C/EBPβ activation in vascular smooth muscle cells promotes hyperlipidemia-induced phenotypic transition and arterial stiffness

Abstract

Arterial stiffness is a critical factor in cardiovascular and cerebrovascular events, yet clinical practice lacks specific therapeutic targets and biomarkers for its assessment. Hyperlipidemia closely correlates with arterial stiffness, and we observed elevated CCAAT/enhancer-binding protein β (C/EBPβ) expression in atherosclerotic mouse arterial walls. As the arterial medial layer predominantly consists of vascular smooth muscle cells (VSMCs), C/EBPβ's role in VSMCs under hyperlipidemia remains unclear. Our findings demonstrate that cholesterol-induced phenotypic transition of contractile VSMCs to macrophage-like cells coincides with C/EBPβ upregulation and activation. The activation of C/EBPβ is closely related to cellular assembly and organization, regulating the cytoskeleton via Disheveled-associated activator of morphogenesis 1 (Daam1). Conditional knockout of C/EBPβ in VSMCs of ApoE^-/- mice alleviated hyperlipidemia-induced vascular remodeling and reduced the elevation of aortic pulse wave velocity. Additionally, C/EBPβ-regulated cytokine platelet-derived growth factor-CC (PDGF-CC) is correlated with brachial-ankle pulse wave velocity in humans. These results indicate that the activation of C/EBPβ promotes the transition of VSMCs from a contractile phenotype to a macrophage-like phenotype by regulating morphological changes, and C/EBPβ activation contributes to hyperlipidemia-induced arterial stiffness. PDGF-CC exhibited a significant association with arterial stiffness and may serve as a promising indicator of arterial stiffness in humans. Our study reveals molecular mechanisms behind hyperlipidemia-induced arterial stiffness and provides potential therapeutic targets and biomarkers.

Fig. 1

Knockdown of C/EBPβ expression inhibited MBD-Chol-induced transition of contractile VSMCs into a macrophage-like phenotype. a Modified Russell-Movat Pentachrome stain was used to show murine aortic arch tissue composition. Black for elastin fibers, yellow for collagen, blue/cyan for proteoglycan, red for collagen fibers, and black/purple for nuclei, scale bar = 100 μm. b Immunofluorescence staining was used to assess protein expression and localization in the murine aortas, blue (DAPI) for nuclei, green for C/EPBβ, and red for CD68 (representative images). The scale bars correspond to 100 μm for low-magnification images and 20 μm for high-magnification views. c Immunofluorescence staining was used to assess protein expression and localization in VSMCs, blue for nuclei, green for ACTA2, and red for CD68 (representative images), scale bar = 50 μm. d Immunofluorescence staining was used to assess protein localization and expression in VSMCs, blue for nuclei, green for C/EBPβ, and red for CD68 (representative images), scale bar = 50 μm. e mRNA levels of contractile phenotype-associated markers (ACTA2, CNN1, MYH11) in VSMCs, N = 3. f mRNA levels of macrophage-like phenotype-associated markers (CD68, LGALS3) and C/EBPβ in VSMCs, N = 3. g Protein expression levels detected by WB in VSMCs (representative images). h Statistical results of protein expression levels, N = 3. *p < 0.05 and ***p < 0.001. NC= scrambled siRNA group, si-C/EBPβ = C/EBPβ siRNA group. Segment of aortic arch was obtained for staining

Fig. 2

The role of C/EBPβ in the functional changes of VSMCs after MBD-Chol stimulation. a Oil Red O staining was used to detect lipid phagocytosis in VSMCs, the top image shows low magnification, the bottom image shows high magnification, scale bar=100μm, red for lipids. b Quantification of lipid area in Oil Red O staining, N = 6. c Functional enrichment analysis of ChIP-seq-identified C/EBPβ targets in control group (GO database). The change in red color from dark to light represents low to high P value, and the change in shape from small to large represents the increase in the number of related genes in the pathway from less to more, horizontal coordinates represent the enrichment score, the higher the score, the greater the effect, vertical coordinates represent the different functions. d Parallel analysis of MBD-Chol-treated specimens showing C/EBPβ‘s pathway engagement. e Distribution and location of peaks on different chromosomes, vertical coordinates represent the different chromosome numbers, horizontal coordinates represent the size of the chromosomes. f Classification of C/EBPβ-related functions in “Molecular and Cellular Functions” and “Cellular Assembly and Organization” by IPA, with P-values in ascending order from the top. *p < 0.05 and ***p < 0.001. NC = scrambled siRNA group, si-C/EBPβ = C/EBPβ siRNA group

