comparative analysis of in vitro angiogenic activities of endothelial cells of heterogeneous origin

Comparative Analysis of in Vitro Angiogenic Activitiesof Endothelial Cells of Heterogeneous Origin

Kevin Harvey,* Zachary Welch,* A. Thomas Kovala,* Joe G. N. Garcia,†and Denis English*,‡,1

*Experimental Cell Research Program, Methodist Research Institute, Indianapolis, Indiana 46202;†Division of Pulmonary and Critical Care Medicine, Johns Hopkins University School of Medicine,Baltimore, Maryland 21224; and ‡Department of Allied Health Sciences,Indiana University School of Medicine, Indianapolis, Indiana 46202

Endothelial cells are dynamic participants in many as-pects of host defense, innate immunity, inflammation,angiogenesis, and vasculogenesis, but the interpretationof studies of their responses is often clouded by thesource of the cells under observation. Thus, it is not clearwhich endothelial cell type should be utilized in in vitrostudies to clarify the basis and physiological relevance ofessential processes, including chemotactic migration andmorphogenic differentiation. In this study, we comparedresponses of endothelial cells of a variety of origins, aswell as an immortalized cell line, using both proteingrowth factors and biologically active lipid mediators asagonists. While cells of divergent origin displayedmarked differences in the extent of their responsiveness,with a few notable exceptions, their pattern of respon-siveness to receptor-dependent stimuli was remarkablysimilar. Moreover, even the immortalized endothelial cellline Py-4-l migrated in a pattern consistent to that seenwith primary cells in culture although the immortalizedcells failed to form capillarylike structures under any ofthe conditions tested. We conclude that although the im-mortalized endothelial cell line Py-4-l is not appropriatefor investigations of endothelial cell morphogenic re-

1 To whom correspondence and reprint requests should be ad-dressed. Fax: (317) 962-5954. E-mail:[email protected].

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sponses, cultured cells from other sources, includingarteries, veins, and capillaries, often provide qualita-tively similar results to divergent metabolic stimuli.© 2002 Elsevier Science (USA)

Key Words: endothelial cell migration; capillary mor-phogenesis; angiogenesis.

INTRODUCTION

Capable of directed migration, prolific multiplica-tion and profound morphogenic alterations, endothe-lial cells are influenced by numerous extracellular fac-tors including phospholipid and protein mediators(for reviews see Folkman, 1995a; Folkman and Shing,1992; English et al., 2001; Kimura et al., 2000; Lee, M. etal., 1999; Lee, O. et al., 1999; Van Belle et al., 1998; Wanget al., 1999). These factors evoke complex biochemicalevents that preserve the integrity of the dynamic bar-rier the endothelial cells form between the vessels andthe tissues they supply, induce the cells to divide torepair tissue injury or disruption that comes with age,and migrate and change their shape in order to form

Received September 7, 2001

Microvascular Research 63, 316–326 (2002)doi:10.1006/mvre.2002.2406, available online at http://www.idealibr
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new vessels when needed for growth and develop-

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ment and to repair wounded tissue (Folkman, 1995a;Folkman and Shing, 1992; English et al., 2001). Under

0026-2862/02 $35.00© 2002 Elsevier Science (USA)

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abnormal circumstances, endothelial cells are stimu-lated to vascularize neoplastic tissues; to disrupt theintegrity of their monolayers, resulting in a devastat-ing breakdown of the crucial barrier functions; and toundergo apoptosis, resulting in ischemia, with conse-quent failure of the ability of the blood and lymph toprovide necessary factors and remove noxious toxinsfrom tissues and organs (Carmeliet and Jain, 2000;English et al., 1999; Garcia et al., 1993; Stevens et al.,2000).

As a result of recent intense research, the functionsof endothelial cells and the biochemical mechanismsthat induce them are becoming clear, as the agoniststhat drive these functions, and the membrane recep-tors that recognize them, are being identified. Thisresearch provides ample evidence that endothelialcells are complex and multifaceted. Complicating thisproblem even further, endothelial cells used for inves-tigations have been derived from a multiplicity ofsources; large arteries or veins, fetal or adult organs;capillaries from skin, lung, and other tissue; and bloodvessels of animals and humans. In attempts to providea standardized starting point, as well as to simplifyother aspects of endothelial cell research, several in-vestigators have attempted to derive information fromvarious immortalized endothelial cell lines. However,no single source of endothelial cells has gained acceptancefor use in experiments aimed at understanding the funda-mental processes they provide. As a result, much confu-sion surrounds comparison of results of studies thatemploy endothelial cells derived from differentsources, and warranted criticism is often the result ofexpansive interpretation of results obtained with cellsof a particular type.

