Vascular Trauma Induces Rapid but Transient Mobilization of VEGFR2+AC133+ Endothelial Precursor Cells
Abstract—Bone marrow (BM)–derived circulating endothelial precursor cells (CEPs) are thought to play a role in postnatal angiogenesis. Emerging evidence suggests that angiogenic stress of vascular trauma may induce mobilization of CEPs to the peripheral circulation. In this regard, we studied the kinetics of CEP mobilization in two groups of patients who experienced acute vascular insult secondary to burns or coronary artery bypass grafting (CABG). In both burn and CABG patients, there was a consistent, rapid increase in the number of CEPs, determined by their surface expression pattern of vascular endothelial growth factor receptor 2 (VEGFR2), vascular endothelial cadherin (VE-cadherin), and AC133. Within the first 6 to 12 hours after injury, the percentage of CEPs in the peripheral blood of burn or CABG patients increased almost 50-fold, returning to basal levels within 48 to 72 hours. Mobilized cells also formed late-outgrowth endothelial colonies (CFU-ECs) in culture, indicating that a small, but significant, number of circulating endothelial cells were BM-derived CEPs. In parallel to the mobilization of CEPs, there was also a rapid elevation of VEGF plasma levels. Maximum VEGF levels were detected within 6 to 12 hours of vascular trauma and decreased to baseline levels after 48 to 72 hours. Acute elevation of VEGF in the mice plasma resulted in a similar kinetics of mobilization of VEGFR2+ cells. On the basis of these results, we propose that vascular trauma may induce release of chemokines, such as VEGF, that promotes rapid mobilization of CEPs to the peripheral circulation. Strategies to improve the mobilization and incorporation of CEPs may contribute to the acceleration of vascularization of the injured vascular tissue.
- circulating endothelial precursor cells\b
- vascular endothelial growth factor \b
- vascular endothelial growth factor receptor 2
Vascular trauma, induced by burn or by mechanical disruption, such as during surgical procedures, leads to a cascade of events that result in the chemoattraction of inflammatory cells and other cell types to the site of injury.1 Production and release of proangiogenic factors such as VEGF and basic fibroblast growth factor 2 (FGF-2) may potentiate the recruitment of inflammatory cells including monocytes,2 as well as other nonhematopoietic cell types such as circulating endothelial precursor cells (CEPs),3 4 to the site of injured vascular tissue, accelerating vascular healing.
Vascular healing is a complex process and may require rapid endothelialization to prevent fatal complications such as thrombosis or bleeding. Two possible sources of endothelialization are (1) migration and co-option of preexisting vascular wall endothelial cells (ECs) or (2) recruitment of CEPs from the stem-cell reservoirs such as bone marrow (BM). CEPs may reflect the phenotype of embryonic angioblasts, which are migratory ECs with the capacity to circulate, proliferate, and differentiate into mature ECs but have neither acquired characteristic markers of mature ECs nor formed lumina.
We have shown that allogeneic sex-mismatched BM transplantation resulted in the transfer of CEPs to recipient dogs.5 In humans, evidence for CEPs originates from patients implanted with a left ventricular assist device (LVAD), where the surface of the titanium housing of LVADs is colonized with BM-derived circulating CD34+ EC-like cells.6 However, because BM contains both mature ECs as well as BM-derived CEPs, these studies did not conclusively demonstrate the existence of a phenotypically and functionally distinct population of CEPs. Moreover, discrimination between CEPs and mature ECs or hematopoietic cells is complicated by the fact that subsets of hematopoietic cells express markers similar to those of ECs.
