A robust endothelial injury model inducing medial arterial calcification in C57BL/6J mice on a standard diet

Arterial calcification is an age-associated process that develops in association with atherosclerosis, type 2 diabetes and chronic kidney disease (CKD). Arterial calcification can affect the intimal and/or medial layers of large arteries. Intimal calcification is associated with atherosclerosis, a condition that affects virtually all adults; it is the most common and the most extensively studied form of arterial calcification (1). In atherosclerotic plaques, the earliest microcalcifications can already be observed in early plaques, likely arising within necrotic cellular debris, and subsequently expand into larger calcified deposits that can occupy a substantial portion of the plaque volume (2). Calcifications in human plaques can present with different morphologies, ranging from microcalcifications, to sheet-like calcifications, nodular calcifications and, in relatively rare cases, osteoid metaplasia (3). All of these calcification morphologies are associated with dysregulated expression of osteoblast differentiation markers and/or calcification inhibitors (4).

Despite the large body of clinical data available, the impact of plaque calcification on plaque stability remains poorly understood and, in fact, somewhat controversial (1). Medial arterial calcification (MAC) begins in the third decade of life and increases in prevalence to affect the majority of individuals over the age of 65 years (5). MAC is further exacerbated in two common and often comorbid age-related diseases: CKD and type 2 diabetes mellitus (T2DM). Histologically, MAC typically originates as microcalcifications located near the internal elastic lamina (6). It appears to progress through areas of cartilage metaplasia, characterized by chondrocyte-like cells and expression of chondrocyte markers (7,8). In advanced MAC, up to two-thirds of the medial layer may be calcified, with occasionally presence of bone structures (6). MAC is believed to impair arterial function and adversely affect cardiovascular morbidity and mortality (5,9,10).

Since the early 2000s, an increasing number of laboratories have established animal models to investigate the pathophysiological mechanisms underlying arterial calcification and to evaluate the effects of potential therapeutic interventions. Among the available experimental species (including rodents, pigs, and non-human primates), mice remain by far the most widely used due to their ease of manipulation and the extensive range of genetic models they provide. Nonetheless, while relatively robust models of intimal calcification have been developed in mice, medial calcification is more challenging to induce and is strongly influenced by both the mouse strain and the specific disease-inducing protocol.

Although imperfect, mouse models of intimal calcification have been available for many years and are now well characterized. Apolipoprotein E-deficient (ApoE-KO) mice represent the most widely used model, as ApoE deficiency induces atherosclerosis that recapitulates most key features of the human disease (11,12). As in human plaques (13), calcification is observed in virtually all atherosclerotic lesions in ApoE-KO mice (14). However, plaque calcification in these mice differs in important ways from that in humans. In ApoE-deficient mice, calcification develops through a process resembling endochondral ossification, characterized by the formation and subsequent mineralization of cartilaginous metaplasia (14,15). In contrast, ossification is less common in human plaques, and when present it more closely resembles intramembranous ossification, in which osteoblasts mineralize the tissue without a chondrocytic intermediate (2). Whether such species-specific differences in ossification pathways contribute to the much higher incidence of plaque rupture in humans compared with mice remains speculative, but it is an intriguing possibility that warrants further investigation (11,12).

