Proc. Nati. Acad. Sci. USAVol. 89, pp. 12018-12022, December 1992Genetics

Adhesion and incorporation of lacZ-transduced endothelial cellsinto the intact capillary wall in the rat

(gene therapy/skeetal mucke/retroviral vectors/c ry endothelum)

Louis M. MESSINA*t, RACHEL M. PODRAZIK*, THOMAS A. WHITEHILL*, DARYOUSH EKHTERAE*,THOMAS E. BROTHERS*, JAMES M. WILSONf, WILLIAM E. BURKEL§, AND JAMES C. STANLEY**Jobst Vascular Research Laboratories, Department of Surgery, tDepartments of Biological Chemistry and Internal Medicine and Howard Hughes MedicalInstitute, and IDepartment of Anatomy and Cell Biology, University of Michigan, Ann Arbor, MI

Communicated by Horace W. Davenport, August 31, 1992

ABSTRACT Use of the capillary bed of skeletal muscle asan in vivo recipient site to tnlt autologous end alcells that have undergone gene transfer ex vivo has considerablepotenial s a technique of somatic gene therapy. Here wedocument a previously unrecognized capacity of endothelialcells to adhere and Incorporate spontaneously into confluentendothelial cell monolayers in vitro and in vivo. This sponta-neous adhesion and incorporation of endothellal cells enabledus to seed lacZ-transduced endothelial cells into the wall ofskeletal muscle capiies of the hindlimb of the rat. Certaintransduced endothelial cells became incorporated within thecapillary wall, whereas others remained within the capillarylumen where they formed focal, electron-dense, contacts withhost endothelium. lacZ expression in the capillary bed wasdocumented for up to 1 month after transplantation. Use of theintact capillary bed of skeletal muscle as an in vivo recipient sitefor transduced, autologous endothelial cells holdsp i as astrategy for somatic gene therapy to treat various genetic andacquired human diseases.

Advances in the understanding and sequencing of the humangenome and development of molecular techniques for genetransfer into a target cell have made gene therapy technicallyfeasible. One approach to somatic gene therapy is to removethe cells targeted for gene transfer from an affected individualand transfer the recombinant gene into those cells ex vivo.These transduced cells are then reimplanted at a specificrecipient site in vivo. Identification of the optimal target celland recipient site has not been established for most forms ofsomatic gene therapy.

Recently, endothelial cells have been identified as anexcellent target cell for gene transfer (1-11). Endothelial cellsare durable, pluripotent cells that are located strategically atthe blood-surface interface. The optimal in vivo recipient sitefor endothelial cells that have undergone ex vivo gene transferhas not been identified. Genetically modified endothelial cellshave been seeded successfully onto synthetic vascular graftsas well as onto the walls of large muscular arteries after theexisting endothelium had been denuded by enzymatic ormechanical techniques (2, 3, 10). However, muscular arteriesform only a small portion ofthe surface area ofthe circulatorysystem, which may be inadequate for the purpose of systemicgene therapy.Because capillaries constitute >80%o of the surface area of

the circulatory system, they are a logical recipient site forseeding genetically transduced endothelial cells in vivo. How-ever, use of the capillary wall as a recipient site for trans-duced endothelial cells is problematic. One reason is that invitro and in vivo endothelial cells exist in tightly organized

monolayers in which cell density is fixed, and mechanicallystrong interactions exist between endothelial cells and theirbasement membrane to regulate solute and protein transportand to maintain an antiadhesive surface (12-14). Unlikeendothelium of muscular arteries, denudation of capillaryendothelium, as a means of facilitating all transplantation,might disrupt important normal microcirculatory hemody-namics and functions as well as initiate a vessel wall injuryresponse.To circumvent the need for denudation of the capillary

surface, we undertook in vitro experiments that tested thehypothesis that activation ofconfluent endothelial cell mono-layers by cytokines would promote adhesion and incorpo-ration of endothelial cells seeded onto these monolayers. Weused cytokines to activate endothelial cells because cyto-kines increase the expression of various surface adhesionmolecules, cause loss ofjunctional integrity between endo-thelial cells, and increase the metabolism and proliferation ofendothelial cells (15-19). These effects may enable seededendothelial cells to adhere and incorporate into cytokine-activated confluent monolayers.

