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【摘要】
Objective- Sphingosine-1-phosphate (S1P) is a bioactive lipid, which influences migration and proliferation of endothelial cells through activation of S1P receptors and has been shown to support SDF-1 induced migration and bone marrow homing of CD34 + progenitors.
Methods and Results- Here, we show that incubation of patient-derived endothelial progenitor cells (EPCs) with S1P or its synthetic analog FTY720 improved blood flow recovery in ischemic hind limbs. Likewise, recovery of blood flow was dramatically reduced after induction of hindlimb ischemia in mice deficient for the S1P receptor 3 (S1P 3 ). S1P 3 -/- bone marrow-derived mononuclear cells (BMCs) failed to augment neovascularization after hind limb ischemia. Of note, treatment of BMCs derived from S1P 3 -/- mice with S1P did not rescue blood flow recovery. Mechanistically, S1P and FTY720 induced phosphorylation of CXCR 4, activated the Src kinase, and stimulated phosphorylation of JAK2. The contribution of CXCR 4 for S1P-mediated effects was further supported by the findings that S1P preincubation failed to stimulate invasion capacity and in vivo blood flow recovery of BMCs from CXCR 4 +/- mice. The activation of CXCR 4 was dependent on the Src kinase family as demonstrated by preincubation with the Src inhibitor PP2. The activation of the CXCR 4 signaling by S1P is mediated via the S1P 3 receptor, since S1P-induced Src phosphorylation was abrogated in EPC from S1P 3 -/- mice.
Conclusions- S1P agonists might serve as sensitizers of CXCR 4 -mediated signaling and may be applied in clinical progenitor cell therapy to improve EPC or BMC function in patients with coronary artery disease.
Treatment of progenitor cells with sphingosine-1-phosphate (S1P), a bioactive lipid, improved blood flow recovery in ischemic hind limbs via the S1P 3 receptor. Mechanistically, S1P induced phosphorylation of the CXCR 4 receptor, the Src kinase, and JAK2. S1P failed to stimulate in vivo blood flow recovery of bone marrow-derived mononuclear cells from CXCR 4 +/- mice. The activation of the CXCR 4 receptor is mediated via the S1P 3 receptor because S1P did not induce Src phosphorylation in progenitor cells from S1P 3 -/- mice.
【关键词】 SP receptor crosstalk progenitor cells
Introduction
Sphingosine-1-phosphate (S1P) is a bioactive lipid, which influences proliferation, differentiation, migration, and survival of endothelial cells, but also of smooth muscle cells or bone marrow cells through activation of the G protein-coupled S1P receptors. 1-6 In addition, S1P has been shown to play a critical role in vascular development, determine vessel maturation, 7,8 and promote HDL-induced vascular reactivity. 9 Migratory responses or angiogenic activities of endothelial cells have been shown to be promoted via the receptors, S1P 1 or S1P 3. 1,10-12 A synthetic analog, FTY720, originally developed as an immunosuppressive agent for kidney transplantation, activates 4 of the 5 S1P receptors 13 and induces lymphocyte migration and homing in secondary lymphatic organs by regulating egress from lymph nodes. 14,15 FTY720 was recently shown to support SDF-1-induced migration and bone marrow homing of CD34 + progenitors. 16 See page 263
Transplantation of culture-expanded progenitor cells or selected bone marrow mononuclear cells successfully promotes therapeutic neovascularization in both ischemic hind limbs as well as acute myocardial infarction models. 17-21 Mechanistically, these cells can either induce angiogenesis by incorporation into vascular structures depicting phenotypes of endothelial cells or may induce angiogenesis by production of growth factors acting in a paracrine manner (for review see 22 ).