Angiogenesis is the process in which endothelialcells are recruited to generate new capillaries frompreexisting blood vessels; a process that is required forsuccessful tumor growth, wound healing, as well asnormal growth and development (Carmeliet and Jain,2000; English et al., 1999, 2000, 2001; Folkman, 1995a,b;Folkman and Shing, 1992). As such, much of the re-search involving endothelial cell functions has re-cently been directed at understanding the fundamen-tal processes involved in the angiogenic response.Indeed, these processes may be involved in other crit-ical functions of endothelial cells as well. While angio-

genesis is composed of several important stages, twosteps are key to successful neovascularization. First,the directed, chemotactic migration of endothelialcells is an early, critical component responsible forrecruiting cells in the angiogenic response. Thus, en-dothelial cells must mobilize in order to reach the siteof new capillary formation. Second, capillary morpho-genesis or angiogenic differentiation must be initiatedfor blood vessel generation to occur. Two notable invitro assays are commonly used to provide an indica-tion of the potential of endothelial cells to migrate anddifferentiate in response to various lipid and proteinfactors. First, the chemotaxis assay, which involvesquantitation of the directed migration of the endothe-lial cells through a porous membrane in response tosoluble factors, is designed to mimic the recruitmentprocess (Kimura et al., 2000; Lee, O. et al., 1999; Nehlset al., 1998; Wang et al., 1999). Second, an in vitrocapillary morphogenesis assay is designed to monitorthe ability of the endothelial cells to differentiateinto capillarylike structures upon exposure to angio-genic factors (Bootle-Wilbragam et al., 2000; Lee, M. etal., 1999; Wilde et al., 2000). These two assaysare designed to imitate the mechanisms involved inthe early onset of the angiogenic process, thereby lay-ing the groundwork for mature blood vessel forma-tion.

Although many factors have been demonstrated toinitiate endothelial cell angiogenic responses, currentresearch has focused, for the most part, on a fewwell-characterized lipid and protein factors which areknown to play pivotal roles in the angiogenic process.These factors include biologically active, phosphory-lated lipids (see English et al., 2001 for review), basic-fibroblast growth factor (Nguyen et al., 1994; Gerwinset al., 2000), hepatocyte growth factor (Van Belle et al.,1998), and vascular endothelial growth factor (Folk-man, 1995a; Gerwins et al., 2000). To date, no studieshave comparatively evaluated the angiogenic poten-tial of various sources of endothelial cells with respectto cell migration and capillary morphogenesis. Thepresent study was undertaken in an attempt to closethis gap and determine how endothelial cells derivedfrom heterogeneous origins migrate and differentiatein response to select angiogenic factors.

317In Vitro Angiogenic Potential of Endothelial Cells

© 2002 Elsevier Science (USA)All rights reserved.

MATERIALS AND METHODS

Reagents

Chemicals and reagents were purchased from SigmaChemical Co. (St. Louis, MO) unless otherwise noted.Growth-factor-reduced Matrigel Matrix was obtainedfrom Becton–Dickinson Labware (Bedford, MA).Transwell chemotaxis chambers (6.5-mm diameter,8-�m pore size) were purchased from Costar, Inc.(Cambridge, MA). Costar 24-well tissue-culture-treated plates were acquired from Coming Incorpo-rated (Coming, NY). Sphingosine-1-phosphate (SPP)and sphingosylphosphorylcholine (SPPC) were pur-chased from Calbiochem (La Jolla, CA). Lysophospha-tidic acid (C18:1) was obtained from Avanti PolarLipids, Inc. (Alabastar, AL). The protein factors usedin this study were acquired as follows: recombinanthuman basic fibroblast growth factor (bFGF), bovinederived bFGF, human-derived platelet-derived growthfactor (PDGF), and recombinant human hepatocytegrowth factor (HGF) were obtained from R&D Sys-tems, Inc. (Minneapolis, MN) and vascular endothelialcell growth factor (VEGF, 165) and endothelial cellgrowth supplement were purchased from UpstateBiotechnology Inc. (Lake Placid, NY). Fetal bovineserum was purchased from Hyclone Laboratories, Inc.(Logan, UT). Antibiotic-antimycotic and amino acidsolutions as well as Dulbecco’s modified essential me-dium were obtained from Life Technologies, Inc.(Rockville, MD). Human umbilical vein endothelialcells, human aortic endothelial cells, human microvas-cular endothelial cells of lung origin, the EGM-2MV“bullet kits,” and the RPMI 1640 medium containingHEPES and l-glutamine were purchased from Clonet-ics (Walkersville, MD). Human dermal microvascularendothelial cells (HDMECs), bovine pulmonary aorticendothelial cells (BPAECs), and the CS-C completemedium kits were provided by Cell Systems Corpo-ration, Inc. (Kirkland, WA). The immortalized endo-thelial cell line Py-4-l was a gift from Dr. VictoriaBautch, University of North Carolina (Chapel Hill,NC). Human serum was obtained from normal donorsin accordance with institutional guidelines.

