Pleiotrophin Induces Transdifferentiation of Monocytes Into Functional Endothelial Cells
Objective— Pleiotrophin (PTN) is a cytokine that is expressed by monocytes/macrophages in ischemic tissues and that promotes neovascularization, presumably by stimulating proliferation of local endothelial cells. However, the effect of PTN on monocytes/macrophages remains unknown. We investigated the role of PTN in regulating the phenotype of monocytes/macrophages.
Methods and Results— RT-PCR, real-time PCR, and fluorescence-activated cell sorter analysis revealed that the expression of PTN by monocytic cells led to a downregulation of CD68, c-fms, and CD14 monocytic cell markers and an upregulation of FLK-1, Tie-2, vascular endothelial-cadherin, platelet endothelial cell adhesion molecule-1, endothelial NO synthase, von Willebrand factor, CD34, GATA-2, and GATA-3 endothelial cell markers. Fibrin gel assays showed that the treatment of mouse and human monocytic cells with PTN led to the formation of tube-like structures. In vivo studies showed that PTN-expressing monocytic cells incorporated into the blood vessels of the quail chorioallantoic membrane. The intracardial injection of PTN-expressing monocytic cells into chicken embryos showed that cells integrated only into the developing vasculature. Finally, the injection of PTN-expressing monocytes into a murine ischemic hindlimb model significantly improved perfusion of the ischemic tissue.
Conclusions— PTN expression by monocytes/macrophages led to a downregulation of their monocytic cell markers and an upregulation of endothelial cell characteristics, thus inducing the transdifferentiation of monocytes into functional endothelial cells.
Neovascularization is a hallmark of chronic inflammatory diseases. By releasing a wide array of cytokines such as pleiotrophin (PTN), monocytes/macrophages play a key role in this neovascularization.
PTN is a developmentally regulated 136-aa (15.3 kDa) secreted growth/differentiation cytokine that is expressed during embryogenesis but rarely in adults (eg, few sites in the brain). PTN is differentiation or growth factor for various cell types (thus named PTN); it has mitogenic, antiapoptotic, transforming, angiogeneic, and chemotactic biological activities that can differ between its target cells.1 Cells transformed by PTN develop into highly vascularized, aggressive tumors when implanted into nude mice (Jackson Laboratories, Bar Harbor, Me). In ischemic tissues, PTN is expressed by macrophages within an area of exuberant neovasculature that is formed at the margins of the infarct and in endothelial cells of the newly formed vessels,2,3 suggesting a role for PTN in the neovascularization of ischemic tissue. Nothing is known about the effect of PTN on monocytes/macrophages.
Monocytes/macrophages display a high degree of plasticity, as demonstrated by their ability to transdifferentiate into endothelial cells in vitro and in vivo.4–14 Because PTN stimulates different progenitor cells to enter lineage-specific differentiation pathway, we hypothesize that the expression of PTN by activated monocytes/macrophages in ischemic tissue may affect fate of the cells in an autocrine fashion by altering their phenotype into endothelial cells. The data presented here support this concept.
Materials and Methods
Cloning of full-length PTN, preparation of bicistronic retrovirus, primer sequences, reaction conditions, RT-PCR, real-time PCR, histology, cell culture, and fibrin gel assay are described in detail in the online supplement (available at http://atvb.ahajournals.org).
Quail Chorioallantoic Membrane Assay
The quail chorioallantoic membrane (CAM) assay used fertilized Japanese quail eggs, which were cultured ex-ovo essentially as described.15 Fertilized Japanese quail eggs (Coturnix coturnix japonica) were obtained primarily from Boyd’s Bird Co. They were maintained at 37°C under ambient atmosphere, cracked in a sterile laminar flow hood at embryonic stage 3 (E3), transferred into 10-cm2 wells of polystyrene tissue culture dishes, and cultured further at 37°C. Cells expressing PTN/green fluorescence protein (GFP) were placed on the surface of each E7 CAM; the CAMs were incubated for 3 days and then fixed. A total of 60 CAM specimens were used, 12 CAMs per group. The CAM of a fixed specimen was dissected and mounted between a glass slide and a cover slip. Fluorescent and confocal images of terminal arterial vessels from the middle region of the CAM were acquired in gray scale.
