Bone Marrow AT1 Augments Neointima Formation by Promoting Mobilization of Smooth Muscle Progenitors via Platelet-Derived SDF-1α
Objectives— Bone marrow (BM)-derived endothelial progenitor cells (EPCs) and vascular smooth muscle progenitor cells (VPCs) contribute to neointima formation, whereas the angiotensin II (Ang II) type 1 receptor (AT1)-mediated action on BM-derived progenitors remains undefined.
Methods and Results— A wire-induced vascular injury was performed in the femoral artery of BM-chimeric mice whose BM was repopulated with AT1-deficient (BM-Agtr1−/−) or wild-type (BM-Agtr1+/+) cells. Neointima formation was profoundly reduced by 38% in BM-Agtr1−/− mice. Although the number of circulating EPCs (Sca-1+Flk-1+) and extent of reendothelialization did not differ between the 2 groups, the numbers of both circulating VPCs (c-Kit−Sca-1+Lin−) and tissue VPCs (Sca-1+CD31−) incorporated into neointima were markedly decreased in BM-Agtr1−/− mice. The accumulation of aggregated platelets and their content of stromal cell–derived factor-1α (SDF-1α) were significantly reduced in BM-Agtr1−/− mice, accompanied by a decrease in the serum level of SDF-1α. Thrombin-induced platelets aggregation was dose-dependently inhibited (45% at 0.1 IU/mL, P<0.05) in Agtr1−/− platelets compared with Agtr1+/+ platelets, accompanied by the reduced expression and release of SDF-1α.
Conclusions— The BM-AT1 receptor promotes neointima formation by regulating the mobilization and homing of BM-derived VPCs in a platelet-derived SDF-1α–dependent manner without affecting EPC-mediated reendothelialization.
Bone marrow (BM)-derived progenitors have been shown to contribute to vascular repair and remodeling in both human and animals.1,2 BM-derived progenitors are mobilized from BM after vascular injury, home into the sites of healing, and differentiate into endothelial-like cells or vascular smooth muscle–like cells, thereby contributing to reendothelialization or neointima formation.1,2,3,4 Stromal cell-derived factor-1α (SDF-1α) and its receptor CXCR4 were shown to play a crucial role in the mobilization and homing of BM-derived progenitors after injury.3–7 However, the underlying mechanisms that regulate the serum level of SDF-1α and CXCR4 expression on BM-derived progenitors remain poorly understood.8
Angiotensin II (Ang II)-mediated biological actions are involved in the pathogenesis of neointimal hyperplasia after vascular injury.9,10 Ang II type1 (AT1) receptor–deficient (Agtr1−/−) mice showed attenuated cuff-induced neointima formation.11 Ang II receptor blocker (ARB) also reduced neointimal hyperplasia in both animal experiments and clinical trials.12–14 Ohtani et al showed that peripheral blood mononuclear cells (MNCs) isolated from ARB-treated animals showed a decrease in transdifferentiation into smooth muscle-like progenitors.12 Yamada et al also reported that ARB treatment inhibited neointimal hyperplasia by reducing the accumulation of smooth muscle-like progenitors in neointima.13 However, the precise mechanisms for AT1-mediated actions on the mobilization/homing kinetics of BM-derived endothelial progenitor cells (EPCs) and vascular smooth muscle progenitor cells (VPCs) after injury remain poorly defined.
In this study, BM cells of wild-type (WT) were repopulated with Agtr1−/− or Agtr1+/+ cells to elucidate the underlying mechanism for BM-AT1-mediated actions on vascular repair. The results demonstrated for the first time that BM-AT1 is closely involved in neointima formation by causing the mobilization and homing of VPCs rather than EPCs, in which aggregated platelet-derived SDF-1α plays a crucial role. These findings provide a novel understanding regarding the effect of BM-AT1 on the kinetics of BM-derived vascular-lineage progenitors after vascular injury especially through platelets AT1 receptor, and suggest that the BM renin–angiotensin system could be a potential therapeutic target for the vascular remodeling.
A full description of all methods can be found in the supplemental materials (available online at http://atvb.ahajournals.org).
