Atherosclerotic lesions are not uniformly distributed

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1 Albumin and Cr-EDTA Uptake by Systemic Arteries, Veins, and Pulmnary Artery f Rabbit M. Jhn Lever and Mark T. Jay Experiments have been perfrmed bth in viv and In vitr t measure the steady-state uptake f labeled albumin and Cr-ethylenedlamlnetetraacetate by varius bld vessels f the rabbit the ascending and descending prtins f the thracic arta, the cartid artery, the pulmnary artery, and the Inferir vena cava. The In vitr experiments Indicated that the wall tissues f the pulmnary artery and the vena cava have much greater distributin vlumes fr albumin than d the systemic arteries. This may In part explain the differences In wall tissue cncentratins In viv and, In turn, the differences between vessels In their susceptibility t athersclersis. (Arterisclersis 10: , July/August 1990) Athersclertic lesins are nt unifrmly distributed amng bld vessels. They ccur mst cmmnly In certain thick-walled arteries 12 but are nrmally absent frm veins and frm pulmnary arteries unless there is pulmnary hypertensin. 3 One prcess that appears t be imprtant in lesin develpment is the mvement f material between the bld and the wall tissue. This prcess als shws reginal variatin; uptake f labeled prtein frm the bld ccurs t a greater extent in the prximal than in the distal parts f the arta, 456 while uptake by the pulmnary artery trunk greatly exceeds that by the arta. 7 The level t which a tracer Is taken up by a bld vessel wall is determined by varius factrs, including the rate f tracer mvement thrugh the endthelium and the deeper layers f the wall and als by the distributin vlumes f the tracer within the different layers. The main purpse f the present study was t investigate hw the distributin vlumes fr tracers vary between different bld vessels. We measured the values fr a small tracer, 51 Cr-ethylenediaminetetraacetate (EDTA), which is cmmnly used fr the estimatin f the ttal extracellular space In tissues, 8 and als fr 12S l-bvlne serum albumin (BSA), which is excluded t a cnsiderable degree frm the media f the cartid artery. 9 BSA is nt excluded t the same extent as lipprteins, 10 but its distributin vlume appears t be a sensitive index f differences in interstitial prperties. Measurements have been made by incubating vessels in labeled slutins fr a sufficient time fr tracer mlecule cncentratins in the tissues t apprach equilibrium values. We studied bth relaxed vessel segments and als air-pressurized vessels, since tissue strain appears t mdify distributin vlumes. 11 We have als determined the steady-state wall tissue Frm the Physilgical Rw Studies Unit Imperial Cllege, Lndn, U.K. This wrk was supprted by the Natinal Heart Research Fund and Schwartz Pharmaceuticals Limited. Address fr reprints: M. Jhn Lever, Physilgical Flw Studies Unit, Imperial Cllege, Lndn SW7 2AZ, U.K. Received August 18, 1989; revisin accepted February 16, levels that are attained In the same vessels when labeled albumin is intrduced int the circulatin in viv. Methds In Wv Studies Eight New Zealand White rabbits (2 t 2.5 kg) were anesthetized by injectin f 30 mg/kg f pentbarbitne (Sagatal, May & Baker FaJrfield, Hampshire) int the marginal ear vein. During the experiment this was supplemented t maintain a cnstant level f anesthesia. 12S I-BSA (150 fic\ prepared by the chlramine T methd and purified by repeated dialysis at 4 C) was injected int the same vein. Bld was sampled at 5 minutes, 1 hur, and 3 hurs after injectin via a cannula in the femral artery. The plasma was separated by centrifugatin, and radiactivity was measured in 40 /il aliquts in a gamma cunter. After 3 hurs, an incisin was made in the abdmen and a 16 G needle was placed in the abdminal arta s that it pinted tward the heart Istnic saline and then, after 30 secnds, 4% glutaraldehyde in saline were flushed thrugh the needle int the arta frm reservirs placed 100 cm abve the animal. Bld and excess fluid left the circulatin thrugh veins damaged during expsure f the abdminal arta After a 20-minute fixatin at a perfusin pressure f 100 cm H 2 O, varius vessels were excised. These included tw prtins f the ascending arta (the inner and uter surfaces f curvature); a segment f the descending thracic arta between the secnd and fifth intercstal arteries, which was divided int drsal and ventral prtins; the cmmn cartid artery; the pulmnary artery trunk between its rigin and its divisin int separate pulmnary arteries; and the inferir vena cava between the diaphragm and the right atrium. The fixed, excised vessels were washed in saline fr several hurs, then were pened axially and placed intimal surface dwn nt lightly greased slides. After freezing, a micrtme was used t cut 10 ^m, en face sectins thrugh the whle thickness f the wall frm the luminal t the adventjtial surfaces. 9 After drying, the 551

