Considerations of aortic elastin chemistry in the genesis of the intimal plaque (Broad-Breasted White turkey)

EXPEHIXIEXTAL

AND

MOLECULAR

PATHOLOGY

Considerations of Aortic the lntimal Plaque ROBERT

J.

BOUCEK,

‘,*

( 1979)

Elastin Chemistry in the Genesis (Broad-Breasted White Turkey)

NANCY

AND

31,400412

CHARLES

L. NoBLE,t ZEENAT F. SIMPSON $

GUN

of

JA-SMITH,*

* Department of Medicine, Unir;ersity of Miami School of Medicine, Miami, Florida 33101; t Departments of Biochemistry and hledicine, University of hliami School of Medicine, Miami, Florida 33101; $ College of Veterinary hledicine, Unicersity of Florida, Gainesville, Florida 32610 Received July 30, 1979, and in reoised form Azgu.rt 31, 1979 Fragmentation of internal and external elastic membranes occurs in the abdominal but not in the thoracic aorta of the Broad-Breasted White turkey during the early developmental period. In this early period, isolated elastin from the abdominal aorta contains more polar amino acid residues than elastin of the thoracic aorta. Additionally, the relative amount of a chemically unstable elastin cross-link, dehydrolysinonorleucine, is greater in the abdominal as compared to the thoracic aorta during the early developmental and adult periods. Cells of the abdominal aorta exposed to a higher arterial pressure and a greater rate of arterial pressure rise (dp/dt) and in a region with a higher collagen:elastin ratio (reduced compliance to pulsepressure-volume deformations) may synthesize a chemically unstable elastin which breaks down in regions of accentuated wall stress. Through these breaks of the internal elastic membranes, modified smooth muscle cells may migrate, proliferate and ultimately synthesize new elastin in forming the intimal plaque.

INTRODUCTION Enlargement of the fenestrae and fragmentation of the internal elastic membrane along with intimal hyperplasia are early age-related arterial tissue changes occurring in man (Ehrich et al., 1931; Gross et nE., 1934; Levene, 1956; Velican and Velican, 1976). These histologic changes are not uniform along the course of arteries, and in one species, the Broad-Breasted turkey, they appear as early as 6 weeks of age in the abdominal but not in the thoracic aorta (McDonald et al., 1964). The question arises whether the fragmentation of the elastic membrane simply reflects a focal accentuation of a hemodynamic stress causing a mechanical disruption of elastic membranes or whether regional differences in chemical properties of elastin predispose the elastin to accelerated elastin degradation. To develop information on this question, we determined the amino acid composition and the profile of lysyl-derived cross-links of abdominal and thoracic aortic elastins in immature and mature Broad-Breasted White (BBW) turkeys. That arterial elastin varies chemically along the course of the aorta is sug’ To whom

correspondence

should

he nddressetl. 400

0014-4800/79/060400-13$02.00/O Copyright AII rights

0 1979 by Academic Press, Inc. of reproduction in arly form reserved.

ELASTIN

CHEMISTRY

AND ELASTIC

MEMBRANE

FRAGMENTATION

401

gested by differences in histological staining (Wolff, 1928; Gillman, 1959), in the afllnity for calcium salts (Lansing et al., 1950) and in amino acid composition( Lansing et al., 1952). Grant found increased amounts of hydroxyproline in purified aortic elastin from the pig, sheep, goat and human as the vessel was traversed from the arch to the subrenal section, and also higher values for glutamic and aspartic acids and basic amino acids in the abdominal as compared to the thoracic aortic elastin in the pig and human (Grant, 1966; Grant, 1967). McCullagh et al. (1973) found higher values of polar amino acid residues in elastin of the abdominal than the thoracic aorta of the African elephant. These data suggest chemical difference in different aortic elastins; however, variations in the cross-link composition of aortic elastin have not been reported. Lysyl-derived cross-links form as the soluble elastin precursor, tropoelastin (Smith et al., 1968; Smith et al., 1972; Sandberg et al., 1971), is converted into an insoluble protein (Narayanan and Page, 1976) that is highly resistant to inorganic and organic solvents (Lowry et al., 1941). These cross-linking compounds develop after enzymatic conversion of specific peptidyl-lysyl residues to cu-aminoadipic-y-semialdehyde ( allysine) (Pinnell and Martin, 1968) which in turn condenses with a neighboring peptidyl-lysyl-derived aldehyde in forming an aldol condensation product (Lent et aZ., 1969), or three peptidyl-allysines fuse with a peptidyl-lysyl residue to form a pyridinium ring structure (isodesmosine and desmosine) (Thomas et al., 1963; Partridge et al., 1963; Anwar and Oda, 1966), or a peptidyl-allysine interacts with an c-amino group of a neighboring peptidyl-lysyl residue (lysinonorleucine) ( Franzblau et al., 1965; Lent and Franzblau, 1967), or the aldol condensation product of two peptidylallysine interacts with a peptidyl-lysyl group in forming merodesmosine (Starcher et al., 1967). A comparison of the chemical nature of the elastin isolated from two regions of the aorta of the BBW turkey reveals larger amounts of potentially polar amino acid residues in elastin from the abdominal than from the thoracic aorta. Regional differences in the levels of lysyl-derived cross-links of elastin are also found in aortic tissue. With increasing age, the amino acid composition of elastin becomes more uniform along the course of the aorta; however, differences in the cross-links persist. The chemical properties of the two aortic elastins suggest a greater polarity and a less stable lysyl-cross-linking in abdominal than in thoracic aortic elastin. These chemical differences may influence the susceptibility of elastin to elastolysis leading to fragmentation and loss of elastic Iamina and, ultimately, to intimal plaque formation in the abdominal aorta of the turkey. MATERIALS

