Tail Vein Injections of Recombinant Human Thioredoxin Prevents High Fat-induced Endothelial Dysfunction in Mice

Background Obesity is a serious risk factor for cardiovascular diseases. A high fat diet results in cellular oxidative stress and endothelial dysfunction in resistance-sized arteries, characterized by reduced nitric oxide (NO) and endothelium-dependent hyperpolarizing (EDH) responses. Thioredoxin-1, a sulfo-oxidoreductase protein that cleaves disulfide bridges between two adjacent cysteine residues in oxidized proteins, has been shown to lower blood pressure and improve endothelium-dependent relaxing responses in aged C57Bl6/J mice. Methods and Results Young (∼ 3 month-old) male C57Bl6/J mice were fed a high fat diet (42% kcal from fat; obese) or a normal chow (lean) for 3 months. Mice were administered recombinant human thioredoxin-1 (rhTrx; 25 mg/kg) or saline (0.9% NaCl) via tail vein injection at the start, after one month, and after two months. Body weight (BW) was comparable between lean/rhTrx1 and lean/saline at the time of euthanasia (32 ±1 g versus 32 ± 1 g). The high fat regimen resulted in a comparable BW between obese/saline and obese/rhTrx mice (47 ± 1 g versus 45 ± 2 g, respectively). Small (second-order branches) mesenteric arteries (MA2), coronary and femoral arteries were isolated and mounted on the wire-myograph. MA2 and femoral arteries from obese/saline had blunted acetylcholine (10−9 – 10−5 M)-mediated relaxations compared to lean/saline mice, but not to the NO donor sodium nitroprusside. NO and EDH-mediated relaxing responses were blunted in MA2 from obese/lean mice compared to the three other groups. Conclusion Tail vein injections with rhTrx prevented endothelial dysfunction in obese mice by improving NO and EDH relaxing responses in MA2.


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eNOS and endothelial K Ca channels. In this study it was hypothesized that tail vein 67 injections of recombinant human Trx (rhTrx) would protect against a high fat diet-induced 68 impairment in endothelium-dependent relaxation in resistance-sized arteries in C57Bl6/J 69 mice. In this study, attention was focused on the role of endothelium-derived NO and EDH 70 relaxing responses in mediating ACh-and NS309-induced relaxations in murine arteries 71 derived from lean and obese mice with or without intervention with rhTrx. 74 Male C57Bl6/J mice (10 -12 weeks) were placed on either a normal chow (lean group) 75 or a high fat diet (obese group). The high fat (42% kcal from fat) diet was purchased from 76 Harlan Laboratories (Teklad Custom Research Diet TD.88137). Mice were divided in four 77 groups: lean/saline, obese/saline, lean/rhTrx, and obese/rhTrx. In the saline groups, mice 78 were injected via the tail vein with saline (100 L of a 0.9% NaCl sterile solution), and for 79 the rhTrx group with recombinant human Thioredoxin-1 (R&D Systems; 2.5 mg/kg in 100 80 L solution in 0.9% NaCl solution). Mice were briefly placed in a holding chamber and 81 anesthetized with isoflurane (1.5% delivered in 100% O 2 ). The tail was heated with a light 82 source in order to dilate the tail vein. Tail vein injections were performed with insulin 83 syringes (Exel, 30G). After one and two months the tail vein injections were repeated. 84 After three months mice were euthanized. All procedures were approved by the IACUC 85 at Campbell University and were consistent with the Guide for the Care and Use of 86 Laboratory Animals published by the National Institute of Health. All animals were 87 maintained on a standard 12-h light/12-h dark cycle, in a temperature-controlled barrier 88 facility.

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Isolation of arteries and isometric wire-myography 90 Mice were euthanized via CO 2 inhalation and the mesentery and heart were dissected. 91 From the left upper leg a 2 mm segment of the femoral artery was dissected. The 92 mesentery was placed in a Petri dish fill with black silicon and ice-cold Krebs Ringer Buffer 93 (KRB) with the following composition (in mM): 118.5 NaCl, 4.7 KCl, 2.5 CaCl 2 , 1.2 MgSO 4 , 94 1.2 KH 2 PO 4 , 25.0 NaHCO 3 , and 5.5 D-glucose. Second-order branches of the superior 95 mesenteric artery (MA2) were dissected. From the heart a 1.5 − 2 mm-segment of the left 96 descending coronary artery was dissected. Segments were mounted on a wire-myograph 97 (Danish Myotechnology Inc, Model 620M, Aarhus, Denmark) and stretched to their 98 optimal internal circumference as described earlier [9]. Force (mN) generated by stretch 99 was corrected for vessel length to obtain tension values in mN/mm. Vessel length was 100 measured in the myograph chamber with the help of a scale bar in the ocular of the stereo 101 dissecting microscope. After an incubation period of 60 minutes, arteries were "woken 102 up" by replacing KRB with 60 mM KCl in KRB (replacing equimolar NaCl with KCl), thus 103 generating a stable tension after a few minutes. This tension level (minus the baseline 104 tension) was set as 100% contraction (as % of K 60 ). Cumulative concentration-response 105 curves (CRC) were performed with phenylephrine (PHE; 0.01 -30 M) for MA2, and  Student's t test. P < 0.05 was considered to be statistically significant. Sensitivity (pEC 50 ) 136 to ACh and NS309 was determined in GraphPad Prism (version 7) using nonlinear 137 regression (variable slope with four parameters; constrains: TOP: 100, BOTTOM, 0).

