Zhongbiao Wang

NF-κB is required for TNF-α-directed smooth muscle cell migration

Migration of vascular smooth muscle cells (VSMC) is a crucial event in the formation of vascular stenotic lesions. Tumor necrosis factor‐α (TNF‐α) is elaborated by VSMC in atherosclerosis and following angioplasty. We investigated the role of nuclear factor‐κB (NF‐κB) in human VSMC migration induced by TNF‐α. Adenoviral expression of a mutant form of the inhibitor of NF‐κB, IκB‐αM, suppressed TNF‐α‐triggered degradation of cellular IκB‐α, inhibited activation of NF‐κB, and attenuated TNF‐α‐induced migration. Further, IκB‐αM suppressed TNF‐α‐stimulated release of interleukin‐6 and ‐8 (IL‐6 and IL‐8). Neutralization of IL‐6 and IL‐8 with appropriate antibodies reduced TNF‐α‐induced VSMC migration. Addition of recombinant IL‐6 and IL‐8 stimulated migration. Collectively, our data provide initial evidence that TNF‐α‐mediated VSMC migration requires NF‐κB activation and is associated with induction of IL‐6 and IL‐8 which act in an autocrine manner.

Smooth muscle cell migration stimulated by interleukin 6 is associated with cytoskeletal reorganization

BACKGROUND Interleukin 6 (IL-6) is elevated in the arterial wall in atherosclerosis and restenosis after angioplasty. An important contributor to these pathologies is migration of vascular smooth muscle cells (VSMC), which is often associated with cytoskeletal reorganization initiated by growth factors and chemokines. We recently reported that IL-6 stimulated migration of VSMC. Here, we examined the cytoskeleton of VSMC and cytoskeletal associated proteins to determine potential mechanisms associated with IL-6 induced migration. MATERIALS AND METHODS Studies were performed in VSMC cultured from rat aortas. RESULTS IL-6 significantly stimulated VSMC migration. IL-6 induced actin polymerization, and tyrosine phosphorylation of focal adhesion-associated cytoskeletal proteins including focal adhesion kinase (FAK) and paxillin. Cytochalasin D, an inhibitor of actin polymerization, blocked phosphorylation of FAK and paxillin as well as cell motility induced by the cytokine. CONCLUSIONS Collectively, these data demonstrate for the first time that IL-6 stimulates VSMC motility which correlated with induction of actin cytoskeletal reorganization and tyrosine phosphorylation of FAK and paxillin.

Interleukin-lβ Induces Migration of Rat Arterial Smooth Muscle Cells Through a Mechanism Involving Increased Matrix Metalloproteinase-2 Activity

BACKGROUND Interleukin-lβ (IL-lβ) is associated with vascular smooth muscle cell (VSMC) migration during neointimal formation following arterial injury, of which matrix metalloproteinase-2 (MMP-2) may have an important role. We investigated whether IL-lβ stimulated migration and MMP-2 production in VSMC, and, if so, whether migration correlated with MMP-2 activity. MATERIALS AND METHODS Modified Boyden chamber assay quantified cultured rat aorta VSMC migration. Methyl-thiazolyl-tetrazolium assay assessed cell growth. Gelatin zymography and Western blotting determined MMP-2 activity and protein levels, respectively. RESULTS IL-lβ (0.1 - 10 ng/mL) induced migration of VSMC in a concentration-dependent manner without cell proliferation. VSMC released increasing levels of active MMP-2 in a dose-response fashion at IL-1β 1-10 ng/mL (P < 0.05) while significantly increased levels of latent MMP-2 (pro-MMP-2) were attained more gradually (10 ng/mL, P < 0.05). There was a dose-dependent increase in the ratio of active MMP-2 to pro-MMP-2 in response to IL-1β (1-10 ng/mL, P < 0.05), suggesting extracellular activation of pro-MMP-2. Protein levels on Western blot paralleled enzyme activity, with the synthesis of more active MMP-2 than pro-MMP-2 in response to IL-1β. IL-lβ-stimulated VSMC migration was significantly attenuated by both the pan-selective MMP inhibitor GM6001 and cis-9-octadecenoyl-N-hydroxylamide, a MMP-2-selective inhibitor. CONCLUSIONS IL-lβ increases MMP-2 activity in VSMC through increased protein synthesis and activation of pro-MMP-2. VSMC migration induced by IL-lβ requires active MMP-2. IL-lβ may play a role in arterial remodeling following injury.