Cell Maintenance

Human-derived pulmonary microvascular cells(HMVEC-L) and human aortic endothelial cells(HAEC) were maintained in EBM-2 medium contain-ing the bullet kit materials as specified by the manu-facturer. HUVECs were cultured in RPMI 1640 me-dium containing 20% fetal bovine serum, 1%antibiotic-antimycotic solution (100�), 90 �g/mL ofheparin, and 15 �g/mL of endothelial cell growthsupplement. Human dermal microvascular endothe-lial cells (HDMVEC) were grown in Cell Systems Cor-poration (CSC) medium supplemented with CSCgrowth factors as well as 10% fetal bovine serum and1% antibiotic-antimycotic solution (100�). BPAECswere maintained in DMEM containing 20% fetal bo-vine serum, 1% antibiotic-antimycotic solution (100�),15 �g/mL endothelial cell growth supplement, and1% nonessential amino acid solution. Finally, the im-mortalized endothelial cell line Py-4-l was maintainedin DMEM containing 10% fetal bovine serum and 1%antibiotic-antimycotic solution (100�).

Endothelial Cell Migration Assay

As previously described (English et al., 2000), endo-thelial cells (up to passage 12) were dislodged fromthe flasks by trypsinization. The cells were washedand resuspended in serum-free media at a concentra-tion of 1 � 106 cells/mL. The source of serum-freemedia was dependent on the media required to main-tain the various endothelial cell lines in culture. Cells(1 � 105) were placed onto gelatin-coated Transwellchambers and incubated at 37°C for 30 min to allowthe endothelial cells enough time to adequately anchorto the filters. The endothelial monolayers were thenexposed to 300 �L of media containing specified che-moattractants located within the lower compartmentof the Transwell chambers. Lipid and protein che-moattractants were assessed in this study in compar-ison to dilute human serum. Endothelial migrationwas halted following a 2-h incubation period by re-moving the Transwell inserts from the chambers andcarefully extracting the remaining cells from the upperportion of the filter using a cotton swab. Transwellinserts were then placed into a 5% formaldehyde so-

318 Harvey et al.


lution in order to fix the endothelial cells that hadmigrated to the lower surface of the filter. The filtercontaining the fixed endothelial cells was carefullydetached from the Transwell unit and subsequentlystained with hematoxylin for visualization of the mi-grated cells. Endothelial cell migration was quanti-tated by microscopic enumeration to determine theaverage cell number/field of view (200�) using anOlympus BH-2 microscope by counting four randomfields of view on each of three separate filters. Exper-iments were repeated at least three times.

In Vitro Endothelial Morphogenesis Assay

Endothelial cell differentiation into capillarylikestructures was determined using a Matrigel-basedsupport assay as previously described (English et al.,2000; Lee, M. et al., 1999). Initially, growth-factor-re-duced Matrigel was thawed on ice, and 200 �L of thematerial was plated in each well of 24-well tissue-culture-treated plates. The Matrigel-coated plateswere placed at 37° for at least 20 min for solidification.Trypsinized endothelial cells were washed and resus-pended in serum-free medium. Since various endothe-lial cell lines were examined in this study, each endo-thelial cell line was assessed using serum-free mediumderived from the same source of media required forcell culture maintenance. The cells (2.5–5 � 104), var-ious protein growth factors, lipids, or human serumwere placed onto the Matrigel supports in a totalvolume of 200 �L/well at the final concentrationsindicated. For certain cell lines, up to 5.0 � 104 endo-thelial cells/well were required to generate consistentcapillarylike structures. The capillarylike structureswere examined microscopically (40�) on an invertedOlympus CK40 microscope following a 16- to 20-hincubation at 37° in the presence of 5% CO2. Theexcess medium, which contained nonadherent cells,was removed to facilitate visualization of the multi-cellular structures. To quantitate the extent of the for-mation of the capillarylike structures, photomicro-graphs were taken and the number of nodes/field wasdetermined, a node being defined as an intersectionwhereby at least three multicellular structures adjoin.Each sample was assayed in triplicate and the exper-iments were repeated at least three times.

