Bone Marrow-Derived Cells Do Not Incorporate Into the Adult Growing Vasculature

Bone Marrow–Derived Cells Do Not Incorporate Into theAdult Growing Vasculature

Tibor Ziegelhoeffer, Borja Fernandez, Sawa Kostin, Matthias Heil, Robert Voswinckel,Armin Helisch, Wolfgang Schaper

Abstract—Bone marrow–derived cells have been proposed to form new vessels or at least incorporate into growing vesselsin adult organisms under certain physiological and pathological conditions. We investigated whether bone marrow–derived cells incorporate into vessels using mouse models of hindlimb ischemia (arteriogenesis and angiogenesis) andtumor growth. C57BL/6 wild-type mice were lethally irradiated and transplanted with bone marrow cells fromlittermates expressing enhanced green fluorescent protein (GFP). At least 6 weeks after bone marrow transplantation,the animals underwent unilateral femoral artery occlusions with or without pretreatment with vascular endothelialgrowth factor or were subcutaneously implanted with methylcholanthrene-induced fibrosarcoma (BFS-1) cells. Sevenand 21 days after surgery, proximal hindlimb muscles with growing collateral arteries and ischemic gastrocnemiusmuscles as well as grown tumors and various organs were excised for histological analysis. We failed to colocalize GFPsignals with endothelial or smooth muscle cell markers. Occasionally, the use of high-power laser scanning confocalmicroscopy uncovered false-positive results because of overlap of different fluorescent signals from adjacent cells.Nevertheless, we observed accumulations of GFP-positive cells around growing collateral arteries (3-fold increaseversus nonoccluded side, P�0.001) and in ischemic distal hindlimbs. These cells were identified as fibroblasts,pericytes, and primarily leukocytes that stained positive for several growth factors and chemokines. Our findings suggestthat in the adult organism, bone marrow–derived cells do not promote vascular growth by incorporating into vessel wallsbut may function as supporting cells. (Circ Res. 2004;94:230-238.)

Key Words: arteriogenesis � angiogenesis � bone marrow–derived cells

In the embryo, the vascular system develops from meso-dermal precursor cells called angioblasts, which invade the

different embryonic organ primordia and ensemble in situ toform the primary capillary plexus. This process has beentermed vasculogenesis. It is followed by sprouting andnonsprouting angiogenesis of the primary capillary plexusand additional differentiation, maturation, and remodeling,which finally result in the mature vascular system.1,2

Recently, the concept that vasculogenesis is restricted toembryonic life has been questioned. Bone marrow–derivedstem or endothelial progenitor cells have been proposed tocirculate in adult organisms and to be recruited to and toincorporate into sites of physiological and pathological neo-vascularization.3,4 Furthermore, transplantation of these cellsseems to augment recovery of perfusion and function inmodels of myocardial and peripheral ischemia.5–9 To differ-entiate endothelial progenitor or circulating/bone marrow–derived cells from native endothelial cells in vivo, fluorescentcarbocyanine DiI–labeled cells, as well as sex-mismatchedand reporter gene–labeled cells isolated from bone marrow orfrom circulating blood, have been transplanted into recipient

animals.3,5,10,11 The reported relative contribution of trans-planted cells to the endothelium of growing vessels varieswidely, from almost no incorporation to �50%.5,12 Moreover,some recent reports have raised doubts about the extent towhich bone marrow–derived cells trans-differentiate intoorgan-specific cells in adult organisms.13,14 Taking into ac-count the methodological difficulties, the varying or evencontradictory results, and the many unanswered questionsconcerning the possible mechanisms, the role of bone mar-row–derived cells in organ or vascular repair and growth hasremained enigmatic.