2 552 ARTERIOSCLEROSIS VOL 10, N 4, JULY/AUGUST 1990 sectins were cunted fr radiactivity and were weighed. The tissue water cntent was estimated by using the dry sectin weights and the sectin vlume (btained frm the width and length f the tissue blck and the sectin thickness). Tissue label cncentratin (Ct) was estimated frm the cunts and sectin vlumes and was expressed as a rati f the average plasma cncentratin (C p ). Prfiles f label cncentratin acrss the thickness f the wall were btained by pltting the C,/C p value f each sectin as a functin f its distance frm the luminal surface. Mean prfiles fr each vessel were btained by averaging the data frm different experiments. Transverse sectins were btained in fur experiments and were used t assess the thickness f the medial and adventitial layers f the wall. The estimates f medial thickness were used t select C,/C p values fr calculatin f a mean medial value fr each vessel. In Vitr Studies Relaxed Vessels Rabbits were killed with an verdse f pentbarbitne, and segments f the vessels listed abve were quickly excised. After a saline rinse, the segments were incubated in Krebs slutin cntaining 4% BSA and dialyzed either 126 I-BSA r 61 Cr-EDTA. The ph was adjusted t 7.4, and the slutin was kept at 37 C and was bubbled with 95% O 2 /5% CO 2. Aliquts f the incubating slutin were taken at intervals fr estimatin f label cncentratin (Cp). After 1 r 3 hurs, segments were remved and briefly rinsed in saline t wash ff the surface label. Thse segments that had been incubated in 12S I-BSA were immersed in 4% glutaraldehyde fr 20 minutes and then in saline fr several hurs t remve the free label that was nt bund t the albumin. All segments were sectined en face at 10 ^m intervals as described abve t btain C, /C p prfiles and water cntents. Segments incubated in 61 Cr-EDTA were rinsed, frzen, and sectined withut fixatin. Transverse sectins were used t estimate the psitin f the medialadventitial bundary in each vessel. Because glutaraldehyde fixatin is knwn t cause tissue shrinkage, we assessed the effects f fixatin n tissue water cntents and labeled albumin cncentratins by perfrming measurements n each f fur cartid arteries and artas. After incubatin in labeled slutin, each vessel was cut int tw prtins, ne f which was fixed befre sectining, while the ther was frzen and sectined immediately. Air-pressurized Vessels Rabbits were anesthetized as described abve, and while the lungs were artificially ventilated, the thrax was pened by a mid-sternal split. Heparin (1000 IU) was injected intravenusly t prevent bld cagulatin. The cmmn cartid arteries and the arta were expsed, and ligatures were placed arund the rts f the right cartid, right and left subclavian, the three mst prximal pairs f intercstal arteries, and arund the great veins where they enter the right atrium. A cannula was intrduced int the right ventricle thrugh an incisin at the apex f the heart, and Krebs slutin cntaining 4% BSA was gently injected t flush bld frm the heart and pulmnary arteries. After a ligature was placed arund the left atrium, a secnd cannula was placed in the left ventricle, and bld was flushed frm the arta and the left cartid artery. The cannulated heart was then excised with the attached pulmnary artery, arta, and cartid artery. Adhering fat was carefully remved frm the vessels s that their uter surfaces wuld be fully expsed t the incubating slutins. Air was flushed thrugh bth cannulas t displace the Krebs slutin frm the vessels, which were then tied at their distal ends. The cartid artery and the arta were air-pressurized via the cannula in the left ventricle t 100 cm H 2 O, and the pulmnary artery trunk was air-pressurized t 20 cm H a O via the cannula in the right ventricle. The pressurized arteries were then immersed in Krebs slutin cntaining 2% BSA