Thrombopoietin Gene TransfereCMediated Enhancement of Angiogenic Responses to Acute Ischemia
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循环研究杂志 2005年第8期
the Department of Genetic Medicine (H.A., S.R., R.G.C.) and Belfer Gene Therapy Core Facility (N.R.H., R.G.C.), Weill Medical College of Cornell University, New York.
Abstract
The development of new blood vessels is a complex process, likely requiring the synergy of multiple angiogenic mediators. This study focuses on the proximal angiogenic response using the platelet as a complex carrier of critical mediators of angiogenesis. Platelet levels are controlled by circulating levels of thrombopoietin (TPO) functioning to activate megakaryocyte differentiation and platelet release through the c-mpl receptor. We hypothesized that TPO gene transfer should enhance correction of experimental ischemia by providing increased levels of platelets and hence platelet-derived mediators of angiogenesis. To evaluate this hypothesis, we dissected the role of the TPOeCc-mpleCmegakaryocyteeCplatelet pathway in the angiogenic response using a model of acute hindlimb ischemia of wild-type, TPOeC/eC, and c-mpleC/eC mice. The data demonstrate that infusion of platelets will enhance the angiogenic response in wild-type mice and that the endogenous angiogenic response is blunted in TPOeC/eC and c-mpleC/eC mice. Consistent with this observation, adenovirus (Ad)-mediated transfer of TPO (AdTPO) enhanced the correction of ischemia in wild-type and TPOeC/eC, but not c-mpleC/eC, mice. Local versus systemic administration of AdTPO showed that the effect of TPO gene transfer was systemic, not local, and it could be replaced by gene transfer of VEGF, one of the many mediators of angiogenesis carried by the platelets, even in the absence of components in the TPOeCc-mpleCmegakaryocyteeCplatelet pathway.
Key Words: angiogenesis thrombopoietin platelets megakaryocyte gene therapy
Introduction
Gene transfer of single angiogenic mediators to treat ischemia involves local delivery of the gene for an angiogenic factor to stimulate the development of new blood vessels in the local milieu.1eC5 Among the most intensively studied genes for angiogenic gene therapy are those for various forms of vascular endothelial growth factor (VEGF)-A (most commonly VEGF121 or VEGF165; usually referred to as VEGF), VEGF-B, VEGF-D, fibroblast growth factor (FGF)-2 and FGF-4.3,5eC7 Although the results of many of the studies are encouraging, there is growing evidence that gene transfereCmediated delivery of mixtures of angiogenic proteins may yield better vessel morphology and less leakage.8eC10 This synergy between various angiogenic growth factors likely reflects the complexity of physiological angiogenesis that involves the coordinated action of many mediators.
It is uncertain whether optimal therapeutic angiogenesis requires the use of multiple angiogenic mediators or whether one proximal mediator will initiate the complex cascade of events required to augment angiogenesis in response to ischemia. This study focuses on the proximal angiogenic response using the platelet as a carrier of multiple mediators of angiogenesis. Platelets are critical to hemostasis and subsequent angiogenesis in wound healing, and transfusion of platelets enhances the angiogenic recovery of blood flow in models of ischemia.11eC13 Platelets are a major source of angiogenic growth factors including VEGF, platelet-derived growth factor, and FGF-2, as well as other factors that play a role in angiogenesis, including lipids, such as sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidate.12,14eC19 Consistent with these concepts, Iba et al20 have shown that local injection of platelets, either alone or in combination with blood mononuclear cells, into an ischemic hindlimb can promote recovery of blood flow.
Based on these concepts, and the knowledge that platelet levels are controlled by circulating levels of thrombopoietin (TPO) functioning through the c-mpl receptor to activate megakaryocyte differentiation and platelet release,21,22 we hypothesized that TPO gene transfer should correct experimental ischemia by providing increased levels of platelets and hence platelet-derived mediators of angiogenesis. To evaluate this hypothesis, we have dissected the role of the TPOeCc-mpleCmegakaryocyteeCplatelet pathway in the angiogenic response using the model of acute hindlimb ischemia of wild-type, TPOeC/eC, and c-mpleC/eC mice. The data demonstrate that infusion of platelets will enhance the angiogenic response and that the endogenous angiogenic response is blunted in TPOeC/eC and c-mpleC/eC mice. Consistent with this observation, adenovirus (Ad)-mediated transfer of TPO enhances the correction of ischemia in wild-type and TPOeC/eC, but not c-mpleC/eC mice in a process mediated through platelets. The effect of TPO gene transfer is systemic, not local, and it can be replaced by gene transfer of VEGF, one of the many mediators of angiogenesis carried by the platelets, even in the absence of components in the TPOeCc-mpleCmegakaryocyteeCplatelet pathway.
Materials and Methods
Adenovirus Gene Transfer Vectors
AdTPO is a E1eCE3eC Ad vector coding the murine cDNA for TPO expressed from the cytomegalovirus promoter.23 AdVEGF121 is similar with the cDNA for the 121-aa isoform of human VEGF.24 A similar Ad with no transgene (AdNull) was used as control.25 Vectors were propagated on 293 cells and purified by 2 cesium chloride density gradients.26,27 Dosing was performed in particle units (pu).28
Model of Acute Hindlimb Ischemia
All animal experiments were executed after approval from the Institutional Animal Care and Use Committee. TPOeC/eC29 and c-mpleC/eC mice,30 all on the C57Bl/6/sv129 hybrid background, were obtained from Fred Sauvage (Genentech, San Francisco, Calif). Wild-type C57Bl/6 mice were obtained from Jackson Laboratories (Bar Harbor, Maine). The model of hindlimb ischemia was previously described.31 Eight-week-old mice were anesthetized, and a midline incision was made in the abdominal skin permitting exposure of the external iliac artery in the upper left limb. The artery was then ligated both proximally and distally and an intervening 6-mm section excised. The vectors (total dose, 108 pu) were injected (100 e蘈) into the underlying ipsilateral quadriceps muscle. In some experiments, the vectors were administered to the contralateral quadriceps muscle or intravenously.
Blood flow to the hindlimbs was assessed by scanning the lower abdomen and limbs of the mouse with a Lisca (PIM II) scanning laser Doppler (Perimed). The ratio of blood flow in the ischemic (left) to the control (right) limb was calculated by dividing the integrated blood flow in an area of the image that included the left-foot pad by the integrated blood flow for an area of the same size that included the right-foot pad. Blood flow measurements were assessed both pre- and postoperatively and on days 3, 7, 14, 21, and 28. Platelets counts were assessed by collecting blood from the tail vein, using a capillary (Unopette; Becton Dickinson) and counting using a Neubauer hemocytometer (Fisher Scientific). To determine plasma VEGF levels, the blood was collected using microhematocrit capillary tubes. The blood was centrifuged at 6000g for 5 minutes, the plasma was collected, and VEGF levels were assessed by ELISA specific for murine VEGF (R&D Systems).
Platelet Administration
Anesthetized donor wild-type C57Bl/6 mice were bled via heart puncture into heparinized microtubes. The blood was pooled and centrifuged at 220g for 5 minutes, and the platelet-rich plasma was collected. The platelets were adjusted to 5x107/mL in PBS (pH 7.4). Platelet suspension (50 e蘈) was injected into the tail vein on induction of ischemia and at 4 day intervals afterward.
Morphological Quantification of Blood Vessel Number
The ipsilateral quadricep muscle was excised and fixed with 4% paraformaldehyde for 3 hours. The muscle was then transferred into 70% ethanol overnight and embedded in paraffin, and 5-e sections were mounted and deparaffinized. Sections were stained with platelet-endothelial cell adhesion molecule (PECAM)-1 polyconal antibody (H300; Santa Cruz Biotechnology, Santa Cruz, Calif) followed by a horseradish peroxidaseeCconjugated secondary antibody (K1490; Dako, Carpinteria, Calif) and the diaminobenzidine substrate. The number of PECAM-positive cells for 20 muscle fibers chosen at random in 1 slide for each of 5 animals per treatment group was counted.
Quantification of Bone Marrow Megakaryocyte Number
Megakaryocyte number in sections of bone marrow was assessed by counting the number of megakaryocytes in 5 random fields per slide (one H&E-stained 5-e bone marrow section from each animal in each group).
Statistics
Data are expressed as mean±SD. Groups were compared by unpaired Student t test with correction for multiple comparisons if required.
