Diverse Origin and Function of Cells With Endothelial Phenotype Obtained From Adult Human Blood
Cells with endothelial phenotype generated from adult peripheral blood have emerging diagnostic and therapeutic potential. This study examined the lineage relationship between, and angiogenic function of, early endothelial progenitor cells (EPCs) and late outgrowth endothelial cells (OECs) in culture. Culture conditions were established to support the generation of both EPCs and OECs from the same starting population of peripheral blood mononuclear cells (PBMCs). Utilizing differences in expression of the surface endotoxin receptor CD14, it was determined that the vast majority of EPCs arose from a CD14+ subpopulation of PBMCs but OECs developed exclusively from the CD14− fraction. Human OECs, but not EPCs, expressed key regulatory proteins endothelial nitric oxide synthase (eNOS) and caveolin-1. Moreover, OECs exhibited a markedly greater capacity for capillary morphogenesis in in vitro and in vivo matrigel models, tube formation by OECs being in part dependent on eNOS function. Collectively, these data indicate lineage and functional heterogeneity in the population of circulating cells capable of assuming an endothelial phenotype and provide rationale for the investigation of new cell-therapeutic approaches to ischemic cardiovascular disease.
Accumulating evidence suggests circulating precursor cells may contribute to endothelial replacement in areas of postnatal neovascularization.1 The identification and characterization of such precursors are matters of ongoing investigation. In this regard, Lin et al2 generated outgrowth endothelial cells (OECs) from peripheral blood of bone marrow transplant recipients. OEC colonies, developing after 3 weeks in culture, were of donor karyotype, appeared typically endothelial, and exhibited an extraordinary growth capacity providing compelling evidence for a stem or progenitor origin.
Using similar yet distinct culture conditions, others have obtained endothelial progenitor cells (EPCs) after 4 to 7 days of peripheral blood mononuclear cell (PBMC) culture.3 Based on these techniques, an array of EPC characteristics have been evaluated and related to the role of vascular stem cells in biology.4–6 Yet central to the interpretation of these studies has been an assumption that the reported EPCs are indeed stem-like, capable of further differentiation to functional, proliferating endothelial cells such as OECs. Remarkably, little data exist to support this assumption. In fact, in one of the few long-term studies of EPC culture, Murasawa et al5 were unable to obtain differentiated OECs in the absence of genetic intervention. Moreover, others have demonstrated that EPCs express markers of monocytic differentiation,7 suggesting but not excluding a lack of progenitor capability.6,8
Regardless of phenotype, the therapeutic potential of EPC transplantation in promoting tissue vascularity is well established1,9,10 but may be limited by absolute numbers of EPCs obtainable.5 Alternatively, OECs, which have yet to be evaluated in models of angiogenesis, offer a near endless supply of potentially therapeutic cells obtainable from a relatively small blood volume. The present study was designed to critically examine the lineage relationship between, and angiogenic potency of, early peripheral blood–derived EPCs and late differentiated OECs in culture, hypothesizing origins from different subpopulations of PBMCs.
Materials and Methods
Human buffy-coat PBMCs were cultured on fibronectin in EGM-2 (Clonetics) for the first week either as previously described by Kalka et al3 or using a modification of a protocol detailed by Lin et al.2 Cells were maintained in EGM-2 for 3 further weeks. Further details and methods for cell separation, characterization, NO fluorimetry, and tube formation are available in the online data supplement at http://www.circresaha.org.
PBMC culture for 7 days using techniques described by Kalka et al3 generated EPCs as expected, continued culture-producing proliferating OEC colonies in only 2 of 20 experiments. By using a modification of other published techniques (fibronectin substrate, 2% serum EGM, daily medium change),2 phenotypically identical EPCs were generated at 7 days; this protocol additionally generated OECs in all experiments (n=20). Thus, the latter method was used to generate EPCs and OECs for subsequent studies.
Day 7–attached cells were dominantly of spindle morphology (Figure 1A), exhibited previously defined characteristics of EPCs (lectin binding, acetylated LDL incorporation),3–7 expressed endothelial antigens CD31, KDR, and Tie-2, but not VE-cadherin (Figure 1B, top), and were strongly positive for the monocytic antigen CD14 (Figure 1B, top). OECs, developing at 2 to 3 weeks, exhibited more pronounced endothelial morphology (cobblestone monolayer, contact inhibition) and proliferated >20 passages consistent with a progenitor origin2 (Figure 1A). The endothelial surface antigen profile was more distinct, but cells were negative for CD14 (Figure 1B, bottom).
To investigate whether this marked difference in CD14 expression was a consequence of EPC differentiation to OEC (ie, downregulation), or whether it implied a difference in EPC and OEC origin, CD14+ and CD14− fractions were separated from PBMCs on day 0 (>98% purity by FACS) and cultured independently (n=8). Most EPCs, defined as acetylated LDL/lectin+ cells, arose from the CD14+ starting fraction and retained CD14 expression at 7 days; those few EPCs arising from CD14− PBMCs remained CD14-negative (Figures 1C and 1D). OEC colonies subsequently developed in all CD14− cultures but never from CD14+ fractions. To confirm that CD14− cells gave rise to OECs without a CD14+ intermediary, additional studies were performed (n=3), reseparating cells on day 7. The doubly isolated CD14− populations generated OECs while remaining CD14+ populations did not. Finally, to investigate whether a CD14+ population could be induced to differentiate into OECs under influence of CD14− cells (eg, cell contact, secreted factors), PBMCs were cultured after differential labeling for CD14 before remixing on day 0. Again, although the vast majority of early EPCs developed from CD14+ cells, OEC colonies arose exclusively from the fraction that was initially CD14− (Figure 1E).
