Intimal Ultrastructure of Human Umbilical...

Intimal Ultrastructure of Human Umbilical Arteries

OBSERVATIONS ON ARTERIES FROM NEWBORN CHILDREN OF SMOKING ANDNONSMOKING MOTHERS

By Inger Asmussen and Knud Kjeldsen

ABSTRACTThe umbilical artery was chosen as a possible model for evaluating the vascular injury

provoked by tobacco smoking in humans. Cords from newborn children delivered by 15nonsmoking and 13 smoking mothers were studied in the transmission and the scanningelectron microscope. Pronounced intimal changes were seen in the arteries from smokingmothers; the most important findings were degenerative changes of the endothelium suchas swelling, blebbing, contraction, and subsequent opening of the endothelial junctionswith formation of subendothelial edema. Other observations included dilation of theendoplasmic reticulum in the endothelium and reparative changes such as a considerablewidening of the basement membrane. Since similar changes can be induced in arteries ofanimals by exposure to carbon monoxide or perfusion with nicotine, we conclude that thepresent study supports the concept that tobacco smoking is harmful to the vascularendothelium. This study also contributes to an understanding of the mechanism throughwhich vascular injury is provoked in heavy smokers.

KEY WORDS pregnancy placenta carbon monoxide nicotinetissue hypoxia vascular injury

• Clearly, a close connection exists between to-bacco smoking and development of arterial dis-eases, especially coronary heart disease and periph-eral arterial disease (1). The question is how doestobacco smoke provoke arterial damage. Evidenceaccumulated during the last decade indicates thatthe carbon monoxide in tobacco smoke is harmfulto the arterial wall (2). Exposure to low doses ofcarbon monoxide accelerates atherogenesis in cho-lesterol-fed animals (3-5) and produces significantultrastructural changes in the aortic and the coro-nary endothelium of rabbits and primates that areindistinguishable from early atherosclerosis (6, 7).For obvious reasons similar exposure studies andsubsequent vascular biopsies cannot be performedin humans. Moreover, a comparison of vascularbiopsy studies in human smokers and nonsmokershas not been published to date. As a possible modelfor evaluating the vascular damage provoked bytobacco smoking in humans, the umbilical artery ofbabies of smoking and nonsmoking mothers wasused for ultrastructural studies in the presentpaper.

From the Department of Obstetrics and Gynecology and theDepartment of Clinical Chemistry A, Rigshospitalet, Universityof Copenhagen, DK-2100 Copenhagen, Denmark.

This work was supported by The Danish Heart Foundationand The Danish Medical Research Council.

Received February 4, 1974. Accepted for publication January31, 1975.

MethodsPATIENTS

Pregnant women admitted to the Department ofObstetrics and Gynecology at Rigshospitalet were se-lected for the study. All patients were examined by thesame investigator, and each subject completed a ques-tionnaire on smoking habits before and during preg-nancy. Twenty-eight patients took part in the study, 13smokers and 15 nonsmokers. All smokers were inhalingcigarette smokers. One subject smoked 40-60 cigarettesdaily.

Women suffering from hypertension, diabetes, andother diseases and those with Rh negative blood typeswere not used in this study. The patients chosen for thestudy were normal before and during the pregnancy: alllaboratory investigations (hemoglobin, blood, sugar,etc.) including urinary analyses for sugar, protein, andestriol were normal. Therefore, the only difference in thetwo experimental groups was smoking habits.

The clinical data for these women are presented inTable 1. All of the patients were white, had normalpregnancies, and delivered babies at full term. All of thechildren were mature, and malformations were notfound.

The average weight of children born to smokers was3,370 g and that of children born to nonsmokers was3,695 g, a difference of 325 g. A similar difference of 123 gwas found in the weights of the placentas. Macroscopi-cally, placentas from smokers were less loose and morefibrotic in appearance than were those from the controlgroup. Neonatal icterus was found in five cases—four inthe control group. Ten of the 13 children born to smokerswere female.

