Impaired therapeutic vasculogenesis by transplantation of OxLDL-treated endothelial progenitor cells

Previous in vitro studies have revealed that oxidized low density lipoprotein (OxLDL) has negative effects on the proliferation and activity of endothelial progenitor cells (EPCs). Here, we evaluated the effect of OxLDL on the therapeutic potential of EPCs in ischemia-induced neovascularization. EPCs derived from mobilized human peripheral blood mononuclear cells were cultured without or with OxLDL before transplantation. Hindlimb ischemia models were surgically induced in athymic nude mice, which then received an intracardiac injection of 3 × 105 EPCs. By laser Doppler perfusion image and ischemia damage score, we found that blood perfusion and ischemia damage were less well recovered in the OxLDL-treated EPC transplantation group than in controls. Histological examination showed fewer transplanted EPCs and lower capillary density in ischemic tissue. Local delivery of Stromal cell-derived factor (SDF-1) restored this defect and improved blood perfusion by recruiting OxLDL-treated EPCs to the ischemic area and increasing host capillary density. These results provide for the first time direct evidence that OxLDL impaired the therapeutic potential of EPCs in ischemia-induced neovascularization through an inhibitory effect on the migration, adhesion, and incorporation of EPCs into vasculature and/or entrapment in the perivascular region in vivo. A therapeutic strategy based on SDF-1 administration ameliorated such defects and improved postischemic neovascularization. Previous in vitro studies have revealed that oxidized low density lipoprotein (OxLDL) has negative effects on the proliferation and activity of endothelial progenitor cells (EPCs). Here, we evaluated the effect of OxLDL on the therapeutic potential of EPCs in ischemia-induced neovascularization. EPCs derived from mobilized human peripheral blood mononuclear cells were cultured without or with OxLDL before transplantation. Hindlimb ischemia models were surgically induced in athymic nude mice, which then received an intracardiac injection of 3 × 105 EPCs. By laser Doppler perfusion image and ischemia damage score, we found that blood perfusion and ischemia damage were less well recovered in the OxLDL-treated EPC transplantation group than in controls. Histological examination showed fewer transplanted EPCs and lower capillary density in ischemic tissue. Local delivery of Stromal cell-derived factor (SDF-1) restored this defect and improved blood perfusion by recruiting OxLDL-treated EPCs to the ischemic area and increasing host capillary density. These results provide for the first time direct evidence that OxLDL impaired the therapeutic potential of EPCs in ischemia-induced neovascularization through an inhibitory effect on the migration, adhesion, and incorporation of EPCs into vasculature and/or entrapment in the perivascular region in vivo. A therapeutic strategy based on SDF-1 administration ameliorated such defects and improved postischemic neovascularization. The ubiquitous blood vessel system is vulnerable to many pathological processes, such as atherosclerosis and diabetes mellitus (1Cao Y. Hong A. Schulten H. Post M.J. Update on therapeutic neovascularization. Cardiovasc. Res. 2005; 65: 639-648Crossref PubMed Scopus (91) Google Scholar). In the late stage of these diseases, patients usually suffer from severe limb ischemia. Thus, prevention and treatment of tissue ischemia is important. The classical mechanism of postnatal neovascularization had been considered to be limited to angiogenesis until endothelial progenitor cells (EPCs) were discovered (2Kawamoto A. Asahara T. Losordo D.W. Transplantation of endothelial progenitor cells for therapeutic neovascularization. Cardiovasc. Radiat. Med. 2002; 3: 221-225Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The finding that EPCs are home to sites of neovascularization and differentiate into endothelial cells (ECs) in situ is consistent with “vasculogenesis,” a critical paradigm well described for embryonic neovascularization but recently proposed in adults (3Asahara T. Murohara T. Sullivan A. Silver M. van der Zee R. Li T. Witzenbichler B. Schatteman G. Isner J.M. Isolation of putative progenitor endothelial cells for angiogenesis. Science. 