Statistical Analysis

Data represent the mean � the standard deviation ofat least three determinations. The Student’s t test wasperformed in order to determine statistical signifi-cance between groups. Where the calculated P value isless than 0.05, statistical significance in illustrated ex-periments is indicated by an asterisk.

RESULTS

Chemotaxis

Figure 1 illustrates the how endothelial cells of di-verse origin migrate toward various protein and lipidchemoattractants. Each panel depicts the results of aspecific cell line derived from large vessels (BPAECs,HUVECs, and HAECs), capillaries (HDMVECs, HM-VEC-L), as well as the immortalized cell line Py-4-l. Asshown in Fig. 1A, human dermal microvascular endo-thelial cells vigorously responded to dilute serum,with optimal responses observed using a serum con-centration of 5%. These cells also responded vigor-ously to SPP at a concentration of 500 nM. Addition-ally, SPPC triggered migration at all concentrationswith a peak response occurring at 1 �M. Lysophos-phatidic acid was virtually ineffective in inducing mi-gration of HDMVECs. Among the protein angiogenicfactors tested, only VEGF elicited a chemotactic re-sponse, which occurred at concentrations greater than10 ng/mL. As seen in Fig. 1B, human umbilical veinendothelial cells responded similarly, with the excep-tion that migration to SPP was observed at levels aslow as 100 nM, whereas microvascular cells appearedto require higher levels of this lipid angiogenic factorsfor effective chemotactic migration.

Figure 1C depicts the chemotactic migration of hu-man aortic endothelial cells. These cells displayed anoverwhelming response to 500 nM SPP when com-pared to dilute human serum. This response wasgreater than twice the quantity of migrated cells incomparison to 5% human serum, the optimal effectiveconcentration. SPPC-challenged human aortic endo-thelial cells migrated similarly to that of dilute human



FIG. 1. Chemotactic migration of endothelial cells. Endothelial cells (1 � 105 cells/well) were loaded into Transwell chambers and challengedto various lipid and protein factors. After migration, cells were stained with hematoxylin and enumerated under �200 magnification. Eachpanel depicts an endothelial cell line derived from a different source as follows: (A) human dermal microvascular endothelial cells (HDMVEC),(B) human umbilical vein endothelial cells (HUVEC), (C) human aortic endothelial cells (HAEC), (D) pulmonary-derived human microvascularendothelial cells (HMVEC-L), (E) immortalized endothelial cell line Py-4-l, and (F) bovine pulmonary aortic endothelial cells (BPAEC).

320 Harvey et al.


serum. Lysophosphatidic acid and the protein angio-genic factors induced no significant directional migra-tion of HAECs when compared to levels of migrationobserved in the absence of stimulus.

As shown in Fig. 1D, HMVEC-L migrated less ag-gressively to all factors when compared to the othersources of endothelial cells described in this study. Inrelative terms, these cells proved to be the most re-sponsive to SPP but mounted a conservative chemo-tactic response to dilute human serum. A small num-ber of these pulmonary-derived cells migrated inresponse to 5 �M SPPC and 10 ng/mL of VEGF, butno detectable differences existed in response to anyother factors when compared to control.

The immortalized endothelial cell line Py-4-l mi-grated to dilute human serum, SPP, SPPC, and to avery limited extent lysophosphatidic acid (Fig. 1E). Nosignificant migration was observed to the protein an-giogenic factors assessed in this study.

When compared to the other endothelial cell typesassessed in this study, bovine pulmonary aortic endo-thelial cells (Fig. 1F) migrated in very high numbers toseveral key chemotactic factors. Dilute fetal bovineserum induced chemotactic migration of approxi-mately 400–500 cells/field, a level nearly three timesgreater than that observed with the next most respon-sive cell line, HAEC. Furthermore, 500 nM sphin-gosine-1-phosphate induced substantial migration ofbovine-derived endothelial cells with numbers ap-proaching that of dilute serum. SPPC-challenged mi-gration provoked a relatively high number of respon-dent endothelial cells as observed previously(Boguslawski et al., 2000). Finally, lysophosphatidicacid induced significant migration of BPAECs at con-centrations as low as 1 �M. No other endothelial celltype examined in this study displayed such a highresponse to lysophosphatidic acid. However, of theprotein factors tested, none proved able to inducesignificant migration of BPAECs when compared tocontrol.

