Pharmacologic prophylactic treatment for perioperative protection of skeletal muscle from ischemia-reperfusion injury in reconstructive surgery.

BACKGROUND: In autogenous muscle transplantation, unpredictable complications can cause prolonged ischemia, resulting in ischemia-reperfusion injury. The authors investigated the efficacy and mechanism of nicorandil, a nitrovasodilator and adenosine triphosphate-sensitive potassium channel opener, in inducing perioperative protection of muscle flaps from ischemia-reperfusion injury. METHODS: Pigs (18.2 ± 2.4 kg) were assigned to one control and eight treatment groups. Bilateral latissimus dorsi muscle flaps were raised after saline administration (control) and 0, 4, 8, 12, 24, 48, 72, and 96 hours after nicorandil administration. Subsequently, flaps were subjected to 4 hours of ischemia and 48 hours of reperfusion. Viability was assessed, and biochemical probes were used to study nicorandil-induced infarct protection. RESULTS: Protection by nicorandil was biphasic. Infarction reduced from 40.2 ± 1.9 percent (control) to 27.3 ± 1.7 percent and 24.0 ± 2.3 percent (p < 0.05) 0 and 4 hours after nicorandil administration, respectively (early phase protection). No difference was seen between control and treatment groups between 8 and 12 hours after nicorandil administration compared with the control. Infarct protection increased again (p < 0.05) at 24 (22.4 ± 2.0 percent), 48 (25.1 ± 2.1 percent), and 72 hours (28.5 ± 2.1 percent) but not at 96 hours (43.9 ± 4.6 percent) compared with control (late phase protection). The sarcolemmal and mitochondrial channels played a central role in the trigger and mediator mechanisms, respectively. Late protection was associated with lower myeloperoxidase activity and mitochondrial calcium overload and higher adenosine triphosphate content (p < 0.05). CONCLUSIONS: Nicorandil induced 48-hour uninterrupted muscle infarct protection, starting 24 hours after intravenous administration. This category of clinical drug is a potential prophylactic treatment against skeletal muscle ischemia-reperfusion injury in reconstructive surgery.

Combination of hypoxic preconditioning and postconditioning does not induce additive protection of ex vivo human skeletal muscle from hypoxia/reoxygenation injury.

We previously demonstrated that hypoxic preconditioning (HPreC) or postconditioning (HPostC) protected ex vivo human skeletal muscle from hypoxia/reoxygenation injury. Here, we investigated if combined HPreC and HPostC could convey additive protection. Human rectus abdominis muscle strips were cultured in normoxic Krebs buffer for 5 hours (control) or in 3 hours hypoxic/2 hours normoxic buffer (treatment groups). HPreC and HPostC were induced by 1 cycle of 5 minutes hypoxia/5 minutes reoxygenation immediately before or after 3 hours hypoxia, respectively. Muscle injury, viability, and adenosine triphosphate (ATP) synthesis were assessed by measuring lactate dehydrogenase release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction, and ATP content, respectively. Hypoxia/reoxygenation caused lactate dehydrogenase to increase and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide reduction and ATP content to decrease (P < 0.05; n = 7). HPreC, HPostC, and combination of both were equally effective in protection of muscle from hypoxia/reoxygenation injury. Atractyloside (5 × 10 M), a mitochondrial permeability transition pore opener, abolished the protective effect of HPreC or HPostC. We conclude that HPreC and HPostC protect ex vivo human skeletal muscle against hypoxia/reoxygenation injury by closing the mitochondrial permeability transition pore. For that reason, they are equally effective and do not demonstrate an additive effect. Moreover, the potent effect of HPostC indicates ischemic postconditioning as an effective clinical intervention against reperfusion injury in autogenous skeletal muscle transplantation and replantation surgery.

Efficacy and mechanism of hypoxic postconditioning in salvage of ex vivo human rectus abdominis muscle from hypoxia/reoxygenation injury.