Most animal studies involving resection or ligation of afemoral artery have focused on the incorporation of precursorcells into capillaries in the distal ischemic hindlimbs.3,5,11

However, there is much evidence to suggest that afterocclusion of a major artery, the growth of true bypasscollateral arteries is necessary to restore bulk blood flow.15–17

This process, called arteriogenesis, occurs by outward remod-eling of preexistent interarterial connections. Monocytes/macrophages that have been found to accumulate aroundgrowing collateral vessels have long been speculated to be

Original received June 16, 2003; revision received November 21, 2003; accepted November 21, 2003.From Max-Planck-Institut for Clinical & Physiological Research (T.Z., B.F., S.K., M.H., A.H., W.S.), Bad Nauheim, Germany, and Department of

Internal Medicine (R.V.), University Hospital Giessen, Giessen, Germany.Correspondence to Tibor Ziegelhoeffer, MD, Max-Planck-Institute for Clinical & Physiological Research, Benekestrasse 2, 61231, Bad Nauheim,

Germany. E-mail [email protected]© 2004 American Heart Association, Inc.

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involved in arteriogenesis, and more recent evidence hascorroborated their role.17–19 They have been hypothesized tobe the source of growth factors, cytokines, and variousproteases.17,20–22 More recently, infusion of other circulatingcells, including even platelets, has been found to augmentrestoration of flow after arterial occlusion.23 Thus, a varietyof circulating cells may play a role in collateral artery growth.

The present study was designed to test whether circulatingbone marrow–derived cells incorporate into collateral arteriesafter femoral artery ligation. Furthermore, the incorporationof these cells into vessels in the distal ischemic hindlimb andin growing tumors was also studied. Additionally, we infusedvascular endothelial growth factor (VEGF) to increase mobi-lization of bone marrow–derived stem cells and investigateits possible effect on the incorporation rate. To distinguishcirculation-derived cells from tissue-resident cells, we trans-planted bone marrow from transgenic donors constitutivelyexpressing enhanced green fluorescent protein (enhancedGFP) into lethally irradiated wild-type hosts. High-resolutionlaser scanning confocal microscopy was used to assessGFP-positive bone marrow–derived cell incorporation intothe vasculature. We show that GFP-positive cells of bonemarrow origin accumulate around growing collateral arteries,in ischemic tissues, and in growing tumors. However, wehave not found any incorporation into the endothelium or thetunica media of the vessels.

Materials and MethodsThe present study was performed with permission of the State ofHessen, Regierungspräsidium Darmstadt, according to section 8 ofthe German Law for the Protection of Animals. It conforms with theNIH Guide for the Care and Use of Laboratory Animals.

Bone Marrow Transplantation andTransgenic MiceWild-type recipient mice 11 to 14 weeks old (n�35) were lethallyirradiated, and 3 to 5�106 bone marrow donor cells from C57BL/6-TgN(ACTbEGFP)1Osb transgenic littermates (Jackson Laborato-ries, Bar Harbor, Maine) were transplanted via tail vein injection. Inthis transgenic line, with an enhanced GFP cDNA under the controlof a chicken �-actin promoter and cytomegalovirus enhancer (cac/enhanced GFP), all of the tissues, with the exception of erythrocytesand hair follicle cells, appear green under excitation light.24 Successof bone marrow transplantation was evaluated in each transplantedmouse by flow cytometry analysis using a panel of monoclonalantibodies against CD3, CD4, CD8, CD11b, CD19, and F4/80. Totest whether transplanted mice maintained the appropriate pool ofprogenitor/stem cells and responded adequately to physiologicalstimuli, we performed endothelial progenitor cell assays5 (EPCassay), colony-forming unit assays (CFU assay), and flow cytometryanalysis (CD34�lin� cells) with and without stimulation withrecombinant human VEGF (rhVEGF).

Animal Model

Mouse Model of Hindlimb IschemiaAll surgical procedures were performed as described previous-ly.15,17,19 Briefly, the recipient mice were anesthetized, and thefemoral artery was ligated just distal to the origin of the deep femoralartery and proximal to the popliteal artery. Additionally, one groupof animals was pretreated with 10 �g/d rhVEGF IP for 1 week beforethe surgical procedure. Relative blood flow and hemoglobin oxygensaturation measurements in the mouse feet were performed before,immediately after, and on the third and seventh postoperative dayusing a laser Doppler perfusion imager and AbTisSpec TM spec-

trometer, respectively. The right-to-left, ie, ligated-to-nonligatedside, perfusion and hemoglobin oxygen saturation ratios were cal-culated for each mouse, and the results were expressed asmean�SEM.