In the course of these experiments we identified a previ-ously unrecognized capacity of endothelial cells to adhereand incorporate spontaneously into untreated postconfluentendothelial cell monolayers in vitro. In subsequent in vivoexperiments we studied the fate of microvascular endothelialcells injected intraarterially into the capillary bed of skeletalmuscle of the rat hindlimb. In this report we document thespontaneous adhesion and incorporation of lacZ-transducedendothelial cells into the intact capillary wall in vivo withoutthe need for denudation of the preexisting endothelium.

METHODSEndothelial Cell Derivation. Macrovascular endothelial

cells were derived from adult canine jugular veins by stan-dard enzymatic techniques (20). Rat epididymal fat pads weredigested in collagenase type 1A. The suspension was seriallyfiltered across a glass bead column (150-212 ,um) and theretained blood vessel fragments were digested in trypsin/EDTA (0.05%, 0.53 mM Na4EDTA). The microvascularendothelial cells were plated onto gelatin-coated flasks.Comatn of Identity ofEnd l Cels. Identity ofthe

derived endothelial cells was confirmed by their character-istic cobblestone growth pattern and the presence of recep-tors for acetylated form of low density lipoprotein (LDL) asjudged by uptake of fluorescent AcLDL (21).

Endotheial Cell blg with PKH-95 and PKH-26. Toeach T-25 flask of confluent endothelial cells, 2 ml of Diluent

Abbreviations: X-Gal, 5-bromo4chloro-3-indolyl B-D-galactoside;TEM, transmission electron microscopy.tTo whom reprint requests should be addressed at: UniversityHospital, 2210 THCC, 1500 East Medical Center Drive, Ann Arbor,MI 48109-0329.

12018

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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C containing 6.5 mCi (1 Ci = 37 GBq) ofPKH-95 and/or 2 mlofPKH-26 was added (Zynaxis Cell Science, Malverne, PA).Three minutes after labeling, 2 ml of bovine plasma-derivedserum was added for 1 min. After unattached label wasaspirated, each flask was washed five times with completemedium containing 20% bovine plasma-derived serum andthen was refed medium. PKH-26 is a fluorescent lipophilicdye (peak excitation, 595 nm; peak emission, 640 nm) thatbinds irreversibly within the endothelial cell membrane anddoes not leak into the surrounding medium nor transfer toother cells (22).

Transduction of Endothelial Cells. T-25 flasks of endothe-lium at <50% confluence were exposed for 12 hr each day toa murine amphotrophic retroviral vector containing the genesfor f3-galactosidase and neomycin resistance (J3-galactosidaseafter gag) (104-105 G418-resistant clones per ml) in Polybrene(10 Ag/ml), Dulbecco's modified essential medium, and 10%ocalf serum at 370C and 5% CO2. Daily exposures werecontinued until endothelial cells reached confluence. Afterreaching confluence, the endothelial cells were selected in aneomycin analog, G418 (1.0 mg/ml). Percent transductionwas determined by histochemical staining with 5-bromo-4-chloro-3-indolyl 8-D-galactoside (X-Gal) according to themethods ofDannenberg and Suga (23). Northern blot analysisalso confirmed lacZ gene expression by the transduced cellsin vitro.In Vitro Endothelial Cell Adhesion Assay of [3HIThymidine-

Labeled Endothelial Cells and Cytokine Pretreatment ofMono-layers. Canine venous endothelial cells were grown to conflu-ence on six-well culture dishes. Complete medium [M199supplemented with L-glutamine (2 mM), penicillin G (100units/ml), streptomycin (100 mg/ml), endothelial cell growthsupplement (25 ,ug/ml), heparin (15 units/ml), and 5% bovineplasma-derived serum] was replaced by complete mediumcontaining tumor necrosis factor a (Cetus) at concentrations(50 units/ml to 800 units/ml) known to increase leukocyte andtumor cell adhesion to endothelial cell monolayers (15-19).After 4 hr ofincubation, the medium containing tumor necrosisfactor a and control well medium were removed. Five thou-sand endothelial cells, labeled with [3H]thymidine, were addedin medium to each well and incubated for 1 hr. After incuba-tion, the medium and nonadherent cells were aspirated and thewells were washed three times with Hanks' balanced saltsolution. Wash solution and medium from each well weresaved. Adherent endothelial cells were harvested by adding1% SDS in 0.5 M NaOH for 2 hr at 37°C and were thenneutralized with 510 ul of 1 M HCl per well. Adherent andnonadherent cell suspensions were then dissolved separatelyin aqueous scintillation fluid. Endothelial cell adhesion (% oftotal) was calculated as [adherent cell cpm]/[adherent plusnonadherent cell cpm] x 100, after cell counting in a ,( counter.Data are expressed as mean ± SD for three experiments. Eachcondition was examined in replicates of three.In Vitro Endothelial Cell Adhesion Assay ofPKH-26-Labeled

