Susceptibility to Spontaneous Atherosclerosis in Pigeons: An Autosomal Recessive Trait

Abstract

The inheritance pattern for susceptibility to spontaneous (noninduced) aortic atherosclerosis in pigeons was determined by crossbreeding and backcrossing experiments with atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer breeds. Susceptibility, assessed by the presence of grossly visible lesions at the celiac bifurcation of the aorta at 3 years of age, demonstrated an inheritance pattern consistent with an autosomal recessive Mendelian trait. Cell culture studies indicated that susceptibility is a constitutive property of aortic cells as evidenced by vacuole formation and lipid content in smooth muscle cells from various tissues in susceptible pigeons.

Atherosclerotic cardiovascular disease is the leading cause of death in the United States and other economically developed countries. Genetic factors are currently recognized as major determinants of this pathology (Galton and Ferns 1989), which is considered the most prevalent genetic disorder affecting humans (Funke and Assman 1999). Susceptibility to atherosclerosis is believed to have a complex phenotype probably involving a number of linkages, and this complexity has made attempts to characterize the genetic mechanisms problematic. It is now believed that in addition to numerous genes, significant gene-environment interactions are likely (Breslow 2000). Therefore an understanding of the role of inheritance appears crucial to significantly reducing the death rate.

Since genetic studies of atherosclerosis in humans are difficult, much work has been directed toward animal models, especially the laboratory mouse. The mouse is technically advantageous because of its small size, short generation time, and the availability of many inbred strains (Breslow 2000). However, laboratory mice fed a chow diet do not develop spontaneous atherosclerotic lesions; atherosclerosis must be induced by feeding a diet containing 15% fat, 1.25% cholesterol, and 0.5% cholic acid. This dietary manipulation presents serious limitations for comparison with humans because of the atypical vascular location of the lesions and their lack of pathologic progression (Smithies and Maeda 1995).

Consequently, from the perspective of pathology, a more relevant animal model than the mouse would be desirable for genetic studies. The White Carneau (WC) pigeon (Columba livia) develops spontaneous (naturally occurring) atherosclerosis without elevated plasma cholesterol levels and in the absence of other known risk factors (Clarkson et al. 1959). These noninduced atherosclerotic lesions are morphologically and ultrastructurally similar to those seen in humans (Cooke and Smith 1968; Santerre et al. 1972) and occur at similar geographic sites along the arterial tree (Kjaernes 1981). A variety of studies have clearly demonstrated that susceptibility in the WC resides at the level of the arterial wall (reviewed by St. Clair 1983). Under identical diet and housing conditions, and with similar blood cholesterol levels, the Show Racer (SR) pigeon is resistant to the development of atherosclerosis (Clarkson et al. 1959).

Although atherosclerosis in the pigeon model has been studied for more than 40 years, the inheritance mechanism has not been elucidated. Several early reports (Goodman and Herndon 1963; Herndon et al. 1962; Wagner et al. 1973) suggested a “polygenic mechanism with dominance of factors for resistance” and that factors responsible for lesion initiation were largely independent of factors responsible for lesion progression from initial to advanced stages. Unfortunately the one crossbreeding study reported was confounded by feeding the pigeons a high-fat, high-cholesterol diet to “accelerate” the atherosclerotic pathology, and the individual F₁ and F₂ progeny were not examined (Wagner et al. 1973). The pathologic processes involved in spontaneous atherosclerosis differ dramatically from those of diet-induced or diet-aggravated atherosclerosis in pigeons (Gosselin 1979; St. Clair 1983).

The objective of this study was to define the mechanism for inheritance of susceptibility or resistance to spontaneous atherosclerosis in pigeons by classical breed ing studies—that is, crossbreeding and backcrossing. From an examination of the presence or absence of grossly visible, spontaneous celiac lesions at 3 years of age in individual atherosclerosis-suscepti ble WC, atherosclerosis-resistant SR, and in F₁, F₂, and backcross progeny, we report that susceptibility to spontaneous ather osclerosis is inherited in a pattern consis tent with a single-gene, autosomal recessive, Mendelian trait. Smooth muscle cells cultured from several tissues of WC, SR, and F₁ pigeons demonstrate that lipid ac cumulation in susceptible aorta cells is a constitutive property of WC.

