Aims

Expression of SM22 (also known as SM22alpha and transgelin), a vascular smooth muscle cells (VSMCs) marker, is down-regulated in arterial diseases involving medial osteochondrogenesis. We investigated the effect of SM22 deficiency in a mouse artery injury model to determine the role of SM22 in arterial chondrogenesis.

Methods and results

Sm22 knockout (Sm22−/−) mice developed prominent medial chondrogenesis 2 weeks after carotid denudation as evidenced by the enhanced expression of chondrogenic markers including type II collagen, aggrecan, osteopontin, bone morphogenetic protein 2, and SRY-box containing gene 9 (SOX9). This was concomitant with suppression of VSMC key transcription factor myocardin and of VSMC markers such as SM α-actin and myosin heavy chain. The conversion tendency from myogenesis to chondrogenesis was also observed in primary Sm22−/− VSMCs and in a VSMC line after Sm22 knockdown: SM22 deficiency altered VSMC morphology with compromised stress fibre formation and increased actin dynamics. Meanwhile, the expression level of Sox9 mRNA was up-regulated while the mRNA levels of myocardin and VSMC markers were down-regulated, indicating a pro-chondrogenic transcriptional switch in SM22-deficient VSMCs. Furthermore, the increased expression of SOX9 was mediated by enhanced reactive oxygen species production and nuclear factor-κB pathway activation.

Conclusion

These findings suggest that disruption of SM22 alters the actin cytoskeleton and promotes chondrogenic conversion of VSMCs.

Resveratrol (RSVL), a polyphenolic antioxidant present in red wine, has been shown to provide cardiovascular protection by improving endothelial function and reducing myocardial ischemia. However, little is known about how RSVL affects vascular smooth muscle cells (VSMC) differentiation. RSVL blocks VSMC proliferation in vitro and neointimal formation following artery injury in vivo. Thus, one might expect that RSVL will promote VSMC differentiation. Unexpectedly, our results in this study show that RSVL induces VSMCs phenotypic modulation; this is characterized by suppressed transcription of SMC-specific marker genes Tagln, Acta2, Myh11, and Smtn in a dose-dependent and time-dependent manner in cultured VSMCs. Consistent with previous studies, RSVL induces the nuclear translocation of p53 and the expression of p53-responsive genes such as Cdkn1a, Gadd45a, Gadd45, and Fas. In an effort to identify the molecular mechanisms whereby RSVL represses VSMC differentiation, we found that RSVL inhibits the transcription of Myocd and Srf, the key VSMC transcriptional regulators. However, knockingdown and overexpressing p53 did not affect RSVL-induced VSMCs phenotypic modulation: this suggests that RSVL may induce VSMC dedifferentiation via p53-independent mechanisms. This study provides the first evidence showing that RSVL induces VSMC dedifferentiation by regulating Myocd and SRF-mediated VSMC gene transcription.

Background

The infrarenal abdominal aorta exhibits increased disease susceptibility relative to other aortic regions. Allograft studies exchanging thoracic and abdominal segments showed that regional susceptibility is maintained regardless of location, suggesting substantial roles for embryological origin, tissue composition and site-specific gene expression.

Results

We analyzed gene expression with microarrays in baboon aortas, and found that members of the HOX gene family exhibited spatial expression differences. HOXA4 was chosen for further study, since it had decreased expression in the abdominal compared to the thoracic aorta. Western blot analysis from 24 human aortas demonstrated significantly higher HOXA4 protein levels in thoracic compared to abdominal tissues (P < 0.001). Immunohistochemical staining for HOXA4 showed nuclear and perinuclear staining in endothelial and smooth muscle cells in aorta. The HOXA4 transcript levels were significantly decreased in human abdominal aortic aneurysms (AAAs) compared to age-matched non-aneurysmal controls (P < 0.00004). Cultured human aortic endothelial and smooth muscle cells stimulated with INF-γ (an important inflammatory cytokine in AAA pathogenesis) showed decreased levels of HOXA4 protein (P < 0.0007).

Conclusions

Our results demonstrated spatial variation in expression of HOXA4 in human aortas that persisted into adulthood and that downregulation of HOXA4 expression was associated with AAAs, an important aortic disease of the ageing population.

Rationale

SM22 (or transgelin), an actin-binding protein abundant in vascular smooth muscle cells (VSMC), is downregulated in atherosclerosis, aneurysm and various cancers. Abolishing SM22 in apolipoprotein E knockout mice accelerates atherogenesis. However, it is unclear whether SM22 disruption independently promotes arterial inflammation.

Objective

To investigate whether SM22 disruption directly promotes inflammation upon arterial injury and to characterize the underlying mechanisms.

Methods and Results

Using carotid denudation as an artery injury model, we showed that Sm22 knockout (Sm22−/−) mice developed enhanced inflammatory responses with higher induction of pro-inflammatory genes, including Vcam1, Icam1, Cx3cl1, Ccl2 and Ptgs2. Higher expression of these genes was confirmed in primary Sm22−/− VSMCs and in PAC1 cells after Sm22 knockdown, while SM22 recapitulation in primary Sm22−/− VSMCs decreased their expression. NF-κB pathways were prominently activated in both injured carotids of Sm22−/− mice and in PAC1 cells after Sm22 knockdown and may mediate upregulation of these pro-inflammatory genes. As a NF-κB activator, reactive oxygen species (ROS) increased in primary Sm22−/− VSMCs and in PAC1 cells after Sm22 knockdown. ROS scavengers blocked NF-κB activation and induction of pro-inflammatory genes. Furthermore, Sm22 knockdown increased Sod2 expression and activated p47phox, reflecting contributions of mitochondria and NADPH oxidase to the augmented ROS production; this may result from actin and microtubule cytoskeletal remodeling.

Conclusions

Our findings show that SM22 downregulation in VSMCs can independently promote arterial inflammation through activation of ROS-mediated NF-κB pathways. This study provides initial evidence linking VSMC cytoskeleton remodeling with arterial inflammation.

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 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.