Agtr1−/− mice (C57BL/6 background) were obtained from Tanabe Seiyaku Co Ltd (Osaka, Japan). Vascular injury was performed by inserting a spring-wire into the femoral artery of BM-chimeric mice whose BM was repopulated with AT1-deficient (BM-Agtr1−/−) or wild-type (BM-Agtr1+/+) cells. All animal experiments were conducted in accordance with the Guidelines for Animal Experiments at Kyoto Prefectural University School of Medicine.
BM-AT1 Deficiency Attenuates Neointima Formation After Vascular Injury
The intimal area and intima/media ratio were significantly reduced in BM-Agtr1−/− mice (38% and 33%, respectively, P<0.05), and the lumen dimension was increased (47%, P<0.05) (Figure 1A and 1B). Hemodynamic data and peripheral blood counts data did not differ between the 2 groups (supplemental Tables I and II).
BM-AT1 Deficiency Does Not Affect Circulating EPCs or Reendothelialization
The number of circulating Sca-1+Flk-1+ EPCs was similarly increased ≈2-fold in both BM-Agtr1+/+ and BM-Agtr1−/− mice at day 3 after injury (Figure 1C). The extent of reendothelialization was also equivalent between the 2 groups at day 7 and day 14 after injury (Figure 1D and 1E), suggesting that attenuated neointima formation in BM-Agtr1−/− mice was not attributable to the accelerated re-endothelialization by BM-derived EPCs.
BM-AT1 Deficiency Inhibits the Mobilization of VPCs
BM-derived VPCs have been shown to contribute to neointima formation after arterial injury,3,4,12,13 in which circulating VPCs were defined as c-Kit−Sca-1+Lin− cells.4 We found that the number of circulating VPCs (c-Kit−Sca-1+Lin−) was markedly increased by 117% in BM-Agtr1+/+ mice at day 3 after injury (P<0.05), whereas it was completely diminished in BM-Agtr1−/− mice (Figure 2A and 2B). The numbers of BM-VPCs at day 3 after injury were equivalent between BM-Agtr1+/+ and BM-Agtr1−/− mice (Figure 2C and 2D), suggesting that the mobilization of VPCs from BM into the circulation was likely to be attenuated in BM-Agtr1−/− mice.
BM-AT1 Deficiency Reduces Platelet Aggregation and SDF-1α Release
SDF-1α/CXCR4 axis has been shown to play a crucial role in the mobilization of VPCs.4 The expression levels of CXCR4 on the surface of BM-VPCs did not differ between the 2 chimeric mice (supplemental Figure I). We next examined the vascular expression of SDF-1α and found that SDF-1α–positive staining was remarkably declined in BM-Agtr1−/− mice compared with BM-Agtr1+/+ mice 3 days after injury (Figure 3A and 3E). Because aggregated platelets have been shown to secrete SDF-1α on the surface of injured arteries,15 the extent of aggregated platelets and their colocalization with SDF-1α were examined. One day after injury, the inner surface of the injured artery was uniformly covered by platelets, as indicated by CD41 (platelet integrin αIIb)-positive staining, which was almost equivalent between the BM-Agtr1−/− and BM-Agtr1+/+ mice (Figure 3B and 3F), suggesting that primary platelet adhesion was not affected by platelet AT1 deficiency. In contrast, fibrinogen-positive staining, which reflects the fibrinogen trapped by aggregated platelets, was broadly detected in BM-Agtr1+/+ mice 3 days after injury, whereas it was apparently diminished in BM-Agtr1−/− mice (Figure 3C and 3G). The expression level of GPIIb mRNA was remarkably reduced by 36% in BM-Agtr1−/− mice compared with BM-Agtr1+/+ mice (Figure 3H), supporting the notion that platelets aggregation in the injured vessels is attenuated in BM-Agtr1−/− mice. Moreover, SDF-1α–positive staining was mostly colocalized with fibrinogen-positive staining (Figure 3D), suggesting that diminished platelets aggregation contributes to the decreased content of SDF-1α at sites of injured vessel in BM-Agtr1−/− mice. We also examined the relationship between eNOS and SDF-1α, because SDF-1α has been reported to have a deep relationship to NO synthase.8 Immunohistochemical analysis showed that CD31-positive endothelium was hardly observed in the inner layer of the injured vessels 3 days after injury, in which colocalization of aggregated platelets and SDF-1α was observed (supplemental Figure IIA). Consistent with this finding, the expression level of eNOS mRNA was much lower in the wire-injured vessels compared with the uninjured contralateral vessels, and the expression levels of eNOS mRNA in the wire-injured vessels did not differ between BM-Agtr1+/+ and BM-Agtr1−/− mice (supplemental Figure IIB). These findings suggest that eNOS is unlikely involved in the production of aggregated platelet-derived SDF-1α in our wire-mediated endothelium injury model, compared with the artery ligation model in which endothelium is preserved.8