ARTERIAL BIOPSIES AND PREPARATION FOR MICROSCOPY

All specimens were selected and handled through theinitial preparation by Dr. Asmussen. Immediately after

Circulation Research, Vol. 36, May 1975 579

by guest on May 4, 2018

http://circres.ahajournals.org/D

ownloaded from

http://circres.ahajournals.org/

580 ASMUSSEN, KJELDSEN

Clinical Data

Cigarettes/day

000000000000000

10101015152020202020253060

Mother'sage

(years)

32302229212826263225341823362432221721292628272319203031

Mother'sweightbefore

pregnancy(kg)

65567655536454486450554555526060555175506092756352506069

Child'sbirth

weight

(g)

3760283038504150345532853900334038503900400033003800390041003550315034503745355031253500265034003343312039803250

Child'slength(cm)

52505054505354505052555150525350525252505151515051515450

TABLE

Sex

FFMMFFMFMMFMMMMMMFFFFFFFMFFF

1

Apgarscore

10/109/10

10/109/109/10

10/1010/1010/1010/1010/109/10

10/1010/103/10

10/1010/1010/1010/1010/1010/1010/1010/108/10

10/1010/1010/1010/1010/10

Icterus

+

_++___

_

+_______

_

___+-

Placentaweight

(g)

620840840

1100870650600730710750950530750950850

630780680900570665440660750450780610

± Daysto term

+2- 7

+ 10- 1

+28+21+8

+40?+5- 2+6

0+ 14

- 2+ 10+8

- 2 1- 3+2

+ 15+8- 4

+ 18- 1 0

- 60

- 1 10

Duration oflabor

(hours)

<12<12<12Cold section<12 (section)<12<12<12<12<12<12 (section)<12<12<12<12

18<12<12

13<12<12<12<12<12

12<12<12<16

delivery of the placenta, the umbilical cord was cutabout 10 cm from the placenta. A soft plastic catheterwas placed in one of the umbilical arteries, and about 50ml of cold Ringer's solution was infused from a syringewith a light pressure. Perfusion was continued with 4.5%cold purified glutaraldehyde containing 2% acrolein andbuffered to pH 7.4 with 0.2M phosphate buffer for at least5 minutes to prevent contraction during the followingcutting procedures.

For transmission electron microscopy, the middleportion of the fixed artery was carefully dissected out,cut into small blocks, and immediately transferred to thesame glutaraldehyde for 1 hour. Postfixation was per-formed in 1% buffered osmium tetroxide (pH 7.4) for 1hour at 4°C. Tissue blocks were dehydrated in gradedethanols, cleared in propylene oxide, and embedded inEpon 812 or Araldite (Durcupan ACM). Sections 0.5-1.0urn thick were stained with toluidine blue for lightmicroscopy. Ultrathin sections were cut on glass kniveswith an LKB Ultrotome III, mounted on uncoated coppergrids, and contrasted with magnesium uranyl acetate (8)and lead citrate (9). These sections were examined andphotographed in a Zeiss EM 9S-2 electron microscope.

Tissue for scanning electron microscopy was obtainedin the following way. After perfusion, the artery was cutlongitudinally and immediately transferred to a flat

vessel containing 4.5% cold glutaraldehyde. Samplesmeasuring about 3 x 3 mm were cut out, fixed withneedles to a cork plate, and placed with the endotheliumdownward in a small beaker with the same glutaralde-hyde for 1 hour at 4°C. Stretching, bending, and touch-ing of the inner surface were carefully avoided. Postfixa-tion was performed in 1% buffered osmium tetroxide (pH7.4) at 4°C. After dehydration with acetone, the speci-mens were taken through graded mixtures of acetone andbenzene to pure benzene, freeze-dried, and mounted onaluminum stubs with silver paste; a conduction coat ofabout 200 A of gold was deposited in a vacuum with anEdwards 306 coater. The endothelial surface was studiedin a Cambridge Steroscan S-600 scanning electron mi-croscope and photographed with a Hasselblad camera.

