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• W2145868785 abstract "Genetics plays an important role in determining peripheral arterial disease (PAD) pathology, which causes a spectrum of clinical disorders that range from clinically silent reductions in blood flow to limb-threatening ischemia. The cell-type specificity of PAD pathology, however, has received little attention. To determine whether strain-dependent differences in skeletal muscle cells might account for the differential responses to ischemia observed in C57BL/6 and BALB/c mice, endothelial and skeletal muscle cells were subjected to hypoxia and nutrient deprivation (HND) in vitro, to mimic ischemia. Muscle cells were more susceptible to HND than were endothelial cells. In vivo, C57BL/6 and BALB/c mice displayed strain-specific differences in myofiber responses after hindlimb ischemia, with significantly greater myofiber atrophy, greater apoptosis, and attenuated myogenic regulatory gene expression and stress-responsive signaling in BALB/c mice. Strain-specific deficits were recapitulated in vitro in primary muscle cells from both strains after HND. Muscle cells from BALB/c mice congenic for the C57BL/6 Lsq-1 quantitative trait locus were protected from HND-induced atrophy, and gene expression of vascular growth factors and their receptors was significantly greater in C57BL/6 primary muscle cells. Our results indicate that the previously identified specific genetic locus regulating strain-dependent collateral vessel density has a nonvascular or muscle cell-autonomous role involving both the myogenic program and traditional vascular growth factor receptor expression. Genetics plays an important role in determining peripheral arterial disease (PAD) pathology, which causes a spectrum of clinical disorders that range from clinically silent reductions in blood flow to limb-threatening ischemia. The cell-type specificity of PAD pathology, however, has received little attention. To determine whether strain-dependent differences in skeletal muscle cells might account for the differential responses to ischemia observed in C57BL/6 and BALB/c mice, endothelial and skeletal muscle cells were subjected to hypoxia and nutrient deprivation (HND) in vitro, to mimic ischemia. Muscle cells were more susceptible to HND than were endothelial cells. In vivo, C57BL/6 and BALB/c mice displayed strain-specific differences in myofiber responses after hindlimb ischemia, with significantly greater myofiber atrophy, greater apoptosis, and attenuated myogenic regulatory gene expression and stress-responsive signaling in BALB/c mice. Strain-specific deficits were recapitulated in vitro in primary muscle cells from both strains after HND. Muscle cells from BALB/c mice congenic for the C57BL/6 Lsq-1 quantitative trait locus were protected from HND-induced atrophy, and gene expression of vascular growth factors and their receptors was significantly greater in C57BL/6 primary muscle cells. Our results indicate that the previously identified specific genetic locus regulating strain-dependent collateral vessel density has a nonvascular or muscle cell-autonomous role involving both the myogenic program and traditional vascular growth factor receptor expression. Peripheral arterial disease (PAD) results from atherosclerosis of peripheral arteries, most commonly in the lower extremities, and causes a spectrum of clinical disorders that range from clinically silent reductions in blood flow to limb-threatening ischemia for which amputation is often required.1Norgren L. Hiatt W.R. Dormandy J.A. Nehler M.R. Harris K.A. Fowkes F.G. TASC II Working GroupInter-Society consensus for the management of peripheral arterial disease (TASC II).J Vasc Surg. 2007; 45: S5-S67Abstract Full Text Full Text PDF PubMed Scopus (4441) Google Scholar In patients with intermittent claudication, arterial occlusive disease results in reduced blood flow manifested as pain with exertion, whereas in patients with critical limb ischemia (CLI) the blood flow is inadequate to meet the resting demands of the limb and results in pain at rest and/or tissue necrosis. Although less common than claudication, CLI results in significantly higher morbidity and mortality; patients with CLI have a risk of major