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Comparable body weight after 3 months of a high fat diet regimen 141 The average age at euthanization was similar for all four mice groups (28 ± 1 g for 142 lean/saline, 28 ± 1 g for obese/saline, 28 ± 1 g for lean/rhTrx, and 28 ± 2 g for 143 obese/rhTrx). Body weight progressively increased during the 3-month high fat regimen 144 or normal diet in both saline-infused and rhTrx-injected mice ( Figure 1A). The body weight 145 gain was roughly 15 g for mice placed on a high fat diet for 13 weeks ( Figure 1B Figure 1C; 168 ± 5 m versus 184 ± 5 m, respectively). Infusion 156 of rhTrx prevented this inward remodeling after a high-fat diet, since optimal diameters 157 were comparable for both lean and obese mice that were given rhTrx ( Figure 1C; 183 ± 158 5 m versus 182 ± 4 m, respectively). Active wall tension (in mN/mm) in response to a 159 depolarizing KRB solution containing 60 mM KCl were comparable in MA2 for all 160 experimental mice groups ( Figure 1D). Coronary artery optimal diameter ( Figure 1E) and 161 active wall tension ( Figure 1F) were similar for the four experimental mice groups, 162 although wall tensions tended to be larger in the obese/rhTrx group ( Figure 1F). Femoral 163 artery optimal diameter tended to be smaller in the obese/saline group compared to the 164 other three groups ( Figure 1G), but active wall tension were statistically significantly 165 reduced compared to the other three groups ( Figure 1H).  but not coronary arteries, derived from obese mice injected with saline. In MA2, pEC 50 181 was decreased 10-fold in obese/saline compared to lean/saline (5.45 ± 0.08 versus 6.55 182 ± 0.05; P < 0.05; Figure 3A). E max was significantly diminished in obese/saline compared 183 to lean/saline (58 ± 6% versus 85 ± 3%; P < 0.05; Figure 3A). ACh-induced relaxations 184 were comparable in MA2 between lean/saline and lean/rhTrx mice ( Figure 3A). Strikingly, 185 tail vein infusion of rhTrx completely prevented the high fat-induced ACh-induced 186 impairment, with pEC 50 and E max values similar to lean/saline values (6.25 ± 0.08 and 75 187 ± 7%, respectively; Figure 3A). A similar trend was observed in femoral arteries, but the 188 differences were not as pronounced as in MA2 ( Figure 3C). ACh-induced relaxations in    Historically, the EDH-mediated response is analyzed in the presence of L-NAME and 228 indomethacin. Figure 6A shows that ACh-induced EDH responses were smallest in MA2 229 from obese/saline mice. AAC values were significantly reduced in MA2 from obese/saline 230 compared to the other groups ( Figure 6B). Sensitivity for ACh was significantly lower in 231 MA2 from obese/saline mice compared to the other groups ( Figure 6C). NS309-induced 232 relaxing responses were reduced in MA2 from obese/saline mice compared to the other 233 groups ( Figure 6D). AAC ( Figure 6E) and sensitivity ( Figure 6F The present isometric myograph functional data support the hypothesis that tail vein 239 injections of human recombinant thioredoxin completely protects against high fat-induced 240 endothelial dysfunction in small mesenteric and femoral arteries. In small mesenteric 241 arteries this protection is characterized by an increased NO and EDH relaxing response, 242 the latter via enhanced endothelial K Ca channel opening.

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Obesity is a major risk factor for the development of cardiovascular and metabolic 244 complications such as hypertension and type 2 diabetes [13,14]. This mouse strain is 245 well suited because of its high sensitivity for obesity and type 2 diabetes in response to a 246 high fat diet [15]. The objective of this study was not to assess plasma triglycerides and  The mesenteric arterial bed is prone to high fat-induced impairment in 279 endothelium-dependent relaxation, probably due to the close proximity of intestinal 280 absorption of fatty acids. The small mesenteric artery is a preferred choice of resistance-281 sized artery for the vascular biologist due to its abundance and relative ease of dissection.

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A pharmacological approach was used to assess the role of NO and the