Korean J Physiol Pharmacol.  2022 Nov;26(6):519-530. 10.4196/kjpp.2022.26.6.519.

Lactate promotes vascular smooth muscle cell switch to a synthetic phenotype by inhibiting miR-23b expression

• Hu Y
• Zhang C
• Fan Y
• Zhang Y
• Wang Y
• Wang C

• KMID: 2534527
• DOI: http://doi.org/10.4196/kjpp.2022.26.6.519

Abstract

Recent research indicates that lactate promotes the switching of vascular smooth muscle cells (VSMCs) to a synthetic phenotype, which has been implicated in various vascular diseases. This study aimed to investigate the effects of lactate on the VSMC phenotype switch and the underlying mechanism. The CCK-8 method was used to assess cell viability. The microRNAs and mRNAs levels were evaluated using quantitative PCR. Targets of microRNA were predicted using online tools and confirmed using a luciferase reporter assay. We found that lactate promoted the switch of VSMCs to a synthetic phenotype, as evidenced by an increase in VSMC proliferation, mitochondrial activity, migration, and synthesis but a decrease in VSMC apoptosis. Lactate inhibited miR-23b expression in VSMCs, and miR-23b inhibited VSMC's switch to the synthetic phenotype. Lactate modulated the VSMC phenotype through downregulation of miR-23b expression, suggesting that overexpression of miR-23b using a miR-23b mimic attenuated the effects of lactate on VSMC phenotype modulation. Moreover, we discovered that SMAD family member 3 (SMAD3) was the target of miR-23b in regulating VSMC phenotype. Further findings suggested that lactate promotes VSMC switch to synthetic phenotype by targeting SMAD3 and downregulating miR-23b. These findings suggest that correcting the dysregulation of miR-23b/ SMAD3 or lactate metabolism is a potential treatment for vascular diseases.

Keyword

Lactate; miR-23b; Phenotype switch; SMAD3; Smooth muscle

Figure

• 

  Fig. 1 Lactate-induced synthetic phenotype in vascular smooth muscle cells (VSMCs). (A) Lactate treatment caused VSMCs to lose their spindle shape and acquire an irregular morphology. Scale bar = 200 μm. (B) CCK-8 assay reveals the cell viability of VSMCs treated with the indicated lactate. (C) VSMC apoptosis was determined using the Tunel assay after treatment with 4 mM lactate (magnification 200×). After treatment with 4 mM lactate, the mitochondrial membrane potential of VSMCs was measured by flow cytometry (E) and quantified (D). (F) The protein levels of apoptosis-associated markers in VSMCs treated with 4 mM lactate were measured. (G) A representative image of the transwell assay was used to determine the migration of VSMCs following treatment with 4 mM lactate (magnification 400×). (H) Using Western blotting, the protein levels of contractile phenotype and synthetic phenotype markers were determined in lactate-treated VSMCs. Values in this figure are presented with mean ± SD. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001.

• 

  Fig. 2 Lactate decreased miR-23b expression in vascular smooth muscle cells (VSMCs). (A) Lactate treatment (4 mM) altered the expression of microRNAs in VSMCs. (B) qRT-PCR was used to determine the expression of the top three downregulated miRNAs: miR-222, miR-23b, and miR-133a in VSMCs transfected with miRNA inhibitors. (C–E) qRT-PCR analysis of the expression of VSMC phenotypic markers in VSMCs transfected with miR-222 (C), miR-23b (D), or miR-133a (E) inhibitors. (F) VSMC proliferation was measured with CCK-8 after transfection with miR-23b inhibitors. (G) After transfection with miR-23b inhibitors, the migration of VSMC was measured using the transwell assay (magnification 400×). Values in this figure are presented with mean ± SD. n = 3. NC, negative control. *p < 0.05, **p < 0.01, and ***p < 0.001.

• 

  Fig. 3 miR-23b mimic attenuated the effects of lactate on vascular smooth muscle cell (VSMC) phenotype switch. (A) qRT-PCR analysis of the expression of miR-23b in VSMCs after transfection with a miR-23b mimic. (B–F) VSMCs cells were treated with 4 mM lactate with or without overexpression of a miR-23b mimic. (B) VSMC cell viability was determined using CCK-8. (C) The Tunel assay was used to determine the apoptosis of VSMSCs (magnification 200×). (D) Using JC-1 staining, the mitochondrial membrane potential of VSMCs was determined. (E) The migration of VSMCs was determined using a transwell assay, and the quantitative results were presented (magnification 400×). (F) The effects of lactate on the protein levels of α-SMA, SM22, SM-MHC, vimentin, and collagen 1 in VSMCs were attenuated using a Western blot assay. Values in this figure are presented with mean ± SD. n = 3. NC, negative control. *p < 0.05, **p < 0.01, and ***p < 0.001.

• 

  Fig. 4 miR-23b targeting of SMAD3. (A) Target genes of miR-23b have been predicted using bioinformatics. (B) The mRNA expression of WEE1, CNOT2, HMGA2, and SMAD3 in vascular smooth muscle cells (VSMCs) after miR-23b mimic transfection. (C, D) The protein expression of WEE1, CNOT2, HMGA2, and SMAD3 in VSMCs after miR-23b mimic transfection. The representative image was displayed (C), followed by statistical analysis in triplicate (D). (E) Complementary sequences of Wt/Mut-SMAD3 3UTR and miR-23b. (F) Dual-luciferase reporter assay demonstrating that SMAD3 is an miR-23b target. (G) In VSMCs, the level of SMAD3 protein was decreased by the miR-23b mimic and increased by lactate. Values in this figure are presented with mean ± SD. n = 3. NC, negative control. *p < 0.05, **p < 0.01, and ***p < 0.001.

• 

  Fig. 5 Lactate modulates vascular smooth muscle cell (VSMC) phenotype switch via regulating miR-23b/SMAD3 axis. Following transfection with SMAD3 OE or/and miR-23b mimic, VMSCs were treated with lactate. (A) Expression of miR-23b and SMAD3 in VSMCs as determined by qRT-PCR and Western blotting, respectively. (B) CCK-8 assay determines the B cell viability of VSMCs. (C) The VSMC apoptosis as determined by the Tunel assay (magnification 200×). (D) By JC-1 staining, the mitochondrial membrane potential of VSMCs is determined. (E) A transwell assay was used to determine VSMC migration (magnification 400×). (F) The Western blot-detected expression levels of phenotype switch-associated makers. Values in this figure are presented with mean ± SD. n = 3. *p < 0.05, **p < 0.01, and ***p<0.001.

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