Capillary Morphogenesis

Figure 2 represents typical capillarylike structuresgenerated in the in vitro Matrigel assay. Each of thefour panels portrays the capillarylike structures found

in a typical experiment. Endothelial cells were ex-posed to 2.5% serum, the lowest concentration wefound to consistently promote morphogenesis for eachof the cell types that responded in this system. Al-though the four endothelial cell lines illustrated in Fig.2 consistently produced capillarylike structures,BPAECs formed structures distinctly different fromthose obtained with the other cell types. These struc-tures were composed of a greater number of cells,thereby creating a larger, thicker capillarylike frame-work. Results with the three other cell lines (HDM-VEC, HMVEC-L, and HUVEC) each appeared verysimilar, with a distinct, delicate array of cells produc-ing tubelike formation evident after 16 h of culture. Incontrast, HAECs did not consistently generate typicalcapillarylike structures in this overnight assay, evenwhen up to 5 � 104 cells/well were plated (notshown); therefore, they were excluded from the exper-iments illustrated. Furthermore, the immortalized en-dothelial cell line Py-4-l was found not to be capable offorming capillarylike structures within the allotted20-h incubation period despite the fact that greaterthan 5.0 � 104 cells/well were analyzed (data notshown).

Figure 3 depicts the results of the in vitro capillarymorphogenesis induced by various agonists. Asshown in Fig. 3A, HDMVECs significantly respondedto 2.5% human serum when compared to control.Physiologically relevant levels of SPP (500 nM) andbFGF (10 ng/mL) promoted vigorous capillary mor-phogenesis as well. VEGF induced approximately atwofold increase in the number of capillarylike struc-tures over controls; however, the dermal microvascu-lar cells were not responsive to PDGF or HGF in thisassay. In Fig. 3B, HUVECs clearly demonstrated thegeneration of abundant capillarylike structures in thepresence of dilute human serum. SPP and bFGF in-duced a morphogenic response similar to that seenwith dilute human serum. Although VEGF and PDGFprovided little to no effect, HGF-stimulated endothe-lial cells generated a significant number of capillary-like structures from HUVECs.

In order to successfully generate capillarylike struc-tures from pulmonary-derived microvascular endo-thelial cells, a total of 3.5 � 104 cells/well were re-quired. SPP induced a response similar to that of the



dilute (2.5%) human serum (Fig. 3C). HGF and bFGFinfluenced these endothelial cells to generate capil-larylike structures numbering roughly two-thirds thequantity generated in the wells containing serum,while the remaining protein factors invoked no signif-icant increase over control. BPAECs required the pres-ence of 5.0 � 104 cells/well for consistent formation ofdifferentiated capillarylike structures (Fig. 3D). Nextto dilute human serum, these cells responded mostfavorably to HGF followed closely by SPP. When com-pared to control, bFGF was able to induce the forma-tion of only twice as many capillary-like structures.

DISCUSSION

The experiments depicted in this report provide ananalysis of how endothelial cells derived from different

sources respond to various endothelial cell stimuli intwo important and relevant in vitro assays. With respectto both chemotactic migration and capillary morphogen-esis, all of the endothelial cell lines assessed in this studyresponded dramatically to dilute serum and SPP. SPPC,a lipid structurally related to SPP, elicited a substantialchemotactic response from HUVEC, HDMVEC, the im-mortalized cell line Py-4-1, and—to a limited extent—BPAEC. Other endothelial cell lines, including HAECand HMVEC-L, failed to migrate to SPPC under theconditions used. Importantly, lysophosphatidic acid in-duced a chemotactic response 10 times greater than un-challenged control of BPAECs. The only other cell line togenerate a significant response to LPA, albeit diminutivemigration when compared to that of unstimulated cells,was the immortalized cell line Py-4-1. A previous reporthas demonstrated substantial migration of fetal aorticendothelial cells to LPA (Panetti et al., 2000). Fetal cellresponses were not examined in the present study.

FIG. 2. Capillary morphogenesis of endothelial cells. Endothelial cells were placed onto Matrigel supports in an overnight assay andstimulated with 2.5% serum. Once the endothelial cells had differentiated into capillarylike structures, photomicrographs were obtained using�40 magnification with an inverted microscope. Each panel represents a typical field of view within one well of a 24-well plate. Depicted inthe figure are examples obtained from the following endothelial cell lines: (A) human dermal microvascular endothelial cells, (B) human umbilicalvein endothelial cells, (C) pulmonary-derived human microvascular endothelial cells, and (D) bovine pulmonary aortic endothelial cells.