and 12S I-BSA. The slutin was maintained at 37 C and was bubbled with 95% 0^5% CO 2. After incubatin fr 3 r 6 hurs, the heart and vessels were rinsed quickly with saline, were fixed fr 20 minutes in 4% glutaraldehyde while still pressurized, and were then incubated in saline. The individual vessels were sectined as described abve. Statistical Analysis Analyses f variance were used t investigate the differences amng the water cntents and the mean medial C,/C p values measured in the different vessels when studied under each f the three experimental cnditins: in viv, relaxed in vitr, and air-pressurized in vitr. Multiple cmparisns between pairs f vessels were then made by using the Bnferrni methd. 12 Further analyses f variance were perfrmed t assess the effect f the three experimental cnditins n the values btained with each f the vessels. Results Tissue Levels f 1i5 l-bvine Serum Albumin In Vessels In Viv The average 125 I-BSA C,/C p prfiles thrugh the thickness f the walls f each vessel are shwn in Figure 1, and the mean medial Ct /C p values fr each vessel are shwn in Table 1 tgether with medial thicknesses and medial water cntents. Despite a twfld difference in the medial thickness f the prximal and distal prtins f the pulmnary artery, the mean medial C,/C p values fr the tw regins were nt significantly different. N significant differences were seen either between the inner and uter walls f curvature f the ascending arta r between the ventral and drsal walls f the descending thracic arta. Analysis f variance shwed there t be significant differences between the C,/C p fr the five different vessels. The values fr the pulmnary artery and the vena cava were significantly higher than thse f the ascending arta (p<0.05 in bth cases), which, in turn, were higher than thse f the descending thracic arta and the cartid artery (p<0.05 in bth cases).

3 TRACER UPTAKE BY DIFFERENT BLOOD VESSELS Lever and Jay 553 ASCENDING AORTA Outtr wait Inntr vatl 40 Distance frm luminal surface Figure 1. Graphs shwing mean C/C p values in viv fr 125!-bvlne serum albumin acrss the thickness f the vessel walls frm the luminal t the adventitial surfaces. Vertical bars Indicate the standard deviatins f the values, and the hrizntal bars indicate the means and standard deviatins f the psitins f the medial-adventitial bundary estimated frm transverse sectins. The data were btained frm the vessels frm seven rabbits. Table 1. Medial Thickness, Water Cntents, and 3-hur C,/C p Values fr 12S l-bvlne Serum Albumin fr Different Vessels In Viv Vessel Ascending arta Descending thracic arta Cartid artery Pulmnary artery (upstream) Pulmnary artery (dwnstream) Inferir vena cava n Medial thickness (jim) 214±43 105±16 48±6 97±43 54±24 16±5 Medial water cntent (%) 69±2 64±3 66±5 76±3 76±2 81 ±3 Values are the means±sd fr vessel frm seven rabbits, n-number f vessel segments. BSA=bvine serum albumin, C,-tissue label cncentratin, C p =piasma cncentratin. Medial C/C p fr 12B I-BSA 0.029± ± ± ± ± ±0.019 Tissue Levels f 129 l-bvlne Serum Albumin and 51 Cr-EDTA In Relaxed Arteries In Vitr The average 12S I-BSA C,/Cp prfiles after 3 hurs f incubatin are shwn in Figure 2 and thse fr "Cr-EDTA, in Figure 3. The mean medial C,/C p values are shwn in Table 2. In the experiments perfrmed t test the effects f fixatin, the rati f the 126 I-BSA C,/C p values in the fixed tissue t thse in the nnfixed tissue was 0.95±0.09 (n=8). This rati is nt significantly different frm 1, but lwer values might be expected in the fixed tissue because f the washut f tracer, which is nt bund t albumin. There were n differences between the 125 I-BSA CyC p values fr the prximal and distal prtins f the pulmnary artery r between the ventral and drsal prtins f the descending thracic arta, but thse fr the inner wall f curvature f the ascending arta (0.111, SD=0.019) were greater than thse fr the uter wall (0.080, SD=0.015, p<0.05). Analysis f variance shwed that the mean medial 12S I-BSA Q/Cp values were lwer after 1 hur than after 3-hur incubatins fr the pulmnary artery (p<0.05), but that the 1- and 3-hur values fr the descending thracic arta and the cartid arteries were nt significantly different The values fr the vena cava were greater than thse fr the pulmnary artery (p<0.001), which in turn, were greater than thse f the ther arteries studied (p<0.001 in all cases).