Results
To confirm that platelets are critical players in angiogenesis, the impact of platelet infusion on the recovery from experimental hindlimb ischemia in C57Bl/6 mice was assessed. Excision of a 6-mm section of the external iliac artery resulted in a decrease in blood flow ratio (ischemic/control limb), as assessed by laser Doppler scanning from 0.98±0.04 to 0.10±0.01 immediately postsurgery (Figure 1A). Seven transfusions with 106 platelets on days 0, 4, 8, 12, 16, 20, and 24 postsurgery increased recovery of flow ratio by day 28 from 0.49±0.08 with PBS infusion to 0.78±0.07 with transfusion of platelets (P<0.005). The platelet-dependent increase in flow was confirmed to reflect angiogenesis by histochemical examination. Sections of the quadriceps were stained by an anti-PECAM antibody to identify endothelial cells (Figure 1B through 1D). The average number of PECAM-positive cells per muscle fiber was 1.86±0.13 for the PBS-infused mice and 2.36±0.26 for the platelet-infused mice (P<0.005).
Impact of Reduced Platelet Levels on Angiogenesis in Acute Ischemia
If platelets play a role in enhancing the angiogenic response to acute ischemia, it follows that platelet-deficient mice should have an impaired angiogenic response. Recovery from acute hindlimb ischemia, as assessed by the flow ratio between the treated ischemic limb and the untreated control right limb, was assessed at intervals in wild-type, TPOeC/eC, and TPO receptor (c-mpleC/eC) knockout mice (Figure 2). The initial excision of the iliac artery dropped the flow ratio from 0.93±0.04 before surgery to 0.06±0.03 immediately postsurgery (mean values for all 3 groups combined). Recovery in wild-type mice reached a flow ratio of 0.51±0.03 by 28 days compared with 0.31±0.04 in TPOeC/eC mice and 0.31±0.06 in c-mpleC/eC mice. At all time points studied (days 7, 14, 21, 28), the flow ratio in both groups of knockout mice was significantly lower (P<0.05) than for the wild-type mice. The mean platelet levels in naive mice of these strains are 870 000±110 000 platelets/e蘈 for wild-type mice, 290 000±34 000 for TPOeC/eC mice, and 260 000±53 000 for c-mpleC/eC mice (Figure 2B). There was no significant change in platelet count in any strain as a function of time after induction of acute ischemia in the hindlimb. Thus, deletions of the gene coding for TPO or the TPO receptor resulted in reduced platelet counts, consistent with the impact of these gene deletions on the angiogenic response to acute hindlimb ischemia.
Impact of TPO Gene Transfer on Bone Marrow, Platelet Levels, and Angiogenesis
If platelets are critical players in angiogenesis, it follows that TPO gene transfer, which results in elevated platelet levels,23,32,33 should enhance angiogenesis. To show that TPO gene transfer resulted in enhanced production of platelets from progenitors, the bone marrow of wild-type, TPOeC/eC, and c-mpleC/eC mice was examined for accumulation of megakaryocytes (Figure 3). Intramuscular administration of AdTPO resulted in the accumulation of megakaryocytes in bone marrow at 28 days postvector to a level of 3.1±0.7 megakaryocytes/field in wild-type mice and 2.4±0.8 megakaryocytes/field in TPOeC/eC mice. The dependency of megakaryocyte levels on the TPOeCc-mpl axis was shown by the lower number of megakaryocytes in bone marrow of mice injected with AdNull, the control vector with no transgene, in both wild-type mice (1.7±0.5 megakaryocytes/field; P<0.001 compared with AdTPO-treated wild-type mice) and TPOeC/eC mice (0.6±0.7 megakaryocytes/field; P<0.001 compared with AdTPO-treated TPOeC/eC mice). However, in the case of c-mpleC/eC mice, which are predicted to be nonresponsive to TPO, there was no difference in the number of bone marrow resident megakaryocytes in AdTPO-treated (0.2±0.4 megakaryocytes/field) and AdNull-treated (0.7 megakaryocytes/field±0.4 megakaryocytes/field) animals (P>0.05).
The increased levels of megakaryocytes in bone marrow in response to AdTPO were parallel to the platelet counts (Figure 4). In wild-type mice, the level rose from 0.93±0.06x106/e蘈 before induction of ischemia to a peak of 2.30±0.55x106/e蘈 at 7 days posteCvector administration and remained significantly (P<0.05) higher than background for 28 days (Figure 4A). Similarly, in the TPOeC/eC mice, platelets increased from 0.27±0.04x106/e蘈 before induction of ischemia to a peak of 1.62±0.11x106/e蘈 at 7 days posteCvector administration and remained significantly (P<0.05) higher than background for 28 days (Figure 4B). In both of these mouse strains, there was no change in platelet count as a result of AdNull treatment (P>0.05 comparing AdNull treated wild-type and TPOeC/eC mice preischemia to all other time points). As expected, the c-mpleC/eC mice were nonresponsive to TPO gene transfer, with a similar platelet count at all time points in the AdTPO- and AdNull-treated groups (Figure 4C; P>0.05).
To show that the changes in megakaryocytes in bone marrow and level of platelets correlated with the enhanced angiogenic response to acute ischemia, the blood flow ratio between the ischemic and nonischemic hindlimb was assessed in the same mice (Figure 5). Wild-type, TPOeC/eC, and c-mpleC/eC mice were administered AdTPO or AdNull, and the flow ratio was assessed as a function of time. In both wild-type and TPOeC/eC mice, there was a time-dependent increase in flow ratio in the AdTPO group, becoming significantly higher than the flow ratio in the AdNull group by 7 days (Figure 5A and 5B; P<0.01). By contrast, in the c-mpleC/eC mice, there was no difference (P>0.1) between the AdNull and AdTPO group at any time point (Figure 5C).
The correlation of the extent of the angiogenic response to acute ischemia with platelet levels was also confirmed by histological studies quantifying the number of PECAM-positive cells in the ischemic quadriceps (Figure 6). In wild-type and TPOeC/eC mice, AdTPO treatment gave an increased frequency of PECAM-positive cells, whereas in c-mpleC/eC mice, the PECAM-positive staining was similar for the AdTPO- and AdNull-treated mice. Counting the number of PECAM-positive cells and normalizing to the number of muscle fibers showed a 1.6-fold increase in the frequency of PECAM-positive cells in the AdTPO-treated wild-type mice relative to the AdNull group and a 1.7-fold increase in the TPOeC/eC mice (P<0.001 comparing AdTPO-treated mice with AdNull-treated mice for both strains). In contrast, in the c-mpleC/eC mice, there was no significant difference in the frequency of PECAM-positive cells in the ischemic hindlimb between the AdTPO- and AdNull-treated group (P>0.4).
Effect of Systemic TPO Gene Transfer on the Angiogenic Response to Acute Ischemia
The effects of TPO gene transfer may be mediated by its systemic effect (eg, by increasing platelet levels) or local effects of the TPO in the ischemic limb. To distinguish these possibilities, a comparison was made among the angiogenic responses to acute ischemia of local intramuscular injection of AdTPO into the ischemic limb, injection of AdTPO into the contralateral quadriceps, and intravenous injection (which results in expression in the liver).34,35 The data show that AdTPO results in a similar recovery from hindlimb ischemia independent of route of administration of vector (Figure 7). At 28 days, the flow ratio in the mice that received vector in the ischemic limb was 0.80±0.07 compared with 0.81±0.08 in the mice receiving injections into the contralateral limb and 0.80±0.14 in the mice receiving intravenous vector (P>0.8, all pairwise comparisons). In all cases, the recovery was higher than in the group treated with AdNull by the same route (P<0.01).
In parallel with the flow data, administration of AdTPO by various routes also resulted in elevation of platelet levels regardless of the route of administration (Figure 7). At day 14 postvector, the mice injected with the AdTPO vector into the ischemic muscle had a 2.7-fold higher platelet count than the AdNull group compared with a 2.8-fold higher level in the contralateral intramuscular group and 1.8-fold in the intravenous group (P<0.05 comparing AdNull-treated mice of each group to the AdTPO mice receiving injections by the same route). In all groups, the elevation of platelet levels persisted at >2-fold higher than the AdNull control group for at least 28 days posteCvector administration.
Mediation of the Angiogenic Effects of Platelets by VEGF
Among the potential role of platelets is to provide growth factors such as VEGF for induction of the angiogenic process.11eC19 In that situation, the recovery of blood flow to hindlimb induced by AdTPO should be suppressed by inhibitors of the VEGF pathway. Therefore, the AdTPO-dependent recovery from hindlimb ischemia was assessed in wild-type mice treated with anti-VEGFR1 antibody (35 e IP every 3 days). Injection of control antibody led to a flow ratio of 0.88±0.04 at 28 days, whereas injection of an anti-VEGFR1 antibody led to a flow ratio of 0.37±0.03 at 28 days (P<0.05 compared with control).
If VEGF is a dominant downstream factor delivered by platelets, then angiogenic gene therapy with VEGF should mimic the effects of increased platelets on the enhanced angiogenesis in response to the acute ischemia, and this effect should be unaffected by mutations in the TPOeCc-mpl pathway. To assess this hypothesis independently, the effects of intramuscular AdVEGF on recovery from acute hindlimb ischemia was assessed in wild-type, TPOeC/eC, and c-mpleC/eC mice (Figure 8). In all 3 strains, AdVEGF led to an increase in flow ratio with a significantly better flow ratio at all time points beyond 14 days (P<0.05 compared with AdNull group).