1997; 275: 964-967Google Scholar). Our previous work indicated that transplantation of ex vivo expanded EPCs from either peripheral or cord blood could enhance neovascularization in hindlimb ischemia of nude mice (4Kalka C. Masuda H. Takahashi T. Kalka-Moll W.M. Silver M. Kearney M. Li T. Isner J.M. Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. Sci. USA. 2000; 97: 3422-3427Crossref PubMed Scopus (1753) Google Scholar, 5Yang C. Zhang Z.H. Li Z.J. Yang R.C. Qian G.Q. Han Z.C. Enhancement of neovascularization with cord blood CD133+ cell-derived endothelial progenitor cell transplantation. Thromb. Haemost. 2004; 91: 1202-1212Google Scholar). However, the number of circulating EPCs and their migratory activity were reported to be reduced in patients with risk factors for ischemic cardiovascular disease or to be negatively correlated with the Framingham cardiovascular risk factor score (6Vasa M. Fichtlscherer S. Aicher A. Adler K. Urbich C. Martin H. Zeiher A.M. Dimmeler. S. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ. Res. 2001; 89: E1-E7Google Scholar, 7Hill J.M. Zalos G. Halcox J.P. Schenke W.H. Waclawiw M.A. Quyyumi A.A. Finkel. T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N. Engl. J. Med. 2003; 348: 593-600Google Scholar). Circulating EPCs serve as a biological marker for vascular function (7Hill J.M. Zalos G. Halcox J.P. Schenke W.H. Waclawiw M.A. Quyyumi A.A. Finkel. T. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N. Engl. J. Med. 2003; 348: 593-600Google Scholar), and reduction of the number of circulating EPCs predicts future cardiovascular events (8Schmidt-Lucke C. Rossig L. Fichtlscherer S. Vasa M. Britten M. Kamper U. Dimmeler S. Zeiher A.M. Reduced number of circulating endothelial progenitor cells predicts future cardiovascular events: proof of concept for the clinical importance of endogenous vascular repair. Circulation. 2005; 111: 2981-2987Google Scholar). Given the concept of therapeutic vasculogenesis contributed by EPCs, further understanding of the regulation of EPC kinetics may bring new insights into the pathogenesis of vasculogenesis. Oxidized low density lipoprotein (OxLDL) is one of factors that influence the growth and bioactivity of EPCs. Previous work indicated that OxLDL has negative effects on the number and activity of EPCs in vitro, inhibiting EPC differentiation and inducing EPC senescence, leading to cellular dysfunction (9Imanishi T. Hano T. Matsuo Y. Nishio I. Oxidized low-density lipoprotein inhibits vascular endothelial growth factor-induced endothelial progenitor cell differentiation. Clin. Exp. Pharmacol. Physiol. 2003; 30: 665-670Google Scholar, 10Imanishi T. Hano T. Sawamura T. Nishio I. Oxidized low-density lipoprotein induces endothelial progenitor cell senescence, leading to cellular dysfunction. Clin. Exp. Pharmacol. Physiol. 2004; 31: 407-413Google Scholar, 11Wang X. Chen J. Tao Q. Zhu J. Shang Y. Effects of ox-LDL on number and activity of circulating endothelial progenitor cells. Drug Chem. Toxicol. 2004; 27: 243-255Google Scholar). Recently, we showed that OxLDL inhibited EPC survival and impaired its function, and this action was attributable to an inhibitory effect on endothelial nitric oxide synthase (12Ma F.X. Zhou B. Chen Z. Ren Q. Lu S.H. Sawamura T. Han Z.C. Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase. J. Lipid Res. 2006; 47: 1227-1237Google Scholar). However, the exact therapeutic potential of these OxLDL-treated EPCs (OxLDL-EPCs) in ischemic settings is little known. We hypothesize that OxLDL not only directly injures ECs but also impairs the repair process by EPCs (therapeutic vasculogenesis) in vivo. Therefore, we studied the ability of OxLDL-EPCs in treating severe hindlimb ischemia to demonstrate the impact of OxLDL on EPCs both in vivo and in vivo. Mobilized peripheral blood mononuclear cells (M-PBMNCs) were collected from healthy volunteers who received 600 μg/day recombinant human granulocyte colony-stimulating