Materials and Methods

White Carneau and Show Racer pigeons were obtained from the University of New Hampshire (UNH) colonies which are housed in fly coops at ambient tempera ture and allowed free access to water, Purina Pigeon Chow Checkers, and Palmetto Pigeon Health Grit. The colonies were established in 1962 with birds obtained from the Palmetto Pigeon Plant (Sumter, SC) and have been inbred ever since. The col onies are maintained under the supervi sion of the UNH Animal Care and Use Com mittee. For crossbreeding, 40 males and females of each breed were paired in 30 inch × 30 inch × 30 inch breeding cages containing a roost and two nest boxes. F₁ progeny were removed at 4 weeks of age, banded, placed in a fly coop, and allowed to pair randomly to produce F₂ progeny. Sixteen F₁ males and females were selected and backcrossed with parental WC and SR birds in breeding cages. The backcross progeny were removed at 4 weeks of age, banded, and placed in a fly coop.

At 3 years of age, birds were sacrificed for necropsy. Aortas were removed from the heart to the sciatic trifurcation, opened longitudinally along the dorsal side, and the celiac branch region was dissected free. The most prominent and widely studied spontaneous atherosclerotic lesion in susceptible pigeons occurs at the celiac bifurcation of the aorta and reaches a size that is easily visible on gross examination by 3 years of age (San terre et al. 1972; Wagner et al. 1973). Lesions were observed as raised, yellow areas under lighted magnification at 1.2×. The location, pathology, and age progression of these lesions have been described in detail by Santerre et al. (1972).

Primary aortic smooth muscle cell cultures were prepared from 18-day WC, SR, and F₁ embryos according to the method de veloped in this laboratory (Smith et al. 1965). In addition, smooth muscle cell cul tures were prepared from the small intestine and crop of WC and SR embryos. Thirty cells were examined at 310× magnification in each of four fields around 10 explants for each culture to determine a vacuole grade for that culture. Vacuole grades were as signed as follows: (1) no Oil Red O-positive vacuoles; (2) less than ⅓ cytoplasm occupied by Oil Red O-positive vacuoles; (3) ⅓ to ½ cytoplasm occupied by Oil Red O-positive vacuoles; and (4) more than ⅔ cyto plasm occupied by Oil Red O-positive vacuoles. Lipid content was calculated from a standard curve based on at least 40 largescale cultures from the parental breeds. Un fortunately, insufficient F₂ and backcross progeny were available for culture since larger numbers would have been needed to observe the expected segregation ratios. In addition, pigeons are reported to be monog amous and to pair for life (Levi 1957), and we observed that up to 1 year was neces sary for rematched pairs to produce off spring in backcross matings. Even when reproducing well, one pair of pigeons produces offspring at a maximal rate of two progeny every 6 weeks from April through September in New England, and rematched pairs produced at lower rates.

Results

Figure 1 shows the lesion-free celiac region of an aorta, which is typical of 3-year-old resistant pigeons, while Figures 2 and 3 illustrate typical atherosclerotic lesions in the celiac region of susceptible pigeons. The presence or absence of lesions at this age was unmistakable, and, when present, lesions corresponded to at least a grade 3 lesion as described by Santerre et al. (1972), with 50% being grade 4 or 5. Grade 3 lesions are characterized by appreciable protrusion into the lumen, large amor phous lipid pools, some central necrosis, and involvement of the underlying medial layers. Grade 3 lesions contain 3.5 times more esterified cholesterol on a DNA basis than does the normal, unaffected aorta. Grade 4 lesions exhibit significant luminal occlusion (up to 50%) and have a prominent fibrous cap over an extensive central necrotic area. These lesions have a 30-fold greater cholesteryl ester content than the normal aorta. Grade 5 lesions cause massive luminal occlusion (50–90%) and contain thromboses and areas of calcification in the necrotic lesion core (Nicolosi et al. 1972; Santerre et al. 1972).

Figure 1.