In Vivo Model of Tumor GrowthMethylcholanthrene-induced fibrosarcoma (BFS-1) cells25 weregrown in RPMI containing 10% FCS, 1% penicillin/streptomycin,1% pyruvate, and 2% glutamine. Confluent monolayers were washedwith PBS and trypsinized, and the cell suspension was collected bycentrifugation. The cells were resuspended in DMEM�, and1.5�106 cells/50 �L were injected subcutaneously into the back ofthe recipient mice. Three weeks after injection, the tumors wereexcised and processed for histological analysis.

Perfusion Fixation and Tissue SamplingAt days 7 and 21 after surgery, the mice were euthanized and thethoracic aorta was cannulated and perfused at 100 mm Hg with PBSbuffer containing 0.1% adenosine plus 0.05% BSA followed byfixative (3% buffered paraformaldehyde). In this model, collateralarteries follow a constant course on the surface of the adductormuscles, which allows their identification in histological prepara-tions.15,17,19 Thus, the adductor muscles containing the growingcollateral arteries as well as gastrocnemius muscles from the ligatedand nonligated sides were excised and processed for histologicalexamination.

Histological AnalysisTo assess incorporation of bone marrow–derived cells into thevasculature, we looked for GFP-positive cells in paraffin or cryo-sections of adductor and gastrocnemius muscles, tumors, spleen,intestine, heart, lung, liver, and kidney of transplanted animals. Thetissue samples were postfixed and either embedded in paraffin orcryopreserved. Five- to ten-micrometer-thick sections were immu-nostained with cell-specific (Bandeiraea simplicifolia [BS-1] lectin,�-actin, vimentin, CD45, F4/80, CD3, CD34, and CD31) or cyto-kine-specific (monocyte chemoattractant protein-1 [MCP-1],granulocyte-macrophage colony-stimulating factor [GM-CSF], pla-centa growth factor [PlGF], fibroblast growth factor-2 [FGF-2], andVEGF) antibodies, and the immunoreactions were visualized usingLeica DMLD fluorescence microscope or Leica TCS SP laserscanning confocal microscope. In some cases, after immunostainingand fluorescence analysis, the sections were stained with H&E toadditionally identify GFP-positive cells. In addition, the numbers ofGFP-positive cells in the adventitia of collateral arteries of mice withoccluded and nonoccluded femoral arteries were quantified inarterial segments (minimum of 0.3 mm) using consecutive transver-sal paraffin sections.

An expanded Materials and Methods section can be found in theonline data supplement available at http://www.circresaha.org.

StatisticsStatistical analyses were performed with Student’s t test. Differenceswere considered to be statistically significant if P�0.05.

ResultsBone Marrow TransplantationTo test the efficiency of bone marrow transplantation intolethally irradiated mice, we analyzed blood from recipients up to6 months after transplantation. As measured by their fluores-cence intensity (flow cytometry), 79% to 86% of nucleated cellsexpressed eGFP 6 weeks after bone marrow transplantation and85% to 93% after 6 months, indicating that most of the originalstem-cell population was replaced by donor cells (Figures 1Aand 1B). Furthermore, we compared the proportion of leukocytesubpopulations between transplanted and nontransplanted mice(flow cytometry). No difference between both groups was

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observed, confirming that the cell counts were within a physio-logical range at the time of surgery. To additionally evaluatewhether transplanted mice do have an appropriate pool ofstem/progenitor cells, we performed CFU assays, EPC assays,and flow cytometry analyses of peripheral blood with or withoutstimulation of bone marrow with rhVEGF. In CFU assay, thenumber of colonies obtained from bone marrow of transplantedmice did not differ from those from nontransplanted control

mice (Figures 1E and 1F). Using EPC assays, we observed acomparable number of adherent cells staining positive forendothelial cell markers (BS-1 lectin and CD31) in samplesfrom transplanted mice versus samples from control animals.The number of EPCs in peripheral blood increased after treat-ment with rhVEGF (Figure 1G). Flow cytometry analysisconfirmed the presence of circulating CD34�lin� progenitorcells in the blood of transplanted mice (Figures 1C and 1D).