322 Harvey et al.


In comparison with sphingolipids and serum, vas-cular endothelial cell growth factor induced only aslight chemotactic response to the majority of the celllines used; however, a two- to threefold increase inmigration was detected to the HUVEC and HDMVECwith VEGF at the relatively high concentration of 100ng/mL. Additionally, pulmonary microvascular cells(HMVEC-L) displayed a fivefold increase over that ofunstimulated cells to 10 ng/mL VEGF. It should benoted that the chemotactic migration of HMVEC-L

yielded the lowest number of cells per field next to thePy-4-1 cells to all stimuli examined by a substantialmargin. With respect to capillary morphogenesis,VEGF induced a small, but significant, increase in thehuman dermal microvascular endothelial cells in con-trast to the other cell lines, which displayed no notableincrease in capillary morphogenesis upon exposure toVEGF.

Not only was platelet-derived growth factor unableto promote chemotactic migration, PDGF was also

FIG. 3. Quantification of capillary morphogenesis of endothelial cells. Endothelial cells were placed onto Matrigel supports in an overnightassay and stimulated with various protein and lipid factors. Once the endothelial cells had differentiated into capillarylike structures,photomicrographs were taken and subsequently quantitated. The number of nodes per field was determined by defining a node as anintersection whereby at least three multicellular structures adjoin. Each panel depicts an endothelial cell line derived from a different sourceas follows: (A) human dermal microvascular endothelial cells, (B) human umbilical vein endothelial cells, (C) pulmonary-derived humanmicrovascular endothelial cells, and (D) bovine pulmonary aortic endothelial cells.



unable to induce differentiation of these endothelialcells, resulting in capillary morphogenesis on Matrigelsupports.

Another well-known protein angiogenic factor, b-FGF, was relatively ineffective as a chemoattractant inrecruiting the migration of any of the cell lines ad-dressed in this study under the conditions used. Onthe other hand, b-FGF was a potent stimulus of endo-thelial cell differentiation in order to generate capillarymorphogenesis. In this respect, HDMVECs respondedparticularly well to b-FGF, demonstrating an 8-foldincrease in the generation of the tubelike structureswhen compared to that of unstimulated cells.HUVECs yielded a 5-fold increase in the number ofnodes/field when exposed to b-FGF. Although theincrease in capillary morphogenesis was significant,both the BPAE and HMVEC-L cells responded weaklyto b-FGF compared to the HDMVECs and HUVECs,with only a 1.5- to 2-fold increase over control.

Finally, HGF induced little, if any, migration fromany of the cell lines assessed in this study. Whenassessed as a differentiation factor in the capillarymorphogenesis assay, notable increases in the numberof nodes per field were observed in several cell lines.HGF induced the most striking response withBPAECs, effecting a sevenfold increase in the forma-tion of capillarylike structures. The HUVEC and HM-VEC-L cells additionally had a significant response toHGF, albeit considerably less intense with a less thantwofold increase ascertained in both cell lines.

This study was undertaken to determine the rela-tionship between various endothelial cell lines in theirability to respond to lipid and protein factors withinfunctional in vitro assays associated with the earlyonset of angiogenesis. Clearly, BPAECs respondedmost vigorously in terms of chemotactic mobility to allthe agents that induced a response. Furthermore, theHDMVEC and HUVEC cells were far superior in theirability to generate capillarylike structures. StimulatedBPAEC and HMVEC-L cells did in fact generate asignificant number of these structures; however, asubstantially greater number of cells per well wasrequired in order to generate a similar capillarylikenetwork on the Matrigel supports. It is additionallyinteresting to note that the protein factors assessed inthis study were poor chemoattractants, whereas the

lipid factors were far superior in their ability to inducea chemotactic response. With respect to capillary mor-phogenesis, the protein factors generally elicited atleast a measurable, if not impressive, response, withthe exception of PDGF. Regarding the lipid and pro-tein factors analyzed in this study, only SPP had theability to potently induce both chemotaxis and capil-lary morphogenesis.

In contrast to the striking differences in quantitativeresponses of the divergent cell types used to proteinand lipid growth factors, the data clearly indicate thaton a qualitative basis, responsiveness was remarkablyconsistent. Thus, with few important exceptions, thepattern of responsiveness of all of the cell types usedwas quite similar. As mentioned above, all of the celllines migrated most vigorously to serum and sphin-golipids; weaker responses were consistently ob-served with lysophosphatidic acid and protein growthfactors. Similarly, morphogenic differentiation of en-dothelial cells propagated from large and small ves-sels induced by protein and lipid factors were quali-tatively similar; factors effective in promoting tubeformation of BPAECs also effectively promoted tubeformation of HDMVECs. The exceptions to this con-sistency are notable. The immortalized cell line Py-4-lfailed to form capillarylike structures under any con-dition. Thus, these cells may not possess the biochem-ical machinery necessary to initiate this important as-pect of the angiogenic response and may therefore notbe suitable for analysis of events leading to a response.In addition, lysophosphatidic acid, a lipid mediatorimplicated in angiogenesis, induced migration of onlyone of the cell types used, namely BPAECs. Finally, itis of interest that HAECs displayed a marked differ-ence in the optimal extent of migration to serum ver-sus SPP. Studies with HUVECs and BPAECs havedemonstrated that the most abundant chemoattractantfor these cells present in serum is, in fact, plateletderived SPP (English et al., 2000). This finding may nothold for endothelial cells of all origins. Indeed, thedivergence of response of HAECs to serum versus SPPindicates that other factors may be involved. In addi-tion, it is possible that serum possesses factors thatlimit the extent of migration of endothelial cells ofcertain vessels, thereby slowing the response. How-ever, taking all these factors into account, the results of