ResultsNONSMOKERS

In the scanning electron microscope, the endo-thelial surface of the umbilical arteries from non-smokers showed a regular pattern of spindle-shaped cells (Fig. 1) oriented longitudinally andfollowing the spiraling course of the vessel. Withthe present technique, the width of the cells was

Circulation Research, Vol. 36, May 1975



ownloaded from


ULTRASTRUCTURE OF UMBILICAL ARTERIES IN SMOKERS 581

FIGURE 1

Surface of an umbilical artery from a child of a nonsmokingmother showing the spindle-shaped endothelial cells arrangedlongitudinally. Bar indicates 40 pm.

approximately 5 nm and the length varied consid-erably, probably depending on the degree of con-traction in the individual cells. The shortest endo-thelial cells measured about 50 pm and the longestabout 100 ^m.

In the transmission electron microscope, theendothelium lining the luminal surface was of thecontinuous type with closed intercellular junctions.The cytoplasm was rich in the organelles usuallyfound in arterial endothelium: rough endoplasmicreticulum, mitochondria, and Golgi complexes(Fig. 2). Although the number of mitochondria wasmoderate, the Golgi complexes and rough endo-plasmic reticulum were often highly developed.The nuclei were usually crenated and containedwell-developed nucleoli.

Pinocytotic vesicles were seen dispersed in thecytoplasm; they were especially numerous at theluminal surface and at the base of the endothelialcells. A regular feature in this area was the pres-ence of small bundles of fine filaments oriented inthe longitudinal axis of the cells; these filamentsresembled the myofilaments of the underlyingsmooth muscle cells. Centrosomes and lysosomeswere also observed.

No continuous endothelial basement membranewas present. The basal lamina appeared to consistof fragments of homogeneous material with amedium electron density.

The subendothelial area contained a sparseCirculation Research, Vol. 36, May 1975

ground substance and smooth muscle cells, often inclose apposition to the endothelial lining, and wasrich in bundles of collagen fibers. Since no internalelastic membrane was present, there was no sharpdemarcation between the intima and the tunicamedia. Occasionally, lymphocytes were seen justbeneath the endothelial cells.SMOKERS

Many pathological changes in the intima wereobserved in the arterial samples from smokers. Inall of the specimens observed in the scanningelectron microscope, the most conspicuous findingwas large areas of swollen and irregular endothelialcells with a peculiar cobblestone appearance (Fig.3). At higher magnifications, small cytoplasmicprocesses (blebs) were often seen protruding fromthe surface of these cells (Fig. 4).

In the transmission electron microscope, edema-tous swelling and degenerative changes were char-acteristic features. Endothelial swelling was seen inareas of the cobblestone endothelium. As a rule,the rough endoplasmic reticulum in such cells wasalso enormously dilated by a fluid with a moderateelectron density (Fig. 5). Lysosomes of two typeswere often seen: one type contained marginallyarranged, electron-dense, round bodies withopaque dots, giving the organelle a wheellikeappearance (Fig. 5) and the other type exhibited acharacteristic striation (Fig. 6). In some areas, thebasal myoid filaments were considerably con-tracted, giving the endothelial cells a balloonlikeappearance.

Even in areas without swelling of the endothe-lium, extensive edema of the subendothelial spacewas a regular finding (Fig. 7). The basementmembrane was always considerably thickened—often equal in width to the endothelial lining; itconsisted of a fine reticular material with a feltliketexture (Figs. 6-8). The smooth muscle cells in theedematous subendothelial space often showedvacuolization. Cytoplasmic protrusions of the cellsurface (blebs) were also seen in the transmissionelectron microscope (Figs. 5, 8). In edematousareas, focal loss of closed intercellular junctionswas a characteristic finding (Fig. 6). In addition tothese observations, a decreased amount of collagenfibers and an absence of lymphocyte infiltration ofthe luminal part of the arteries were seen.

The morphological findings have been correlatedwith tobacco consumption in Table 2.

Discussion

Sheppard and Bishop (10) recently described thefine structure of sheep umbilical vessels, but the



ownloaded from


582 ASMUSSEN. KJELDSEN

FIGURE 2

Longitudinal section of an endothelial cell from an umbilical artery from a child of a nonsmoking mother. L = lumen, M =mitochondrion, F = filaments, G = Golgi apparatus, ER = endoplasmic reticulum, and BM = basement membrane. Bar indicates1 fim.

only report on the ultrastructure of human umbili-cal vessel endothelium at term seems to be that ofParry and Abramovich (11). Although doubleclamping of the selected arterial segments andimmersion in cold fixative was used in both of thesestudies to prevent artifacts due to arterial contrac-tion, Sheppard and Bishop (10) also injected coldfixative into the lumen to retain the fine structureof the intima. In our experience, satisfactory inti-mal ultrastructure can also be obtained by perfus-ing glutaraldehyde containing formaldehyde oracrolein to ensure fast penetration of the fixative.