In search of a BM-derived, CEP-specific marker, we have found that AC133, a novel hematopoietic stem-cell marker, is also expressed on subsets of CD34+ cells.7 AC133+ CEPs express vascular endothelial growth factor receptor 2 (VEGFR2) and are dependent on VEGF for survival and in vitro expansion. AC133+VEGFR2+ CEPs isolated from cord blood, BM, or fetal liver proliferate in vitro in an anchorage-independent manner and can be induced to undergo migration or differentiate into mature adherent AC133− mature ECs.7
Several studies have capitalized on the in vitro growth capacity and differentiation profile of CEPs to distinguish between mature fallout vessel-derived endothelium and BM-derived CEPs. Using fluorescent in situ hybridization analysis to detect the Y chromosome in blood samples from BM transplant recipients who received gender-mismatched stem cells, Lin et al8 could distinguish between vessel wall and BM-derived endothelial cells. They show that in vitro–derived ECs form adherent colonies during early phases of culture (9 days after plating) and undergo 6-fold expansion and are derived predominantly from the recipient vessel wall, whereas ECs derived from late-outgrowth endothelial colonies (CFU-ECs, 30 days after culture) undergo 98-fold expansion and mostly generate from transplanted donor BM cells. In this regard, the potential of forming late-outgrowth colonies and expression of AC133 provide for surrogate markers to identify and quantify the numbers of human BM-derived CEPs.
Because rapid endothelialization of denuded injured vessels is essential to avoid fatal complications, we hypothesized that elevation of plasma VEGF levels immediately after vascular trauma may promote CEP mobilization into the peripheral circulation. In the present study, we demonstrate that in burn patients and those who undergo coronary artery bypass grafting (CABG), there is a rapid elevation of VEGF levels followed by immediate mobilization, within 6 to 12 hours of vascular trauma, of VEGFR2+AC133+ cells into the peripheral circulation. Given that a significant number of VEGFR2+ cells have the capacity to form late-outgrowth colonies and also express the stem-cell marker AC133, they can be considered as BM-derived CEPs. These data suggest that CEPs may act as first-aid angiogenic modules rapidly mobilized to enhance the endothelialization of denuded prothrombotic injured vasculature. Isolation and ex vivo expansion of these cells may facilitate the development of strategies to augment and accelerate vascular healing in patients with profound vascular insufficiencies.
Materials and Methods
Collection of Peripheral Blood Samples and Isolation of Mononuclear Cells
Permission to perform these studies was approved by the investigational review board at Cornell University Medical College. Samples were taken from patients who had >15% burn injury (n=8). Peripheral blood (4 to 5 mL) was taken at 12, 24, 48, and 72 hours and on the 7th and 14th days. Blood (4 to 5 mL) was also taken from patients (n=7) who underwent CABG. Samples were taken preoperatively at 6, 12, 24, 48, and 72 hours and on the 4th day.
The blood samples from burn and CABG patients were collected in heparinized tubes. Blood (5 mL) was diluted with HBSS (Life Technologies) and overlaid on top of a Ficoll preparation (Accue Prep Lymphocytes, Accurate Chemical & Scientific Corp). The buffy coat, containing the peripheral blood mononuclear cells (PBMNCs), was isolated and washed twice in HBSS at 1800g for 10 minutes. Finally, the PBMNCs were resuspended in serum-free medium (X-vivo 20, BioWhittaker). These cells were studied for the expression of endothelial-specific markers and formation of late-outgrowth endothelial colonies.
Flow Cytometry Studies
PBMNCs, cultured in serum-free medium X-vivo 20, were incubated with 1.5 μL FITC (green fluorescence) labeled high-affinity nonneutralizing monoclonal antibody (MoAb) to VEGFR2 (clone 6.64, Imclone Systems Inc) and 1.5 μL phycoerythrin (PE; red fluorescence) labeled anti-CD15 antibody (Sigma), for 20 minutes at 4°C. Nonviable cells were excluded by propidium iodide (PI; 30 μmol/L, Becton Dickinson). The number of positive cells was compared with IgG isotype control (FITC, PE; Immunotech) and quantified using a Coulter Elite flow cytometer (Coulter). Other endothelial markers studied were vascular endothelial cadherin (VE-cadherin) (BV9 clone, Imclone Systems Inc) and AC133 (Miltenyi Biotech). For analysis in burn patients, samples taken from normal age and gender-matched subjects were used as control, and preoperative samples were used as control in the CABG patients.