amputation or death that approaches 40% in 1 year.2Hirsch A.T. Criqui M.H. Treat-Jacobson D. Regensteiner J.G. Creager M.A. Olin J.W. Krook S.H. Hunninghake D.B. Comerota A.J. Walsh M.E. McDermott M.M. Hiatt W.R. Peripheral arterial disease detection, awareness, and treatment in primary care.JAMA. 2001; 286: 1317-1324Crossref PubMed Scopus (2219) Google Scholar, 3Taylor S.M. Cull D.L. Kalbaugh C.A. Senter H.F. Langan 3rd, E.M. Carsten 3rd, C.G. York J.W. Snyder B.A. Gray B.H. Androes M.P. Blackhurst D.W. Comparison of interventional outcomes according to preoperative indication: a single center analysis of 2,240 limb revascularizations.J Am Coll Surg. 2009; 208 (discussion 778–780): 770-778Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 4Dormandy J. Heeck L. Vig S. The fate of patients with critical leg ischemia.Semin Vasc Surg. 1999; 12: 142-147PubMed Google Scholar Although it was long held that CLI represents the natural progressive deterioration of PAD in patients with claudication, this appears not to be the case. In fact, only a small percentage of patients with claudication eventually develop symptoms of CLI, and a substantial number of patients with CLI deny prior symptoms of claudication.5Mätzke S. Lepäntalo M. Claudication does not always precede critical leg ischemia.Vasc Med. 2001; 6: 77-80PubMed Google Scholar Furthermore, patients with the same degree of lower extremity athero-occlusive disease can present with either intermittent claudication or CLI. For these reasons, it appears that intermittent claudication and CLI represent distinct phenotypic manifestations of the same underlying atherosclerotic disease process, likely because of differences in genetic susceptibility. Thus, identifying the genetic modifiers that predispose individuals to develop CLI remains an important area of investigation in PAD. Mouse models of limb ischemia provide useful tools with which to investigate the mechanisms regulating the ischemic response.6Couffinhal T. Silver M. Kearney M. Sullivan A. Witzenbichler B. Magner M. Annex B. Peters K. Isner J.M. Impaired collateral vessel development associated with reduced expression of vascular endothelial growth factor in ApoE-/- mice.Circulation. 1999; 99: 3188-3198Crossref PubMed Scopus (240) Google Scholar, 7Couffinhal T. Silver M. Zheng L.P. Kearney M. Witzenbichler B. Isner J.M. Mouse model of angiogenesis.Am J Pathol. 1998; 152: 1667-1679PubMed Google Scholar It is well established that different inbred strains of mice display markedly different responses to surgically induced hindlimb ischemia (HLI).8Chalothorn D. Clayton J.A. Zhang H. Pomp D. Faber J.E. Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains.Physiol Genomics. 2007; 30: 179-191Crossref PubMed Scopus (149) Google Scholar, 9Dokun A.O. Keum S. Hazarika S. Li Y. Lamonte G.M. Wheeler F. Marchuk D.A. Annex B.H. A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.Circulation. 2008; 117: 1207-1215Crossref PubMed Scopus (95) Google Scholar, 10Helisch A. Wagner S. Khan N. Drinane M. Wolfram S. Heil M. Ziegelhoeffer T. Brandt U. Pearlman J.D. Swartz H.M. Schaper W. Impact of mouse strain differences in innate hindlimb collateral vasculature.Arterioscler Thromb Vasc Biol. 2006; 26: 520-526Crossref PubMed Scopus (182) Google Scholar In particular, the C57BL/6 (BL6) and BALB/c strains have frequently been compared because of their markedly different responses to ischemia: BL6 mice display significantly better collateral artery formation and limb perfusion and less tissue damage than BALB/c mice after HLI.10Helisch A. Wagner S. Khan N. Drinane M. Wolfram S. Heil M. Ziegelhoeffer T. Brandt U. Pearlman J.D. Swartz H.M. Schaper W. Impact of mouse strain differences in innate hindlimb collateral vasculature.Arterioscler Thromb Vasc Biol. 2006; 26: 520-526Crossref PubMed Scopus (182) Google Scholar Nonetheless, little is known about the genetic mechanisms responsible for these differences in phenotype. Chalothorn et al8Chalothorn D. Clayton J.A. Zhang H. Pomp D. Faber J.E. Collateral density, remodeling, and VEGF-A expression differ widely between mouse strains.Physiol Genomics. 