4 554 ARTERIOSCLEROSIS VOL 10, N 4, JULY/AUGUST 1990 ASCENDING AORTA Oufrtr vail laa«r veil 1-0 DESCENDING THORACIC AORTA Mr PULMONARY ARTERY HIT INFERIOR VENACAVA <K 1 tt T tt " 05 <» Distance frm luminal surface Figure 2. Graphs shwing mean Ct/Cp values fr 12S l-bvine serum albumin acrss the thickness f relaxed vessel walls in vitr frm the luminal t the adventitial surfaces. Vertical bars indicate the standard deviatins f the values, and the hrizntal bars indicate the means and standard deviatins f the psitins f the medlaj-adventitial bundary estimated frm transverse sectins. Trie data were btained frm the vessels frm 11 rabbits. ASCENDING AORTA 1-0 F DESCENDING THORACIC AORTA f I i 1 s 05 { M + tt+*l + M* H r PULMONARY ARTERY INFERIOR VENACAVA Distance frm luminal surface pm Figure 3. Graphs shwing mean C/C p values fr B1 Cr-ethylenedlamlnetetraacetate acrss the thickness f relaxed vessel walls in vitr frm the luminal t the adventttial surfaces. Vertical bars indicate the standard deviatins f the values, and the hrizntal bare indicate the means and standard deviatins f the psitins f the medlaj-adventitial bundary estimated frm transverse sectins. The data were btained frm the vessels frm fur rabbits. The mean medial 125 I-BSA C,/C p values fr each bld vessel were greater when measured in the relaxed state in vitr than in viv in the ascending arta by 3.3 times, in the descending thracic arta by 11.9 times, in the cartid artery by 20 times, in the pulmnary artery by 4.5 times, and in the vena cava by 15.1 times (p< in all cases). TTie mean medial 51 Cr-EDTA C,/C p values after 1- and 3-hur incubatins were fund t be indistinguishable and were, therefre, pled. Analysis f variance shwed there t be significant differences between the values fr the five vessels. The values fr the vena cava and the pulmnary artery were greater than thse f the ther vessels (p<0.01 andp<0.05, respectively), but the mag-

5 TRACER UPTAKE BY DIFFERENT BLOOD VESSELS Lever and Jay 555 Table 2. Medial Thickness, Water Cntents, and 3-hur Ct/C p Values fr 12S l-bvlne Serum Albumin and "Cr-EDTA fr Relaxed Vessel Segments In Vitr Medial Medial C/Cp, fr 125 I-BSA Media) C/Cp fr»r B1 Tr PITTA 1 Vessel (jim) cntent (%) 1-hur 3-hur and 3 hurs Ascending arta 279±56 73±2(14) ±0.023 (14) 0.45±0.02 (6) Descending thracic arta 181 ±34 72±3(16) 0.092±0.038 (6) 0.107±0.021 (10) 0.43±0.01 (11) Cartid artery 91 ±8 76±5(12) 0.094±0.018 (6) 0.104±0.017 (6) 0.41 ±0.02 (6) Pulmnary artery 155±40 81 ±4 (26) 0.174±0.053 (9) 0.234±0.038(17) 0.46±0.01 (11) Inferir vena cava 16±12 81 ±6 (19) 0.508±0.21 (10) ±0.08 (6) Values are means±sd. Vessels were excised frm 11 rabbits. Values in parentheses indicate number f vessel segments. BSA=bvine serum albumin, EDTA-ethyienedlamlnetetraacetate. (0 I M U Mr "ASCENDING Outtr wall Innir wait AORTA f * r CAROTID ARTERY Distance frm Iuminai surface DESCENDING THORACIC AORTA Figure 4. Graphs shwing mean C, /C p values fr 125 l-bvlne serum albumin acrss the thickness f air-pressurized vessel walls In vitr frm the Iuminai t the adventltial surfaces. Vertical bars indicate the standard deviatins f the values, and the hrizntal bars indicate the means and standard deviatins f the psitins f the medlat-adventitial bundary estimated frm transverse sectins. The data were btained frm the vessels frm 10 rabbits. nitudes f the differences between the vessels were small cmpared with the values fr 123 I-BSA. Tissue Levels f 1u l-bvlne Serum Albumin In Air-pressurized Arteries In Vitr The average prfiles f Ct/C p values fr 12S I-BSA acrss the walls f each vessel are shwn in Figure 4. The prfiles indicate a steeper increase in medial Ct/C p values away frm the Iuminai surface and tward the adventrtial surface cmpared t thse in relaxed vessels r fr thse studied In viv. A similar bservatin was made in an earlier study n the air-pressurized descending thracic arta 11 This