BM-AT1 Deficiency Decreases the Serum Level of SDF-1α
To further elucidate the causal relationship between aggregated platelet-derived SDF-1α and the mobilization of VPCs, we examined the localization of SDF-1α in the injured vessels and the time course of serum levels of SDF-1α. The serum SDF-1α levels in both BM-Agtr1+/+ and BM-Agtr1−/− mice were significantly increased as rapid as 6 hours after injury and thereafter decreased (Figure 4A). Consistent with the findings demonstrated by Zernecke et al, our immunohistochemical analysis at 6 hours after injury showed that SDF-1α–positive staining was observed in the medial wall (data not shown), suggesting that medial smooth muscle cells were the major source of SDF-1α at the acute phase after injury. Thereafter, the serum SDF-1α level in BM-Agtr1+/+ mice gradually declined but was still higher than the baseline level 24 hours after injury and thereafter reverted to the baseline at 3 days. In contrast, the serum SDF-1α level in BM-Agtr1−/− mice declined more rapidly and normalized 24 hours after injury and thereafter further decreased. SDF-1α–positive staining at 3 days after injury was mostly colocalized with fibrinogen-positive staining at the inner surface of the injured artery but not the medial wall (Figure 3D). These findings suggest that a main source of serum SDF-1α after injury was medial smooth muscle cells in the early phase, and that in the late phase is derived from aggregated platelets. We also examined the serum SDF-1α levels in sham operated animals 3 days after injury, and found that they were equivalent to those (baseline in Figure 4A) in unoperated animals (data not shown). These findings strongly support the notion that attenuated platelet aggregation followed by the reduced release of SDF-1α is closely involved in a rapid decline in the serum SDF-1α level, resulting in impaired mobilization of VPCs in BM-Agtr1−/− mice.
Impaired Mobilization of Agtr1−/− VPCs Is Restored by SDF-1α
We examined whether administration of SDF-1α restored the impaired mobilization of VPCs in BM-Agtr1−/− mice. An injection of SDF-1α into vascular-injured BM-Agtr1−/− mice increased the number of circulating VPCs by 44% (P<0.05), whereas vascular injury alone did not increase the number of circulating VPCs (Figure 4B). We also examined the effect of anti–SDF-1α antibody (to block CXCR4 axis) on the number of circulating VPCs. Three-day pretreatment with anti–SDF-1α antibody completely abolished the difference in the number of circulating VPCs between the 2 chimeric mice (supplemental Figure III). These findings strongly support the notion that the rapid decline in the serum SDF-1α level caused by the attenuated platelet aggregation seems to be attributable to the impaired mobilization of VPCs in BM-Agtr1−/− mice.
BM-AT1 Deficiency Attenuates the Homing of VPCs
To examine the homing of VPCs, the vascular localization of VPCs was evaluated at day 7 after injury. As shown in Figure 4C and 4E, vascular Sca-1+CD31− cells (arrows), corresponding to VPCs,8 were colocalized with aggregated platelets which express SDF-1α. The number of VPCs, was markedly reduced by 42% in BM-Agtr1−/− mice compared with BM-Agtr1+/+ mice (P<0.05) (Figure 4D). In contrast, the number of Sca-1+CD31+ cells, corresponding to EPCs,7 did not differ between the 2 groups, suggesting that BM-AT1 deficiency attenuates the homing of BM-derived progenitor cells concomitant with the impaired mobilization of VPCs.