Endothelial Cells to Postconfluent Monolayers. Canine venousendothelial cells from 12 different cell lines were grown toconfluence on six-well culture dishes in complete mediumsupplemented with 20% calf serum. Fourteen days afterplating and at least 7 days after reaching confluence, fluo-rescently labeled endothelial cells (PKH-26) were seededonto postconfluent monolayers at the following densities:1:100 (5000 cells), 1:10 (50,000 cells), 1:2 (250,000 cells) (24).After 1 hr of incubation at 37°C and 5% CO2, the supernatantwas aspirated and each well was washed twice with phos-phate-buffered saline (PBS). Adherent endothelial cells andthe underlying monolayer endothelial cells were harvestedwith trypsin (0.5%). One 200-,u sample was taken from eachwell for counting in a Coulter Counter (Coulter), and theremaining well contents as well as the aspirates and cellwashings underwent fluorescence-activated cell sorter deter-

mination of percent labeled cells. Percent cell adhesion wascalculated after determination of cell counts and percentlabeled cells as described above as [adherent cells]/[adherentplus nonadherent cells]. Data are expressed as the mean +SD for 12 experiments. Each condition was examined inreplicates of three.To determine the effect of time and genetic transduction of

endothelial cells on endothelial cell adhesion to postconfluentmonolayers in vitro, percent adhesion of seeded cells wasquantitated at 1, 2, 4, 12, 24, 48, and 72 hr after seeding.Parallel monolayers at identical time points were fixed forexamination by light and transmission electron microscopy.Data are expressed as the mean ± SD for two experiments.The relative quiescence ofthese postconfluent monolayers

was documented by [3H]thymidine uptake and incorporationinto DNA as assessed by autoradiography. Among 10-daypostconfluent cells, only 1% (11 of 1091 cells counted) hadincorporated [3H]thymidine, substantiating that these post-confluent monolayers were truly growth quiescent and there-fore that cell shedding and proliferation did not account forthe adhesion and incorporation of seeded endothelial cellsinto these postconfluent monolayers.In Vivo Endothelial Cell Adhesion to the Capillary Wall. The

fate of microvascular endothelial cells injected intraarteriallywas studied in a group of syngeneic Wistar F-455 rats(Harlan-Sprague-Dawley). After the induction of anesthesiawith ketamine (13 mg/kg) and zylazine (87 mg/kg) givenintramuscularly, the saphenous artery branch of the femoralartery was isolated and cannulated with a 26-gauge stainlesssteel cannula attached to a polyethylene tube and stopcock.Microvascular endothelial cells were doubly labeled with afluorescent label, PKH-26 (Zynaxis Cell Science), for ana-tomical localization of cells, and a radioactive label, 125I1labeled PKH-95 (125I-PKH-95), for quantitative tracking ofendothelial cells in specific tissues and organs.These doubly labeled endothelial cells (1-2 x 106) were

injected into the capillary bed of skeletal muscle of the rathindlimb through the femoral artery. Before the cells wereinjected, the femoral artery was clamped proximal to the originsaphenous artery to reduce pressure and flow rates in thecapillary bed. One hour after injection of the cells the clampwas removed. Groups of four rats were sacrificed at 1 hr, 1day, 7 days, and 28 days after injection. At the time of sacrificeall muscles and other tissues of both hindlimbs were excisedand counted individually. Segments of the gastrocnemius andtibialis anterior muscles were removed for microscopy todetermine the anatomical localization ofthe cells. The visceralorgans of the abdomen and thorax as well as the residualcarcass and head were subdivided further and counted indi-vidually. Animal care was in strict compliance with the Uni-versity of Michigan guidelines, state and federal laws, andNational Institutes of Health (NIH) Standards No. 85-23.