In reconstructive surgery, skeletal muscle may endure protracted ischemia before reperfusion, which can lead to significant ischemia/reperfusion injury. Ischemic postconditioning induced by brief cycles of reperfusion/reocclusion at the end of ischemia has been shown to salvage skeletal muscle from ischemia/reperfusion injury in several animal models. However, ischemic postconditioning has not been confirmed in human skeletal muscle. Using an established in vitro human skeletal muscle hypoxic conditioning model, we tested our hypothesis that hypoxic postconditioning salvages ex vivo human skeletal muscle from hypoxia/reoxygenation injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP) and preservation of ATP synthesis. Muscle strips (~0.5×0.5×15mm) from human rectus abdominis muscle biopsies were cultured in Krebs-Henseleit-HEPES buffer, bubbled with 95%N(2)/5%CO(2) (hypoxia) or 95%O(2)/5%CO(2) (reoxygenation). Samples were subjected to 3h hypoxia/2h reoxygenation. Hypoxic postconditioning was induced by one or two cycles of 5min reoxygenation/5min hypoxia after 3h hypoxia. Muscle injury, viability and ATP synthesis after 2h of reoxygenation were assessed by measuring lactate dehydrogenase (LDH) release, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) reduction and ATP content, respectively. Hypoxic postconditioning or treatment with the mPTP-opening inhibitors Cyclosporine A (CsA, 5×10(-6)M) or N-Methyl-4-isoleucine Cyclosporine (NIM811, 5×10(-6)M) 10min before reoxygenation decreased LDH release, increased MTT reduction and increased muscle ATP content (n=7 patients; P<0.05). Conversely, treatment with the mPTP opener Atractyloside (5×10(-6)M) 10min before hypoxic postconditioning abolished its protective effect (n=7 patients; P<0.05). We conclude that hypoxic postconditioning effectively salvages human skeletal muscle from hypoxia/reoxygenation injury by inhibition of mPTP opening and preservation of ATP synthesis during reoxygenation.

Vasorelaxation effect and mechanism of action of vascular endothelial growth factor-165 in isolated perfused human skin flaps.

BACKGROUND: Experimental evidence is accumulating to indicate that local acute vascular endothelial growth factor-165 (VEGF(165)) therapy is effective in attenuation of skin ischemia and increase in skin viability in rat skin flap surgery and the mechanism involves vasodilation induced by VEGF(165). So far, the vasodilator effect and mechanism of action of VEGF(165) have not been studied in human skin. The objective of this project is to test the hypothesis that VEGF(165) is also a potent vasodilator in human skin vasculature. MATERIALS AND METHODS: We used an established isolated perfused human skin flap model and pharmacologic probes to demonstrate that VEGF(165) is a potent vasodilator in human skin vasculature and the mechanism involves activation of receptors and postreceptor signaling pathway, which in turn stimulates local synthesis/release of endothelial vasodilators. RESULTS: We observed that VEGF(165) induced a concentration-dependent vasorelaxation in human skin flaps preconstricted with norephinephrine (8 × 10(-7)M; n = 7) or endothelin-1 (3 × 10(-9)M; n = 6). The vasorelaxation potency of VEGF(165) (pD(2) = 12.02 ± 0.25; n = 7) was higher (P < 0.05) than that of acetylcholine (pD(2) = 6.76 ± 0.06; n = 5) in human skin flaps preconstricted with 8 x 10(-7)M of norepinephrine. Using pharmacologic probes, we also detected that the vasorelaxation effect of VEGF(165) in the isolated perfused human skin flaps (n = 4) was triggered by activation of VEGF receptor-2. Furthermore, the postreceptor signaling pathway involved activation of Src family tyrosine kinase, phospholipase C, protein kinase C, an increase in inositol 1,4,5-triphosphate activity, a release of the intracellular Ca(2+) store, and finally synthesis/release of the endothelial nitric oxide (eNO) and prostacyclin and eNO predominantly mediated the vasodilator effect of VEGF(165) in the effector mechanism. CONCLUSION: These findings support our hypothesis that VEGF(165) is a potent vasodilator in human skin vasculature and also provide important insights into the clinical study of local acute VEGF(165) therapy for prevention/treatment of skin ischemia in skin flap surgery.

Na+/H+ exchange inhibitor cariporide attenuates skeletal muscle infarction when administered before ischemia or reperfusion.