The findings of Sheppard and Bishop (10) and ofParry and Abramovich (11) of a metabolicallyactive arterial endothelium with closed junctions, athin fragmented basal lamina, and sparse elastictissue in the intima are very similar to the observa-tions in the nonsmoking controls of the presentstudy. No reports of ultrastructural pathological

changes in the umbilical vessels can be found in theliterature.

Pathological intimal changes identical to thoseseen in the specimens of the umbilical arteries fromsmoking mothers have previously been reportedfrom our laboratory in rabbits exposed for 2 weeksto a moderate degree of carbon monoxide or arterialhypoxia (7, 8). An edematous reaction of theendothelium has also been described by otherauthors following exposure to severe grades ofhypoxia (12-14) and to various compounds such ascatecholamines, angiotensin, endotoxin, and cer-tain amines (15,16). Therefore, it seems reasonableto assume that edematous swelling or blebbing andcontraction with subsequent opening of the endo-thelial junctions is a characteristic reaction of theendothelium to injury.

The oxygen saturation of fetal hemoglobin in theumbilical vein is about 70%; in the umbilical artery




ownloaded from



it is about 26% (17). Fetal carboxyhemoglobinsaturations due to maternal smoking vary between2.5% and 10% depending on the degree of inhala-tion of the mother (18, 19). On the average, fetalcarboxyhemoglobin saturations are about 1.8 timeshigher than the corresponding maternal values andare in agreement with the values calculated accord-ing to Haldane's original equation with due regardto the fetal and maternal blood oxygen tensions.

Due to the low fetal oxygen saturation, thepresence of carboxyhemoglobin has a more seriouseffect on fetal blood oxygen transport than it doeson maternal blood oxygen transport. For example,a maternal carboxyhemoglobin level of 6%, corre-sponding to a fetal carboxyhemoglobin level ofabout 11%, will reduce fetal blood oxygen transportapproximately 15% and therefore significantly in-terfere with fetal tissue oxygenation.

In the wall of the umbilical artery, the competi-tion between carbon monoxide and oxygen due tothe low oxygen tensions in the blood will be morepronounced than it is in the wall of the maternalarteries and may subsequently induce greater ul-trastructural changes. Although the intra-arterialpressure is only 50 mm Hg in the human umbilicalartery, the ultrastructural findings reported in thisstudy show striking similarities to those reported inthe aorta and the coronary arteries of animals afterlight in vivo exposure to carbon monoxide for 2weeks (6) or perfusion for 40 seconds with a solutionof 100 g/ml of nicotine in buffer (20). Endothelialswelling and degeneration along with opening of

FIGURE 3

Surface of an umbilical artery from a child of a smoking mother(20 cigarettes/day). Note the cobblestone appearance of theendothelium due to swelling and contraction of the endothelialcells. Bar indicates 20 fim.Circulation Research, Vol. 36, May 1975

FIGURE 4

Surface of an umbilical artery from a child of a smoking mother(20 cigarettes/day). The endothelium is studded with cytoplas-mic blebs. The endothelial cells appear swollen and irregular.Bar indicates 5 \im.

endothelial cell junctions and formation of a largeedema in the subendothelial space were character-istic features in these two animal studies. However,the pronounced dilation of the endoplasmic reticu-lum and the wide basement membrane reported inthe present study were not observed in the animalexperiments; these morphological dissimilaritiesmay reflect differences in metabolism and adapta-tion in fetal and adult endothelium that render thefetal tissue more sensitive to this type of hypoxia.With these distinctions in mind, the observationthat practically no overlapping of the pathologicalfindings was present in this study suggests that theumbilical artery may prove to be a useful model ofthe vascular injury produced not only by tobaccosmoking in humans but also by other agents toxicto the vascular system.