In contrast to the intimal calcification of atherosclerotic plaques—which can be studied using several well-established mouse models (14-16)—MAC is more difficult to induce in mice, and the feasibility largely depends on whether MAC is associated with CKD, T2DM, or aging. For CKD-related MAC, two principal mouse models are commonly used (17). The first is adenine supplementation, which induces CKD (18) and triggers extensive MAC in C57BL/6 mice, accompanied by bone-like tissue formation, with no evidence of cartilage differentiation (19). The second model, 5/6 nephrectomy, also mimics human CKD, but it does not reliably induce MAC—particularly in the C57BL/6 strain, where medial calcification is modest and often entirely absent in some mice (20-22). One way to increase the severity of MAC after nephrectomy is to use mice on the DBA/2 background (23). In nephrectomized DBA/2 mice, as in C57BL/6 mice fed adenine, MAC appears to depend on osteoblast-like rather than chondrocyte-like differentiation (23). The DBA/2 strain allows the use of nephrectomy to induce MAC instead of the more aggressive adenine protocol, but switching to this background may require several generations of backcrossing when working with genetically modified mice originally produced on the C57BL/6 background (24). The greater susceptibility of DBA/2 mice to MAC compared with C57BL/6 mice is attributed to lower circulating levels of the mineralization inhibitors inorganic pyrophosphate and magnesium, resulting respectively from a hypomorphic mutation in the Abcc6 gene and from impaired regulation of the Trpm6 magnesium transporter (25). Another strategy to induce MAC in nephrectomized C57BL/6 mice is to use ApoE-deficient animals (26). However, nephrectomy in ApoE-KO mice amplifies intimal plaque calcification and induces MAC both at atherosclerotic intimal sites and at medial regions free of atheroma (26), which complicates the analysis and quantification of medial calcification specifically. In summary, investigators studying CKD-associated MAC have several options, including adenine supplementation in C57BL/6 mice or 5/6 nephrectomy in DBA/2 mice. Unfortunately, fewer models are available for studying MAC associated with T2DM or aging. One model of arterial calcification associated with T2DM is the low-density lipoprotein receptor-deficient (Ldlr-KO) mouse. However, when fed a high-fat diet, these mice develop both intimal calcification within atherosclerotic plaques and medial calcification (27,28), which complicates the specific quantification of medial calcification. Finally, to our knowledge, no mouse model exists that develops MAC specifically as a consequence of aging. Although MAC does occur in several genetic mouse models—such as matrix Gla protein–deficient mice (29) or transgenic mice overexpressing alkaline phosphatase in vascular smooth muscle cells (30)—these models, by definition, exhibit a constitutive dysregulation of calcification pathways.

In a recently published study (31), Hu et al. used an established model of endothelial regeneration—the mouse carotid artery wire-injury model (32)—and adapted it to develop a reliable model of MAC. Notably, this model was created in the C57BL/6 background, which is, as mentioned above, a strain known to be relatively resistant to ectopic calcification (25), and it did not require a high-fat or diabetogenic diet. In two-month-old male mice, the carotid arteries were surgically exposed under anesthesia. The left common and internal carotid arteries were temporarily occluded, and the external carotid artery (ECA) was ligated. A small incision was then made in the ECA with microscissors, and endothelial injury was induced using a stainless-steel wire. Sham-operated mice underwent the same procedure except for the mechanical wire injury. Three months later, mice subjected to mechanical injury—but not sham controls—developed medial calcification accompanied by chondroid and bone metaplasia, together with increased expression of Runt-related transcription factor 2 (RUNX2), the transcription factor that drives the differentiation of mineralizing hypertrophic chondrocytes and osteoblasts. This is an important finding since MAC in humans has also been reported to involve chondrocyte-like differentiation, a feature that appears absent in MAC induced in adenine-fed C57BL/6 mice (19) and in nephrectomized DBA/2 mice (23). This new surgical model may therefore represent a unique system for investigating chondrocyte differentiation in the context of MAC.

The fact that MAC is induced by endothelial injury, and not by CKD, T2DM or aging, may appear as a negative point, but it may not be without interest. Indeed, endothelial injury and internal elastic lamina disruption appear as important risk factors for the progression of vascular calcification in CKD patients (33,34). Moreover, in patients with T2DM, endothelial injury is positively associated with the progression of coronary artery calcification, and with mortality (35). In aging patients, calcification of the intracranial carotid artery, which is more predictive of stroke risk than that of extracranial arteries (36), appears to be mostly unrelated to atherosclerosis but associated with internal elastic lamina (37). Several non-exclusive mechanisms have been hypothesized to explain how endothelial injury and/or internal lamina disruption may lead to MAC: induction of endothelial-to-mesenchymal transition, exacerbation of inflammatory stimulation of vascular smooth muscle cells, or calcification of elastin fragments (38-40). The model proposed by Hu et al. may therefore be useful for gaining insight into these mechanisms. In particular, it would be very interesting to determine to what extent endothelial injury also compromises the integrity of the internal elastic lamina, and whether this may contribute to medial calcification. Deeper histological studies will be important to address this question.