AND METHODS

One-day-old male and female domesticated turkeys of the BBW strain were fed a 22% protein diet until 4 weeks of age and a 16% protein diet until sacrificed at 4 or 10 weeks or 8 months of age. The diets were supplemented with minerals and vitamins in accordance with the nutritional recommendations of the National Research Council for turkeys. The birds were weighed biweekly. Two days prior to sacrifice, heart rate and arterial pressures were recorded on a linear-core P-166 transducer (Narco Bio-Systems Inc., Houston, TX) through a plastic catheter inserted through procaine-infiltrated skin and then

402

BOUCEK ET AL.

into the carotid artery. In addition, a 0.62” OD catheter was passed through locally anesthetized skin and threaded through the femoral artery to the thoracic aortic arch, and the arterial pressure and maximum rate of pressure rise mm were recorded on a differentiation coupIer 730 (Narco BioWsec (dp/W Systems Inc.) at several sites as the catheter was withdrawn. At sacrifice the aorta was removed, trimmed of extraneous tissue and sections removed for histologic studies. The remainder was frozen for biochemical analyses. Histologic samples were fixed in Bodian’s solution and stained with Gomori aldehyde fuchsin stain for elastic fibers (Gomori, 1950). Elastin was isolated from the abdominal and thoracic aortae according to the method of Starcher and Galione (1976). Weighed tissue samples (60 to 80 mg, ww) were homogenized in phosphate buffer (0.05 M NaZHPOJ, pH 7.6, and extracted with the buffer for 72 hr containing 1% NaCl and 0.1% EDTA) with changes twice daily. After washing twice with water, the residue was freeze-dried, autoclaved and then treated with trypsin, cyanogen bromide : 97% formic acid, and urea:/+mercaptoethanol as outlined in the procedure. The final product was defatted with ether and acetone and freeze-dried. The product was hydrolyzed in zjucz1.0 (6 N HCI, 48 hr, 110°C) and the amino acids, isodesmosine, desmosine and lysinonorleucine, were separated and quantitated using the Jeol-5AH Automatic Amino Acid Analyzer and a sodium citrate buffer system. To determine the borohydride-reducible cross-links of the elastin of aortic tissue, weighed samples of abdominal and thoracic aortae (40 to 120 mg) were homogenized in water, freeze-dried, placed in 0.2 M phosphate buffer, pH 7.4, and stirred at 4°C for 16 hr and at room temperature for 1 hr. [HS]NaBHA (310 mCi/mmole, New England Nuclear, Boston, M4) dissolved in dimethylformamide (I5 mg/2.5 ml) was added to the tissue in three equal aliquots at 3-min intervals to give a total of 68 pg of NaBHi per mg of estimated elastin and collagen. Initial results indicated that approximately 10% of the wet weight of the abdominal and 20% of the thoracic aortic tissue was elastin and collagen. The reduction was stopped by the addition of glacial acetic acid to pH 5-6. The reduced tissues were dialyzed exhaustively against water (4”C), freezedried and then hydroIyzed in ~jncuo in 3 N p-toluene sulfonic acid (24 hr at 110°C). The hydrolysate was filtered through a sintered gIass funne1 and radioactivity and hydroxyproline were determined. A sample containing 2 x 10” cpm was applied to a cation-exchange column and the cross-links eluted with sodium citrate buffers as described previously (Boucek et aZ., 1979). To further resolve the labeled cross-links, fractions containing dihydroxylysinonorleucine, hydroxylysinonorleucine, merodesmosine and lysinonorleucine were individuaIIy pooled, placed on a second cation-exchange column and eluted with 0.35 M sodium citrate buffer, pH 5.28, containing 0.55% benzyl alcohol as described by Mechanic ( 1974). To quantitate isodesmosine, desmosine and lysinonorleucine, another sample of the tosyl hydrolysate was analyzed on the JeoI-5AH Automatic Amino Acid Analyzer. . -__----.FIG. 1. (A) Abdominal aorta of the Broad-Breasted White (BBW) turkey plaques (IP), adventitia {AD) and media (M): G6mGri aldehyde fuchsin Fragmented internal elastic lamina (IEL) separating the media from the Note the smooth muscle (SM) and the stratified elastic lamina in the plaque. designated (L) : GGmiiri aldehyde fuchsin ( X180,0).

showing (~112.5). intimal \‘ascular

intimal (B) plaque. lumen

ELASTIN

CHEMISTRY

AND

ELASTIC

MEMBRANE

FRAGMENTATION

403

404

BOUCEK

AORTICdRGH -DISTANCE

ET AL.

