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Fatty acid composition of cultured aortic cells from white carneau and show racer pigeons
379
JOURNAL OF ATHEROSCLEROSIS RESEARCH
F A T T Y A C I D C O M P O S I T I O N OF C U L T U R E D A O R T I C CELLS F R O M WHITE CARNEAU AND SHOW RACER PIGEONS
S. C. SMITH, R. G. STROUT, W. R. D U N L O P AND E. C. SMITH
Biomo!ecular Hygiene Section, University of New Hampshire, Durham, New Hampshire (U.S.A ) (Received April 20, 1964. Revised manuscript received September 2nd, 1964)
INTRODUCTION
Tissue culture studies of intimal cells from atherosclerotic h u m a n arteries have d e m o n s t r a t e d a cellular inability to metabolize t e m p o r a r y lipid overloads 1. This finding was correlated with intimal changes in foetal aortas to suggest a pre-atherosclerotic state or a metabolic error in lipid transport regulation 2. Since all h u m a n s are reported to be susceptible to f a t t y streaks in the arteries 3, h u m a n intimal cells might generally be expected to contain this metabolic defect, possibly in a latent form. White Carneau pigeons have been shown to be susceptible to atherosclerosis while Show Racer pigeons are highly resistanO. Lesions found in arteries of White Carneaux are reported to be the only ones among common experimental animals which closely resemble h u m a n lesions and conform to the World Health Organization definition of atherosclerosis 5. An atherosclerosis susceptible-resistant animal species in which the disease approximates the h u m a n condition provides a unique source of aortic intimal cells for study of metabolic phenomena pre-disposing to atherosclerosis. This c o m m u n i c a t i o n presents a method for culture of aortic intimal cells from the aforementioned breeds of pigeons and the higher f a t t y acid composition of the cells cultured. MATERIALS AND METHODS Colonies of the two breeds of pigeons were maintained under normal conditions 5 to provide a supply of eggs for artificial incubation. Aortas were dissected from 17-day e m b r y o s a n d rinsed in sterile H a n k ' s balanced salt solution. E x t r a n e o u s material was r e m o v e d and the aortas opened longitudinally. Endothelium a n d subendothelial i n t i m a were peeled from the tissue adjacent to the lumen while adventitia and m o s t of the media were peeled from the outer surface of the tissue. The remaining intimal tissue was cut into pieces approx. 1 m m 2 and explanted without plasma clots on T-15 flasks, three-ounce bottles, 30 ml " F a l c o n " tissue culture j . Atheroscler. Res., 5 (1965) 379-387
380
S. C. SMITH, R. G. STROUT, "~V. R. DUNLOP, E. C. SMITH
Fig. 1. Cross section of dissected W h i t e C a r n e a u i n t i m a l tissue. V e r h o e f f - V a n Gieson; X 600.
Fig. 2. Cross section of dissected S h o w R a c e r i n t i m a l tissue. Verhoeff V a n Gieson; × 600.
.]. Atheroscler. Res., 5 (1965) 379-387
F A T T Y A C I D S O F C U L T U R E D A O R T I C C E L L S FROM T W O B R E E D S O F P I G E O N S
381
flasks, or 6 × 30 m m coverslips in tubes. Dissection of the intimal tissue was verified histologically b y paraffin sections and Verhoeff-Van Gieson staining (Figs. 1-4). Intimal tissue as used for explants was homogenized with methanol in a microtissue grinder and treated with boron trichloride gas to form m e t h y l esters of the f a t t y acids present s . The following culture media were evaluated for ability to elicit proliferation of cells from explants and for maintenance of cells through passage b y trypsinization, versene or mechanical removal. NCTC 109 s u p p l e m e n t e d with 10 or 20 % horse serum, 10 % calf serum, 10 % bovine serum, or 2 or 10 % homologous pigeon serum. Medium 199 s u p p l e m e n t e d with 10 % calf serum or 1 % peptone and 3 rag/ 100 ml arginine 7. W a y m o u t h ' s m e d i u m MB-752/1 supplemented with 10 % calf serum. Lactoalbumin hydrolysate (0.5 %) in H a n k ' s BSS supplemented with I0 °/o or 20 % bovine serum. Chick e m b r y o extraetso (2 %) in H a n k ' s BSS supplemented with 10, 20, or 40 % homologous pigeon serum. Homologous pigeon e m b r y o extract50 (2, 10, or 20 %) in H a n k ' s BSS supplemented with 40 % homologous pigeon serum. Eagle's m e d i u m supplemented with 10, 25, or 50 % calf serum or with 15 % chick embryo extract~o, 15 % horse serum and 0.146 mg L-glutamine s. Routine examinations of cells growing out from explants were performed using phase-contrast microscopy or osmium tetroxide-May-Grtinwald-Giemsa staining. Ex-
Fig. 3. Cross section of White Carneau aorta; lumen at upper right. Verhoeff-Van Gieson; × 400. J. Atheroscler. Res., 5 (1965) 379-387
382
S.C. SMITH, R. G. STROUT, W. R. DUNLOP, E. C. SMITH
amination of stained cells under high magnification was possible on coverslips or " F a l c o n " flasks opened with a heated knife.
