the pulmonary endothelial cell - bmj

Thorax, 1979, 34, 200-208

The pulmonary endothelial cell

DONALD HEATH AND PAUL SMITH

From the Department of Pathology, University of Liverpool, Liverpool, UK

ABSTRACT The surface of the endothelial cells of the pulmonary trunk of the Wistar albino ratwas studied by means of silver preparations and by scanning and transmission electronmicroscopy. This surface is the site of cytoplasmic projections and the opening of caveolaewhich together appear to be features associated with the active metabolic role of thepulmonary endothelial cell.

When one looks at a transverse section of pul-monary trunk, the cytoplasm of the endothelialcells is spread out so thinly as to be barely dis-cernible. Indeed, the endothelial layer is detectedlargely through the prominent nuclei that bulgefrom the inner surface of the vessel (fig 1). Theapparently dull nature of the pulmonary endo-thelial cell has led histologists and pathologiststo pay it but scant attention. In recent years,however, it has become clear that the surface ofpulmonary endothelial cells has an importantmetabolic role. For this reason we thought thatas pathologists we should take a closer look atthe structure of the pulmonary endothelial cellas shown by silver preparations and by scanningand transmission electron microscopy.

Silver preparations

TechniqueThe margins of endothelial cells were stainedwith silver deposits by a modification of one of themethods described by Poole et al (1958) and Floreyet al (1959). A male Wistar albino rat was killedby inhalation of ether. After removal of attachedfat the pulmonary trunk was removed and openedlongitudinally. Excess blood was washed from theendothelial surface with 0 85% saline, and thespecimen was pinned on to clean cork using steelpins. The specimen was then rinsed in distilledwater and exposed to osmium tetroxide vapourfor 10 minutes, stained with a fresh solution of025% silver nitrate for 30 seconds, and rinsed

.......::.....

Fig 1 Transverse section of pulmonaryq trunk of Wistar albino rat. Bulk of tissue

.l seen comprises smooth muscle cells,nuclei of which are clearly visualised.Elastic fibres form clear undulatingstrands in media. Surface of pulmonarytrunk is lined by endothelial cells that

| have very thin extensions of cytoplasmbut prominent nuclei (arrow). Were itnot for latter endothelial layer would bebarely visible (Haematoxylin and eosin,X635).

200

on January 11, 2022 by guest. Protected by copyright.

http://thorax.bmj.com

/T

horax: first published as 10.1136/thx.34.2.200 on 1 April 1979. D

ownloaded from

http://thorax.bmj.com/


again in distilled water and placed in Caulfield'smedium (a fixative containing osmic acid at pH7-4 with added sucrose) for 30 minutes. This wasfollowed by washing in several changes of distilledwater for two hours, then dehydrating in ascend-ing grades of ethyl alcohol, clearing in xylol, andmounting in DPX.

ResultsWhen the margins of the pulmonary endothelialcells are stained in this manner by silver deposits,the inner surface of the pulmonary trunk is seento be lined by a mosaic of cells. The silver pre-parations show the shape, size, and arrangementof the cells (fig 2) and allow one to determinetheir dimensions and surface area. Most of theendothelial cells are roughly rectangular, althoughsome have a more ovoid or triangular shape(fig 2a). The cell margins have a wavy outline,and in some cases there are finger-like projectionsfrom one cell fitting into a corresponding recessin another (fig 2a). Presumably these aid a tighterlocking together of the endothelium. The greaterdiameter of the cells is usually in the region of30 to 35 ,um and the shorter diameter some 15 to20 ,um. The range of surface area of the individualendothelial cells, as determined by planimetry in24 cells, is 369 to 627 ,um2 (mean 503 tLm2). Someareas of the inner surface of the pulmonary trunkshow more elongated cells in which the diameters

cLb

are in the region of 50 and 8 ,um (fig 2b). The"waves"l may be few and large so that the cell hasa dumb-bell shape (fig 2b).