Activated Monocytes/Macrophages Express PTN
PTN is expressed by activated macrophages in the ischemic rat brain. To investigate the expression of PTN by macrophages in vitro, mouse peritoneal macrophages and the mouse RAW monocytic cell line were treated with tumor necrosis factor-α, and the expression of PTN was measured by Northern blot. We found that although resting monocytes/macrophages did not express PTN, their activation with tumor necrosis factor-α markedly upregulated PTN expression (see online supplement), suggesting that activated macrophages express PTN, and, in turn, this cytokine can affect activity of these cells in an autocrine fashion.
PTN Downregulates the Expression of Monocytic Cell Markers
To investigate the autocrine impact of PTN expression on monocytes, human THP-1 and mouse RAW monocytic cell lines were transduced with a bicistronic retroviral vector expressing PTN and GFP (see online supplement). The presence of GFP allowed us to track the fate of the cells in vivo. The transduced cells were analyzed for the expression of monocytic cell markers by RT-PCR. Uninfected THP-1 cells (Figure 1A, lane 2), cells treated with phorbol 12-myristate 13-acetate (Figure 1A, lane 3), cells infected with GFP retrovirus (Figure 1A, lane 4), or PTN antisense strand (Figure 1A, lane 6) expressed monocytic cell markers c-fms and CD68. Retroviral transduction of cells with the PTN sense strand markedly downregulated expression of c-fms and CD68 (Figure 1A, lane 5). These markers were not detected in the negative control human coronary artery endothelial cells (Figure 1A, lane 7). GAPDH amplification showed that the RT-PCRs proceeded efficiently for all tested samples. Real-time PCR analysis confirmed the RT-PCR analysis and showed that the expression of CD68 was downregulated by ≈6.4 to 7.6-fold when compared with uninfected THP-1 cells, cells transduced with antisense PTN, or GFP (see online supplement). In addition, fluorescence-activated cell sorter (FACS) analysis revealed that PTN downregulated the expression of CD14 by ≈76%, similar to the level found in the negative control endothelial cells when compared with untransduced THP-1 cells or cells transduced with control GFP or PTN sense strand (Figure 1B).
PTN Coaxes Monocytic Cells to Acquire an Endothelial Cell Phenotype
We then asked whether PTN affects the endothelial commitment of monocytes. To explore this, the expression of endothelial cell markers in human and mouse monocytic cells that had been transduced with PTN sense, PTN antisense, or GFP were investigated by RT-PCR. The untransduced human monocytic THP-1 cells (Figure 2A, lane 1), mouse monocytic RAW cells (Figure 2A, lane 2), and human promonocytic U937 cells (Figure 2A, lane 3) did not express endothelial cell markers. However, cells that were transduced with the PTN sense strand (Figure 2A, lane 9) expressed vascular endothelial growth factor receptor-2 (FLK-1), Tie-2, vascular endothelial-cadherin (VE-cadherin), platelet endothelial cell adhesion molecule-1, endothelial NO synthase, von Willebrand factor (vWF), and CD34, similar to that of positive control human coronary artery endothelial cells (Figure 2A, lane 6). In contrast, these markers were not detected in THP-1 cells transduced with the PTN antisense strand (Figure 2A, lane 10) or the GFP control vector (Figure 2A, lane 11). Likewise, endothelial cell markers were not detected in nonmonocytic cells, such as NIH 3T3 cells (Figure 2A, lane 4), human coronary artery smooth muscle cells (Figure 2A, lane 5), RPMI 8226 B lymphocyte plasmacytoma cell line (Figure 2A, lane 7), and human skin fibroblasts (Figure 2A, lane 8). The weak expression of FLK-1 in smooth muscle cells (Figure 2A, lane 5) is consistent with the expression of this endothelial cell marker in human smooth muscle cells.16
Real-time PCR analysis was used to compare the expression level of the VE-cadherin, vWF, and platelet endothelial cell adhesion molecule-1 genes in the PTN-transduced THP-1 cells with those of positive control human endothelial cells. The expression levels of these genes were 0.8×105, 2.9×105, and 1.3×105 copies/100 ng of RNA, respectively (P value all <0.001). These levels of expression are similar to those of the positive control human endothelial cells (0.6×105, 3.2×105, and 1.4×105 copies/100 ng endothelial cell RNA; P value all <0.001, respectively).
