Therapeutic Angiogenesis

Is peripheral arterial disease treatment evolving from stents to stem cells?

By Toshio Matsuhashi, MD; Dara L. Kraitchman, VMD, PhD; and Lawrence V. Hofmann, MD
 

Medicine is experiencing a molecular and cellular revolution, which has been spurred by the sequencing of the human genome and the development of research tools that permit high-throughput analysis of a variety of molecular and cellular systems. The endovascular specialist has begun to feel the effects of this revolution in the treatment of atherosclerotic occlusive disease with the introduction of drug-eluting stents for the prevention of in-stent restenosis. The next potential molecular or cellular therapy for treating peripheral arterial disease (PAD) is therapeutic angiogenesis, possibly involving stem cells. The purpose of this article is to familiarize the reader with this burgeoning field.

THE EXTENT OF PAD

The spectrum of symptomatic PAD ranges from intermittent claudication to chronic limb ischemia (CLI). Current practice treats only the patients in the middle of the spectrum (ie, disease severity that would warrant an invasive procedure, but within patients in whom revascularization is an option). It is estimated that approximately 5 million people1 have intermittent claudication.2 The vast majority of patients with intermittent claudication are typically managed conservatively with a walking program and cilostazol therapy.2 Despite these therapies, most patients continue to have pain with ambulation, which affects their quality of life. The most severe manifestation of PAD is CLI, defined as rest pain and/or tissue loss. CLI develops in 500 to 1,000 individuals per million per year.3 Psychologic testing of patients with CLI demonstrates quality-of-life indices similar to patients with terminal cancer.4 Twenty percent of patients with limb-threatening ischemia have disease that is so extensive that revascularization, such as bypass surgery or angioplasty/stent placement, is not feasible.5 The only option for these patients is amputation. Although the endovascular specialist is able to treat a portion of patients with PAD, there is a larger segment of PAD patients that currently receive suboptimal therapy.

THERAPEUTIC ANGIOGENESIS

There are two key processes that dictate the symptomatology of a patient with PAD. The first is the degree of the arterial occlusion. The second is the degree to which an endogenous arteriogenic response is mounted to compensate for the occlusion. Our current endovascular techniques can treat the former, but have no effect on the latter. In an attempt to augment the endogenous arteriogenic response, therapeutic angiogenesis is the process of administering exogenous agents to improve and sustain blood flow to the limb. However, it is important to differentiate angiogenesis and arteriogenesis. Angiogenesis is the process of developing new vessels, typically capillaries, resulting in a 1.5- to 1.7-fold increase in blood flow, which is important for local tissue perfusion. Arteriogenesis is the process of developing, by way of maturation of pre-existing collaterals or de novo formation, mature conductance vessels, such as arterioles. Arteriogenesis is important for regional perfusion and results in a 10- to 20-fold increase in blood flow.6 Although the goal of this therapy is focused on improving regional perfusion, for historical reasons, this field has been termed therapeutic angiogenesis instead of therapeutic arteriogenesis.

There are three broad categories of therapeutic angiogenic agents: (1) protein-based therapies; (2) gene-based therapies; and (3) cell-based therapies (Figure 1). The only randomized, controlled patient study using protein showed marginal efficacy.7 A phase 2 gene therapy study showed no benefit in patients treated with an adenoviral vector encoding vascular endothelial growth factor-121.8 Moreover, the current consensus is that a single growth factor is unlikely to generate a clinically significant arteriogenic response. Cell-based therapy has the advantage that the implantation of a single agent may result in the sustained production of multiple arteriogenic growth factors,9,10 and these cells may possibly incorporate into the vessel wall. A small randomized clinical study using bone marrow-mononuclear cells showed a sustained arteriogenic response with cell-based therapy.11

Cell-based Therapy

Cell-based therapy for a variety of disease states has become the subject of significant political and media attention. Most cell-based therapies focus on stem cells. A stem cell is an undifferentiated cell that is able to produce multiple different cell types. Equally important is a stem cell's ability for self-renewal. This property would enable stem cell therapy to be a one-time therapy, obviating the need for multiple administrations.