Scanning electron microscopy

TechniqueFour female Wistar albino rats were anaesthetisedwith ether vapour and the thorax and abdomenwere opened by a mid-line incision. The carotidarteries and superior venae cavae were ligated. Asmall incision was made in the inferior vena cavabetween the origins of the renal veins, and a poly-thene cannula was passed into the vessel towardsthe heart. The cannula was tied firmly into place.The abdominal aorta was also similarly cannulatedclose to its bifurcation. The cannula within theinferior vena cava was connected to a flask con-taining heparinised saline, which entered the in-ferior vena cava at a pressure of 20 cm water. Thisflushed out the inferior vena cava, pulmonaryarteries, and pulmonary veins, the blood and sur-plus saline being collected via the aortic cannula.When a three-way stopcock was turned the salinewas replaced by chilled, buffered glutaraldehyde.After this had filled the pulmonary vasculatureand had started to flow from the aorta, the aorticcannula was clamped to reduce the flow of fixativeto about 1 ml min-'. By this means the full hydro-static pressure of 20 cm water was applied to the

Fig 2a and b. Silver preparations ofendothelial surface of pulmonary trunkof Wistar albino rat. Barely discernibleendothelial layer of fig 1 is now seen asan extensive mosaic of cells, outlines ofwhich are shown by silver deposits.(a) Roughly rectangular endothelial cellswith wavy outlines. One cell shows afinger-like projection (arrow). (b) An areashowing more elongated, narrow cells,one of which has a dumb-bell shape(Osmium tetroxide vapour and silver

~~~nitrate solution, both X777).

E

201



/T


ownloaded from


Donald Heath and Paul Smith

pulmonary trunk to fix it as close to its dimensionsin vivo as possible. Fixation was aided by pouringglutaraldehyde on to the outside surface of thepulmonary trunk. It was left to fix for two hours.The thoracic organs were then removed, and thepulmonary trunk was carefully dissected free andstored overnight in glutaraldehyde at 40C.An intact cylinder of pulmonary trunk 3 mm

long was dehydrated in ascending grades of ethanoland stored in amyl acetate. It was then transferredto a critical-point drying apparatus and the amylacetate flushed out under pressure with liquidcarbon dioxide. After soaking in liquid carbondioxide for one hour it was dried by the critical-point technique. The dried cylinder was then cut

into two segments by two longitudinal cuts madeunder a dissecting microscope. The two halveswere mounted on stubs, endothelium uppermost,using silver dag as the cement. The mountedvessels were coated with gold-palladium in vacuoand examined with a Cambridge Stereoscan scan-ning electron microscope at a voltage of 7 kV.

ResultsScanning electron micrographs of erythrocytes inclose apposition to the surface of the pulmonaryendothelial cells (fig 3) serve as a reminder of theintimate approximation of blood cells and plasmaon the one hand and the endothelium on theother, and of the potential metabolic significance

_y~~~ ~ufc ofpumoay:rnko

laal...meWtaboataE.. seni. cecont..atw.i....h el. s r ,.ES.~~~~~~~~~~~~~~~~......,

Fig 3 This figure and figs 4 to6 are scanning electron

; ~~~~~micrographs of endothelialt;: * ~~~surface of pulmonary trunk of-2- 4 ~~~afemale Wistar albino rat.t .- xF: ~Biconcave red blood cells, RBC,

I:.X are seen in close contact withW --w: X ~~the endothelial surface, ES.

v This surface is roughened by a< ^- ~~networkof projections(X3200).

202



/T


ownloaded from



of this. The pulmonary trunk does not have asmooth lining (fig 4). Instead it has a "cobblestonesurface" that is produced by the protuberance ofthe nuclei of the endothelial cells (fig 4). Super-imposed on the cobblestone lining produced bythe nuclei is a shaggy appearance brought aboutby large numbers of cytoplasmic flecks and pro-jections (figs 4 to 6). Some are round or oval andothers linear and finger-like. They may be singleor may be aggregated into complexes in which asmany as 15 individual projections can be seen(fig 6). Some of the projections are branched andothers show small buds arising from the side ofprojections. Others assume leaf-like flaps andfolds; these comprise the largest projections andthey predominate along the margins of cells(fig 6). The endothelial projections range in lengthup to 2700 nm and in width up to 430 nm. Theyare seen all over the surface of the pulmonarytrunk but are especially numerous over the nuclearprominences. They tend to be large and leaf-likeover the cell margins and this facilitates delin-eation of one cell from another by the observer(fig 6).The pulmonary endothelial cells tend to be

