A novel approach for therapeutic angiogenesis : low doses of ionizing radiation

Tese de doutoramento, Medicina (Cirurgia Vascular), Universidade de Lisboa, Faculdade de Medicina, 2018Peripheral arterial disease (PAD) is a vascular occlusive disease accompanied by an insufficient angiogenic response, resulting in hypoperfusi on of the affected extremities. When arterial lesions impair blood flow to such an extent that the nutritive requirements of the tissues cannot be met, limb pain is chronically present at rest, frequently with trophic and necrotic skin lesions. This condition corresponds to the most advanced clinical stage of PAD, known as critical limb ischemia (CLI). Although just a minority of PAD patients progress to CLI, as PAD is highly prevalent among diabetics, smokers and people over 70 years, this condition remains very common, with 500 to 1000 new cases per million inhabitants every year, in the developed countries. The goals of CLI treatment are ischemic pain relief, ulcer healing, improvement of muscle function and quality of life, limb loss prevention, and overall survival increase. The most important aspect of CLI is the poor prognosis, no matter what treatment is employed. Any kind of surgical/endovascular revascularization, the therapy of choice in CLI patients, should be done whenever technically possible. Attempts to manipulate and normalize the microcirculatory flow pharmacologically may enhance the results of revascularization or be one option in patients in whom revascularization is impossible or has failed. In patients with CLI not eligible for arterial revascularization, the “non-option” patients, most pharmacological agents have reduced or no real effect. Amputation is often recommended, even if it is associated to morbidity and mortality. The need for alternative treatment in CLI patients is therefore compelling, and therapeutic angiogenesis is an undoubtedly promising tool to treat these patients. There are three major processes that contribute to neovascularization and are essential for the establishment of functional collateral networks, namely angiogenesis, arteriogenesis and vasculogenesis. While angiogenesis describes the process of growth of new blood vessels from pre-existing ones, arteriogenesis is characterized by the enlargement of arteriolar anastomoses to collateral vessels through growth and proliferation and vasculogenesis is de novo formation of blood vessels. These vessels can grow considerably, enough even to take over the role of a large artery when occluded. Several strategies have been pursued to stimulate therapeutic angiogenesis, ranging from recombinant protein and gene transfer therapies to the use of progenitor cell therapies. During this research, in collaboration with the biotechnology company ECBio (Amadora, Portugal), cell based therapeutic angiogenesis was addressed, as we evaluated the possibility of UCX®, a specific population of human umbilical cord derived-mesenchymal stromal cells (UC-MSCs), to induce therapeutic angiogenesis. Using a C57BL/6 mouse model of hindlimb ischemia (HLI), we demonstrated that UCX® delivered via intramuscular injection enhance blood perfusion (evaluated by laser Doppler imaging) by stimulating angiogenesis (capillary density determined by CD31 immunohistochemistry) and arteriogenesis (collateral vessel density determined by diaphonization) in ischemic muscles. This is achieved through a new mechanism in which durable and simultaneous up-regulation of several proangiogenic genes in endothelial cells (ECs) are induced by UCX®. Cryopreservation and subsequent thawing, both in vitro and in vivo, did not impair phenotype, immunomodulatory and angiogenic potencials of this specific UC-MSCs population. These data demonstrate that UCX® improve the angiogenic potency of ECs in the murine ischemic limb, suggesting the potential of UCX® as a new therapeutic tool for CLI. This study also suggests that potency impairment related to cryopreservation in a given tissue source can be avoided by the production process. The results have positive implications for the development of an advanced therapy medicinal product. However, the use of UCX® in patients with CLI is conditioned by the lack of studies to ensure its safety and the absence of significant adverse effects. The scope of our work, in the search for a new therapeutic approach that could overcome the current lack of available medical treatments for a large number of CLI patients, led us to consider therapeutic angiogenesis beyond its cellular component, in a unique perspective based on the use of low-dose ionizing radiation (LDIR). Our previous research has demonstrated that LDIR (<0.8 Gy) induces a proangiogenic phenotype in ECs in vitro, modulating endothelial dysfunction, promoting survival, migration and preventing ECs apoptosis. Likewise, LDIR promotes neovascularization in vivo by inducing angiogenic sprouting in the transgenic fluorescent zebrafish Tg (fli1:EGFP) embryos and by increasing vessel density in adult fli1:EGFP zebrafish after caudal fin regeneration. Therefore, according to our previous results, LDIR induces angiogenesis in vitro and in vivo; however, there is no evidence so far, that it enhances neovascularization in vascular occlusive disease, our overall objective. After surgical induction of unilateral HLI, both hindlimbs of female C57BL/6 mice were sham-irradiated or irradiated with four daily fractions of 0.3 Gy, in consecutive days and limb perfusion, capillary density and collateral vessel formation were measured. We found that LDIR (4 x 0.3 Gy) improves limb perfusion by enhancing arteriogenesis through EPCs recruitment to sites of collateral vessel development, an effect dependent on exposure of the ischemic niche to LDIR, but not on the local recruitment of myeloid cells. Likewise, LDIR also favours angiogenesis through simultaneous activation of a repertoire of pro-angiogenic factors in mature ECs in a mechanism dependent of VEGFR signaling, with no short-term side effects and no effects on resting vasculature, opening a possibility to new therapeutic strategies in lower limb vascular insufficiency. The vasculature in an irradiated non-ischemic bed was not affected and after 52-wk of LDIR exposure no differences in the incidence of morbidity and mortality were noticed. Moreover, it was found that a dose of 0.3 Gy administered during 4 consecutive days does not induce toxicity in C57BL/mice and this dose fraction was identified as the lowest dose that is still able to induce therapeutic angiogenesis. The outcome of these in vivo experiments performed in a mice model suggests that LDIR may have a potential clinical use in the treatment of lower limb vascular insufficiency, emerging as a novel approach in the treatment of CLI patients. Accordingly, we designed an already approved and ongoing clinical trial – Low-dose ionizing radiation modulates the expression of pro-angiogenic genes in Critical Limb Ischemia Patients – and report our preliminary results. To date, 16 “non-option” CLI patients were enrolled in the study, but only 12 patients, corresponding to 13 limbs, were considered for analysis. As the expected amputation number (12 major limb amputations) for analysis of the primary endpoint was not reached, the trial is still ongoing. Concerning the primary endpoint, preliminary results suggest that LDIR induces a pro-angiogenic effect through the modulation of several pro-angiogenic factors in ECs collected from “non-option” CLI gastrocnemius muscles. The primary endpoint is corroborated by the finding that LDIR is associated with an increase in capillary density and a significant increase (P = 0.03) in vessel density (VD), 30 days post-intervention. It is also interesting to report that in the non-amputated patients, 6 months after irradiation, only the one muscle exposed to LDIR showed a persistent and continuous increase of VD. Regarding the surrogate clinical endpoints of ischemia and as expected, no significant differences were found between LDIR limbs and controls. LDIR has a biological rationale widely investigated and discussed in the work developed by our research group. Therefore, based on these preliminary results and considering that up to 40% of CLI patients are not candidates to revascularization, LDIR may be considered as a novel approach for the management of these patients.Quadro de Referência Estratégico Nacional (QREN), LISBOA-01-0202-FEDER-030196: ClinUC