Fig. 4. Cross section of Show Racer aorta; intima at upper right corner. Verhoeff-Van Gieson; × 400.
Prior to analysis of the cellular f a t t y acids, explants were r e m o v e d with a capillary pipette and the cell layer harvested b y trypsinization. Cells were washed with buffered saline (pH 7.4) and interesterified with boron trichloride 6. Gas-liquid chromatographic analyses were performed on a 6-ft., ethyleneglycol succinate column (12 % on 80/90-mesh A n a k r o m A) in an argon ionization system (90St detector). The column was maintained at 187°C with 15 psi argon inlet pressure. Under these conditions f a t t y acid-methyl esters below m y r i s t a t e were eluted with the solvent. The column was calibrated with N I H Metabolism Study Section methylester mixtures A, B and D, analytical precision being within the 2 °/o limit specified. RESULTS Tissue used for explanting contained few, if any, elastic fibers (cf. Figs. 1 and 2) and closely a p p r o x i m a t e d the intimal regions of intact aortas (Figs. 3 and 4). The higher f a t t y acid compositions of dissected intimal tissue from each breed are presented in Table I. Of the nutrients evaluated only m e d i u m 199 failed to support proliferation from the explant. The others promoted growth of cells to varying limited degrees, but only one was able to maintain the cells for more t h a n four passages. Eagle's j . Atheroscler. Res., 5 (1965) 379-387
FATTY ACIDS OF CULTURED
AORTIC CELLS FROM TWO BREEDS OF PIGEONS
383
b a s a l m e d i u m s u p p l e m e n t e d w i t h 15 % chick e m b r y o extracts0, 15 % horse s e r u m , a n d 0.146 m g L - g l u t a m i n e as u s e d in t h e c u l t u r e of r a b b i t a o r t a cells 8 was f o u n d t o y i e l d cell p r o l i f e r a t i o n f r o m t h e e x p l a n t a p p r o a c h i n g a m o n o l a y e r in 9-12 d a y s . T h e cells c o u l d b e s u c c e s s f u l l y t r y p s i n i z e d a t this t i m e a n d c a r r i e d for a t least 18 p a s sages (at i n t e r v a l s of 3 - 6 days). Some difficulty w a s f o u n d in r e m o v i n g cells f r o m glass surfaces, b u t n o p r o b l e m was e n c o u n t e r e d in r e m o v a l from " F a l c o n " ( p o l y s t y r e n e ) flasks. O r i g i n a l g r o w t h from t h e e x p l a n t was s o m e w h a t m o r e e x t e n s i v e in p l a s t i c flasks. I n o r d e r t o m i n i m i z e possible c h r o m o s o m a l a n d / o r m e t a b o l i c c h a n g e s on passage, f a t t y a c i d c o m p o s i t i o n s of cells f r o m e x p l a n t s were d e t e r m i n e d a f t e r 8 days' proliferation. TABLE I HIGHI~R FATTY ACID COMPOSITION OF AORTIC INTIMA*
Fatty acid
White Carneau
Show Racer
Myristic (C14)
3.1% (1.6-5.4) 1.8 (0.6-1.9) 25.5 (21.3-34.3) 3.4 (2.0-5.7) 24.0 (20.8-27.2) 22.7 (16.4-31.4) 8.5 (8.0-8.9) not detected
1.7 % ( 1.1-2.5) 2.3 0.4-5.3) 22.5 (19.5-26.8) 5.5 ( 1.6-9.9) 19.6 (12.5-28.7) 30.0 (29.0-31.2) 7.8 (5.1-10.1) 1.4 (0.0-5.1) 0.9 (0.0-1. l) 8.5 (3.2-16.8) 100.2 %
Unidentified Palmitic (C16) Palmitoleic (C16 : 1) Stearic (C18) Oleie (C18 : 1) Linoleic (C18 : 2) Arachidic (C20) Linolenic (C18 : 3) Arachidonic (C20:4) Total
2.1 (2.1-2.2) 8.7 (5.8-12.5) 99.8 %
* Figures are means of values over the ranges indicated. Five analyses were performed for each breed, a separate tissue pool from 3-5 aortas being used for each analysis. Components eluted between C14 and C20 : 4 which constituted approximately 1.0 % or more were detected. A l t h o u g h e x t e n s i v e m o r p h o l o g i c a l s t u d i e s were n o t p e r f o r m e d , cells c u l t u r e d b y t h e t e c h n i q u e d e s c r i b e d in this r e p o r t a p p e a r e d to r e s e m b l e t h o s e f o u n d in t h e i n t a c t tissue (Figs. 5 a n d 6). H i g h e r f a t t y a c i d c o m p o s i t i o n s of c u l t u r e d i n t i m a l cells from b o t h b r e e d s of pigeons are p r e s e n t e d in T a b l e I I . j . Atheroscler. Res., 5 (1965) 379-387
384
S . C . SMITH, R. G. STROUT, W. R. DUNLOP, E. C. SMITH
Fig. 5. I n t i m a l cells f r o m e x p l a n t e d W h i t e C a r n e a u t i s s u e . G i e m s a ; × 400.