roughly rectangular, oval, or even triangular in

shape, the greater diameter being up to 30 ,um inthe tissue fixed and processed for scanning electronmicroscopy, the smaller diameter being 10 to19 ym (fig 5). The nuclei have a round or ovaloutline (fig 5). The surface of the pulmonaryendothelial cells is studded with small pits (fig 6).These are openings on to the cell surface of thecaveolae intracellulares.

Transmission electron microscopy

TechniqueIsolation and fixation of the pulmonary trunk wereperformed according to the method of Smith etal (1978) described above. Small arcs of pulmonarytrunk were trimmed off the vessel, post-fixed forone hour in 1% osmium tetroxide, stained withuranyl acetate, and embedded in Araldite. Semi-thin sections, 1 ,um in thickness, were stained withtoluidine blue for the selection of a suitable field.Fine sections were cut with an LKB UltrotomeIII, stained with lead citrate, and examined withan AEI EM 6B electron microscope.

ResultsTransmission electron microscopy of the pul-

Fig 4 Oblique view of endothelial surface ofpulmonary trunk showing a cobblestoneappearance due to prominences formed by bulgingnuclei, N. Cell margins (arrows) are picked outclearly by endothelial projections at this site.Numerous endothelial projections, p, are also seenover bulging nuclei and over surrounding flattercytoplasm (X2228).

203



/T


ownloaded from



Fig 5 Surface of endothelialcells of pulmonary trunk.Central, or slightly excentric,nuclei show as pale ovoidstructures, N. Surroundingflattened areas of endothelialcytoplasm appear dark.Endothelial projections areprominent over nuclei but arealso seen over surroundingcytoplasmic sheets. They alsooccur prominently at cellmargins picking out thesestructures clearly. Dark cracksare artefacts induced bytechnique of preparation(X2836).

")AA



/T


ownloaded from



Fig 6 Cytoplasmid surface hasbeen photographed at a highermagnification to show itsdistinctive features more clearly.Endothelial projections, p, areseen over cytoplasmic surfaceand especially at cell margins,pm. Luminal openings ofcaveolae intracellulares (arrows)appear like small craters onsurface of endothelial cell(X7047).

205



/T


ownloaded from



monary endothelial cells confirms that the "cobble-stone appearance" of the lining of the pulmonarytrunk is due to the nuclei bulging into the lumen(fig 7). At this point the depth of cytoplasm andnucleoplasm is in the region of 4-7 ,um. The depthof the flat, squamous extremities of the cytoplasmof the endothelial cell falls to a value as low as0 4 ym. The cells are held closely and firmly to-gether to produce the mosaic shown in fig 2 by"tight junctions" (fig 8). The endothelial projec-tions seen on scanning electron microscopy arealso clearly visible in transmission electron micro'graphs (fig 9). Some contain caveolae intra-cellulares and ribosomes.The surface of the pulmonary endothelial cells is

studded with very large numbers of caveolae intra-cellulares (fig 10). They open on to the luminalsurface by a stoma covered by a diaphragm com-posed of a single lamella. Sections through thestoma show dense osmiophilic bodies at themeeting point of the plasma and caveolar mem-branes and the caveolar diaphragm (arrow infig 10). Caveolae are 50 to 90 nm in diameter. Thewall around their cavity is of unit membrane typewith two lamellae. This wall is not uniform inappearance but is granular with the formation ofsmall nodules. Sometimes several caveolae fusetogether to form a complex structure (fig 9) but indoing so the shape and identity of the individual

caveolae are maintained so that the whole comesto resemble a bunch of grapes.

Microtubules are common in the cytoplasm ofpulmonary endothelial cells (fig 10) but, it shouldbe noted, there were no bundles of fine filamentsakin to the actomyosin filaments of smoothmuscle cells.

Discussion

The view that the microscopist customarily has ofthe endothelium of a transverse section of thepulmonary trunk is that of an immensely thinsheet of cytoplasm all but invisible. Indeed itsexistence is largely inferred by the protuberancesformed by the nuclei of the endothelium (fig 1).This being the case, it is hardly surprising that thepulmonary endothelial cell has received scantattention from histologists and pathologists.When one looks at the surface of the pulmonary