Discussion
In these studies, a combined gene transfer and murine gene knockout approach has been used to dissect the ability of platelets to enhance angiogenesis in response to acute ischemia. Commencing by confirmation of the observation that platelets are angiogenic in vivo,20 we have expanded the concept to show the converse: that mutations in the TPOeCc-mpleCmegakaryocyteeCplatelet pathway that result in low endogenous levels of platelets led to a subdued angiogenic response to acute ischemia in the hindlimb. If platelets enhance angiogenesis in vivo, then it follows that TPO gene transfer, which we have previously shown to result in elevated platelet levels,23,32,33 would also enhance angiogenesis in response to acute ischemia. The data demonstrate that local or systemic TPO gene transfer promotes angiogenic responses to acute hindlimb ischemia. The angiogenic effect of gene transfer is most likely mediated by platelets delivering angiogenic factors, especially VEGF. Studies with gene transfer of VEGF in mice with defects in the TPOeCc-mpleCmegakaryocyteeCplatelet pathway suggest that VEGF can substitute for TPO, suggesting VEGF is the major mediator carried by platelets that is responsible for the enhanced angiogenesis in response to acute ischemia that is observed with platelet administration or with gene transfereCmediated administration of TPO.
TPO-Mediated Angiogenesis
The effects of TPO gene transfer in the angiogenic response to acute ischemia could result from at least 3 pathways: direct effects of TPO on the endothelium, effects of TPO on platelets, or angiogenic effects of the platelets synthesized in response to TPO. It is clear that TPO has direct effects on endothelium. Human umbilical vascular endothelial cells express the c-mpl receptor for TPO and have an intact signaling pathway that results in phosphorylation of signal transducers and activators of transcription proteins in response to the addition of TPO.36 Moreover, TPO causes endothelial migration in vitro and angiogenesis in vivo in the context of a matrigel implant. Both of these effects appeared to be mediated by platelet-activating factor, a biologically active phospholipid. However, this mechanism is unlikely to account for the angiogenic effects of TPO gene transfer to hindlimb. First, local and systemic administration of AdTPO were equally effective in restoring hindlimb blood flow, suggesting that local effects of TPO on endothelium are not the major mediator of the enhanced angiogenesis. Second, platelet infusions, a therapy that would not directly deliver TPO to the ischemic hindlimb, was effective in promoting recovery in the hindlimb ischemia model.
TPO is known to have direct effects on megakaryocytes and to enhance VEGF synthesis and secretion.37 It is unknown if the TPO produced after gene transfer may enhance release of VEGF or other angiogenic growth factors by platelets. Because platelet infusion alone (ie, in the absence of increased TPO levels) is angiogenic, it is difficult to assign a relative proportion of TPO versus platelet effect in the angiogenic effect of TPO gene transfer.
It is more likely that the TPO gene transfer is angiogenic because of its effects on bone marrow, where it results in differentiation of megakaryocytes and subsequent increases in platelet numbers. Previous studies from our laboratory have shown that subcutaneous or intratracheal administration of AdTPO results in a transient, dose-dependent increase in platelet levels mediated by effects on bone marrow.23,32,33 That data are extended in the current study showing that intramuscular and intravenous injection are equally effective with enhanced platelet levels achieved with administration of only 108 pu of the AdTPO vector. The accumulation of megakaryocytes in bone marrow and subsequent increase in platelet levels was observed in wild-type and TPOeC/eC mice but not in c-mpleC/eC mice, in which platelet levels were, as expected, nonresponsive to TPO gene transfer. The evidence suggests that the combination of increased platelet level in combination with local ischemia provides the angiogenic milieu. Platelets alone are probably not angiogenic because the capillary density in TPOeC/eC and c-mpleC/eC mice was similar to that in wild-type mice. However, platelets are know to accumulate at sites of endothelial injury,38eC40 and this pathway is likely enhanced by increased platelet count, thereby enhancing the local delivery of angiogenic factors by the platelets.
Effects of TPO on Bone Marrow
TPO was originally discovered as a factor that caused the differentiation of megakaryocytes from stem cells in the bone marrow and subsequent platelet production. However, the phenotype of mice that lack either the TPO or c-mpl gene pointed to a second effect of TPO, the promotion of stem cell survival and self-renewal.41eC45 The data shown here, specifically the effects of platelet infusion on recovery from acute hindlimb ischemia, suggest that the major mechanism by which TPO gene transfer promotes angiogenesis is via megakaryocyte differentiation in bone marrow and an increase in systemic platelet levels. An additional role of TPO in angiogenesis may be through its effects on raising platelet levels, with secondary effects on raising VEGF (or other platelet mediator) levels and the consequent effects of these mediators on bone marroweCderived stem cells leading to enhanced production of endothelial progenitors.45
Angiogenic Factors Delivered by Platelets
The effects of TPO on the enhancement of angiogenesis in response to acute ischemia are likely mediated by the delivery of angiogenic factors to the ischemic tissue by platelets, functioning locally or systemically or both. For example, platelet-derived lipids have been reported to be angiogenic. Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidate are all biologically active lipids that act on cells via G-protein coupled receptors.12 One of these receptors designated EDG1 (endothelial differentiation gene), is highly expressed in activated endothelium and is thought to mediate the effects of sphingosine 1-phosphate on endothelial cells.12 These effects include the adherens junction assembly and induction of tube-forming morphology, effects that are thought to be mimics of angiogenesis in vivo.36,46
However, in addition to lipids with a role in angiogenesis, platelets also deliver angiogenic proteins. One of the most potent angiogenic mediators delivered to sites of ischemia by platelets is VEGF. The blockage of the angiogenesis resulting from TPO gene transfer by antibodies against the VEGF receptor suggests that VEGF is the primary mediator of the angiogenic effects of platelets. The VEGF content of platelets is estimated to be 0.5 pg/106 platelets.47 Studies in patients with malignancy show a strong correlation of plasma VEGF levels and platelet count. Furthermore, activation of platelets is known to result in the release of large quantities of VEGF.48 The current study supports the concept that VEGF is a major angiogenic mediator released by platelets, either via direct effects or secondarily via induction of other angiogenic-mediated pathways and by mobilization of stem cells from bone marrow. Whatever the specific mechanisms, the data show that the effect is downstream from the effects of TPO.
Acknowledgments
These studies were supported, in part, by grant P01 HL59312 from the National Institutes of Health and The Will Rogers Memorial Fund, Los Angeles, Calif. We thank Barbara Ferris, Rafael Tejada, and Koji Shido for scientific and technical assistance; and N. Mohamed for help in preparing this manuscript.
References
Freedman SB, Isner JM. Therapeutic angiogenesis for coronary artery disease. Ann Intern Med. 2002; 136: 54eC71.
Isner JM. Myocardial gene therapy. Nature. 2002; 415: 234eC239.
Khan TA, Sellke FW, Laham RJ. Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia. Gene Ther. 2003; 10: 285eC291.
Morishita R. Recent progress in gene therapy for cardiovascular disease. Circ J. 2002; 66: 1077eC1086.
Syed IS, Sanborn TA, Rosengart TK. Therapeutic angiogenesis: a biologic bypass. Cardiology. 2004; 101: 131eC143.
Ferrara N, Gerber HP, Le Couter J. The biology of VEGF and its receptors. Nat Med. 2003; 9: 669eC676.
Cao R, Eriksson A, Kubo H, Alitalo K, Cao Y, Thyberg J. Comparative evaluation of FGF-2-, VEGF-A-, and VEGF-C-induced angiogenesis, lymphangiogenesis, vascular fenestrations, and permeability. Circ Res. 2004; 94: 664eC670.
Hao X, Mansson-Broberg A, Gustafsson T, Grinnemo KH, Blomberg P, Siddiqui AJ, Wardell E, Sylven C. Angiogenic effects of dual gene transfer of bFGF and PDGF-BB after myocardial infarction. Biochem Biophys Res Commun. 2004; 315: 1058eC1063.
Lubiatowski P, Gurunluoglu R, Goldman CK, Skugor B, Carnevale K, Siemionow M. Gene therapy by adenovirus-mediated vascular endothelial growth factor and angiopoietin-1 promotes perfusion of muscle flaps. Plast Reconstr Surg. 2002; 110: 149eC159.
Siddiqui AJ, Blomberg P, Wardell E, Hellgren I, Eskandarpour M, Islam KB, Sylven C. Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium. Biochem Biophys Res Commun. 2003; 310: 1002eC1009.
Pinedo HM, Verheul HM, D’Amato RJ, Folkman J. Involvement of platelets in tumour angiogenesis Lancet. 1998; 352: 1775eC1777.