[cm]-

ILIAC ARTtRY

FIG. 2. Peak systolic pressure, mm Hg (0 - - - 0) and rate of arterial pressure rise, mm Hg/sec, dp/dt ( O---O ) along the course of the BBW turkey aorta. Pressures recorded through a linear-core P-100 transducer and the dp/dt on a differentiation coupler 730 (Narco Biosystems Inc. ) . To calculate the elastin composition of abdominal and thoracic aortae, the amount of isodesmosine and desmosine in the whole tissue hydrolysate was converted to elastin weight equivalents using a factor derived from the weight percent of the two cross-links in elastin isolated from the same tissue. The amount of hydroxyproline in this elastin value (calculated using the factor derived from the weight percent of hydroxyproline in the isolated elastin) was subtracted from the total hydroxyproline of the tissue. The resultant hydroxyproline value was converted to a collagen equivalent using the factor of 7.46 (Neuman and Logan, 1950). Radioactive samples were analyzed in the Packard 3003 TriCarb Liquid Scintillation Spectrometer in a solvent system of Triton X-100 and toluene ( 1:2 v/v) containing 5.0 g of 2,5-diphenyloxazole and 0.1 g of 1,4-bis-2-(4-methyl-5phenyloxazolyl)-benzene per liter. To dissolve the buffer salts in the aliquots (0.1 to 0.5 ml) from the column fractions, 1 ml of water was added to 14 ml of the solvent system. Results were expressed as cpm and corrected for background.

RESULTS Intimal hyperplasia of elastin-staining material, the intimal plaque, is irregularly dispersed around the lumen of the abdominal aorta from a lo-week-old turkey as shown in Fig. 1A. An intimal plaque consistently develops in the abdominal aorta of the BBW turkey and contains fragmented internal elastic membrane, modified smooth muscle cells (Simpson, 1972) and fibers that stain as elastin (Fig. 1B). The plaque increases in size with age (Simpson, 1972). Fragmentation of the internal elastic membrane and intimal plaque formation are not seen in the thoracic aorta. Because these regional histologic changes may reflect different mechanical stressesin the artery wall, the arterial pressure and the rate of pressure rise (dp/dt) were recorded from the aortic arch to the iliac artery. As shown in Fig. 2, both the systolic pressure and the dp/dt increase along the course of the aorta and reach their highest levels in the iliac region of the distal abdominal aorta.

ELASTIN

Amino

Amino

CHEMISTRY

Acid

AND

Composit,ion

ELASTIC

MEMBRANE

TABLE

I

Isolated (Females)

from

of Elnst~ins

acid

Residue/1000 -~.