Lesion-free celiac region of atherosclerosis-resistant pigeon aorta. The celiac bifurcation is the hole near the center of the segment.

Figure 2.

Grade 3 atherosclerotic lesion in the celiac region of a susceptible pigeon aorta.

Figure 3.

Grade 4 atherosclerotic lesion in the celiac region of a susceptible pigeon aorta.

Frequencies of atherosclerotic lesions in 3-year-old pigeons in each mating category (parentals and F₁, F₂, and backcross progeny) are presented in Table 1 along with the frequencies expected if susceptibility is an autosomal recessive trait. Chi-squared analysis showed that the observed frequencies were not significantly different (Table 1) from the expected values. The one lesion found in an F₁ pigeon appeared to be much smaller than the typical lesions seen in other birds, and was different in color and orientation. This pigeon had not been used in a backcross mating. Since resistance to atherosclerotic lesion development in pigeons can be overwhelmed by stress (Lewis 1983), it is possible that this F₁ pigeon was at the low end of the social order in that coop. There is also the possibility of a mutation, but this seems remote.

Table 1.

Incidence of spontaneous aortic lesions in matings of White Carneau (WC) and Show Racer (SR) pigeons demonstrating autosomal recessive inheritance for susceptibility to lesion formation

Mating type	Total	Aortic lesions n (%)	Expected n	Lesion scorea (mean ± SEM)	No aortic lesions n (%)	Expected n
White Carneau (WC)	40	40 (100)	40	3.8 ± 0.1	0 (0)	0b
Show Racer (SR)	40	0 (0)	0	0	40 (100)	40b
F1 (WC {♀} × SR {♂})	26	1c (3.8)	0	2	25 (96.2)	26b
F1 (SR {♀} × WC {♂})	18	0 (0)	0	0	18 (100)	18b
Total F1	44	1c (2.3)	0	2	43 (97.7)	44b
F2	11	2 (18.2)	2.75	4.0 ± 0	9 (81.2)	8.25d
F1 × SR	16	0 (0)	0	0	16 (100)	16b
F1 × WC	40	19 (47.5)	20	3.4 ± 0.1	21 (52.5)	20e

^aLesion score as described by Santerre et al. (1972).

^bNo deviation from expected value.

^cAtypical lesion.

^dχ² = 0.27, df = 1, P = .60.

^eχ² = 0.10, df = 1, P = .75.

Vacuole grades and lipid contents for smooth muscle cell cultures are presented in Table 2. Values within each breed were similar for cells from all three tissues, but were significantly different between WC and SR. There was no difference in vacu olization and lipid content between aorta smooth muscle cell cultures prepared from F₁ pigeon embryos and SR cultures, but the F₁ cultures were different from WC cultures.

Table 2.

Vacuole grade and lipid content in smooth muscle cells cultured from embryonic White Carneau (WC) and Show Racer (SR) pigeon tissues^a

	Smooth muscle cells
Mating type	n	Aorta	n	Small intestine	n	Crop
	Vacuole grade
White Carneau (WC)	8	2.7 ± 0.1ax	8	2.6 ± 0.1ax	8	2.6 ± 0.1ax
Show Racer (SR)	10	2.0 ± 0.1bx	7	2.0 ± 0.1bx	8	2.0 ± 0.1bx
F1	9	2.0 ± 0.1bx		nd		nd
μg lipid/μg DNA
White Carneau (WC)	8	60.4 ± 1.9ax	6	59.0 ± 1.7ax	8	59.3 ± 1.2ax
Show Racer (SR)	10	50.5 ± 1.4bx	7	49.8 ± 1.7bx	8	50.1 ± 1.2bx
F1	9	50.5 ± 1.6bx		nd		nd

^a,bMeans within a variable column with different superscripts differ (P ≤ .05) significantly for comparisons among mating types. ^x,yMeans across a single row with different superscripts differ (P ≤ 0.05) significantly for comparisons among smooth muscle cells. nd = not done.

^aValues are mean ± SEM.