Endothelial nitric oxide synthase (eNOS) protein was not detectable in human EPCs by immunostaining or immunoblotting. OECs demonstrated clear expression, albeit at lower levels than mature human aortic endothelial cells (Figures 2A and 2B). eNOS function was indicated by generation of NO on calcium stimulation, this being abrogated by pretreatment with NG-nitro-l-arginine methyl ester (L-NAME) (Figure 2C). Caveolin-1, a coat protein of caveolae and key regulator of eNOS in mature endothelial cells, was similarly undetectable in EPCs but present in OECs (Figure 2A). Likewise, at the ultrastructural level, caveolae were only detectable in OECs (Figure 2D).
Given the phenotypic differences, it was hypothesized that EPCs and OECs may differ in their ability to form neovessels. Morphogenesis into capillary tubes was dramatically more evident in OECs compared with EPCs in vitro (online Figures 1A and 1B). Serum concentration did not affect EPC tube-forming ability. Tube-forming capacity of OECs, but not EPCs, was significantly impaired by preincubation with L-NAME, suggesting an OEC angiogenic mechanism dependent in part on eNOS function. Subcutaneously implanted OEC matrigel plugs similarly formed a significantly greater total number of capillaries and markedly more blood-containing channels than EPC plugs (online Figures 1C and 1D). Although indirect mechanisms were not excluded, these data suggest that OECs have a greater angiogenic capacity than EPCs, related to their intrinsic tube-forming ability.
The present study demonstrates that the vast majority of cells currently reported to be EPCs express CD14, originate from CD14+ PBMCs, and, critically, do not serve as precursors to OECs. OEC precursors are shown to reside in the small subset of adherent PBMCs that lack CD14 expression. Moreover, comparative functional studies reveal OECs to possess a more pronounced endothelial phenotype than EPCs and to exhibit greater intrinsic angiogenicity.
Despite numerous studies exhorting the stem or progenitor attributes of peripheral blood–derived EPCs, emerging evidence suggests a CD14+/monocytic origin.7,11,12 While certain CD14+ subsets may possess multipotency8 and EPCs may be capable of remarkable nonendothelial differentiation,6 there is little data to support their endothelial stem or progenitor status: namely the ability to give rise to proliferating, functional endothelial cells such as OECs. In this regard, Rehman et al7 recently reported only minimal EPC expression of stem cell antigens such as CD133. The present study extends these findings by demonstrating OECs to arise from a population distinct from that which gives rise to the majority of EPCs, indicating heterogeneity in the population of circulating cells capable of assuming an endothelial phenotype. This identified heterogeneity will influence the interpretation of and comparisons among studies that aim to correlate circulating endothelial cell/progenitor numbers with specific disease processes.4,13
Transplanted EPCs appear to augment tissue vascularity in part through direct channel formation3,9,10 (which is not precluded by a monocytic origin14) but probably also through indirect paracrine mechanisms.7 The present findings of a markedly greater intrinsic angiogenic capacity of OECs together with near limitless cell availability may thus serve to advance autologous cell-based therapeutic approaches to ischemic cardiovascular disease.
We recognize research support from the NIH (HL65191 to R.D.S.), the American Heart Association (0325543Z to R.G.), and the Mayo Foundation. R.D.S. is an Established Investigator of the American Heart Association. We extend appreciation to Traci Paulson for secretarial support and to Laurel Kleppe for technical expertise.
↵*Both authors contributed equally to this study.
Original received September 30, 2003; revision received October 23, 2003; accepted October 28, 2003.
Kalka C,Masuda H,Takahashi T,Kalka-Moll WM,Silver M,Kearney M,Li T,Isner JM,Asahara T.Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization.Proc Natl Acad Sci U S A. 2000;97:3422–3427.
Murasawa S,Llevadot J,Silver M,Isner JM,Losordo DW,Asahara T.Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells.Circulation. 2002;106:1133–1139.
Badorff C,Brandes RP,Popp R,Rupp S,Urbich C,Aicher A,Fleming I,Busse R,Zeiher AM,Dimmeler S.Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes.Circulation. 2003;107:1024–1032.
Rehman J,Li J,Orschell CM,March KL.Peripheral blood “endothelial progenitor cells” are derived from monocyte/macrophages and secrete angiogenic growth factors.Circulation. 2003;107:1164–1169.
Zhao Y,Glesne D,Huberman E.A human peripheral blood monocyte-derived subset acts as pluripotent stem cells.Proc Natl Acad Sci U S A. 2003;100:2426–2431.
Kawamoto A,Gwon H-C,Iwaguro H,Yamaguchi J-I,Uchida S,Masuda H,Silver M,Ma H,Kearney M,Isner JM,Asahara T.Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia.Circulation. 2001;103:634–637.
Assmus B,Schachinger V,Teupe C,Britten M,Lehmann R,Dobert N,Grunwald F,Aicher A,Urbich C,Martin H,Hoelzer D,Dimmeler S,Zeiher AM.Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE).Circulation. 2002;106:3009–3017.
Schmeisser A,Garlichs CD,Zhang H,Eskafi S,Graffy C,Ludwig J,Strasser RH,Daniel WG.Monocytes coexpress endothelial and macrophagocytic lineage markers and form cord-like structures in matrigel and angiogenic conditions.Cardiovasc Res. 2001;49:671–680.
Moldovan NI,Goldschmidt-Clermont PJ,Parker-Thornburg J,Shapiro SD,Kolattukdy PE.Contribution of monocytes/macrophages to compensatory neovascularization:the drilling of metalloelastase-positive tunnels in ischemic myocardium.Circ Res. 2000;87:378–384.