This study demonstrated that RSVL downregulates the transcription of SMC key regulators Myocd and Srf, and hence decreases SMC maker gene expression. VSMCs phenotypic switch from contractile to synthetic phenotype play important roles in the pathogenesis of vascular diseases such as atherosclerosis and aneurysm.
RSVL is known for its beneficial cardiovascular and cancer preventive effects. However, RSVL may also have adverse pathological effect such as renal toxicity in rat (Crowell et al., 2004
). The present study shows that RSVL induces VSMC dedifferentiation in vitro
. Since SMC dedifferentiation is usually regarded as a pathological process participating in the pathogenesis of cardiovascular diseases, such as hypertension, atherosclerosis, and aneurysm, it is possible that RSVL may have the potential of inducing VSMCs phenotypic modulation in vivo
, thus aggravating the pathogenesis of cardiovascular diseases.
In this study the cell models used to carry out the VSMC phenotypic modulation induced by RSVL were cultured PAC1 cells (a rat pulmonary artery-derived SMC line), and 10T1/2 cells (a mouse embryonic mesenchymal progenitor cell line). The PAC1 cells has been shown to retain the VSMC phenotype even after extensive passages as evident by the spindle shaped morphology, the expression of functional surface receptors for angiotensin II, norepinephrine, and alpha-thrombin as well as the expression of smooth muscle markers such as isoforms of alpha-actin, myosin heavy chain, myosin regulatory light chain, and alpha-tropomyosin (Rothman et al., 1992
). Nevertheless, it should be noted that cultured VSMC lines, VSMC progenitor cell line, as well as primary VSMCs could behave differently from VSMC and their progenitor cells in vivo. In view of this, in future studies the potential adverse effect of RSVL on the vascular system should be examined using animal model of vascular diseases.
VSMC dedifferentiation is characterized by the down regulation of VSMC contractile gene expression, which has been associated with the pathogenesis of vascular diseases such as atherosclerosis and aneurysms (Ailawadi et al., 2009
; Lenk et al., 2007
; Regan et al., 2000
; Shanahan et al., 1994
). However, the functional roles of VSMC dedifferentiation in VSMC phenotypic modulation remain unknown. Studies of SM22 knockout mice showed that SM22 deficiency promotes VSMC inflammation in response to high fat diet and carotid artery injury in vivo
(Feil et al., 2004
; Shen et al., 2010
). Therefore, downregulation of cytoskeleton proteins may play an active role in promoting VSMC phenotypic modulation in response to injury. Consistent with this notion, the down regulation of smooth muscle cell marker genes occurs early before aneurysm formation (Ailawadi et al., 2009
). More evidence suggests that VSMC dedifferentiation may be a phenotype precondition for VSMC remodeling into other subtypes in response to environmental stimuli.
The phenotypic modulation of different VSMC subtypes is controlled by complex regulatory mechanisms involving the regulatory circuits of transcription factors and the crosstalks of intracellular pathways (Yoshida and Owens, 2005
). Myocd, and SRF are the key regulators in coordinating the transcriptional regulation of VSMC marker genes. The expression of Myocd, and Srf
has been associated with VSMC phenotypic modulation in response to a variety of intracellular signal pathways and extracellular stimuli such as treatment of PDGF-BB, cyclosporine and loss of tensile stress (Deaton et al., 2009
; Garvey et al., 2010
; Liu et al., 2005
; Wang and Olson, 2004
; Yoshida and Owens, 2005
; Zheng et al., 2010
). To explore the molecular mechanisms mediating RSVL-induced VSMC phenotypic modulation, we examined the expression of Myocd
in response to RSVL. We found that RSVL suppresses the transcription of Myocd
without affecting their mRNA stabilities ().
Although the direct molecular target of RSVL has not yet been identified, RSVL affects the activation of a variety of intracellular signal pathways. The present data showed that RSVL induces p53 activation in VSMCs. Unlike the recent study showing that p53 decreased myocardin expression and hence inhibited differentiation from mesenchymal cells to SMC (Molchadsky et al., 2008
), p53 activation does not appear to participate directly in the RSVL-induced VSMC phenotypic modulation (). The effect of p53 on Myocd-mediated SMC differentiation may depend on the cell models.
To determine the molecular mechanisms underlying RSVL-induced VSMCs phenotypic modulation, we also explored the potential participation of several RSVL regulated targets such as Sirt1
and β-catenin in SMC phenotypic modulation (Gracia-Sancho et al., 2010
) (Zhou et al., 2009
). We found that knockingdown Sirt1
or β-catenin is not sufficient to block RSVL-induced VSMC dedifferentiation by the siRNA knockdown approach (data not shown). Further studies are needed to understand the molecular mechanisms underlying RSVL-induced VSMCs phenotypic modulation.
It is worth noting that microRNAs (miRNAs) have emerged as central players in governing the transcriptional regulatory programs essential for cardiovascular development and diseases (Liu and Olson, 2010
). It is possible that RSVL may regulate the expression of miRNAs to inhibit VSMC marker genes expression. It is likely that genome-wide gene profile analyses using microarray and microRNA arrays may reveal the molecular mechanisms mediating RSVL-induced VSMC phenotypic modulation.