In ongoing political debates, the term stem cell has been used to refer primarily to embryonic stem cells. Embryonic stem cells are pluripotent cells (able to give rise to all three major tissue types: ectoderm, mesoderm, endoderm) with the capability to differentiate into any cell type in the body (Figure 2). Therefore, embryonic stem cells are theoretically considered the optimal stem cell for cell-based therapy, although there is concern regarding immunogenicity and the risk of teratoma formation.12 Embryonic stem cells are derived from the inner cell mass of the blastocyst (250 cells) at 5 to 7 days after fertilization.13 Currently, the federal government will not fund embryonic stem cell research beyond the pre-existing stem cell lines available in 2001. Thus, many of the recent advances and improvements that have been developed in the ensuing years cannot be pursued. Moreover, the federal government funding level of embryonic stem cell research has been approximately $25 million per year. Patient groups, frustrated by the lack of federal funds in this area, sought state support to circumvent the federal government. The most noteworthy example is the state of California's Proposition 71. This proposition, passed last November, authorized the use of $3 billion over 10 years for stem cell research. California's $300 million/year support of this field, all of which could be used for embryonic stem cell research, dwarfs the federal funding.

It is unlikely that all of the California funds will focus solely on embryonic stem cells because stem cells derived from bone marrow, cord blood, and other systems offer promise for regenerative therapies. These other stem cells for therapeutic angiogenesis include endothelial progenitor cells, hematopoietic stem cells, bone marrow mononuclear cells, mesenchymal stromal cells (MSCs), and skeletal myoblasts, among others. These cells are harvested from either the bone marrow or the peripheral blood of adults, are cultured expanded, and are then administered.

In ischemic hindlimb experiments, MSCs have been the most extensively studied. MSCs are multipotent cells with the ability to transdifferentiate into a variety of cell types of mesenchymal origin (eg, skeletal muscle, fibroblasts, cardiomyocytes, etc.). MSCs are known to express a variety of arteriogenic cytokines: vascular endothelial growth factor, fibroblastic growth factor, placental growth factor, monocyte chemoattractant protein, insulin-like growth factor, interleukins 1 and 6, plasminogen activator, and hepatocyte growth factor.14

Compared to other cell types, MSCs have a number of desirable attributes, which include (1) ease of isolation, (2) high expansion potential, and (3) reproducible characteristics from isolate to isolate.15 MSCs are exceedingly rare in the bone marrow, representing <.01% of the cell population. Thus, in practice, MSCs are expanded in culture for approximately 21 to 28 days and then frozen for up to 2 years for later use. These cells could be administered to the donor or to the recipient. Intuitively, one would expect allogeneic MSCs would initiate an immune response because they express many cell surface markers recognized by T cells. However, the response to MSCs co-cultured with activated T cells is the opposite of expectation, such that a more immune tolerant phenotype is induced.16-18 Thus, it is likely that “MSC banks” akin to blood banks will be developed to provide a ready source of allogeneic MSCs that have been screened and tested in advance.

Another immunologic concern is the expression of major histocompatibility complex II upon MSC differentiation. As borne out by numerous studies that have demonstrated persistence of allogeneic MSCs for many months in animals, it would appear that even differentiated MSCs are not targeted by the immune system for destruction.15,19,20 Recently, Heeschen et al have shown that the ability to recruit stem cells from the bone marrow may be impaired in certain older adults.21 Thus, exogenous administration of allogeneic MSCs offers a method to expand the available number of progenitor cells in patients who have an impaired ability to recruit endogenous cells, either due to age or other lack of the appropriate cytokine response.

The role that these administered cells have in promoting arteriogenesis is unclear. There is debate about whether the administered cells have the leading role and incorporate and/or fuse with the enlarging vessel wall, or whether they function in a supporting role and produce multiple cytokines in the perivascular tissue in order to enlarge the blood vessel.14,22,23