Moreover, recent clinical studies suggest that restoration of blood flow in peripheral artery disease and recovery of left ventricular function can be enhanced after autologous transplantation of bone marrow-derived cells or cultured EPCs in patients with coronary artery disease. 23-26 However, EPCs or BMCs derived from patients are functionally impaired 27-29 compared with EPCs from healthy donors. Recent data indicate that the therapeutic success is determined by functional properties of transplanted cells, 30,31 providing the basis for improvement of functional activities, eg, by pharmacological stimulation of surface receptors in order to enhance homing of progenitor or stem cells. One important family of surface receptors is the family of the S1P receptors. Seitz et al demonstrated that sustained activation of the S1P1 receptor by an agonist during the homing process resulted in increased engraftment in vivo. 32 Furthermore, S1P plays a crucial role in the cardiovascular system: it acts as a proangiogenic molecule, 12 improves endothelial function, 9 myocardial perfusion, 33 and is involved in cardiovascular development. 6,34 Additional studies demonstrate that S1P modulates homing and trafficking of cells. 14,35
Therefore, we investigated whether S1P or the synthetic analog, FTY720, enhances the efficiency of transplanted EPCs or BMCs for therapeutic neovascularization. Furthermore, we attempted to elucidate the underlying mechanisms.
Materials and Methods
For detailed descriptions of the materials and methods, please see supplemental material (available online at http://atvb.ahajournals.org).
Study Population and Patient Characteristics
Peripheral blood mononuclear cells or bone marrow mononuclear cells were isolated from peripheral blood of healthy volunteers or patients with coronary artery disease as documented by angiographic evidence of coronary lesions. Patients with signs of acute myocardial ischemia documented by classical symptoms of chest pain, ECG alterations, elevation of creatine kinase or Troponin T were excluded. Further exclusion criteria were the presence of active or chronic infection, surgical procedures, stroke or trauma within the last three months, or evidence for malignant diseases (for details see supplementary Table I). The ethics review board of the Johann Wolfgang Goethe University of Frankfurt, Germany approved the protocol, and the study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient.
Pharmacological Agents: S1P and FTY720
S1P (Sigma) and a synthetic analog FTY720 (Novartis Institute for Biomedical Research, Basel, Switzerland, kindly provided by V.Brinkmann) were used.
Mice Strain
CXCR 4 +/- mice (B6.129X- Cxcr4 tm1Qma /J) and C57Bl6/J background mice were purchased from Jackson Laboratories (Charles River, Germany). S1P 3 -/- mice were kindly provided by J. Chun (La Jolla, Calif). 36
Results
S1P Receptor Agonists Promote EPC-Mediated Neovascularization in Hindlimb Ischemia
Patient-derived EPCs and BMCs showed significantly impaired capacity for neovascularization in a mouse model of hind limb ischemia. 37,38 Because the S1P receptor family has been shown to play an important role in homing and trafficking of cells, we wanted to investigate whether S1P or FTY720 enhance neovascularization. 35 Therefore, we preincubated patient-derived EPCs for 2 hours with S1P or FTY720 before intravenous infusion in a mouse model of hindlimb ischemia. Preincubation of human EPCs with S1P and FTY720 followed by i.v. transplantation into the ischemic nude mice model resulted in significantly improved blood flow recovery in ischemic hind limbs as measured by Laser Doppler at 2 weeks after ligation of the femoral artery and almost completely restored blood flow recovery compared with EPCs from healthy volunteers ( Figure 1a and 1 b).
Figure 1. S1P and FTY720 promote in vivo neovascularization after EPC transplantation. a, Representative photographs from laser Doppler perfusion imaging at day 14 of ischemic (right) and nonischemic limbs (left) in nude mice after ligation of femoral artery in mice without EPC transplantation (control) and in mice receiving injections of 5 x 10 5 EPC pre-incubated with or without S1P (200 nM) or FTY720 (10 nM), N=6 to 7 per group. (b) Quantification of laser Doppler-derived blood flow at day 14. Hindlimb perfusion was significantly improved at day 14 in animals receiving EPC incubated with S1P or FTY720 compared with untreated EPC. *, P <0.01 versus control animals without cell transplantation; #, P <0.01 versus untreated EPC from patients.