Platelet AT1 Receptor Deficiency Inhibits Platelet Aggregation
Thrombin-stimulated Agtr1+/+ platelets showed an apparent increase in fibrinogen binding in a dose-dependent manner, whereas fibrinogen binding in Agtr1−/− platelets was significantly reduced (45% at 0.1 IU/mL, P<0.05; supplemental Figure IVA and IVB), which was consistent with the previous data showing that ARB treatment significantly reduced platelet aggregation.16 Platelets produce reactive oxidative species (ROS) via NAD(P)H oxidase activation, and their function is tightly regulated by the redox state.17 We examined whether production of ROS is actually reduced in Agtr1−/− platelets. Intracellular ROS production, which was detected by flow cytometric analysis with H2DCF-DA, showed a significant decrease in Agtr1−/− platelets by 37% (P<0.01) compared with Agtr1+/+ platelets (supplemental Figure V). This finding strongly supports the notion that attenuated oxidative stress in Agtr1−/− platelets is, at least in part, responsible for the impaired platelet aggregation.
Platelet AT1 Receptor Deficiency Inhibits the Expression and Release of SDF-1α
Immunohistochemical analysis revealed that the number of CD41+SDF-1α+ platelets was markedly increased (3.2-fold) in the Agtr1+/+ platelets after thrombin stimulation, whereas it was severely inhibited (1.8-fold) in Agtr1−/− platelets (Figure 5A). Furthermore, the SDF-1α protein in the conditioned medium after thrombin stimulation was significantly reduced in Agtr1−/− platelets compared with Agtr1+/+ platelets (Figure 5B), suggesting that impaired platelets aggregation contributed to the decreased serum level of SDF-1α.
The present study provides new evidence that deficiency of the BM-AT1 receptor inhibits neointimal formation after vascular injury by affecting the mobilization and homing of BM-derived VPCs (rather than EPCs) in a SDF-1α–dependent manner. Platelet-derived SDF-1α at the sites of injury and the serum level of SDF-1α were profoundly impaired in chimeric mice with Agtr1−/− BM cells, followed by the reduction of both circulating VPCs and vascular VPCs incorporated into neointima. Inhibition of platelet aggregation by ADP receptor blocker ticlopidine markedly suppressed platelet-derived SDF-1α production, which resulted in a decrease in circulating VPCs and attenuation of neointima formation (supplemental Figure VI). These findings provide a new insight into the action of BM-AT1 on the mobilization and homing kinetics of BM-derived vascular-lineage progenitors in the vascular repair.
The SDF-1α/CXCR4 axis has been shown to be implicated in the mobilization and homing of EPCs as well as VPCs.7 Stellos et al18 reported that platelet-derived SDF-1α enhanced the accumulation of CD34+ cells at sites of injury after intravenously injection of CD34+ cells. Likewise, Xiao et al2 reported that local transplantation of embryonic stem cell–derived EPCs inhibited neointimal hyperplasia after wire-induced femoral arterial injury. However, the direct effect of platelet-derived SDF-1α on the mobilization and homing of EPCs was not investigated in these experiments. Zernecke et al4 showed that blocking of SDF-1α after injury reduced the percentage of gated events of VPCs rather than those of EPCs. Taken together, SDF-1α appears to be preferentially involved in the mobilization and homing of VPCs rather than EPCs in the pathogenesis of neointima formation after vascular injury.
The effect of AT1 blockade on the kinetics of BM-derived VPCs after vascular injury remains poorly understood. Yamada et al13 recently investigated the effect of ARB on the homing of BM-derived smooth muscle–like cells using BM chimeric mice whose BM was repopulated with apoE−/−/GFP+ cells. ARB treatment has been shown to reduce vascular oxidative stress and the redox-sensitive gene expression of chemokines and other inflammation-promoting factors,9,10 all of which are critically involved in the mobilization and homing of BM-derived VPCs. In this study, BM chimeric mice were newly generated, which enabled the investigation of AT1-mediated effects on BM-derived progenitor cells independently of vascular AT1-mediated actions. Although the number of BM-VPCs did not differ between BM-Agtr1−/− and BM-Agtr1+/+ mice, circulating VPCs after injury were markedly reduced in BM-Agtr1−/− mice. This finding suggests that the mobilization of VPCs after vascular injury was impaired by BM-AT1 deficiency independent of vascular AT1-mediated actions. Ohtani et al12 found that trasnsdifferentiation of peripheral blood MNCs to smooth muscle progenitor cells was severely suppressed in ARB-treated animals, consistent with our present observation that ARB treatment significantly reduced the number of circulating VPCs after injury (supplemental Figure VII). Considering that platelet-derived SDF-1α causes the homing of BM-derived progenitor cells into injured arteries,15 these findings suggest for the first time that AT1 blockade attenuates the mobilization and homing of BM-derived VPCs into neointima by inhibiting platelet aggregation and the production of vascular SDF-1α without any effect on EPC-mediated reendothelialization.
