Nathalie F. Worth

A role for rho in smooth muscle phenotypic regulation.

Abstract: The role of the small GTP‐binding protein Rho in the process of smooth muscle cell (SMC) phenotypic modulation was investigated using cultured rabbit aortic SMCs. Both Rho transcription and Rho protein expression were high for the first 3 days of culture (“contractile” state cells), with expression decreasing after change to the “synthetic” state and peaking upon return to the contractile phenotype. Activation of Rho (indicated by translocation to the membrane) also peaked upon return to the contractile state and was low in synthetic state SMCs. Transient transfection of synthetic state rabbit SMCs with constitutively active Rho (val14rho) caused a dramatic decrease in cell size and reorganization of cytoskeletal proteins to resemble those of the contractile phenotype; α‐actin and myosin adopted a tightly packed, highly organized arrangement, whereas vimentin localized to the immediate perinuclear region and focal adhesions were enlarged. Conversely, specific inhibition of endogenous Rho, by expression of C3 transferase, resulted in the complete loss of actin and myosin filaments without affecting the distribution of vimentin. Focal adhesions were reduced in number. Thus, Rho plays a key role in regulating SMC phenotypic expression.

Leukaemia inhibitory factor retards the progression of atherosclerosis

OBJECTIVE Our previous studies showed that the pleiotropic cytokine leukaemia inhibitory factor (LIF) inhibits the de novo formation of experimental atherosclerotic lesions. The present study examined whether LIF also inhibits progression of pre-existing atheroma. METHODS Balloon angioplasty was performed on the right carotid arteries of 18 rabbits immediately before placing animals on a cholesterol-enriched diet. After 4 weeks, at which time the intima:media ratio (I:M) was 0.99+/-0.12 (n=6), osmotic minipumps containing LIF (n=6) or saline control (n=6) were inserted into the peritoneal cavity of each of the remaining rabbits for a further 4 weeks. Arteries were then harvested for analysis. RESULTS Continuous administration of LIF for the final 4 weeks of an 8-week cholesterol-enriched diet completely inhibited lesion progression in injured carotid arteries (I:M 1.05+/-0.16) compared with the saline-treated group at 8 weeks (1.62+/-0.13; P<0.05). Similarly in contralateral uninjured carotid arteries, LIF treatment prevented an increase in I:M from a baseline of 0.11+/-0.01 at 4 weeks to 0.15+/-0.02 at 8 weeks compared with 0.40+/-0.04 for the saline-treated group at 8 weeks (P<0.05). LIF reduced the number of macrophages in the neointima of uninjured arteries, but had no effect on the cellular composition of injured arteries. LIF treatment normalised smooth muscle-dependent vasoreactivity to phenylephrine and sodium nitroprusside in both injured and uninjured arteries. Expression and activity of inducible nitric oxide synthase (iNOS) were up-regulated in response to hypercholesterolemia with levels further increased following endothelial denudation. With LIF treatment, iNOS expression was increased in uninjured arteries but marginally reduced in injured arteries. LIF receptors were expressed in both uninjured and injured arteries, with LIF treatment having no significant effect on expression levels. CONCLUSION LIF prevents progression of pre-formed atherosclerotic plaques, affecting lesion size and vascular reactivity. LIF treatment has differential effects within the artery wall, depending on the presence or absence of de-endothelialisation injury.

Heterogeneous Distribution of Isoactins in Cultured Vascular Smooth Muscle Cells Does Not Reflect Segregation of Contractile and Cytoskeletal Domains

We have previously demonstrated that α-smooth muscle (α-SM) actin is predominantly distributed in the central region and β-non-muscle (β-NM) actin in the periphery of cultured rabbit aortic smooth muscle cells (SMCs). To determine whether this reflects a special form of segregation of contractile and cytoskeletal components in SMCs, this study systematically investigated the distribution relationship of structural proteins using high-resolution confocal laser scanning fluorescent microscopy. Not only isoactins but also smooth muscle myosin heavy chain, α-actinin, vinculin, and vimentin were heterogeneously distributed in the cultured SMCs. The predominant distribution of β-NM actin in the cell periphery was associated with densely distributed vinculin plaques and disrupted or striated myosin and α-actinin aggregates, which may reflect a process of stress fiber assembly during cell spreading and focal adhesion formation. The high-level labeling of α-SM actin in the central portion of stress fibers was related to continuous myosin and punctate α-actinin distribution, which may represent the maturation of the fibrillar structures. The findings also suggest that the stress fibers, in which actin and myosin filaments organize into sarcomere-like units with α-actinin-rich dense bodies analogous to Z-lines, are the contractile structures of cultured SMCs that link to the network of vimentin-containing intermediate filaments through the dense bodies and dense plaques.

Rho and smooth muscle cell phenotype

This study determined the influence of Rho in regulating phenotype in early passage rabbit aortic smooth muscle cell (SMC) culture. Extended confluence and serum-deprivation induced SMC differentiation, indicated by increased expression of contractile markers (α-SM actin, SM-MHC and SM-2). Activation of Rho by sphingosine-1-phosphate (S-1-P) further enhanced the expression of contractile proteins and their reorganisation such that cells adopted a more ‘contractile’ phenotype. Inhibition of Rho induced an extreme ‘synthetic’ morphology including increased vimentin expression.