English D, Garcia JG, Brindley DN. Platelet-released phospholipids link haemostasis and angiogenesis. Cardiovasc Res. 2001; 49: 588eC599.
Sierko E, Wojtukiewicz MZ. Platelets and angiogenesis in malignancy. Semin Thromb Hemost. 2004; 30: 95eC108.
Sekido Y, Morishima Y, Ohya K. Activity of platelet-derived growth factor (PDGF) in platelet concentrates and cryopreserved platelets determined by PDGF bioassay. Vox Sang. 1987; 52: 27eC30.
Wartiovaara U, Salven P, Mikkola H, Lassila R, Kaukonen J, Joukov V, Orpana A, Ristimaki A, Heikinheimo M, Joensuu H, Alitalo K, Palotie A. Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation. Thromb Haemost. 1998; 80: 171eC175.
Gunsilius E, Petzer A, Stockhammer G, Nussbaumer W, Schumacher P, Clausen J, Gastl G. Thrombocytes are the major source for soluble vascular endothelial growth factor in peripheral blood. Oncology. 2000; 58: 169eC174.
Ma L, Elliott SN, Cirino G, Buret A, Ignarro LJ, Wallace JL. Platelets modulate gastric ulcer healing: role of endostatin and vascular endothelial growth factor release. Proc Natl Acad Sci U S A. 2001; 98: 6470eC6475.
Salgado R, Benoy I, Bogers J, Weytjens R, Vermeulen P, Dirix L, Van Marck E. Platelets and vascular endothelial growth factor (VEGF): a morphological and functional study. Angiogenesis. 2001; 4: 37eC43.
Lacoste E, Martineau I, Gagnon G. Platelet concentrates: effects of calcium and thrombin on endothelial cell proliferation and growth factor release. J Periodontol. 2003; 74: 1498eC1507.
Iba O, Matsubara H, Nozawa Y, Fujiyama S, Amano K, Mori Y, Kojima H, Iwasaka T. Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs. Circulation. 2002; 106: 2019eC2025.
de Sauvage FJ, Carver-Moore K, Luoh SM, Ryan A, Dowd M, Eaton DL, Moore MW. Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin. J Exp Med. 1996; 183: 651eC656.
Kaushansky K. Thrombopoietin. N Engl J Med. 1998; 339: 746eC754.
Cannizzo SJ, Frey BM, Raffi S, Moore MA, Eaton D, Suzuki M, Singh R, Mack CA, Crystal RG. Augmentation of blood platelet levels by intratracheal administration of an adenovirus vector encoding human thrombopoietin cDNA. Nat Biotechnol. 1997; 15: 570eC573.
Mack CA, Patel SR, Schwarz EA, Zanzonico P, Hahn RT, Ilercil A, Devereux RB, Goldsmith SJ, Christian TF, Sanborn TA, Kovesdi I, Hackett N, Isom OW, Crystal RG, Rosengart TK. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Thorac Cardiovasc Surg. 1998; 115: 168eC176.
Hersh J, Crystal RG, Bewig B. Modulation of gene expression after replication-deficient, recombinant adenovirus-mediated gene transfer by the product of a second adenovirus vector. Gene Ther. 1995; 2: 124eC131.
Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fukayama M, Stier LE, Paakko PK, Gilardi P, Stratford-Perricaudet LD, Perricaudet M, Jallet S, Pavirani A, Le cocq J-P, Crystal RG. Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science. 1991; 252: 431eC434.
Rosenfeld MA, Yoshimura K, Trapnell BC, Yoneyama K, Rosenthal ER, Dalemans W, Fukayama M, Bargon J, Stier LE, Stratford-Perricaudet L, Perricaudet M, Guggino WB, Pavirani A, Le cocq J-P, Crystal RG. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell. 1992; 68: 143eC155.
Mittereder N, March KL, Trapnell BC. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol. 1996; 70: 7498eC7509.
Bunting S, Widmer R, Lipari T, Rangell L, Steinmetz H, Carver-Moore K, Moore MW, Keller GA, de Sauvage FJ. Normal platelets and megakaryocytes are produced in vivo in the absence of thrombopoietin. Blood. 1997; 90: 3423eC3429.
Gurney AL, Carver-Moore K, de Sauvage FJ, Moore MW. Thrombocytopenia in c-mpl-deficient mice. Science. 1994; 265: 1445eC1447.
Whitlock PR, Hackett NR, Leopold PL, Rosengart TK, Crystal RG. Adenovirus-mediated transfer of a minigene expressing multiple isoforms of VEGF is more effective at inducing angiogenesis than comparable vectors expressing individual VEGF cDNAs. Mol Ther. 2004; 9: 67eC75.
Ohwada A, Rafii S, Moore MA, Crystal RG. In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation-and chemotherapy-induced bone marrow suppression. Blood. 1996; 88: 778eC784.
Suzuki M, Singh R, Moore MA, Song WR, Crystal RG. Similarity of strain- and route-dependent murine responses to an adenovirus vector using the homologous thrombopoietin cDNA as the reporter genes. Hum Gene Ther. 1998; 9: 1223eC1231.
Herz J, Gerard RD. Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci U S A. 1993; 90: 2812eC2816.
Worgall S, Wolff G, Falck-Pedersen E, Crystal RG. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration. Hum Gene Ther. 1997; 8: 37eC44.
Brizzi MF, Battaglia E, Montrucchio G, Dentelli P, Del Sorbo L, Garbarino G, Pegoraro L, Camussi G. Thrombopoietin stimulates endothelial cell motility and neoangiogenesis by a platelet-activating factor-dependent mechanism. Circ Res. 1999; 84: 785eC796.
Mohle R, Green D, Moore MA, Nachman RL, Rafii S. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci U S A. 1997; 94: 663eC668.
Massberg S, Enders G, Matos FC, Tomic LI, Leiderer R, Eisenmenger S, Messmer K, Krombach F. Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo. Blood. 1999; 94: 3829eC3838.
Nieuwenhuis EE, Matsumoto T, Exley M, Schleipman RA, Glickman J, Bailey DT, Corazza N, Colgan SP, Onderdonk AB, Blumberg RS. CD1d-dependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung. Nat Med. 2002; 8: 588eC593.
Roberts AM, Ovechkin AV, Mowbray JG, Robinson TW, Lominadze D. Effects of pulmonary ischemia-reperfusion on platelet adhesion in subpleural arterioles in rabbits. Microvasc Res. 2004; 67: 29eC37.
Drachman JG. Role of thrombopoietin in hematopoietic stem cell and progenitor regulation. Curr Opin Hematol. 2000; 7: 183eC190.
Sawai N, Koike K, Mwamtemi HH, Ito S, Kurokawa Y, Sakashita K, Kinoshita T, Higuchi T, Takeuchi K, Shiohara M, Kamijo T, Higuchi Y, Miyazaki H, Kato T, Kobayashi M, Miyake M, Yasui K, Komiyama A. Thrombopoietin enhances neutrophil production by bone marrow hematopoietic progenitors with the aid of stem cell factor in congenital neutropenia. J Leukoc Biol. 2000; 68: 137eC143.
Fox N, Priestley G, Papayannopoulou T, Kaushansky K. Thrombopoietin expands hematopoietic stem cells after transplantation. J Clin Invest. 2002; 110: 389eC394.
Perlingeiro RC, Kyba M, Bodie S, Daley GQ. A role for thrombopoietin in hemangioblast development. Stem Cells. 2003; 21: 272eC280.
Avecilla ST, Hattori K, Heissig B, Tejada R, Liao F, Shido K, Jin DK, Dias S, Zhang F, Hartman TE, Hackett NR, Crystal RG, Witte L, Hicklin DJ, Bohlen P, Eaton D, Lyden D, de Sauvage F, Rafii S. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med. 2004; 10: 64eC71.
Kim HK, Song KS, Chung JH, Lee KR, Lee SN. Platelet microparticles induce angiogenesis in vitro. Br J Haematol. 2004; 124: 376eC384.
Salven P, Orpana A, Joensuu H. Leukocytes and platelets of patients with cancer contain high levels of vascular endothelial growth factor. Clin Cancer Res. 1999; 5: 487eC491.