Light and Electron Microscopy. Cell culture wells werefixed in 0.5% glutaraldehyde in PBS for 5 min, washed inPBS, and incubated in X-Gal for 18 hr at 37°C according tothe methods of Dannenberg and Suga (23). They were post-fixed in 2% paraformaldehyde/2% glutaraldehyde in 0.1 Msodium cacodylate buffer and stained in 0.05% uranyl acetatein 0.05 M maleate buffer at pH 5.2 followed by staining in 1%OS04 in 0.1 M sodium cacodylate buffer. The sections weredehydrated, embedded in epon, and then stained in leadacetate for electron microscopy. Thin sections of muscle orliver were also fixed in 1.5% glutaraldehyde in PBS for 15 minand then incubated in X-Gal, postfixed, stained, embedded,sectioned, and stained as described above. Tissues for lightmicroscopy were treated similarly to those for electronmicroscopy with the exception that they were embedded inglycol methylacrylate and stained in eosin instead of stainingin uranyl, acetate, OS04, and lead acetate. The X-Gal reac-tion product by light microscopy appeared as blue-green

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A 100-0

* 80

= 60

.! 40

o 20LU

* - Fluorescently labeled cells-@- Fluorescently labeled

BAG-transduced cells

12 24 36 48 60 72Time After Seeding

[Hr]

B

~ ~ ~ '

At

-I.1.() Rapi adeso of enohla el t otof4:~~~~~~~~~~~~~~~~~~~~~~~4f

monolayers in vitro. Mean ± SD for two experiments. BAG,/3-galactosidase after gag. *, P < 0.05 by Student's t test vs.fluorescently labeled nontransduced cells. (B) Light micrograph ofincorporated, lacZ-transduced endothelial cells 72 hr after beingseeded onto monolayer surface. (x220.)

granules in transduced cells. The reaction product by elec-tron microscopy appeared as distinct square or rectangularcrystals of moderate electron density in the cytosome andaround the nuclear envelope (25).

RESULTSEndothelial Cell Adhesion and Incorporation into Intact

Confluent Monolayers. After 4 hr of tumor necrosis factor aYpretreatment of endothelial cell monolayers, between 32%o

100 -i

and 47% of the seeded cells adhered to the pretreatedmonolayers. Of greater importance, 43% of the seeded en-dothelial cells adhered to the control untreated monolayers.This magnitude of adhesion of seeded endothelial cells tountreated confluent endothelial cell monolayers was an un-expected finding because the surface of confluent endothelialcells is antiadhesive and the cell density of endothelial cellmonolayers in vitro and in vivo is tightly regulated (12-14).To substantiate this magnitude of spontaneous adhesion of

endothelial cells onto confluent monolayers in vitro, werepeated these experiments using postconfluent monolayers,which are manyfold less adhesive to inflammatory cells thanare proliferating or newly confluent endothelial cells (24).Endothelial cells were seeded at densities representing ratiosof seeded cells to monolayer endothelial cells of 1:100, 1:10,and 1:2. Adhesion of endothelial cells seeded at these threedensities onto untreated postconfluent monolayers was 34%,38%, and 39%o, respectively, thus substantiating a magnitudeof spontaneous adhesion similar to that reported above. Earlypassaged endothelial cells (passages 1-4) had an adhesionrate twice that of late passaged cells (passages 6-10).We then determined the effects of time and genetic trans-

duction of endothelial cells on the rate of endothelial celladhesion to postconfluent monolayers over a 72-hr timecourse. In these in vitro time course experiments, the rate ofendothelial cell adhesion onto postconfluent monolayers wasrapid (Fig. 1A). Nearly 90% of the seeded endothelial cellsbecame adherent within 6 hr after seeding onto the surface ofthe monolayers and nearly 100%o of the seeded cells wereadherent by 24 hr. This rate of adhesion resulted in a 50%increase in the monolayer cell density by 24 hr. lacZ-transduced endothelial cells became adherent at a signifi-cantly slower rate over the first 12 hr of the experiment, butby 24 hr their rate of adhesion was similar to that ofnontransduced endothelial cells (Fig. 1B).Transmission electron microscopy (TEM) of the monolay-

ers at 1 hr revealed that most of the seeded endothelial cellsinitially became adherent near endothelial cell junctions,where most functional endothelial-endothelial and tumor-endothelial cell receptors are known to be located (26-28). At4 hr TEM documented unambiguously that some of theadherent cells were already incorporated into the monolayeritself. This rapid incorporation was consistent with the re-quirement for polar cells, such as endothelial cells, to attachto a substrate for survival and proliferation.