Angiotensin II induces proliferation of human cerebral artery smooth muscle cells through a basic fibroblast growth factor (bFGF) dependent mechanism.

Remodeling of cerebral arteries in hypertension produces thickened vessel walls associated with atherosclerotic plaque formation. In both thickening and plaque formation, proliferation of vascular smooth muscle cells is a hallmark. Genetically hypertensive rats treated with an angiotensin II (Ang II) AT1 receptor antagonist inhibited thickening of cerebral arteries suggesting a mitogenic action of Ang II on cerebral arterial VSMC (CVSMC). However, in studies using smooth muscle cells cultured from peripheral arteries, Ang II causes cell hypertrophy, but not proliferation. We determined the effect of Ang II on proliferation of cultured human CVSMC. CVSMC were cultured from the basilar artery obtained at autopsy. Ang II (10(-7) M) stimulated proliferation determined by counting cells and mitochondrial activity assay. Synthesis and release of basic fibroblast growth factor (bFGF) was essential for Ang II-stimulated proliferation. These findings are consistent with the notion that Ang II stimulates CVSMC proliferation thereby contributing to vessel remodeling.

Stimulation of β-adrenergic receptors inhibits the release of tumor necrosis factor-α from the isolated rat heart

OBJECTIVES: Beta-adrenergic receptor agonists such as isoproterenol inhibit production of tumor necrosis factor (TNF)-alpha in a number of cell types. Because the heart is a source of TNF-alpha, we hypothesized that isoproterenol would inhibit cardiac production of the cytokine. DESIGN: Analysis of cardiac release of TNF-alpha. SETTING: Medical research laboratory. SUBJECTS: Rats. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: With the approval of the Institutional Animal Care and Use Committee, rats were anesthetized and hearts were removed and perfused. After 30 mins, bacterial lipopolysaccharide (LPS) with or without isoproterenol was infused for 60 mins. At 30, 60, 90, 120, and 150 mins, coronary flow was measured and coronary effluent was analyzed for TNF-alpha. Cardiac production of TNF-alpha was expressed as pg/min. Cyclic adenosine monophosphate (AMP) in the coronary effluent was measured. TNF-alpha messenger RNA was determined in ventricular tissue. After 30 mins, TNF-alpha was undetectable in the coronary effluent However, 60 mins after the initiation of LPS infusion, TNF-alpha release was 875+/-255 pg/min and increased to 2164+/-721 pg/min at 150 mins. Simultaneous infusion of isoproterenol with LPS stimulated cyclic AMP release and inhibited TNF-alpha production. For instance, at 60 and 150 mins, TNF-alpha release was 75+/-38 and 58+/-29 pg/min, respectively (p < .05 vs. LPS alone). Simultaneous infusion of isoproterenol with LPS blocked the induction of TNF-alpha messenger RNA by LPS. Isoproterenol, begun 30 mins after the initiation of LPS infusion, still suppressed LPS-stimulated TNF-alpha release by 95% at 150 mins. Similar results were obtained with norepinephrine. CONCLUSIONS: Activation of beta-adrenergic receptors inhibits cardiac TNF-alpha release. This implies that cytokine production by the heart is inhibited by the sympathetic nervous system. In heart failure, the cardiac response to the sympathetic nervous system is impaired. This impairment may play a role in the high plasma levels of TNF-alpha found in heart failure.