Verheul HM, Hoekman K, Luykx-de Bakker S, Eekman CA, Folman CC, Broxterman HJ, Pinedo HM. Platelet: transporter of vascular endothelial growth factor. Clin Cancer Res. 1997; 3: 2187eC2190.(Hideki Amano, Neil R. Hac)
Abstract
The development of new blood vessels is a complex process, likely requiring the synergy of multiple angiogenic mediators. This study focuses on the proximal angiogenic response using the platelet as a complex carrier of critical mediators of angiogenesis. Platelet levels are controlled by circulating levels of thrombopoietin (TPO) functioning to activate megakaryocyte differentiation and platelet release through the c-mpl receptor. We hypothesized that TPO gene transfer should enhance correction of experimental ischemia by providing increased levels of platelets and hence platelet-derived mediators of angiogenesis. To evaluate this hypothesis, we dissected the role of the TPOeCc-mpleCmegakaryocyteeCplatelet pathway in the angiogenic response using a model of acute hindlimb ischemia of wild-type, TPOeC/eC, and c-mpleC/eC mice. The data demonstrate that infusion of platelets will enhance the angiogenic response in wild-type mice and that the endogenous angiogenic response is blunted in TPOeC/eC and c-mpleC/eC mice. Consistent with this observation, adenovirus (Ad)-mediated transfer of TPO (AdTPO) enhanced the correction of ischemia in wild-type and TPOeC/eC, but not c-mpleC/eC, mice. Local versus systemic administration of AdTPO showed that the effect of TPO gene transfer was systemic, not local, and it could be replaced by gene transfer of VEGF, one of the many mediators of angiogenesis carried by the platelets, even in the absence of components in the TPOeCc-mpleCmegakaryocyteeCplatelet pathway.
Key Words: angiogenesis thrombopoietin platelets megakaryocyte gene therapy
Introduction
Gene transfer of single angiogenic mediators to treat ischemia involves local delivery of the gene for an angiogenic factor to stimulate the development of new blood vessels in the local milieu.1eC5 Among the most intensively studied genes for angiogenic gene therapy are those for various forms of vascular endothelial growth factor (VEGF)-A (most commonly VEGF121 or VEGF165; usually referred to as VEGF), VEGF-B, VEGF-D, fibroblast growth factor (FGF)-2 and FGF-4.3,5eC7 Although the results of many of the studies are encouraging, there is growing evidence that gene transfereCmediated delivery of mixtures of angiogenic proteins may yield better vessel morphology and less leakage.8eC10 This synergy between various angiogenic growth factors likely reflects the complexity of physiological angiogenesis that involves the coordinated action of many mediators.
It is uncertain whether optimal therapeutic angiogenesis requires the use of multiple angiogenic mediators or whether one proximal mediator will initiate the complex cascade of events required to augment angiogenesis in response to ischemia. This study focuses on the proximal angiogenic response using the platelet as a carrier of multiple mediators of angiogenesis. Platelets are critical to hemostasis and subsequent angiogenesis in wound healing, and transfusion of platelets enhances the angiogenic recovery of blood flow in models of ischemia.11eC13 Platelets are a major source of angiogenic growth factors including VEGF, platelet-derived growth factor, and FGF-2, as well as other factors that play a role in angiogenesis, including lipids, such as sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidate.12,14eC19 Consistent with these concepts, Iba et al20 have shown that local injection of platelets, either alone or in combination with blood mononuclear cells, into an ischemic hindlimb can promote recovery of blood flow.
Based on these concepts, and the knowledge that platelet levels are controlled by circulating levels of thrombopoietin (TPO) functioning through the c-mpl receptor to activate megakaryocyte differentiation and platelet release,21,22 we hypothesized that TPO gene transfer should correct experimental ischemia by providing increased levels of platelets and hence platelet-derived mediators of angiogenesis. To evaluate this hypothesis, we have dissected the role of the TPOeCc-mpleCmegakaryocyteeCplatelet pathway in the angiogenic response using the model of acute hindlimb ischemia of wild-type, TPOeC/eC, and c-mpleC/eC mice. The data demonstrate that infusion of platelets will enhance the angiogenic response and that the endogenous angiogenic response is blunted in TPOeC/eC and c-mpleC/eC mice. Consistent with this observation, adenovirus (Ad)-mediated transfer of TPO enhances the correction of ischemia in wild-type and TPOeC/eC, but not c-mpleC/eC mice in a process mediated through platelets. The effect of TPO gene transfer is systemic, not local, and it can be replaced by gene transfer of VEGF, one of the many mediators of angiogenesis carried by the platelets, even in the absence of components in the TPOeCc-mpleCmegakaryocyteeCplatelet pathway.
Materials and Methods
Adenovirus Gene Transfer Vectors
AdTPO is a E1eCE3eC Ad vector coding the murine cDNA for TPO expressed from the cytomegalovirus promoter.23 AdVEGF121 is similar with the cDNA for the 121-aa isoform of human VEGF.24 A similar Ad with no transgene (AdNull) was used as control.25 Vectors were propagated on 293 cells and purified by 2 cesium chloride density gradients.26,27 Dosing was performed in particle units (pu).28
Model of Acute Hindlimb Ischemia
All animal experiments were executed after approval from the Institutional Animal Care and Use Committee. TPOeC/eC29 and c-mpleC/eC mice,30 all on the C57Bl/6/sv129 hybrid background, were obtained from Fred Sauvage (Genentech, San Francisco, Calif). Wild-type C57Bl/6 mice were obtained from Jackson Laboratories (Bar Harbor, Maine). The model of hindlimb ischemia was previously described.31 Eight-week-old mice were anesthetized, and a midline incision was made in the abdominal skin permitting exposure of the external iliac artery in the upper left limb. The artery was then ligated both proximally and distally and an intervening 6-mm section excised. The vectors (total dose, 108 pu) were injected (100 e蘈) into the underlying ipsilateral quadriceps muscle. In some experiments, the vectors were administered to the contralateral quadriceps muscle or intravenously.
Blood flow to the hindlimbs was assessed by scanning the lower abdomen and limbs of the mouse with a Lisca (PIM II) scanning laser Doppler (Perimed). The ratio of blood flow in the ischemic (left) to the control (right) limb was calculated by dividing the integrated blood flow in an area of the image that included the left-foot pad by the integrated blood flow for an area of the same size that included the right-foot pad. Blood flow measurements were assessed both pre- and postoperatively and on days 3, 7, 14, 21, and 28. Platelets counts were assessed by collecting blood from the tail vein, using a capillary (Unopette; Becton Dickinson) and counting using a Neubauer hemocytometer (Fisher Scientific). To determine plasma VEGF levels, the blood was collected using microhematocrit capillary tubes. The blood was centrifuged at 6000g for 5 minutes, the plasma was collected, and VEGF levels were assessed by ELISA specific for murine VEGF (R&D Systems).
Platelet Administration
Anesthetized donor wild-type C57Bl/6 mice were bled via heart puncture into heparinized microtubes. The blood was pooled and centrifuged at 220g for 5 minutes, and the platelet-rich plasma was collected. The platelets were adjusted to 5x107/mL in PBS (pH 7.4). Platelet suspension (50 e蘈) was injected into the tail vein on induction of ischemia and at 4 day intervals afterward.
Morphological Quantification of Blood Vessel Number
The ipsilateral quadricep muscle was excised and fixed with 4% paraformaldehyde for 3 hours. The muscle was then transferred into 70% ethanol overnight and embedded in paraffin, and 5-e sections were mounted and deparaffinized. Sections were stained with platelet-endothelial cell adhesion molecule (PECAM)-1 polyconal antibody (H300; Santa Cruz Biotechnology, Santa Cruz, Calif) followed by a horseradish peroxidaseeCconjugated secondary antibody (K1490; Dako, Carpinteria, Calif) and the diaminobenzidine substrate. The number of PECAM-positive cells for 20 muscle fibers chosen at random in 1 slide for each of 5 animals per treatment group was counted.
Quantification of Bone Marrow Megakaryocyte Number
Megakaryocyte number in sections of bone marrow was assessed by counting the number of megakaryocytes in 5 random fields per slide (one H&E-stained 5-e bone marrow section from each animal in each group).
Statistics
Data are expressed as mean±SD. Groups were compared by unpaired Student t test with correction for multiple comparisons if required.
Results
To confirm that platelets are critical players in angiogenesis, the impact of platelet infusion on the recovery from experimental hindlimb ischemia in C57Bl/6 mice was assessed. Excision of a 6-mm section of the external iliac artery resulted in a decrease in blood flow ratio (ischemic/control limb), as assessed by laser Doppler scanning from 0.98±0.04 to 0.10±0.01 immediately postsurgery (Figure 1A). Seven transfusions with 106 platelets on days 0, 4, 8, 12, 16, 20, and 24 postsurgery increased recovery of flow ratio by day 28 from 0.49±0.08 with PBS infusion to 0.78±0.07 with transfusion of platelets (P<0.005). The platelet-dependent increase in flow was confirmed to reflect angiogenesis by histochemical examination. Sections of the quadriceps were stained by an anti-PECAM antibody to identify endothelial cells (Figure 1B through 1D). The average number of PECAM-positive cells per muscle fiber was 1.86±0.13 for the PBS-infused mice and 2.36±0.26 for the platelet-infused mice (P<0.005).
