Introduction: We previously identified Notch2 in smooth muscle cells (SMC) in human atherosclerosis and found that signaling via Notch2 suppressed human SMC proliferation. Thus, we tested whether loss of Notch2 in SMC would alter atherosclerotic plaque progression using a mouse model. Methods: Atherogenesis was examined at the brachiocephalic artery and aortic root in a vascular SMC null (inducible smooth muscle myosin heavy chain Cre) Notch2 strain on the ApoE−/− background. We measured plaque morphology and size, as well as lipid, inflammation, and smooth muscle actin content after Western diet. Results: We generated an inducible SMC Notch2 null on the ApoE−/− background. We observed ∼90% recombination efficiency with no detectable Notch2 in the SMC. Loss of SMC Notch2 did not significantly change plaque size, lipid content, necrotic core, or medial area. However, loss of SMC Notch2 reduced the contractile SMC in brachiocephalic artery lesions and increased inflammatory content in aortic root lesions after 6 weeks of Western diet. These changes were not present with loss of SMC Notch2 after 14 weeks of Western diet. Conclusions: Our data show that loss of SMC Notch2 does not significantly reduce atherosclerotic lesion formation, although in early stages of plaque formation there are changes in SMC and inflammation.

Notch is an important signaling molecule in vascular development and repair and regulates atherosclerosis in a tissue- and receptor-/ligand-dependent manner. Proinflammatory cytokine treatment of endothelial cells suppressed Notch1 expression, and hemizygous deletion of Notch1 in endothelial cells increased predisposition to atherosclerosis [1, 2]. The delta-like ligand 4 (Dll4)/Notch1 axis promoted the M1 pro-inflammatory phenotype in macrophages [3, 4], and inhibition of signaling using neutralizing anti-Dll4 antibodies reduced accumulation of macrophages, decreased fat mass, and attenuated insulin resistance [5]. Notch regulation of vascular smooth muscle cell (SMC) phenotype and function is also important in vascular SMC proliferative diseases [6, 7]. The effects of Notch signaling in the adult vascular SMC are complex and receptor dependent, and while many cells are present in complex plaques, lineage tracing studies have shown that the SMC represents up to 70% of cells representing a wide spectrum of phenotypes [8‒11].

Vascular SMC-expressed Notch1 promotes proliferation and cell survival, Notch2 promotes contractile differentiation and inhibits proliferation, and Notch3 promotes both contractile differentiation and proliferation [7]. We previously showed that Jagged1 activation of Notch2 mediated a unique suppression of proliferation in the human vascular SMC [12]. Jagged1 activation of Notch2 in the atherosclerotic vascular SMC derived from endarterectomy samples also suppressed proliferation but failed to induce smooth muscle actin expression as it does in the healthy vascular SMC [13]. SMC deletion of Notch effector, CBF1/RBPjκ/Su(H)/Lag1, prevented the cap SMC from reacquiring contractile smooth muscle actin expression, while overexpression of Notch intracellular domain to produce forced Notch signaling inhibited medial SMC contribution to plaques [14]. However, these previous studies did not specifically address the Notch protein responsible for these effects. Based on our previous data that Notch2 suppresses human vascular SMC proliferation, we hypothesized that Notch2 may be an endogenous inhibitor of atherogenesis via suppression of SMC growth. Thus, we engineered loss of Notch2 from the vascular SMC in a mouse model of atherosclerosis. Our findings indicate that suppression of Notch2 in the SMC does not significantly change the overall progression of atherosclerosis in the mouse.

Mouse Models

Studies were approved by the Institutional Animal Care and Use Committee of Maine Medical Center. All mice were on the C57BL/6 background. Athero-susceptible mice with inducible smooth muscle myosin heavy chain (SMMHC)-specific Notch2 deletion were generated by crossing Notch2flox/flox [15] × SMMHC-CreERT2 mice [16, 17] (Jackson Laboratory, #01052, #019079) with ApoE−/− mice [18] (Jackson Laboratory #002052). In Notch2flox/flox mice, loxP sites flank exon 3 of Notch2 [15]. ApoE−/− × Notch2flox/flox × SMMHC-CreERT2Cre/0 mice were randomized to receive either 1 mg tamoxifen i.p. in 100 μL corn oil or vehicle control daily for 5 days at 5–7 weeks of age. The ApoE−/− × Notch2flox/flox × SMMHC-CreERT2Cre/0 mice are referred to as ApoE−/−;N2SMC-null, and corn oil-treated ApoE−/− × Notch2flox/flox × SMMHC-CreERT2Cre/0 littermates are referred to as ApoE−/−;N2SMC-ctr.

Genotyping

Genomic DNA was isolated by alkaline lysis. PCR was performed using 5PRIME MasterMix for the ApoE allele at a 68°C annealing temperature using the primers (5′-GCCTAGCCGA­GGGAGAGCCG-3′), (5′-TGTGACTTGGGAGCTCTGCAGC-3′), and (5′-GCCGCCCCGACTGCATCT-3′). This resulted in a 155 bp band from the wildtype and a 245 bp product from the mutant allele. PCR to amplify the Notch2 allele was performed at an annealing temperature of 60°C. Notch2fl/fl primers (5′-TAGGAAGCAGCTCAGCTCACAG-3′, 5′-ATAACGCTAAACGTGCACTGGAG-3′) yielded a 161 bp band from the wildtype and a 20 bp product from the floxed allele. The SMMHC-CreERT2 genotyping was performed at an annealing temperature of 58°C using primers (5′-TCCAACCTGCTGACTGTG-3′, 5′-TCAGAGTTCTCCATCAGGG-3′), yielding a 455 bp transgenic product.

Induction of Atherosclerosis and Tissue Collection

ApoE−/−;N2SMC-ctr and ApoE−/−;N2SMC-null littermates were fed Western diet (Research Diets D12079B, 41% calories from fat) for 6 weeks, or fed Western diet (Envigo Teklad TD.88137, 42% calories from fat) for 14 weeks. Feeding started 7 days after the last injection of tamoxifen/vehicle. Mice were fasted for 12 h prior to blood collection to measure fasting glucose (ZOETIS AlphaTRAK® 2) and serum cholesterol (Molecular Probes Amplex Red Cholesterol Assay). Blood was collected by submandibular vein puncture with a sterile lancet (Braintree Scientific) followed by cervical dislocation and perfusion with 10% formalin. The brachiocephalic artery was fixed overnight in 10% formalin and processed for paraffin embedding. The heart was weighed, fixed overnight in 10% formalin, and stored in 30% sucrose in PBS. Hearts were bisected horizontally at the atria and the upper portion frozen in OCT (Tissue-Tek) prior to cryosectioning. The spleen was weighed, and left tibia length measured.

