Transplantation Of Normal Adipose Tissue Improves Blood Flow And Angiogenesis In High Fat Fed Mice With Hindlimb Ischemia

Objective: Fat deposition is associated with peripheral arterial disease.Adipose tissue has recently been implicated in vascular remodeling and angiogenic activity.But the molecular mechanisms for adipose tissue transplantation regulating blood vessel function and maturation remain unclear.We hypothesized that the transplantation of adipose tissues from normal mice improves blood flow perfusion and neovascularization in high-fat diet fed mice.Further analysis the pathophysiologic mechanisms by which local transplanted adipose tissue influences the development of vascular disease.Methods : 1.HFD mice feeding: high fat(HFD;45% fat by kcal)and high glucose diet was fed to normal C57/BL6 mice for about 14 weeks.The weight and blood glucose were recorded regularly.2.The establishment of the model of lower limb ischemia: Unilateral hindlimb ischemia was induced in mice by ligation and excision of a segment of the left femoral artery,as previously described.Mice were anesthetized using intraperitoneal sodium pentobarbital(60 mg/kg body weight).A subcutaneous dose of buprenorphine hydrochloride(0.1 mg/kg)was administered for analgesia.Additional sodium pentobarbital(12 mg/kg body weight)or 5% isoflurane was given as needed to maintain anesthesia.The left femoral artery was exposed,ligated proximally and distallywith 5-0 silk ligatures,and the femoral bifurcation with all branches was excised.Mice were euthanized by cervical dislocation at the end of the experiment while still under anesthesia.3.Adipose tissue transplantation: For the transplantation experiments,subcutaneous WAT and BAT fat pads were harvested from 8-week-old male enhanced Green Fluorescent Protein(eGFP)mice(background strain of C57BL/6J),cut into one 50-mg piece and subcutaneously transplanted over the region of the adductor muscles of20-week-old recipient HFD mice fed a high-cholesterol diet for 14 weeks(n=9male mice per group).Each recipient HFD mice received an equivalent transplanted fat mass.Transplanted mice underwent surgery by ligation and excision of a segment of the left femoral artery,as described above.Finally,the musculofascial and skin incisions were sutured.Sham surgeries of control animals were performed in the same manner,but without fat pad transplantation.Three weeks after transplantation,the flow perfusion of the hindlimb was evaluated.In order to verify the importance of macrophages in ischemia-perfusion,transplanted mice were injected with macrophage depletion(Clodronate liposomes),and the blood perfusion was observed after removing macrophages.HFD limb ischemia mice were transplanted with WAT,and then randomly divided into two groups of 6 mice each.Intraperitoneal injection of Clodronate liposomes or placebo at 0 and 4 days postoperatively,0.2 ml / body.Thus,WAT-inh group(Clodronate liposomes group),WAT-sham group(placebo group)were established.4.In vivo optical imaging: Anesthetized mice wereplaced in an in vivo FX PRO(BRUKER Corporation,Billerica,MA,USA).Scanning was performed on the transplanted fat of mice.Fluorescent images were recorded using two-step scan(GFP 30 s plus white light 0.175s)at 3,7,10,14,and 21 days after ligation.eGFP was obtained with an excitation wavelength of 480 ± 10 nm and emission wavelength of 510 ± 10 nm.5.Perfusion of the ischemic and non-ischemic hindlimb was measured in each mouse by laser-Doppler imaging(LDI)immediately before surgery,immediately after ligation,and at 3,7,11,14,and 21 days after ligation using a scanning moorLDI2-HIR(Moor Instruments,Wilmington,Del)high-resolution laser Doppler imager.6.Histological Assessment: Mouse adductor and gastrocnemius muscles were obtained 3 weeks after hind limb ischemia surgery and fixed with 4%(wt/vol)paraformaldehyde in PBS overnight.Then,the samples were embedded in paraffin compound and serially sectioned(6μm).Cross-sections were prepared for immunofluorescence analysis.Capillary density,vascular smooth muscle cells/pericytes,and macrophages were determined by immunostaining using anti-PECAM-1(Santa Cruz Biotechnology Inc.,Santa Cruz,CA,USA),anti-NG2(Santa Cruz Biotechnology Inc.,Santa Cruz,CA,USA),and anti-F4/80(Abcam,Cambridge,UK)antibodies,respectively.The secondary antibodies were goat anti-rabbit IgG Alexa Fluor 568-conjugated antibodies(Molecular Probes,Invitrogen).Images were captured with a fluorescence microscope(Leica).Numbers were quantified in 5 microscopic fields in each of 3 cross-sections ofeach implant using ImagePro Plus software.7.Real-time quantitative RT-PCR detection: Extraction of RNA from the adductor and gastrocnemius muscle tissue of mice,and detect mRNA expression levels of anti-inflammatory M2markers: CD206,transforming growth factor-β(TGF-β1),fibronectin-1(Fn1)and interleukin-10(IL-10)and mRNA expression levels of Pro-inflammatory M1markers: interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),monocyte chemotactic protein-1(MCP-1)and CD11 c.Then analyze the differences of inflammatory related factors,macrophage infiltration and polarization.Extraction of RNA from the adductor and gastrocnemius muscle tissue of mice,and detect mRNA expression levels of secretory factor angiopoietin like protein-4(ANGPTL-4),vascular endothelial growth factor-A(VEGF-A)and platelet-derived growth factor-B(PDGF-B).Then analyze the differences among vascular growth factors and do a further analysis of the differences in angiogenesis.8.Glucose tolerance test and insulin tolerance test : After an overnight fast,a glucose tolerance test(GTT)was performed following an intraperitoneal(IP)injection of D-glucose(Roth,Karlsruhe,Germany)(2 g of glucose/kg body mass).Insulin tolerance testing was performed using IP injections of insulin(0.75 U insulin/kg body mass)after a 4-h fast.Blood samples were then obtained from the caudal vein,and could measured the blood glucose value at 0,30,60,and 120 min after D-glucose injection using a One Touch? Vita? glucometer(Zug,Switzerland).Results: 1.The transplantation of WAT derived from normal miceimproved functional blood flow in HFD-fed mice compared to mice transplanted with BAT and sham-treated mice.WAT transplantation increased the recruitment of pericytes associated with nascent blood vessels,but did not affect capillary formation.2.Transplantation of WAT ameliorated HFD-induced insulin resistance,M2 macrophage predominance and the release of arteriogenic factors in ischemic muscles.Mice receiving WAT also displayed a marked reduction in several proinflammatory cytokines.In contrast,mice transplanted with BAT were glucose intolerant and demonstrated increased IL-6levels in ischemic muscles.Conclusions: 1.These results indicate that transplantation of adipose tissue elicits improvements in blood perfusion and beneficial effects on systemic glucose homeostasis.2.Fat grafts increase glucose tolerance and improve insulin resistance.3.To study the application of fat transplantation for the treatment of obesity,metabolic-related diseases and peripheral arterial disease,provide a theoretical basis and potential treatment and could be a promising therapeutic option for the treatment of diabetic peripheral arterial disease.