Angiogenic Factor AGGF1Promotes Therapeutic Angiogenesis In A Mouse Limb Ischemia Model

Peripheral arterial disease (PAD) is caused by atherosclerosis, which results in progressive narrowing and occlusion of the peripheral arteries and inhibits blood flow to the lower extremities. The prevalence of PAD is increasing in the modern aging society, and reaches about12%of the adult population. In the US, approximately8to12million people are affected with PAD. In Germany, the prevalence rate of PAD for women and men aged≥65years was17%and20%, respectively. In severe cases, PAD significantly affects quality of life, and increases morbidity and mortality. Approximately25%patients with critical limb ischemia die in one year. Moreover, among patients with PAD, the prevalence of coronary artery disease (CAD) is about46%to71%, and at least10%of them suffer cerebrovascular disease.Some PAD patients may require leg amputation to relieve the unbearable pain and life-threatening situation. To avoid amputation or other serious prognosis, angioplasty, stenting and peripheral artery bypass surgeries are used to restore blood flow to the legs in some patients. However, PAD patients undergoing vascular surgeries had a significantly worse long-term prognosis than CAD patients with similar procedures. Furthermore, many PAD patients are not suitable candidates for interventional or revascularization surgeries. Therefore, therapeutic angiogenesis, i.e. promotion of angiogenesis and microcirculation using angiogenic factors, has been proposed as a new and potential treatment strategy for PAD patients in the last decade. The administration of angiogenic factors, either as naked plasmid DNA or recombinant proteins, may promote neovascularization, augment the collateral circulation, and enhance blood perfusion to ischemic tissues.In this study, I assessed the potential of a new angiogenic factor AGGF1for therapeutic angiogenesis in a critical limb ischemia model in mice for PAD. The following results have been generated:(1) Direct injection of naked plasmid DNA for an AGGF1expression plasmid (pcDNA3.1-AGGF1-FLAG) into gastrocnemius muscle successfully results in a high level of expression of both AGGF1mRNA and protein. The increased AGGF1expression remained high for two weeks after the injection, but decreased to near the basal level four weeks after the injection.(2) Increased AGGF1expression stimulated an increase of blood flow in the ischemic hindlimb. I measured the change of blood flow in the hindlimb ischemia model using high-resolution microultrasound. One day after the surgery (ligation of arteries), the ratio of blood flow in the ischemic leg over that in the non-ischemic leg decreased sharply to10%of the level before the surgery. Color PW Doppler echocardiography showed that the blood flow in ischemic hindlimbs was significantly increased in the AGGF1group compared to control mice at time points of7,14, and28days after DNA administration (n=9/group, P=0.016,0.0034, and0.0012, respectively).I evaluated ischemic limb functions by scoring limb tissue necrosis and ambulatory impairment. Both scores decreased markedly in mice injected with pcDNA3.1-AGGF1-FLAG compared with those injected with control vector pcDNA3.1-FLAG.(3) Increased AGGF1expression inhibited necrosis in the ischemic hindlimb. H&E histological staining was carried out for ischemic hindlimb muscle sections for both pcDNA3.1-AGGF1-FLAG and control pcDNA3.1-FLAG groups of mice7days after DNA injection. Ischemic gastrocnemius muscle sections from mice with overexpression of AGGF1showed less gross tissue necrosis than that from mice without AGGF1overexpression.(4) Overexpression of human AGGF1induces angiogenesis in ischemic gastrocnemius muscles. Density of CD31-positive vessels was significantly higher in mice injected with pcDNA3.1-AGGF1-FLAG than control pcDNA3.1-FLAG group of mice (0.934±0.413/mm2vs.0.538±0.388/mm2, P<0.01). Increased AGGF1expression induced angiogenesis in the ischemic hindlimb.I also found that no significant difference was detected for the expression level of VEGFA between tissues injected with the AGGF1expression plasmid DNA and those with an empty vector DNA. The results suggest that the observed effects in PAD mice were specific for increased AGGF1expression, but not due to indirect effects such as increased expression of other agiogenic factors like VEGFA.In conclusion, the data in this study suggest that gene transfer using AGGF1naked plasmid DNA significantly increases blood flow by promoting angiogenesis and inhibiting tissue necrosis in a mouse model of critical hindlimb ischemia for PAD. Thus, I have established a new potential therapeutic method for treating PAD. Therapeutic angiogenesis with AGGF1may be beneficial to patients with not only PAD, but also other ischemic conditions such as ischemic heart disease, MIs, and strokes.