key concept that hypoxia was the trigger for the release of VEGF which then leads to angiogenesis.

DEFINITION OF ANGIOGENESIS

Angiogenesis may also be defined as the formation of new capillaries from postcapillary venules and is mainly stimulated by hypoxia-inducible factor (HIF-1) expression.^27,28 Several terminologies are often interchangeable with angiogenesis; however, some have specific definitions such as arteriogenesis and vasculogenesis. Arteriogenesis, also called collateral growth, is defined as the growth of developed arteries as visualized by conventional angiography.^29,30 On the other hand, vasculogenesis is the process of blood vessel in situ formation from endothelial progenitor cells and vascular progenitor cells.^31,32 Until recently, vasculogenesis was once thought to be only existent in embryonic development but is now known to play a role in post-ischemic vascular regeneration.

GROWTH FACTOR TRIALS

Clinical trials in CLI use several parameters in assessing the efficacy of therapeutic angiogenesis. These parameters include evaluating the resolution of ischemic pain, ABI, appearance of visible collateral vessels by digital subtraction angiography (DSA), pain free walking time, transcutaneous oxygen tension (TcPO2), and magnetic resonance imaging for quantification of flow.^33,34

Fibroblast growth factor (FGF), hepatocyte growth factor (HGF), Angiopoetin (Ang-1), VEGF, and HIF-1 are the growth factors that are most widely studied in angiogenesis trials (Table 1). These growth factors are produced using gene coding to stimulate angiogenesis and promote endothelial cell proliferation and mainly target the endothelial cells.

Table 1: Gene Therapy Trials in Therapeutic Angiogenesis

Trial	Year	Treatment	Patients	Follow up	Primary Endpoint	Outcome
Makinen et al.34	2002	VEGF	54 (14 CLI)	3 mo	Digital subtraction angiography analysis of vascularity	No significant difference in amputation rate, ulcer healing or rest pain but increased vascularity in VEGF group
Kusumanto et al.36	2006	VEGF	54	100 d	Amputation rate at 100 days	No significant difference in amputation rate but improved wound healing and ABI
TALISMAN investigators37	2008	FGF-1	125	25 wk	Complete healing of at least 1 ulcer at week 25	No significant difference in ulcer healing but with a significant reduction in amputations and a trend towards increased survival
HGF-STAT40	2008	HGF (high vs. mid vs. low dose)	104	6 mo	Safety, ABI, Amputation, wound healing, and TcPO2	Higher TcPO2 in the high dose group but no difference in ABI, pain relief, and wound healing among groups
TAMARIS38	2011	FGF-1	525	1 year	Major amputation or death at 1 year	No difference in amputation rates and mortality between groups

Vascular Endothelial Growth Factor

Among all the growth factors, VEGF and FGF are the most widely studied in clinical trials. Isner et al. demonstrated the intra-arterial gene transfer of a plasmid that encodes for VEGF on a patient with an ischemic right leg. Using an angioplasty balloon coated with phVEGF165, the balloon was inflated in the distal popliteal artery. Four weeks after gene therapy, collateral vessels were noted at the knee to the ankle levels supporting the hypothesis that administration of endothelial cell mitogens can promote angiogenesis with limb ischemia.^32,33,35 Another study using phVEGF165 was done on 54 diabetic patients with CLI. Kusumanto et al. evaluated the amputation rate (primary endpoint) of these patients after 100 days. Although there was no significant reduction in amputation, there was meaningful improvement in the secondary endpoints such as ABI and wound ulcer healing.³⁶