Adhesion and Incorporation of Seeded Endothelial Cells intothe Capillary Wail in Vivo. To determine whether the former

1 hour 24 hours

Uver Lungs/ Other Excretion InjectedSpleen Leg

Liver Lungs/ Other ExcretionSpleen

28 days

eF FIG. 2. Distribution of 1251-PKH-95-labeled endothelial cellsin tissues and organs at 1 hr, 24 hr,7 days, and 28 days after injection

1721V-779 of cells into rat femoral artery.Injected Liver Lungs/ Other Excretion Values are expressed as mean ±

Leg Spleen SD for four rats at each time point.

InjectedLeg

I

80 -

60 -

40 -

20

:0 0 -

co

60

40'

20'

0

7 days

Injected LiverLeg

It/ row mzLungs/ Other ExcretionSpleen

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FIG. 3. Photomicrograph of an X-Gal-stained cross section oftibialis anterior muscles. lacZ-transduced endothelial cells are innumerous capillaries 1 hr after injection of transduced endothelialcells. (x70.)

in vitro property ofspontaneous endothelial cell adhesion andincorporation into monolayers ofendothelium cells occurs invivo and to determine whether this property could be used asa strategy for somatic gene therapy, we studied the fate ofradiolabeled microvascular endothelial cells injected intraar-terially into syngeneic Wistar F-455 rats (Fig. 2). One hourafter the clamp was removed, 74% of the injected radioac-tivity was detected in the injected hindlimb. At 24 hr afterinjection, 27% of the injected radioactivity was present in thehindlimb, and at 7 days, 24% ofthe injected radioactivity wasdetected. At 28 days, 12% of the injected radioactivity wasstill present in the hindlimb.

Light microscopic analysis of the gastrocnemius and tibi-alis anterior muscle segments at each time point showedregions of discrete fluorescence localized to the capillariesbetween skeletal muscle fibers. These discrete areas offluorescence often appeared tubular, suggesting incorpo-ration of fluorescently labeled endothelial cells into thecapillary wall. The number of fluorescently labeled cellsobserved in the muscle biopsies at the different time pointsafter injection appeared to correlate with the results ofthe 125Icell tracking studies.The l251 cell tracking studies revealed that the remainder of

the injected radioactivity resided in organs containing aportion ofthe reticuloendothelial system: the liver, lungs, and

spleen. Selected histological examination of the liver, lungs,and spleen did not reveal discrete areas of fluorescenceindicating intact endothelial cells. But definitive interpreta-tion of the histology of the liver was complicated by thestrong autofluorescence of liver tissue at the same wave-length as that of the PKH-26 endothelial cell label.

Histochemical staining for P-galactosidase documentedlacZ gene expression within the skeletal muscle capillaries ofthe injected hindlimb at 1 day (four rats), 1 week (four rats),and 1 month (four rats) after injection (Fig. 3). The lacZ-transduced cells exhibited the same pattern of localization tothe capillary wall as did the fluorescently labeled cells.TEM of skeletal muscle sections from the hindlimbs re-

vealed that certain transduced endothelial cells were incor-porated into the capillary wall (Fig. 4 A and B). Musclebiopsies from 24-hr and 7-day groups documented that themijority of transduced cells identified remained within thecapillary lumen. These transduced endothelial cells formedmultiple areas of focal electron-dense contacts with theunderlying endothelium ofthe capillary walls. Although theseendothelial cells had no major surface interaction with thecapillary basal lamina identified, they appeared healthy andviable (Fig. 4 C and D).

DISCUSSIONThese experiments show that endothelial cells adhere andincorporate spontaneously into confluent monolayers in vitroand into the intact capillary wall of skeletal muscle in vivo.This spontaneous adhesion and incorporation of endothelialcells enabled us to seed lacZ-transduced endothelial cellsonto the capillary wall without denuding the preexistingendothelium. This previously unrecognized capacity of en-dothelial cells to adhere and incorporate spontaneously intoquiescent, intact monolayers in vitro and in vivo was asurprising and unexpected finding in view of the normallyantiadhesive nature of the surface of endothelial monolayersas well as the tight regulation ofthe density ofendothelial cellmonolayers. A second surprising observation ofthese studieswas the ability to increase substantially the density of post-confluent monolayers in vitro. In the in vitro experiments, thedensity ofthe postconfluent monolayers was increased by upto 50%o (Fig. 1). Unlike other cell types such as fibroblasts, atconfluence endothelial cell saturation density is independent

