Korean J Physiol Pharmacol.  2024 Sep;28(5):449-456. 10.4196/kjpp.2024.28.5.449.

Biophysically stressed vascular smooth muscle cells express MCP-1 via a PDGFR-ββ-HMGB1 signaling pathway

• Kim JW
• Kim JY
• Bae HE
• Kim CD

• KMID: 2559137
• DOI: http://doi.org/10.4196/kjpp.2024.28.5.449

Abstract

Vascular smooth muscle cells (VSMCs) under biophysical stress play an active role in the progression of vascular inflammation, but the precise mechanisms are unclear. This study examined the cellular expression of monocyte chemoattractant protein 1 (MCP-1) and its related mechanisms using cultured rat aortic VSMCs stimulated with mechanical stretch (MS, equibiaxial cyclic stretch, 60 cycles/ min). When the cells were stimulated with 10% MS, MCP-1 expression was markedly increased compared to those in the cells stimulated with low MS intensity (3% or 5%). An enzyme-linked immunosorbent assay revealed an increase in HMGB1 released into culture media from the cells stimulated with 10% MS compared to those stimulated with 3% MS. A pretreatment with glycyrrhizin, a HMGB1 inhibitor, resulted in the marked attenuation of MCP-1 expression in the cells stimulated with 10% MS, suggesting a key role of HMGB1 on MCP-1 expression. Western blot analysis revealed higher PDGFR-α and PDGFR-β expression in the cells stimulated with 10% MS than 3% MS-stimulated cells. In the cells deficient of PDGFR-β using siRNA, but not PDGFR-α, HMGB1 released into culture media was significantly attenuated in the 10% MS-stimulated cells. Similarly, MCP-1 expression induced in 10% MS-stimulated cells was also attenuated in cells deficient of PDGFR-β. Overall, the PDGFR-β signaling plays a pivotal role in the increased expression of MCP-1 in VSMCs stressed with 10% MS. Therefore, targeting PDGFR-β signaling in VSMCs might be a promising therapeutic strategy for vascular complications in the vasculatures under excessive biophysical stress.

Keyword

HMGB1; Mechanical stretch; Monocyte chemoattractant protein 1; PDGF receptor; Vascular smooth muscle cells

Figure

• 

  Fig. 1 Force- and time-dependent effects of mechanical stretch (MS) on monocyte chemoattractant protein 1 (MCP-1) expression in vascular smooth muscle cells (VSMCs). (A) Rat aortic VSMCs were stressed with MS (0, 3, 5 and 10% strain, 60 cycles/min) using Flexcell Tension Plus FX-4000T system for 12 h. MCP-1 expression in VSMCs was measured by Western blot, and the blots are representative of 5 independent experiments. β-Actin was used as an internal control. Quantitative results were expressed as the mean ± SEM of 5 independent experiments. **p < 0.01 vs. control. (B) The cultured VSMCs were stressed with 10% MS for 12 h, and MCP-1 expression in VSMCs was measured by Western blotting. β-Actin was used as an internal control. The blots are representative of 4 independent experiments. Quantitative results were expressed as the means ± SEM of 4 independent experiments. *p < 0.05; **p < 0.01 vs. control.

• 

  Fig. 2 High mobility group box 1 (HMGB1) release and its role on monocyte chemoattractant protein 1 (MCP-1) expression in vascular smooth muscle cells (VSMCs) stimulated with mechanical stretch (MS). (A) Force- and time-dependent effects of MS on HMGB1 release in VSMCs. Rat VSMCs were stimulated with 3% and 10% MS for 0–12 h, and then HMGB1 released into cell culture media was measured by ELISA. The concentration of HMGB1 released into culture media was quantified and expressed as the means ± SEM of 4 independent experiments **p < 0.01 vs. corresponding value in the 3% MS group. (B) Role of HMGB1 on MCP-1 expression in VSMCs stimulated with MS. Rat VSMCs were pretreated with an inhibitor for HMGB1 (glycyrrhizin, 0–10 nM) for 24 h, and the cells were then stimulated with 10% MS for 12 h. The expression of the MCP-1 protein was determined by Western blot. β-Actin was used as an internal control. The blots are representative of 4 independent experiments. Relative intensities to β-actin were quantified and expressed as the means ± SEM of 4 independent experiments. **p < 0.01 vs. control. ##p < 0.01 vs. vehicle.

• 

  Fig. 3 Effects of mechanical stretch (MS) on PDGFR expression in vascular smooth muscle cells (VSMCs). The cultured rat VSMCs were stimulated with MS (3 and 10%) for 6 h, and then the expression levels of PDGFR-α and PDGFR-β were determined by Western blot. β-Actin was used as an internal control. The blots are representative of 5 independent experiments. Relative intensities to β-actin were quantified and expressed as the means ± SEM of 5 independent experiments. **p < 0.01 vs. corresponding value in control.

• 

  Fig. 4 Individual roles of the PDGFR isoforms on high mobility group box 1 (HMGB1) release in mechanical stretch (MS)-stimulated vascular smooth muscle cells (VSMCs). The cultured VSMCs were transfected with siRNAs (200 nM) for PDGFR-α (A) or PDGFR-β (B) for 24 h, and then stimulated with 10% MS for 6 h. The expression levels of PDGFR-α and PDGFR-β isoforms were determined by Western blotting using β-actin as an internal control. Relative intensities were quantified and expressed as the means ± SEM of 4 independent experiments. **p < 0.01 vs. control. ##p < 0.01 vs. corresponding value in negative control. (C, D) VSMCs transfected with PDGFR-α or PDGFR- β siRNA (200 nM) for 24 h were stimulated with 10% MS for 6 h. The HMGB1 released in the culture media were quantified by ELISA. The concentration of HMGB1 were quantified and expressed as the means ± SEM of 4 independent experiments. **p < 0.01 vs. control. ##p < 0.01 vs. corresponding value in negative control.

• 

  Fig. 5 Role of PDGFR- β signaling on monocyte chemoattractant protein 1 (MCP-1) expression in vascular smooth muscle cells (VSMCs) stressed with mechanical stretch (MS). The cultured VSMCs were transfected with PDGFR-β siRNA (200 nM) for 24 h, and then stimulated by 10% MS for 6 h. The expression levels of PDGFR-β and MCP-1 were determined by Western blotting using β-actin as an internal control. The blots are representative of 4 independent experiments (A). Relative intensities of PDGFR-β (B) and MCP-1 (C) to β-actin were quantified and expressed as the means ± SEM of 4 independent experiments. **p < 0.01 vs. control. ##p < 0.01 vs. corresponding value in negative control. (D) MCP-1 expression in 10% MS-stimulated cells was compared with that in control cells, and the relative increase rate was quantified. Data were expressed as the means ± SEM of 4 independent experiments. **p < 0.01 vs. negative control.

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