factor (Kirin Pharmaceuticals, Tokyo, Japan) subcutaneously for 5 days to mobilize stem/progenitor cells. All volunteers had no risk factors of coronary artery diseases, including hypertension, diabetes, smoking, positive family history of coronary artery disease, and hypercholesterolemia, and were free of wounds, ulcers, retinopathy, recent surgery, inflammation, malignant diseases, or medications that may influence EPC kinetics. Human umbilical cord was manipulated according to the standard protocol approved by the Institutional Review Board of the Institute of Hematology and the Hospital of Blood Diseases, Chinese Academy of Medical Sciences and Peking Union Medical College. All involved volunteers were well informed and provided signed informed consent. Blood samples for LDL preparation were taken from healthy volunteers after 12 h of fasting. Written informed consent was obtained from all volunteers in accord with the Institutional Ethics Committee. LDL was separated by density gradient ultracentrifugation as described previously (12Ma F.X. Zhou B. Chen Z. Ren Q. Lu S.H. Sawamura T. Han Z.C. Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase. J. Lipid Res. 2006; 47: 1227-1237Google Scholar). After isolation by density gradient ultracentrifugation, LDL was oxidized by exposure to 5 μmol/l CuSO4 for 18 h at 37°C. LDL was totally oxidized, because there is only one band by electrophoresis on agarose gels. Compared with native LDL, the OxLDL showed increased electrophoretic mobility and an increased level of thiobarbituric acid-reactive substances (TBARS). The level of TBARS was 2.23 ± 1.56 and 24.37 ± 8.14 nmol/mg protein in native LDL and OxLDL, respectively. Compared with the native LDL, OxLDL showed increased electrophoretic mobility on agarose gels of 1.8 ± 0.4 times. OxLDL was sterilized by passing it through a 0.22 mm filter. Mobilized peripheral blood mononuclear cells were isolated from blood of human volunteers by density gradient centrifugation with Histopaque-1077 (Sigma). Cells were plated on culture dishes coated with human fibronectin (Sigma) and maintained in EC Basal Medium-2 (EBM-2) (Clonetics, Cambrex), supplemented with EGM-2 MV SingleQuots containing 5% FBS, human vascular endothelial growth factor (VEGF), human fibroblast growth factor-2, human epidermal growth factor, insulin-like growth factor-1, ascorbic acid, and hydrocortisone. After 4 days in culture, nonadherent cells were removed and new medium was added. The culture was maintained through day 7. After 7 days of culture, EPCs were stimulated with or without OxLDL (50 μg/ml) for 24 h. Then, OxLDL-EPCs were used in the following in with EPCs. Human umbilical endothelial cells were isolated by umbilical with for at and were cultured in with and in were the effect of SDF-1 on we supplemented the culture with SDF-1 for 12 with medium as a After 7 days in culture, cells × were with and were used to on EPCs. as a Cells were and the of this cells that showed of low density lipoprotein and of were considered EPCs. cells were with at for 4 h and with for After cells were then with (Sigma) at for cells were for the with and The incorporation of and of were by All were on athymic nude mice Institute of according to Peking Union Medical and were with before and its were to hindlimb ischemia T. Silver M. Kearney M. Witzenbichler B. Isner J.M. of angiogenesis. J. Scholar). day after the of hindlimb mice received an intracardiac injection of 3 × 105 EPCs or OxLDL-EPCs in the of the cells, mice in group received cells with cells were and with for 5 at and on After with cells were in and into mice before these mice received an intracardiac injection of either of or the therapeutic potential of athymic nude mice in group received a injection of of SDF-1 in the of the lower by an injection of 3 × 105 OxLDL-EPCs or EPCs as a Doppler perfusion was used to the of blood in mice after surgery, as described previously (4Kalka C. Masuda H. Takahashi T. Kalka-Moll W.M. Silver M. Kearney M. Li T. Isner J.M. Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. Sci. USA. 2000; 97: 3422-3427Crossref PubMed Scopus (1753) Google Scholar). were before and at days and after The were to of blood and the perfusion was as the of to limb blood A of the ischemic limb was in a a of a clinical score (1Cao Y. Hong A. Schulten H. Post M.J. Update on therapeutic neovascularization. Cardiovasc. Res. 2005; 65: 639-648Crossref PubMed Scopus (91) Google no but no 3 damage was also no 3 4 T. M. S. S. Local delivery of cells perfusion through Circulation. 2004; Scholar), and all was by a from the lower of ischemic and healthy were on days and and in of 5 mm were on and for h. EPC incorporation at time after transplantation days and from the mice with EPCs and were for A of in 5 from were and the EPCs were from ischemic and hindlimb on days 3 and 7 were also for the of were with and and with The effect of and SDF-1 on OxLDL-EPCs was by the in vitro in a or SDF-1 to or in 600 of containing was in the lower and 105 EPCs were in the After for h at the were and the cells in the lower were obtained and with for at a The migratory was by the of cells into the lower events for cell events for × All were studied in EPC induced by was by as described previously (12Ma F.X. Zhou B. Chen Z. Ren Q. Lu S.H. Sawamura T. Han Z.C. Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase. J. Lipid Res. 2006; 47: 1227-1237Google Scholar). cells, only and cells were We also used to in situ according to the protocol were with and the of cells was evaluated in a the ability of EPCs after OxLDL either or ECs were used in EPCs were with at for 4 h and with of EPCs and OxLDL-EPCs in containing were culture for h at and then to nonadherent cells. of EPC to a of was h before the by × 105 cells in well of were for 12 h with A of 5 × EPCs with were to well and for 3 h at 37°C. cells were removed with EPCs were with with and in The EPCs on were by and of well were by the ability of EPCs to into vascular EPCs were with on was at and on a culture at for h to EPCs without or with OxLDL were and with × were with × with (Sigma) and in EGM-2 MV at for 24 h. was as a a its The of EPCs in was in samples were at 3 or 7 days after for were to and the was with the protein were separated with the of gels and with to or of was with the of are as ± The of was by by for for which was by was at a of mobilized peripheral blood mononuclear cells isolated and cultured for in a the endothelial of the EPCs used in this The of cultured EPCs ± ± ± ± and ± and the by cells, such as and or These cells were also to and to consistent with endothelial cells The impact of EPC administration on therapeutic neovascularization was in a of hindlimb ischemia T. Silver M. Kearney M. Witzenbichler B. Isner J.M. of angiogenesis. J. Scholar). blood was T. Asahara T. Silver M. C. Masuda H. C. Kearney M. Chen oxide synthase angiogenesis in to tissue ischemia. J. Clin. Scholar). examination of hindlimb perfusion by at days and in limb perfusion days after the of limb ischemia. are in or 3 days limb perfusion was reduced in both the blood were in mice EPCs. perfusion in mice OxLDL-EPCs and was than in the but to a than that of the EPC group By day the of ischemic to blood in transplanted mice was ± ± in mice EPCs perfusion hindlimb and with EPCs, had of function at day ischemic score ± ± and severe ischemic with ischemia damage score was reported that the and incorporation of transplanted EPCs into the capillary in the ischemic were for the improved ischemia T. H. J. S. K. H. I. K. T. cord endothelial cells postnatal neovascularization. J. Clin. 2000; Scholar). the of the impaired therapeutic potential of OxLDL-EPCs in we the incorporation and/or entrapment of transplanted OxLDL-EPCs in the or perivascular area of ischemic and the number of host OxLDL-EPCs were in tissue by host ECs with were in Histological examination reduced of OxLDL-EPCs with ± ± day ± ± day ± ± a number of capillary ECs was in mice with mice ± ± day ± ± 24 day ± ± the of EPCs to ischemic and differentiation into EC cells in vivo. human EPCs were artery at 7 days after We also the of of these EPCs to ischemic By we found that there were protein for as well as in ischemic at 3 or 7 days after surgery, but no for hindlimb by could