.~

FRAGMENTATION

Abdominal

and Thoracic

a-Month -

Lys His

3.94 1.39 8.52 13.6 6.31 10.0 6.89 17.8 123 366 172 153

k HYP Asx Thr Ser Glx Pro GUY Ala Val Met Ilu Leu

2.08 0.57 8.37 14.5 3.99 8.25 4.07 13.7 118 383 175 155

-

TY~ Phe Ide Des LNI, Glx, Lys, Thr,

Thoracic

f 0.14 f 0.07 f 0.26 f 0.9 f 0.37 f 0.4 i 0.50 f 0.4 f5 dz5 fl f 1

Asx His, Arg Ser, Tyr

a Each value separated and sodium citrate

Aortae

residues5 __-

lo-Week Abdominal

405

_Abdominal

f f f f f & * 5 fl f2 fl z&

0.07 0.10 0.69 1.0 0.1-i 0.14 0.22 0.3

1

1.69 0.78 6.33 13.2 3.01 10.0 4.28 13.5 135 366 197 1.53

f f f f f & zk f f8 f z!z f2

Thoracic 0.18 0.09 O.-i7 l.T, 0.23 0.3 0.23 0.9 7 18

1.67 0.62 7.65 18.1 3.24 7.74 3.42 14.4 124 378 172 154

-

zlz 0.12 31 0.13 f 0.3H f 1.0 zt 0.06 f 0.19 f 0.06 f 1.5 f 11 f 4 +3 f4 -

24.1 52.0 14.3 20.8 1.05 1.25 052

f f f f f f f

0.6 2.8 0.2 0.4 0.03 0.02 0.04

23.5 .a.7 13.3 22.6 1.03 0.97 0.52

f f f i f f f

0.4 2.2 0.1 0.9 0.02 0.01 0.01

22.0 53.1 12.8 19.9 0.87 0.89 0.54

f f zt f f f f

0.8 1.7 0.6 1.0 0.08 0.09 0.05

23.0 54.9 13.4 20.7 0.87 0.93 0.61

f f f f f f f

0.2 0.3 0.3 0.7 0.05 0.05 0.02

24.1 13.8 31.2

f f f

0.6 0.1 1.0

17.7 11.0 25.6

f f f

0.4 0.6 0.5

16.5 8.99 27.1

f f f

1.1 0.77 1.1

17.6 f 9.94 f 24.6 f

1.5 0.37 0.5

is the mean quantitated system.

for three tissues % standard error of the mean (SEM). using the Jeol-5AH Automatic Amino Acid Analyzer

Residues were and a B-buffer

A comparison of the amino acid composition of elastin isolated from the abdominal and thoracic aortae of the lo-weekand &month-old BBW turkey can be made from the data in Table 1. While the amounts of the numerically dominant non-polar amino acids (glycine, alanine, valine and proline) are generally similar for each of the four elastins, differences are seen in the amounts of the polar amino acid residues in the two elastins from the lo-week-old turkey. The elastin from the abdominal aorta contains in 1000 residues 24.1 residues of Asx and Glx (aspartic and glutamic residues in the acid and amide forms), 13.8 residues of lysine, histidine and arginine and 31.2 residues of amino acids with unionized but polar substituents (threonine, serine, and tyrosine) as compared to 17.7, 11.0, and 25.6 respectively for thoracic aortic elastin (P < 0.02 for comparisons in each of the three groups of residues). These differences in amino acid composition are not seen in elastins isolated from the 8-month turkey. The amounts of the lysyl-derived cross-links (isodesmosine, desmosine and lysinonorleucine) are similar in the elastins of the lo-week-old birds. Aortic elastins from the Smonth-old animals have lower levels of the desmosines,

406

BOUCEK

ET

AL.

while the level of lysinonorleucine remains unchanged (abdominal aorta) or increases (thoracic aorta) when compared to elastins from the lo-week-old animals. Comparisons of the borohydride-reducible lysyl-derived cross-links of elastin and collagen of the abdominal and thoracic aortae are shown in Fig. 3. Using the two-column chromatographic system to resolve the cross-links, the most prominent reducible cross-links in the thoracic aortic tissue are merodesmosine and lysinonorleucine, while dihydroxylysinonorleucine (resolution of Peak D; inset, Fig. 3) of collagen is the most prominent peak of the abdominal aorta. Since borohydride-reducible lysinonorleucine exists in both cohagen and elastin, the amount in elastin was estimated by subtracting the collagen-derived [3H]lysinonorleucine calculated by using the 10 : 1 13H] hydroxylysinonorltucine : [“H]lysinonorleucine ratio reported for collagen (Kang et al., 1970; Boucek et al., 1979) from the total [3H]lysinonorleucine. Measurements of the borohydride-reduced aldol condensation product were not included in this study since this elastin cross-link is partially destroyed by acid hydrolysis. The relative concentration of borohydride-reducible lysyl-derived cross-links in the elastins of the aortic tissue of the lo-weekand S-month-old turkeys are given in Table II. At both ages, the estimated concentration of elastin dehydrolysinonorleucine relative to the total elastin lysinonorleucine or to the desmosines is higher in the abdominal than in the thoracic aorta, and this difference becomes larger with age (approximately 20% at 10 weeks increasing to approximately 50% at 8 months). The concentration of dehydromerodesmosine, on the other hand, is significantly lower in the abdominal than in the thoracic aorta at both age periods. Both dehydro-forms of elastin cross-links are markedly lowered at 8 months of age in the two aortic regions. The elastin and collagen concentrations of the abdominal and thoracic aortae at three developmental periods are shown in Table III. At each period, the collagen : elastin ( C/E ) ratio is greater (4 to 6 times) in the abdominal than TABLE NaB3H4-Reducible

Elast.in

Cross-links

11

of Intact

Abdominal

LNL vpm/mg

and Thoracic

LNL LNL

Aortae AID

cpm/mg

(Ide

+ DPS)

x IO-3 lo-Week old ( 0 ) Abdominal (3) Thoracic (3)

2.12 & 0.10 1.53 + 0.03 <0.02

3.88 3x 0.29 6.50 + 0.07
+ 0.46

0.31

0.17 f

1.62 zt 0.07

0.20

20.0 16.1

f i

1.02 2.1

P g-Month old Abdominal (2 03:3 0) Thoracic (3 oT:3 0)

3.18

P

Mean value in the tissues.

f

SEM


of turkeys

indicated

f

0.04

zt 0.01 <0.02

in parenthesis.

