The JCR:LA-cp rat: a novel rodent model of cystic medial necrosis

Triggers for cystic medial necrosis (CMN) have been difficult to study due to lack of animal models to recapitulate the pathologies seen in humans. Our study is the first description of CMN in the rat. Thus the JCR:LA-cp rat represents a useful model to investigate the underlying molecular changes leading to the development of CMN.

Abstract

Although there are multiple rodent models of the metabolic syndrome, very few develop vascular complications. In contrast, the JCR:LA-cp rat develops both metabolic syndrome and early atherosclerosis in predisposed areas. However, the pathology of the normal vessel wall has not been described. We examined JCR:LA control (+/+) or cp/cp rats fed normal chow diet for 6 or 18 mo. JCR:LA-cp rats developed multiple features of advanced cystic medial necrosis including “cysts,” increased collagen formation and proteoglycan deposition around cysts, apoptosis of vascular smooth muscle cells, and spotty medial calcification. These appearances began within 6 mo and were extensive by 18 mo. JCR:LA-cp rats had reduced medial cellularity, increased medial thickness, and vessel hypoxia that was most marked in the adventitia. In conclusion, the normal chow-fed JCR:LA-cp rat represents a novel rodent model of cystic medial necrosis, associated with multiple metabolic abnormalities, vascular smooth muscle cell apoptosis, and vessel hypoxia.

NEW & NOTEWORTHY Triggers for cystic medial necrosis (CMN) have been difficult to study due to lack of animal models to recapitulate the pathologies seen in humans. Our study is the first description of CMN in the rat. Thus the JCR:LA-cp rat represents a useful model to investigate the underlying molecular changes leading to the development of CMN.

the metabolic syndrome is characterized by obesity, hypertension, dyslipidemia, insulin resistance, impaired glucose tolerance, and a high risk of cardiovascular disease (6). While there are multiple rodent models of the metabolic syndrome, few demonstrate the vascular complications without a second genetic defect, e.g., loss of apolipoprotein-E (ApoE) or low-density lipoprotein receptor. For example, rats homozygous for the ob or fa gene (leptin receptor) become obese, insulin resistant, and hypertriglyceridemia (5); however, most rat strains show no vasculopathy. In contrast, the JCR:LA-cp rat is unique in developing spontaneous intimal lesions resembling early atherosclerosis and evidence of myocardial infarction [reviewed in Russell et al. (9)]. Aortas in these rats show intimal vascular smooth muscle cells (VSMCs) with foam cell formation, hyperresponsiveness to vasoconstrictors, and defective endothelial function (7, 9). Although such “fatty streaks” occur at sites predisposed to atherosclerosis, the structure of the vessel wall at nonaffected sites has not been described.

Cystic medial necrosis (CMN) is a pathology characterized by degeneration of elastin fibers accompanied by loss of medial VSMCs (producing holes or “cysts”) and changes in extracellular matrix (ECM) composition (1). CMN vessels show increased Alcian blue staining of glycosaminoglycans around the cysts and speckled calcification of the media. Although CMN is associated with genetic vascular degeneration in humans (e.g., Erdheim disease, Marfan syndrome), the triggers for medial degeneration in CMN are not known. In addition, CMN has not been described in rat rodent models. We demonstrate that the JCR:LA-cp rat shows multiple features of CMN, associated with markers of VSMC apoptosis and hypoxia. CMN may be a generalized arterial response to the loss of VSMCs associated with metabolic abnormalities in rodents.

METHODS AND RESULTS

Male JCR:LA rats (both +/+ and cp/cp) were obtained from the Charles River (Wilmington, MA) and housed under pathogen-free conditions. The use of animals and experimental procedures were approved by the Institutional Animal Care and Use Committee at NEOMED (Rootstown OH). Animal room was maintained on a reversed 12-h:12-h light/dark cycle with temperature maintained at 25°C. We examined JCR:LA control +/+ or cp/cp rats fed normal chow for 6 or 18 mo. As described previously, JCR:LA-cp rats developed glucose intolerance, hyperinsulinemia, and hyperlipidemia as early as 3 mo (Table 1) and early atherosclerosis at 6 mo (Fig. 1, A and B) (3, 4, 8–10).