Impaired Angiogenesis in S1P 3 -/- Mice
To investigate the role of S1P 1-4 receptors in blood flow recovery after cell transplantation into ischemic hind limbs, we first assessed the expression of S1P receptors on cultured human EPC by fluorescence-activated-cell sorter (FACS)-analysis. The predominantly expressed receptors in endothelial cells, S1P 1 and S1P 3, were also expressed on EPCs ( Figure 2 a). Therefore, we made use of mice deficient in the S1P 3 (knock down of the other major receptor, S1P 1, is embryonically lethal) and characterized the phenotype of isolated and cultivated EPCs derived from spleen of S1P 3 -/- mice. EPC numbers as counted by Dil-acetylated LDL positive cells were lower in S1P 3 -/- mice compared with wild-type mice. In addition, colony forming units were also reduced in EPC cultures derived from S1P 3 -/- mice (data not shown). Furthermore, S1P-induced migration was abolished in EPC derived from S1P 3 -/- mice ( Figure 2 b). Having demonstrated that EPCs derived from S1P 3 -/- mice were functionally impaired, we investigated the recovery of blood flow in these mice after hindlimb ischemia. Indeed, recovery of blood flow was dramatically reduced after induction of hindlimb ischemia in S1P 3 -/- mice, compared with ischemic wild-type litter mates ( Figure 2 c). Likewise, the capacity to restore blood flow recovery in the ischemic hindlimb model of nude mice was also significantly impaired using BMCs derived from S1P 3 -/- mice ( Figure 2 d). Of note, only treatment of wild-type BMCs with S1P enhanced blood flow recovery in nude mice after hindlimb ischemia ( Figure 2 d). Accordingly, capillary density was induced by S1P-stimulated BMC derived from wild-type mice (capillary/myocyte ratio increased with S1P to 122±13%, P =0.032, N=4). In contrast, capillary density in ischemic hindlimb muscles of nude mice was not different after transplantation of BMCs derived from S1P 3 -/- mice with or without S1P incubation (99±10% of capillary/myocyte ratio from S1P 3 -/- without S1P).
Figure 2. Impaired angiogenic capacities in murine EPCs derived from S1P 3 -/- mice. a, Sphingosine 1 phosphate receptor expression levels of S1P 1 and S1P 3 4 per group). b, Migration capacity toward SDF-1 of EPCs derived from spleen. S1P stimulates migratory capacity in wild-type mice, but failed to stimulate migration in EPCs derived from S1P 3 -/- mice, n=3 to 4. c, Laser doppler perfusion at 14 days after induction of endogenous hindlimb ischemia in wild-type vs S1P 3 -/- mice (n=6 to 7) indicating dramatically reduced recovery of blood flow in S1P 3 -/- mice, P <0.001. d, Infusion of BMCs derived from wild-type mice or S1P 3 -/- mice into nude mice after induction of hindlimb ischemia. S1P incubation induced blood flow recovery (* P <0.001 vs control and wild-type cells and S1P 3 -/- cell groups, n 3).
Activation of the CXCR 4 -Receptor and the Downstream Signaling Pathway by S1P
Next, we attempted to investigate the mechanisms underlying the effects of S1P or FTY720 on EPC function. S1P agonists have been shown to stimulate SDF-induced migration 16 and CXCR 4 importantly contribute to EPC migration and neovascularization. 38 Moreover, CXCR4-driven transendothelial migration of peripheral lymphnode T cells is stimulated by S1P and FTY720. 39 Thus, we postulated that S1P stimulates the activation of CXCR 4 and/or its downstream signaling. To assess whether stimulation of S1P receptors activates tyrosine phosphorylation of CXCR 4, we immunoprecipitated CXCR 4. Tyrosine phosphorylation of CXCR 4 by S1P agonists occurred rapidly reaching a 2-fold induction after 30 minutes incubation (1.96±0.57, P <0.01; Figure 3 a). Because JAK2 is a known downstream target of the CXCR 4 receptor, 38 we investigated whether JAK2 phosphorylation was induced by S1P. Indeed, immunoblotting revealed that JAK2 phosphorylation was significantly increased after preincubation of EPCs with S1P or FTY720 ( Figure 3 b). Taken together, these data suggest that S1P and FTY720 induce CXCR 4 -mediated signaling.