Figure 1. Flow cytometry analysis of pe-ripheral blood showing the success ofeGFP-transgenic bone marrow trans-plantation. Representative histograms ofthe percentage of eGFP-positive cells inperipheral blood in wild-type mice A,Before bone marrow transplantation. B, 6weeks after transplantation. C and D,Repopulation of CD34�lin� progenitorcells in peripheral blood from irradiatedand transplanted mice as measured byflow cytometry. Only lineage-negativecells were gated and are shown in theblots: C, Isotype control; D, CD34� cellsare shown in R3. E and F, CFU assaywas performed by culturing 2�105 bonemarrow cells from irradiated/transplantedmice and control mice in semisolidmethylcellulose-based culture medium.E, Typical colony formation of cells fromtreated mice. F, Quantitative analysis ofCFU-GM showing no difference betweenirradiated/transplanted and control mice.G, Quantification of EPC assays. Thenumber of EPCs from the blood of trans-planted mice increased after the treat-ment with rhVEGF. H, Serial measure-ments of laser Doppler perfusion in thehindlimbs of transplanted and nontrans-planted/nonirradiated mice.

232 Circulation Research February 6, 2004



After femoral artery occlusion, the gross appearance andfunctionality of the limbs of transplanted and nontransplantedmice were similar. Measurements of perfusion of the distalhindlimbs by laser Doppler imaging revealed no significantdifferences between the nontransplanted wild-type and bonemarrow–transplanted groups of animals before (0.99�0.03versus 0.99�0.04), immediately after (0.23�0.07 versus0.26�0.07), and on days 3 (0.49�0.09 versus 0.53�0.12)and 7 (0.91�0.07 versus 0.93�0.02) after the femoral arteryligation, indicating that neither the irradiation nor the bonemarrow transplantation affected blood flow recovery at thetime of histological evaluation (Figure 1H). Hemoglobinoxygen saturation measurements of the mice feet (1.01�0.01versus 0.99�0.03 before, 0.35�0.11 versus 0.39�0.11 im-mediately after, 0.81�0.03 versus 0.83�0.02 on day 3, and0.86�0.07 versus 0.92�0.03 on day 7 after femoral arteryligation) confirmed these findings.

Bone Marrow–Derived Stem Cellsin ArteriogenesisImmunofluorescent examination of adductor muscles fromtransplanted mice containing growing collateral arteries fromthe limb with femoral artery occlusion (Figure 2A) andquiescent collateral arteries from the limb without occlusion(Figure 2B) revealed numerous GFP-positive cells in theinterstitium of adductor muscles. GFP-positive cells werefrequently present in the adventitia of collateral arteries andclose perivascular space in both occluded and nonoccludedlimbs. However, 21 days after surgery, the amount of GFP-positive cells in the perivascular space of growing collateralarteries on the occluded side was significantly higher comparedwith quiescent collateral arteries from the hindlimb withoutfemoral artery ligation (Figures 2C through 2E). In addition,numerous GFP-positive cells were found around the femoralartery and vein, close to the area of occlusion (Figure 2F).

The perivascular GFP-positive cells formed clusters thatwere in close contact with the vascular walls. However, wewere not able to find any GFP-positive cells in the tunicamedia (colocalization with actin antibodies) nor in the endo-thelial layer (colocalization with BS-1 lectin or CD34 anti-bodies) of vessels. Although the number of EPCs in periph-eral blood increased after VEGF treatment, VEGF-pretreatedanimals showed similar results in terms of GFP-positive cellincorporation into the vascular wall. In some cases, as shownin Figures 3A and 3B, false-positive results were detectedwhen 7-�m-thick sections were analyzed (Figure 3A), prob-ably because of overlapping of signals from adjacent cells.Closer examination of 1-�m-thick confocal slices revealedlack of colocalization in all of the areas analyzed (Figure 3B).