Table 1.

Body weight, glucose and lipid profiles of the JCR:LA-cp rats

	Age, mo
	1		3		6		9		18
Male	+/+	cp/cp	+/+	cp/cp	+/+	cp/cp	+/+	cp/cp	+/+	cp/cp	References
Weight, g					343 ± 9	617 ± 24*			481 ± 23	1033 ± 38*
Fasting plasma glucose, mg/dl	105 ± 21	138 ± 43	116 ± 10	144 ± 18*	136 ± 12	137 ± 18	134 ± 24	125 ± 30			(8)
Plasma insulin concentrations, mU/l	5.2 ± 1.8	32 ± 16	23 ± 8	425 ± 85*	24 ± 1	350 ± 115*	52 ± 14	239 ± 80*			(8)
Plasma glucagon concentrations, ng/dl	186 ± 15	289 ± 90	103 ± 9	289 ± 13*	189 ± 22	296 ± 24*	207 ± 29	143 ± 27*			(8)
VLDL
VLDL cholesterol, mg/100 ml	1.1 ± 1.5	8.0 ± 2.6*	0.47 ± 1.4	7.7 ± 0.8*	0.49 ± 0.09	4.6 ± 0.9*	0.7 ± 0.3	3.1 ± 0.5*			(3, 4, 10)
Cholesterol esters, mg/100 ml	2.7 ± 3.9	16.6 ± 9.2	0.88 ± 0.20	11.3 ± 1.7*	0.71 ± 0.32	9.0 ± 1.2*	1.3 ± 0.7	6.5 ± 0.6*			(3, 4, 10)
Triacylglycerols, mg/100 ml	5.9 ± 5.2	145 ± 19*	6.4 ± 2.3	274 ± 27*	3.5 ± 1.6	169 ± 9.2*	12.3 ± 6.3	118 ± 13.8*			(3, 4, 10)
Phospholipids, mg/100 ml	4.2 ± 5.0	38.9 ± 10.3*	2.1 ± 0.47	52.8 ± 6.0*	0.88 ± 0.39	29.1 ± 2.5*	3.6 ± 1.6	18.5 ± 2.1*			(3, 4, 10)
HDL
HDL cholesterol, mg/100 ml	5.5 ± 1.1	6.2 ± 0.7	2.5 ± 0.6	4.3 ± 0.3*	2.9 ± 0.2	3.9 ± 0.6	2.6 ± 0.3	4.3 ± 0.4*			(3, 4, 10)
Cholesterol esters, mg/100 ml	63.0 ± 5.4	90.0 ± 8.9*	46.0 ± 9.5	96.9 ± 5.6*	57.8 ± 1.6	115.9 ± 7.3*	72.4 ± 6.0	96.3 ± 7.2*			(3, 4, 10)
Triacylglycerols, mg/100 ml	2.0 ± 1.8	0.3 ± 0.2	0.72 ± 0.96	1.1 ± 0.5	0.99 ± 0.52	2.7 ± 0.8*	0.88 ± 0.65	8.9 ± 0.7*			(3, 4, 10)
Phospholipids, mg/100 ml	47.2 ± 8.8	71.2 ± 5.9*	20.1 ± 5.6	69.4 ± 3.4*	19.2 ± 1.6	61.7 ± 4.1*	29.0 ± 6.0	56.4 ± 4.0*			(3, 4, 10)

Values are means ± SD, except weight (means ± SE).

^*P < 0.05 control vs. cp/cp male; n = 3–6.

Fig. 1.

Representative images of JCR:LA +/+ and cp/cp rats at 6 (A and B) and 18 (C and D) mo stained with Masson’s trichrome. JCR:LA cp/cp develops atherosclerotic lesion and mild cystic medial necrosis (CMN) at 6 mo. At 18 mo, both lean and obese rats develop CMN with higher severity in the obese. JCR:LA cp/cp has larger lumen size and thicker vessel wall as compared with the JCR:LA +/+. Arrow, atherosclerotic lesion; arrowhead, CMN. Magnification, ×4.