Impact of Reduced Platelet Levels on Angiogenesis in Acute Ischemia
If platelets play a role in enhancing the angiogenic response to acute ischemia, it follows that platelet-deficient mice should have an impaired angiogenic response. Recovery from acute hindlimb ischemia, as assessed by the flow ratio between the treated ischemic limb and the untreated control right limb, was assessed at intervals in wild-type, TPOeC/eC, and TPO receptor (c-mpleC/eC) knockout mice (Figure 2). The initial excision of the iliac artery dropped the flow ratio from 0.93±0.04 before surgery to 0.06±0.03 immediately postsurgery (mean values for all 3 groups combined). Recovery in wild-type mice reached a flow ratio of 0.51±0.03 by 28 days compared with 0.31±0.04 in TPOeC/eC mice and 0.31±0.06 in c-mpleC/eC mice. At all time points studied (days 7, 14, 21, 28), the flow ratio in both groups of knockout mice was significantly lower (P<0.05) than for the wild-type mice. The mean platelet levels in naive mice of these strains are 870 000±110 000 platelets/e蘈 for wild-type mice, 290 000±34 000 for TPOeC/eC mice, and 260 000±53 000 for c-mpleC/eC mice (Figure 2B). There was no significant change in platelet count in any strain as a function of time after induction of acute ischemia in the hindlimb. Thus, deletions of the gene coding for TPO or the TPO receptor resulted in reduced platelet counts, consistent with the impact of these gene deletions on the angiogenic response to acute hindlimb ischemia.
Impact of TPO Gene Transfer on Bone Marrow, Platelet Levels, and Angiogenesis
If platelets are critical players in angiogenesis, it follows that TPO gene transfer, which results in elevated platelet levels,23,32,33 should enhance angiogenesis. To show that TPO gene transfer resulted in enhanced production of platelets from progenitors, the bone marrow of wild-type, TPOeC/eC, and c-mpleC/eC mice was examined for accumulation of megakaryocytes (Figure 3). Intramuscular administration of AdTPO resulted in the accumulation of megakaryocytes in bone marrow at 28 days postvector to a level of 3.1±0.7 megakaryocytes/field in wild-type mice and 2.4±0.8 megakaryocytes/field in TPOeC/eC mice. The dependency of megakaryocyte levels on the TPOeCc-mpl axis was shown by the lower number of megakaryocytes in bone marrow of mice injected with AdNull, the control vector with no transgene, in both wild-type mice (1.7±0.5 megakaryocytes/field; P<0.001 compared with AdTPO-treated wild-type mice) and TPOeC/eC mice (0.6±0.7 megakaryocytes/field; P<0.001 compared with AdTPO-treated TPOeC/eC mice). However, in the case of c-mpleC/eC mice, which are predicted to be nonresponsive to TPO, there was no difference in the number of bone marrow resident megakaryocytes in AdTPO-treated (0.2±0.4 megakaryocytes/field) and AdNull-treated (0.7 megakaryocytes/field±0.4 megakaryocytes/field) animals (P>0.05).
The increased levels of megakaryocytes in bone marrow in response to AdTPO were parallel to the platelet counts (Figure 4). In wild-type mice, the level rose from 0.93±0.06x106/e蘈 before induction of ischemia to a peak of 2.30±0.55x106/e蘈 at 7 days posteCvector administration and remained significantly (P<0.05) higher than background for 28 days (Figure 4A). Similarly, in the TPOeC/eC mice, platelets increased from 0.27±0.04x106/e蘈 before induction of ischemia to a peak of 1.62±0.11x106/e蘈 at 7 days posteCvector administration and remained significantly (P<0.05) higher than background for 28 days (Figure 4B). In both of these mouse strains, there was no change in platelet count as a result of AdNull treatment (P>0.05 comparing AdNull treated wild-type and TPOeC/eC mice preischemia to all other time points). As expected, the c-mpleC/eC mice were nonresponsive to TPO gene transfer, with a similar platelet count at all time points in the AdTPO- and AdNull-treated groups (Figure 4C; P>0.05).
To show that the changes in megakaryocytes in bone marrow and level of platelets correlated with the enhanced angiogenic response to acute ischemia, the blood flow ratio between the ischemic and nonischemic hindlimb was assessed in the same mice (Figure 5). Wild-type, TPOeC/eC, and c-mpleC/eC mice were administered AdTPO or AdNull, and the flow ratio was assessed as a function of time. In both wild-type and TPOeC/eC mice, there was a time-dependent increase in flow ratio in the AdTPO group, becoming significantly higher than the flow ratio in the AdNull group by 7 days (Figure 5A and 5B; P<0.01). By contrast, in the c-mpleC/eC mice, there was no difference (P>0.1) between the AdNull and AdTPO group at any time point (Figure 5C).
The correlation of the extent of the angiogenic response to acute ischemia with platelet levels was also confirmed by histological studies quantifying the number of PECAM-positive cells in the ischemic quadriceps (Figure 6). In wild-type and TPOeC/eC mice, AdTPO treatment gave an increased frequency of PECAM-positive cells, whereas in c-mpleC/eC mice, the PECAM-positive staining was similar for the AdTPO- and AdNull-treated mice. Counting the number of PECAM-positive cells and normalizing to the number of muscle fibers showed a 1.6-fold increase in the frequency of PECAM-positive cells in the AdTPO-treated wild-type mice relative to the AdNull group and a 1.7-fold increase in the TPOeC/eC mice (P<0.001 comparing AdTPO-treated mice with AdNull-treated mice for both strains). In contrast, in the c-mpleC/eC mice, there was no significant difference in the frequency of PECAM-positive cells in the ischemic hindlimb between the AdTPO- and AdNull-treated group (P>0.4).
Effect of Systemic TPO Gene Transfer on the Angiogenic Response to Acute Ischemia
The effects of TPO gene transfer may be mediated by its systemic effect (eg, by increasing platelet levels) or local effects of the TPO in the ischemic limb. To distinguish these possibilities, a comparison was made among the angiogenic responses to acute ischemia of local intramuscular injection of AdTPO into the ischemic limb, injection of AdTPO into the contralateral quadriceps, and intravenous injection (which results in expression in the liver).34,35 The data show that AdTPO results in a similar recovery from hindlimb ischemia independent of route of administration of vector (Figure 7). At 28 days, the flow ratio in the mice that received vector in the ischemic limb was 0.80±0.07 compared with 0.81±0.08 in the mice receiving injections into the contralateral limb and 0.80±0.14 in the mice receiving intravenous vector (P>0.8, all pairwise comparisons). In all cases, the recovery was higher than in the group treated with AdNull by the same route (P<0.01).
In parallel with the flow data, administration of AdTPO by various routes also resulted in elevation of platelet levels regardless of the route of administration (Figure 7). At day 14 postvector, the mice injected with the AdTPO vector into the ischemic muscle had a 2.7-fold higher platelet count than the AdNull group compared with a 2.8-fold higher level in the contralateral intramuscular group and 1.8-fold in the intravenous group (P<0.05 comparing AdNull-treated mice of each group to the AdTPO mice receiving injections by the same route). In all groups, the elevation of platelet levels persisted at >2-fold higher than the AdNull control group for at least 28 days posteCvector administration.
Mediation of the Angiogenic Effects of Platelets by VEGF
Among the potential role of platelets is to provide growth factors such as VEGF for induction of the angiogenic process.11eC19 In that situation, the recovery of blood flow to hindlimb induced by AdTPO should be suppressed by inhibitors of the VEGF pathway. Therefore, the AdTPO-dependent recovery from hindlimb ischemia was assessed in wild-type mice treated with anti-VEGFR1 antibody (35 e IP every 3 days). Injection of control antibody led to a flow ratio of 0.88±0.04 at 28 days, whereas injection of an anti-VEGFR1 antibody led to a flow ratio of 0.37±0.03 at 28 days (P<0.05 compared with control).
If VEGF is a dominant downstream factor delivered by platelets, then angiogenic gene therapy with VEGF should mimic the effects of increased platelets on the enhanced angiogenesis in response to the acute ischemia, and this effect should be unaffected by mutations in the TPOeCc-mpl pathway. To assess this hypothesis independently, the effects of intramuscular AdVEGF on recovery from acute hindlimb ischemia was assessed in wild-type, TPOeC/eC, and c-mpleC/eC mice (Figure 8). In all 3 strains, AdVEGF led to an increase in flow ratio with a significantly better flow ratio at all time points beyond 14 days (P<0.05 compared with AdNull group).