Fig. 3

Regulatory role of C/EBPβ in cytoskeleton in VSMCs after MBD-Chol stimulation. a Phalloidin staining of VSMCs cytoskeleton in different groups. Blue for nuclei, and green for F-actin (representative images), scale bar = 50 μm. b Quantification of cytoskeleton area in Phalloidin staining, N = 6. c Results of ChIP-seq showing C/EBPβ-binding genes, horizontal coordinates represent fold change (log2), vertical coordinates represent P value (log 10). d Protein expression levels detected by WB in VSMCs (representative images). e Statistical results of protein expression levels, N = 3. f Immunofluorescence staining was used to assess protein localization and expression in VSMCs, blue for nuclei, green for Daam1, and red for C/EBPβ. Scale bar=50 μm. *p < 0.05 and ***p < 0.001. NC = scrambled siRNA group, si-C/EBPβ = C/EBPβ siRNA group

Fig. 4

Changes in lipids levels, arterial stiffness and plaque formation at different times in ApoE^−/− mice fed with 60% high-fat diet. a Small animal ultrasound was used to assess aPWV in vivo (representative images). b Statistical results of changes in aPWV of mice at different feeding times, N = 10. c Small animal ultrasound was used to assess aortic contractile function in vivo (representative images). d Statistical results of changes in aortic contractile function of mice at different feeding times, N = 10. e Changes in blood lipids levels of mice at different feeding times, N = 10. f Changes in body weight of mice at different feeding times, N = 10. g Plaque formation in murine aortas at different feeding times (representative images). h Statistical results of plaque formation in murine aortas at different feeding times, N = 10. TC total cholesterol, TG triglycerides, LDL-c low-density lipoprotein cholesterol, HDL-c high-density lipoprotein cholesterol. Control=ApoE^−/− mice fed with chow food, HF (ApoE^−/−) ApoE^−/− mice fed with a 60% high-fat diet. *p < 0.05 and ***p < 0.001 compared with 0-week, ^#p < 0.05 and ^###p < 0.001 compared with the control group

Fig. 5

Knockout of C/EBPβ in VSMCs alleviated hyperlipidemia-induced arterial stiffness. a Statistical result of changes in aPWV, N = 6. b aortic contractile function in vivo, N = 6.c Flow cytometry results of the Control group and the HF (ApoE^−/−) group, N = 6. d Modified Russell–Movat Pentachrome stain was used to show murine arterial tissue composition. Black for elastin fibers, yellow for collagen, blue/cyan for proteoglycan, red for collagen fibers, and black/purple for nuclei (representative images), scale bars correspond to 100 μm for low-magnification images and 20 μm for high-magnification views. e Immunofluorescence staining was used to assess protein expression and localization in the murine aortas, blue for nuclei, green for C/EBPβ, and red for CD68 (representative images), scale bars correspond to 100 μm for low-magnification images and 20 μm for high-magnification views. f From left to right, respectively, are statistical results of vascular wall/lumen measured ex vivo, N = 6, statistical results of elastic fibers, N = 6; Statistical results of proteoglycan, N = 6, Statistical results of CD68 fluorescence area, N = 6. ACTA2 + = ACTA2-positive cells, CD68 + = CD68-positive cells, CD68 + /CEBPb + = CD68/C/EBPβ double-positive cells. Control = ApoE^−/− mice fed with chow food, HF (ApoE^−/−) = ApoE^−/− mice fed with a 60% high-fat diet, HF (C/EBPβ^CKO/ApoE^−/−) = ApoE^−/−/C/EBPβ^fl/fl--Tagln^cre mice fed with a 60% high-fat diet. *p < 0.05 and ***p < 0.001. Segment of aortic arch was obtained for staining

Fig. 6

Downstream secretory factor regulated by C/EBPβ—PDGF-CC levels increased. a Screening for exocytosis factor genes that could be bound by C/EBPβ in VSMCs after MBD-Chol stimulation, horizontal coordinates represent fold change (log₂), vertical coordinates represent P value (log ₁₀). b Levels of PDGF-CC in murine VSMCs supernatants, N = 3. c Levels of TNFSF11 in murine VSMCs supernatants, N = 3. d Volcano plot showed the difference in genes expression levels in human VSMCs after 10 μg/ml MBD-Chol treatment for 0 h and 72 h, blue for genes down-regulated expression, red for genes up-regulated expression, horizontal coordinates represent fold change (log₂), vertical coordinates represent P value (log₁₀), the threshold for P-value is 0.05, the threshold for fold change is 0.8. e C/EBPβ gene expression levels in VSMCs of human species. f PDGF-C gene expression levels in VSMCs of human species. g Protein expression levels detected by WB in murine VSMCs (representative images). h Statistical results of protein expression levels, N = 3. *p < 0.05 and ***p < 0.001

Fig. 7

Correlation between PDGF-CC and baPWV in population. a Comparison of plasma PDGF-CC levels between participants with baPWV ≥1400 cm/s and baPWV < 1400 cm/s. b Relationship between plasma PDGF-CC levels and baPWV in the whole cohort. c Receiver operative characteristic curves for distinguishing baPWV ≥1400 cm/s by plasma PDGF-CC levels