Fig. 6. I n t i m a l cells f r o m e x p l a n t e d S h o w R a c e r tissue. G i e m s a ; × 600.
J. Atheroscler. Res., 5 (1965) 379 387
FATTY ACIDS OF CULTURED AORTIC CELLS FROM TWO BREEDS OF PIGEONS
385
T A B L E II HIGHER
FATTY
ACID COMPOSITION
OF CELLS CULTURED
FROM AORTIC INTIMA*
Fatty acid
White Carneau
Show Racer
Myristic (C14)
3.5 (1.1-3.6)
3.5 (0.0-5.1)
P a l m i t i c (C16)
17.5 (11.8-22.7)
(8.5-13.5)
Palmitoleic (C16 : I)
3.5 (1.3-5.8)
2.8 (0.5-5.3)
Stearic (C18)
30.3 (27.3-33.2)
29.3 18.5-38.9)
Oleic (C18 : l)
22.3 (21.0-23.6)
21.9 20.3-23.6)
Linoleic (C18 : 2)
19.8 (18.6-21.0)
22.3 21.5-23.1)
Arachidic (C20)
1.9 (0.0-3.0)
< 1.0 trace)
Linolenic (C18 : 3)
1.4 (0.0-2.2)
1.2 0.0-2.9)
Arachidonic (C20 : 4)
not detected
7.7 7.3-8.1)
Total
100.2 ~o
99.7+ %
ll.O
*Figures are m e a n s of values o v e r t h e ranges indicated. F o u r a n a l y s e s were p e r f o r m e d on cells f r o m each breed. S e p a r a t e c u l t u r e pools from 1-3 b o t t l e s c o n t a i n i n g 3-12 a o r t a s were used for each analysis. C o m p o u n d s e l u t e d b e t w e e n C14 and C 2 0 : 4 w h i c h c o n s t i t u t e d a p p r o x i m a t e l y 1.0 % or m o r e were d e t e c t e d .
DISCUSSION
Culture and passage of aortic intimal cells from White Carneau and Show Racer pigeons without the use of plasma clots was possible in only one of the culture media evaluated. Absence of a clot facilitates biochemical analyses of the cells. Previous reports concerning culture of aortic intimal cells9,10 d e m o n s t r a t e d the presence of b o t h endothelial- and fibroblast-type cells. However, no a t t e m p t s were m a d e to remove the endothelium from aortic tissue before culture. In the present s t u d y the cells cultured are v e r y similar to those in the intimal tissue used for explanting. Cells of the intimal and sub-intimal layers are of particular relevaltce in the development of h u m a n atherosclerosis, whereas involvement of sub-endothelial cells is characteristic of the experimental process in animals 11. Differences between stearate and oleate composition in intimal tissues of White Carneau and Show R a c e r pigeons cannot be explained at present. However, the differences in p a l m i t a t e and arachidonate between intimal cells cultured from these j . Atheroscler. Res., 5 (1965) 379-387
386
s.c.