This phenotypic modulation of monocytic cells by PTN was further substantiated by investigating the expression of endothelial cell-specific αvβ3 integrin by flow cytometry. FACS analysis revealed that 80±4% of THP-1 cells expressing PTN are positive for αvβ3 (Figure 2B, bottom left panel) compared with 1±3% of THP-1 cells expressing GFP (Figure 2B, top left panel). The level of αvβ3 integrin in the PTN-expressing THP-1 cells (80±4%) is similar to those of human coronary artery endothelial cells (Figure 2B, bottom right panel). The omission of the anti-αvβ3 antibody reduced positivity to 4±3% (Figure 2B, top right panel). Further, FACS analysis revealed that 72±5% of THP-1 cells transduced with the PTN sense strand expressed Tie-2 compared with 7±3% of cells transduced with GFP retroviral vector.
PTN Mediates the Phenotypic Modulation of Monocytes at the Transcriptional Level
Next, we asked whether the phenotypic modulation of monocytic cells by PTN is regulated at the transcriptional level. To accomplish this, the expression of the transcription factors GATA-2 and GATA-3 that affects endothelial cell commitment17–20 was measured. RT-PCR analysis showed that untransduced THP-1 (Figure 2C, lane 1), monocytic RAW (Figure 2C, lane 2), and U937 cells (Figure 2C, lane 3) as well as THP-1 cells transduced with either the PTN antisense strand (Figure 2C, lane 10) or the GFP control vector (Figure 2C, lane 11) did not express these transcription factors. In contrast, THP-1 cells infected with the PTN sense strand (Figure 2C, lane 9) expressed both GATA-2 and GATA-3 similar to the control human coronary artery endothelial cells (Figure 2C, lane 6). Nonmonocytic cells such as mouse NIH 3T3 cells (Figure 2C, lane 4), smooth muscle cells (Figure 2C, lane 5), RPMI 8226 B lymphocyte plasmacytoma cells (Figure 2C, lane 7), and human dermal fibroblasts (Figure 2C, lane 8) did not express GATA-2 and GATA-3. Figure 2 of the supplement further demonstrates the colocalization of GATA-2 expression and VE-cadherin in the transdifferentiated cells.
The monocytic cell lines that we used (THP-1 and RAW cells) are established cell lines with known monocytic cell characteristics that do not exhibit characteristics of multipotent cells. The expression of differentiated cell markers such as CD68, c-fms, and CD14 in THP-1 cells support this concept. However, to investigate the maturity/immaturity of THP-1 cells and RAW cells in more detail the expression of AC133 and Oct-4, 2 well-known markers of stem/progenitor cells21–23 were studied (Figure 2C). Among the examined cells, AC133 was expressed only in human U937 cells (Figure 2C, lane 3), and Oct-4 was expressed in mouse NIH 3T3 cells (Figure 2C, lane 4) and human RPMI 8226 B lymphocyte plasmacytoma cells (Figure 2C, lane 7).
PTN Induces Functional Transdifferentiation of Monocytic Cells Into Endothelial Cells
In Vitro Studies
To determine whether the transdifferentiated cells function like endothelial cells, they were cultured in a fibrin gel assay and tube formation was investigated. This showed that cultured THP-1 cells expressing PTN (Figure 3A) or RAW cells expressing PTN (Figure 3C) formed capillary-like structures. In contrast, the morphology of THP-1 cells (Figure 3B) or RAW cells (Figure 3D) that expressed GFP did not change.
To determine whether PTN affects the phenotype of primary monocytes, peritoneal macrophages were isolated from mice and cultured in a fibrin gel in the presence of conditioned media derived from either RAW cells expressing PTN/GFP or GFP. PTN induced the formation of capillary-like structures in the primary cells (Figure 3E), whereas cells exposed to the GFP-derived media did not form such structures (Figure 3F). To assess whether the activity found in the conditioned media is specifically related to PTN, the media were preabsorbed with an anti-PTN neutralizing antibody or an isotype-matched nonspecific antibody before its addition to cells. The nonspecific antibody had no effect on capillary formation (Figure 3G), whereas the anti-PTN neutralizing antibody markedly downregulated capillary formation (Figure 3H).