2007; 30: 179-191Crossref PubMed Scopus (149) Google Scholar demonstrated significantly lower expression of vascular endothelial growth factor A (VEGF-A) in response to HLI in BALB/c mice compared with BL6, suggesting that insufficient angiogenesis or collateralization is responsible for the poor recovery of BALB/c mice. In that study, a bioinformatics approach was used to identify a putative expression quantitative trait locus (QTL) for VEGF-A expression on mouse chromosome 17, suggesting a polymorphism in BALB/c mice that may be responsible for reduced VEGF-A expression. To investigate the genetic mechanisms responsible for the ischemic response in more detail, our group recently performed genome-wide scanning with polymorphic markers in BL6×BALB/c offspring.9Dokun A.O. Keum S. Hazarika S. Li Y. Lamonte G.M. Wheeler F. Marchuk D.A. Annex B.H. A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.Circulation. 2008; 117: 1207-1215Crossref PubMed Scopus (95) Google Scholar A QTL linked to perfusion recovery and limb necrosis was identified on chromosome 7 (Lsq-1) with an extremely high degree of statistical significance [logarithm of the odds (LOD) score > 7.9]. Using haplotype mapping in BL6, BALB/c, and A/J mice, in which the ischemic response is similar to that of BALB/c, this QTL was narrowed to a region encompassing 37 genes. The same locus was subsequently linked to the regulation of pial collateral vessel number and limitation of cerebral infarct size after middle cerebral artery occlusion.11Wang S. Zhang H. Dai X. Sealock R. Faber J.E. Genetic architecture underlying variation in extent and remodeling of the collateral circulation.Circ Res. 2010; 107: 558-568Crossref PubMed Scopus (67) Google Scholar, 12Keum S. Marchuk D.A. A locus mapping to mouse chromosome 7 determines infarct volume in a mouse model of ischemic stroke.Circ Cardiovasc Genet. 2009; 2: 591-598Crossref PubMed Scopus (49) Google Scholar Notably, however, none of the candidate genes in this QTL has a previously defined role in vascular growth, suggesting an as yet undiscovered vascular function for at least one of these genes, and/or that genetic influences on other cellular processes play an important role in the response to ischemia. Numerous studies of the effects of ischemia on muscle tissue have centered on vascular cell responses or collateral vessel density, perhaps in part because targeting the vasculature through therapeutic angiogenesis holds promise as a potential treatment for ischemic diseases such as PAD.13Simons M. Angiogenesis: where do we stand now.Circulation. 2005; 111: 1556-1566Crossref PubMed Scopus (366) Google Scholar Although studies have examined the effects of ischemia on other aspects of the limb muscle response, including mitochondrial biogenesis and the transition of muscle fibers to more ischemia-tolerant phenotypes,14Arany Z. Foo S.Y. Ma Y. Ruas J.L. Bommi-Reddy A. Girnun G. Cooper M. Laznik D. Chinsomboon J. Rangwala S.M. Baek K.H. Rosenzweig A. Spiegelman B.M. HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha.Nature. 2008; 451: 1008-1012Crossref PubMed Scopus (845) Google Scholar, 15van Weel V. Deckers M.M.L. Grimbergen J.M. van Leuven K.J.M. Lardenoye J.H.P. Schlingemann R.O. van Nieuw Amerongen G.P. van Bockel J.H. van Hinsbergh V.W.M. Quax P.H.A. Vascular endothelial growth factor overexpression in ischemic skeletal muscle enhances myoglobin expression in vivo.Circ Res. 2004; 95: 58-66Crossref PubMed Scopus (61) Google Scholar, 16Williams R.S. Annex B.H. Plasticity of myocytes and capillaries: a possible coordinating role for VEGF.Circ Res. 2004; 95: 7-8Crossref PubMed Scopus (20) Google Scholar little is known about genetic modifiers of the skeletal muscle cell response to ischemia. Furthermore, it is unclear whether vascular cells or muscle cells are more susceptible to ischemia-induced injury. In the present study, we used both in vitro and in vivo models to investigate the genetic influence on the skeletal muscle cell response to ischemia. Here we demonstrate mouse strain-dependent differences in the myogenic regulatory program in response to HLI in vivo and show that these differences are recapitulated in isolated primary skeletal muscle cells in vitro. Furthermore, we demonstrate that the muscle cell-specific expression of vascular growth factors and their cognate receptors in vivo and in vitro in response to ischemia