SMITH, R. G. S T R O U T , W. I~. D U N L O P , E. C. SMITH
pigeons m a y be of importance. I t is interesting t h a t cells from the atherosclerosissusceptible White Carneau are devoid of arachidonate while those from the resistant breed a p p a r e n t l y contain the normal amount. Essential f a t t y acid deficiency in the arterial wall is reported to decrease adenosine triphosphatase production and in t u r n lead to localized excesses of long chain f a t t y acids 12, Highly unsaturated f a t t y acids are necessary in mitochondrial phospholipids for normal oxidative metabolism 13. In early, h u m a n atherosclerosis WATTS 14 d e m o n s t r a t e d intracellular lipid accumulation in and around mitochondria. He further associated this accumulation with defects in mitochondrial adenosine triphosphatase, f a t t y acid oxidase, and oxidative enzymes involved in electron transport. F r o m these reports it seems likely t h a t the lack of arachidonate in intimal cells cultured from susceptible pigeons m a y be related to the development of atherosclerotic lesions in these pigeons. Presence of similar amounts of arachidonate in intimal tissue from b o t h breeds of pigeons suggests t h a t a cellular defect leading to arachidonate deficiency in susceptible-pigeon cells m a y become apparent when the cells are exposed to unusual conditions such as those in tissue culture. Possibly, the arachidonate in White Carneau intimal tissue m a y be formed elsewhere in the bird, and the intimal cells of this breed m a y be defective in arachidonate synthesis. Elucidation of mechanisms responsible for the lack of arachidonate in cells cultured from White Carneau intima must await further study. However, it a p p e a r s t h a t the conversion of linoleate to arachidonate m a y be impaired since cells from b o t h breeds have increased a m o u n t s of linoleate in tissue culture. Chromosome studies in the cultured a o r t a cells were not performed. However, i t seems unlikely that a chromosomal change in p r i m a r y White Carneau cultures would be responsible for the arachidonate deficiency since the cells were not subcultured before analysis.
ACKNOWLEDGEMENTS This investigation was supported in part by the New Hampshire Heart Association, and the New Hampshire Agricultural Experiment Station (Scientific Contribution No. 353). The authors are indebted to DR. T. E. YOUNG for examinations of dissected tissue and cultured cells. The technical assistance of MMES. HELEN LANGLEY, EMILY DONOVAN, ROSE THOMAS AND MR. TAKIS PAPAS is gratefully acknowledged. SUMMARY Cell cultures have been established from aortic intimal tissue of White Carneau and Show Racer pigeons. Analyses of the higher f a t t y acid ( > l a u r a t e ) c o m positiomof the cultures revealed the absence o f arachidonate in aortic cells from A thero gg-lev. Res.,
5 (~965) 379-387
387
F A T T Y ACIDS OF C U L T U R E D AORTIC CELLS FROM TWO BREEDS OF PIGEONS atherosclerosis-susceptible arachidonate
White
Carneaux.
by
these
c e l l s is s u g g e s t e d
sclerosis-resistant
Show
Racers
have
A metabolic since
the normal
aortic amount
defect
in the formation
cells cultured
from
of
athero-
of arachidonate.
REFERENCES 1 A. LAZZARINI--ROBERTSON, JR., Arch. Surg., 84 (1962) 41. 2 A. LAZZARINI--ROBERTSON, JR., In: A b s t r a c t s of t h e S i x t h I n t e r n a t i o n a l C o n g r e s s of Ger o n t o l o g y , C o p e n h a g e n , 1963, Excerpta bled., 57 (1963) 270. 3 R. L. HOLMAN, H. C. McGILL, JR., J. P. STRONG AND J. C. GEER, Am. J. Pathol., 34 (1958) 209. 4 T. B. CLARKSON, R. W. PRICHARD, M. G. Ni~TSKY AND H. B. LOFLAND, Arch. Pathol., 68 (1959). 143. s R. W . PRICHARD, T. B. CLARKSON, H. O. GOODMAN AND H. B. LOFLAND, Arch. Pathol., 77 (1964) 244. 6 K. ABEL, H . DE SCI~MERTZlNG AND J. I. PET:~RSON, J. Bacteriol., 85 (1963) 1039. 7 A. LAZZARINI ROBERTSON, JR., In: S. GARATTINI AND R. PAOLETTI (Eds.), Proceedings S3,mposium on Drugs Affecting Lipid Metabolism, Elsevier, A m s t e r d a m , 1961, p. 306. s T. KOKUBU AND O. J. POLLAK, J. Atheroscler. Res., 1 (1961) 229. 9 A. LAZZARINI ROBERTSON, JR., Studies on the Effects of Lipoid Emulsions on Arterial Intimal Cells in Tissue Culture in Relation to A therosclerosis, Thesis, G r a d u a t e Medical School of Cornell U n i v e r s i t y , 1959. 10 R. KOIDE, O. J. POLLAK AND D. A. BURNS, J. Atheroscler. Res., 3 (1963) 32. la ~V. B. ROBERTSON, J. C. GEER, J. P. STRONG AND ]7I. C. McGILL, JR., Exptl. Mol. Pathol., S u p p l . No. 1 (1963) 28. 12 M. SANDLER AND G. ]7t. ]BOURNE, Nature, 193 (1962) 1184. 13 D. ]~. GREEN AND S. FLEISCHER, Biochim. Biophys. Ac!a, 70 (1963) 554. 14 H. F. WATTS, I n : \V. LIKOFF AND J. H. MOYER (Eds.) Coronam, Heart Disease, G r u n e a n d S t r a t t o n , N e w Y o r k , 1963, p. 98.