Administration of Na(+)/H(+) exchange isoform-1 (NHE-1) inhibitors before ischemia has been shown to attenuate myocardial infarction in several animal models of ischemia-reperfusion injury. However, controversy still exists as to the efficacy of NHE-1 inhibitors in protection of myocardial infarction when administered at the onset of reperfusion. Furthermore, the efficacy of NHE-1 inhibition in protection of skeletal muscle from infarction (necrosis) has not been studied. This information has potential clinical applications in prevention or salvage of skeletal muscle from ischemia-reperfusion injury in elective and trauma reconstructive surgery. The objective of this research project is to test our hypothesis that the NHE-1 inhibitor cariporide is effective in protection of skeletal muscle from infarction when administered at the onset of sustained ischemia or reperfusion and to study the mechanism of action of cariporide. In our studies, we observed that intravenous administration of cariporide 10 min before ischemia (1 or 3 mg/kg) or reperfusion (3 mg/kg) significantly reduced infarction in pig latissimus dorsi muscle flaps compared with the control, when these muscle flaps were subjected to 4 h of ischemia and 48 h of reperfusion (P < 0.05; n = 5 pigs/group). Both preischemic and postischemic cariporide treatment (3 mg/kg) induced a significant decrease in muscle myeloperoxidase activity and mitochondrial-free Ca(2+) content and a significant increase in muscle ATP content within 2 h of reperfusion (P < 0.05; n = 4 pigs/group). Preischemic and postischemic cariporide treatment (3 mg/kg) also significantly inhibited muscle NHE-1 protein expression within 2 h of reperfusion after 4 h of ischemia, compared with the control (P < 0.05; n = 3 pigs/group). These observations support our hypothesis that cariporide attenuates skeletal muscle infarction when administered at the onset of ischemia or reperfusion, and the mechanism involves attenuation of neutrophil accumulation and mitochondrial-free Ca(2+) overload and preservation of ATP synthesis in the early stage of reperfusion.

Postconditioning for salvage of ischemic skeletal muscle from reperfusion injury: efficacy and mechanism.

We tested our hypothesis that postischemic conditioning (PostC) is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mitochondrial permeability transition pore (mPTP). In bilateral 8x13 cm pig latissimus dorsi muscle flaps subjected to 4 h ischemia, muscle infarction increased from 22+/-4 to 41+/-1% between 2 and 24 h reperfusion and remained unchanged at 48 (38+/-6%) and 72 (40+/-1%) h reperfusion (P<0.05; n=4 pigs). PostC induced by four cycles of 30-s reperfusion/reocclusion at the onset of reperfusion after 4 h ischemia reduced muscle infarction from 44+/-2 to 22+/-2% at 48 h reperfusion. This infarct protective effect of PostC was mimicked by intravenous injection of the mPTP opening inhibitor cyclosporin A or NIM-811 (10 mg/kg) at 5 min before the end of 4 h ischemia and was abolished by intravenous injection of the mPTP opener atractyloside (10 mg/kg) at 5 min before PostC (P<0.05; n=4-5 pigs). PostC or intravenous cyclosporin A injection at 5 min before reperfusion caused a decrease in muscle myeloperoxidase activity and mitochondrial free Ca2+ concentration and an increase in muscle ATP content after 4 h ischemia and 2 h reperfusion compared with the time-matched controls. These effects of PostC were abolished by intravenous injection of atractyloside at 5 min before PostC (P<0.05; n=6 pigs). These observations support our hypothesis that PostC is effective in salvage of ischemic skeletal muscle from reperfusion injury and the mechanism involves inhibition of opening of the mPTP.

Radiation-induced craniofacial bone growth inhibition: in vitro cytoprotection in the rabbit orbitozygomatic complex periosteum-derived cell culture.