Reverse Transcriptase–Polymerase Chain Reaction (RT-PCR) Analysis of PBMNCs
Total RNA from PBMNCs of burn and CABG patients at different time points was obtained using Tri-Zol (Life Technologies), according to the manufacturer’s instructions. The first-strand cDNA was synthesized using the T-Primed First Strand Kit (Amersham Pharmacia Biotech Inc), amplified by Taq DNA polymerase (Advantage cDNA Polymerase Mix, Clontech) in a 25-μL reaction mixture, including 10 mmol/L dNTP (MBI Fermentas Inc) and 10 μmol/L of each primer (Genelink). PCR was performed using a PCR thermal cycler (MWG Biotech). The PCR program used to amplify VEGFR2, VE-cadherin, and β-actin had the following parameters: 5 minutes initial denaturation at 94°C, annealing at 60°C for 1 minute, and 30 seconds of elongation at 72°C. This initial cycle was followed by 35 cycles of denaturation at 94°C for 45 seconds, annealing at 60°C for 45 seconds, and 2 minutes of elongation at 72°C, followed by 7 minutes of extension at 72°C. Subsequently, PCR products were visualized in 1.5% ethidium bromide–stained agarose gels. Human umbilical vein endothelial cell (HUVEC) cDNA was used as a positive control. RNA from normal donors for the burn patients and preoperative samples from the CABG patients were used as a control for baseline mRNA expression.
β-actin: β-actin primer sequence (513 bp): sense: TCA TGT TTG AGA CCT TCA A; antisense: GTC TTT GCG GAT GTC CAC G
VEGFR2: VEGFR2 primer sequence (790 bp): sense: CTG GCA TGG TCT TCT GTG AAG CA; antisense: AAT ACC AGT GGA TGT GAT GCG G
VE-cadherin: VE-cadherin primer sequence (596 bp): sense: ACG GGA TGA CCA AGT ACA GC; antisense: ACA CAC TTT GGG CTG GTA GG
Late-Outgrowth Endothelial Colony Assay
To evaluate the potential of mobilized PBMNCs as CEPs, we used a protocol developed by Lin et al,8 with minor modifications. Freshly isolated PBMNCs, obtained 12 hours after trauma, were cultured in endothelial growth medium (M199, Gibco BRL) supplemented with 20% FBS (HyClone), endothelial cell growth factor (30 μg/mL), FGF (5 ng/mL, human recombinant basic FGF [Sigma]), heparin (5 U/mL), penicillin (100 U/mL), streptomycin (100 μg/mL), and fungizone (0.25 μg/mL). These cells were placed on 12-well plates and coated with 0.2% gelatin. The plates were incubated at 37°C in a humidified environment with 5% CO2. This process resulted in attachment of cells consisting mostly of monocytes or mature endothelial colonies on the well plates. Nonadherent cells were transferred after 4 to 5 days to other wells coated with 0.2% gelatin and grown in endothelial growth medium. Each week, for up to 2 weeks, the nonadherent cells were transferred to fresh plates to assess for the formation of late-outgrowth colonies.
Endothelial colonies were identified by metabolic uptake of DiI-acetylated-LDL (DiI-Ac-LDL) by incubating the cells with 1 μg/mL of human DiI-Ac-LDL (PerImmune Inc) at 37°C for 5 hours and visualizing by fluorescence microscopy. For von Willebrand factor (vWF) staining, cells were rinsed with HBSS and fixed immediately with 4% paraformaldehyde for 10 minutes. A primary mouse antibody against human vWF (Dako) was used at 1:250 dilution. Biotinylated rat anti-mouse IgG (Vector Laboratories Inc) was used at 1:200 dilution. Streptavidin linked to FITC (Jackson ImmunoResearch Laboratories Inc) was used at a dilution of 1:200. Cells were visualized by fluorescence microscopy. HUVECs were used as positive control for DiI-Ac-LDL uptake and vWF staining. Adherent colonies incorporating DiI-Ac-LDL and coexpressing vWF, representing early outgrowths, were quantified every week. DiI-Ac-LDL+vWF+ colonies derived from passaged nonadherent cells, which formed between 6 to 8 weeks, were considered as late-outgrowth colonies (CFU-ECs). Samples from the healthy donors were used as control in the burn patients and preoperative samples were used as control in the CABG patients.
