Atherosclerosis, the leading cause of cardiovascular disease, cannot be sufficiently explained by established risk factors including hypercholesterolemia. Elevated plasma homocysteine (Hcy) is an independent risk factor for atherosclerosis, increases cardiovascular risk in combination with hypercholesterolemia, and is strongly linked to cardiovascular mortality. Here we show Scanning Transmission Electron Microscopy data (STEM) of aortic specimens from rabbits fed diet deficient in B vitamins and choline required for Hcy degradation in the absence and presence of intravenous injections of Hcy and hypercholesterolemia. Our data show that rabbits fed a diet deficient in B vitamins and choline (VCDD) required for Hcy degradation exhibit a cholesterol-independent accumulation of macrophages and lipids in the aorta as well as disorganization of the aortic collagen and elastin; and a combination of VCDD and high cholesterol diet (VCD/HCD) leads to aggravation of atherosclerosis compared to high cholesterol diet (HCD) alone (Almer et al, 2022, Biomed Pharmacother, DOI:10.1016/j.biopha.2022.113640). Furthermore, our data show that intravenous injections of Hcy into VCDD-fed rabbits lead to fragmentation of aortic elastin, accumulation of elastin-specific electron-dense inclusions, collagen disorganization, lipid degradation, and autophagolysosome formation as well as appearance of enlarged smooth muscle cells (SMCs) (Tehlivets et al, 2024, Biomed Pharmacother, DOI:10.1016/j.biopha.2024.117244) and intravenous injections of Hcy into VCD/HCD-fed rabbits lead to even more pronounced fragmentation of aortic elastin, accumulation of collagen, degradation of aortic lipids, as well as diminished abundancy and smaller SMCs compared with either group (Tehlivets et al, in preparation). Of note, aortic specimens from rabbits in VCD/HCD and  VCD+Hcy groups exhibited void bodies defined as electron bright cells with reduced organelle content, while VCD/HCD+Hcy showed decreased number of aortic cells in media. Furthermore, aortic specimens from rabbits in VCD/HCD+Hcy group exhibited very pronounced foam cell accumulation in neointima and in media in close proximity to neointima. In addition,  rabbits in VCD/HCD+Hcy group displayed very prominent elastin degradation and most striking SMCs phenotype alteration compared to other groups. Shown are representative aortic specimens from rabbits fed standard diet (SD), vitamin and choline deficient diet (VCDD), VCDD combined with intravenous injections of Hcy (VCDD+Hcy), high cholesterol diet (HCD), VCDD combined with high cholesterol diet  (VCD/HCD) and VCD/HCD combined with intravenous injections of Hcy (VCD/HCD+Hcy).

1. Animal experiments

All animal experiments were approved by the Austrian Federal Ministry of Education, Science and Research (BMWF 66.010_0070-V_3b_2018). For the study, 4- to 6-month old male rabbits (New Zealand White rabbits, Austria) were used. Prior to the studies, rabbits were acclimatized for 2-4 weeks. Rabbits were set on special diets after or before surgery. The diets used included a complete chow diet (standard diet, SD) for full nutritional conditions and purified special diets either deficient in vitamins and choline required for Hcy metabolization (no vitamin B₁₂, 20% folate (2 mg/kg), 20% vitamin B₆ (6 mg/kg) and 10% choline (0.128 mg/kg) (VCDD), enriched with 1% cholesterol (high cholesterol diet, HCD), or combined diet deficient in vitamins and choline and enriched with 1% cholesterol (vitamin and choline deficient/high cholesterol diet, VCD/HCD). Special purified diet was acquired from Sniff, Germany. Injury of the adult rabbit aorta abdominalis was performed with the automated JURY-controlled balloon catheter injury device with one retraction at 1.8 bar and retraction speed of 2 mm/s. For preconditioning of the animals before the surgical intervention VCDD was initiated two weeks and HCD one week before vessel wall injury. One day after the intervention, the special diets were resumed and fed for another 8 weeks. To further elevated plasma Hcy levels rabbits from the VCDD+Hcy and VCD/HCD+Hcy  groups received in total 60 µmol/kg L-Hcy by intravenous injections over 8 weeks. Eight weeks after the surgery, the rabbits were sacrificed and the aortas dissected. Sections from the same aortic area of each animal were used for further analyses.

2. Electron microscopy

Aortic tissue was fixed in 2.5% (w/v) glutaraldehyde and 2% PFA (w/v) in 0.1 M cacodylate buffer, pH 7.4, for 2 h, and then post-fixed in 2% (w/v) osmium tetroxide for 2 h at room temperature (RT). After dehydration (in graded series of ethanol), tissues were infiltrated (ethanol and TAAB Embedding Resin, pure TAAB Embedding Resin) and placed in TAAB Embedding Resin (8 h), transferred into embedding molds, and polymerized (48 h, 60°C). Ultrathin sections (70 nm) were cut with a UC 7 Ultramicrotome (Leica Microsystems, Austria) and stained with lead citrate for 5 min and platinum blue for 15 min. Electron micrographs were taken using a Tecnai G2 transmission electron microscope (FEI, Netherlands) with a Gatan Ultrascan 1000 charge coupled device (CCD) camera (-20°C, acquisition software Digital Micrograph, Gatan, Germany and Serial EM). Acceleration voltage was 120 kV. Scanning transmission electron microscopy (STEM) was done by stitching TEM images with “Atlas 5” (ZEISS, Oberkochen, Germany). STEM was done for three biological replicates.