BACKGROUND: Under normoxic conditions, hypoxia-inducible factor (HIF)-1α is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure. METHODS AND RESULTS: PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouse C2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1α protein and angiogenesis genes, respectively. CONCLUSIONS: We demonstrated that HIF-1α upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.
BACKGROUND: Under normoxic conditions, hypoxia-inducible factor (HIF)-1α is rapidly degraded by 2 hydroxylases: prolyl hydroxylase (PHD) and factor-inhibiting HIF-1 (FIH). Because HIF-1α mediates the cardioprotective response to ischemic injury, its upregulation may be an effective therapeutic option for ischemic heart failure. METHODS AND RESULTS:PHD and FIH were cloned from mouse embryonic stem cells. The best candidate short hairpin (sh) sequences for inhibiting PHD isoenzyme 2 and FIH were inserted into novel, nonviral, minicircle vectors. In vitro studies after cell transfection of mouseC2C12 myoblasts, HL-1 atrial myocytes, and c-kit(+) cardiac progenitor cells demonstrated higher expression of angiogenesis factors in the double-knockdown group compared with the single-knockdown and short hairpin scramble control groups. To confirm in vitro data, shRNA minicircle vectors were injected intramyocardially after left anterior descending coronary artery ligation in adult FVB mice (n=60). Functional studies using MRI, echocardiography, and pressure-volume loops showed greater improvement in cardiac function in the double-knockdown group. To assess mechanisms of this functional recovery, we performed a cell trafficking experiment, which demonstrated significantly greater recruitment of bone marrow cells to the ischemic myocardium in the double-knockdown group. Fluorescence-activated cell sorting showed significantly higher activation of endogenous c-kit(+) cardiac progenitor cells. Immunostaining showed increased neovascularization and decreased apoptosis in areas of injured myocardium. Finally, western blots and laser-capture microdissection analysis confirmed upregulation of HIF-1α protein and angiogenesis genes, respectively. CONCLUSIONS: We demonstrated that HIF-1α upregulation by double knockdown of PHD and FIH synergistically increases stem cell mobilization and myocardial angiogenesis, leading to improved cardiac function.
Authors: J Li; M Post; R Volk; Y Gao; M Li; C Metais; K Sato; J Tsai; W Aird; R D Rosenberg; T G Hampton; F Sellke; P Carmeliet; M Simons Journal: Nat Med Date: 2000-01 Impact factor: 53.440
Authors: H Kamihata; H Matsubara; T Nishiue; S Fujiyama; Y Tsutsumi; R Ozono; H Masaki; Y Mori; O Iba; E Tateishi; A Kosaki; S Shintani; T Murohara; T Imaizumi; T Iwasaka Journal: Circulation Date: 2001-08-28 Impact factor: 29.690
Authors: K A Vincent; K G Shyu; Y Luo; M Magner; R A Tio; C Jiang; M A Goldberg; G Y Akita; R J Gregory; J M Isner Journal: Circulation Date: 2000-10-31 Impact factor: 29.690
Authors: K A Jackson; S M Majka; H Wang; J Pocius; C J Hartley; M W Majesky; M L Entman; L H Michael; K K Hirschi; M A Goodell Journal: J Clin Invest Date: 2001-06 Impact factor: 14.808
Authors: D Orlic; J Kajstura; S Chimenti; F Limana; I Jakoniuk; F Quaini; B Nadal-Ginard; D M Bodine; A Leri; P Anversa Journal: Proc Natl Acad Sci U S A Date: 2001-08-14 Impact factor: 11.205
Authors: Min Cheng; Junlan Zhou; Min Wu; Chan Boriboun; Tina Thorne; Ting Liu; Zhifu Xiang; Qiutang Zeng; Toshikazu Tanaka; Yao Liang Tang; Raj Kishore; Michael H Tomasson; Richard J Miller; Douglas W Losordo; Gangjian Qin Journal: Circ Res Date: 2010-09-16 Impact factor: 17.367
Authors: C Kalka; H Masuda; T Takahashi; R Gordon; O Tepper; E Gravereaux; A Pieczek; H Iwaguro; S I Hayashi; J M Isner; T Asahara Journal: Circ Res Date: 2000-06-23 Impact factor: 17.367
Authors: Michael Keeney; Sang-Ging Ong; Amanda Padilla; Zhenyu Yao; Stuart Goodman; Joseph C Wu; Fan Yang Journal: ACS Nano Date: 2013-07-16 Impact factor: 15.881
Authors: Sang-Ging Ong; Bruno C Huber; Won Hee Lee; Kazuki Kodo; Antje D Ebert; Yu Ma; Patricia K Nguyen; Sebastian Diecke; Wen-Yi Chen; Joseph C Wu Journal: Circulation Date: 2015-08-25 Impact factor: 29.690