j . Atheroscler. Res., 5 (1965) 3 7 9 - 3 8 7
JOURNAL OF ATHEROSCLEROSIS RESEARCH
F A T T Y A C I D C O M P O S I T I O N OF C U L T U R E D A O R T I C CELLS F R O M WHITE CARNEAU AND SHOW RACER PIGEONS
S. C. SMITH, R. G. STROUT, W. R. D U N L O P AND E. C. SMITH
Biomo!ecular Hygiene Section, University of New Hampshire, Durham, New Hampshire (U.S.A ) (Received April 20, 1964. Revised manuscript received September 2nd, 1964)
INTRODUCTION
Tissue culture studies of intimal cells from atherosclerotic h u m a n arteries have d e m o n s t r a t e d a cellular inability to metabolize t e m p o r a r y lipid overloads 1. This finding was correlated with intimal changes in foetal aortas to suggest a pre-atherosclerotic state or a metabolic error in lipid transport regulation 2. Since all h u m a n s are reported to be susceptible to f a t t y streaks in the arteries 3, h u m a n intimal cells might generally be expected to contain this metabolic defect, possibly in a latent form. White Carneau pigeons have been shown to be susceptible to atherosclerosis while Show Racer pigeons are highly resistanO. Lesions found in arteries of White Carneaux are reported to be the only ones among common experimental animals which closely resemble h u m a n lesions and conform to the World Health Organization definition of atherosclerosis 5. An atherosclerosis susceptible-resistant animal species in which the disease approximates the h u m a n condition provides a unique source of aortic intimal cells for study of metabolic phenomena pre-disposing to atherosclerosis. This c o m m u n i c a t i o n presents a method for culture of aortic intimal cells from the aforementioned breeds of pigeons and the higher f a t t y acid composition of the cells cultured. MATERIALS AND METHODS Colonies of the two breeds of pigeons were maintained under normal conditions 5 to provide a supply of eggs for artificial incubation. Aortas were dissected from 17-day e m b r y o s a n d rinsed in sterile H a n k ' s balanced salt solution. E x t r a n e o u s material was r e m o v e d and the aortas opened longitudinally. Endothelium a n d subendothelial i n t i m a were peeled from the tissue adjacent to the lumen while adventitia and m o s t of the media were peeled from the outer surface of the tissue. The remaining intimal tissue was cut into pieces approx. 1 m m 2 and explanted without plasma clots on T-15 flasks, three-ounce bottles, 30 ml " F a l c o n " tissue culture j . Atheroscler. Res., 5 (1965) 379-387
380
S. C. SMITH, R. G. STROUT, "~V. R. DUNLOP, E. C. SMITH
Fig. 1. Cross section of dissected W h i t e C a r n e a u i n t i m a l tissue. V e r h o e f f - V a n Gieson; X 600.
Fig. 2. Cross section of dissected S h o w R a c e r i n t i m a l tissue. Verhoeff V a n Gieson; × 600.
.]. Atheroscler. Res., 5 (1965) 379-387
F A T T Y A C I D S O F C U L T U R E D A O R T I C C E L L S FROM T W O B R E E D S O F P I G E O N S
381
flasks, or 6 × 30 m m coverslips in tubes. Dissection of the intimal tissue was verified histologically b y paraffin sections and Verhoeff-Van Gieson staining (Figs. 1-4). Intimal tissue as used for explants was homogenized with methanol in a microtissue grinder and treated with boron trichloride gas to form m e t h y l esters of the f a t t y acids present s . The following culture media were evaluated for ability to elicit proliferation of cells from explants and for maintenance of cells through passage b y trypsinization, versene or mechanical removal. NCTC 109 s u p p l e m e n t e d with 10 or 20 % horse serum, 10 % calf serum, 10 % bovine serum, or 2 or 10 % homologous pigeon serum. Medium 199 s u p p l e m e n t e d with 10 % calf serum or 1 % peptone and 3 rag/ 100 ml arginine 7. W a y m o u t h ' s m e d i u m MB-752/1 supplemented with 10 % calf serum. Lactoalbumin hydrolysate (0.5 %) in H a n k ' s BSS supplemented with I0 °/o or 20 % bovine serum. Chick e m b r y o extraetso (2 %) in H a n k ' s BSS supplemented with 10, 20, or 40 % homologous pigeon serum. Homologous pigeon e m b r y o extract50 (2, 10, or 20 %) in H a n k ' s BSS supplemented with 40 % homologous pigeon serum. Eagle's m e d i u m supplemented with 10, 25, or 50 % calf serum or with 15 % chick embryo extract~o, 15 % horse serum and 0.146 mg L-glutamine s. Routine examinations of cells growing out from explants were performed using phase-contrast microscopy or osmium tetroxide-May-Grtinwald-Giemsa staining. Ex-
Fig. 3. Cross section of White Carneau aorta; lumen at upper right. Verhoeff-Van Gieson; × 400. J. Atheroscler. Res., 5 (1965) 379-387
382
S.C. SMITH, R. G. STROUT, W. R. DUNLOP, E. C. SMITH
amination of stained cells under high magnification was possible on coverslips or " F a l c o n " flasks opened with a heated knife.