In our opinion, the results of this study supportthe concept that tobacco smoking is harmful to thevascular endothelium. These findings also contrib-ute to an understanding of the mechanism throughwhich vascular injury is provoked in heavy smok-ers.



ownloaded from



" :&

FIGURE S

Endothelial cell from an umbilical artery from a child of a smoking mother (60 cigarettes I day). Notethe swelling of the cell with formation ofluminal blebs and the dilation of the endoplasmic reticulum.Beneath the nucleus is a wheellike lysosome. L = lumen, B = cytoplasmic process forming a surfacebleb, ER = endoplasmic reticulum, Ly = lysosome, and IJ = intercellular junction. Bar indicates 1




ownloaded from



BM

BM

.

FIGURE 6

Intima of an umbilical artery from a child of a smoking mother (20 cigarettes/day). Photomontage showing two endothelial cells withfocal loss of closed intercellular junctions. Note the wide, continuous basement membrane. L = lumen, E = endothelial cell. IJ = inter-cellular junction, BM = basement membrane, F = filaments, G = Golgi apparatus. Ly = lysosome, ER = endoplasmic reticulum, andM = mitochondrion. Bar indicates I ^m.




ownloaded from



FIGURE 7

Intima of an umbilical artery from a child of a smoking mother (25 cigarettes/day). Note theedematous subendothelial space containing smooth muscle cells and the wide basement membrane.L = lumen, E = endothelium, BM = basement membrane, SES = subendothelial space, and SM =smooth muscle cells. Bar indicates 5 nm.




ownloaded from



FIGURE 8

Intima of an umbilical artery from a child of a smoking mother (25 cigarettes/day). Note blebbing ofthe cell surface and the wide basement membrane. L = lumen, E = endothelium, B = cytoplasmicbleb, M = mitochondrion with condensation of the matrix, BM = basement membrane, C = collagenfibers, and SM = smooth muscle cell. Bar indicates 1 nm.




ownloaded from


588 ASMUSSEN, KJELDSEN

TABLE 2

Morphological Changes

Cigarettes/day

000000000000000

10101015152020202020253060

Continuousbasementmembrane

___

( + )( + )___

( + )( + )__

_

+( + )++++++++++

Basementmembrane

average width(M)

0.10.10.10.10.30.30.90.10.10.10.20.10.10.10.1

0.60.80.91.20.91.51.22.21.62.82.53.5

EdemaOpening ofjunctions

Pathologicalmyocytes

Dilation ofendoplasmic

reticulum

The morphological changes were graded on a scale from + to + + + + . The most marked difference between the smokers and thenonsmokers was the reparative changes in the basement membrane, which exhibited a considerable widening, and the formation of acontinuous membrane. (+) implies that only short patches of continuous basement membrane were seen in the artery. Edemaincludes both intimal and subintimal edema. The degree of opening of the junctions refers to the focal loss of tight intercellularjunctions. The changes in the smooth muscle cells were classified according to the degree of vacuolization, ameboid polymorphism, and"woolly" outer surface. The dilation of the endoplasmic reticulum due to an electron-dense homogeneous mass was only pronounced inthe one smoker who consumed 60 cigarettes/day.

Acknowledgment

The authors are indebted to Poul Astrup, M.D., Dyre Trolle,M.D., and J«rgen Falck-Larsen, M.D., for valuable discussionsleading to the design of this study. We also want to express ourgratitude to Morten Nielsen, M.D., for his kind interest in ourwork and for his practical help in many ways and to NinaLorentzen and Hanne Hansen for their skillful technical assist-ance. The photographic work by Erik Jensenius and LennartLarsen is also gratefully acknowledged.