0.06

1.54 f 0.04
occurred

ELASTIN

CHEMISTRY

AND

ELASTIC

MEMBRANE

FRAGMENTATION

407

FIG. 3. Chromatographic separation of sodium borohydride ( NaB3Ha) -reducible cross-links of the thoracic and abdominal aorta of the BBW turkey through a cation-exchange column. Resolution of Peak D by a second cation-exchange column is shown in the inset. Abbreviations: DHLNL + dihydroxylysinonorleucine; HNL + hydroxynorleucine; HLNL + hydroxylysinonorleucine; MD * merodesmosine; LNL -+ lysinonorleucine; HHMD + histidinohydroxymerodesmosine. Amino acid markers at the top of the chromatograms: Hyp + hydroxyproline; Pro -+ proline; Phe + phenylalanine; His + histidine; Lys + lysine.

in the thoracic aorta. From the 4-week period to 8 months of age, elastin concentration increases in both aortic regions with relatively minor age-related changes in collagen concentration. DISCUSSION Certain features of aortic elastin having potential bearing on elastic membrane fragmentation and intimal plaque formation are suggested by these studies in the BBW turkey. Fragmentation and loss of elastic fibers occur in the abdominal but not in the plaqueless thoracic aorta during the growth and developmental period of the turkey, and the elastin from the abdominal aorta contains higher concentrations of potentially polar amino acid residues and borohydride-reducible lysinonorleucine than the thoracic aorta. These chemical characteristics of the aortic elastins suggest a greater susceptibility of the elastin to degradation in the abdominal than in the thoracic region during the early growth and developmental period. With maturity, the chemical characteristics of the elastins change in a manner that may decrease their susceptibility to degradation; the abdominal aortic elastin becomes less polar, and both the abdominal and thoracic aortic elastins contain less dehydrolysinonorleucine and dehydromerodesmosine. In studying the chemistry of aortic elastin, the method of elastin isolation and purification is a major concern. This concern is particularly important when con-

408

BOUCEK

ET

TABLE El&in

and Collagen

in Intact

III Abdominal

Elastin

26.6 f 86.4 f 28.5 120

&Month old Abdominal (2 c?:2 PI Thoracic (3 c7:3 0)

Mean seen.

value

Collagen tissue

Aortae (C)

C/E

ww

(3 c? : 3 P f

IO-Week old (3 0 ) Abdominal Thoracic

Abdominal 4-Week Thoracic 4-Week

and Thoracic

(E) rg/mg

4-Week old Abdominal Thoracic

AL.

73.0 126

3.8 8.4

52.3 97.1

zt f

9.3 2.2

1.98 f 0.34 *

1.1 8

58.9 41.5

zt f

1.6 5.3

2.08 0.3-l

zt 11.3

86.9

f. 18.5

1.19 *

0.12

f

35.0

&

0.28

0.01

zk +

6

vs. 8-month

P < 0.01

vs. g-month

P < 0.01

f

SEM

for

number

of turkeys

indicated

1.8

0.17 0.06

f 0.13 + 0.03

f
<0.05 in parenthesis.

Xo sex differences

were

trasting the polar amino acids of elastin from arterial regions at different periods of development since varying amounts of contaminating microfibrillar proteins, collagen and other non-scleroproteins may be present, particularly in tissues from immature animals. After comparing the hydroxyproline, lysine, methionine and histidine composition of elastins isolated from the same tissue by several procedures, the method of Starcher and Galione (1976) was selected because autoclaving followed by trypsin digestion and cyanogen bromide treatment gave the lowest hydroxyproline value, and digestion with trypsin and treatment with urea and P-mercaptoethanol are generally accepted procedures for removing glycoprotein-rich microfibrillar protein. Turkey aortic elastin isolated by the Starcher and Galione (1976) procedure has no detectable methionine and one or lessresidue of histidine per 1000 amino acid residues (Table I). With the Starcher and Galione (1976) procedure, higher concentrations of potentially polar amino acids are found in the isolated elastin from the abdominal than from the thoracic aorta (Table I), and with maturity, the number of these polar amino acid residues in the abdominal aortic elastin declines. These developmental changes in the number of polar amino acid residues resemble the findings of Keeley (1971) for aortic elastin from chick embryos as compared with the SO-day adult chicken. A higher number of potentially polar amino acid residues in elastin of immature animals could affect its degradation. Given a constant level of aortic tissue elastase (Hornebeck and Robert, 1976) and elastase-inhibitors (Janoff and Scherer, 1968; Turin0 et al., 1974), polarity of elastin, particularly that arising from anionic residues, may play a decisive role in binding elastase to its substrate prior to peptide bond cleavage and elastolysis (Hall and Czerkawski, 1961; Gertler, 1971; Kagan et al., 1972; Kagan et al. 1976). The possibility of a greater susceptibility to digestion by elastase