VEGF Plasma Determination
Quantitative determination of VEGF in the plasma of patients or AdVEGF165-injected mice was determined by a specific human or murine VEGF ELISA using the Quantikine VEGF-ELISA kit (R&D Systems Inc), according to the manufacturer’s instructions. Peripheral blood plasma samples were obtained at different time points from the burn and CABG patients. Plasma samples from healthy donors were used to determine the baseline VEGF levels for the burn patients, whereas in CABG patients, this was determined by analyzing the preoperative plasma levels of the patients. Plasma of mice injected with AdNull was used as control. Both assays have a sensitivity limit of 7 pg/mL. Samples were analyzed in triplicate.
In Vivo Adenoviral Delivery of VEGF
Immunocompetent mice (on BALB/C background), aged 8 weeks (>20 g) and sex-matched, were purchased from the Jackson Laboratory (Bar Harber, Maine) and maintained in germ-free conditions. Mice (n=6) received AdVEGF165 (1.5×108 pfu) or AdNull (1.5×108 pfu) in a volume of 100 μL by single intravenous administration on day 0. AdVEGF165 is an Ad5-derived, E1a-, E3-deficient (E1a–E3–E4+) vector with an expression cassette in the E1a region containing the mouse AdVEGF165 cDNA and driven by the cytomegalovirus major immediate/early promoter/enhancer. The control vector, AdNull, is similar in design, except that it contains no transgene in the expression cassette.
Flow Cytometry Studies on Mouse PBMNCs
Cells were costained with 1.5 μL of FITC-labeled high-affinity MoAb to VEGFR2 (clone DC101, Imclone Systems Inc) and 1 μL of PE (red fluorescence) labeled anti-CD11b antibody (Pharmingen) for 20 minutes at 4°C. The number of positive cells was compared with IgG isotype control (FITC, PE; Pharmingen). PI (30 μmol/L) was used to exclude the nonviable cells. The cells were analyzed by using the Coulter Elite flow cytometer.
A standard Student’s t test was used to determine statistical differences.
VEGF Plasma Levels Increase After Vascular Trauma
VEGF plasma levels from both burn and CABG patients increased immediately after vascular trauma, which peaked between 6 to 12 hours after injury, and then decreased gradually over a 2-day period (Figures 1A⇓ and 1B⇓). VEGF levels were lower after 3 to 4 days of injury. These results suggest that vascular trauma induces a rapid but transient increase in plasma VEGF levels after trauma.
Vascular Trauma Induces Mobilization of PBMNCs Expressing mRNA for VEGFR2 and VE-Cadherin
We investigated whether the rapid increase in VEGF levels correlated with an increase in the number of circulating VEGFR2+ PBMNCs. Peripheral blood samples from burn and CABG patients demonstrated VEGFR2 and VE-cadherin expression at the mRNA level, as determined by RT-PCR (Figures 2A⇓ and 2B⇓). VEGFR2 and VE-cadherin mRNA were present in the PBMNCs between 6 and 12 hours and then gradually decreased over time. Control samples for burn and CABG patients had virtually undetectable mRNA levels for any of these markers.
Characterization of Mobilized CEPs
The RT-PCR analysis of PBMNCs from burn and CABG patients suggested that after vascular trauma, a population of cells expressing VEGFR2 and VE-cadherin may be rapidly mobilized into the peripheral circulation. These results were subsequently confirmed by analysis of surface expression using flow cytometry (FACS).