Fig. 4. Cross section of Show Racer aorta; intima at upper right corner. Verhoeff-Van Gieson; × 400.
Prior to analysis of the cellular f a t t y acids, explants were r e m o v e d with a capillary pipette and the cell layer harvested b y trypsinization. Cells were washed with buffered saline (pH 7.4) and interesterified with boron trichloride 6. Gas-liquid chromatographic analyses were performed on a 6-ft., ethyleneglycol succinate column (12 % on 80/90-mesh A n a k r o m A) in an argon ionization system (90St detector). The column was maintained at 187°C with 15 psi argon inlet pressure. Under these conditions f a t t y acid-methyl esters below m y r i s t a t e were eluted with the solvent. The column was calibrated with N I H Metabolism Study Section methylester mixtures A, B and D, analytical precision being within the 2 °/o limit specified. RESULTS Tissue used for explanting contained few, if any, elastic fibers (cf. Figs. 1 and 2) and closely a p p r o x i m a t e d the intimal regions of intact aortas (Figs. 3 and 4). The higher f a t t y acid compositions of dissected intimal tissue from each breed are presented in Table I. Of the nutrients evaluated only m e d i u m 199 failed to support proliferation from the explant. The others promoted growth of cells to varying limited degrees, but only one was able to maintain the cells for more t h a n four passages. Eagle's j . Atheroscler. Res., 5 (1965) 379-387
FATTY ACIDS OF CULTURED
AORTIC CELLS FROM TWO BREEDS OF PIGEONS
383
b a s a l m e d i u m s u p p l e m e n t e d w i t h 15 % chick e m b r y o extracts0, 15 % horse s e r u m , a n d 0.146 m g L - g l u t a m i n e as u s e d in t h e c u l t u r e of r a b b i t a o r t a cells 8 was f o u n d t o y i e l d cell p r o l i f e r a t i o n f r o m t h e e x p l a n t a p p r o a c h i n g a m o n o l a y e r in 9-12 d a y s . T h e cells c o u l d b e s u c c e s s f u l l y t r y p s i n i z e d a t this t i m e a n d c a r r i e d for a t least 18 p a s sages (at i n t e r v a l s of 3 - 6 days). Some difficulty w a s f o u n d in r e m o v i n g cells f r o m glass surfaces, b u t n o p r o b l e m was e n c o u n t e r e d in r e m o v a l from " F a l c o n " ( p o l y s t y r e n e ) flasks. O r i g i n a l g r o w t h from t h e e x p l a n t was s o m e w h a t m o r e e x t e n s i v e in p l a s t i c flasks. I n o r d e r t o m i n i m i z e possible c h r o m o s o m a l a n d / o r m e t a b o l i c c h a n g e s on passage, f a t t y a c i d c o m p o s i t i o n s of cells f r o m e x p l a n t s were d e t e r m i n e d a f t e r 8 days' proliferation. TABLE I HIGHI~R FATTY ACID COMPOSITION OF AORTIC INTIMA*
Fatty acid
White Carneau
Show Racer
Myristic (C14)
3.1% (1.6-5.4) 1.8 (0.6-1.9) 25.5 (21.3-34.3) 3.4 (2.0-5.7) 24.0 (20.8-27.2) 22.7 (16.4-31.4) 8.5 (8.0-8.9) not detected
1.7 % ( 1.1-2.5) 2.3 0.4-5.3) 22.5 (19.5-26.8) 5.5 ( 1.6-9.9) 19.6 (12.5-28.7) 30.0 (29.0-31.2) 7.8 (5.1-10.1) 1.4 (0.0-5.1) 0.9 (0.0-1. l) 8.5 (3.2-16.8) 100.2 %
Unidentified Palmitic (C16) Palmitoleic (C16 : 1) Stearic (C18) Oleie (C18 : 1) Linoleic (C18 : 2) Arachidic (C20) Linolenic (C18 : 3) Arachidonic (C20:4) Total
2.1 (2.1-2.2) 8.7 (5.8-12.5) 99.8 %
* Figures are means of values over the ranges indicated. Five analyses were performed for each breed, a separate tissue pool from 3-5 aortas being used for each analysis. Components eluted between C14 and C20 : 4 which constituted approximately 1.0 % or more were detected. A l t h o u g h e x t e n s i v e m o r p h o l o g i c a l s t u d i e s were n o t p e r f o r m e d , cells c u l t u r e d b y t h e t e c h n i q u e d e s c r i b e d in this r e p o r t a p p e a r e d to r e s e m b l e t h o s e f o u n d in t h e i n t a c t tissue (Figs. 5 a n d 6). H i g h e r f a t t y a c i d c o m p o s i t i o n s of c u l t u r e d i n t i m a l cells from b o t h b r e e d s of pigeons are p r e s e n t e d in T a b l e I I . j . Atheroscler. Res., 5 (1965) 379-387
384
S . C . SMITH, R. G. STROUT, W. R. DUNLOP, E. C. SMITH
Fig. 5. I n t i m a l cells f r o m e x p l a n t e d W h i t e C a r n e a u t i s s u e . G i e m s a ; × 400.