Discussion
In these studies, a combined gene transfer and murine gene knockout approach has been used to dissect the ability of platelets to enhance angiogenesis in response to acute ischemia. Commencing by confirmation of the observation that platelets are angiogenic in vivo,20 we have expanded the concept to show the converse: that mutations in the TPOeCc-mpleCmegakaryocyteeCplatelet pathway that result in low endogenous levels of platelets led to a subdued angiogenic response to acute ischemia in the hindlimb. If platelets enhance angiogenesis in vivo, then it follows that TPO gene transfer, which we have previously shown to result in elevated platelet levels,23,32,33 would also enhance angiogenesis in response to acute ischemia. The data demonstrate that local or systemic TPO gene transfer promotes angiogenic responses to acute hindlimb ischemia. The angiogenic effect of gene transfer is most likely mediated by platelets delivering angiogenic factors, especially VEGF. Studies with gene transfer of VEGF in mice with defects in the TPOeCc-mpleCmegakaryocyteeCplatelet pathway suggest that VEGF can substitute for TPO, suggesting VEGF is the major mediator carried by platelets that is responsible for the enhanced angiogenesis in response to acute ischemia that is observed with platelet administration or with gene transfereCmediated administration of TPO.
TPO-Mediated Angiogenesis
The effects of TPO gene transfer in the angiogenic response to acute ischemia could result from at least 3 pathways: direct effects of TPO on the endothelium, effects of TPO on platelets, or angiogenic effects of the platelets synthesized in response to TPO. It is clear that TPO has direct effects on endothelium. Human umbilical vascular endothelial cells express the c-mpl receptor for TPO and have an intact signaling pathway that results in phosphorylation of signal transducers and activators of transcription proteins in response to the addition of TPO.36 Moreover, TPO causes endothelial migration in vitro and angiogenesis in vivo in the context of a matrigel implant. Both of these effects appeared to be mediated by platelet-activating factor, a biologically active phospholipid. However, this mechanism is unlikely to account for the angiogenic effects of TPO gene transfer to hindlimb. First, local and systemic administration of AdTPO were equally effective in restoring hindlimb blood flow, suggesting that local effects of TPO on endothelium are not the major mediator of the enhanced angiogenesis. Second, platelet infusions, a therapy that would not directly deliver TPO to the ischemic hindlimb, was effective in promoting recovery in the hindlimb ischemia model.
TPO is known to have direct effects on megakaryocytes and to enhance VEGF synthesis and secretion.37 It is unknown if the TPO produced after gene transfer may enhance release of VEGF or other angiogenic growth factors by platelets. Because platelet infusion alone (ie, in the absence of increased TPO levels) is angiogenic, it is difficult to assign a relative proportion of TPO versus platelet effect in the angiogenic effect of TPO gene transfer.
It is more likely that the TPO gene transfer is angiogenic because of its effects on bone marrow, where it results in differentiation of megakaryocytes and subsequent increases in platelet numbers. Previous studies from our laboratory have shown that subcutaneous or intratracheal administration of AdTPO results in a transient, dose-dependent increase in platelet levels mediated by effects on bone marrow.23,32,33 That data are extended in the current study showing that intramuscular and intravenous injection are equally effective with enhanced platelet levels achieved with administration of only 108 pu of the AdTPO vector. The accumulation of megakaryocytes in bone marrow and subsequent increase in platelet levels was observed in wild-type and TPOeC/eC mice but not in c-mpleC/eC mice, in which platelet levels were, as expected, nonresponsive to TPO gene transfer. The evidence suggests that the combination of increased platelet level in combination with local ischemia provides the angiogenic milieu. Platelets alone are probably not angiogenic because the capillary density in TPOeC/eC and c-mpleC/eC mice was similar to that in wild-type mice. However, platelets are know to accumulate at sites of endothelial injury,38eC40 and this pathway is likely enhanced by increased platelet count, thereby enhancing the local delivery of angiogenic factors by the platelets.
Effects of TPO on Bone Marrow
TPO was originally discovered as a factor that caused the differentiation of megakaryocytes from stem cells in the bone marrow and subsequent platelet production. However, the phenotype of mice that lack either the TPO or c-mpl gene pointed to a second effect of TPO, the promotion of stem cell survival and self-renewal.41eC45 The data shown here, specifically the effects of platelet infusion on recovery from acute hindlimb ischemia, suggest that the major mechanism by which TPO gene transfer promotes angiogenesis is via megakaryocyte differentiation in bone marrow and an increase in systemic platelet levels. An additional role of TPO in angiogenesis may be through its effects on raising platelet levels, with secondary effects on raising VEGF (or other platelet mediator) levels and the consequent effects of these mediators on bone marroweCderived stem cells leading to enhanced production of endothelial progenitors.45
Angiogenic Factors Delivered by Platelets
The effects of TPO on the enhancement of angiogenesis in response to acute ischemia are likely mediated by the delivery of angiogenic factors to the ischemic tissue by platelets, functioning locally or systemically or both. For example, platelet-derived lipids have been reported to be angiogenic. Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidate are all biologically active lipids that act on cells via G-protein coupled receptors.12 One of these receptors designated EDG1 (endothelial differentiation gene), is highly expressed in activated endothelium and is thought to mediate the effects of sphingosine 1-phosphate on endothelial cells.12 These effects include the adherens junction assembly and induction of tube-forming morphology, effects that are thought to be mimics of angiogenesis in vivo.36,46
However, in addition to lipids with a role in angiogenesis, platelets also deliver angiogenic proteins. One of the most potent angiogenic mediators delivered to sites of ischemia by platelets is VEGF. The blockage of the angiogenesis resulting from TPO gene transfer by antibodies against the VEGF receptor suggests that VEGF is the primary mediator of the angiogenic effects of platelets. The VEGF content of platelets is estimated to be 0.5 pg/106 platelets.47 Studies in patients with malignancy show a strong correlation of plasma VEGF levels and platelet count. Furthermore, activation of platelets is known to result in the release of large quantities of VEGF.48 The current study supports the concept that VEGF is a major angiogenic mediator released by platelets, either via direct effects or secondarily via induction of other angiogenic-mediated pathways and by mobilization of stem cells from bone marrow. Whatever the specific mechanisms, the data show that the effect is downstream from the effects of TPO.
Acknowledgments
These studies were supported, in part, by grant P01 HL59312 from the National Institutes of Health and The Will Rogers Memorial Fund, Los Angeles, Calif. We thank Barbara Ferris, Rafael Tejada, and Koji Shido for scientific and technical assistance; and N. Mohamed for help in preparing this manuscript.
References
Freedman SB, Isner JM. Therapeutic angiogenesis for coronary artery disease. Ann Intern Med. 2002; 136: 54eC71.
Isner JM. Myocardial gene therapy. Nature. 2002; 415: 234eC239.
Khan TA, Sellke FW, Laham RJ. Gene therapy progress and prospects: therapeutic angiogenesis for limb and myocardial ischemia. Gene Ther. 2003; 10: 285eC291.
Morishita R. Recent progress in gene therapy for cardiovascular disease. Circ J. 2002; 66: 1077eC1086.
Syed IS, Sanborn TA, Rosengart TK. Therapeutic angiogenesis: a biologic bypass. Cardiology. 2004; 101: 131eC143.
Ferrara N, Gerber HP, Le Couter J. The biology of VEGF and its receptors. Nat Med. 2003; 9: 669eC676.
Cao R, Eriksson A, Kubo H, Alitalo K, Cao Y, Thyberg J. Comparative evaluation of FGF-2-, VEGF-A-, and VEGF-C-induced angiogenesis, lymphangiogenesis, vascular fenestrations, and permeability. Circ Res. 2004; 94: 664eC670.
Hao X, Mansson-Broberg A, Gustafsson T, Grinnemo KH, Blomberg P, Siddiqui AJ, Wardell E, Sylven C. Angiogenic effects of dual gene transfer of bFGF and PDGF-BB after myocardial infarction. Biochem Biophys Res Commun. 2004; 315: 1058eC1063.
Lubiatowski P, Gurunluoglu R, Goldman CK, Skugor B, Carnevale K, Siemionow M. Gene therapy by adenovirus-mediated vascular endothelial growth factor and angiopoietin-1 promotes perfusion of muscle flaps. Plast Reconstr Surg. 2002; 110: 149eC159.
Siddiqui AJ, Blomberg P, Wardell E, Hellgren I, Eskandarpour M, Islam KB, Sylven C. Combination of angiopoietin-1 and vascular endothelial growth factor gene therapy enhances arteriogenesis in the ischemic myocardium. Biochem Biophys Res Commun. 2003; 310: 1002eC1009.
Pinedo HM, Verheul HM, D’Amato RJ, Folkman J. Involvement of platelets in tumour angiogenesis Lancet. 1998; 352: 1775eC1777.
English D, Garcia JG, Brindley DN. Platelet-released phospholipids link haemostasis and angiogenesis. Cardiovasc Res. 2001; 49: 588eC599.
Sierko E, Wojtukiewicz MZ. Platelets and angiogenesis in malignancy. Semin Thromb Hemost. 2004; 30: 95eC108.
Sekido Y, Morishima Y, Ohya K. Activity of platelet-derived growth factor (PDGF) in platelet concentrates and cryopreserved platelets determined by PDGF bioassay. Vox Sang. 1987; 52: 27eC30.