In Vivo Studies
The quail CAM was used to determine whether the transdifferentiated cells incorporate into blood vessels. Fertilized Japanese quail eggs were cultured ex-ovo at E3, and then RAW cells expressing PTN were transplanted onto the surface of the CAM at E7 (see online supplement). RAW cells or 293 cells expressing GFP were used as controls in addition to PBS. After 3 days, at E10 (see online supplement), CAMs were analyzed by fluorescence and confocal microscopy.
RAW cells expressing PTN/GFP integrated into large and small CAM blood vessels at various branches in 9 of 12 quail embryos (Figure 4A; see online supplement). In contrast, RAW cells expressing GFP did not get incorporated into any blood vessels of the CAM (Figure 4B). To further demonstrate the specificity of the incorporation of RAW cells, the confocal image of the newly formed fluorescent-labeled blood vessels was overlapped with a differential interference contrast image. This improved the contrast, and the incorporation of cells into the blood vessels was examined in the context of a full view of the CAM vasculature. Although the expression of GFP per se was insufficient for the incorporation of RAW cells into the CAM (Figure 4C), expression of PTN was sufficient for the integration of cells into CAM vasculature (Figure 4D). This integration generated chimeric quail–mouse blood vessels.
However, the CAM assay did not reveal whether the cells incorporated into already established or developing blood vessels. To determine this, RAW cells expressing either PTN/GFP or GFP were injected intracardially into stage 16-17 chicken embryos. At this stage, the chicken brain and ocular system are being developed, whereas their cardiovascular system is already established. The embryos were collected 2 to 3 days after injection, fixed, and sectioned (n=10 embryos/cell types). The sections were stained with either anti-GFP or anti–Tie-2 antibodies. In embryos injected with RAW cells expressing PTN/GFP, most of the positive staining appeared along the developing vessels in the head, eyes, and intersomitic regions (Figure 5, PTN panel). In contrast, embryos injected with GFP-expressing RAW cells did not stain (Figure 5, GFP panel). This demonstrates that only the RAW cells expressing PTN had the ability to incorporate into developing blood vessels.
Finally, the ability of transdifferentiated cells to improve blood flow into ischemic tissue was measured. Hindlimb ischemia was induced in BALB/C mice followed by the injection of RAW cells expressing PTN 1 day after surgery. Laser Doppler perfusion imaging monitored blood flow at days 7, 14, and 21 after surgery. BALB/C background mice were selected for these experiments because RAW cells are congenic to this mouse strain, thus avoiding a potential graft-versus-host complication. Figure 6A shows that blood flow recovery at 7 days after surgery was significantly higher in mice injected with PTN-expressing RAW cells when compared with control mice injected with GFP-expressing RAW cells. Fluorescence images of frozen sections from the mice injected with the PTN/GFP-RAW cells show that the cells were incorporated into blood vessels of ischemic hindlimb (Figure 6B), whereas such incorporation was not detected in the control mice (data not shown). Cumulative laser Doppler perfusion imaging data show that mice injected with cells expressing PTN had 60±3% increase in blood flow at 7 days, 50±4% at 14 days, and 30±3% at 21 days compared with control animals injected with PBS at similar time points (Figure 6C).