Bone Marrow–Derived Stem Cells in AngiogenesisOn the nonligated side, a few GFP-positive cells were foundin the gastrocnemius and the adductor muscles with a similarfrequency (data not shown). On the ligated side, denseinfiltrates of GFP-positive cells were found. These musclesshowed signs of ischemic alterations, including leukocyteinfiltration and fibrosis. Many GFP-positive cells were foundscattered in the tissue, often around arteries and capillaries(Figure 3C). However, no GFP-positive smooth muscle or

endothelial cells could be detected after a serial examinationof the tissue stained with actin and CD31 antibodies or BS-1lectin by confocal microscopy.

To additionally evaluate the contribution of bone marrow–derived cells to angiogenesis, we used a model of tumorgrowth. The vasculature of the tumors was composed ofvessels with different sizes deprived of a smooth musclelayer. GFP-positive endothelial cells were never found inthese vessels (Figure 3D). Around the tumors, many feedingvessels showed GFP-positive cells in the adventitia, butagain, they were never found incorporated into the media orendothelium (not shown).

Cell Type of Bone Marrow–Derived CellsMost of the GFP-positive cells detected in the differentorgans analyzed were found scattered in the interstitium andaround vessels. The shape, localization, distribution, andmorphology of these cells suggested that they are leukocytes,fibroblasts, and pericytes. Immunofluorescent staining withvimentin antibodies revealed that some interstitial spindle-shape GFP-positive cells are fibroblasts (Figures 4A and 4B).However, most GFP-positive cells accumulating in ischemicgastrocnemius muscles (Figures 4C and 4D) and in theadventitia of growing collateral arteries (Figures 4E and 4F)were identified as leukocytes by their positive staining withthe pan-leukocyte marker CD45. Additional characterizationof CD45-positive cells revealed that most leukocytes areF4/80-positive monocytes/macrophages (Figures 5A and 5B),whereas some are CD3-positive T-lymphocytes (Figures 5Cand 5D). Immunolabeling for CD34 revealed specific signalsconfined occasionally to some endothelial cells and perivas-cular cells (Figures 6A and 6B). CD31 signals were detectedin all vascular endothelial cells and rarely in some GFP-positive perivascular cells (Figures 6C and 6D).

Given the prevalent association of bone marrow–derivedcells with growing collateral arteries and the previouslyreported stimulatory effect of chemokines like MCP-126 andGM-CSF27 on arteriogenesis, we explored the expression ofseveral cytokines and growth factors in growing collateralarteries and perivascular leukocytes by immunoconfocal mi-croscopy. Specific FGF-2 immunofluorescent signals weremainly detected in GFP-positive perivascular cells accumu-lating around growing collateral arteries (Figures 7A and 7B).Likewise, VEGF immunofluorescent signals were also de-tected in perivascular GFP-positive leukocytes (Figures 7Cand 7D). However, the number of VEGF-positive cells wasclearly lower than FGF-2. Despite the previously reportedinfluence of PlGF on arteriogenesis,28 we did not detect thisgrowth factor by immunofluorescent methods (data notshown). Likewise, GM-CSF was not found in growingcollateral arteries or surrounding tissue (data not shown).However, we found strong and specific localization ofMCP-1 in endothelium and adventitia of growing collateralarteries (Figures 7E and 7F). In addition, most GFP-positiveperivascular leukocytes showed strong MCP-1immunoreactivity.

DiscussionSeveral studies have suggested that a variety of circulatingcells, including monocytes and monocytoid potential stem or




precursor cells as well as leukocytes and platelets, promotethe recovery of perfusion in ischemic animal mod-els.3,5,7,8,11,23,29,30 The main objective of this study was toinvestigate whether these positive effects are related to theincorporation of circulating cells into growing vessels, par-ticularly into collateral arteries, which are essential forrestoring bulk blood flow after arterial occlusion. To trackand distinguish the cells of bone marrow origin from thetissue-resident ones, we transplanted lethally irradiated

C57BL/6J mice with bone marrow from their eGFP-transgenic littermates. Using flow cytometry and histologicalanalysis, we confirmed that all of the cells of transgenicanimals, except those of hair cells and erythrocytes, consti-tutively express GFP (cac/eGFP), as previously reported.24