may be due in part t greater cmpactin f the inner layers f the wall cmpared with the uter layers. Hwever, anther factr in the present study, which might cntribute t the bserved prfile shape, is a variatin in medial thickness as measured frm transverse sectins. This variatin ccurred bth circumferentially and axialty, particularly in the ascending arta and in the pulmnary artery, and sme en face sectins frm these vessels included bth medial and adventrtial tissue. The mean medial C,/C p values fr each vessel shwn in Table 3 were, therefre, calculated by using data frm sectins assumed (frm measurements f medial thickness in transverse sectins) t cntain nly medial tissue. There was n difference in the values btained after 3 r

6 556 ARTERIOSCLEROSIS VOL 10, N 4, JULY/AUGUST 1990 Table 3. Medial Thickness, Water Cntents, and 3-hur C,/C p Values fr 12S l-bv1ne Serum Albumin fr Segments f Air-pressurized Vessels In Vitr Vessel Ascending arta Medial 170±49 cntent (%) 65±6 (20) Medial C,/C p fr BSA 3-hur 0.035±0.017(16) 6-hur 0.033±0.013(4) Descending thracic arta Cartid artery Pulmnary artery 101±16 49±10 53±7 61 ±8 (30) 67±7 (8) 73 ±9 (34) ±0.019 (22) 0.043±0.001 (4) 0.142±0.073 (28) (8) (4) (6) Values are means±sd In vessels frm 10 rabbits. Values in parentheses indicate number f vessel segments. BSA=bvine serum albumin. 6 hurs f incubatin. The value fr each air-pressurized vessel was lwer than fr the crrespnding relaxed vessel (p<0.001 fr all vessels except the cartid artery, fr which p<0.05). The value fr the pulmnary artery was greater than thse fr the ther vessels (p< 0.001). Medial Water Cntents f Bld Vessels The medial water cntents f the different vessels measured under the different experimental cnditins are shwn with the ther data in Tables 1 t 3. Fixatin caused significant shrinkage f the tissues; the rati f the water cntent f the fixed tissue relative t that f the unfixed tissue was the same in the cartid artery and the arta and averaged 0.90±0.04 (n=8). Values fr Vessels Fixed In Viv Analysis f variance shwed significant differences between the water cntents f the vessels (p<0.001). The mean medial water cntent f the vena cava was greater than that f the pulmnary artery (p<0.05), which was greater than that f the ascending arta (p<0.01). The ascending arta had a greater value than the descending thracic arta (p<0.05). Values fr Relaxed Vessels Fixed In Vitr The mean medial water cntents f the vena cava and the pulmnary artery were greater than thse f the ther vessels (p<0.05 in all cases). The medial water cntents f sme f the vessels incubated in the relaxed state and fixed in vitr were greater than thse f the same vessels fixed in viv (pulmnary artery, p<0.05; cartid artery and descending thracic arta, p<0.001). Values fr Air-pressurized Vessels Fixed In Vitr The medial water cntent f the pulmnary artery was greater than thse f the ther vessels (p<0.01 fr the ascending arta and the descending thracic arta and p<0.05 fr the cartid artery), and thse f the ascending arta and the cartid artery were greater than that f the descending thracic arta (p<0.05). Each f the values in the air-pressured vessels was lwer than thse in relaxed vessels (p< fr all vessels except the cartid artery, fr which p<0.05). Only in the ascending arta was there a significantly lwer water cntent in the air-pressurized vessels than in viv (p<0.05). Discussin Tissue Levels f 12B I-Bvlne Serum Albumin In Viv Data frm previus studies in the rabbit suggest that steady-state tissue cncentratins f 12S I-BSA wuld be apprached in the cartid artery and in the drsal thracic arta within the 3-hur duratin f this experiment Since the vena cava and pulmnary artery have thinner walls, it is expected that steady-state values wuld als have been attained in these vessels. Hwever, because the ascending arta has thicker walls than the ther vessels, it is pssible that the true