Figure 3. Activation of CXCR 4 receptor signaling by S1P. a, EPCs were serum depleted for 12 hours. Immunoprecipitation of CXCR 4 : Representative immunoblot shows tyrosine receptor phosphorylation which was induced by preincubation of EPCs with S1P agonists, n=5. b, Immunoblotting of JAK2 phosphorylation (upper panel) and JAK2 (lower panel) after preincubation of EPCs with S1P (200 nmol/L) or FTY720 (10 nmol/L), n=4 to 6. For quantification see also Figure 5.
Figure 5. Involvement of Src kinases in S1P mediated JAK2 phosphorylation of cultured EPCs. a, Representative JAK2 protein phosphorylation by immunoblotting in cultured EPCs with or without stimulation by S1P. EPCs were serum depleted for 12 hours before stimulation. b, Equal loading was confirmed by JAK2 protein expression and quantification expressed as pJAK2/JAK2 ratio, n=4. * P <0.001 vs control, # P <0.01 vs control or S1P stimulation. c, Representative JAK2 protein phosphorylation by immunoblotting in BMCs from wild-type mice with or without stimulation by S1P and with or without coincubation with PP2; PP2 (Calbiochem) was used at 1 µmol/L for 30 minutes preincubation. d, Equal loading was confirmed by JAK2 protein and quantification expressed as pJAK2/JAK2 ratio. * P <0.001 vs control, # P =ns vs control. e, Invasion of wild-type BMCs, stimulated with S1P or S1P and PP2, n=3 to 4. * P <0.05 vs control, # P <0.01 vs S1P.
To further strengthen this hypothesis, we next investigated the effects of S1P in CXCR 4 +/- mice. We previously demonstrated that BMCs and EPCs from CXCR 4 +/- mice showed impaired angiogenic activities. 38 In line with these findings, S1P-mediated JAK2 phosphorylation was significantly reduced in BMCs derived from heterozygous CXCR 4 +/- mice ( Figure 4a and 4 b). Likewise, S1P incubation failed to stimulate the invasion capacity of BMCs of heterozygous CXCR 4 +/- mice ( Figure 4 c). Finally, S1P preincubation of transplanted BMCs from CXCR 4 +/- mice failed to improve in vivo blood flow recovery in the ischemic hindlimb model of nude mice (blood flow ratio 26±11% versus 30±18%), further supporting the concept that the activation of the CXCR 4 receptor signaling by S1P is required to improve the functional capacities of EPCs or BMCs. Similar findings were observed with FTY720 (data not shown).
Figure 4. S1P does not activate JAK2 phosphorylation in CXCR 4 +/- mice. a and b, JAK2 phosphorylation in BMCs from wild-type and CXCR 4 +/- mice. BM cells were serum depleted for 6 hours and treated with S1P agonists for the indicated time points. Quantification expressed as pJAK2/JAK2 ratio. * P <0.01 vs basal, n=4. c, Quantification of SDF-1-induced invasion in BM-MNCs from background and CXCR 4 +/- mice with or without S1P stimulation, n=7. * P <0.05.
Involvement of the Src Kinase Family in the Transactivation of CXCR 4 Signaling
Src-family tyrosine kinases have been implicated in the S1P-induced activation of several growth factor receptors like the VEGF-receptor 2 and the PDGF receptor. 40,41 Therefore, we investigated whether the Src kinase family is involved in S1P-stimulated activation of CXCR 4 receptor signaling in EPCs. To elucidate the mechanism by which S1P or FTY720 activate CXCR 4, we preincubated EPCs with the Src kinase family inhibitor PP2. Basal and S1P-induced JAK2 phosphorylation was reduced by PP2 in human EPCs ( Figure 5a and 5 b). Likewise, JAK2 phosphorylation was inhibited by coincubation of PP2 with S1P in murine BMCs ( Figure 5c and 5 d). PP2 incubation also reduced S1P-or FTY720-induced invasion of BMC toward SDF-1 ( Figure 5 e).
S1P Induced Activation of Src Kinases Is Dependent on S1P 3
Next, we investigated whether S1P induced the activation of Src. As demonstrated in Figure 6 a, concomitantly with the time-dependent phosphorylation of active Src at tyrosine 416 in human EPCs, phosphorylation of the inactive form of Src at tyrosine 527 was reduced after S1P incubation. Finally, we evaluated whether Src kinase activation is detectable in EPC from S1P 3 -/- mice. Src phosphorylation at tyrosine 416 was induced by S1P in EPC derived from wildtype mice, whereas Src phosphorylation and activation were abrogated in EPC derived from S1P 3 -/- mice ( Figure 6 b).