However, by 6 mo, there was increased collagen deposition in both the media and adventitia of the thoracic aortas of JCR:LA-cp versus control rats, which was increased further by 18 mo; this was accompanied by marked medial degeneration with formation of “cysts,” seen to a more minor extent in JCR:LA control rats (Fig. 1, C and D; Fig. 2, A–D). Cystic degeneration in JCR:LA-cp rats at 18 mo was accompanied by increased Alcian blue staining for glycosaminoglycan, most marked around the cysts (Fig. 2, E–H). Increased collagen deposition was confirmed using Masson’s trichrome staining (Fig. 2, I, J, and R), and spotty medial calcification was evident on Von Kossa staining (Fig. 2, K, L, and S) in 18-mo JCR:LA-cp rats. We used transmission electron microscopy to examine the ultrastructure of vessels of LCR:LA rats at 18 mo and estimated medial thickness. As described before JCR:LA-cp rats showed intimal VSMCs (9) and also marked medial thickening (Fig. 2, M, N, and Q). This medial thickening was due to increased ECM and collagen between elastic laminae. Aortic sections of Zucker +/+ and fa/fa rats were stained with Picrosirius red at 12 mo and examined (Fig. 2, O and P). The Zucker fa/fa rats did not exhibit the features of CMN as observed in the JCR:LA-cp rat.

Fig. 2.

JCR:LA cp/cp rats show cystic medial necrosis. Picrosirius red for collagen (A–D) and Alcian blue (E–H) staining in 6- or 18-mo-old JCR:LA control +/+ or cp/cp rats are shown. Masson’s trichrome (I, J), Von Kossa staining (K, L), and transmission electron microscopy (M, N) of 18-mo JCR:LA +/+ (I, K, and M) or cp/cp rats (J, L, and N) are shown. O–P: Picrosirius red for collagen staining of 12-mo-old Zucker control and fa/fa rats. Q: medial thickness of JCR:LA +/+ and cp/cp rats at 18 mo as measured from M and N. R: relative collagen quantitation of JCR:LA +/+ and cp/cp rats at 18 mo as measured from I and J. S: relative calcification quantitation of JCR:LA +/+ and cp/cp rats at 18 mo as measured from K and L. iel, internal elastic lamina; smc, smooth muscle cell; m, media; e, endothelium; L, lumen; v, vacuole. Arrow, CMN; arrowhead, collagen deposits; white arrowhead, glycosaminoglycans; star, calcium deposits. Histology magnification , ×20 for JCR:LA cp/cp rats and ×40 for Zucker fa/fa rats. Scale bars = 5 mm. *P < 0.05, statistical significant difference between JCR:LA +/+ and cp/cp rats, n = 3.

VSMC apoptosis induces CMN in fat-fed ApoE^−/− mice, with cysts appearing where cells have died (2). We therefore examined vessels for evidence of apoptosis. The prolonged time course (18 mo) and consequent low incidence excludes the use of TUNEL/cleaved caspases for detection of apoptosis. In contrast, caspase cleavage of other nuclear substrates such as poly-ADP-ribose polymerase 1 (PARP-1) is a longer-lasting marker when there is accumulation of cell debris. JCR:LA-cp rats showed markedly increased labeling for the p85 (cleaved) form of PARP-1 by 18 mo (Fig. 3, A–E). Finally, we examined the medial degeneration and increased medial thickness on hypoxia in these arteries by administration of the hypoxia marker pimonidazole into the tail vein just before euthanasia. Pimonidazole accumulated in the adventitia of JCR:LA-cp rats at 6 and 18 mo but did not colocalize with PARP-1 p85 (Fig. 3, B and D). Medial cellularity was reduced in JCR:LA-cp rats at both 6 and 18 mo as evidenced from the DAPI staining (Fig. 3F).

Fig. 3.

JCR:LA-cp/cp rats show markers of vascular smooth muscle cell (SMC) apoptosis and hypoxia. Immunohistochemistry for poly-ADP-ribose polymerase 1 (PARP-1) p85, pimonidazole, or DAPI with merged images on the right of JCR:LA +/+ or cp/cp rats at 6 (A and B) or 18 (C and D) mo. E: apoptotic cells (PARP-1 p85 positive-cells/area) of JCR:LA +/+ and cp/cp rats at 6 and 18 mo. F: medial cellularity (cells/area) of JCR:LA +/+ and cp/cp rats at 6 and 18 mo. Arrow, pimonidazole staining in adventitia; arrowhead, PARP-1 p85 staining in SMC. *P < 0.05, statistical significant difference between JCR:LA +/+ and cp/cp rats, n = 3.

