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. 2023 Sep;24(5):e69.
doi: 10.4142/jvs.23148.

Kalkitoxin attenuates calcification of vascular smooth muscle cells via RUNX-2 signaling pathways

Saroj K Shrestha  1 , Se-Woong Kim  1 , Yunjo Soh  2
Affiliations

Kalkitoxin attenuates calcification of vascular smooth muscle cells via RUNX-2 signaling pathways

Saroj K Shrestha et al. J Vet Sci. 2023 Sep.

Abstract

Background: Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease.

Objectives: Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect.

Methods: VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit.

Results: Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca++-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runt-related transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca2+-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta.

Conclusions: This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.

Keywords: BMP-2; Kalkitoxin; RUNX-2; vascular calcification; vascular smooth muscle cell.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Fig. 1
Fig. 1. KT inhibits calcification in VSMC. For 7 days, VSMCs were grown in DMEM/F12 or Ca++ (3.6 mM) with or without 10 nM and 20 nM KT. (A) Alizarin red and von Kossa, staining, and (B) calcium levels in mice were noted. The results are presented as mean ± SD (n = 3). (C) For 48 h, VSMCs were cultivated in DMEM/F12 or Ca++ (3.6 mM) with or without KT (5, 10, 20, 40, and 80 nM). The MTT test was used to determine cell viability. The results are the mean ± SD (n = 3). As a control, Ca++ (3.6 mM) was used.
VSMC, vascular smooth muscle cell; DMEM/F12, Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12; KT, kalkitoxin; SD, standard deviation; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. *p < 0.05 shows that the Ca++ (3.6 mM)-treated group differed significantly.
Fig. 2
Fig. 2. KT inhibits osteoblastic genes during vascular calcification. For 7 days, VSMCs were grown in DMEM/F12 or Ca++ (3.6 mM) with or without 10 nM and 20 nM KT. (A) The gene expression of osteoblastic-related genes was examined using RT-PCR. The quantitative band solidity of the genes (B) RUNX-2, (C) SMAD4, (D) OPN, (E) collagen 1α, (F) OCN, and (G) OSX expression were measured and compared to Ca++ (3.6 mM). The results are provided as mean ± SD (n = 3). B-actin was used as a standard control.
KT, kalkitoxin; VSMC, vascular smooth muscle cell; DMEM/F12, Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12; RT-PCR, real-time polymerase chain reaction; RUNX-2, runt-related transcription factor 2; SMAD4, SMAD family member 4; OPN, osteopontin; OCN, osteocalcin; OSX, osterix; SD, standard deviation. *p < 0.05, **p < 0.001 denotes statistically significant changes from the Ca++ (3.6 mM)-treated group.
Fig. 3
Fig. 3. KT attenuates RUNX-2 pathways. For 7 days, VSMCs were grown in DMEM/F12 or Ca++ (3.6 mM) with or without 10 nM and 20 nM KT. (A) The expression of osteoblastic-related proteins was determined using a western blot. The quantitative band density of proteins (B) SMA, (C) OPG, (D) collagen 1α, (E) BMP-2, and (F) RUNX-2 expression were measured and compared in Ca++-treated and -untreated cells. The results are provided as mean ± SD (n = 3). β-actin was used as a standard control.
KT, kalkitoxin; RUNX-2, runt-related transcription factor 2; VSMC, vascular smooth muscle cell; DMEM/F12, Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12; SMA, smooth muscle actin; OPG, osteoprotegerin; BMP-2, bone morphogenetic protein 2; SD, standard deviation. *p < 0.05, **p < 0.001 denotes statistically significant change.
Fig. 4
Fig. 4. KT suppresses vascular calcification under ex vivo conditions. Three mice aortic ring sections were extracted and incubated for 7 days in DMEM/F12 or Ca++ (3.6 mM) with or without 10 nM and 20 nM KT. (A) The calcified area was identified using Alizarin red staining and von Kossa staining. (B) The amount of calcium deposition found in the aortic ring. The results are provided as means ± SDs (n = 5).
KT, kalkitoxin; DMEM/F12, Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12; SD, standard deviation; H&E, hematoxylin and eosin. *p < 0.05 shows that the Ca++ (3.6 mM)-induced group differed significantly from the control group.
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