324 Harvey et al.


the present study amply demonstrate that responseswith endothelial cells of large and microvessel origincan often be appropriately compared when dissectingthe basis and relevance of these responses. We ac-knowledge that some of the differences observed maybe a result of differing conditions and media used topropagate the cells and thereby not a reflection of truebiological differences in cellular reactivity. However,the importance of our results lies more in the demon-stration of similar patterns of migration and morpho-genesis of diverse cell lines to protein and lipid ago-nists than in the differences in the extent of responsesof the various cell types. These findings help justifythe use, for example, of BPAECs or HUVECs for as-says of certain angiogenic responses and the applica-tion of results gained with these cells to those expectedfor cells of microvascular origin.

In conclusion, these data provide a direct compara-tive analysis of the angiogenic potential of varioussources of endothelial cells to protein and lipid factorswith respect to cell migration and capillary morpho-genesis. The experiments of this study do not neces-sarily reflect the in vivo angiogenic potential of thevarious cell lines used to each agonist; they merelyreflect what results may be expected in vitro, as manyfactors, including culture conditions, growth factorsupplements, and other additions, may influence theresults we obtain. Further studies need to be carriedout in order to determine what role these variousendothelial cells would play in an in vivo assay. Ad-ditionally, capillary morphogenic assays involvingthree-dimensional matrixes over longer periods oftime may result in subsequent differences not seen inan overnight assay. This study was not intended tocover every in vitro angiogenesis-associated assay, norwas it intended to demonstrate with certainty therelative angiogenic potential of endothelial cells ofdifferent origin. However, the data presented providesignificant insight into the inherent similarities anddifferences that could play a vital role in determiningwhat type of endothelial cell line may appropriatelybe utilized in a particular investigation. The fact that,in many instances, the pattern of responses of endo-thelial cells of microvascular vs macrovascular originwere similar justifies the use of the latter cells forstudies of angiogenic responses.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health GrantsR01 HL 61751 and PO 1 HL 58064 and by a generous grant from thePhi Beta Psi Sorority awarded to D.E. Additional support wasprovided by the Methodist Heart Institute–Cardiovascular Trustawarded to A.T.K.

REFERENCES

Boguslawski, G., Lyons, D., Harvey, K. A., Kovala, A. T., andEnglish, D. (2000). Sphingosylphosphorylcholine induces endo-thelial cell migration and morphogenesis. Biochem. Biophys. Res.Comm., doi: 10.1006/bbrc.2000.2822.

Bootle-Wilbragam, C. A., Tazzyman, S., Marshall, J. M., and Lewis,C. E. (2000). Fibrinogen E-fragment inhibits the migration andtubule formation of human dermal microvascular endothelialcells in vitro. Cancer Res. 60, 4719–4724.

Carmeliet, P., and Jain, R. K. (2000). Angiogenesis in cancer andother diseases. Nature 407, 249–257.

English, D., Cui, Y., Siddiqui, R. A., Patterson, C., Natarajan, V.,Brindley, D. N., and Gargia, J. G. N. (1999). Induction of endo-thelial monolayer permeability by phosphatidate. J. Cell. Biochem.75, 104–117.

English, D., Kovala, A. T., Welch, Z., Harvey, K. A., Siddiqui, R. A.,Brindley, D. N., and Garcia, J. G. N. (1999). Induction of endo-thelial cell chemotaxis by sphingosine 1-phosphate and stabiliza-tion of endothelial monolayer permeability by lysophosphatidicacid, potential mediators of hematopoietic angiogenesis. J. Hema-tother. Stem Cell Res. 8, 627–634.

English, D., Welch, Z., Kovala, A. T., Harvey, K., Volpert, O. V.,Brindley, D. N., and Garcia, J. G. N. (2000). Sphingosine 1-phos-phate released from platelets during clotting accounts for thepotent endothelial cell chemotactic activity of blood serum andprovides a novel link between hemostasis and angiogenesis.FASEB J. 14, 2255–2265.