is genetically determined. These results provide novel insights into the genetic determinants of severe limb ischemia, such as that caused by CLI, by demonstrating that the same genetic locus linked to strain-dependent collateral vessel density also has a nonvascular or muscle cell-autonomous role. These findings establish that muscle–specific responses play a greater role than previously thought in determining pathological outcomes in response to ischemia. Experiments were conducted on 6- to 8-week-old adult male C57BL/6 or BALB/c mice (Jackson Laboratory, Bar Harbor, ME) and were approved by the Duke University Institutional Animal Care and Use Committee. Surgical hindlimb ischemia was performed as described previously.7Couffinhal T. Silver M. Zheng L.P. Kearney M. Witzenbichler B. Isner J.M. Mouse model of angiogenesis.Am J Pathol. 1998; 152: 1667-1679PubMed Google Scholar, 9Dokun A.O. Keum S. Hazarika S. Li Y. Lamonte G.M. Wheeler F. Marchuk D.A. Annex B.H. A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.Circulation. 2008; 117: 1207-1215Crossref PubMed Scopus (95) Google Scholar Briefly, ischemia was induced by anesthetizing mice by injection of ketamine (90 mg/kg i.p.) and xylazine (10 mg/kg i.p.), and unilateral hindlimb ischemia was surgically induced by ligation and excision of the femoral artery from its origin just above the inguinal ligament to its bifurcation at the origin of the saphenous and popliteal arteries. The inferior epigastric, lateral circumflex, and superficial epigastric artery branches were also isolated and ligated. Mice were closely monitored during the postoperative period, and perfusion in the ischemic and contralateral nonischemic limbs was measured immediately after surgery to verify successful ischemia. The extent of necrosis in ischemic hindlimbs, if any, was recorded postoperatively using a previously described semiquantitative scale9Dokun A.O. Keum S. Hazarika S. Li Y. Lamonte G.M. Wheeler F. Marchuk D.A. Annex B.H. A quantitative trait locus (LSq-1) on mouse chromosome 7 is linked to the absence of tissue loss after surgical hindlimb ischemia.Circulation. 2008; 117: 1207-1215Crossref PubMed Scopus (95) Google Scholar: grade 0, no necrosis in ischemic limb; grade I, necrosis limited to toes; grade II, necrosis extending to dorsum pedis; grade III, necrosis extending to crus; and grade IV, necrosis extending to thigh or complete limb necrosis. Immunofluorescence microscopy was used for visualization of muscle morphology, capillary density, apoptosis, and monocyte infiltration in ischemic and control skeletal muscle. Transverse sections (8 μm thick) were cut from tibialis anterior (TA) muscle frozen in liquid nitrogen-cooled isopentane in optimum cutting temperature (OCT) medium. Sections were allowed to come to room temperature and then were fixed/permeabilized with ice-cold acetone for 10 minutes at 4°C. Sections were allowed to air dry for 5 minutes at room temperature and then were rehydrated in 1× PBS before blocking in 5% normal goat serum (Sigma-Aldrich, St. Louis, MO) in 1× PBS at room temperature for 45 minutes. Slides were then incubated overnight at 4°C in a primary antibody solution containing either rat anti-CD31 (1:50; MCA-1364; AbD Serotec, Raleigh, NC) or mouse IgG2b anti-CD11 (1:100; MAS 034; Harlan Laboratories, Indianapolis, IN) and mouse IgG2a anti-dystrophin supernatant (1:5; MANDYS1 3B7; developed by G.E. Morris and obtained from the Developmental Studies Hybridoma Bank under the auspices of the NIH National Institute of Child Health and Human Development and maintained by the University of Iowa Department of Biology, Iowa City, IA). Slides were then washed three times in 1× PBS at room temperature and incubated for 1 hour at room temperature in the dark in a secondary antibody solution containing Alexa Fluor 488-, 568-, or 633-conjugated secondary antibodies in blocking solution (all at 1:250 dilution). Sections were then washed three times for 5 minutes each in the dark with 1× PBS at room temperature. Coverslips were mounted using Vectashield HardSet mounting medium with DAPI (H-1500; Vector Laboratories, Burlingame, CA). TUNEL immunofluorescence staining (Invitrogen; Life Technologies, Carlsbad, CA) was performed according to the manufacturer's