steady-state C /C p value fr 12S I-BSA is smewhat greater than the value measured after 3 hurs. The cncentratin f tracer in the plasma fell by apprximately 20% during the 3-hur perid f the experiments, but an average value (dented C p ) was used t calculate the C,/C p values fr each vessel. Vessels with a very shrt time cnstant fr uptake may, therefre, have lst tracer during the later part f the experiment, and s the Q/Cp values given may be slight underestimates f the true values. The vessels were fixed in situ befre excisin in an attempt t remve any lw mlecular weight radiactive cntaminants frm the tissues. 14 The fixative was intrduced via the arterial system, but it was apparent in the utflw frm cut veins within 1 minute. The vena cava and the pulmnary artery shuld, therefre, have been fixed almst as rapidly as the ther vessels. Large differences were fund between the C,/C p values measured in each vessel. Greater uptake by the media f the ascending arta cmpared with the descending thracic arta has been bserved in several previus studies invlving different experimental animate and varius prtein tracers. 4-6 ' Christensen et al. 7 measured albumin uptake by the pulmnary artery f the pig and, as in the present study, fund greater tissue cncentratins than in systemic arteries. These researchers interpreted their findings by assuming that, within the perid f the experiment, albumin entered the pulmnary artery mre rapidly than the arta and that saturatin levels had been reached in the frmer, but nt in the latter. Hwever, as discussed belw, it is unlikely that saturatin levels as indicated by the distributin vlumes are ever attained in viv. Tissue Levels f 12a l-bvlne Serum Albumin Uptake In Relaxed Vessels In Vitr Mean medial Ct/C p values in the cartid artery and in the descending thracic artery were the same whether

7 TRACER UPTAKE BY DIFFERENT BLOOD VESSELS Lever and Jay 557 the incubatin had been fr 1 r 3 hurs, implying that equilibrium (saturatin) levels had been reached in these vessels. The increase with time in the Ct/C p values fr the pulmnary artery, althugh small, is surprising, in view f the relatively thin walls f these vessels. We cannt exclude the pssibility that certain cmpnents f the interstitjum that influence the degree f exclusin might have diffused ut f these vessels during the prlnged incubatin. Tissue swelling may als have cntributed t the rise in the medial C,/C p values with incubatin time. The data shw that the water cntent f bld vessels appears t increase when they are taken frm the stretched, pressurized state t the relaxed state, 11 but the time curse fr this increase is unclear. Albumin exclusin is strngly dependent n tissue hydratin, and the steady state C,/C p values fund in the excised, relaxed vessels may therefre exceed the distributin vlumes that prevail in viv. With the vena cava, nly ne r tw en face sectins cntained medial tissue. The data cnfirm previus findings that the distributin vlume fr albumin is very much greater in the adventitja than in the media, and even small quantities f adventitial tissue in the "medial" sectins wuld have given rise t an verestimate f the mean medial CyC p value. A technique with a better spatial reslutin is needed t reslve this prblem. Tissue Levels f 125 l-bvlne Serum Albumin In Air-pressurized Vessels In Vitr In the experiments with air-pressurized vessels, the tracer was nly able t enter the wall frm the uter surface, and the time cnstant fr uptake may have been lnger than in thse cases in which the tracer culd enter frm bth the luminal and adventitial surfaces. It appears, hwever, that an equilibrium state crrespnding t saturatin f the tissue was reached in all vessels within 3 hurs, since n differences were fund between the values fr 3- and 6-hur incubatins. The vessels studied are nt unifrmly cmpliant and are expsed t different ranges f transmural pressures in viv. The