English, D., Garcia, J. G. N., and Brindley, D. N. (2001). Platelet-released phospholipids link haemostasis and angiogenesis. Car-diovasc. Res. 49, 588–599.

Folkman, J., and Shing, Y. (1992). Angiogenesis. J. Biol. Chem. 267,10931–10934.

Folkman, J. (1995a). Clinical applications of research on angiogene-sis. New Engl. J. Med. 333, 1757–1763.

Folkman, J. (1995b). Angiogenesis in cancer, vascular, rheumatoidand other diseases. Nat. Med. 1, 27–31.

Garcia, J. G. N., Paterson, C., Bahler, C., Aschner, J., Hart, C. M., andEnglish, D. (1993). Thrombin receptor activating peptides induceCa2� mobilization, barrier dysfunction, prostaglandin synthesis,and platelet derived growth factor mRNA expression in culturedendothelium. J. Cell. Physiol. 156, 541–549.



Gerwins, P., Skoldenberg, E., and Claesson-Welsh, L. (2000). Func-tion of fibroblast growth factors and vascular endothelial growthfactors and their receptors in angiogenesis. Crit. Rev. Oncol. He-matol. 34, 185–194.

Kimura, T., Watanabe, T., Sato, K., Kon, J., Tomura, H., Tamama, K.,Kuwabara, A., Kanda, T., Kobayashi, I., Ohta, H., Ul, M., andOkajima, F. (2000). Sphingosine 1-phosphate stimulates prolifer-ation and migration of human endothelial cells possibly throughthe lipid receptors, Edg-1 and Edg-3. Biochem. J. 348, 71–76.

Lee, M., Thangada, S., Claffey, K. P., Ancellin, N., Liu, C. H., Kluk,M., Volpi, M., Sha’afi, R., and Hla, T. (1999). Vascular endothelialcell adherens junction assembly and morphogenesis induced bysphingosine-1-phosphate. Cell 99, 301–312.

Lee, O., Kim, Y., Lee, Y. M., Moon, E., Lee, D., Kim, J., Kim, K., andKwon, Y. (1999). Sphingosine 1-phosphate induces angiogenesis:Its angiogenic action and signalling mechanism in human umbil-ical vein endothelial cells. Biochem. Biophys. Res. Comm., doi:10.1006/bbrc. 1999.1586.

Nehls, V., Herrmann, R., and Huhnken, M. (1998). Guided migra-tion as a novel mechanism of capillary network remodeling isregulated by basic fibroblast growth factor. Histochem. Cell Biol.109, 319–329.

Nguyen, M., Watanabe, H., Budson, A. E., Richie, J. P., Hayes, D. F.,and Folkman, J. (1994). Elevated levels of an angiogenic peptide,

basic fibroblast growth factor, in the urine of patients with a widespectrum of cancers. J. Natl. Cancer Inst. 86, 356–361.

Panetti, T. S., Nowlen, J., and Mosher, D. F. (2000). Sphingosine-1-phosphate and lysophosphatidic acid stimulate endothelial cellmigration. Arterioscler. Thromb. Vascul. Biol. 20, 1013–1019.

Stevens, T., Garcia, J. G., Shasby, D. M., Bhattacharya, J., and Malik,A. B. (2000). Mechanisms regulating endothelial cell barrier func-tion. Am. J. Physiol. Lung Cell. Mol. Physiol. 279, L419–L422.

Van Belle, E., Witzenbichler, B., Chen, D., Silver, M., Chang, L.,Schwall, R., and Isner, J. M. (1998). Potentiated angiogenic effectof scafter factor/hepatocyte growth factor via induction of vas-cular endothelial growth factor: The case for paracrine amplifi-cation of angiogenesis. Circulation 97, 381–390.

Wang, F., Van Brocklyn, J. R., Hobson, J. P., Movafagh, S.,Zukowska-Grojec, Z., Milstien, S., and Spiegel, S. (1999). Sphin-gosine 1-phosphate stimulates cell migration through a Gi-cou-pled cell surface receptor: Potential involvement in angiogenesis.J. Biol. Chem. 50, 35343–35350.

Wild, R., Ramakrishnan, S., Sedgewick, J., and Griffioen, A. W.(2000). Quantitative assessment of angiogenesis and tumor vesselarchitecture by computer-assisted digital image analysis: Effectsof VEGF-toxin conjugate on tumor microvessel density. Micro-vasc. Res. 59, 368–376.

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