recommendations. Images were captured using a Zeiss Axio Observer inverted laser scanning microscope LSM 510 using Zeiss LSM 510 software version 4.2 and were analyzed offline using ImageJ software version 1.43u (NIH, Bethesda, MD). Frozen sections from TA muscle samples were stained with H&E. Digital images were obtained at ×20 magnification, and myofiber cross-sectional area (in μm2) was quantified by a single blinded investigator from approximately 300 myofibers per animal, using NIH ImageJ image analysis software version 1.43u. Immortalized murine C2C12 and rat L6 skeletal muscle cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Cells were propagated in growth medium [Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% penicillin/streptomycin and 0.2% amphotericin B, and 10% fetal bovine serum]. Differentiation was stimulated by serum withdrawal in differentiation medium (DMEM supplemented with 2% horse serum, 1% penicillin/streptomycin, 0.2% amphotericin B, and 0.01% human insulin/transferrin/selenium). Immortalized EC-RF24 (ECRF) cells (described by Fontijn et al17Fontijn R. Hop C. Brinkman H.J. Slater R. Westerveld A. van Mourik J.A. Pannekoek H. Maintenance of vascular endothelial cell-specific properties after immortalization with an amphotrophic replication-deficient retrovirus containing human papilloma virus 16 E6/E7 DNA.Exp Cell Res. 1995; 216: 199-207Crossref PubMed Scopus (94) Google Scholar) were a gift from Ruud Fontijn and were propagated in DMEM supplemented with 1% penicillin/streptomycin, 0.2% amphotericin B, and 10% fetal bovine serum. Human umbilical vein endothelial cells (HUVECs) were isolated from donor placental umbilical veins and were used before passage 6. HUVECs were propagated on 0.1% gelatin-coated dishes in endothelial basal medium (Lonza, Walkersville, MD) supplemented with 20% fetal bovine serum, 1% penicillin/streptomycin and 0.2% amphotericin B, and an EGM-MV bullet kit (Lonza, Walkersville, MD) containing epidermal growth factor, bovine brain extract, hydrocortisone, and gentamicin. To evaluate the effects of ischemia/hypoxia in vitro, we used an established model of cellular hypoxia in which cells are subjected to 0% O2 and deprived of nutrients in Hank's balanced salt solution14Arany Z. Foo S.Y. Ma Y. Ruas J.L. Bommi-Reddy A. Girnun G. Cooper M. Laznik D. Chinsomboon J. Rangwala S.M. Baek K.H. Rosenzweig A. Spiegelman B.M. HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha.Nature. 2008; 451: 1008-1012Crossref PubMed Scopus (845) Google Scholar to mimic the local environment resulting from severe ischemia in PAD, referred to hereafter as hypoxia plus nutrient deprivation (HND). Primary murine muscle precursor cells (mouse myoblasts) derived from diaphragm and hindlimb muscles were prepared as described previously.18Lees S.J. Rathbone C.R. Booth F.W. Age-associated decrease in muscle precursor cell differentiation.Am J Physiol Cell Physiol. 2006; 290: C609-C615Crossref PubMed Scopus (51) Google Scholar, 19Mitchell P.O. Pavlath G.K. Skeletal muscle atrophy leads to loss and dysfunction of muscle precursor cells.Am J Physiol Cell Physiol. 2004; 287: C1753-C1762Crossref PubMed Scopus (93) Google Scholar Briefly, hindlimb and diaphragm muscles were extracted from 4- to 6-week-old female C57BL/6 or BALB/c mice, digested with pronase (0.2%) for 60 minutes at 37°C, and then triturated to release precursor cells. Individual cells were washed in DMEM and PBS and then preplated for 3 hours on tissue culture polystyrene dishes; the cellular supernatant was transferred into collagen-coated (1 mg/mL in DMEM) plates and maintained in growth medium (Ham's F10 medium supplemented with 1% penicillin/streptomycin and 0.2% amphotericin B, 20% fetal bovine serum, and 2.5 ng/mL hFGF) for 4 days, which allowed myoblasts to attach to the substratum and proliferate. Myoblasts were then propagated in growth medium and used in experiments within four passages of isolation. Differentiation was stimulated by serum withdrawal in differentiation medium (DMEM supplemented with 2% horse serum, 1% penicillin/streptomycin, 0.2% amphotericin B, and 0.01% human insulin/transferrin/selenium) and verified by light microscopy and quantitative RT-PCR for the myogenic regulatory factor