Harada et al19 previously reported that neointima formation after vascular injury was not suppressed in AT1-deficient mice compared with wild-type mice, and they also described the possibility that growth factors and vasoactive peptides, such as FGF-2, PDGF-B, TGF-β1, and endotheline-1, etc, might substitute fully for AT1-mediated actions in AT1-deficient mice.19 We therefore compared the expression levels of FGF-2, PDGF-B, and TGF-β1 mRNAs between wild-type and AT1-deficient mice. We found that the relative expression levels of FGF-2 and PDGF-B mRNAs in the injured vessels were actually higher in AT1-deficient mice than wild-type mice, whereas TGF-β1 mRNA levels were equivalent between the 2 groups (supplemental Figure VIII). FGF-2 mRNA expression in BM-Agtr1−/− mice was also significantly higher than BM-Agtr1+/+ mice, however the extent was much lower than AT1-deficient mice. The mRNA expression levels of PDGF-B and TGF-β1 did not show any difference between BM-Agtr1+/+ and BM-Agtr1−/− mice. These findings confirm the involvement of growth factors in neointima formation after vascular injury in AT1-deficient mice, and provide evidence that BM-AT1 rather than vascular AT1 plays a more important role in the formation of neointima.
AT2 receptor has also been reported to play a crucial role in neointima formation after vascular injury,11 therefore we newly generated bone marrow chimeric mice whose bone marrow cells were repopulated with Agtr2+/+ (BM-Agtr2+/+) or Agtr2−/− (BM-Agtr2−/−) cells. In contrast to the effect of BM-AT1, neointimal area and neointima/media ratio were significantly increased by 40% and 39% in BM-Agtr2−/− mice compared with BM-Agtr2+/+ mice (supplemental Figure IXA and IXB), which was consistent with the previous findings that neointimal hyperplasia was exaggerated in Agtr2−/− mice.11 Reendothelialization and the numbers of VPCs did not differ between BM-Agtr2+/+ and BM-Agtr2−/− mice (supplemental Figures IXC, IXD, and X). Similarly, the extent of aggregated platelets and their colocalization with SDF-1α showed no discernable difference between the two groups of mice (supplemental Figure XI). In agreement with these findings, serum concentrations of SDF-1α 3 days after injury and the number of Sca-1+CD31− cells incorporating into the injured artery in BM-Agtr2−/− mice were equivalent with those in BM-Agtr2+/+ mice (supplemental Figure XII). These findings suggest that BM-AT2 receptor counteracts the effect of BM-AT1 on the development of neointimal formation, however underlying mechanisms for BM-AT2-mediated vasoprotective actions are different from those of BM-AT1, and further studies will be required to clarify it.
We also studied the effects of BM-AT1 and BM-AT2 on apoptosis in the development of neointimal formation. The TUNEL index, which was calculated as the ratio of TUNEL-positive nuclei to total nuclei in the neointima and media, showed a slightly increase in BM-Agtr1−/− mice, whereas it was modestly, but not significantly, reduced in BM-Agtr2−/− mice compared with control mice (supplemental Figure XIII). Because all of the cells in neointima and media are not derived from bone marrow, the effects of BM-AT1 or BM-AT2 on apoptosis might be modest compared with the previous study in which total cells are AT1-deficient or AT2-deficient cells.11
This study provides novel important evidence that BM-AT1 contributes to neointima formation after vascular injury by promoting the mobilization and homing of BM-derived VPCs in a platelet-derived SDF-1α–dependent manner, and that AT1 deficiency inhibits SDF-1α release by blocking aggregation of platelets. Such relationship between BM vascular-lineage progenitors and BM renin-angiotensin system, especially in terms of platelet-SDF-1α/VPCs interaction, may provide a new insight into the role of renin–angiotensin system in the pathogenesis of vascular repair.
We thank Prof T. Todo and J. Kobayashi, and the Radiation Biology Center Kyoto University (H18-17) for assistance with bone marrow transplantation.
Sources of Funding
This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (00240036).
Received May 27, 2009; revision accepted September 29, 2009.
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