Given that monocytes express Fc receptors and may give rise to false-positive results, the PBMNCs were also costained with CD15 to exclude mobilized cells of myeloid-monocytic lineage. Confirming the RT-PCR results, FACS analysis of PBMNCs obtained from burn and CABG patients showed a rapid increase in circulating VEGFR2+ cells after 6 to 12 hours (Figures 3⇓ and 4⇓). These cells were negative for the myeloid marker CD15 (see Figures 3A⇓ and 3B⇓ for a typical FACS profile). Samples from burn patients showed an average of 20 655±4077 VEGFR2+CD15− cells (5.1±1.04% of the total PBMNCs) mobilized 12 hours after burn injury (Figure 4A⇓) and decreasing after 72 hours. On the other hand, healthy donors had 0.10%±0.01 (535±127 cells) circulating VEGFR2+CD15− cells (P<0.05). Similarly, in CABG patients, although the preoperative samples showed 915±107 (0.02±0.02% of the total PBMNCs) VEGFR2+CD15− cells, there was an average of 34 843±13 515 (8.4±3.2% of the total PBMNCs) VEGFR2+CD15− cells mobilized 6 hours after surgery (Figure 4B⇓). In these patients, at 12 and 24 hours after surgery, there was a sustained elevation of VEGFR2+CD15− cells with an average of 17 800±5823 (4.5±1.45% of the total PBMNCs) and 11 467±4097 (2.8±1% of the total PBMNCs) cells (Figure 4B⇓), respectively. The number of VEGFR2+CD15− cells gradually decreased over a 3- to 4-day period, in both groups of patients. The last peripheral blood sample from burn patients was taken 14 days after injury and had 664±257 (0.20±0.03% of the total PBMNCs) VEGFR2+CD15− cells (Figure 4A⇓). Similarly, the last sample from CABG patients was taken on the fourth day after surgery and showed 1329±408 (0.3±0.06% of the total PBMNCs) VEGFR2+CD15− cells (Figure 4B⇓). These results demonstrate the presence of a small, but distinct, population of VEGFR2+CD15− cells within the PBMNC population of vascular trauma patients, which rapidly gets mobilized within 6 to 12 hours after injury and then decreases gradually over a period of days.
Phenotypic Analysis of the VEGFR2+CD15− Cells
The peripheral blood samples from burn and CABG patients were further analyzed for the presence of other hematopoietic and endothelial markers by flow cytometry. The 12-hour time point was chosen, because it consistently showed, in both groups of patients, the highest number of mobilized CEPs. Twelve hours after injury, in both burn and CABG patients, there was a maximum increase in cells expressing VE-cadherin and AC133 (Table⇓), which accompanied the maximum increase in VEGFR2 expression. There was a mean increase of 15 987±4555 (5.1±1.04% of the total PBMNCs) and 15 544±3390 (4.5±1.45% of the total PBMNCs) VE-cadherin+ cells at 12 hours, in burn and CABG patients, respectively (Table⇓). Similarly, the increase in AC133+ cells in burn and CABG patients was 8769±2019 (2.2±0.5% of the total PBMNCs) and 8342±2109 (2.1±0.51% of the total PBMNCs), respectively (Table⇓). Furthermore, as shown for VEGFR2, expression of these markers in the PBMNC population gradually decreased over a 2- to 3-day period (data not shown). These results suggest that the cell population mobilized after vascular trauma consists of VEGFR2+ cells expressing VE-cadherin and AC133.
BM-Derived CEPs Are Present in a Significant Number of Mobilized PBMNCs
Emerging data suggest that late-outgrowth endothelial cells differentiating from the nonadherent population of plated PBMNCs represent a population of BM-derived anchorage-independent BM-derived CEPs characterized by high proliferative potential.
