Hyaluronic acid and proteoglycan link protein 1 suppresses platelet‑derived growth factor-BB-induced proliferation, migration, and phenotypic switching of vascular smooth muscle cells

Abstract

The development of atherosclerotic cardiovascular disease is associated with the phenotypic switching of vascular smooth muscle cells (SMCs) from a contractile to a synthetic state, leading to cell migration and proliferation. Platelet‑derived growth factor‑BB (PDGF‑BB) modulates this de-differentiation by initiating a number of biological processes. In this study, we show that gene expression of hyaluronic acid (HA) and proteoglycan link protein 1 (HAPLN1) was upregulated during differentiation of human aortic SMCs (HASMCs) into a contractile state, but downregulated upon during PDGF-BB-induced dedifferentiation. This is the first study showing that the treatment of HASMCs with full-length recombinant human HAPLN1 (rhHAPLN1) significantly reversed PDGF-BB-induced decrease in the protein levels of contractile markers (SM22α, α-SMA, calponin, and SM-MHC), and inhibited the proliferation and migration of HASMCs induced by PDGF-BB. Furthermore, our results show that rhHAPLN1 significantly inhibited the phosphorylation of FAK, AKT, STAT3, p38 MAPK and Raf mediated by the binding of PDGF-BB to PDGFRβ. Together, these results indicated that rhHAPLN1 can suppress the PDGF-BB-stimulated phenotypic switching and subsequent de-differentiation of HASMCs, highlighting its potential as a novel therapeutic target for atherosclerosis and other vascular diseases. [BMB Reports 2023; 56(8): 445-450].

Fig. 1

rhHAPLN1 inhibits PDGF-BB-induced de-differentiation of HASMCs. (A) Expression levels of smooth muscle-specific markers and endogenous expression levels of HAPLN1 in HASMCs cultured in SMC differentiation media for 6 days, with or without the addition of PDGF-BB. CON: Cells that were not incubated in SMC differentiation medium; Untreated: Cells that were not treated with PDGF-BB; PDGF-BB: Cells that were treated with 10 ng/ml PDGF-BB. (B) Expression levels of smooth muscle-specific markers in HASMCs cultured in SMC differentiation media for 6 days, with or without the addition of rhHAPLN1. Untreated: Cells that were not treated with rhHAPLN1; rhHAPLN1: Cells that were treated with 20 ng/ml rhHAPLN1. (C) Expression levels of smooth muscle-specific markers determined by western blots. GAPDH was used as a positive control. Cells were exposed to 10 ng/ml PDGF-BB for 48 h with or without the indicated concentrations of rhHAPLN1. P-values indicated by ^# were calculated relative to control or untreated cells, whereas P-values indicated by * were calculated relative to untreated or PDGF-BB treated cells. * or ^# indicates P < 0.05, ** or ^## indicates P < 0.001, *** or ^### indicates P < 0.0005, and **** or ^#### indicates P < 0.0001 (N = 3 per group).

Fig. 2

rhHAPLN1 inhibits PDGF-BB elevated HASMCs proliferation. (A) Ki-67 (green) and DAPI (blue) staining of HASMCs after treatment with 20 ng/ml rhHAPLN1 or 10 ng/ml PDGF-BB for 48 h. Representative images (left) and corresponding quantification of Ki-67-positive HASMCs (right) are shown. Scale bar: 100 μm. (B, C) Western blots examining the protein expression levels of FAK, p53, p21^Cip1, p27^Kip1, Cyclin D, CDK4, Cyclin E, and CDK2 in HASMCs after treatment with rhHAPLN1 (concentrations indicated on figure) or 10 ng/ml PDGF-BB for 48 h. GAPDH was used as a positive control. P-values indicated by ^# were calculated relative to untreated cells, whereas P-values indicated by * were calculated relative to PDGF-BB treated cells. * or ^# indicates P < 0.05, ** or ^## indicates P < 0.001, *** or ^### indicates P < 0.0005, and **** or ^#### indicates P < 0.0001 (N = 3 per group).

Fig. 3

rhHAPLN1 inhibits PDGF-BB elevated HASMCs migration, and secretion of inflammatory factors. (A) Inverted microscopy images of confluent HASMCs 8 hours after wounding. The percentage of area covered by migrated cells over time was calculated using ImageJ, and these results are presented in the panel on the right. Scale bar: 100 μm. (B) The protein expression level of MMP-9 in HASMCs after treatment with rhHAPLN1 (concentrations indicated on figure) or 10 ng/ml PDGF-BB for 48 h. (C) qRT-PCR results of mRNA expression levels of inflammatory factors, TNF-a, IL-1β, and IL-8 in HASMCs after treatment with 20 ng/ml rhHAPLN1 or 10 ng/ml PDGF-BB for 48 h. (D) The protein expression levels of p-AKT/AKT, p-NF-κB/NF-κB was determined by Western blot analysis. P-values indicated by ^# were calculated relative to untreated cells, whereas P-values indicated by * were calculated relative to PDGF-BB treated cells. * or ^# indicates P < 0.05, ** or ^## indicates P < 0.001, *** or ^### indicates P < 0.0005, and **** or ^#### indicates P < 0.0001 (N = 3 per group).

Fig. 4

rhHAPLN1 suppresses PDGFRβ, FAK and Ras/MAPK/STAT3 pathways activated by PDGF-BB in HASMCs. Western blots conducted on HASMCs after treatment with rhHAPLN1 (concentrations indicated on figure) for 48 h or 10 ng/ml PDGF-BB for 5 min. The following protein levels were measured: (A) p-PDGFRβ/PDGFRβ, (B) p-FAK/FAK, (C) p-JNK/JNK, p-p38/p38, and p-ERK/ERK, p-STAT3/STAT3, (D) Ras/p-c-Raf/c-Raf. P-values indicated by ^# were calculated relative to untreated cells, whereas P-values indicated by * were calculated relative to PDGF-BB treated cells. * or ^# indicates P < 0.05, ** or ^## indicates P < 0.001, *** or ^### indicates P < 0.0005, and **** or ^#### indicates P < 0.0001 (N = 3 per group).