Blood Pressure

Systolic and diastolic blood pressure (BP) were measured for 5 days prior to start of diet and 5 days prior to completion of treatment regime using an automated computer-controlled Hatteras SC1000 (Hatteras Instruments) analysis system. The first 3 days of analysis were training and data were collected the last 2 days as previously described [19]. Each session consisted of 4 preliminary reads and 10 measurements.

Analysis of Genomic Recombination

Two ApoE−/−;N2SMC-null and two ApoE−/−;N2SMC-ctr mice were collected 1 week after tamoxifen or corn oil injection. The brachiocephalic artery, brain, and aorta were collected. Brachiocephalic arteries and brain were fixed in 10% formalin before paraffin embedding. For PCR and immunoblot, the aortic media were isolated by removing endothelium with a cotton swab and pulling a thin film of SMC away from the adventitia. Aorta and brain were flash frozen. Genomic DNA was isolated by EDTA lysis of 10 mg of tissue for 3 h or overnight at 55°C, precipitated with 3 M sodium acetate, washed with 70% ethanol. PCR of the intact Notch2 floxed allele with genotyping primers results in amplification of a 201 bp product, while PCR of excised Notch2 floxed does not, such that after normalizing to an internal control, the relative amplification between samples indicates excision efficiency. Intercellular adhesion molecule 1 (ICAM1) primers (5′-GAAATCATGTCTTGT­GGAACTGA-3′, 5′-CTCCTTCAACAGAGAAGCCAG-3′) provide control reactions with matching efficiency. Quantitative PCR was performed using Green Fast qPCR Mix LoRox (Azura) using a CFX384 thermocycler (Bio-Rad). Cycling was performed with an annealing temperature of 60°C for 40 cycles. Notch2 CT values were normalized to ICAM1 CT values and relative presence of intact Notch2 calculated by the comparative CT method (2^−ΔΔCT) [20].

Tissue Lysates and Immunoblot

Tissues were ground in liquid nitrogen using RIPA buffer plus protease inhibitors (Sigma), sonicated, and protein quantified using the DC protein assay (Bio-Rad), mixed with Laemmli sample buffer containing 100 mM dithiothreitol, and heated at 95°C for 10 min. SDS-PAGE was performed using TGX FastCast acrylamide 10% and 12% gels (Bio-Rad) and 15–50 µg protein/lane. Gels were transferred to polyvinylidene difluoride membranes using the TransBlot Turbo Transfer System (Bio-Rad). Membranes were blocked for 10 min in 5% milk/PBS with 0.01% Tween-20. Primary antibodies were diluted in 5% milk and incubated overnight at 4°C: 0.2 μg/mL CST 5732 rabbit anti-Notch2, 0.3 μg/mL CST 3700 rabbit anti-β-actin, and 0.5 μg/mL Abcam Ab53219 mouse anti-SMMHC. Membranes were incubated for 1 h with HRP-linked mouse or rabbit secondary antibodies (CST) diluted in 5% milk. Signal was detected with Luminata Chemiluminescent HRP substrate (Millipore) and imaged on a ChemiDoc MP system (Bio-Rad).

Histology and Immunofluorescence Staining

Brachiocephalic arteries were sectioned at 5 μm from the aortic arch for ∼100 μm and stained with H&E. Hearts were cryosectioned through the aortic root, post-fixed with 10% formalin fume, and stained with oil red O (ORO). Slides were washed in 85% propylene glycol and then stained with 0.7% ORO (Sigma) in propylene glycol for 15 min at 55°C. Slides were washed in 85% propylene glycol and then distilled water, counterstained with hematoxylin, and coverslipped with Aquatex® aqueous mounting media (Millipore).

Formalin fixed, paraffin embedded sections were rehydrated and underwent sodium citrate antigen retrieval, 45 min permeabilization with 0.5% Triton X-100 (EM Scientific), blocking in PBS with 0.5% Tween-20, 2% BSA (Sigma), and 5% goat serum (Jackson ImmunoResearch) for 2 h at room temperature, and with Mouse-on-Mouse Block (Vector Labs) overnight at 4°C. Sections were incubated overnight with primary antibodies in PBS/2% BSA: 1.8 μg/mL rabbit anti-Notch2 (CST 4530), 1.0 μg/mL rabbit anti-Notch3 (Abcam ab23426), 10 μg/mL mouse anti-smooth muscle actin (SMA; Abcam ab7817), rabbit anti-IgG (CST 3900), and 10 μg/mL mouse anti-IgG1 (CST 5415), 10 μg/mL mouse anti-smooth muscle actin (Abcam ab7817), 2 μg/mL rat anti-Mac2 (Cedarlane CL8942AP), 10 μg/mL mouse anti-IgG1 (CST 5415), and 2 μg/mL rat anti-IgG2a (BioLegend 400502). Sections were washed 3× for 15 min in TBS-T and incubated with Alexa Fluor-conjugated secondary antibodies (Invitrogen) in 1% BSA/TBS-T for 1 h at room temperature: 2 μg/mL goat anti-rabbit-AF488 (A11034) and 2 μg/mL goat anti-mouse-AF568 (A11077), 2 μg/mL goat anti-mouse-AF488 (A11001), and 2 μg/mL goat anti-rat-AF568 (A11004).

Aortic root sections were permeabilized for 5 min with 0.1% Tween-20 and then treated with 0.01 mol/L sodium citrate buffer at 50°C 15 min. Sections were blocked in 1% BSA/TBS-T for 20 min before overnight incubation at 4°C with primary antibodies in 1% BSA/TBS-T: 0.12 μg/mL rabbit anti-smooth muscle actin (CST 19245), 2 μg/mL rat anti-Mac2 (Cedarlane CL8942AP), 0.12 μg/mL rabbit anti-IgG (CST 3900), and 2 μg/mL rat anti-IgG2a (BioLegend 400502). Secondary antibodies (Invitrogen) were used in 1% BSA/TBS-T for 1 h at room temperature: 2 μg/mL goat anti-rabbit-AF488 (A11034) and 2 μg/mL goat anti-rat-AF568 (A11004). Sections were washed and incubated for 2 min with TrueVIEW Autofluorescence Quenching Kit and coverslipped using Vectashield Hard Set anti-fade mounting medium (Vector Labs). Confocal images were captured using a Leica TCS SP8 laser scanning confocal microscope with a 10×/0.40 dry or a 63x/1.40 oil objective.