endothelium stained by silver salts rather than at asection of it, however, the appearance is trans-formed, and one realises that the endothelium ofthe pulmonary trunk forms an extensive area com-prising a mosaic of numerous cells with which theblood comes into intimate contact during itspassage through the lung (fig 2).Scanning electron microscopy reinforces this

impression and one appreciates the intimate con-

Fig 7 Transmission electronmicrograph of endothelialsurface of pulmonary trunk ofa Wistar albino rat. Endothelialcells consist of a centralprotuberant part due to thenucleus with surroundingflattened plates of cytoplasm(X5217).

Fig 8 Transmission electronmicrograph of peripheralflattened plates of cytoplasm oftwo endothelial cells ofpulmonary trunk from Wistaralbino rats. Cells are keptclosely apposed by tightjunctions (arrow) (X16666).

206



/T


ownloaded from



Fig 9 Transmission electron micrograph of superficialportion of an endothelial cell of pulmonary trunk ofa Wistar albino rat. Three endothelial projections,whose surface appearance on scanning electronmicdroscopy is shown in figs 4, 5, and 6, are seen.Luminal surface of cell is site of numerous caveolaeintracellulares. At one place several caveolae havefused (arrow), and it should be noted that individualcaveolae have retained their shape and structure(X60 000).

tact between the luminal surface of the pulmonaryendothelial cells and the erythrocytes and plasmaof the blood (fig 3). This view of the endothelialsurface at ultrastructural level brings with it thesomewhat surprising realisation that it is notsmooth but roughened by a network of finger-likeprojections and strands (figs 3-6). Some of theseprojections may be artefact and derived fromfibrin in the blood, but there is no doubt that mostare true ultrastructural features of the surface ofthe pulmonary endothelial cell (figs 5 and 6). Theycan be identified readily in transmission electronmicrographs (fig 9). They appear to have beenfirst described by Smith et al in 1971 who re-ported their presence in the pulmonary arteries

Fig 10 Transmission electron micrograph ofsuperficial portion of an endothelial cell of pulmonarytrunk of a Wistar albino rat. Opening on to luminalsurface of cell are several caveolae intracellulares.Example indicated by an arrow shows characteristicstructure with two osmiophilic knobs either side ofstoma, which is covered by a thin diaphragm. Wall ofcaveola has a granular appearance. Microtubules, m,run through cytoplasm (X82 758).

of the dog. These authors ascribe a haemodynamiceffect to these endothelial projections. They sug-gest that the dense, irregular meshwork of finger-like processes could produce an eddying flow ofcell-free plasma along the surface of the en.do-thelial cells. This is probably of importance inlarge pulmonary arteries where nutrient capillariesenter the adventitia and outer media but do notpenetrate to the endothelium. A retarded flow ofplasma along the surface of the cell could providefavourable conditions for the exchange of meta-bolites and possibly for the metabolism of cir-culating hormones. Since the projections are sonumerous they must greatly increase the surfacearea of the cell.Our study confirms in the pulmonary trunk the

same ultrastructure of caveolae intracellularesreported by Smith and Ryan (1972) as occurring inthe endothelial cells of the capillaries of the ratlung. Smith and Ryan (1972, 1973) believed that

207



/T


ownloaded from



the dense, osmiophilic knobs seen in transversesection around the stoma represent a circularskeletal structure that maintains the patency ofthe stoma and the integrity of the diaphragm. Ourown observations support this idea for we wereimpressed by the fact that when several caveolaefused together to form one complex, the indi-vidual caveolae still retained their identity andshape so that the whole came to resemble a bunchof grapes (fig 9).The granular, nodular nature of the wall sur-