BACKGROUND: Radiotherapy for the management of head and neck cancer in pediatric patients results in severe inhibition of craniofacial bone growth. Previously, the infant rabbit orbitozygomatic complex was established as an experimental model. Amifostine, a cytoprotective agent, was found effective in preventing radiation-induced bone growth inhibition. This study was designed to investigate the effects radiation on osteogenic cells from infant rabbit orbitozygomatic complex periostea and to assess the effects of cytoprotection in vitro. METHODS: Infant New Zealand White rabbits (n = 18) were randomized into three groups and received radiation (0, 10, or 15 Gy) to both orbitozygomatic complexes. Cell cultures were developed from orbitozygomatic complex periostea, and cell numbers, proliferation, alkaline phosphatase, and collagen type I expression and mineralization were assessed. Subsequently, rabbits (n = 18) were randomized into three groups to receive either radiation at the effective dose, pretreatment with amifostine (300 mg/kg, intravenously, 20 minutes before irradiation) with the effective radiation dose, or no treatment. Cell cultures were developed and tested for proliferation and alkaline phosphatase expression. RESULTS: Irradiation resulted in a significant inhibition of cell numbers (p < 0.001) and proliferation (p < 0.01) at the 15-Gy dose and no statistically significant changes in alkaline phosphatase activity. Collagen type I expression and mineralization were also significantly reduced at the 15-Gy dose. Pretreatment with amifostine significantly (p < 0.05) enhanced the number of surviving cells. CONCLUSIONS: Amifostine is capable of protecting orbitozygomatic complex periosteum-derived osteogenic cells from the deleterious effects of radiation. This study provides the basis for understanding the cellular mechanisms of radiation-induced craniofacial bone growth inhibition and cytoprotection by amifostine.

An in vitro model of radiation-induced craniofacial bone growth inhibition.

Radiation-induced craniofacial bone growth inhibition is a consequence of therapeutic radiation in the survivors of pediatric head and neck cancer. Previously, the infant rabbit orbitozygomatic complex (OZC) was established as a reliable animal model. The purpose of this study was to develop a cell culture model from the rabbit OZC to study the effects of radiation in the craniofacial skeleton. Infant (7-week-old) New Zealand white rabbits were used in this study. Periostea from both OZC were harvested in sterile conditions, introduced into cell culture by way of sequential digestion, and subcultured at confluence. Cultures were analyzed for cellular proliferation (methylthiazoletetrazolium assay), alkaline phosphatase activity, collagen type I expression, and mineralization. Electron microscopy was performed to reveal the in vitro ultrastructure. Subsequently, rabbits were irradiated with sham or 15 Gy radiation, and cell cultures were developed and analyzed for cell numbers. Cell cultures, grown from OZC periostea, expressed osteoblast-like phenotype, with high alkaline phosphatase activity, collagen type 1 expression, and mineralization in an osteogenic environment. Electron microscopy confirmed the characteristic ultrastructural features of osteogenesis in vitro. Finally, significantly (P < 0.01) fewer cells were obtained from animals treated with 15 Gy radiation compared with those from control animals.A primary cell culture with osteoblast-like cellular phenotype was developed from infant rabbit OZC periosteum. This cell culture system responded to in vivo administered radiation by a significant decrease in cell numbers. This in vitro model will be subsequently used to study the cellular mechanisms of radiation and radioprotection in craniofacial osteoblast-like cells.

Development of an in vitro model for study of the efficacy of ischemic preconditioning in human skeletal muscle against ischemia-reperfusion injury.

Ischemia-reperfusion (I/R) injury causes skeletal muscle infarction and ischemic preconditioning (IPC) augments ischemic tolerance in animal models. To date, this has not been demonstrated in human skeletal muscle. This study aimed to develop an in vitro model to investigate the efficacy of simulated IPC in human skeletal muscle. Human skeletal muscle strips were equilibrated in oxygenated Krebs-Henseleit-HEPES buffer (37 degrees C). Aerobic and reperfusion phases were simulated by normoxic incubation and reoxygenation, respectively. Ischemia was simulated by hypoxic incubation. Energy store, cell viability, and cellular injury were assessed using ATP, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and lactate dehydrogenase (LDH) assays, respectively. Morphological integrity was assessed using electron microscopy. Studies were designed to test stability of the preparation (n = 5-11) under normoxic incubation over 24 h; the effect of 1, 2, 3, 4, or 6 h hypoxia followed by 2 h of reoxygenation; and the protective effect of hypoxic preconditioning (HPC; 5 min of hypoxia/5 min of reoxygenation) before 3 h of hypoxia/2 h of reoxygenation. Over 24 h of normoxic incubation, muscle strips remained physiologically intact as assessed by MTT, ATP, and LDH assays. After 3 h of hypoxia/2 h of reoxygenation, MTT reduction levels declined to 50.1 +/- 5.5% (P < 0.05). MTT reduction levels in HPC (82.3 +/- 10.8%) and normoxic control (81.3 +/- 10.2%) groups were similar and higher (P < 0.05) than the 3 h of hypoxia/2 h of reoxygenation group (45.2 +/- 5.8%). Ultrastructural morphology was preserved in normoxic and HPC groups but not in the hypoxia/reoxygenation group. This is the first study to characterize a stable in vitro model of human skeletal muscle and to demonstrate a protective effect of HPC in human skeletal muscle against hypoxia/reoxygenation-induced injury.