Morphometry and Image Analysis

Quantification was performed while blinded to the treatment group. All brachiocephalic artery sections were quantified, while for aortic roots, sections 100–300 μm from appearance of first leaflet were used. Images were analyzed for plaque area, medial area, and necrotic core, and ORO images were also analyzed for lipid content. Images were acquired as 18 μm z-stacks composed of 7 slices interspaced by 3 μm; then sum slices was applied to generate 32-bit greyscale sum projections in ImageJ. Images were also analyzed for smooth muscle actin and Mac2 immunostaining. Plaque and medial areas were normalized to vessel circumference. Morphometry was performed by tracing plaques, necrotic cores, and the internal and external elastic lamina. The region of interest manager was used to distinguish signal intensity within a specified area from total signal. For aortic roots, medial area was traced to include only areas of the vessel wall with dense elastic fiber. For ORO images, color thresholding within red hued pixels allowed for selection of ORO-positive area. The smooth muscle actin and Mac2 sum projections were thresholded using the Otsu algorithm.

Statistical Analysis

The 6-week studies included 2 cohorts with 6–11 mice per cohort; the 14-week studies included 10 cohorts with 2–5 mice per cohort. Tissue sections from each mouse were utilized for each of the staining protocols. If sections from a given mouse did not survive the staining procedure, data for that stain on that mouse were not available. Statistical analysis was completed in GraphPad Prism v8.4.3, with outliers removed by ROUT testing (Q = 5%). Data are reported as mean ± SEM and compared by 2-way ANOVA and Sidak’s multiple comparison test. Physiological measures were compared by 2-way ANOVA and Tukey’s multiple comparison. Differences were considered significant at p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***), or p ≤ 0.0001 (****).

Confirmation of SMC-Specific Notch2 Deletion in ApoE−/− Mice

The use of the Notch2fl/fl model [15] in combination with the SMMHC-CreERT2 driver strain [17] resulted in reduced Notch2 protein in the vascular SMC [16]. We validated this model and assessed SMC Notch2 deletion on the ApoE−/− background. Aorta, brachiocephalic artery, and brain were collected from 8-week-old ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null male mice 1 week after the last tamoxifen or corn oil injection. Smooth muscle was expected to show high efficiency of recombination and reduced Notch2 protein while neural tissue was not expected to be significantly affected by SMMHC Cre. PCR with primers flanking the upstream loxP site of the Notch2 floxed locus results in a 201 bp band when no recombination occurs, absence of product when Cre recombination is successful, or a 161 bp wildtype band (Fig. 1a, wildtype band not present). Because amplification product results from wildtype Notch2 and nonrecombined Notch2 floxed, but not from recombined Notch2 floxed, the relative amplification was used to calculate recombination efficiency. Amplification was significantly reduced for ApoE−/−;Notch2SMC-null aorta-derived DNA compared to all other conditions (Fig. 1b).

Fig. 1.

Notch2 deletion in vascular SMC in ApoE−/− mice. a Genomic DNA was collected 1 week after Cre induction from brain cortex and aorta for PCR amplification. Shown are representative PCR products from ApoE−/−;Notch2SMC-ctr (N2ctr) and ApoE−/−;Notch2SMC-null (N2null) mice. The Notch2floxed allele generates a 201 bp fragment, which was specifically lost from the aorta in N2null mice, indicating Cre recombination. b PCR amplification data were quantified, and technical replicates were compared in two-way ANOVA. Significant results of Dunnett’s multiple comparisons test of each condition to ApoE−/−;Notch2SMC-ctr brain are indicated by asterisks. Amplification from the N2null aorta was significantly lower, ****p≤ 0.0001 compared to the other three groups. c Representative immunoblot for SMMHC and Notch2 with brain cortex and aorta lysates from ApoE−/−;Notch2SMC-ctr (N2ctr) and ApoE−/−;Notch2SMC-null (N2null) mice, with β-actin as a loading control. Molecular weights indicate molecular weight markers. d Brachiocephalic artery and brain cortex sections were immunostained for Notch2/SMA or Notch3/SMA with DAPI nuclear staining. Nonimmune IgG and IgG1 stained sections are shown as negative controls. L = lumen. Red arrowhead highlights Notch2-negative SMC next to Notch2-positive endothelial cells (white arrowhead). Scale bar, 50 μm.

Fig. 1.

Notch2 deletion in vascular SMC in ApoE−/− mice. a Genomic DNA was collected 1 week after Cre induction from brain cortex and aorta for PCR amplification. Shown are representative PCR products from ApoE−/−;Notch2SMC-ctr (N2ctr) and ApoE−/−;Notch2SMC-null (N2null) mice. The Notch2floxed allele generates a 201 bp fragment, which was specifically lost from the aorta in N2null mice, indicating Cre recombination. b PCR amplification data were quantified, and technical replicates were compared in two-way ANOVA. Significant results of Dunnett’s multiple comparisons test of each condition to ApoE−/−;Notch2SMC-ctr brain are indicated by asterisks. Amplification from the N2null aorta was significantly lower, ****p≤ 0.0001 compared to the other three groups. c Representative immunoblot for SMMHC and Notch2 with brain cortex and aorta lysates from ApoE−/−;Notch2SMC-ctr (N2ctr) and ApoE−/−;Notch2SMC-null (N2null) mice, with β-actin as a loading control. Molecular weights indicate molecular weight markers. d Brachiocephalic artery and brain cortex sections were immunostained for Notch2/SMA or Notch3/SMA with DAPI nuclear staining. Nonimmune IgG and IgG1 stained sections are shown as negative controls. L = lumen. Red arrowhead highlights Notch2-negative SMC next to Notch2-positive endothelial cells (white arrowhead). Scale bar, 50 μm.

Close modal

We also confirmed loss of vascular SMC Notch2 protein. Aorta and brain cortex from ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice were analyzed by immunoblot (Fig. 1c). As expected, significant SMMHC protein was detected in aorta but not brain, and Notch2 protein was present endogenously in both tissues. SMMHC expression in the vascular SMC led to selective loss of Notch2 protein in the aorta from ApoE−/−;Notch2SMC-null mice. Immunofluorescence staining was also performed in brain and brachiocephalic arteries from ApoE−/−;Notch2SMC-ctr and ApoE−/−; Notch2SMC-null mice to visualize Notch2, Notch3, and smooth muscle actin protein location within tissues (Fig. 1d). All proteins were abundantly expressed in ApoE−/−;Notch2SMC-ctr brachiocephalic artery samples, while Notch2 was specifically absent in the SMC in ApoE−/−;Notch2SMC-null mice. Conversely, Notch2 protein was unchanged in neuronal cells of brain tissue regardless of genotype. Notch3 protein was assayed to test whether loss of SMC Notch2 would impact Notch3 levels. Notch3 protein was abundant in vessel walls for both ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null and absent in neuronal cells of brain tissue.