Figure 6. S1P activates Src kinase via S1P 3. a, Immunoblot showing active (tyrosine 416) or inactive (tyrosine 527) form of Src. S1P incubation-induced Src phosphorylation at tyrosine 416 in human EPCs as early as 10 minutes. Concomitantly, the inactive form of Src was reduced. b, Immunoblot detecting the active (tyrosine 416) or inactive (tyrosine 527) form of Src. After stimulation of cultured EPCs derived from spleen of each of 15 mice, protein extracts were analyzed for Src activation. Src phosphorylation was induced by S1P in EPCs derived from wild-type mice, whereas no induction of Src phosphorylation was detectable in EPC derived from S1P 3 -/- mice.
Discussion
The results of the present study demonstrate that S1P and FTY720 improved EPC-mediated blood flow recovery in ischemic hind limbs. Specific deletion of S1P 3 blocked the S1P effects on progenitor cells and lead to severe impairment of neovascularization. Mechanistically, S1P stimulates the transactivation of the CXCR 4 receptor leading to the activation of Src and JAK2 signaling.
S1P plays a crucial role in the cardiovascular system: it acts as a proangiogenic molecule, 12 improves endothelial function, 9 myocardial perfusion, 33 and is involved in cardiovascular development. 6,34 Additional studies demonstrate that S1P modulates homing and trafficking of cells. 14,35 In accordance, the present study documents that S1P influences the functional activity of endothelial progenitor cells to improve neovascularization after ischemia. S1P exerts its effects by activating the G protein-coupled receptors S1P 1-5. S1P 1-3 are widely expressed and have been shown to be involved in the development of a mature vascular system during embryonic development. S1P 1 and S1P 3 are considered to be the major receptors in endothelial cells contributing to angiogenic processes. Using S1P 3 -/- cells, we demonstrated that specifically the S1P 3 receptor is required for progenitor cell-mediated migration in vitro, neovascularization improvement in vivo, and is essential for S1P-mediated activation of down stream signaling pathway. Although S1P 1 and S1P 3 have been shown to act cooperatively, 42 distinct functions can be attributed to both receptor subtypes. Previous studies revealed that selective S1P 1 agonists control lymphocyte recirculation, whereas S1P 3, and not S1P 1, regulates heart rate. 11 The specific role of S1P 3 in proangiogenic signaling was underscored by the finding that vascular sprouting can also be induced by a synthetic peptide derived from the second intracellular loop of S1P 3. 12 This is in line with the data of the present study demonstrating that S1P 3 -deficient mice show a severe impairment of neovascularization after ischemia. However, other studies using knockdown strategies by RNA interference clearly identified S1P 1 as a critical component of tumor angiogenic responses. 43 Because S1P 1 is also expressed in EPC and S1P 1 knock out mice are embryonically lethal, one may speculate that the combined deletion of both receptors may even more severely affect EPC function.
The signaling downstream of the S1P 3 receptor to mediate the S1P effects on EPCs involves the transactivation of the CXCR 4 receptor. Previous studies demonstrated that S1P transactivates several receptors involved in angiogenesis such as the PDGF receptor, VEGF receptor-2 and EGF receptor, which mediate migratory activities in endothelial cells or smooth muscle cells. 40,44-46 The present study now extends these findings by demonstrating that tyrosine phosphorylation of the CXCR 4 receptor is stimulated by S1P in EPCs. CXCR 4 plays a crucial role for endothelial cell migration and is essential for homing and functional integration of EPCs to ischemic tissues. 38,47,48 Moreover, CXCR 4 +/- EPCs failed to augment neovascularization in a previous experimental study. 38 Consistently, the S1P-mediated stimulation of EPC function and signaling was abolished in CXCR 4 +/- mice. S1P-mediated activation of the CXCR 4 receptor signaling was sensitive to the Src inhibitor PP2, implicating that Src family kinases mediate the signaling between the S1P 3 receptor and the CXCR 4 receptor. Because nothing is known about an intrinsic tyrosine kinase activity of the S1P receptors, it is tempting to speculate that Src is required for CXCR 4 tyrosine phosphorylation. Indeed, Src-family tyrosine kinases are activated by S1P stimulation and have previously been implicated in S1P-mediated activation of several growth factor receptors. 40,41,49,50 Although our data showed a Src-dependent activation of the CXCR 4 receptor signaling and a requirement of CXCR 4 expression for S1P responses, we cannot rule out that Src activation improves EPC function by additional pathways. Thus, Src may activate the VEGF receptor 2, which plays an important role for endothelial progenitor cell mobilization, survival, and function (for review see 51,52 ). Moreover, Src has been shown to activate the PI3K/Akt/eNOS-signaling pathway 53 and by this means may exert beneficial effects on endothelial progenitor cell function and homing.