Efficacy and mechanism of adenovirus-mediated VEGF-165 gene therapy for augmentation of skin flap viability.

Skin ischemic necrosis due to vasospasm and/or insufficient vascularity is the most common complication in the distal portion of the skin flap in reconstructive surgery. This project was designed to test our hypothesis that preoperative subdermal injection of adenoviral vectors encoding genes for vascular endothelial growth factor-165 (Ad.VEGF-165) or endothelial nitric oxide (NO) synthase (Ad.eNOS) effectively augments skin viability in skin flap surgery and that the mechanism of Ad.VEGF-165 gene therapy involves an increase in synthesis/release of the angiogenic and vasodilator factor NO. PBS (0.5 ml) or PBS containing Ad.VEGF-165, Ad.eNOS, or adenovirus (Ad.Null) was injected subdermally into the distal half of a mapped rat dorsal skin flap (4 x 10 cm) 7 days preoperatively, and skin flap viability was assessed 7 days postoperatively. Local subdermal gene therapy with 2 x 10(7)-2 x 10(10) plaque-forming units of VEGF-165 increased skin flap viability compared with PBS- or Ad.Null-injected control (P < 0.05). Subdermal Ad.VEGF-165 and Ad.eNOS gene therapies were equally effective in increasing skin flap viability at 5 x 10(8) plaque-forming units. Subdermal Ad.VEGF-165 therapy was associated with upregulation of eNOS protein expression, Ca2+ -dependent NOS activity, synthesis/release of NO, and increase in capillary density and blood flow in the distal portion of the skin flap. Injection of the NOS inhibitor Nomega-nitro-L-arginine (15 mg/kg im), but not the cyclooxygenase inhibitor indomethacin (5 mg/kg im), 45 min preoperatively completely abolished the increase in skin flap blood flow and viability induced by Ad.VEGF-165 injected subdermally into the mapped skin flap 7 days preoperatively. We have demonstrated for the first time that 1) Ad.VEGF-165 and Ad.eNOS mapped skin flap injected subdermally into the mapped skin flap 7 days preoperatively are equally effective in augmenting viability in the rat dorsal skin flap compared with control, 2) the mechanism of subdermal Ad.VEGF-165 gene therapy in augmenting skin flap viability involves an increase in NO synthesis/release downstream of upregulation of eNOS protein expression and Ca2+ -dependent NOS activity, and 3) the vasodilating effect of NO may predominantly mediate subdermal Ad.VEGF gene therapy in augmenting skin flap blood flow and viability.

Inducing late phase of infarct protection in skeletal muscle by remote preconditioning: efficacy and mechanism.

