Dual Roles Of Tumor Necrosis Factor Receptor-1 In A Mouse Model Of Hindlimb Ischemia

Background:Peripheral arterial disease (PAD) is chronic arterial occlusive disease of the body arteries other than the coronary and intracranial vessels. It's prevalence increases with age. Schroll and Munck reported that the prevalence of PAD was 16% in men and 13% in women aged 60 years. As the disease progresses, eventually 25% of PAD patients will require revascularization and 5% will evolve into critical limb ischemia (CLI). There are limited therapeutic options for CLI patient nowadays. And amputation of lower extremities needs to be performed in part of these patients due to severe ischemia. However, the prognosis of patients after amputation is poor. Occlusion of the femoral artery leads to acute ischemia of the limbs and accounts for a wide variety of complications, culminating very often in limb loss and even death. The two main causes of peripheral artery occlusion (PAO) are embolism and thrombosis. Recent epidemiological data estimated that PAO occurs 14 in every 100,000 individuals and accounts for 10-16% of the vascular pathologies. Patients with PAO are more susceptible to heart attacks and strokes. In contrast to extensive investigations on brain and heart ischemia, PAO has received less attention and its cellular and molecular mechanisms are poorly understood. There is currently littleeffective pharmacological treatment for PAO.Tumor necrosis factor (TNF) is a multifunctional cytokine that has been implicated as mediator of diverse physiologic and pathophysiologic events, including cell survival, growth, differentiation, apoptosis, inflammation, and angiogenesis. There are two high-affinity receptors of TNF, i.e. type 1 or TNFR-1 (also named as p55) and type 2 or TNFR-2 (p75) . TNFR-1 is the main receptor subtype in most cell types, including the heart; its complex and divergent downstream signaling system has been extensively studied. The transduction of signals from TNFR-2 and its role in TNF signaling remains less characterized. After activation by TNF, TNFR-1 may couple with the adaptor Fas-associated death-domain protein (FADD) and lead to the recruitment and activation of apoptotic proteases with subsequent progression to programmed cell death . However, TNFR-1 activation is not always associated with apoptosis. It can also activate NFKB-mediated transcriptional events, inducing the expression of protective genes involved in cellular growth, survival and proliferation. Signals in the TNF-TNFR-1 pathway are upregulated after acute hindlimb ischemia. It is unclear, however, whether this upregulation is a pathophysiological consequence or a compensatory response. For example, TNF activity may either increase or inhibit apoptosis and angiogenesi, and the mechanism of the TNF-TNFR-1-mediated post-ischemic angiogenesis is obscure.Objective:Signals in the TNF pathway are upregulated after acute limb ischemia, yet its role remains unclear. In the present study, we investigate the role of TNFR-1 in cell death and post-ischemia angiogenesis in a hindlimb ischemia model in TNFR-1-/- and WT mice.Methods:C57BL/6 (Harlan, Indianapolis, IN) and C57BL/6 TNFR-1-/- mice (Jackson Laboratory, Bar Harbor, Maine) weighing 18 to 22 g (8- to 10-week-old) were anesthetized with intraperitoneal injections of 4% chloral hydrate. Under sterile conditions, the proximal portion of the left femoral artery and vein including the superficial and the deep branch as well as the distal portion of the saphenous artery were exposed and ligated with 7-0 silk sutures. All arterial branches between the ligation were ligated. A sham procedure (dissection of vessels without ligation) was performed on the contralateral right leg. For mitotic labeling of cell proliferation, BrdU was administered by i.p. injection to animals at a dosage of 50 mg/kg body weight once daily until the day of sacrifice. After surgery, a semiquantitative assessment of the ischemic damage to the limb was performed with a modification of a ischemic score (0=no change, 1=mild discoloration, 2=moderate/severe discoloration, 3=necrosis, 4=amputation; n=8 mice per group). We measured blood flow in both limbs using a Laser Doppler blood perfusion scanner. Animals were euthanized at designated time points after surgery by cervical dislocation under deep anesthesia, and perfused with normal saline via left ventricle. Equal amount of adductor muscles of both legs were harvested to prepare protein and DNA samples. Calf muscles were dissected and snap-frozen in O.C.T. solution over dry ice. All the samples were transferred to a -80oC freezer and ready for electrophoresis, Western blot or immunohistochemistry staining. The DNA samples from equal amounts of adductor muscles were analyzed by electrophoresis in a 1.5% agarose slab gel containing 0.2 μg/ml ethidium bromide and visualized under UV illumination. A TUNEL staining kit was used to visualize cell death in 8-um frozen sections of calf muscle (n=6 per group). Vessels were identified by immunostaining with an anti-CD31 antibody. To double-stain vessels with BrdU, brain slides were post-fixedand the BrdU staining procedure was performed. Expression of caspase-3, bax and angiogenic proteins including Tie-2, angiopoietin-1, angiopoietin-2 and VEGF was examined by Western blot.Results:Hind limb ischemia was induced in TNFR-1 knockout (TNFR-1^-/-) and wild type (WT) mice. Laser Doppler scanning showed that limb perfusion was significantly higher in TNFR-1^-/- mice until 7 days after operation. Consistently, TUNEL positive cells, DNA fragmentation, and the calculated ischemic score were lower in TNFR-1^-/- group. Western blot showed less expression of Bax and cleaved caspase 3 in TNFR-1^-/- mice 1 day after ischemia, suggesting a reduced apoptotic process in the absence of TNFR-1. The rate of post-ischemia amputation was 50% in WT mice versus 0% in TNFR-1^-/- mice. However, 21 days post ischemia, co-localization assay of CD31 and BrdU indicated impaired angiogenic activity in TNFR-1^-/- mice. Western blot showed downregulated expression of angiopoietin-1 and its receptor tie-2 in TNFR-1^-/- mice.Conclusions:1. These results suggest that TNFR-1 deficiency prevents the activation of death-related proteins down streaming to TNF-α and attenuated apoptotic cell death in acute limb ischemia, thus reduces the auto-amputation rate.2. TNFR-1 is critically involved in the angiogenesis process after hindlimb ischemia, lack of TNFR-1 signaling attenuates the belated angiogenesis mediated by angiopoietin-1 and tie-2 pathway.