exclusin f albumin frm wall tissue appears t depend n wall strain, 11 and the air-pressurizatin experiments were undertaken in an attempt t measure distributin vlumes in the vessel walls under cnditins mre clsely apprximating nrmal physilgical strain. Hwever, as had been fund previusly, the absence f transmural fluid flux appears t cause the wall tissue t cmpact, with a fall in tissue water cntent cmpared t that measured in freshly excised vessels. The net effect f the tissue dehydratin caused by the impsed wall stresses and cmpactin is a reductin f the C,/C p values t belw thse fund fr relaxed vessels. Despite these effects, the value fr the pulmnary artery greatly exceeds thse f the ther vessels even thugh it is mre cmpliant and underges greater strain n pressurizatin. It is presumed that the true distributin vlume in viv lies smewhere between the C,/C p values fund fr the relaxed and air-pressurized vessels. The true distributin vlume will exceed the steady state C,/C p values fund in viv because there is a cntinuus flux f prtein acrss the wall. If this flux is mainly diffusinal, it wuld be driven by a chemical ptential gradient acrss the tissue between the luminal plasma and the adventitial lymph necessitating wall cncentratins belw saturatin levels. If the flux is cnvective, wall cncentratins will be belw saturatin levels because f the lwer permeability f the intimal surface t prteins cmpared with fluid. Tissue Levels f s1 Cr-EDTA Measurements with this small tracer, which is assumed t ccupy the whle f the extracellular space, were nly made n relaxed vessels in vitr. The medial CyC p values and, therefre, the extracellular space was slightly greater in the vena cava and pulmnary artery than in the systemic vessels, and this cntributed in part t the bserved differences in albumin distributin vlume between the vessels. Hwever, applicatin f simple fiber matrix thery 18 leads t the cnclusin that the differences in extracellular space are nt large enugh t explain the much larger differences in the prtein distributin vlumes. It therefre appears likely that the biplymers within the interstitium f the vena cava and the pulmnary artery cause less exclusin than thse in the interstitium f the systemic arteries. Factrs Affecting In Viv Wall Cncentratins f Tracers Varius mechanisms have been advanced in the past fr the variatin in the bserved patterns f tracer uptake exhibited by different vessels. These include fluid mechanical factrs, 19 ' 20 differences in endthelial cell turnver 21 r mrphlgy, 2223 differences in the prperties f the glyccalyx, 24 and differences in the numbers f plasmalemmal vesicles. 26 Anther cntributry factr hwever, must be the capacity f the tissue fr the tracers; an index f tissue capacity is the distributin vlume. Fr the reasns given abve, it is difficult t estimate the abslute values f the distributin vlumes f prteins in viv, but it appears frm this study that the values fr albumin in the pulmnary artery and vena cava exceed thse f the systemic arteries. The high distributin vlume may, therefre, be an imprtant cntributin t the higher wall tissue cncentratins bserved fr these vessels in viv. The lw Ct/C p values in the cartid artery in viv was surprising. It is unlikely t have been due t the washut f the tracer befre fixatin, since the transprt rate in the media is quite lw. 9 If the rate f drainage frm the media int adventitial lymphatics is imprtant in determining the steady-state cncentratin in the wall, then the thinness f the cartid artery wall may accunt fr the lw values. The pssibility remains, hwever, that the cartid endthelium has a lwer permeability than that f the arta. The degree f accumulatin f material in the intima f the vessel wall during the develpment f athersclersis may be influenced by hindrance t the utward passage f the material thrugh the media. The lw exclusin f prtein and hence the presumed high prsity f the media f the vena cava and pulmnary artery may permit easy drainage f such materials, preventing their accumulatin in the tissue and thereby cntributing t the lw susceptibility f these vessels t disease. Further exper-