Six weeks and 6 months after transplantation, most (79% to86% and 93%, respectively) nucleated cells in peripheralblood expressed GFP, thus confirming the successful bonemarrow engraftment and hematopoietic reconstitution. More-

Figure 2. A and B, Microphotographs of adductor muscles containing collateral arteries filled with bismuth/gelatin contrast agent. A,Typical corkscrew pattern of growing collateral arteries (arrowheads) connecting the deep femoral artery (red arrow) with the femoralartery (white arrow) 21 days after femoral artery ligation (circle indicates ligation site). B, Preexisting collateral vessels (arrowheads) onthe contralateral nonligated side. C through E, Accumulation of bone marrow–derived cells around growing collateral arteries. Red indi-cates BS-1 lectin; blue, nuclei. C, Numerous GFP-positive cells are clustered around growing collateral artery (CA) 3 weeks after femo-ral artery occlusion. D, Only a few GFP-positive cells are found around quiescent collateral arteries. E, Graph showing quantification ofthe number of GFP-positive cells around collateral arteries. A 3-fold, significant (P�0.001) increase was found in growing (3 weeks aftersurgery) compared with quiescent collateral arteries. Sixteen collateral arteries were subjected to quantification, belonging to a total of8 animals. F, GFP-positive cells were frequently found in the perivascular space between the femoral artery (FA) and femoral vein (FV).Red indicates �-actin; blue, nuclei.




over, data obtained by CFU assays, EPC assays, and flowcytometry analysis indicate that progenitor and stem cellswere present in the blood and the bone marrow of trans-planted animals with proportions similar to nontransplantedmice and respond adequately to VEGF stimuli.

We performed a rigorous inspection of adductor muscleswith growing collateral arteries, gastrocnemius muscles withischemic and normoxic tissue, end-stage tumors, spleen,liver, heart, lung, kidney, and intestine using conventionalfluorescence and high-power laser scanning microscopy toverify whether bone marrow–derived cells transform intovascular cells under different physiological conditions. Endo-thelial and smooth muscle cell markers were used to confirmincorporation of these cells into growing or quiescent vascu-lature. Overall, at least 3000 sections were analyzed. Wenever found colocalization of GFP-signals with endothelial orsmooth muscle cell markers. GFP-positive cells were fre-quently found in perivascular space and interstitium. Thesecells were identified as leukocytes, fibroblasts, and pericytesby shape and distribution in the tissue as well as by colocal-ization with specific markers (CD45 and vimentin). Addition-ally, we characterized the subsets of GFP- and CD45-positiveleukocytes and found these cells to be mainly monocytes/macrophages and T lymphocytes, as identified by positivestaining for F4/80 and CD3.

In the present study, we identified occasional GFP-positivecells colocalizing with CD31 and CD34 markers. It has beendescribed that CD34 is expressed by hematopoietic progeni-tor cells, particularly by myelomonocytic colony-forming

cells, endothelial progenitor cells, some mature endothelialcells, and monocytes. CD31 has been reported to be ex-pressed by mature endothelial cells, myeloid precursor cells,CFU-macrophage precursor cells, and precursor cells forgranulocytes. However, we failed to find GFP�CD34� andGFP�CD31� cells incorporated into the vascular wall. Thus,our data provide no evidence for the natural recruitment ofbone marrow–derived CD31/CD34-positive cells in the pro-cess of collateral artery growth. In some small arteries,conventional fluorescence microscopy and confocal micros-copy using thick sections showed colocalization of theendothelial marker BS-1 lectin or the smooth muscle cellmarker �-actin with GFP signals. However, using high-resolution confocal microscopy, we show that overlapping ofsignals is attributable to the convolution of perivascular cellsaround vessels rather than colocalization of these signals inthe same cell. Thus, in our experimental models of hindlimbischemia and tumor growth, we have not found any evidenceof postnatal vasculogenesis or an integration of bone mar-row–derived or circulating cells into the endothelium ortunica media of vessels.