DISCUSSION

Erdheim-Gsell cystic medial degeneration is a characteristic vascular pathology seen in some genetic vasculopathies (e.g., Erdheim disease and Marfan syndrome). Focal CMN is also a moderately common feature associated with idiopathic arterial dissections in older patients. CMN shows degeneration of the media with loss of medial VSMCs, associated with accumulation of glycosaminoglycans and destruction of elastin fibers. Although CMN may be associated with mutations of ECM proteins, the pathological triggers of CMN are unknown.

We demonstrate that JCR:LA-cp rats show progressive and multiple features of CMN, resulting in marked medial destruction by 18 mo of age. The cystic changes and reduction in medial cellularity are particularly striking, whereas the increased collagen and glycosaminoglycan contents result in a thickened vessel wall. Both markers of VSMC apoptosis and vessel hypoxia are present, the latter most likely due to the thickened vessel wall reducing oxygen diffusion from the lumen. We had also examined the vasculature of another metabolic syndrome rat model, the Zucker fa/fa rat, up to 12 mo. The Zucker fa/fa did not exhibit the features of cystic medial necrosis as observed in the JCR:LA-cp rat. Thus we believe that the differences in the genetic make-up between these two strains might have contributed to the pathology.

The causes of CMN have been difficult to study, in part because we lack of good animal models that recapitulate the features seen in humans. Our study is the first description of CMN in the rat, and the first detailed description of medial pathology in JCR:LA-cp rats fed with normal chow. Thus, in addition to being a model of early atherosclerosis, the JCR:LA-cp rat may be a useful model of CMN. Indeed, the medial degeneration in JCR:LA-cp rats is far more prominent than their modest changes of early atherosclerosis.

Impaired glucose tolerance, insulin resistance, and diabetes are associated with multiple vascular pathologies including atherosclerosis, arteriosclerosis and Monkeberg’s medial calcinosis. However, this study is the first to link the multiple metabolic defects and aging seen in JCR:LA-cp rats with CMN. Our work also provides clues as to the primary pathological events in CMN. To date CMN has been described in fat-fed ApoE^−/− mice with forced induction of VSMC apoptosis (2) and in the mouse models of Hutchison-Gilford progeria and RECS1 (11, 12). The Hutchison-Gilford progeria mouse model shows progressive loss of VSMCs, accumulation of cellular debris, elastin breakage, and accumulation and disorganization of collagen and glycosaminoglycans resulting in thickened media and adventitia, and diminished vascular responsiveness to NO-independent vasodilators (11). These three studies suggest that CMN is a primary VSMC pathology, with secondary defects in collagen synthesis and glycosaminoglycan accumulation possibly being an abortive attempt at healing. Subsequent vessel thickening may exacerbate the medial degeneration, for example by generating hypoxia.

In conclusion, we show that JCR:LA-cp rats develop multiple features of advanced CMN. This propensity to develop CMN is most likely a combination of a strain-specific VSMC abnormality, coupled with both exposure to multiple metabolic defects and aging. The JCR:LA-cp rat represents an useful model to study CMN.

GRANTS

This work was supported by National Institutes of Health Grants HL-32788, R01-83366, RC1-HL-100828 (to W. M. Chilian), British Heart Foundation Grant RG/08/009/25841 (to M. R. Bennett), and American Heart Association Postdoctoral Fellowship 09POST2290021 and Malaysia Ministry of Higher Education Grant FRGS/1/2015/SKK08/UNIM/03/2 (to Y. F. Pung).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Y.F.P. and N.F. performed experiments; Y.F.P., W.M.C., and M.R.B. analyzed data; Y.F.P., W.M.C., M.R.B., and M.H.K. interpreted results of experiments; Y.F.P. prepared figures; Y.F.P., W.M.C., and M.R.B. drafted manuscript; Y.F.P., W.M.C., and M.R.B. approved final version of manuscript; W.M.C., M.R.B., and M.H.K. edited and revised manuscript.