We have previously demonstrated that remote ischemic preconditioning (IPC) by instigation of three cycles of 10-min occlusion/reperfusion in a hindlimb of the pig elicits an early phase of infarct protection in local and distant skeletal muscles subjected to 4 h of ischemia immediately after remote IPC. The aim of this project was to test our hypothesis that hindlimb remote IPC also induces a late phase of infarct protection in skeletal muscle and that K(ATP) channels play a pivotal role in the trigger and mediator mechanisms. We observed that pig bilateral latissimus dorsi (LD) muscle flaps sustained 46 +/- 2% infarction when subjected to 4 h of ischemia/48 h of reperfusion. The late phase of infarct protection appeared at 24 h and lasted up to 72 h after hindlimb remote IPC. The LD muscle infarction was reduced to 28 +/- 3, 26 +/- 1, 23 +/- 2, 24 +/- 2 and 24 +/- 4% at 24, 28, 36, 48 and 72 h after remote IPC, respectively (P < 0.05; n = 8). In subsequent studies, hindlimb remote IPC or intravenous injection of the sarcolemmal K(ATP) (sK(ATP)) channel opener P-1075 (2 microg/kg) at 24 h before 4 h of sustained ischemia (i.e., late preconditioning) reduced muscle infarction from 43 +/- 4% (ischemic control) to 24 +/- 2 and 19 +/- 3%, respectively (P < 0.05, n = 8). Intravenous injection of the sK(ATP) channel inhibitor HMR 1098 (6 mg/kg) or the nonspecific K(ATP) channel inhibitor glibenclamide (Glib; 1 mg/kg) at 10 min before remote IPC completely blocked the infarct- protective effect of remote IPC in LD muscle flaps subjected to 4 h of sustained ischemia at 24 h after remote IPC. Intravenous bolus injection of the mitochondrial K(ATP) (mK(ATP)) channel inhibitor 5-hydroxydecanoate (5-HD; 5 mg/kg) immediately before remote IPC and 30-min intravenous infusion of 5-HD (5 mg/kg) during remote IPC did not affect the infarct-protective effect of remote IPC in LD muscle flaps. However, intravenous Glib or 5-HD, but not HMR 1098, given 24 h after remote IPC completely blocked the late infarct-protective effect of remote IPC in LD muscle flaps. None of these drug treatments affected the infarct size of control LD muscle flaps. The late phase of infarct protection was associated with a higher (P < 0.05) muscle content of ATP at the end of 4 h of ischemia and 1.5 h of reperfusion and a lower (P < 0.05) neutrophilic activity at the end of 1.5 h of reperfusion compared with the time-matched control. In conclusion, these findings support our hypothesis that hindlimb remote IPC induces an uninterrupted long (48 h) late phase of infarct protection, and sK(ATP) and mK(ATP) channels play a central role in the trigger and mediator mechanism, respectively.

Oleate-mediated stimulation of microsomal triglyceride transfer protein (MTP) gene promoter: implications for hepatic MTP overexpression in insulin resistance.

Hepatic lipoprotein overproduction in a fructose-fed hamster model of insulin resistance was previously shown to be associated with a significant elevation of intracellular mass of microsomal triglyceride transfer protein (MTP) and elevated plasma levels of free fatty acids (FFA). Here, we further establish that fructose feeding and development of an insulin resistant state result in higher levels of MTP mRNA, protein mass, and lipid transfer activity. MTP protein mass was increased in fructose-fed hamster hepatocytes to 161 +/- 35.8% of control (p < 0.05), while MTP mRNA levels and MTP lipid transfer activity were increased to 147.5 +/- 30.8% (p < 0.05) and 177.5 +/- 14.5% (p < 0.05) of control levels, respectively. To identify underlying mechanisms, we also investigated the potential link between enhanced FFA flux and hepatic MTP gene expression. Direct modulation of MTP gene transcription by fatty acids was investigated by transfecting HepG2 cells with a reporter (luciferase) construct containing various base pair regions of the human MTP promoter including pMTP124 (with the sterol response element (SRE)), pMTP116, pMTP109 and pMTP100 (no SRE), and pMTP124SREKO (SRE sequences mutated). Treatment of HepG2 cells with oleic acid (360 muM) significantly increased luciferase activities in cells transfected with pMTP124 (136.6 +/- 11.0%, p < 0.05) and pMTP124SREKO (153.9 +/- 11.1%, p < 0.01) compared with control. Luciferase activity was also increased in a time and dose-dependent manner in the presence of oleic acid when transfected with pMTP124SREKO but not pMTP109 and pMTP100. Furthermore, long-term oleic acid treatment of HepG2 cells (10 days) induced higher levels of MTP mRNA (p < 0.05) confirming transcriptional stimulation of the MTP gene by oleic acid. In contrast, palmitate, arachidonic acid or linoleic acid did not significantly stimulate pMTP124 or pMTP124SREKO luciferase activity (p > 0.05). These data demonstrate that (1) MTP gene transcription may be directly up-regulated by oleic acid; (2) up-regulation of MTP gene transcription by oleic acid is SRE sequence independent; and (3) the sequence -116 to -109 in the MTP promoter region is essential for oleic acid-mediated stimulation. Stimulation of MTP gene expression may be a novel mechanism by which certain FFAs can induce hepatic lipoprotein secretion in insulin resistant states.

Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction.

We previously demonstrated in the pig that instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (approximately 300 mmHg) elicited protection against ischemia-reperfusion injury (infarction) in multiple distant skeletal muscles subsequently subjected to 4 h of ischemia and 48 h of reperfusion, but the mechanism was not studied. The aim of this project was to test our hypothesis that mitochondrial ATP-sensitive potassium (KATP) (mKATP) channels play a central role in the trigger and mediator mechanisms of hindlimb remote ischemic preconditioning (IPC) of skeletal muscle against infarction in the pig. We observed in the pig that hindlimb remote IPC reduced the infarct size of latissimus dorsi (LD) muscle flaps (8 x 13 cm) from 45 +/- 2% to 22 +/- 3% (n = 10; P < 0.05). The nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-hydroxydecanoate (5-HD, 5 mg/kg), but not the selective sarcolemmal KATP (sKATP) channel inhibitor HMR-1098 (3 mg/kg), abolished the infarct-protective effect of hindlimb remote IPC in LD muscle flaps (n = 10, P < 0.05) when these drugs were injected intravenously at 10 min before remote IPC. In addition, intravenous bolus injection of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) at the end of hindlimb remote IPC also abolished the infarct protection in LD muscle flaps (n = 10; P < 0.05). Furthermore, intravenous injection of the specific mKATPchannel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of ischemia protected the LD muscle flap against infarction to a similar extent as hindlimb remote IPC, and this infarct-protective effect of BMS-191095 was abolished by intravenous bolus injection of 5-HD (5 mg/kg) at 10 min before or after intravenous injection of BMS-191095 (n = 10; P < 0.05). The infarct protective effect of BMS-191095 was associated with a higher muscle content of ATP at the end of 4 h of ischemia and a decrease in muscle neutrophilic myeloperoxidase activity at the end of 1.5 h of reperfusion compared with the time-matched control (n = 10, P < 0.05). These observations led us to conclude that mKATP channels play a central role in the trigger and mediator mechanisms of hindlimb remote IPC of skeletal muscle against infarction in the pig, and the opening of mKATP channels in ischemic skeletal muscle is associated with an ATP-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.

Acute local subcutaneous VEGF165 injection for augmentation of skin flap viability: efficacy and mechanism.

Distal skin ischemic necrosis is a common complication in skin flap surgery. The pathogenesis of skin flap ischemic necrosis is unclear, and there is no clinical treatment available. Here, we used the 4 x 10 cm rat dorsal skin flap model to test our hypothesis that subcutaneous injection of vascular endothelial growth factor 165 (VEGF165) in skin flaps at the time of surgery is effective in augmentation of skin flap viability, which is associated with an increase in nitric oxide (NO) production, and the mechanism involves 1) an increase in skin flap blood flow in the early stage after surgery and 2) enhanced angiogenesis subsequently to sustain increased skin flap blood flow and viability. We observed that subcutaneous injection of VEGF165 in skin flaps at the time of surgery increased skin flap viability in a dose-dependent manner. Subcutaneous injection of VEGF165 at the dose of 2 microg/flap increased skin flap viability by 28% (P < 0.05; n = 8). Over 80% of this effect was blocked by intramuscular injection of the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine (13 mg/kg) 45 min before surgery (P < 0.05; n = 8). The VEGF165 treatment also increased skin flap blood flow (2.68 +/- 0.63 ml x min(-1) x 100 g(-1)) compared with the control (1.26 +/- 0.10 ml x min(-1) x 100 g(-1); P < 0.05, n = 6) assessed 6 h postoperatively. There was no change in skin flap capillary density at this time point. VEGF165-induced increase in capillary density (32.2 +/- 1.1 capillaries/mm2; P < 0.05, n = 7) compared with control (24.6 +/- 1.4 capillaries/mm2) was seen 7 days postoperatively. There was also evidence to indicate that VEGF165-induced NO production in skin flaps was stimulated by activation of NOS activity followed by upregulation of NOS protein expression. These observations support our hypothesis and for the first time provide an important insight into the mechanism of acute local VEGF165 protein therapy in mitigation of skin flap ischemic necrosis.

Vasodilator effect and mechanism of action of vascular endothelial growth factor in skin vasculature.