Fibroblast Growth Factor

Fibroblast growth factor -1 (FGF-1) was studied by the TALISMAN (Tenecteplase Versus Alteplase in Ischemic Stroke Management) investigators in 2008. They observed the occurrence of complete healing of at least one ulcer in the treated limb after 25 weeks of follow up. A total of 8 injections of FGF-1 were given the treatment group on days 1, 15, 30, and 45 for a total of 16mg of NV1FGF, a plasmid-based angiogenic gene delivery system for local expression of FGF-1. There were significant improvements in ulcer healing in patients who received NV1FGF (19.6%) versus placebo (14.3%; P = 0.514). In addition, there was also a trend for increased in survival with the use of NV1FGF (HR 0.460; P = 0.105).³⁷ However, in 2011, TAMARIS (Efficacy and Safety of XRP0038/NV1FGF in Critical Limb Ischemia Patients With Skin Lesions), a phase 3 randomized clinical trial demonstrated contrasting results from the TALISMAN investigators. A total of 525 patients from 30 countries were studied with a primary endpoint of major amputation or death after 1 year. Similar to the previous trial, patients received eight intramuscular injections of NV1FGF or placebo in the index leg on days 1, 15, 29, and 43. There was no significant difference between the placebo group and the treatment group with regards to the primary endpoint (HR 1.11, 95% CI 0.83-1.49; p=0.48). The group concluded that there was no evidence that FGF-1 was effective in patients with CLI with regards to amputation rate and survival.³⁸

Hepatocyte Growth Factor

HGF or scatter factor (SF) was studied by Van Belle et al. in vivo in a rabbit model of hind limb ischemia. They demonstrated that HGF stimulates the proliferation and migration of endothelial cells via the c-Met receptor. They demonstrated a significant improvement in collateral formation and regional blood flow and as well as the reduction of muscle atrophy in the rabbit hind limbs.³⁹ Interestingly, these findings were more evident with the concurrent administration of VEGF postulating the synergistic effects of growth factors. In human trials, the Hepatocyte Growth Factor Plasmid to Improve Limb Perfusion in Patients with Critical Limb Ischemia (HGF-STAT) was designed to assess the effect of HGF on limb perfusion as measured by TcPO2. Patients were given intramuscular (IM) injections of high vs. mid vs. low dose of HGF. The high dose group was noted to have higher TcPO2 compared to the other groups. However, no difference was noted in ABIs, pain relief, and wound healing.⁴⁰

Hypoxia-Inducible Factor

The discovery by Keshet and Plate of elevated VEGF levels in hypoxic areas of glioblastoma multiforme tumors led to the hypothesis of hypoxia as a potent trigger for VEGF. HIF-1 belongs to a group of transcriptional regulatory proteins called hypoxia-inducible factors.⁴¹ HIF-1 is the most prevalent and most studied in the group. It has been studied that HIF-1 induces numerous genes including the expression of VEGF and other angiopoietins.^26,41 Several studies are currently investigating the benefit of targeting the expression of active HIF-1 transgenes to ischemic tissue as another method in therapeutic angiogenesis.⁴¹

Novel Growth Factors

Despite discoveries of the different growth factors, the mechanism of angiogenesis in skeletal muscle remains unclear. In 2014, several studies investigated proliferator-activated receptor gamma coactivator (PGC)-1. Its presence was found to be required for the full hypoxic induction of VEGF in skeletal muscle cells. In a study by Thom et al. in 2014, transgenic expression of PGC-1 alpha 4 skeletal muscle in mice induces angiogenesis in vivo.⁴² It is interesting to note that PGC-1 was noted to replicate the mechanisms of angiogenesis similar to exercise. The induced blood vessels were more organized, non-leaky, and functional nascent vessels.⁴³ This is compared to the disorganized structure of blood vessels induced by VEGF. This was studied by Rowe et al. in diabetic mice and is a promising new discovery in gene therapy. In addition, norepinephrine was also discovered to regulate endothelial progenitor cells (EPCs) mobilization in the ischemic hind limb of mice via its different receptors including alpha-adrenoceptor and beta-2-adrenoceptor.⁴⁴

Stromal Derived Factor

Chemokines are a subgroup of cytokines that are potent regulators of leukocyte trafficking in both inflammatory and homeostatic processes. To date, there are more than 50 chemokines that have been discovered and 20 chemokine receptors that have been cloned.⁴⁵ Chemokines as a rule bind to multiple receptors while receptors usually bind to multiple chemokines at a time.

