Disruption of actin cytoskeleton mediates loss of tensile stress induced early phenotypic modulation of vascular smooth muscle cells in organ culture

Abstract

Aorta organ culture has been widely used as an ex vivo model for studying vessel pathophysiology. Recent studies show that the vascular smooth muscle cells (VSMCs) in organ culture undergo drastic dedifferentiation within the first few hours (termed early phenotypic modulation). Loss of tensile stress to which aorta is subject in vivo is the cause of this early phenotypic modulation. However, no underlying molecular mechanism has been discovered thus far. The purpose of the present study is to identify intracellular signals involved in the early phenotypic modulation of VSMC in organ culture. We find that the drastic VSMC dedifferentiation is accompanied by accelerated actin cytoskeleton dynamics and downregulation of SRF and myocardin. Among the variety of signal pathways examined, increasing actin polymerization by jasplakinolide is the only one hindering VSMC dedifferentiation in organ culture. Moreover, jasplakinolide reverses actin dynamics during organ culture. Latrunculin B (disrupting actin cytoskeleton) and jasplakinolide respectively suppressed and enhanced the expression of VSMC markers, SRF, myocardin, and CArG-box-mediated SMC promoters in PAC1, a VSMC line. These results identify actin cytoskeleton degradation as a major intracellular signal for loss of tensile stress-induced early phenotypic modulation of VSMC in organ culture. This study suggests that disrupting actin cytoskeleton integrity may contribute to the pathogenesis of vascular diseases.

Fig. 1

The expression of VSMC-specific genes and key transcriptional regulators SRF and myocardin in organ culture (OC). The real time RT-PCR assay shows that mRNA levels of VSMC-specific gene (SMA, SM22, SM-MHC) and transcriptional regulator (SRF and myocardin) steadily decreases at 1, 2, 4 and 24 hours in aorta organ culture. “*” and “**” indicate p<0.05 and p<0.01 relative to the fresh control group.

Fig. 2

The effect of chemicals on OC-induced downregulation of SMC marker genes. qRT-PCR was used to measure the expression of SM22 from aorta segments freshly isolated and in OC for 24 hours with the indicated chemicals. The values are expressed as comparison with the freshly isolated aorta.

Fig. 3

Role of actin cytoskeleton dynamics in organ culture (OC)-induced dedifferentiation of VSMC. (A) the downregulation of VSMC-specific genes can be partially blocked by Jasplakinolide (Jasp), an actin polymerization inducer. (B) The actin cytoskeleton dynamics in organ culture was measured by the G-/F-actin ratio using western blot analysis and the intensity of each band was quantified by the Photoshop software. * and ** indicate p<0.05 and p<0.01 vs fresh group; ### indicates p<0.001 vs OC (24 h) group.

Fig. 4

Manipulation of actin cytoskeleton affects VSMC differentiation in vitro. (A) latrunculin B (LatB, 0.5 µM for 6 hours) and Jasplakinolide (Jasp, 0.3 µM for 6 hours) induce significant morphology changes of PAC1 cells. (B–C) LatB and Jasp respectively reduce and enhance the transcription of VSMC-specific markers, SRF and myocardin by the real-time RT-PCR assay (B) and VSMC promoter activities (C) in PAC1 cells treated with LatB or Jasp for 6 hours. * and ** indicate p<0.05 and p<0.01 vs control.

Fig. 5

A potential mechanism for organ culture-induced early phenotypic modulation of VSMC. During organ culture, loss of tensile stress induces actin cytoskeleton dissociation that then leads to decreased expression of SRF and Myocd and the downregulation of VSMC gene transcription.