Physiological Responses to Western Diet in ApoE−/− Mice Are Not Affected by SMC Notch2 Levels

To determine whether loss of SMC Notch2 had a systemic effect on physiological measures that can determine risk for cardiovascular disease, we measured BP, weight, serum glucose and cholesterol, as well as heart weight relative to mouse size (tibia length) as an indicator of cardiac hypertrophy and spleen weight as a rough indicator of systemic inflammation [21] (Table 1). There was no difference upon loss of SMC Notch2 for any of these parameters after 6 or 14 weeks of Western diet, providing support that changes in atherogenesis were due to the local cellular environment rather than systemic changes induced by SMC Notch2 deletion. There were age-related or diet duration-related differences for systolic BP, body weight post-diet, fasting cholesterol, heart weight, and spleen weight.

Table 1.

Physiological measurements in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 and 14 weeks of Western diet

 Physiological measurements in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 and 14 weeks of Western diet
 Physiological measurements in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 and 14 weeks of Western diet

SMC Notch2 Deletion in ApoE−/− Mice Does Not Affect Lesion Size or Medial Area

Cross-sectional plaque area was measured by morphometric tracing of H&E and phase-contrast micrographs for brachiocephalic arteries (Fig. 2a) and ORO and phase-contrast micrographs for aortic roots (Fig. 2b) and then normalized to vessel circumference. Plaque area was significantly greater after 14 weeks compared to 6 weeks of Western diet for both ApoE−/−;Notch2SMC-ctr and ApoE−/−; Notch2SMC-null mice in both regions (Fig. 2a, b, top graphs). The relative change in size of plaque between 6 and 14 weeks of Western diet was greater in the brachiocephalic artery compared to the aortic root. Grouping ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null to find average plaque size for a given timepoint, plaque area was 6.5 times greater after 14 weeks compared to 6 weeks at the brachiocephalic artery while it was only 1.5 times greater at the aortic root, indicative of temporal differences in plaque accumulation in the two vessel regions. There was no significant difference in relative plaque area for ApoE−/−;Notch2SMC-ctr compared to ApoE−/−;Notch2SMC-null mice at either timepoint in either vessel.

Fig. 2.

Overall plaque size does not change with loss of SMC Notch2. Mice with the genotypes indicated were fed a Western diet for 6 or 14 weeks. a Representative brachiocephalic artery images with morphometric quantification of relative plaque size and medial area in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice. Scale bar, 100 μm. No significant results were found by Sidak’s multiple comparisons test (genotype). Significant differences between 6 weeks and 14 weeks of diet found using 2-way ANOVA are indicated by asterisks: **p≤ 0.01, ****p≤ 0.0001. b Representative aortic root images with morphometric quantification of relative plaque size and medial area in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 or 14 weeks of Western diet. The thin solid black lines demarcate the medial layer. Scale bar, 200 μm. No significant results were found by Sidak’s multiple comparisons test based on genotype. Significant differences between 6 weeks and 14 weeks of diet found using 2-way ANOVA are indicated by asterisks: ***p≤ 0.001.

Fig. 2.

Overall plaque size does not change with loss of SMC Notch2. Mice with the genotypes indicated were fed a Western diet for 6 or 14 weeks. a Representative brachiocephalic artery images with morphometric quantification of relative plaque size and medial area in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice. Scale bar, 100 μm. No significant results were found by Sidak’s multiple comparisons test (genotype). Significant differences between 6 weeks and 14 weeks of diet found using 2-way ANOVA are indicated by asterisks: **p≤ 0.01, ****p≤ 0.0001. b Representative aortic root images with morphometric quantification of relative plaque size and medial area in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 or 14 weeks of Western diet. The thin solid black lines demarcate the medial layer. Scale bar, 200 μm. No significant results were found by Sidak’s multiple comparisons test based on genotype. Significant differences between 6 weeks and 14 weeks of diet found using 2-way ANOVA are indicated by asterisks: ***p≤ 0.001.

Close modal

We observed an expansion of the media wall immediately underlying plaque compared to media with no overlying plaque. Medial expansion is a deleterious process and has been observed in acute vessel injury models [22]. Cross-sectional media area was measured to determine if medial hyperplasia was present in this disease model. We performed morphometric tracing of H&E and phase-contrast images for brachiocephalic arteries and ORO and phase-contrast images for aortic roots and normalized areas to vessel circumference. The elastic lamina guided identification of the borders of the tunica media in the brachiocephalic arteries and aortic roots. Medial area was significantly greater after 14 weeks compared to 6 weeks of Western diet for both ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice in brachiocephalic arteries (Fig. 2a). There was no significant difference in relative medial area for ApoE−/−;Notch2SMC-ctr compared to ApoE−/−;Notch2SMC-null mice at either timepoint in either vessel. The increase in medial area and circumference after 14 weeks is likely due to a combination of mouse age and plaque formation.

Atherosclerotic Lesion Necrosis and Lipid Content Are Independent of Notch2 in SMC

H&E images of brachiocephalic arteries and ORO-stained images of aortic roots were examined for regions of low cellularity that contained cholesterol clefts in standard pathological assessment [23]. These necrotic areas were traced and percent of total plaque area that was necrotic was calculated. Positive ORO staining within the plaque was quantified by color thresholding in ImageJ and percent of total plaque area that was lipid was calculated. There was no change in necrotic content for brachiocephalic plaques with loss of SMC Notch2, though necrosis did increase with time on Western diet (Fig. 3a). There was greater necrotic content and lipid content in aortic root plaques after 6 weeks compared to 14 weeks of Western diet (Fig. 3b).

Fig. 3.

Cellularity and lipid content in the plaque are not affected by loss of Notch2 in SMC. Mice with the genotypes indicated were fed a Western diet for 6 or 14 weeks. a Representative brachiocephalic artery histological images with morphometric quantification of lesion necrosis in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 or 14 weeks. Scale bar, 100 μm. No significant results were found by Sidak’s multiple comparisons test based on genotype. Significant differences between 6 weeks and 14 weeks of diet were found using 2-way ANOVA, ***p≤ 0.001. b Representative aortic root sections stained with ORO. Scale bar, 200 μm. Noncellular areas and lipid-containing areas were quantified. No significant results were found by Sidak’s multiple comparisons test comparing genotype. Significant differences between 6 weeks and 14 weeks of diet found using 2-way ANOVA are indicated by asterisks: *p≤ 0.05, **p≤ 0.01.