Fig. 6. I n t i m a l cells f r o m e x p l a n t e d S h o w R a c e r tissue. G i e m s a ; × 600.
J. Atheroscler. Res., 5 (1965) 379 387
FATTY ACIDS OF CULTURED AORTIC CELLS FROM TWO BREEDS OF PIGEONS
385
T A B L E II HIGHER
FATTY
ACID COMPOSITION
OF CELLS CULTURED
FROM AORTIC INTIMA*
Fatty acid
White Carneau
Show Racer
Myristic (C14)
3.5 (1.1-3.6)
3.5 (0.0-5.1)
P a l m i t i c (C16)
17.5 (11.8-22.7)
(8.5-13.5)
Palmitoleic (C16 : I)
3.5 (1.3-5.8)
2.8 (0.5-5.3)
Stearic (C18)
30.3 (27.3-33.2)
29.3 18.5-38.9)
Oleic (C18 : l)
22.3 (21.0-23.6)
21.9 20.3-23.6)
Linoleic (C18 : 2)
19.8 (18.6-21.0)
22.3 21.5-23.1)
Arachidic (C20)
1.9 (0.0-3.0)
< 1.0 trace)
Linolenic (C18 : 3)
1.4 (0.0-2.2)
1.2 0.0-2.9)
Arachidonic (C20 : 4)
not detected
7.7 7.3-8.1)
Total
100.2 ~o
99.7+ %
ll.O
*Figures are m e a n s of values o v e r t h e ranges indicated. F o u r a n a l y s e s were p e r f o r m e d on cells f r o m each breed. S e p a r a t e c u l t u r e pools from 1-3 b o t t l e s c o n t a i n i n g 3-12 a o r t a s were used for each analysis. C o m p o u n d s e l u t e d b e t w e e n C14 and C 2 0 : 4 w h i c h c o n s t i t u t e d a p p r o x i m a t e l y 1.0 % or m o r e were d e t e c t e d .
DISCUSSION
Culture and passage of aortic intimal cells from White Carneau and Show Racer pigeons without the use of plasma clots was possible in only one of the culture media evaluated. Absence of a clot facilitates biochemical analyses of the cells. Previous reports concerning culture of aortic intimal cells9,10 d e m o n s t r a t e d the presence of b o t h endothelial- and fibroblast-type cells. However, no a t t e m p t s were m a d e to remove the endothelium from aortic tissue before culture. In the present s t u d y the cells cultured are v e r y similar to those in the intimal tissue used for explanting. Cells of the intimal and sub-intimal layers are of particular relevaltce in the development of h u m a n atherosclerosis, whereas involvement of sub-endothelial cells is characteristic of the experimental process in animals 11. Differences between stearate and oleate composition in intimal tissues of White Carneau and Show R a c e r pigeons cannot be explained at present. However, the differences in p a l m i t a t e and arachidonate between intimal cells cultured from these j . Atheroscler. Res., 5 (1965) 379-387
386
s.c.
SMITH, R. G. S T R O U T , W. I~. D U N L O P , E. C. SMITH
pigeons m a y be of importance. I t is interesting t h a t cells from the atherosclerosissusceptible White Carneau are devoid of arachidonate while those from the resistant breed a p p a r e n t l y contain the normal amount. Essential f a t t y acid deficiency in the arterial wall is reported to decrease adenosine triphosphatase production and in t u r n lead to localized excesses of long chain f a t t y acids 12, Highly unsaturated f a t t y acids are necessary in mitochondrial phospholipids for normal oxidative metabolism 13. In early, h u m a n atherosclerosis WATTS 14 d e m o n s t r a t e d intracellular lipid accumulation in and around mitochondria. He further associated this accumulation with defects in mitochondrial adenosine triphosphatase, f a t t y acid oxidase, and oxidative enzymes involved in electron transport. F r o m these reports it seems likely t h a t the lack of arachidonate in intimal cells cultured from susceptible pigeons m a y be related to the development of atherosclerotic lesions in these pigeons. Presence of similar amounts of arachidonate in intimal tissue from b o t h breeds of pigeons suggests t h a t a cellular defect leading to arachidonate deficiency in susceptible-pigeon cells m a y become apparent when the cells are exposed to unusual conditions such as those in tissue culture. Possibly, the arachidonate in White Carneau intimal tissue m a y be formed elsewhere in the bird, and the intimal cells of this breed m a y be defective in arachidonate synthesis. Elucidation of mechanisms responsible for the lack of arachidonate in cells cultured from White Carneau intima must await further study. However, it a p p e a r s t h a t the conversion of linoleate to arachidonate m a y be impaired since cells from b o t h breeds have increased a m o u n t s of linoleate in tissue culture. Chromosome studies in the cultured a o r t a cells were not performed. However, i t seems unlikely that a chromosomal change in p r i m a r y White Carneau cultures would be responsible for the arachidonate deficiency since the cells were not subcultured before analysis.