ELASTIN

CHEMISTRY

AND

ELASTIC

MEMBRANE

FRAGMENTATION

409

and a greater metabolic turnover of abdominal as compared to thoracic aortic elastin is under current investigation in our laboratories. Recent information developed in our laboratories (Noble et al., 1979) indicates a loss of isodesmosine and desmosine during elastin isolation using the Starcher and Galione (1976) procedure. The loss of desmosineswas suggested by a higher ratio between desmosines and lysinonorleucine in the whole unfractionated aorta than in elastin isolated from a sample of the same tissue, while the levels of lysinonorleucine were shown to be unaffected by the isolation procedure. Furthermore, the loss of the desmosine fraction with elastin isolation is greater in aortic tissues from the &month-old than in the lo-week-old animals, thereby offering an explanation for the age-related reduction of isodesmosine and desmosine (Table 1). Differences in lysyl-derived elastin cross-links of the thoracic and abdominal aortae are seen when the concentrations of borohydride-reducible lysinonorleucine and merodtsmosine are compared. Reducible lysinonorleucine is consistently higher in the abdominal while reducible merodesmosine is higher in the thoracic aorta (Table II). Borohydride-reducible cross-links, usually the Schiff base types of cross-links, are unstable and decrease with age. Reducible lysinonorleucine is probably the intermediate form of lysinonorleucine (Lent and Franzblau, 1967) and may be the initial cross-link in desmosine formation (Franzblau et al., 1977). Reducible merodesmosine also may be an early crosslink in the dimerization of early forms of elastin (Pathrapamkel and Carnes, 1978), but the precise biology of this compound remains uncertain. As expected, the amounts of reducible lysinonorleucine and merodesmosine are greater in the aorta from young (lo-week-old) than from the adult (&month-old) animals. A greater concentration of reducible lysinonorleucine in the abdominal than in the thoracic aortic elastin in the adult animal suggests either a greater synthesis of an unstable elastin or a more limited conversion of the unstable to the stable form of elastin cross-link in the abdominal aorta with age. As shown in Table III, the collagen: elastin ratio is strikingly higher in the abdominal than in the thoracic aorta at three periods of development, 4 and 10 weeks and 8 months, Roach and Burton (1957) reported that static mechanical properties of the artery wall are determined by the proportions of collagen and elastin; vessels with a higher collagen:elastin ratio have greater tensile strength and a reduced distensibility (compliance) to high pressure deformation. Burton ( 1954) estimated the modulus of elasticity for collagen to be in the order of 1 x log dyne/cm2 and for elastin, 3 x lo6 dyne/cm2. As Harkness et al. (1957) pointed out, the modulus of elasticity would be expected to be higher in arteries with a large collagen: elastin ratio (abdominal aorta, Table III) compared with arteries with a low ratio (thoracic aorta, Table III). However, extrapolating the collagen:elastin value to the mechanical properties of the artery wall in situ may be simplistic because the amount of smooth muscle contraction, the anatomic arrangement of collagen and elastin fibers, the degree of longitudinal tethering and the number and location of branch arteries may affect the mechanical response of the vessel to pulse-pressure-volume deformations. Nevertheless the lateral expansion of the abdominal aorta to pulse-pressure-volume deformations is considerably less than that of the thoracic aorta in many species, suggesting a limited compliance for the abdominal aortic wall,

410

BOUCEK

ET

AL.

It is noteworthy that the difference between the collagen: elastin ratios of the thoracic and abdominal aortae of the BBW turkey is exceptionally high when compared to those in other species (Harkness et al., 1957; Fischer and Llaurado, 1966). Taking the results of the histologic, physiologic and chemical studies of the turkey aorta together suggests that fragmentation of elastic membranes and intimal plaque formation (Fig. 1A and B) occur in a region of high hemodynamic stress, dp/dt and arteria1 pressure (Fig. 2), where the compliance to pulse-pressure-volume deformations may be limited by the high collagen : elastin ratio (Table III) and where the amino acid composition of elastin (Table I) and the concentration of unstable lysinonorleucine (Table II) may render the elastin more susceptible to degradation. The findings suggest the formation of a relatively unstable elastin in the abdominal aorta during the early developmental period, and in regions of accentuated aortic wall stress (or stretch), focal elastolysis develops leading to gaps in the internal and external limiting membranes. Through these gaps, modified smooth muscle cells may migrate, prohferate and ultimately synthesize new elastin (Table III) and collagen. With maturity, the amount of polar amino acids and the level of borohydridereducible cross-links decrease, suggesting the replacement of unstable elastin with a more stable elastin but, as judged by the relative concentrations of dehydrolysinonorleucine in the two aortic regions, the stability of elastin even in the adult may be less in the abdominal than in the thoracic aorta. ACKNOWLEDGMENTS HL

This investigation 17909-04, HL

was supported in part by U.S. 17865-04 and HL 22040-01 from

Public Health Service Research Grants the National Heart and Lung Institute.