In addition, in this recent article (31), histological analyses revealed the presence of M1 macrophages and elevated levels of interleukin (IL)-1β and IL-6, suggesting that inflammation contributed to the development of MAC. This finding is particularly noteworthy, as inflammation is considered one of the major drivers of both intimal and medial calcification (38). By translating clinical classification criteria for vascular calcification, authors developed a histopathological grading system for mouse MAC, allowing them to measure the progression of MAC during time, from non-calcified media to grade IV MAC. Grades I to IV MAC were defined according to the maximum diameter of calcium particle aggregation, being between 0.5 and 40 µM for grade I (micro and punctate calcification), up to 100 µM for grade II (fragment calcification), up to 300 µM for grade III (sheet calcification), and more than 300 µM for grade IV. Importantly, when a 0.45-mm wire was used to induce endothelial injury, MAC developed in nearly 90% of mice after three months, with a majority of grades III and IV. Such a high incidence in C57BL/6 mice will facilitate the investigation of the pathophysiological mechanisms driving cartilage and bone formation within the arterial media, including studies using genetically modified mouse models. Beyond enabling mechanistic exploration, this model also provides a valuable platform for testing therapeutic interventions targeting MAC development. Hu et al. demonstrated that two established calcification inhibitors, etidronate and SNF472, significantly reduced calcium deposition in the carotid artery, further validating this model. Finally, if a 100% incidence of MAC is required, the authors showed that administration of vitamin D₃ can reliably achieve this outcome. On the other hand, if the aim is to explore the mechanisms underlying the initiation of calcification, sacrificing mice one month or two months after surgery, when respectively 80% or 50% of arteries remain uncalcified (31), should allow analysis of the molecular events preceding calcification onset. Finally, while endothelial injury can be performed in normal wild-type mice, as described by Hu et al., it could also be used in combination with diets and/or surgical procedures that induce diabetes and/or kidney disease. Indeed, although type 2 diabetes and CKD accelerate the development of MAC in humans, animal models of these diseases often fail to induce medial calcification. For instance, there is so far no good model of MAC induced by diabetogenic diets in mice, and as explained above, chronic insufficiency induced by nephrectomy in C57BL/6 mice only occasionally induces medial calcification. In this context, using this endothelial injury approach in mice with diabetes or renal insufficiency may constitute an interesting way to study the impact of diabetes or renal disease on MAC.

In conclusion, Hu et al. developed a compelling mouse model of MAC on the relatively calcification-resistant C57BL/6 background, achieving nearly 100% incidence of arterial calcification without the need for high-fat or diabetogenic diets, or vitamin D₃ administration. This model is however not without limitations. First, all experiments were conducted with male mice, and it will be important to determine whether female mice calcify their vascular media to the same extent. In addition, the surgical procedure may be technically demanding and may not be easily implemented or rapidly mastered with high reproducibility across laboratories. Furthermore, MAC in this model is induced by mechanical injury, and differs fundamentally from the complex mechanisms driving MAC in T2DM, CKD, or aging. Nonetheless, in the absence of robust models of MAC—particularly for T2DM- or aging-associated calcification—this approach may still prove valuable.

Acknowledgments

None.

Footnote

Provenance and Peer Review: This article was commissioned by the editorial office, Cardiovascular Diagnosis and Therapy. The article has undergone external peer review.

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Conflicts of Interest: The author has completed the ICMJE uniform disclosure form (available at https://cdt.amegroups.com/article/view/10.21037/cdt-2025-1-613/coif). The author has no conflicts of interest to declare.

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