Figure 3. A and B, GFP-positive cells around a growing collat-eral artery of a VEGF-treated animal 7 days after surgery. Astack (7 �m thick) of confocal slices (A) shows some yellow-stained cells (arrows), indicating colocalization of GFP with theendothelial marker BS-1. A single 1-�m-thick section (B)revealed absence of colocalization of fluorescent signals(arrows). C, In the ischemic gastrocnemius muscle, numerousGFP-positive cells distribute throughout the ischemic area.However, they do not colocalize with the endothelial markerBS-1 (red). Yellow signals come from autofluorescence of GFP-positive leukocytes (arrows). D, In tumors, GFP-positive cellssurround vessels of different sizes but do not incorporate intotheir walls. Nuclei are stained in red. L indicates lumen.

Figure 4. A and B, Colocalization of GFP with vimentin (red) inthe gastrocnemius muscle. Typical elongated fibroblasts in thefascia of the gastrocnemius muscle show red vimentin filamentstogether with GFP signals. A higher magnification of the celllabeled with a white arrow is shown in B. Blue indicates nuclei.C through F, Colocalization of GFP with CD45 (red) in the ische-mic gastrocnemius muscle (C and D) and around growing collat-eral arteries (E and F) 3 weeks after femoral artery occlusion.Blue indicates nuclei. Most GFP-positive cells in the ischemicarea of the gastrocnemius muscle (C and D) were identified asleukocytes (big arrows). However, some GFP-positive butCD45-negative cells were also found (small arrows). Inversely,some CD45-positive cells were GFP-negative (arrowheads).Clusters of GFP-positive cells around collateral arteries (CA) (Eand F) were mostly composed of CD45-positive cells, indicatingthat most of the bone marrow cells that accumulate specificallyaround growing collateral arteries are leukocytes. Note thatsome (�10%) of the CD45-positive cells are GFP-negative(arrows), which corresponds well with the data obtained by flowcytometry (see Figure 1).




To test whether the irradiation or bone marrow transplan-tation itself had influenced the incorporation of bone mar-row–derived cells into the vessel wall and thereby deceleratearteriogenesis, we performed relative blood flow and hemo-globin oxygen saturation measurements in the mouse feetbefore, immediately after, on days 3 and 7 after the femoralartery ligation. We did not observe any differences betweentransplanted and nontransplanted animals at any time point.Thus, we provide functional as well as morphological datathat the incorporation of bone marrow–derived cells into thevessel wall of growing collateral arteries and capillary vesselsis not a natural event in the regeneration of the adult vascularsystem required for successful blood flow recovery afterfemoral artery ligation. Moreover, intraperitoneal administra-

tion of rhVEGF, which has been reported to promote mobi-lization of bone marrow–derived stem cells,29,31 also did notlead to any demonstrable incorporation of GFP cells in thevasculature of these mice.

Despite the fact that we did not observe any incorporationof GFP cells into the vessel wall of transplanted mice, we didobserve an accumulation of GFP-positive cells in the adven-titia of growing collateral arteries in the proximal thigh andalso in the ischemic distal hindlimb. Immunostainings with apanel of cell-specific antibodies indicate that these cells aremainly leukocytes, particularly belonging to monocyte/mac-rophage lineage (F4/80-positive cells) and, as we describehere for the first time, to the T-lymphocyte subpopulation(CD3-positive cells). Consequently, the question appears ofwhether these cells may act in a paracrine way, providingsome growth factors or chemokines. Indeed, leukocytes havebeen previously suggested to play a critical role in arterio-genesis,17–21,26 probably by releasing activating cytokines,growth factors, and metalloproteinases and thereby creatingan inflammatory environment necessary for the enhancementof collateral artery growth.15,16,22,28,32 Our present data sup-port this hypothesis. We observed that the bone marrow–derived GFP-positive cells clustered around growing collat-eral vessels secrete growth factors like FGF-2 and VEGF andchemokines like MCP-1, which had been previously shown topromote arteriogenesis.20,26

Our results are in agreement with some recent studies usinglaser-scanning confocal microscopy, suggesting that the

Figure 5. Colocalization of GFP with F4/80 (red in A and B) orCD3 (red in C and D). B and D show specific CD immunofluo-rescence, whereas A and B show GFP colocalization. Arrowspoint to double-stained leukocytes. Arrowheads in C and Dpoint to CD3-positive/GFP-negative lymphocytes found only in alow frequency. CA indicates collateral artery.