have potential to EPCs. EPCs were to to of in a However, the migratory activity of OxLDL-EPCs in to was reduced with that of EPCs ± ± We further adhesion, which is an in the EPC EPCs were reported to a for on ischemic ECs have an in recruiting circulating EPCs to the revealed that ECs in the ischemic area of and In vitro of ability revealed that OxLDL-EPCs were impaired in their ability to to fibronectin or to a with In EPCs with were in the vessel with in vivo was to the ability of EPCs to into vascular OxLDL-EPCs could into the on OxLDL-EPCs were found to less to the with with EPCs OxLDL-EPCs were to to with SDF-1 in a their was with that of EPCs. The impaired ability with SDF-1 was attributable to in OxLDL-EPCs not the impaired therapeutic vasculogenesis of transplantation was to the and to the ischemic we SDF-1 administration in ischemic could such defects and therapeutic vasculogenesis in Compared with mice with OxLDL-EPCs and with mice with OxLDL-EPCs and with SDF-1 showed blood perfusion in ischemic at day after less than the EPCs at days and the SDF-1 effect on of transplanted OxLDL-EPCs from the in we the incorporation of OxLDL-EPCs into of ischemic by OxLDL-EPCs and were discovered in tissue after SDF-1 administration was a increased of OxLDL-EPCs in with the group day day density was also increased after administration of SDF-1 in ischemic tissue with day day In SDF-1 level in was increased in mice with SDF-1 of SDF-1 in culture reduced with that of medium Previous studies have that transplantation of ex vivo expanded EPCs blood and capillary density in ischemic (4Kalka C. Masuda H. Takahashi T. Kalka-Moll W.M. Silver M. Kearney M. Li T. Isner J.M. Asahara T. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. Sci. USA. 2000; 97: 3422-3427Crossref PubMed Scopus (1753) Google and B. C. Britten M. R. N. Aicher A. Urbich C. Martin H. Transplantation of progenitor cells and in Circulation. 2002; Scholar). The transplanted EPCs home to ischemic and into the vasculature Q. S. C. A. Y. S. R. for circulating endothelial cells. Scholar). Thus, this of EPCs is for cell the of ischemic tissue. In this we found of blood in ischemic after which in severe and ischemia The EPCs were found to home to the ischemic and into in with previous results T. C. Masuda H. Chen Silver M. Kearney M. M. Isner J.M. Asahara T. and of endothelial progenitor cells for neovascularization. Med. Scholar, J. A. Silver M. S. M. Masuda H. Losordo D.W. Isner J.M. Stromal cell-derived effects on ex vivo expanded endothelial progenitor cell for ischemic neovascularization. Circulation. 2003; Scholar). The number of OxLDL-EPCs in ischemic sites in vivo as well as in host in ischemic tissue was less than that of EPCs. may be attributable to the reduced of in OxLDL-EPCs that could to increased SDF-1 in ischemic tissue. The impaired of OxLDL-EPCs the of blood and limb A recent also indicated that and entrapment of circulating cells are in angiogenesis M. I. Y. A. S. S. L. R. and of cells. 2006; Scholar). is that EPCs were to ischemic and in by the of Q. S. C. A. Y. S. R. for circulating endothelial cells. fewer EPCs or in ischemic sites may the in vivo of factors that could therapeutic neovascularization. OxLDL-EPCs are in factors in vivo is and further in the has been that there are the of transplanted stem/progenitor cells to C. Zhang Z.H. Li Z.J. Yang R.C. Qian G.Q. Han Z.C. Enhancement of neovascularization with cord blood CD133+ cell-derived endothelial progenitor cell transplantation. Thromb. Haemost. 2004; 91: 1202-1212Google Scholar). The of cells and cell to in with are and T. H. J. S. K. H. I. K. T. cord endothelial cells postnatal neovascularization. J. Clin. 2000; Scholar). In we found that ischemia induced an of which the of this has also been to to sites of neovascularization T. Takahashi T. Masuda H. C. Chen H. Y. Silver M. Isner J.M. to postnatal neovascularization by endothelial progenitor cells. J. Scholar). The EPCs could also Q. S. C. A. Y. S. R. for circulating endothelial cells. Scholar), including Thus, of by progenitor cells may in a the proliferation of both and and recruiting circulating EPCs. However, fewer OxLDL-EPCs were to the ischemic sites in vivo and in to in Thus, transplantation of OxLDL-EPCs was less at capillary blood and of the ischemic These provide evidence that the of is on EPC at an time which was impaired in Previous have that EPCs work in with ECs to than new J.M. C. J.P. Human endothelial progenitor cells from impaired adhesion, and incorporation into vascular Circulation. 