myogenin. Analysis of the effects of recombinant VEGF on myoblast differentiation was performed by plating equal numbers of myoblasts on plates coated with entactin, collagen, and laminin and allowing cells to reach 90% confluence. Cells were then treated with PBS or recombinant human vascular endothelial growth factor (rH-VEGF; 50 ng/mL) in growth medium for 24 hours and analyzed by quantitative RT-PCR. DNA fragmentation was analyzed on 1% agarose gels using 2 μg genomic DNA isolated from cells using an apoptotic-DNA ladder kit (Roche Diagnostics, Indianapolis, IN) according to the manufacturer's instructions. Muscle myotube diameter was quantified as described previously.20Menconi M. Gonnella P. Petkova V. Lecker S. Hasselgren P.O. Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes.J Cell Biochem. 2008; 105: 353-364Crossref PubMed Scopus (133) Google Scholar Briefly, images of myotubes after treatment were acquired by phase contrast microscopy at ×100 magnification on an Olympus IX70 inverted microscope connected to a PAXCam ARC digital camera system (MIS, Franklin Park, IL). Diameters were measured in ∼100 myotubes from at least 10 random fields (a number chosen by determining no additional change in standard deviation) using NIH ImageJ image analysis software version 1.43u. Each myotube analyzed was measured at three points along the length of the myotube in a blinded fashion, and results are expressed as a percentage of the control treatment diameter. Cellular mitochondrial DNA content was analyzed in total cellular DNA by quantitative RT-PCR using genomic DNA isolated from cells using an apoptotic-DNA ladder kit (Roche Diagnostics) according to the manufacturer's instructions. A primer set against NADH dehydrogenase subunit 2 (Nd2) was used to quantify the mitochondrial genome and was corrected for the nuclear-specific gene Nme1 by quantitative real-time PCR using an ABI 7300 Real-Time PCR System (Applied Biosystems; Life Technologies, Foster City, CA) as described previously.21Zhang H. Bosch-Marce M. Shimoda L.A. Tan Y.S. Baek J.H. Wesley J.B. Gonzalez F.J. Semenza G.L. Mitochondrial autophagy is an HIF-1-dependent adaptive metabolic response to hypoxia.J Biol Chem. 2008; 283: 10892-10903Crossref PubMed Scopus (1313) Google Scholar Total RNA was extracted from mouse gastrocnemius muscles, primary murine skeletal myoblasts, C2C12 murine myoblasts, L6 rat myoblasts, ECRF cells, and HUVECs using TRIzol (Invitrogen) phenol/chloroform extraction. RNA (5 μg) was reverse-transcribed using SuperScript III Reverse Transcriptase and random primers (Invitrogen). Reactions were incubated at 50°C for 50 minutes and at 85°C for 5 minutes. Real-time PCR was performed using an ABI 7300 system (Applied Biosystems). Relative quantification of GADD45, p21, MyoD, myogenin, paired box gene (Pax7), Pgc1-α, Ang-1, Ang-2, Tie1, Tie2, VEGF, VEGFR-1/Flt, VEGFR-2/Flk, and NRP-1 mRNA levels were determined using the comparative threshold cycle (ΔΔCT) method using FAM TaqMan gene expression assays (Applied Biosystems) specific for each of these genes run in complex (multiplex) with a VIC-labeled GAPDH control primer. Strain-specific primary myotube hypoxia responses were determined in vitro using an adenoviral hypoxia response element-luciferase reporter construct.22Cao Y. Li C.Y. Moeller B.J. Yu D. Zhao Y. Dreher M.R. Shan S. Dewhirst M.W. Observation of incipient tumor angiogenesis that is independent of hypoxia and hypoxia inducible factor-1 activation.Cancer Res. 2005; 65: 5498-5505Crossref PubMed Scopus (79) Google Scholar, 23Lima B. Lam G.K. Xie L. Diesen D.L. Villamizar N. Nienaber J. Messina E. Bowles D. Kontos C.D. Hare J.M. Stamler J.S. Rockman H.A. Endogenous S-nitrosothiols protect against myocardial injury.Proc Natl Acad Sci USA. 2009; 106: 6297-6302Crossref PubMed Scopus (191) Google Scholar Cells were adenovirally transduced with a hypoxia response element-luciferase reporter construct and subjected to HND; luciferin substrate (Promega, Madison, WI) was added and luciferase activity was measured by bioluminescence imaging (IVIS; Xenogen, Hopkinton, MA). Cell cultures or gastrocnemius muscles were washed twice with ice-cold PBS and lysed on ice in a lysis buffer consisting of 50 mmol/L HEPES, 150 mmol/L NaCl, 100 