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As with all new therapies, there is a tendency to initially restrict the therapy to the no-option population. Indeed, most therapeutic angiogenesis trials have been carried out in symptomatic patients who have exhausted standard therapy modalities.19 These patients tend to be older, with more extensive disease and clinical evidence of not being responsive to standard therapies, thus suggesting defects in intrinsic neovascularization response. These characteristics can make these patients especially poor candidates for neovascularization.

Another issue that has been arduously learned from early clinical trials is the occurrence of a significant placebo effect.20,21 Although placebo effects are well described in many fields of medicine, the sheer magnitude of the effect observed in these trials was surprising. In the placebo group of the end-stage, no-option patients, their exercise capacity increased by 45 to 60 seconds while scores on the Seattle Angina Questionnaire and Short Form-36 as well as pill counts also showed surprising changes. Regardless of the reason why the placebo response is so prominent and significant in this patient population, the importance of this phenomenon clearly indicates that small open label studies can only be used for the assessment of safety and tolerability; assessment of efficacy should be evaluated by a double-blind randomized manner.

6. Emerging Concepts of Therapeutic Angiogenesis

Despite frustrating results of initial large clinical trials, the underlying premise of therapeutic angiogenesis seems still valid: augmentation of blood vessel growth to compensate for insufficient blood supply to the compromised tissue. The currently tested concept of therapeutic angiogenesis is to induce the increased presence of an angiogenic factor or cellular components in the target area. However, such logic may be flawed. The underlying assumption is that the endogenous biological response to ischemia is impaired because of the lack of angiogenic stimuli, thus justifying exogenous supplementation of growth factors. It appears to be more likely that a defective angiogenic response is due to a defective endothelial signaling especially in cases of diseases such as diabetes.22 Furthermore, there is no evidence demonstrating that levels of growth factors are indeed decreased in the ischemic tissue, resulting

in the obstruction of the neovascularization process. Even if this is the case, we need to be more deliberate with regard to which growth factor is missing and responsible for the impaired angiogenic response in individual patients. Moreover, in the current concept we also assume supplementation of the superphysiological amount of growth factor simply augments normal angiogenic response in old, end-stage patients with ischemic diseases.

6.1. Neovascularization responsiveness

Due to long-term, sustained endogenous angiogenic stimuli or defective endothelial function, the angiogenic adaptation process of these patients may have blunted significantly, resulting in the loss of tissue responsiveness to angiogenesis — a situation similar to other conditions such as insulin resistance in type 2 diabetes and tolerance developed by repeated drug usage. It is known that general sensitivity of the endothelium to angiogenic growth factors is an important determinant of angiogenic response. This may explain the discrepancy in the neovascularization response between healthy young animals and diseased patients with systemic illnesses. To overcome this issue, the focus may need to be more on increasing tissue responsiveness rather than increasing angiogenic stimuli.

To address the neovascularization responsiveness issue, we need to clarify the differences between healthy endothelium and dysfunctional endothelium. It is widely accepted that endothelial function progressively declines with age,23 moreover, dysfunction of the endothelium is well described in patients with atherosclerosis, diabetes, and other risk factors for vascular diseases. With accumulating evidence showing a loss of endothelial homeostasis is a prime event leading to cardiovascular diseases, clinical and basic research have focused on elucidating the role of endothelial dysfunction in influencing vascular disease progression.

Endothelial dysfunction, clinically assessed by endotheliumdependent vasodilator responses, is a broad term that implies diminished production or bioavailability of nitric oxide (NO). It is also affected by an imbalance of endothelium-derived relaxing and contracting factors such as endothelin-1, angiotensin-II and antioxidants. In addition to the vasodilatory effect, NO is a versatile biomediator involved in protection against vascular injury, inflammation,

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thrombosis, angiogenesis, and EPC mobilization.24 Although mixed reports exist with regard to NO production in aged arteries, several studies have shown expression of endothelial NO synthase (eNOS) is attenuated in aging, rendering endothelial cells more susceptible to apoptotic death. This may explain why physical activity prevents agerelated impairment in NO availability in elderly people; shear stress is one of the strongest stimuli for the expression of eNOS.25 Moreover, secretion of many growth factors and hormones such as growth hormone and steroid hormones decline with age. Although the relevance to angiogenic responsiveness has not been established, decreased estrogen levels have been implicated as a risk factor of atherosclerotic disease for post-menopausal women.

Furthermore, it is widely recognized that aging is associated with oxidative stress and related cellular damage, which may well result in refractory neovascular development in old patients.26 Endothelium is an important source of reactive oxygen species (ROS) that is required for normal endothelial functions. However, continuous production of ROS and impaired ROS scavenging system may cause mitochondrial dysfunction in the long run by damaging mitochondrial DNA and contribute to age-dependent cellular dysfunction.

Endothelial dysfunction eventually leads to endothelial senescence, a condition in which cells lose the capacity to divide and enter a state of irreversible growth arrest. Impaired wound healing and angiogenesis observed in older people are attributed to endothelial senescence. One of the indices to evaluate endothelial senescence is the length of telomeres which are essential for maintaining genome stability and integrity, contributing to extended proliferative life span both in cultured cells and in organisms. It is suggested that age-dependent telomere shortening occurs in human endothelium, which results in impaired angiogenesis.27 The activity of telomerase reverse transcriptase (TERT), one of the components of telomerase which serves for preserving telomeric DNA length, is attenuated by oxidative stress, facilitating telomerase ablation in aged cells. Constitutive hTRET expression enhances the regenerative capacity of endothelial progenitor cells. The idea of telomerase rescue may provide with a new approach to therapeutic angiogenesis as an animal experiment using hTERT-transduced endothelial progenitors improved neovascularization in ischemic limbs.28