Fig. 3.

Cellularity and lipid content in the plaque are not affected by loss of Notch2 in SMC. Mice with the genotypes indicated were fed a Western diet for 6 or 14 weeks. a Representative brachiocephalic artery histological images with morphometric quantification of lesion necrosis in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 or 14 weeks. Scale bar, 100 μm. No significant results were found by Sidak’s multiple comparisons test based on genotype. Significant differences between 6 weeks and 14 weeks of diet were found using 2-way ANOVA, ***p≤ 0.001. b Representative aortic root sections stained with ORO. Scale bar, 200 μm. Noncellular areas and lipid-containing areas were quantified. No significant results were found by Sidak’s multiple comparisons test comparing genotype. Significant differences between 6 weeks and 14 weeks of diet found using 2-way ANOVA are indicated by asterisks: *p≤ 0.05, **p≤ 0.01.

Close modal

SMC Notch2 Deletion in ApoE−/− Mice Impacts Smooth Muscle Actin in a Time- and Location-Dependent Manner

Cross-sectional plaque area and medial area were determined by morphometric tracing in immunofluorescent images of brachiocephalic arteries and aortic roots; then smooth muscle actin-positive areas within each region were determined by image analysis (Fig. 4a, 5a). After 6 weeks of Western diet, abundance of smooth muscle actin protein within brachiocephalic artery plaque was significantly reduced, and staining intensity was also significantly lower. This was quantified as % of plaque that was positive for smooth muscle actin staining and the smooth muscle actin staining intensity (total pixel grey value normalized to positive area, Fig. 4c). After 14 weeks of Western diet, brachiocephalic artery plaque levels of smooth muscle actin were low in both ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice (Fig. 4b). There was no difference in smooth muscle actin levels within aortic root plaques after 6 weeks of Western diet (Fig. 5a, c) or 14 weeks (Fig. 5b, c). Percent of medial area positive for smooth muscle actin and medial layer smooth muscle actin staining intensity were significantly reduced after 14 weeks compared to 6 weeks of Western diet for both genotypes at both the brachiocephalic artery and the aortic root (Fig. 4c, 5c).

Fig. 4.

Smooth muscle actin is reduced in early brachiocephalic artery plaque with loss of Notch2 in SMC. Mice with the genotypes indicated were fed a Western diet for 6 (a) or 14 (b) weeks. Representative immunofluorescent images of brachiocephalic artery lesions from ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice. SMA is labeled in red, and Mac2 is labeled in green, and each is also shown individually to better show plaque SMA (yellow arrowheads in panel a). Scale bars, 100 μm. c Morphometric quantification of plaque SMA content, plaque Mac2 content, and medial SMA content in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 or 14 weeks of Western diet. Sidak’s multiple comparisons test showed that % SMA+ plaque area was significantly decreased at 6 weeks in the ApoE−/−;Notch2SMC-null mouse, *p≤ 0.05. Significant differences were found between 6 weeks and 14 weeks of diet for all the other measures using 2-way ANOVA: *p≤ 0.05, ***p≤ 0.001, ****p≤ 0.0001.

Fig. 4.

Smooth muscle actin is reduced in early brachiocephalic artery plaque with loss of Notch2 in SMC. Mice with the genotypes indicated were fed a Western diet for 6 (a) or 14 (b) weeks. Representative immunofluorescent images of brachiocephalic artery lesions from ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice. SMA is labeled in red, and Mac2 is labeled in green, and each is also shown individually to better show plaque SMA (yellow arrowheads in panel a). Scale bars, 100 μm. c Morphometric quantification of plaque SMA content, plaque Mac2 content, and medial SMA content in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 or 14 weeks of Western diet. Sidak’s multiple comparisons test showed that % SMA+ plaque area was significantly decreased at 6 weeks in the ApoE−/−;Notch2SMC-null mouse, *p≤ 0.05. Significant differences were found between 6 weeks and 14 weeks of diet for all the other measures using 2-way ANOVA: *p≤ 0.05, ***p≤ 0.001, ****p≤ 0.0001.

Close modal
Fig. 5.

Aortic root plaque Mac2 is increased with loss of SMC Notch2. Representative immunofluorescent images of aortic root lesions from ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 weeks (a) or 14 weeks (b) of Western diet. SMA is in red, and Mac2 is in green. Individual staining patterns are also shown. Scale bars, 200 μm. c Morphometric quantification of plaque SMA content, plaque Mac2 content, and media SMA content in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 or 14 weeks of Western diet. Sidak’s multiple comparisons test was used to compare groups by genotype – the only statistically significant difference was the Mac2 intensity at 6 weeks, ***p≤ 0.001. Significant differences between 6 weeks and 14 weeks of diet found using 2-way ANOVA are indicated by asterisks: *p≤ 0.05, ***p≤ 0.001, ****p≤ 0.0001.

Fig. 5.

Aortic root plaque Mac2 is increased with loss of SMC Notch2. Representative immunofluorescent images of aortic root lesions from ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 weeks (a) or 14 weeks (b) of Western diet. SMA is in red, and Mac2 is in green. Individual staining patterns are also shown. Scale bars, 200 μm. c Morphometric quantification of plaque SMA content, plaque Mac2 content, and media SMA content in ApoE−/−;Notch2SMC-ctr and ApoE−/−;Notch2SMC-null mice after 6 or 14 weeks of Western diet. Sidak’s multiple comparisons test was used to compare groups by genotype – the only statistically significant difference was the Mac2 intensity at 6 weeks, ***p≤ 0.001. Significant differences between 6 weeks and 14 weeks of diet found using 2-way ANOVA are indicated by asterisks: *p≤ 0.05, ***p≤ 0.001, ****p≤ 0.0001.

Close modal

SMC-Specific Notch2 Deletion in ApoE−/− Mice Increased Mac2 Staining in 6-Week Aortic Root Lesions

Morphometric tracing of immunofluorescent images of brachiocephalic arteries and aortic roots was paired with image analysis of Mac2-positive areas (Fig. 4a, 5a). Mac2 is produced by activated macrophages [24, 25] and dedifferentiating SMC [11] and is used here as general measure for the presence of abnormal cells. Aortic root plaque Mac2 staining intensity was significantly higher for ApoE−/−;Notch2SMC-null after 6 weeks of Western diet. There was no difference in Mac2 intensity or percent plaque area between genotypes after 14 weeks at the aortic root (Fig. 5c). After 14 weeks of Western diet, plaque Mac2 staining intensity was significantly lower for both genotypes at both the brachiocephalic artery and the aortic root than 6 weeks of Western diet (Fig. 4c, 5c). Percent of plaque area Mac2-positive was significantly lower at 14 weeks of Western diet compared to 6 weeks in both genotypes at the brachiocephalic artery (Fig. 4c) but not at the aortic root (Fig. 5c).