rounding the cavity of the vesicle is thought torepresent enzyme clusters or binding sites. Thedistribution of lead phosphate deposits formed onreaction of caveolar 5'-nucleotidase with AMP inthe presence of lead nitrate corresponds with thespacing of the globular structures in the wall(Smith and Ryan, 1972). Plasma membrane andcaveolae may be harvested in a highly purifiedstate suitable for study by a series of low-speedcentrifugations of lung homogenates (Ryan andSmith, 1971).The recently introduced immunoperoxidase tech-

nique allowed Ryan and Ryan (1975) to show an"angiotensin converting enzyme" in globularparticles some 6 nm in diameter in the membranelining caveolae and in undifferentiated plasmamembrane of the surface of pulmonary endothelialcells. In this technique, after preliminary incuba-tion of rat lung tissue with normal goat serum tobind non-specific sites, antibodies to this enzymewere coupled to peroxidase and attached to theenzyme sites in the walls of caveolae. Subsequentlythe peroxidase moiety coupled to the antibodieswere allowed to react with hydrogen peroxide anddiaminobenzidine, thus labelling precisely the sitesof the enzyme at ultrastructural level. Their studyshowed that the inactive decapeptide angiotensin Iis converted into the active octapeptide angio-tensin II in the walls of the caveolae of pulmonaryendothelial cells, predominantly in the pulmonarycapillaries and venules. Thus it would appear thatthe active metabolic role of the lung does notreside in enzymes in the blood but in the enzymessituated in the walls of the caveolae of the pul-monary endothelial cells.An important negative finding in our study was

the absence of bundles of fine filaments in thecytoplasm of pulmonary endothelial cells akin tothe actomyosin filaments of smooth muscle cells.Cells possessing such fine cytoplasmic filamentsare very characteristic of the various intimal pro-liferations in hypertensive pulmonary vasculardisease. This negative finding in the present studysuggests that intimal changes in vascular diseaseof the lung are not due to proliferation of endo-

thelial cells but to some other cell type. We discussthis important matter at length elsewhere (Smithand Heath, 1979).More and more we are coming to realise that

the lung has many non-respiratory functions.Some of these probably lie in the unfamiliar cellssuch as the Feyrter and Clara cells of the bron-chial tree whose functions still remain to be dis-covered. Others, however, lie in the pulmonaryendothelial cells which have intimate access tohormones and metabolites as they pass throughthe pulmonary circulation. For this reason wefelt it worth while to have a closer look at theultrastructural features of the surface cells thatshow so little of their nature and potential in theclassical paraffin section stained with haematoxylinand eosin. It seems likely that in the future in-creasing use of the immunoperoxidase techniquewill allow the histologist and pathologist to learnmore about their behaviour and function inhealth and disease.

References

Florey, H W, Poole, J C F, and Meek, G A (1959).Endothelial cells and "cement" lines. Journal ofPathology and Bacteriology, 77, 625436.

Poole, J C F, Sanders, A G, and Florey, H W (1958).The regeneration of aortic endothelium. Journal ofPathology and Bacteriology, 75, 133-143.

Ryan, J W, and Smith, U (1971). A rapid, simplemethod for isolating pinocytotic vesicles and plasmamembrane of lung. Biochimica et Biophysica Acta,249, 177-180.

Ryan, J W, and Ryan, U S (1975). Metabolic activitiesof plasma membrane and caveolae of pulmonaryendothelial cells, with a note on pulmonary prosta-glandin synthetase. In Lung Metabolism, edited byA F Junod and R de Haller, p 399. Academic Press,New York.

Smith, P, Heath, D, and Padula, F (1978). Evaginationof smooth muscle cells in the hypoxic pulmonarytrunk. Thorax, 33, 31-42.

Smith, P, and Heath, D (1979). Electron microscopyof the plexiform lesion. Thorax, 34, 177-186.

Smith, U, and Ryan, J W (1972). Substructuralfeatures of pulmonary endothelial caveolae cellu-lares. Tissue and Cell, 4, 49-54.

Smith, U, and Ryan, J W (1973). Electron microscopyof endothelial and epithelial components of thelungs: correlations of structure and function.Federation Proceedings, 32, 1957-1966.

Smith, U, Ryan, J W, Michie, D D, and Smith, D S(1971). Endothelial projections as revealed byscanning electron microscopy. Science, 173, 925-927.

Requests for reprints to: Professor Donald Heath,Department of Pathology, Duncan Building, RoyalLiverpool Hospital, Liverpool.

208



/T


ownloaded from


the pulmonary endothelial cell - bmj

Documents