To assess the number of mobilized PBMNCs with the capacity of generating late-outgrowth colonies, PBMNCs mobilized from burn and CABG patients 12 hours after trauma were grown in vitro in conditions defined to support the formation of late-outgrowth colonies.8 Typical endothelial colonies, with cobblestone morphology, appeared after 7 to 10 days of culture (Figures 5A⇓ and 5B⇓) and showed both the metabolic incorporation of DiI-Ac-LDL as well as vWF staining (Figure 5C⇓). These were the early-outgrowth endothelial colonies, which are presumably derived form mature fallout circulating ECs. Few endothelial colonies formed between 10 and 14 days of culture (Figures 5A⇓ and 5B⇓) and were positive for both DiI-Ac-LDL and vWF (Figure 5C⇓). These were also scored as the early outgrowths. On day 15, the nonadherent population was passed for a third time. The colonies formed between 6 and 8 weeks (Figures 5A⇓ and 5B⇓) from this passaged nonadherent population were referred to as the late-outgrowth endothelial colonies (CFU-ECs). These late-outgrowth colonies were quantified based on their capacity to metabolically incorporate DiI-Ac-LDL and stain positively with vWF (Figure 5C⇓). PBMNCs derived from control samples were able to form the early outgrowths but not the late outgrowths. We and others7 9 have shown that purified populations of AC133 have the capacity to form late-outgrowth colonies with a plating efficiency of 5%. On the basis of these results, we estimated that close to 12% of the mobilized AC133 cells were capable of forming late CFU-ECs. Collectively, these data show that a small, but significant, number of the mobilized circulating endothelial cells from burn and CABG patients, 12 hours after trauma, were BM-derived CEPs.
Elevation of VEGF Plasma Levels in Mice Resulted in Increased Number of Rapidly Mobilized VEGFR2+CD11b− PBMNCs
Intravenous administration of Ad vectors expressing soluble VEGF165 (AdVEGF165) (1.5×108 pfu) induced a rapid (24 hour) mobilization of VEGFR2+CD11b− (14.7±0.57%) PBMNCs (Figures 6A⇓ and 6B⇓). CD11b/CD18 (Mac-1) is primarily expressed on the myeloid-monocytic cells. In AdVEGF165-inoculated mice, there was a maximum increase in VEGF plasma levels at 24 hours (Figure 7⇓), which correlated with the number of rapidly mobilized VEGFR2+CD11b− cells. VEGF levels gradually decreased over the next 2 weeks (Figure 7⇓). AdNull vector (no transgene), which was used as a control, did not induce significant mobilization of VEGFR2+CD11b− (1.9±0.28%) cells (Figures 6A⇓ and 6B⇓). Circulating VEGFR2+CD11b− cells were still abundant (2.1±0.28%) in the AdVEGF165-injected mice after 7 days compared with control (AdNull-treated) mice (0.6±0.14%) (Figure 6B⇓). Doses above 1.5×108 pfu were toxic and resulted in the demise of the treated mice (data not shown). There were no signs of toxicity in mice treated with AdNull vectors at doses of up to 109 pfu. These data suggest that elevation of VEGF plasma levels induce a rapid mobilization of VEGFR2+CD11b− cells in mice within a similar time frame to that seen for the mobilization of VEGFR2+CD15− cells in burn and CABG patients. Plasma VEGF levels remained low in AdNull-injected mice (Figure 7⇓). These results suggest that, similar to burn or CABG patients, an acute but transient increase in mouse plasma VEGF levels promotes a rapid mobilization of VEGFR2+CD11b− cells into the peripheral circulation.
Postnatal neovascularization is the process of new blood vessel formation from preexisting endothelial cells.10 However, the origin of endothelial cells incorporated into the newly formed blood vessels has been the subject of intensive scrutiny. Emerging data have suggested that a subpopulation of BM-derived CEPs may contribute to new blood vessel formation.5 11 12 13 It has been shown that CEPs have the capacity of being recruited into ischemic tissues or growing tumors.4 13 14 15 In the present study, we have extended these observations by demonstrating that vascular trauma induces a very rapid but transient mobilization of a significant number of BM-derived VEGFR2+AC133+ cells.