REFERENCES ANWAR, R. A., and ODA, G. ( 1966). The biosynthesis of desmosine and isodesmosine. J. Sol. Chem. 241, 46384641. BOUCEK, R. J., NOBLE, N. L., and GUNJA-S~IITH, Z. (1979). A possible role for dehydrodihydroxylysinonorleucine in collagen fibre and bundle formation. Biochem. J. 177, 853-860. BURTON, A. C. ( 1954). Relation of structure to function of the tissues of the wall of blood vessels. Physiol. Res. 34, 619-650. EHRICK, H., DE LA CHAPELLE, C., and COHN, A. F. (1931). Anatomical ontogeny-man. A study of the coronary arteries. Am. .l. Anat. 49, 241-282. FISCHER, G. M., LLAURADO, J. G. ( 1966). Collagen and elastin content in canine arteries selected from funotionally different vascular beds. Circ. Res. 19, 394-399. FRANZBLAU, C., SINEX, F. M., FARIS, B., and LA~~PIDIS, R. (1965). Identification of a new crosslinking amino acid in elastin. Biochem. Biophys. Res. Commun. 21, 575-581. FRANZBLAU, C., FOSTER, J. A., and FARIS, B. (1977). In “Elastin and Elastic Tissue: Advances in Experimental Medicine and Biology,” Vol. 79 (L. B. Sandberg, H. R. Gray, and C. Franzblau, eds.), pp. 313326. Pub. Plenum Press, NY. GERTLER, A. ( 1971). The non-specific electrostatic nature of the adsorption of elastase and other basic proteins on eIastin. Eur. J. Biochem. 20, 541-546. GILLMAN, T. ( 1959). Reduplication, remodeling, regeneration, repair and degeneration of arterial elastic membranes. AMA Arch. Path. 67, 624-642. GBMBRI, G. (1950). Aldehyde fuchsin stain for elastic tissue. Am. J. Clin. Path. 20, 665-667. GRANT, R. A. ( 1966). Variations in the amino acid composition of aortic elastin from different species. Brit. J. Ezpt. Path. 47, 163-167. GRANT, R. A. ( 1967). Content and distribution of aortic collagen, elastin and carbohydrate in different species. J. Atheroscler. Res. 7, 463-472.

