Three-Step Construction of Atherosclerotic Cell Models: A Comprehensive Guide to ox-LDL-Induced Injury, Foam Cell Formation, and Calcification

Atherosclerosis (AS) is a chronic cardiovascular disease that endangers human health and is one of the most common causes of death in the elderly. The main pathological feature of AS is lipid deposition in some arteries, accompanied by the proliferation of smooth muscle cells (SMCs) and fibrous matrix, which gradually develops into AS plaques.

Vascular calcification (VC) is a pathological phenomenon characterized by calcium phosphate deposition in the walls of arteries and veins. Patients with atherosclerosis (AS), diabetes mellitus, and chronic kidney disease are at a high risk of developing VC.

The accumulation of oxidized low-density lipoprotein (ox-LDL) in the vascular wall plays a crucial role in the development of AS and VC. Studies have shown that highly expressed ox-LDL is detected in the serum of AS patients with cerebral infarction, and it is believed that ox-LDL can accelerate the formation of carotid artery plaques, promote the shedding of unstable plaques, and is an independent risk factor for cerebrovascular diseases. Therefore, as a key pathogenic factor, ox-LDL is an effective model for studying AS.

The pathogenesis of AS is complex, and processes such as cell injury, foam cell formation, and calcification are intertwined and interact with each other, without an absolute and unchanging order. However, it is generally believed that endothelial cell injury is the initial step in the occurrence of atherosclerosis. Under the action of various factors such as hyperlipidemia, hypertension, smoking, and diabetes, vascular endothelial cells are damaged, their permeability increases, and lipids and inflammatory cells in the blood can enter the subendothelium.

After endothelial cell injury, lipids such as low-density lipoprotein cholesterol (LDL-C) in the blood enter the subendothelial space and are oxidatively modified to form ox-LDL. ox-LDL is phagocytosed by macrophages, forming a large number of lipid droplets in the cells, thereby transforming macrophages into foam cells. The accumulation of foam cells is an early lesion of atherosclerosis. With the development of atherosclerotic lesions, smooth muscle cells proliferate, migrate, and undergo phenotypic transformation at the lesion site, while producing a large amount of extracellular matrix. During this process, local inflammatory responses, cell death, etc., release some pro-calcification factors, such as alkaline phosphatase, which promote the deposition of calcium salts in the vascular wall, leading to vascular calcification. Calcification usually occurs after the formation of atherosclerotic plaques, making the vascular wall hard and brittle, and further aggravating vascular lesions.

I. ox-LDL-Induced Human Umbilical Vein Endothelial Cells (HUVECs) Injury Model

1. Experimental Cells

Human umbilical vein endothelial cells (HUVECs) (purchased from American Type Culture Collection, ATCC)

2. Methods for Inducing Endothelial Cell Injury

•         After HUVECs seeding, pretreat with different concentrations of PFE (1.5, 3.0, 7.5 μg/mL) for 2 hours (Note: PFE is a flavonoid compound extracted from coriander seeds).

•         ox-LDL stimulation: Remove the pretreatment medium and add fresh medium containing 100 μg/mL ox-LDL (abs47014903, ANT BIO PTE. LTD.). Continue culturing for 24 hours to induce endothelial injury.

This model simulates early endothelial injury in atherosclerosis by inducing endothelial inflammation and adhesion molecule expression through ox-LDL (Figure 1), and is suitable for mechanistic research on anti-atherosclerotic drugs (such as PFE).

II. ox-LDL-Induced Macrophage RAW 264.7 Foam Cell Formation Model

1. Experimental Cells

Macrophage cell line RAW 264.7 (purchased from American Type Culture Collection, ATCC)

2. Methods for Inducing Foam Cell Formation

•         The experimental group was pretreated with PFE (a flavonoid compound extracted from coriander seeds) at concentrations of 1.5, 3.0, and 7.5 μg/mL for 2 hours. The control group was not treated with PFE.

•         ox-LDL stimulation: Add ox-LDL (abs47014903, ANT BIO PTE. LTD.) to a final concentration of 100 μg/mL and incubate with cells for 24 hours.

Detection Indicators: Oil Red O (abs42024259, ANT BIO PTE. LTD.) staining to observe lipid accumulation (degree of foam cell formation). RT-PCR/Western blot to detect the expression of genes related to cholesterol influx (LOX-1, CXCL16) and efflux (ABCA1, ABCG1, SR-B1) (Figure 2).

This model successfully induces the transformation of RAW 264.7 macrophages into foam cells through ox-LDL, and confirms that the drug PFE promotes cholesterol efflux and inhibits foam cell formation through the PPARγ-ABCA1/ABCG1 pathway.

III. ox-LDL-Induced Smooth Muscle Cells (SMCs) Calcification Model

1. Experimental Cells

Human aortic vascular smooth muscle cells (HA-SMCs) (purchased from China Center for Type Culture Collection, CTCC)

2. Methods for Inducing Calcification

•         Divide HA-SMCs into control group and calcification model group. After passage, starve the cells for 24 hours, then replace the medium with complete medium for the control group.

•         For the calcification model group, replace with calcification medium: 10 mmol/L β-glycerophosphate (abs812987, ANT BIO PTE. LTD.) + 50 μg/mL L-ascorbic acid (abs811910, ANT BIO PTE. LTD.) + 100 nmol/L dexamethasone (abs813595, ANT BIO PTE. LTD.); add ox-LDL (abs47014903, ANT BIO PTE. LTD.) at a concentration of 50 μg/mL to induce cells for 14 days.

•         Alizarin Red S (abs42012987, ANT BIO PTE. LTD.) staining results showed that the calcification model group had obvious orange-red calcified nodules compared with the control group, indicating that the calcification model was successfully established.

This model establishes an SMCs calcification model by screening the optimal induction concentration of ox-LDL (Figure 3), and is suitable for studying the effect of specific anti-inflammatory factors (such as IL-37) on the calcification process of this model and its molecular mechanism.

In the long journey of atherosclerosis research, ox-LDL is undoubtedly a "golden key" in our hands. Through the carefully designed cell model construction methods described above, we can deeply explore its mysteries in disease progression. As a solid backing on your scientific research path, ANT BIO PTE. LTD. provides a variety of high-quality reagents and products, which will help you ride the wind and waves in the field of atherosclerosis research and continuously achieve new breakthroughs and achievements.

References:

[1] Liu J, Zhang W, Li Y, Li X, Li Y, Guo F. Flavonoids extract from the seeds of Psoralea corylifolia L. (PFE) alleviates atherosclerosis in high-fat diet-induced LDLR-/- mice. Phytomedicine. 2022;98:153983. doi:10.1016/j.phymed.2022.153983

[2] Ma C Y. Study on IL-37 inhibiting calcification of human aortic vascular smooth muscle cells and participating in suppressing their osteogenic transformation [D]. Anhui Medical University, 2023.

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