*7=i^ar c arm

FIG. 4. (A) Electron micrograph of a transduced endothelial cell (TEC) incorporated into the wall of a muscle capillary 7 days aftertransplantation. Note the erythrocyte (RBC) within the capillary lumen and the pericyte (PC) adjacent to the capillary. (x4350.) (B) Elementof a portion of the capillary in A. The transduced cell (TEC) is incorporated fully into the wall and against the capillary basal lamina (BL).Numerous X-Gal-stained granules (arrowheads) are apparent. (x7500.) (C) Electron micrograph oftransduced endothelial cell (TEC) within thelumen ofa muscle capillary 7 days after transplantation. TheTEC contains numerous X-Gal-stained granules (arrowheads) and is closely apposedto the normal capillary endothelial cell (EC). (x5600.) (D) Enlargement of the rectangular area of C. In many focal regions the cell (TEC) hasmembrane densities (double arrows) adjacent to the normal EC membrane. Note the X-Gal-stained granules (arrowheads). (x25,000.)

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of serum concentration. This density-dependent inhibition ofendothelial cell proliferation accounts for the extremely lowendothelial cell replication rates in vitro and in vivo.The mechanism of adhesion of the seeded endothelial cells

to the monolayer endothelial cells is unknown. The lightmicroscopy and TEM of the in vitro studies revealed that thesite of adhesion of the endothelial cells was usually at theendothelial cell junctions where most tumor-endothelial andendothelial-endothelial cell receptors are located (26-28).The identification ofthe molecular mechanisms mediating theendothelial cell-endothelial cell adhesion described in thisreport may be pivotal to enhancing the seeding and thedurability of recombinant gene expression by transducedendothelial cells in a capillary bed in vivo for human genetherapy.The in vivo studies utilizing the capillary bed of skeletal

muscle of the hindlimb of the rat showed 12% of transducedendothelial cells present at 1 month after injection of thetransduced cells. To put this degree of endothelial cellretention into perspective, <0.1% of invasive tumor cells areestimated to survive an episode of hematogenous dissemina-tion (29). Light microscopic analysis of the gastrocnemiusand tibialis anterior muscles in the present investigationshowed regions ofdiscrete fluorescence localized exclusivelyto the capillaries between the skeletal muscle fibers. Theseregions ofdiscrete fluorescence sometimes appeared tubular,consistent with incorporation into the capillary wall. Histo-chemical staining for f3-galactosidase documented an identi-cal pattern of localization of the transduced endothelial cellsto the capillaries. Although some histochemical staining wasseen in control muscle in the area of nerve-motor endplatesor in macrophages within the muscle fibers, staining was notseen in the capillaries.TEM studies of the hindlimb muscle showed that although

certain transduced cells were incorporated into the capillarywall others remained within the capillary lumen formingmultiple, focal, electron-dense contacts with host endothelialcells. The latter finding was as unexpected as was the findingofincorporation ofcells into the capillary wall. It is surprisingthat a polar cell such as an endothelial cell could remainviable for up to a month without contact to a basal lamina. Toour knowledge, no previous studies have shown similar typesof endothelial cell-endothelial cell interactions as those doc-umented in this study. Whether the transduced cells locatedwithin the capillary lumen will eventually incorporate fullyinto the capillary wall as did-other transduced cells remainsan issue to be resolved. Furthermore, the nature of the focal,electron-dense contact areas between these cells identifiedby transmission electron microscopy will require furtherdelineation.The capillary wall and in particular the capillary bed of

skeletal muscle may be an optimal site to seed transducedendothelial cells. Capillaries comprise >80% of the surfacearea of the circulatory system. In addition, skeletal musclecomprises 40% of body weight and is a durable tissueresistant to ischemic injury (30). The long-term effectivenessof this type of somatic gene therapy will be dependent uponthe durability of recombinant gene expression, the life-spanof the seeded endothelial cell, and the long-term conse-quences of this transplantation technique on capillary bedand skeletal muscle function.

We thank J. S. Ford for technical assistance throughout theexperimentation and in particular for the derivation and culture ofmicrovascular endothelial cells, A. Gardner for technical assistancein the animal preparations, T. Komorowski for preparation of the

electron micrographs, M. Zeiger for editorial review, and C. Blank-enburg and M. Judge-Nolan for manuscript preparation.

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