2002; Scholar). In this we the incorporation and/or entrapment of transplanted EPCs in the vasculature or the perivascular region in vivo and their in in The impaired ability of OxLDL-EPCs to into with that less vessel contributed by OxLDL-EPCs in vivo. on is a cellular involved in blood vessel growth J.M. C. J.P. Human endothelial progenitor cells from impaired adhesion, and incorporation into vascular Circulation. 2002; Scholar), it is that of blood vessel growth may be impaired in we that delivery of SDF-1 restored the therapeutic of OxLDL-EPCs by recruiting OxLDL-EPCs to ischemic these we found that the reduced of OxLDL-EPCs was attributable to both of and increased is that SDF-1 an in ischemia-induced of cells from peripheral blood to ischemic and also in the entrapment of circulating cells in the perivascular area S. A. S. M. SDF-1 in endothelial and ischemia-induced of progenitor cells. 2004; Scholar). Our results that a number of OxLDL-EPCs were to the ischemic tissue and ischemia is because OxLDL-EPCs a of less than cells. less was on to and injection of SDF-1 the number of that an in blood The restored therapeutic may also be to an of after SDF-1 as reported by J. A. Silver M. S. M. Masuda H. Losordo D.W. Isner J.M. Stromal cell-derived effects on ex vivo expanded endothelial progenitor cell for ischemic neovascularization. Circulation. 2003; Scholar), as well as by of circulating SDF-1 SDF-1 the of stem/progenitor cells from M. I. Y. A. S. S. L. R. and of cells. 2006; Scholar), which may for improved In SDF-1 administration could enhance and endothelial nitric oxide synthase which therapeutic neovascularization K. M. K. S. Q. S. M. T. A. K. of cell-derived ischemic vasculogenesis and angiogenesis vascular endothelial growth nitric oxide for therapeutic neovascularization. Circulation. 2004; Scholar). also in to transplanted SDF-1 host ECs from blood and host EPCs derived from are to the ex vivo culture or the influence of the OxLDL on the function of EPCs. However, we were to direct of EPCs from patients with OxLDL, because these patients with usually also have such as hypertension, diabetes and cardiovascular diseases, which all influence EPC kinetics N. S. T. K. A. M. G. Circulating endothelial progenitor cells and cardiovascular N. Engl. J. Med. 2005; Scholar). has also been reported that the of circulating OxLDL were in patients with events than in patients without K. H. T. K. A. K. H. of circulating oxidized LDL for events in patients with coronary artery disease. 2004; 27: Scholar). We were to the impact of OxLDL on we to the effect of OxLDL on ex vivo expanded EPCs as to the in vivo in which EPCs are by increased as OxLDL in be lower in but in of influence EPCs, not the in vivo because OxLDL the proliferation X. Chen J. Tao Q. Zhu J. Shang Y. Effects of ox-LDL on number and activity of circulating endothelial progenitor cells. Drug Chem. Toxicol. 2004; 27: 243-255Google and increased the (12Ma F.X. Zhou B. Chen Z. Ren Q. Lu S.H. Sawamura T. Han Z.C. Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase. J. Lipid Res. 2006; 47: 1227-1237Google of EPCs, there the in to impaired impaired therapeutic vasculogenesis was also attributable to the survival and increased of OxLDL-EPCs in vivo. the of this the first direct proof of the impaired therapeutic potential of OxLDL-EPCs in ischemia-induced neovascularization. defect was through an of cellular impaired and as a of less of and reduced adhesion, and ability in the incorporation and/or entrapment into the vasculature and the perivascular studies are to these results and to the exact of activity in OxLDL-EPCs and of work was by for the and and from the of and of to

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