Wartiovaara U, Salven P, Mikkola H, Lassila R, Kaukonen J, Joukov V, Orpana A, Ristimaki A, Heikinheimo M, Joensuu H, Alitalo K, Palotie A. Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation. Thromb Haemost. 1998; 80: 171eC175.
Gunsilius E, Petzer A, Stockhammer G, Nussbaumer W, Schumacher P, Clausen J, Gastl G. Thrombocytes are the major source for soluble vascular endothelial growth factor in peripheral blood. Oncology. 2000; 58: 169eC174.
Ma L, Elliott SN, Cirino G, Buret A, Ignarro LJ, Wallace JL. Platelets modulate gastric ulcer healing: role of endostatin and vascular endothelial growth factor release. Proc Natl Acad Sci U S A. 2001; 98: 6470eC6475.
Salgado R, Benoy I, Bogers J, Weytjens R, Vermeulen P, Dirix L, Van Marck E. Platelets and vascular endothelial growth factor (VEGF): a morphological and functional study. Angiogenesis. 2001; 4: 37eC43.
Lacoste E, Martineau I, Gagnon G. Platelet concentrates: effects of calcium and thrombin on endothelial cell proliferation and growth factor release. J Periodontol. 2003; 74: 1498eC1507.
Iba O, Matsubara H, Nozawa Y, Fujiyama S, Amano K, Mori Y, Kojima H, Iwasaka T. Angiogenesis by implantation of peripheral blood mononuclear cells and platelets into ischemic limbs. Circulation. 2002; 106: 2019eC2025.
de Sauvage FJ, Carver-Moore K, Luoh SM, Ryan A, Dowd M, Eaton DL, Moore MW. Physiological regulation of early and late stages of megakaryocytopoiesis by thrombopoietin. J Exp Med. 1996; 183: 651eC656.
Kaushansky K. Thrombopoietin. N Engl J Med. 1998; 339: 746eC754.
Cannizzo SJ, Frey BM, Raffi S, Moore MA, Eaton D, Suzuki M, Singh R, Mack CA, Crystal RG. Augmentation of blood platelet levels by intratracheal administration of an adenovirus vector encoding human thrombopoietin cDNA. Nat Biotechnol. 1997; 15: 570eC573.
Mack CA, Patel SR, Schwarz EA, Zanzonico P, Hahn RT, Ilercil A, Devereux RB, Goldsmith SJ, Christian TF, Sanborn TA, Kovesdi I, Hackett N, Isom OW, Crystal RG, Rosengart TK. Biologic bypass with the use of adenovirus-mediated gene transfer of the complementary deoxyribonucleic acid for vascular endothelial growth factor 121 improves myocardial perfusion and function in the ischemic porcine heart. J Thorac Cardiovasc Surg. 1998; 115: 168eC176.
Hersh J, Crystal RG, Bewig B. Modulation of gene expression after replication-deficient, recombinant adenovirus-mediated gene transfer by the product of a second adenovirus vector. Gene Ther. 1995; 2: 124eC131.
Rosenfeld MA, Siegfried W, Yoshimura K, Yoneyama K, Fukayama M, Stier LE, Paakko PK, Gilardi P, Stratford-Perricaudet LD, Perricaudet M, Jallet S, Pavirani A, Le cocq J-P, Crystal RG. Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science. 1991; 252: 431eC434.
Rosenfeld MA, Yoshimura K, Trapnell BC, Yoneyama K, Rosenthal ER, Dalemans W, Fukayama M, Bargon J, Stier LE, Stratford-Perricaudet L, Perricaudet M, Guggino WB, Pavirani A, Le cocq J-P, Crystal RG. In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium. Cell. 1992; 68: 143eC155.
Mittereder N, March KL, Trapnell BC. Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy. J Virol. 1996; 70: 7498eC7509.
Bunting S, Widmer R, Lipari T, Rangell L, Steinmetz H, Carver-Moore K, Moore MW, Keller GA, de Sauvage FJ. Normal platelets and megakaryocytes are produced in vivo in the absence of thrombopoietin. Blood. 1997; 90: 3423eC3429.
Gurney AL, Carver-Moore K, de Sauvage FJ, Moore MW. Thrombocytopenia in c-mpl-deficient mice. Science. 1994; 265: 1445eC1447.
Whitlock PR, Hackett NR, Leopold PL, Rosengart TK, Crystal RG. Adenovirus-mediated transfer of a minigene expressing multiple isoforms of VEGF is more effective at inducing angiogenesis than comparable vectors expressing individual VEGF cDNAs. Mol Ther. 2004; 9: 67eC75.
Ohwada A, Rafii S, Moore MA, Crystal RG. In vivo adenovirus vector-mediated transfer of the human thrombopoietin cDNA maintains platelet levels during radiation-and chemotherapy-induced bone marrow suppression. Blood. 1996; 88: 778eC784.
Suzuki M, Singh R, Moore MA, Song WR, Crystal RG. Similarity of strain- and route-dependent murine responses to an adenovirus vector using the homologous thrombopoietin cDNA as the reporter genes. Hum Gene Ther. 1998; 9: 1223eC1231.
Herz J, Gerard RD. Adenovirus-mediated transfer of low density lipoprotein receptor gene acutely accelerates cholesterol clearance in normal mice. Proc Natl Acad Sci U S A. 1993; 90: 2812eC2816.
Worgall S, Wolff G, Falck-Pedersen E, Crystal RG. Innate immune mechanisms dominate elimination of adenoviral vectors following in vivo administration. Hum Gene Ther. 1997; 8: 37eC44.
Brizzi MF, Battaglia E, Montrucchio G, Dentelli P, Del Sorbo L, Garbarino G, Pegoraro L, Camussi G. Thrombopoietin stimulates endothelial cell motility and neoangiogenesis by a platelet-activating factor-dependent mechanism. Circ Res. 1999; 84: 785eC796.
Mohle R, Green D, Moore MA, Nachman RL, Rafii S. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets. Proc Natl Acad Sci U S A. 1997; 94: 663eC668.
Massberg S, Enders G, Matos FC, Tomic LI, Leiderer R, Eisenmenger S, Messmer K, Krombach F. Fibrinogen deposition at the postischemic vessel wall promotes platelet adhesion during ischemia-reperfusion in vivo. Blood. 1999; 94: 3829eC3838.
Nieuwenhuis EE, Matsumoto T, Exley M, Schleipman RA, Glickman J, Bailey DT, Corazza N, Colgan SP, Onderdonk AB, Blumberg RS. CD1d-dependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung. Nat Med. 2002; 8: 588eC593.
Roberts AM, Ovechkin AV, Mowbray JG, Robinson TW, Lominadze D. Effects of pulmonary ischemia-reperfusion on platelet adhesion in subpleural arterioles in rabbits. Microvasc Res. 2004; 67: 29eC37.
Drachman JG. Role of thrombopoietin in hematopoietic stem cell and progenitor regulation. Curr Opin Hematol. 2000; 7: 183eC190.
Sawai N, Koike K, Mwamtemi HH, Ito S, Kurokawa Y, Sakashita K, Kinoshita T, Higuchi T, Takeuchi K, Shiohara M, Kamijo T, Higuchi Y, Miyazaki H, Kato T, Kobayashi M, Miyake M, Yasui K, Komiyama A. Thrombopoietin enhances neutrophil production by bone marrow hematopoietic progenitors with the aid of stem cell factor in congenital neutropenia. J Leukoc Biol. 2000; 68: 137eC143.
Fox N, Priestley G, Papayannopoulou T, Kaushansky K. Thrombopoietin expands hematopoietic stem cells after transplantation. J Clin Invest. 2002; 110: 389eC394.
Perlingeiro RC, Kyba M, Bodie S, Daley GQ. A role for thrombopoietin in hemangioblast development. Stem Cells. 2003; 21: 272eC280.
Avecilla ST, Hattori K, Heissig B, Tejada R, Liao F, Shido K, Jin DK, Dias S, Zhang F, Hartman TE, Hackett NR, Crystal RG, Witte L, Hicklin DJ, Bohlen P, Eaton D, Lyden D, de Sauvage F, Rafii S. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med. 2004; 10: 64eC71.
Kim HK, Song KS, Chung JH, Lee KR, Lee SN. Platelet microparticles induce angiogenesis in vitro. Br J Haematol. 2004; 124: 376eC384.
Salven P, Orpana A, Joensuu H. Leukocytes and platelets of patients with cancer contain high levels of vascular endothelial growth factor. Clin Cancer Res. 1999; 5: 487eC491.
Verheul HM, Hoekman K, Luykx-de Bakker S, Eekman CA, Folman CC, Broxterman HJ, Pinedo HM. Platelet: transporter of vascular endothelial growth factor. Clin Cancer Res. 1997; 3: 2187eC2190.(Hideki Amano, Neil R. Hac)