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population; instead, expectation here is paracrine function of mononuclear cells that can regulate neovascularization processes in many steps. Some of relatively large, randomized, controlled cell-based trials are shown in Table 3. Most of the trials initially intended cardiac repair in MI patients; however, it has been suggested that beneficial effects are possibly derived from an angiogenic effect which revascularizes the ischemic tissue. At this point, it is premature to conclusively evaluate the beneficial effect of cell therapy with a limited number of large, doubleblind, randomized trials.

5. Issues Regarding Current Strategy

5.1. Choice of biological agent

Blood vessel growth is a complex event which involves the regulation of a number of genes with multiple growth factors, cytokines and modulators acting at different phases. Therefore, the supplementation of a single growth factor that can only trigger the neovascularization process is most likely insufficient, although the rest of the process may be carried out endogenously. The rationale for the multiple growth factor approach is the utilization of different types of biological reagents that can augment neovascularization in a complementary or synergistic fashion. Hence, one agent initiates the growth of new vascular structures while another induces their maturation, thereby ensuring the stability of new blood vessels. This coordinated therapeutic strategy, however, requires a detailed understanding of the kinetics of vessel growth and the ability to assess the state of neovascular response.

The other side of approach is that because there is still a long way to a full understanding of the blood vessel growth process at this point, we should use a “magic bullet” — a mixed bag of biologic agents without a defined composition that demonstrates functional effectiveness. This is, in essence, the premise inherent in the cell therapy approach where the ability to induce a functional benefit has significantly outstripped a thorough understanding of biology. The expectation here is that such biological materials will spontaneously regulate the neovascularization process by releasing all of the factors, and these factors will figure out by themselves how to grow vessels effectively.

354 M. Murakami & M. Simons

With regard to the selection of an agent, as we begin to realize different forms of vascular growth, it may be better to consider the arteriogenic approach rather than the angiogenic approach as arteriogenesis has more impact on the flow recovery in the ischemic vascular bed. An interesting observation implies the weak contribution of angiogenesis in the cardiovascular mortality. Down’s syndrome patients carrying an extra copy of collagen XVIII gene have increased endostatin levels and appear to be protected from the development of cancer. This is attributed to impaired angiogenesis necessary for tumor growth. However, the same study indicates the same mortality rate of ischemic heart disease in the Down’s and non-Down’s population, suggesting the limited role of angiogenesis per se as a determinant of prognosis in ischemic heart disease.7

Shear response of the endothelium is of prime importance in the process of arteriogenesis, which is followed by mononuclear cell influx. Therefore, one means of inducing arteriogenesis may be the restoration of effective endothelial signaling or alteration of mononuclear cell function rather than administration of a growth factor. The other appealing option is the enhancement of an endogenous vasculogenic process. If vasculogenesis plays an important role in adult tissue neovascularization, agents that mobilize progenitor cells and promote this process may prove effective. We need to be, however, cautious that the effectiveness of these agents may be limited in the end-stage patient population because stem cell functionality declines with age and possibly with disease.8

5.2. Pharmacokinetics and delivery mode

Understanding pharmacokinetics of an angiogenic agent is prerequisite for developing a delivery strategy that effectively promotes therapeutic angiogenesis. However, in animal experiments, the point has been mainly focused on the angiogenic outcome, rather than the pharmacokinetics of an agent. Taking into consideration pharmacokinetics, the effective delivery strategy needs to fulfill several criteria: a necessary concentration of the agent that can initiate neovascularization, specific delivery of an agent at the desired site or cell population such as endothelial cells or monocytes, and sustained effectiveness of an

agent for a duration of time (weeks or even longer) sufficient to allow maturation of newly formed blood vessels. Most likely this cannot be achieved with single-dose administration of proteins or peptides with their short half-lives. Instead, a slow-release gel formulation can be an alternative despite the inconvenient administration with invasive means. A sustained benefit of heparin-alginate-based FGF2 delivery provides a strong endorsement of this strategy.9

Gene therapy is one of the promising modalities to deliver a therapeutic agent for a long period of time; however, neither of two-vector systems currently in use, plasmidor adenovirus-based, provide more than a few weeks of high-level expression. More long-lived gene transfer vectors such as adeno-associated virus (AAV) or lentiviruses have not yet been tested. In any vector system, we can only control the amount and duration of expression without fine tuning of expression levels. This will bring about other concerns such as a long, unregulated expression of an angiogenic agent, possibly leading to substantial side effects like development of atherosclerosis and cancer.

The remaining options include the systemic administration of an agent that specifically acts only in the desired tissue or provide therapeutic effect only at the desired site. For example, PlGF appears to induce vessel growth only in the setting of ischemia,10 and endocrine tissue-specific VEGF raises the possibility that tissue-specific growth factors exist that can be used in a systemic fashion.11

In the premise of augmenting arteriogenesis, the delivery modality is a more complex issue because of the limited accessibility to the endothelium of relatively large vessels in comparison to the endothelium of capillaries. GM-CSF has been shown to enhance collateral growth in experimental models by promoting mobilization of bone marrow cells. However, the START trial, in which patients with critical limb ischemia were treated with subcutaneous injection of GM-CSF, failed to demonstrate efficacy.12

Thus, given our understanding of the biology of neovascular development, especially arteriogenesis, a prolonged treatment modality that can influence endothelial cells, monocyte and/or vascular smooth muscle cells appears to be necessary. This will most likely be achieved by an organizer-type growth factor such as FGF family, rather than an endothelial-specific growth factor such as VEGF and angiopoietin