mmol/L NaF, 5 mmol/L EDTA, 0.5% Triton X-100, and protease inhibitors (5 mg/mL aprotinin, 2 mg/mL leupeptin, and 100 mmol/L phenylmethylsulfonyl fluoride). Lysates were centrifuged at 1000 × g for 5 minutes, and the supernatant (30 to 80 μg total protein) was boiled for 5 minutes in Laemmli sample buffer, and proteins were separated by SDS-PAGE and analyzed by Western blotting. Antibodies were against phospho-ERK1/2 (Thr202/Tyr204), total ERK1/2, phospho-Akt (Thr308/Ser473), total Akt, phospho-mTOR (Ser2481), phospho-p70S6K1 (Thr389), total p70S6K1, phospho-FoxO3a (Ser253), Bax, Bcl-2, and total and cleaved caspase-3 (all purchased from Cell Signaling Technology, Danvers, MA). Loading and transfer of equal amounts of protein was confirmed by Ponceau staining and stripping the membranes and reprobing with antibodies against α-tubulin (for in vitro cell lysate analysis) (Sigma-Aldrich) or GAPDH (for in vivo gastrocnemius tissue analysis) (Novus Biologicals, Littleton, CO). Statistical analysis of within-group differences was performed using one-way analysis of variance; between-group comparisons were performed using Student's t-test. A P value of ≤0.05 was considered statistically significant. To evaluate the individual responses of different cell types (endothelial and muscle cells) primarily affected in the ischemic limb, we used an established in vitro model of cellular hypoxia in which cells are subjected to hypoxia and nutrient deprivation (HND) by incubation in 0% O2 and Hank's balanced salt solution,14Arany Z. Foo S.Y. Ma Y. Ruas J.L. Bommi-Reddy A. Girnun G. Cooper M. Laznik D. Chinsomboon J. Rangwala S.M. Baek K.H. Rosenzweig A. Spiegelman B.M. HIF-independent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC-1alpha.Nature. 2008; 451: 1008-1012Crossref PubMed Scopus (845) Google Scholar in an attempt to reproduce the local O2 and nutrient-depleted environment resulting from severe ischemia in PAD. Initially, we examined effects of HND on skeletal myotube and endothelial cell survival. Three hours of HND induced DNA fragmentation (Figure 1A) in two different immortalized muscle lines, as well as in primary skeletal myotubes. DNA fragmentation was not increased in either of two different types of endothelial cells (Figure 1B). Mitochondrial DNA content decreased with HND in all muscle cell types tested (Figure 1C). Notably, this reduced mitochondrial:nuclear DNA ratio was a primary mitochondrial effect, in that there was a loss of mitochondrial Nd2 without change in total genomic DNA (nuclear-specific Nme1). Somewhat surprisingly, mitochondrial DNA content was either unchanged (immortalized ECRF cells) or increased significantly (primary HUVECs) in endothelial cells (Figure 1D). Although this finding is consistent with the lack of apoptosis, it is opposite that of the skeletal muscle response and was due to an increase in copy number of the mitochondrial gene Nd2. In addition, Western blotting revealed an increase in the Bax/Bcl-2 protein expression ratio and caspase-3 cleavage in muscle cells (Figure 1E), but no change in either measure in endothelial cells (Figure 1F), further supporting a lack of endothelial cell injury at this time point. These findings demonstrate that endothelial cells and myotubes display markedly different responses to the same duration of HND insult, including different effects on myonuclear apoptosis and mitochondrial content. To further characterize the cachectic signaling responses of muscle to ischemia such as that occurring in PAD, mature myotubes were subjected to 3 hours HND and then were analyzed morphologically. All three muscle cell lines underwent significant atrophy (Figure 2, A and B). Consistent with this apparent loss of cellular protein, the phosphorylation of signaling proteins important for cell growth and protein synthesis, Akt and p70 S6 kinase," @default.
• W2145868785 created "2016-06-24" @default.
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• W2145868785 date "2012-05-01" @default.
• W2145868785 modified "2023-10-16" @default.
• W2145868785 title "Skeletal Muscle–Specific Genetic Determinants Contribute to the Differential Strain-Dependent Effects of Hindlimb Ischemia in Mice" @default.
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