Temporal Analysis of Lesion Progression

Comparison of lesions between the 6-week and 14-week timepoints revealed that several factors changed predictably with time for both athero-prone vascular regions. As expected with untreated hypercholesteremia, plaques were larger after 14 weeks of Western diet than they were after 6 weeks. Also as expected, plaques at the brachiocephalic artery were more necrotic after 14 weeks of Western diet than they were after 6 weeks. However, there was a surprising decrease in necrotic plaque area and lipid content after 14 weeks at the aortic root. Atherosclerosis is not a simple process of greater and greater necrosis and greater and greater lipid accumulation but rather a complex series of responses from a myriad of vascular cells that cause changes to matrix, cell growth, and remodeling. Indeed, the opposing direction of change in necrotic content between 6 and 14 weeks of Western diet at the aortic root compared to the brachiocephalic artery indicate that not only is atherosclerosis a complex process, but it is also complex in how it impacts specific sites at given timepoints. Remodeling of extracellular matrix and the migration of cells throughout the plaque may contribute to these changes. Interestingly, Mac2-positive area and/or intensity was decreased after 14 weeks. Mac2 is commonly used as an inflammatory marker and because advanced lesions are typically characterized by robust inflammation, this observation was not expected. However, it was recently found that Mac2-positive macrophages were replaced with Mac2-negative macrophages over time and that unstable plaques were more likely to contain greater numbers of Mac2-negative macrophages [26]. Loss of macrophage Mac2 was hypothesized to promote an invasive proinflammatory phenotype that destabilized plaque. It was also recently observed that not only did populations of lesion SMC activate Mac2 expression [8, 27] but that subpopulations then went on to lose Mac2 expression [11]. It is unknown whether loss of Mac2 expression in the lesion SMC stabilizes or destabilizes plaque. Another factor that changed consistently over time at both vessel locations was reduction of contractile marker smooth muscle actin in the medial layer. This was consistent with the finding that smooth muscle actin in the tunica media of the aortic root was reduced in advanced atherosclerosis [28]. This loss of contractile protein in the media may have been a harbinger of increased SMC motility into lesions and reduced vascular contractility. A related finding in brachiocephalic arteries was increased cross-sectional area, or medial thickening, over time. Medial thickening could be caused by SMC transition to a proliferative phenotype.

SMC Notch2 in SMC in Contractile Phenotype

Deletion of SMC Notch2 resulted in lower contractile smooth muscle actin protein level in the brachiocephalic artery after 6 weeks of Western diet. No significant changes to smooth muscle actin content were observed within lesions or media at the aortic root after 6 weeks of Western diet or in either vessel bed after 14 weeks. Previously, we found that the human SMC derived from endarterectomy samples were unable to upregulate smooth muscle actin upon treatment with jagged1 indicating loss of Notch-mediated maintenance of differentiation for the SMC in late atherosclerosis [13]. In this in vivo study, reduced smooth muscle actin in brachiocephalic artery lesions upon loss of Notch2 signaling in the vascular SMC points to functional Notch-mediated maintenance of SMC differentiation early in atherosclerosis. Reduced expression of contractile proteins in the lesion SMC has been shown to be deleterious in two major ways. The SMC in lesions with the contractile phenotype are typically found near the lumenal face and contribute to the lumenal fibrous cap; loss of this protective layer predisposes lesions to rupture [29]. Additionally, permanent or transitory transition of lesion SMC from a contractile phenotype to a macrophage-like phenotype leads to formation of foam cells and cholesterol accumulation [30, 31]. Our results from mice fed Western diet for 14 weeks reveal that Notch2 signaling does not impact late atherosclerosis, though it may have a minor role in plaque stabilization early in atherosclerosis. One potential limitation of our study is the possible re-emergence of newly differentiated SMC after the tamoxifen-induction period. Because the mature SMC is predicted to contribute to the majority of foam cells within atherosclerotic plaques in humans and mice [32], we believe that targeting of mature SMC prior to atheroma formation is a likely strategy to target Notch2 in SMC-derived populations in the plaque.

Recently, it has been shown that cells other than the SMC may express smooth muscle actin within the plaque environment, thus contributing to lesion stability [33]. Newman et al. [33] generated SMC-PDGFRβ null mice with simultaneous SMC lineage tracing on an ApoE−/− background and observed endothelial cells and macrophages that had transitioned to mesenchymal-like cells that expressed smooth muscle actin and contributed to plaque stability. Loss of SMC-PDGFRβ resulted in 90% less investment of SMC into lesions, yet the plaques did not differ in size, stability, or number of smooth muscle actin-positive cells compared to littermate controls. They provide evidence that mesenchymal-like cells compensated for the reduced SMC in atherosclerotic plaques, at least temporarily. In the current study, we saw instances when loss of SMC Notch2 resulted in changes to lesion smooth muscle actin or Mac2 content, yet lesion size never changed. It is possible that the Notch2 null SMC in our model had reduced function that was compensated for by non-SMC cell types. Without lineage tracing, our study provided insight into total smooth muscle actin in lesions. Because the Newman et al. [33] study showed that loss of SMC-derived smooth muscle actin contribution to plaque stability can be temporarily compensated for by endothelial cell and macrophage-derived mesenchymal cells, future studies of Notch signaling in atherosclerotic lesions should include linage tracing. Lesions likely to lose stability due to loss of SMC-specific smooth muscle actin cannot be detected without additional interrogation of cellular origin.

Vessel Beds Have Distinct Response to Loss of SMC Notch2

The SMC of the aortic root derives from the secondary heart field of lateral plate mesoderm, while the brachiocephalic artery SMC comes from neural crest [34]. There is evidence that differences in SMC origin influence the function of the mature vessel and the constituent SMC [35, 36]. If the role of Notch2 in regulating SMC phenotype varies between the SMC of different developmental origins, this might explain why SMC Notch2 maintains the lesion SMC in a contractile phenotype at the brachiocephalic artery yet prevents transition to and/or accumulation of Mac2-positive cells at the aortic root after 6 weeks of Western diet. Alternatively, because a 6-week brachiocephalic lesion is likely to be developmentally younger than a 6-week aortic root lesion [37], these differences in vessel bed response to loss of SMC Notch2 could be a reflection of different roles for SMC Notch2 through disease progress. Overall, our study indicates a minor role for Notch2 in regulating the SMC in atherogenesis in a location- and temporally dependent manner.