PTN is produced by activated monocytes in the highly vascularized region of ischemic brain and is thought to promote angiogenesis by stimulating sprouting/proliferation of endothelial cells. However, nothing is known about the autocrine effect of PTN in ischemic tissues (ie, its effect on monocytes/macrophages). These cells exhibit a high degree of plasticity, as demonstrated by their ability to alter their phenotype into functional endothelial cells. We found that activated monocytes/macrophages express PTN, and, in turn, this cytokine interacts with cells leading to the downregulation of monocytic cells markers and upregulation of fully differentiated endothelial cell markers. Real-time PCR analysis showed that the level of expression of these markers is similar to those found in the human coronary artery endothelial cells, suggesting that the PTN-mediated upregulation of endothelial markers is functionally relevant. This idea is further underscored by the expression of αvβ3, an integrin that is required for the interaction of endothelial cells with the matrix and the formation of tube-like structures. The fibrin gel assay confirmed this and further supported the notion that the transdifferentiated cells exhibit the characteristics of functional endothelial cells in vitro. In vivo studies bore out our in vitro findings and show that the transdifferentiated mouse cells incorporate into blood vessels. In the CAM assay, the RAW cells were implanted onto the CAM, which allowed them to distribute throughout the CAM, meaning that the cells had the opportunity to randomly incorporate into any part of the CAM structure. However, they specifically integrated into the CAM vasculature, suggesting that the cells had all the necessary information required for homing into the vascular tree. The differential interference contrast image further supports this notion and further showed that the homing and integration of the implanted cells was specific to the CAM blood vessels. Chicken embryo experiments further supported this specificity of integration and further demonstrated that although the intracardially injected cells distributed throughout the developing embryo, PTN-expressing cells incorporated only into the developing blood vessels of the brain and eye but not into the already established cardiovascular system of the embryo. This ability of transdifferentiated mouse cells to incorporate into developing blood vessels also appears to be a conserved phenomenon because similar results have been reported with human peripheral blood monocytes in which ex vivo–expanded purified CD14 cells exhibited endothelial cell characteristics in vitro and were incorporated into newly formed blood vessels in vivo.6
This PTN-induced transdifferentiation appears to be regulated at the transcriptional level because both the GATA-2 and GATA-3 transcription factors that are known to regulate the endothelial cell markers are also upregulated by PTN in monocytic cells, suggesting that this phenotypic alteration may be related to the nuclear reprogramming of the monocytic cells. This transcriptional regulation of the endothelial commitment of monocytic cells by PTN may not be related to the pluripotency characteristics of cells because both THP-1 and RAW cells did not express CD133 and Oct-4, 2 well-known markers of stem/progenitor cells. In addition, primary mouse peritoneal macrophages acquired endothelial cell characteristics when exposed to PTN. Together, these data suggest that PTN has the ability to alter the phenotype of fully differentiated monocytes/macrophages into fully differentiated endothelial cells. This phenotypic modulation is consistent with the classical definition of transdifferentiation (ie, an alteration of the phenotype of 1 fully differentiated cell type into another fully differentiated phenotype).24
The ability of monocytes/macrophages to transdifferentiate into endothelial cells does not seem to be related to their proliferative state. Resting primary human peripheral blood monocytes are known to transdifferentiate into endothelial cells.4–14,25 Similarly, we found that PTN induces transdifferentiation of mouse peritoneal cells, a nonproliferating cell type. The proliferating human THP-1 cells and mouse RAW cells also transdifferentiated into endothelial cells. Together, these data suggest that the ability of monocytes to transdifferentiate into endothelial cells is independent of their proliferative activity, suggesting that tissue macrophages found in chronically inflamed tissues may be able to transdifferentiate into endothelial cells in the presence of PTN.
In addition to primary cells, we used THP-1 and RAW clonal cells to investigate the transdifferentiation activity of PTN for 2 reasons. First, these cells are replicating, and therefore they allowed us to label them with GFP and track their fate in vivo. Second, these cells are a homogeneous population of differentiated cells, and they are not contaminated with other cell types. This allowed us to exclude the potential contribution of contaminating cells to the PTN-induced transdifferentiation of monocytes, a possibility that cannot be excluded when primary monocytes are used. In addition, the use of clonal cells excludes cell fusion between 2 cell types as a potential mechanism for PTN-mediated transdifferentiation.
Monocytes/macrophages are known to transdifferentiate into endothelial cells. However, factor(s) that regulate this event remain unknown. PTN is expressed by activated monocytes/macrophages in the highly vascularized regions of ischemic brain. We offered evidence that PTN produced by macrophages interacts with them in an autocrine fashion, coaxing the cells to transdifferentiate into functional endothelial cells. These transdifferentiated cells have the ability to increase blood flow into ischemic tissue. Thus, in addition to previously reported angiogenic activity, our data identified a novel activity for PTN that could be partly responsible for the neovascularization of inflamed tissues.
This work was supported by grants from the National Institutes of Health (ROI HL50566), Eisner Foundation, Heart Funds, and Women’s Cancer Funds of Entertainment Industry Fund. B.G.S. is an established investigator of American Heart Association.
- Received January 17, 2006.
- Accepted March 30, 2006.
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