The stimulation of progenitor cells with S1P or its analog FTY720 may also be an interesting therapeutic tool to augment EPC function. Risk factors for coronary artery disease and severe heart failure have been shown to impair circulating blood-derived EPCs, 27,54,55 limiting the functional capacity of the isolated cells to augment blood flow recovery after infusion in experimental animal models. 38 Likewise, bone marrow-derived cells isolated from patients with chronic ischemic heart disease showed a significantly impaired homing and neovascularization improvement capacity. 29 Although risk factor for coronary artery disease may affect multiple signaling pathways, 37,56 recent data demonstrate that CXCR 4 signaling and the response toward SDF-1 is significantly impaired in patient-derived cells compared with healthy controls. 38 The transactivation of the CXCR 4 receptor signaling cascade by S1P in patient-derived cells may compensate for the reduced activity of this important signaling pathway.
The compound FTY720 is currently in clinical trials for renal transplantation rejection. However, its precise mechanism of action is not entirely clear. After being metabolized by cellular kinase(s), the compound FTY720 bears structural similarities with S1P and was shown to act as S1P analog. Consistently, FTY720 stimulated CD34 + cell migration. 16 However, other studies have demonstrated that FTY720 also can act as antagonist of S1P-induced responses. When given systemically, FTY720 blocked S1P-angiogenesis and VEGF-induced tumor vascularization. 57 The reason for the different responses may be explained by the pretreatment of the animals and cells with proangiogenic stimuli such as S1P or VEGF. Thus, it is tempting to speculate that further incubation of pretreated cells with FTY720 may lead to internalization of S1P receptors and, thereby, inhibiting S1P responses. In the present study, FTY720 was only used to pretreat the unstimulated cells for 2 hours before reinfusion, whereas the antitumor activity was detected in mice which were prestimulated.
In summary, S1P and its analog FTY720 profoundly stimulate the angiogenic activity and neovascularization capacity of cultured EPCs or BMCs. Mechanistically, S1P activates the CXCR 4 dependent JAK2 signaling, involving in part the activity of Src kinases via the S1P 3 receptor. S1P agonists might serve as ideal sensitizers of CXCR 4 -mediated signaling and may be applied in clinical progenitor cell therapy to improve EPC or BMC function in patients with coronary artery disease.
Acknowledgments
We gratefully acknowledge the expert technical assistance of Christine Goy, Carmen Schön, Tina Rasper, Ariane Fischer, Tino Röxe, Marion Muhly-Reinholz, and Kerstin Abouhamed.
Sources of Funding
This work was supported by a research grant from the Deutsche Forschungsgemeinschaft (WA 1461/2-1 and 2-2). J.C. was supported by NIH R01 NS048478. J.H. was supported by DFG HA 2868/2-3. B.L. was supported by DFG LE 940/3-1 and SFB 656, project A1, and the Deichmann Foundation for Atherosclerosis Research.
Disclosures
None.
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作者单位：From Molecular Cardiology, Department of Internal Medicine III (D.H.W., U.R., J.R., F.S., A.A, C.U., I.S., A.M.Z., S.D., J.H.), University of Frankfurt, Germany; the Department of Molecular Biology (J.C.), Helen L. Dorris Child and Adolescent Neuropsychiatric Disorder Institute, The Scripps Research