The authors acknowledge the expert assistance of the Histopathology Core Facility at MaineHealth Institute for Research (Dr. V. Lindner and G. Mangoba) and our Confocal Microscopy Facility (Dr. I. Prudovsky).

This study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Maine Medical Center, protocol 2110 (Liaw PI).

The authors have no conflicts of interest to declare.

This study was supported by NIH/NHLBI grant R01 HL141149 to L.L. J.D.K. was supported by a predoctoral fellowship from the American Heart Association (16PRE29870001). Our institutional Histopathology Core Facility is supported by NIH/NIGMS award 1P20GM12130 to L.L. and NIH/NIGMS award U54GM115516 (C. Rosen and G. Stein, PIs).

Jessica Davis-Knowlton and Lucy Liaw contributed to conception and design of the study, and Jessica Davis-Knowlton completed the majority of the experimental work and data analysis and wrote the first draft of the manuscript. Jacqueline Turner performed histological and immunostaining and some morphometric tracing. Anne Harrington developed breeding strategies, had oversight of mouse husbandry and genotyping, and performed AAV inoculation. All authors contributed to manuscript revision, read, and approved the submitted version. Lucy Liaw provided funding, resources, and oversight of the project.

The data that support the findings of this study are openly available in Figshare at https://doi.org/10.6084/m9.figshare.16671115.v2, reference number 16671115.v2.