Various laboratories have reported that local subcutaneous or subdermal injection of VEGF(165) at the time of surgery effectively attenuated ischemic necrosis in rat skin flaps, but the mechanism was not studied and enhanced angiogenesis was implicated. In the present study, we used the clinically relevant isolated perfused 6 x 16-cm pig buttock skin flap model to 1) test our hypothesis that VEGF(165) is a potent vasodilator and acute VEGF(165) treatment increases skin perfusion; and 2) investigate the mechanism of VEGF(165)-induced skin vasorelaxation. We observed that VEGF(165) (5 x 10(-16)-5 x 10(-11) M) elicited a concentration-dependent decrease in perfusion pressure (i.e., vasorelaxation) in skin flaps preconstricted with a submaximal concentration of norepinephrine (NE), endothelin-1, or U-46619. The VEGF(165)-induced skin vasorelaxation was confirmed using a dermofluorometry technique for assessment of skin perfusion. The vasorelaxation potency of VEGF(165) in NE-preconstricted skin flaps (pD(2) = 13.57 +/- 0.31) was higher (P < 0.05) than that of acetylcholine (pD(2) = 7.08 +/- 0.24). Human placental factor, a specific VEGF receptor-1 agonist, did not elicit any vasorelaxation effect. However, a specific antibody to VEGF receptor-2 (1 microg/ml) or a specific VEGF receptor-2 inhibitor (5 x 10(-6) M SU-1498) blocked the vasorelaxation effect of VEGF(165) in NE-preconstricted skin flaps. These observations indicate that the potent vasorelaxation effect of VEGF(165) in the skin vasculature is initiated by the activation of VEGF receptor-2. Furthermore, using pharmacological probes, we observed that the postreceptor signaling pathways of VEGF(165)-induced skin vasorelaxation involved activation of phospholipase C and protein kinase C, an increase in inositol 1,4,5-trisphosphate activity, release of the intra-cellular Ca(2+) store, and synthesis/release of endothelial nitric oxide, which predominantly triggered the effector mechanism of VEGF(165)-induced vasorelaxation. This information provides, for the first time, an important insight into the mechanism of VEGF(165) protein or gene therapy in the prevention/treatment of ischemia in skin flap surgery and skin ischemic diseases.

Noninvasive remote ischemic preconditioning for global protection of skeletal muscle against infarction.

The aim of this study was to investigate the efficacy and mechanism of action of a noninvasive remote ischemic preconditioning (IPC) technique for the protection of multiple distant skeletal muscles against ischemic necrosis (infarction). It was observed in the pig that three cycles of 10-min occlusion and reperfusion in a hindlimb by tourniquet application reduced the infarction of latissimus dorsi (LD), gracilis (GC), and rectus abdominis (RA) muscle flaps by 55%, 60%, and 55%, respectively, compared with their corresponding control (n = 6, P < 0.01) when they were subsequently subjected to 4 h of ischemia and 48 h of reperfusion. This infarct-protective effect of remote IPC in LD muscle flaps was abolished by an intravenous bolus injection of the nonselective opioid receptor antagonist naloxone (3 mg/kg) 10 min before remote IPC and a continuous intravenous infusion (3 mg/kg) during remote IPC and by an intravenous bolus injection of the selective delta 1-opioid receptor antagonist 7-benzylidenealtrexone maleate (3 mg/kg). However, this infarct-protective effect of remote IPC was not affected by an intravenous bolus injection of the ganglionic blocker hexamethonium chloride (20 mg/kg) or the nonspecific adenosine receptor antagonist 8-(p-sulfophenyl)theophylline (10 mg/kg) or by a local intra-arterial injection of the adenosine1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (3 mg/muscle flap) given 10 min before remote IPC. It was also observed that this remote IPC of skeletal muscle against infarction was associated with a slower rate of muscle ATP depletion during the 4 h of sustained ischemia and a reduced muscle neutrophilic myeloperoxidase activity after 1.5 h of reperfusion. These observations led us to speculate that noninvasive remote IPC by brief cycles of occlusion and reperfusion in a pig hindlimb is effective in global protection of skeletal muscle against infarction. This infarct-protective effect is most likely triggered by the activation of opioid receptors in the skeletal muscle, and remote IPC is associated with an energy-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.