Given the rapid entry of the CEPs into the peripheral circulation, it has been suggested that the chemocytokines released as a result of the vascular injury may induce mobilization of CEPs. Among the known chemocytokines, VEGF has been shown to be effective in mobilizing CEPs into the peripheral circulation.3 Indeed, patients undergoing CABG or those who have suffered extensive burns, have elevated VEGF plasma levels, peaking 6 to 12 hours after vascular insult and gradually decreasing to baseline after 3 to 4 days. Elevated VEGF levels have also been measured in burn patients in a previous study.16 However, it remained unclear whether this increase in circulating VEGF levels had any physiological significance. In this report, we demonstrate that a rise in circulating VEGF levels is accompanied by an increase in PBMNCs comprised of a significant number of BM-derived CEPs. There was a 50-fold increase, compared with preoperative level, in the number of circulating cells 6 to 12 hours after surgery in CABG patients, and a similar result was seen in those patients suffering from extensive burns. Similarly, we demonstrate that acute elevation of plasma VEGF in vivo in mice leads to increased circulation of VEGFR2+ cells. Mice inoculated with adenoviral vectors encoding the VEGF165 transgene showed a rapid but transient increase in circulating VEGFR2+CD11b− PBMNCs. These data suggest that, similar to the murine VEGF-induced mobilization of CEPs, acute elevation of VEGF levels in vascular trauma patients may be the primary factor inducing mobilization of CEPs. However, because vascular trauma also promotes the release of numerous known or as-yet unrecognized chemocytokines, other factors in addition to VEGF may also contribute to the mobilization of CEPs.
Vascular trauma may also result in the nonspecific introduction of mature vascular wall–derived ECs into the circulation. We have used the expression pattern of AC133 and the potential of late-outgrowth CFU-EC colony formation to estimate the number of BM-derived CEPs. We demonstrate that, on average, close to 40% of the mobilized VEGFR2+ cells express AC133. In addition, we estimated that close to 12% of mobilized AC133+ cells form late-outgrowth CFU-ECs. Collectively, these data suggest that a small, but significant, number of the mobilized PBMNCs are BM-derived CEPs. It is intriguing that only the mobilized PBMNCs from earlier phases after the trauma were AC133+ or formed late-outgrowth colonies, suggesting that the rapid increase in circulating BM-derived CEPs is the result of a chemokinetic process, rather than differentiation or maturation of a population of stem cells. Furthermore, the rapid increase in PBMNCs with endothelial precursor potential suggests that the number of CEPs in the adult BM may be greater than expected. Taken together, these results suggest that the rapid mobilization of CEPs, in response to a rise in circulating VEGF levels after trauma, may contribute to the revascularization of the injured tissues.
Strategies to enhance mobilization and incorporation of CEPs into impaired vascular beds may have important therapeutic implications. However, it remains to be demonstrated to what extent these mobilized CEPs contribute to the compromised vascular tissue and whether acceleration of their mobilization and homing may promote the vascular healing process. Nevertheless, as shown in the present study, therapies aimed at blocking mobilization of CEPs and other BM-derived cells into the peripheral circulation and subsequently into neovascularized tissues may have undesirable clinical complications. Finally, VEGF-induced mobilization of VEGFR2+AC133+ cells may facilitate the isolation of an enriched population of CEPs from the peripheral circulation, which may ultimately be used for gene therapy or ex vivo expansion.
S. Rafii is supported by National Heart, Lung, and Blood Institute (NHLBI) Grants R01 HL-58707, R01 HL-61849, P01-HL 66592 (project 2), an American Heart Association Grant-in-Aid, the Dorothy Rodbell Foundation for Sarcoma Research, and the Rich Foundation.
Original received October 3, 2000; resubmission received November 16, 2000; revised resubmission received December 21, 2000; accepted December 21, 2000.
- © 2001 American Heart Association, Inc.
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