1.
Briot
A
,
Civelek
M
,
Seki
A
,
Hoi
K
,
Mack
JJ
,
Lee
SD
,
.
Endothelial NOTCH1 is suppressed by circulating lipids and antagonizes inflammation during atherosclerosis
.
J Exp Med
.
2015
;
212
(
12
):
2147
63
.
2.
Mack
JJ
,
Mosqueiro
TS
,
Archer
BJ
,
Jones
WM
,
Sunshine
H
,
Faas
GC
,
.
NOTCH1 is a mechanosensor in adult arteries
.
Nat Commun
.
2017
;
8
(
1
):
1620
.
3.
Fung
E
,
Tang
SMT
,
Canner
JP
,
Morishige
K
,
Arboleda-Velasquez
JF
,
Cardoso
AA
,
.
Delta-like 4 induces Notch signaling in macrophages
.
Circulation
.
2007 Jun
;
115
(
23
):
2948
56
.
4.
Monsalve
E
,
Ruiz-García
A
,
Baladrón
V
,
Ruiz-Hidalgo
MJ
,
Sánchez-Solana
B
,
Rivero
S
,
.
Notch1 upregulates LPS-induced macrophage activation by increasing NF-κB activity
.
Eur J Immunol
.
2009 Sep
;
39
(
9
):
2556
70
.
5.
Fukuda
D
,
Aikawa
E
,
Swirski
FK
,
Novobrantseva
TI
,
Kotelianski
V
,
Gorgun
CZ
,
.
Notch ligand delta-like 4 blockade attenuates atherosclerosis and metabolic disorders
.
Proc Natl Acad Sci U S A
.
2012
;
109
(
27
):
E1868
77
.
6.
Boucher
J
,
Gridley
T
,
Liaw
L
.
Molecular pathways of notch signaling in vascular smooth muscle cells
.
Front Physiol
.
2012
;
3
:
1
13
.
7.
Baeten
JT
,
Lilly
B
.
Notch signaling in vascular smooth muscle cells
.
Adv Pharmacol
.
2017
;
78
:
351
82
.
8.
Shankman
LS
,
Gomez
D
,
Cherepanova
OA
,
Salmon
M
,
Alencar
GF
,
Haskins
RM
,
.
KLF4 dependent phenotypic modulation of SMCs plays a key role in atherosclerotic plaque pathogenesis laura
.
Nat Med
.
2015
;
21
(
6
):
628
37
.
9.
Albarrán-Juárez
J
,
Kaur
H
,
Grimm
M
,
Offermanns
S
,
Wettschureck
N
.
Lineage tracing of cells involved in atherosclerosis
.
Atherosclerosis
.
2016
;
251
:
445
53
.
10.
Jacobsen
K
,
Lund
MB
,
Shim
J
,
Gunnersen
S
,
Füchtbauer
EM
,
Kjolby
M
,
.
Diverse cellular architecture of atherosclerotic plaque derives from clonal expansion of a few medial SMCs
.
JCI Insight
.
2017 Oct
;
2
(
19
):
1
13
.
11.
Alencar
GF
,
Owsiany
KM
,
Karnewar
S
,
Sukhavasi
K
,
Mocci
G
,
Nguyen
AT
,
.
Stem cell pluripotency genes Klf4 and Oct4 regulate complex SMC phenotypic changes critical in late-stage atherosclerotic lesion pathogenesis
.
Circulation
.
2020 Nov
;
142
(
21
):
2045
59
.
12.
Boucher
JM
,
Harrington
A
,
Rostama
B
,
Lindner
V
,
Liaw
L
.
A receptor-specific function for Notch2 in mediating vascular smooth muscle cell growth arrest through cyclin-dependent kinase inhibitor 1B
.
Circ Res
.
2013
;
113
(
8
):
975
85
.
13.
Davis-knowlton
J
,
Turner
JE
,
Turner
A
,
Damian-Loring
S
,
Hagler
N
,
Henderson
T
,
.
Characterization of smooth muscle cells from human atherosclerotic lesions and their responses to Notch signaling
.
Lab Investig
.
2018 Mar
;
99
(
3
):
290
304
.
14.
Martos-Rodríguez
CJ
,
Albarrán-Juárez
J
,
Morales-Cano
D
,
Caballero
A
,
Macgrogan
D
,
De La Pompa
JL
,
.
Fibrous caps in atherosclerosis form by Notch-dependent mechanisms common to arterial media development
.
Arterioscler Thromb Vasc Biol
.
2021
;
41
(
9
):
E427
39
.
15.
McCright
B
,
Lozier
J
,
Gridley
T
.
Generation of new Notch2 mutant alleles
.
Genesis
.
2006
;
44
(
1
):
29
33
.
16.
Peterson
SM
,
Turner
JE
,
Harrington
A
,
Davis-Knowlton
J
,
Lindner
V
,
Gridley
T
,
.
Notch2 and proteomic signatures in mouse neointimal lesion formation
.
Arterioscler Thromb Vasc Biol
.
2018 Jul
;
38
(
7
):
1576
93
.
17.
Wirth
A
,
Benyó
Z
,
Lukasova
M
,
Leutgeb
B
,
Wettschureck
N
,
Gorbey
S
,
.
G12-G13-LARG-mediated signaling in vascular smooth muscle is required for salt-induced hypertension
.
Nat Med
.
2008
;
14
(
1
):
64
8
.
18.
Piedrahita
JA
,
Zhang
SH
,
Hagaman
JR
,
Oliver
PM
,
Maeda
N
.
Generation of mice carrying a mutant apolipoprotein E gene inactivated by gene targeting in embryonic stem cells
.
Proc Natl Acad Sci U S A
.
1992
;
89
(
10
):
4471
5
.
19.
Jaffe
IZ
,
Newfell
BG
,
Aronovitz
M
,
Mohammad
NN
,
McGraw
AP
,
Perreault
RE
,
.
Placental growth factor mediates aldosterone-dependent vascular injury in mice
.
J Clin Invest
.
2010 Nov
;
120
(
11
):
3891
.
20.
Schmittgen
TD
,
Livak
KJ
.
Analyzing real-time PCR data by the comparative C(T) method
.
Nat Protoc
.
2008
;
3
(
6
):
1101
8
. [cited 2019 Mar 29].
21.
Buchan
L
,
St Aubin
CR
,
Fisher
AL
,
Hellings
A
,
Castro
M
,
Al-Nakkash
L
,
.
High-fat, high-sugar diet induces splenomegaly that is ameliorated with exercise and genistein treatment
.
BMC Res Notes
.
2018 Oct
;
11
(
1
):
752
.
22.
Kumar
A
,
Lindner
V
.
Remodeling with neointima formation in the mouse carotid artery after cessation of blood flow
.
Arterioscler Thromb Vasc Biol
.
1997 Oct
;
17
(
10
):
2238
44
.
23.
Seimon
TA
,
Wang
Y
,
Han
S
,
Senokuchi
T
,
Schrijvers
DM
,
Kuriakose
G
,
.
Macrophage deficiency of p38α MAPK promotes apoptosis and plaque necrosis in advanced atherosclerotic lesions in mice
.
J Clin Invest
.
2009 Apr
;
119
(
4
):
886
.
24.
Sato
S
,
Hughes
RC
.
Regulation of secretion and surface expression of Mac-2, a galactoside- binding protein of macrophages
.
J Biol Chem
.
1994 Feb
;
269
(
6
):
4424
30
.
25.
Liu
FT
,
Hsu
DK
,
Zuberi
RI
,
Kuwabara
I
,
Chi
EY
,
Henderson
WR
.
Expression and function of galectin-3, a β-galactoside-binding lectin, in human monocytes and macrophages
.
Am J Pathol
.
1995
[cited 2021 Feb 27];
147
(
4
):
1016
28
.
26.
Di Gregoli
K
,
Somerville
M
,
Bianco
R
,
Thomas
AC
,
Frankow
A
,
Newby
AC
,
.
Galectin-3 identifies a subset of macrophages with a potential beneficial role in atherosclerosis
.
Arterioscler Thromb Vasc Biol
.
2020
;
40
:
1491
509
.
27.
Feil
S
,
Fehrenbacher
B
,
Lukowski
R
,
Essmann
F
,
Schulze-Osthoff
K
,
Schaller
M
,
.
Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis
.
Circ Res
.
2014 Sep
;
115
(
7
):
662
7
.
28.
Moss
ME
,
DuPont
JJ
,
Iyer
SL
,
McGraw
AP
,
Jaffe
IZ
.
No significant role for smooth muscle cell mineralocorticoid receptors in atherosclerosis in the apolipoprotein-E knockout mouse model
.
Front Cardiovasc Med
.
2018
;
5
:
81
.
29.
Hansson
GK
,
Libby
P
,
Tabas
I
.
Inflammation and plaque vulnerability
.
J Intern Med
.
2015
;
278
(
5
):
483
93
.
30.
Allahverdian
S
,
Chehroudi
AC
,
McManus
BM
,
Abraham
T
,
Francis
GA
.
Contribution of intimal smooth muscle cells to cholesterol accumulation and macrophage-like cells in human atherosclerosis
.
Circulation
.
2014
;
129
(
15
):
1551
9
.
31.
Li
Y
,
Zhu
H
,
Zhang
Q
,
Han
X
,
Zhang
Z
,
Shen
L
,
.
Smooth muscle-derived macrophage-like cells contribute to multiple cell lineages in the atherosclerotic plaque
.
Cell Discov
.
2021 Nov
;
7
(
1
):
1
4
.
32.
Wang
Y
,
Dubland
JA
,
Allahverdian
S
,
Asonye
E
,
Sahin
B
,
Jaw
JE
,
.
Smooth muscle cells contribute the majority of foam cells in ApoE (Apolipoprotein E)-deficient mouse atherosclerosis
.
Arterioscler Thromb Vasc Biol
.
2019 May
;
39
(
5
):
876
87
.
33.
Newman
AAC
,
Serbulea
V
,
Baylis
RA
,
Shankman
LS
,
Bradley
X
,
Alencar
GF
,
.
Multiple cell types contribute to the atherosclerotic lesion fibrous cap by PDGFRβ and bioenergetic mechanisms
.
Nat Metab
.
2021
;
3
(
2
):
166
81
.
34.
Sinha
S
,
Iyer
D
,
Granata
A
.
Embryonic origins of human vascular smooth muscle cells: implications for in vitro modeling and clinical application
.
Cell Mol Life Sci
.
2014 Jun
;
71
(
12
):
2271
88
.
35.
Zhang
J
,
Chen
J
,
Xu
C
,
Yang
J
,
Guo
Q
,
Hu
Q
,
.
Resveratrol inhibits phenotypic switching of neointimal vascular smooth muscle cells after balloon injury through blockade of Notch pathway
.
J Cardiovasc Pharmacol
.
2014
;
63
(
3
):
233
9
.
36.
Jiao
J
,
Xiong
W
,
Wang
L
,
Yang
J
,
Qiu
P
,
Hirai
H
,
.
Differentiation defect in neural crest-derived smooth muscle cells in patients with aortopathy associated with bicuspid aortic valves
.
EBioMedicine
.
2016
;
10
:
282
.
37.
Nakashima
Y
,
Plump
AS
,
Raines
EW
,
Breslow
JL
,
Ross
R
.
ApoE-deficient mice develop lesions of all phases of atherosclerosis throughout the arterial tree
.
Arterioscler Thromb A J Vasc Biol
.
1994
;
14
(
1
):
133
40
.