Stromal derived factor -1 (SDF-1) is a chemokine, which is also known as CXCL12. It has a multifaceted role in human physiology including embryonic development and organ homeostasis. SDF-1 is unique to other chemokines because it binds exclusively to the CXCR4 receptor. SDF-1 has a two-pronged effect in vasculogenesis, the first is through the recruitment of CXCR4 EPCs and the second is a direct angiogenic effect on endothelial cells. During ischemia, thrombopoietin and other chemokines also induce the release of SDF-1 from platelets and thus facilitate the revascularization of ischemic limbs by mobilizing hemangiocytes.^46-48 In both animal in vivo and in vitro studies, SDF-1 has been shown to increase blood flow and perfusion through the recruitment of EPCs in CLI patients. During ischemia, the levels of SDF-1 are up regulated since hypoxia or apoptotic conditions are potent triggers to the induction of chemokine expression. The up-regulation of SDF-1 in ischemic muscles then acts as a chemo attractant and homing signal for CXCR4 EPCs. The EPCs then augment the process of neovascularization in ischemic tissues.^49-51 Yamaguchi et al. studied the effect of SDF-1 in vasculogenesis in ischemic hind limb muscle of mice. They noted an increase in EPCs through microscopic examination of SDF-1 injected muscle. Clinical trials of SDF-1 in CLI patients are underway and a better understanding of the mechanisms of chemokines, especially SDF-1, is crucial in filling the missing link in growth factor studies in therapeutic angiogenesis.

GENE DELIVERY

Angiogenic growth factors are primarily delivered by non-viral vectors or viral vectors. Non-viral vectors include recombinant proteins or a plasmid with a gene that encodes for the angiogenic protein.⁵² Viral vectors utilize known viruses in which adenovirus is the most commonly used. The main objective of gene transfer is the introduction of foreign nucleic acids into its target cells. The result is a localized and sustained overexpression of the selected gene (i.e., growth factors).⁵³ Non-viral gene transfer uses naked or plasmid DNA and is usually coupled with a lipophilic or hydrophobic agent which facilitate the transport of DNA across the target cell membrane. Naked plasmid DNA is a common vector for FGF and HGF studies and has proven to have a low toxicity and low immune response in contrast to viral vectors.^40,54,55 On the other hand, viral vectors such as adenoviruses have been known to have a higher efficiency of DNA transfer. Gounis et al. studied the delivery of VEGF into a rabbit ischemic hind limb model using adenovirus. After one week of VEGF delivery, angiography demonstrated new large and small vessels in the treated rabbits.⁵⁶ However, the use of viral vectors leads to transaminitis and an increase in the formation of antibodies. To date, no evidence supports that these effects lead to malignancy.⁵³

Novel Concepts In Gene Delivery

The ideal mode of delivery of angiogenic growth factors has yet to be determined. The progress of gene therapy in angiogenesis has been slowed down by the concerns of the lack of efficacy of non-viral vector transfection techniques and the safety of using viral vectors. The tedious use of genetic materials and the collection of implanted cells are also major drawbacks for this route of gene delivery. An effective delivery system using non-invasive and non-viral mediated method is currently being sought. A novel concept of using a biodegradable gelatin hydrogel carrying a sustained-release system of basic FGF (bFGF) was studied for CLI. In 2015, a phase I-IIa trial evaluated 10 CLI patients who received a 200-microgram intramuscular injection of bFGF.^57,58 This novel concept was noted to be not only safe but also improved TcPO2, increased walking time, and decreased ischemic pain.^57,58 Another novel technique for gene delivery, called ultrasound mediated sonoporation, uses albumin-shelled microbubbles, which carry genetic material to deliver to the target. These microbubbles are gas filled, acoustic microspheres that burst using ultrasound when they reach the target. This novel delivery system resulted to a 300-fold increment in transgene expression after naked DNA transfections.^59-61 Both the hydrogel and sonoporation techniques are promising tools in gene delivery in angiogenesis that are currently being studied further in human clinical trials.