8 558 ARTERIOSCLEROSIS VOL 10, N 4, JULY/AUGUST 1990 imerrts are, hwever, needed t test this hypthesis, and, in particular, an investigatin f whether the higher prsity f the media f these vessels is assciated with an increase f transmural flux. References 1. Mitchell JRA, Schwartz CJ. Arterial disease. Oxfrd: Blackwell, 1965: Wd N. Pathlgy f athersclersis. Lndn: Butterwrth, 1982: Heath D, Wd EH, Dushane JW, Edwards JE. The relatin f age and bld pressure t atherma in the pulmnary arteries and thracic arta In cngenital heart disease. Lab Invest 1960:9: Duncan LE, Crnfield J, Buck K. The effect f Wd pressure and stretching n the passage f labeled plasma albumin int canine artic wall. J Clin Invest 1962; 41: Packham MA, Rwsell HC, Jrgensen L, Mustard JF. Lcalized prtein accumulatin In the wall f the arta. Exp Ml Pathl 1967;7: Bell FP, Adamsn IL, Schwartz CJ. Artic endthellal permeability t albumin: Fcal and reginal patterns f uptake and transmural distributin f "*l albumin in the yung pig. Exp Ml Pathl 1974;20: Chrlstensen S, Stender S, Nyvad O, Bagger H. In viv fluxes f plasma chlesterl, phsphatidytchline and prtein Int mini-pig artic and pulmnary segments. Athersclersis 1982:41: Snashall PD, Keyes SJ, Mrgan BM, Jnes B, Murphy K. Reginal extravascular and Interstitial lung water In nrmal dgs. J Appl Physil 1977;49: Car CQ, Lever MJ, Laver-Rudlch Z, et al. Net albumin transprt acrss the wall f the rabbit cmmn cartid artery perfused in situ. Athersclersis 1980:37: Smith EB, Staples EM. Irrtimal and medial plasma prtein cncentratins and endthellal functin. Athersclersis 1982:41: Tedgul A, Lever MJ. Effectf pressure and Irrtimal damage n albumin and 14 C sucrse spaces in arta Am J Physil 1987;253:H1530-H1539 Index Terms: vascular permeability athersclersis pulmnary arteries 12. Wallensteln S, Zucker CL, FMss JL Sme statistical methds useful In circulatin research. Circ Res 1980; 47: Bratzler RL, Chlslm QM, Cttn CK, Smith KA, 21- versmlt DB, Lees RS. The distributin f labeled albumin acrss the rabbit thracic arta in viv. Ore Res 1977; 40: Stemerman MB, Mrrel EM, Burke KR, Cttn CK, Smith KA, Lees RS. Lcal variatin in arterial wall permeability t lw density lipprtein In nrmal rabbit arta. Arterisclersis 1986;6: Duncan LE, Buck K, Lynch A. Lipprtein mvement thrugh canine artic wall. Science 1963;142: Bell FP, Callus AS, Schwartz CJ. Fcal and reginal patterns f uptake and the transmural distributin f 12S I fibringen in the pig arta In viv. Exp Ml Pathl 1974; 20: Fry DL Effect f pressure and stirring n In vitr artic transmural 12S I albumin transprt. Am J Physil 1983; 245:H977-H Ogstn AG. The spaces in a unifrm randm suspensin f fibres. Trans Faraday Sc 1958;54: Fry DL Certain histlgical and chemical respnses f the vascular Interface t acutely induced mechanical stress In the arta f the dg. Ore Res 1969;24: Car CG, Rtz-Gerald JM, Schrter RC. Atherma and arterial wall shear bservatin, crrelatin and prpsal f a shear dependent mass transfer mechanism fr athergenesls. Prc R Sc Lnd B 1971; 177: Caplan BA, Schwartz CJ. Increased endthelial cell turnver In areas f Evans blue uptake In the pig arta. Athersclersis 1973;17: Caplan BA, Gerrtty RG, Schwartz CJ. Endthellal cell mrphlgy In fcal areas f In viv Evans blue uptake in the yung pig arta Exp Ml Pathl 1974;21: Klbrta Q, Heath D, Smith D, Blggar R. Pulmnary endthellal pavements. Thrax 1980;35: Qrg P, Bm GVR. Uneven distributin f sialtc acids n the luminal surface f artic endtheflum. Br J Pathl 1983;64: Osterman H, Bm GVR. Cmparisn f the numbers f plasmalemmal vesicles In arterial and venus endthelium in rats. Prc R Sc Lnd B 1986:227:17-20 distributin vlume veins systemic arteries