Discussion

From the data it is clear that susceptibility to spontaneous aortic atherosclerosis in WC pigeons, whose phenotype is charac terized by grossly visible lesions at the celiac bifurcation, is inherited in a single-gene autosomal recessive pattern. Our results correspond with early reports that resistance to atherosclerosis in pigeons is a dominant trait (Goodman and Herndon 1963; Herndon et al. 1962). However, our data indicate a single gene rather than a polygenic mechanism. Since the previous genetic studies examined diet-induced lesions rather than susceptibility to spontaneous (naturally occurring) atherosclerosis, it is quite possible that there is a significant genetic-environment (diet) interaction that overwhelms or complicates the underlying susceptibility-resistance pattern. Wagner (1978) has stated that factors responsible for progression of athero sclerotic lesions in susceptible birds are independent of those responsible for initiation of lesions. His data support the idea that a high-fat, high-cholesterol diet overwhelms the genetic resistance of SR pigeons to some degree. In addition, Wagner et al. (1973) further state that within each breed of pigeon there are hyper- and hyporesponders to dietary cholesterol, as evidenced by the degree of elevated plasma cholesterol produced. Genetic lines of hyper- and hyporesponders within each of the two breeds were subsequently produced (Wagner 1978; Wagner and Clarkson 1974; Wagner et al. 1973), which clearly in dicates an additional genetic relationship to induced lesion development. This mechanism is probably distinct from the initiation of spontaneous lesions, and perhaps even from lesion progression, be cause induced plasma cholesterol levels and lesion severity are poorly correlated. In fact, Wagner (1978) stated that genetic factors related to hyperlipidemia appear to be independent of factors that render the arterial wall susceptible to lesion initiation. Wagner et al. (1973) also reported marked variations in induced lesion incidence between siblings in the F₂ population, which could be explained by the the oretical pattern of 75% resistance/25% susceptibility for lesion formation (as demonstrated in our work), compounded by the genetic differences in response to an atherogenic diet. Since susceptibility to spontaneous atherosclerosis was not studied by Wagner et al., its inheritance pattern could be quite different from susceptibility to diet-induced or diet-aggra vated lesions.

Our cell culture data are in agreement with the spontaneous lesion incidence observed in vivo. It appears that smooth muscle cells from the aortas of susceptible and resistant pigeons express some component(s) of the phenotypes when cultured. Smooth muscle cells cultured from embryonic pigeon aortas have previously been shown to resemble smooth muscle cells in celiac segments of the aorta of corresponding pigeons and, in the case of WC, these cultured cells show degenerative changes identical to those in developing atherosclerotic lesions, but with a greatly compressed time frame in vitro (1 year in vivo to 12 days in vitro) (Smith and Smith 1974). Therefore the properties of primary cultures from F₁ progeny observed in the present study are consistent with the lesion incidence observed in vivo. More important, the cell culture experiments demonstrated that lipid accumulation in aortic smooth muscle cells (an early characteristic of spon taneous atherosclerosis in pigeons; Cooke and Smith 1968; Wight et al. 1977) is a con stitutive property of cells from susceptible pigeons.

In addition to being a good model in which to study atherogenic factors leading to lesion development at the level of the arterial wall, it is now apparent that the pigeon is also a valuable species for identification of the genetic influence on lesion development in a “simplest case” scenario uncomplicated by risk factors. The findings reported herein suggest that subtractive hybridization is the best approach to identifying the gene responsible for susceptibility or resistance. Once the gene responsible for susceptibility or resistance is isolated and characterized, ultimately it should be possible to determine the causative metabolic event and then to describe the biochemical sequence of atherogenic pathology, as well as the perturbations or aggravations induced by environmental conditions.

Corresponding Editor: Stephen E. Bloom

This is Scientific Contribution no. 2074 from the New Hampshire Agricultural Experiment Station. The authors would like to thank Drs. Charles Boyd and Roger Cady for their suggestions and advice in designing and implementing this study. A portion of the cell culture data was provided by A. F. Stucchi, I. C. Farber, and D. P. Hajjar. Thanks also go to Dr. Tom Foxall for preparation of the figures, and to Janet Anderson for assistance in preparing the manuscript. Dedicated commitment and excellent technical assistance by Shirley Ahern is deeply appreciated.