CELL-BASED THERAPY IN CLI

The concept of vasculogenesis created a paradigm shift in the understanding of angiogenesis. Until two decades ago, the formation new blood vessels was believed to be limited to the process of angiogenesis, which is the formation of new blood vessels from previously existing vasculature. Vasculogenesis, on the other hand, is the process of blood vessel in situ formation from EPCs and vascular progenitor cells.³¹ It was in the late 1990s that Asahara et al. isolated EPCs or angioblasts from human blood. Their findings suggest that these EPCs may play a role in augmenting collateral vessel formation in ischemic tissues.^62,63 The discovery of bone marrow derived progenitor cells, which differentiated into endothelial cells (EC), paved the way for cell therapy in therapeutic angiogenesis.

In response to ischemia, bone marrow derived EPCs have a paracrine effect on existing ECs by the secretion of angiogenic growth factors.⁶⁴ In a study by Shintani et al., elevated levels of circulating EPCs were noted to be mobilized to ischemic myocardium in humans.^65,66 EPCs were also noted to incorporate into capillaries and interstitial arteries and initiate vasculogenesis. Since then, the study of stem cell therapy as a potential option for neovascularization of ischemic tissues, including CLI, commenced.

Cell Based Therapy Clinical Trials

Table 2: Cell-Based Therapy Trials in Therapeutic Angiogenesis

Trial	Investigators	Year	Treatment	Patients	Follow up	Primary Endpoint	Outcome
TACT64	TACT study investigators	2002	BM-MNC	25	2 mo	Safety, ABI, rest pain	Improvement in ABI, TcPO2 and pain-free walking time
START68	van Royen et al	2005	GM-CSF mobilized PB-MNC	40	2 wk	MACE, ABI, walking time	Not beneficial in patients with moderate to severe claudication
TACT67	Matoba et al	2008	BM-MNC	85	24 mo	Mortality and amputation free interval	Improvement in leg pain scale, ulcer size and pain free walking distance but not in ABIs and TcPO2
PROVASA69	Walter et al	2011	BM-MNC		3 mo	ABI	Improved ulcer healing and reduced rest pain but no significant increase in ABI
JUVENTAS70	Teraa et al	2015	BM-MNC	160	6 mo	Major amputation	No difference in amputation rates, quality of life, rest pain, ABI, TcPo2

The Therapeutic Angiogenesis by Cell Transplantation (TACT) study was the first study to attempt to evaluate the efficacy of autologous bone marrow mononuclear cells (BM-MNC). It was first published in 2002 with a follow up study published in 2008 to assess the long-term clinical outcome. In the follow up study, the primary endpoints were mortality and amputation free interval. The 3-year amputation free rate was 60% (95%, CI 46-74) in PAD, while the survival rate was 80% (95%, CI 68-91).^64,67 After 2 years, there was significant improvement in the leg pain scale, ulcer size, and pain free walking distance. However, ABIs and TcPO2 of the patients did not differ significantly.⁶⁷ The TACT study concluded that angiogenic cell therapy with BM-MNC leads to long-term improvement in patients with CLI and delaying amputation.

The STimulation of ARTeriogenesis using subcutaneous application of granulocyte-macrophage colony stimulating factor (GM-CSF) (START) trial was a double-blind, randomized, placebo-controlled study on 40 patients with PAD which was done to replicate the studies of GM-CSF in the coronary circulation. GM-CSF is used to mobilize BM-MNC into the peripheral blood into the target tissue. GM-CSF showed beneficial effects on collateral growth using collateral flow index in patients with coronary artery disease. However, the effects were unknown in peripheral artery disease. Unfortunately, the START trial concluded that GM-CSF does not seem to be beneficial in patients with moderate to severe claudication in all aspects including ABI and walking time.⁶⁸

