J Vet Sci.  2023 Sep;24(5):e69. 10.4142/jvs.23148.

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

• Shrestha SK
• Kim SW
• Soh Y

• KMID: 2546797
• DOI: http://doi.org/10.4142/jvs.23148

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 runtrelated 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.

Keyword

Kalkitoxin; RUNX-2; BMP-2; vascular calcification; vascular smooth muscle cell