ELASTIN

CHEMISTRY

AND

ELASTIC

MEMBRANE

FRAGMENTATION

411

GROSS, L., EPSTEIN, E. Z., and KUGEL, M. A. ( 1934). Histology of the coronary arteries and their branches in the human heart. Am. 1. Path. 10, 253-273. HALL, D. A., and C~ERKAWSKI, J. M. ( 1961). The reaction between elastase and elastic tissue. Biochem. J. 80, 128-134. HARKNESS, M. L. R., HARKNESS, R. O., and MCDONALD, D. A. (1957). The collagen and elastin content of the arterial wall in the dog. PTOC. Royal SOC. 146, 541551. HORNEBECK, W., and ROBERT, L. ( 1976). I n “Elastin and Elastic Tissue: Advances in Experimental Medicine and Biology,” Vol. 79 (L. B. Sandberg, W. R. Gray, and C. Franzblau, eds.), pp. 145-162. Pub. Plenum Press, NY. JANOFF, A., and SCHERER, J. (1968). Mediators of inflammation in leukocyte lysosomes. IX. Elastinolytic activity in granules of human polymorphonuclear leukocytes. J. Exp. Med. 128, 1137-1155. KAGAN, H. M., CROMBIE, G. D., JORDAN, R. E., LEWIS, W., and FRANZBLAU, C. (1972). Proteolysis of elastin-ligand complexes. Biochemistry 11, 3412-3418. KAGAN, H. M., JORDAN, R. E., LERCH, R. M., MIJKHERJEE, D. P., STONE, P., and FRANZBLAU, C. (1976). In “Elastin and Elastic Tissue: Advances in Experimental Medicine and Biology,” Vol. 79 (L. B. Sandberg, W. R. Gray, and C. Franzblau, eds.), pp. 209-229. Pub. Plenum Press, NY. KANG, A. H., FARIS, B., and FRANZBLAU, C. ( 1970). Intramolecular cross-link of chick skin collagen. Biochem. Biophys. Res. Commun. 36, 437-444. KEELEY, F. W. (1971). Cross-links in elastin and collagen. In “Biophysical Properties of the Skin” (Harry R. Elder-r, ed.), p. 259. Wiley Interscience. LANSING, A. I., ROSENTHAL, T., and ALEX, M. (1950). Significance of medial age changes in the human pulmonary artery. .I. Gerontol. 5, 211-215. LANSING, A. I., ROSENTHAL, T. B., ALEX, M., and DEIMPSEY, E. W. ( 1952). The structure and chemical characterization of elastic fibers as revealed by elastase and electronmicroscopy. Anat. Rec. 144, 555-575. LENT, R., and FRANZBLAU, C. ( 1967). Studies on the reduction of bovine elastin: Evidence for the presence of A’, ‘-dehydrolysinonorleucine. Biochem. Biophys. Res. Commun. 26, 43-50. LENT, R. W., SMITH, B., SALCEDO, L. L., FARIS, B., and FRANZBLAU, C. (1969). Studies on the reduction of elastin. II. Evidence for the presence of a-aminoadipic acid 6-semialdehyde and its aldol condensation product. Biochemistry 8, 2837-2845. LEVENE, C. I. ( 1956). The early lesions of atheroma in the coronary arteries. J. Puth. Back LXXII, 79-83. LOWRY, 0. H., GILLIGAN, D. R., and KATEHSKY, E. M. (1941). The determination of collagen and elastin in tissues. J. Biol. Chem. 139, 795-804. MCDONALD, B. E., BIRD, H. R., and LALICH, J. J. (1964). The incidence of spontaneous pathologic changes in aortas of growing turkeys. Podtry Sci. 43, 341-352. MCCULLAGH, K. G., DEROUE~, S., and ROBERT, L. ( 1973). Studies on elephant aortic elastic tissue. Exp. Mol. Pathol. 18, 202-213. MECHANIC, G. L. (1974). A two column system for complete resolution of NaBHb-reduced cross-links from collagen. Anal. Biochem. 61, 355-361. NARAYANAN, A. S., and PAGE, R. C. (1976). Demonstration of a precursor-product relationship between soluble and cross-linked elastin, and the biosynthesis of the desmosines in uitro. J. Biol. Chem. 251, 1125-1130. NEUMAN, R. E., and LOGAN, M. A. ( 1950). The determination of hydroxyproline. J. BioZ. Chef% 184, 299-306. NOBLE, N. L., BOUCEK, R. J., and GUNJA-SMITH, Z. ( 1979). Aortic Elastin Lysyl Cross-links in Intact Tissue and Isolated E&in. XIth International Congress of Biochemistry, Toronto, Canada. PARTRIDGE, S. B., ELSDEN, D. F., and THOMAS, J. ( 1963). Constitution of the cross-linkages in elastin. Nature 197, 1297-1298. PAMRAPAMKEL, A. A., and CARNES, W. H. (1978). Isolation of a cross-linked dimer of elastin. J. BioZ. Chem. 253, 7993-7995. of collagen and elastin: EnPINNELL, S. R., and MARTIN, G. R. (1968). Th e cross-linking zymatic conversion of lysine in peptide linkage to or-aminoadipicbsemialdehyde (allysine) by an extract from bone. PTOC. N&Z. Acad. Sci. USA 61, 708-716.

412

BOUCEK

ET

AL.

ROACH, M. R., and BURTON, A. C. (1957). The reason for the shape of the distensibility curves of arteries. Can. J. Rio&. Phys. 35, 681-690. SANDBERG, L. B., ZEXUS, R. D., and COLTRAIN, I. M. (1971). Tropoelastin purification from copper-deficient swine. Biochim. Biophys. Acta 236, 542-546. SIMPSON, C. F. (1972). Comparative morphology of intimal and adventitial hyperplasia of the arteriosclerotic turkey aorta. Exp. Mol. Puthol. 17, 65-76. SMITH, D. W., WEISSMAN, N., and CARNES, W. H. ( 1968). Cardiovascular studies on copperdeficient swine. XII. Partial purification of a soluble protein resembling elastin. Biochem. Biophys. Res. Commun. 31, 309-315. SETH, D. W., BROWN, D. M., and CARNES, W. H. ( 1972). Preparation and properties of salt-soluble elastin. J. Biol. Chem. 247, 2427-2432. STARCHER, B. C., PARTRIDGE, S. M., and ELSDEN, D. F. (1967). Isolation and partial characterization of a new amino acid from reduced elastin. Biochemktry 6, 2425-2432. STARCHER, B. C., and GALIONE, M. J. (1976). Purification and comparison of elastin from different animal sources. Anal. Biochem. 74, 441447. THOMAS, J., ELSDEN, D. F., and PARTRIDGE, S. M. ( 1963). Partial structure of two major degradation products from the cross-linkages in elastin. Nature 200, 651-652. TURINO, G. M., HORNEBECK, W., and ROBERT, B. (1974). In eioo effect of pancreatic elastase. I. Studies on serum inhibitors, PTOC. SOC. Exp. Biol. Med. 146, 712-717. VELICAN, C., and VELICAN, D. (1976). Intimal thickening in developing coronary arteries and its relevance to atherosclerotic involvement. Atherosclerosis 23, 345-355. WOLFF, E. R., (1928). Elastica und pseudoelastica der grossen arterien: Ein beitrag zur frage der neubildung elestischer membranen. Virchow Arch. Path. Anat. Physiol. 270,

37-50.