ACKNOWLEDGEMENTS This investigation was supported in part by the New Hampshire Heart Association, and the New Hampshire Agricultural Experiment Station (Scientific Contribution No. 353). The authors are indebted to DR. T. E. YOUNG for examinations of dissected tissue and cultured cells. The technical assistance of MMES. HELEN LANGLEY, EMILY DONOVAN, ROSE THOMAS AND MR. TAKIS PAPAS is gratefully acknowledged. SUMMARY Cell cultures have been established from aortic intimal tissue of White Carneau and Show Racer pigeons. Analyses of the higher f a t t y acid ( > l a u r a t e ) c o m positiomof the cultures revealed the absence o f arachidonate in aortic cells from A thero gg-lev. Res.,
5 (~965) 379-387
387
F A T T Y ACIDS OF C U L T U R E D AORTIC CELLS FROM TWO BREEDS OF PIGEONS atherosclerosis-susceptible arachidonate
White
Carneaux.
by
these
c e l l s is s u g g e s t e d
sclerosis-resistant
Show
Racers
have
A metabolic since
the normal
aortic amount
defect
in the formation
cells cultured
from
of
athero-
of arachidonate.
REFERENCES 1 A. LAZZARINI--ROBERTSON, JR., Arch. Surg., 84 (1962) 41. 2 A. LAZZARINI--ROBERTSON, JR., In: A b s t r a c t s of t h e S i x t h I n t e r n a t i o n a l C o n g r e s s of Ger o n t o l o g y , C o p e n h a g e n , 1963, Excerpta bled., 57 (1963) 270. 3 R. L. HOLMAN, H. C. McGILL, JR., J. P. STRONG AND J. C. GEER, Am. J. Pathol., 34 (1958) 209. 4 T. B. CLARKSON, R. W. PRICHARD, M. G. Ni~TSKY AND H. B. LOFLAND, Arch. Pathol., 68 (1959). 143. s R. W . PRICHARD, T. B. CLARKSON, H. O. GOODMAN AND H. B. LOFLAND, Arch. Pathol., 77 (1964) 244. 6 K. ABEL, H . DE SCI~MERTZlNG AND J. I. PET:~RSON, J. Bacteriol., 85 (1963) 1039. 7 A. LAZZARINI ROBERTSON, JR., In: S. GARATTINI AND R. PAOLETTI (Eds.), Proceedings S3,mposium on Drugs Affecting Lipid Metabolism, Elsevier, A m s t e r d a m , 1961, p. 306. s T. KOKUBU AND O. J. POLLAK, J. Atheroscler. Res., 1 (1961) 229. 9 A. LAZZARINI ROBERTSON, JR., Studies on the Effects of Lipoid Emulsions on Arterial Intimal Cells in Tissue Culture in Relation to A therosclerosis, Thesis, G r a d u a t e Medical School of Cornell U n i v e r s i t y , 1959. 10 R. KOIDE, O. J. POLLAK AND D. A. BURNS, J. Atheroscler. Res., 3 (1963) 32. la ~V. B. ROBERTSON, J. C. GEER, J. P. STRONG AND ]7I. C. McGILL, JR., Exptl. Mol. Pathol., S u p p l . No. 1 (1963) 28. 12 M. SANDLER AND G. ]7t. ]BOURNE, Nature, 193 (1962) 1184. 13 D. ]~. GREEN AND S. FLEISCHER, Biochim. Biophys. Ac!a, 70 (1963) 554. 14 H. F. WATTS, I n : \V. LIKOFF AND J. H. MOYER (Eds.) Coronam, Heart Disease, G r u n e a n d S t r a t t o n , N e w Y o r k , 1963, p. 98.
j . Atheroscler. Res., 5 (1965) 3 7 9 - 3 8 7