References1. Smoking and Health: Report of the Advisory Committee to

the Surgeon General. Washington, D.C., Public HealthService, Publication no. 1103, 1964

2. ASTRUP P: Some physiological and pathological effects ofmoderate carbon monoxide exposure. Br Med J4:447-452, 1972

3. ASTRUP P, KJELDSEN K, WANSTRUPJ: Enhancing influence of

carbon monoxide on the development of atheromatosis incholesterol-fed rabbits. Atherosclerosis 7:343-354, 1967

4. BIRNSTINGL M, HAWKINS L, MCEWEN T: Experimentalatherosclerosis during chronic exposure to carbon monox-ide. Eur Surg Res 2:92-93, 1970

5. WEBSTER WS, CLARKSON TB, LOFLAND HB: Carbon monox-ide-aggravated atherosclerosis in the squirrel monkey.Exp Mol Pathol 13:36-50, 1970

6. KJELDSEN K, ASTRUP P, WANSTRUP J: Ultrastructural inti-mal changes in the rabbit aorta after a moderate carbonmonoxide exposure. Atherosclerosis 16:67-82, 1972

8. THOMSEN HK: Carbon monoxide-induced atherosclerosis inprimates. Atherosclerosis 20:233-244. 1974

8. FRASCA JM, PARK VR: Routine technique for double-stain-ing ultrathin sections using uranyl and lead salts. J CellBiol 25:157-161, 1965

9. REYNOLDS ES: Use of lead citrate at high pH as anelectron-opaque stain in electron microscopy. J Cell Biol17:208-212, 1963

10. SHEPPARD BL, BISHOP AJ: Electron microscopical observa-




ownloaded from



tions on sheep umbilical vessels. Q J Exp Physiol 58:39-45, 1973

11. PARRY EW, ABRAMOVICH DR: Ultrastructure of humanumbilical vessel endothelium from early pregnancy to fullterm. J Anat 111:29-42, 1972

12. Ts'Ao C-H: Graded endothelial injury of the rabbit aorta.Arch Pathol 90:222-229, 1970

13. CONSTANTINIDES P, ROBINSON M: Ultrastructural injury ofarterial endothelium: I. Effects of pH, osmolarity, anoxiaand temperature. Arch Pathol 88:99-105, 1969

14. Ts'Ao C-H, GLAGOV S: Basal endothelial attachment. LabInvest 23:510-516, 1970

15. CONSTANTINIDES P, ROBINSON M: Ultrastructural injury ofarterial endothelium. II. Effects of vasoactive amines.Arch Pathol 88:106-117, 1969

16. SHIMAMOTO T: New concept on atherogenesis and treatmentof atherosclerotic diseases. Jap Heart J 13:537-562, 1972

17. BRINKMAN C: Umbilical blood flow and fetal oxygen con-sumption. Clin Obstet Gynecol 13:565-578, 1970

18. LONGO LD: Carbon monoxide in the pregnant mother andfetus and its exchange across the placenta. Ann NY AcadSci 174:313-341, 1970

19. ASTRUP P, TROLLE D, OLSEN HM, KJELDSEN K: Effect of

moderate carbon-monoxide exposure on fetal develop-ment. Lancet 2:1220-1222, 1972

20. CONSTANTINIDES P: Important role of endothelial changes inatherogenesis. In Atherogenesis, vol 2, edited by T Shima-moto, F Numano, and GM Addison. Amsterdam, Ex-cerpta Medica 1972, pp 51-65




ownloaded from


I Asmussen and K Kjeldsenchildren of smoking and nonsmoking mothers.

Intimal ultrastructure of human umbilical arteries. Observations on arteries from newborn

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 1975 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

doi: 10.1161/01.RES.36.5.5791975;36:579-589Circ Res.

http://circres.ahajournals.org/content/36/5/579World Wide Web at:

The online version of this article, along with updated information and services, is located on the

http://circres.ahajournals.org//subscriptions/

is online at: Circulation Research Information about subscribing to Subscriptions:

http://www.lww.com/reprints Information about reprints can be found online at: Reprints:

document. Permissions and Rights Question and Answer about this process is available in the

located, click Request Permissions in the middle column of the Web page under Services. Further informationEditorial Office. Once the online version of the published article for which permission is being requested is

can be obtained via RightsLink, a service of the Copyright Clearance Center, not theCirculation Research Requests for permissions to reproduce figures, tables, or portions of articles originally published inPermissions:



ownloaded from

http://circres.ahajournals.org/content/36/5/579

http://www.ahajournals.org/site/rights/

http://www.lww.com/reprints

http://circres.ahajournals.org//subscriptions/


Intimal Ultrastructure of Human Umbilical...

Documents

Transcript of Intimal Ultrastructure of Human Umbilical...