The intra-arterial administration of bone marrow mononuclear cells (BM-MNC) in patients with critical limb ischemia (PROVASA) trial was a phase II double blind, randomized trial on CLI patients. The trial demonstrated that cell therapy was associated with improved ulcer healing (ulcer area, 3.2+/-4.7 cm 2 to 1.89+/-3.5 cm² [P = 0.014] versus placebo, 2.92+/-3.5 cm² to 2.89+/-4.1 cm² [P = 0.5]) and reduced rest pain (5.2+/-1.8 to 2.2+/-1.3 [P = 0.009] versus placebo, 4.5+/-2.4 to 3.9+/-2.6 [P = 0.3]) within 3 months.⁶⁹ Unfortunately, the trial did not achieve its clinical endpoint. It showed an insignificant increase in ABI in the treatment group.

Conflicting results of the efficacy of bone marrow cells in CLI patients led to the recent Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation (JUVENTAS) trial. The study was designed to provide proof for sustainable clinical effects of bone marrow derived cell therapy in CLI. The JUVENTAS trial was a large randomized, double-blind, placebo-controlled trial which was designed to determine whether repeated intra-arterial infusion of autologous BM-MNCs into the common femoral artery benefits CLI patients. The primary endpoint was avoidance of amputation or limb salvage. The 160 CLI patients were randomly assigned to BM-MNC or placebo. The control group received repeated intra-arterial infusion of BM-MNC into the common femoral artery three times in a 3-week interval. The study demonstrated no significant differences between the control group and placebo group with regard to the primary outcome. The major amputation rates in the BM-MNC group were 19% versus 13% in the placebo group. However, the amputation rates did not meet statistical significance (RR 1.46; 95%, CI 0.62-3.42) .The safety outcome (all-cause mortality, occurrence of malignancy, or hospitalization due to infection) was not significantly different between the groups (RR 1.46; 95%, CI 0.63-3.38). Quality of life, rest pain, ABI, and TcPO2 were improved in the control group compared to placebo; however, once again there was no significant difference.^70,71

Cell-Based Therapy On Diabetic Patients With CLI

Diabetic patients with CLI are very common. The rates of mortality of CLI and DM remain elevated and are compounded. A study by Ruiz-Salmeron et al. studied the safety and efficacy of BM-MNC transplantation in DM patients with PAD. Twenty diabetic patients with severe below the knee arterial ischemia were given autologous BM-MNCs (100-400 x 106 cells) intra-arterially. Clinically, there was improvement in ABIs and in the appearance of diabetic wound using the Rutherford-Becker classification; however, there was no beneficial effect on mortality. Despite the lack of mortality benefit, the study quantitatively proved the significant increase of vascular network improvement in the ischemic areas using digital subtraction angiography (DSA).⁷² Using DSA is a validated, novel method of assessing angiogenesis quantitatively being used in several studies.⁷³

Intramuscular Versus Intra-Arterial Delivery Of Stem Cells

The ideal route of delivery of stem cells in CLI is currently unknown. In CLI stem cell trials, delivery routes tend to be varied. Most trials often use intra-arterial (IA) or intramuscular (IM) injections; however, some studies use the combination of both routes. The main principle of IM injection in ischemic muscle is the formation of a cell depot with paracrine activity in the target area. Van Tongeren and colleagues assessed the safety and efficacy of both administration techniques in CLI patients. Their results showed that both IM and combined IM/IA delivery of autologous bone marrow cells are safe. In addition, bone marrow cell treatment in both routes resulted in a sustained and significant improvement with regards to pain free walking distance and ABI, and reduction of ischemic pain.^37,74