Figure 6. Colocalization of GFP with CD34 (red in A) or CD31(red in C). B and D show GFP fluorescence. Arrows point todouble-stained leukocytes. Arrowheads in A and B show aCD34-positive/GFP-negative endothelial cell rarely found in col-lateral arteries. CA indicates collateral artery.

Figure 7. Colocalization of GFP with FGF-2 (red in A and B),VEGF (red in C and D), and MCP-1 (red in E and F). B, D, and Fshow specific growth factor and cytokine immunofluorescence,whereas A, C, and E show GFP colocalization. Arrows point todouble-stained leukocytes, and arrowheads in E point to MCP-1–positive endothelium of growing collateral arteries. CA indi-cates collateral artery.




transdifferentiation of bone marrow–derived cells into organ-specific cells occurs less frequently than anticipated.13,14,33

On the other hand, numerous studies reported positive effectsof endothelial progenitor or bone marrow–derived cells onblood flow recovery using similar or related models ofischemic tissue revascularization.3,5,10,11 These effects wereinterpreted as a result of the incorporation of bone marrow–derived cells into growing capillaries, even though collateralarteries bypassing the site of arterial occlusion are primarilyresponsible for blood flow recovery.15 Most of these studiesused an approach where progenitor/stem cells were isolated,cultured, and infused into target organs and therefore notmimicking strictly the natural course of the cells infused. Ithas to be noted that the amount of naturally present bonemarrow–derived progenitors in the peripheral blood is verylow, representing much less than 1% of cells in the circula-tion. Therefore, although the relatively moderate mobilizationof bone marrow–derived cells by application of VEGF didnot lead to any incorporation of these cells into the vessel wallof growing arteries in our model, the amount of exogenouscells administered intravenously, intra-arterially, or intraperi-toneally would be expected to exceed this number in magni-tude, possibly leading to their incorporation. In addition,results of some recent studies have suggested differences inbone marrow and stem cell recruitment attributable to differ-ent organ lesions,37 suggesting that these cells may behavedifferently depending on the conditions and organs investi-gated. It is also possible that the isolation and subsequentculture of progenitor or bone marrow–derived cells underspecial conditions can change the properties of these cells interms of their capacity to incorporate into target tissue.Recently, it has been reported that in vitro cultured endothe-lial progenitor cells maintain the expression of monocyte/macrophage markers, such as CD11c, CD11b (Mac-1),CD14, and panleukocyte marker CD45,34–36 and are able tosecrete some angiogenic factors, such as VEGF, hepatocytegrowth factor, G-CSF, and GM-CSF. Therefore, it is possiblethat their in vivo observed proangiogenic effect might be atleast partially attributable to the paracrine secretion of growthfactors and chemokines.34

In conclusion, although we did not find any incorporationof circulating bone marrow–derived cells in the endotheliumand tunica media of growing vessels, we found a significantperivascular accumulation of GFP-positive cells in areas ofcollateral artery growth and capillary growth. Because thesecells stained positive for some growth factors and chemo-kines, we suggest that their capability to promote vasculargrowth is unrelated to their structural incorporation but ratherattributable to paracrine effects.

AcknowledgmentsThis study was supported by grants from the Max-Planck-Gesellschaft (to T.Z. and W.S.) and from the Kerckhoff Clinic, BadNauheim (Forschungsprojekt PFOR-371 to S.K.). We thank DrSchalch and Dr Jonas from the University of Giessen for their helpwith animal irradiation.

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Helisch and Wolfgang SchaperTibor Ziegelhoeffer, Borja Fernandez, Sawa Kostin, Matthias Heil, Robert Voswinckel, Armin

Bone Marrow-Derived Cells Do Not Incorporate Into the Adult Growing Vasculature

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Bone Marrow-Derived Cells Do Not Incorporate Into the Adult Growing Vasculature

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