Published preclinical ischemic hindlimb studies and clinical PAD studies have predominantly focused on intramuscular delivery of therapeutic angiogenic agents. However, there has been a paucity of studies examining different routes of delivery. Although intramuscular injections are minimally invasive, a patient may require up to 40 separate injection sites to provide an equal distribution of the cells throughout the lower extremities. Theoretically, once delivered, the administered cells must migrate to perivascular regions to exert their effect.24 If this is in fact the case, it may be more desirable to deliver these cells via an intra-arterial route, ensuring that all of the arterioles and capillaries would be bathed with the cells. However, the large size of stem cells can result in trapping of these cells in the capillaries25,26 and embolization of the microcirculatory bed. Thus, the cellular dose must be carefully titrated to provide cells without further damaging the arterial tree. Determining the most efficacious and least invasive method of cellular delivery in preclinical models is critical to enhancing the likelihood that clinical trials of cellular therapies will be successful.27 The endovascular specialist is well versed in the importance of delivery routes, as is evidenced by the superior outcomes of catheter-directed thrombolysis compared to systemic administration of fibrinolytics for acute arterial occlusions. Similarly, each therapeutic angiogenic agent should be studied to determine the safest and most efficacious route of delivery. Endovascular specialists, with their extensive backgrounds in imaging, are uniquely poised to advance this area of the field.

Current Research and Future Directions

We have developed an endovascular model of rabbit hindlimb ischemia because we believed that the pre-existing surgical models of hindlimb ischemia were suboptimal due to confounding factors.28,29 The surgical model involved dissection and ligation of the superficial femoral artery. The endovascular model involves occlusion of the superficial femoral artery using coils, via a carotid artery cutdown. Our new endovascular model is an improvement over the surgical model for four reasons. First, it is more akin to atherosclerosis because it occludes the vessel from within. Second, there is no wound-healing response to induce stem cell recruitment because cells are known to be home to sites of tissue damage and inflammation.15,26 Third, it will not disrupt pre-existing collateral vessels in the thigh due to surgical dissection. Finally, there is no postoperative pain in the treated limb to impair mobility.

We have used this model to study the functional effects of protein and gene therapy. However, with cell-based therapies, it is possible to exogenously label the cells with a magnetic resonance contrast agent using cationic agents such that the contrast agent is stably maintained in endosomes and can be detected by magnetic resonance imaging for several months after administration.30 Magnetic resonance imaging offers both near cellular (ie, 20-50 µm) resolution and whole-body imaging capability, which permits the monitoring of delivery, trafficking, and engraftment.

We have applied cell-labeling methods31 using ferumoxides injectable solution (Feridex, Berlex Laboratories, Wayne, NJ) to label and track MSCs in several animal models,32-34 including myocardial infarction.34 With use of x-ray delivery of magnetically labeled MSCs, approximately 30% of the injections either failed or were confluent.34 Thus, a logical approach employed by our group, as well as others, has been to develop magnetic resonance fluoroscopic methods and devices for targeting stem cell delivery, as well as measuring the success of magnetic resonance-labeled MSC delivery in the heart.35-37

By using these methods, we have been able to show precise targeting of the MSCs to the peri-infarction tissue in a dog heart (Figure 3). In addition, persistence of the magnetic resonance-labeled MSCs could be demonstrated in this animal model at 8 weeks after injection (Figure 4).37

We have begun early studies of these techniques using our rabbit endovascular hindlimb ischemia model to evaluate different routes of delivery and their effect on cell trafficking, engraftment, and ultimate therapeutic outcome.

CONCLUSION

PAD treatments continue to evolve. It is clear that researchers and industry are learning that molecular approaches to treating PAD are the next therapeutic steps for our patients. However, because of the inherent difficulties in working with biologics compared to devices, these next steps in therapy will likely require more funding and longer time frames, to ensure their safe introduction into clinical practice.

This work was supported by grants from the National Institutes of Health, K08EB004922 (LVH), K02HL05193 (DLK), and R01HL73223 (DLK).

Toshio Matsuhashi, MD, is from The Russell H. Morgan Department of Radiology and Radiological Science, Division of Vascular and Interventional Radiology, The Johns Hopkins Medical Institutions, Baltimore, Maryland. He has disclosed that he holds no financial interest in any product or manufacturer mentioned herein.

Dara L. Kraitchman, VMD, PhD, is from The Russell H. Morgan Department of Radiology and Radiological Science, the Division of MR Research, The Johns Hopkins Medical Institutions, Baltimore, Maryland. She has disclosed that she holds no financial interest in any product or manufacturer mentioned herein.
Lawrence V. Hofmann, MD, is from The Russell H. Morgan Department of Radiology and Radiological Science, Division of Vascular and Interventional Radiology, The Johns Hopkins Medical Institutions, Baltimore, Maryland. He has disclosed that he holds no financial interest in any product or manufacturer mentioned herein. Dr. Hofmann may be reached at lhofmann@jhmi.edu.

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