FUTURE DIRECTIONS AND NEW CONCEPTS

Therapeutic angiogenesis trials in both gene therapy and cell therapy have been developing progressively, albeit slowly, in the last two decades. It has been recommended that some trials divert their focus towards pursuing gene therapy on patients who are at the earlier stages of their disease when the potential benefit can be maximized.⁷⁵ Novel delivery systems are also vital in therapeutic angiogenesis. A "perfect" or ideal growth factor gene or cell-based therapy is futile if it does not reach ischemic tissues effectively. In 2015, Dash and colleagues studied an injectable elastin-based gene delivery platform for CLI patients. The investigators used a dual gene delivery system using an elastin-like polypeptide (ELP). The system was capable of delivering plasmid eNOS and IL-10.⁷⁶ This non-viral delivery system is a very promising adjunct in treating CLI. In addition, sonoporation, using microbubbles commonly used in echocardiography as echo contrast, is an emerging technique in delivering gene therapy into the target ischemic area.^59,61

CONCLUSIONS

The advent of molecular biology research and better understanding of the mechanism of angiogenesis gave birth to the development of therapeutic angiogenesis as a viable and promising strategy to manage CLI. Bench research using animal models in growth factor therapy and cell based therapy for CLI progressed slowly but steadily to human clinical trials. The angiogenic potentials of growth factors such as VEGF, HGF, FGF, HIF-1, and chemokines were evaluated in human clinical trials albeit with conflicting results. The same is true for cell-based therapy clinical trials. The conflicting and inconclusive results of the clinical trials should be an impetus for further bench, translational, and large randomized human clinical trials not only using different growth factors or cell-based therapies but also evaluating the optimal delivery of these therapies. The verdict for the optimal regimen using therapeutic angiogenesis in CLI patients is yet to be determined. Until then, the therapeutic arsenal for the management of CLI patients who are not candidates for conventional revascularization procedures remains limited.

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Keywords: Adenoviridae, Amputation, Angiography, Digital Subtraction, Angioplasty, Angioplasty, Balloon, Laser-Assisted, Angiopoietins, Ankle Brachial Index, Arterioles, Blood Glucose, Blood Platelets, Blood Pressure, Bone Marrow, C-Reactive Protein, Capillaries, Cell Membrane, Cell Proliferation, Cell Transplantation, Cerebrovascular Disorders, Chemokine CXCL12, Comorbidity, Constriction, Pathologic, Coronary Artery Bypass, Coronary Artery Disease, Coronary Circulation, DNA, Delayed-Action Preparations, Diabetes Mellitus, Experimental, Diet, Atherogenic, Drug-Eluting Stents, Dyslipidemias, Echocardiography, Elastin, Epidemiologic Studies, Epoprostenol, Femoral Artery, Fibroblast Growth Factor 1, Fibroblast Growth Factor 2, Fibroblast Growth Factors, Gangrene, Gene Transfer Techniques, Genetic Therapy, Glioblastoma, Granulocyte-Macrophage Colony-Stimulating Factor, Granulocytes, Hemoglobins, Hepatocyte Growth Factor, Homeostasis, Hydrogel, Polyethylene Glycol Dimethacrylate, Hyperhomocysteinemia, Hyperplasia, Hypertension, Iloprost, Infusions, Intra-Arterial, Infusions, Intravenous, Injections, Intramuscular, Interleukin-10, Intermittent Claudication, Leukocytes, Mononuclear, Limb Salvage, Macrophage Colony-Stimulating Factor, Magnetic Resonance Imaging, Microbubbles, Microspheres, Mitogens, Muscle, Skeletal, Muscular Atrophy, Myocardium, Norepinephrine, Nucleic Acids, Ocimum basilicum, Oxygen, Paclitaxel, Pain, Peripheral Arterial Disease, Plasmids, Popliteal Artery, Pregnancy, Prostaglandins E, Quality of Life, Receptors, Adrenergic, Receptors, CXCR4, Recombinant Proteins, Regeneration, Regional Blood Flow, Renal Insufficiency, Chronic, Risk Factors, Smoking, Spinal Cord Stimulation, Stroke, Survival Rate, Thrombopoietin, Ticlopidine, Tissue Plasminogen Activator, Transfection, Transgenes, Ulcer, Up-Regulation, Vascular Endothelial Growth Factor A, Vasodilator Agents, Venules, Wound Healing

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