Isoproterenol Inhibits Transcription of Cardiac Cytokine Genes Induced by Reactive Oxygen Intermediates

Background Cytokines such as tumor necrosis factor &agr; (TNF-&agr;) are produced by the myocardium in heart disease and might be stimulated by reactive oxygen. In some cell types, cyclic adenosine monophosphate (AMP) inhibits TNF-&agr; production. The authors tested the hypothesis that stimulation of cardiac &bgr;-adrenergic receptors would inhibit cytokine gene transcription induced by reactive oxygen. Methods Rat hearts were perfused with buffer containing hypoxanthine. Reactive oxygen intermediates were generated by infusion of xanthine oxidase. Myocardial mRNA encoding 11 cytokines was determined. TNF-&agr;, interleukin-6, and cyclic AMP were measured in the coronary effluent. Results In control hearts, of the screened RNA, only mRNA encoding interleukin-1&bgr;, -4, and -6 was detected. Stimulation with hypoxanthine–xanthine oxidase (HX–XO) induced detectable mRNA for TNF-&agr; and interleukin-5 and increased mRNA band density for interleukin-1&bgr;, -4, and -6. Simultaneous infusion of isoproterenol inhibited HX–XO-stimulated cytokine gene expression and caused release of cyclic AMP into the coronary effluent. In control hearts, TNF-&agr; was not detected in the coronary effluent. After HX–XO administration, TNF-&agr; was reliably detected at 60 min and interleukin-6 at 90 min. Simultaneous infusion of isoproterenol inhibited TNF-&agr; and interleukin-6 release. Inclusion of propranolol in the perfusion buffer blocked the isoproterenol-induced inhibition of HX–XO-stimulated TNF-&agr; release and release of cyclic AMP into the coronary effluent. In addition, elevating myocardial cyclic AMP with forskolin also blocked release of TNF-&agr; stimulated by HX–XO. Finally, delaying infusion of isoproterenol until 30 min after HX–XO administration still suppressed release of TNF-&agr;. Conclusions Reactive oxygen species activate cytokine gene transcription in the myocardium. The sympathetic nervous system, acting through &bgr;-receptors to elevate myocardial cyclic AMP, regulates cardiac cytokine production by inhibition of transcription.

Phenotypic diversity of smooth muscle cells isolated from human intracranial basilar artery.

The present work examined heterogeneity of vascular smooth muscle cells cultured from human cerebral arteries that has not been previously reported. Primary smooth muscle cell cultures were isolated from human intracranial basilar arteries. Using a ring isolation method, multiple clones were generated from the cell cultures. These clones had two distinctly different morphologies: (1) fusiform; and (2) stellate. At confluence the fusiform-shaped clones grew in compact clusters with overlapping cells while the stellate-shaped clones were contact-inhibited growing in a monolayered pattern. The smooth muscle differentiation markers, alpha-smooth muscle-actin, calponin and smooth muscle-myosin heavy chains were expressed in all these clones. In response to serum stimulation, the stellate-shaped clones had a higher growth rate than the fusiform clones. This study reports that smooth muscle cells derived from human basilar arteries are heterogeneous.

Distribution and expression of Kirre, an IgSF molecule, during postnatal development of rat cerebellum

Immunoglobulin superfamily (IgSF) molecules are actively involved in cell-cell adhesion, neuronal migration, axonal guidance and synapse formation in the nervous system. Kirre, as a member of this family, has been implicated in mammalian neuronal differentiation and development. Although the distribution of rKirre (a rat homologue of Drosophila Kirre) mRNA was previously analyzed in adult rat cerebellum by in situ hybridization, the expression levels of transcript and protein were not well studied. Here, we showed that the expressions of rKirre mRNA and protein significantly increased during postnatal development of rat cerebellum. rKirre mRNA was mainly expressed in the granular layers and Purkinje cell layer in the developing cerebellum, revealing a possible involvement of rKirre in granule cell migration and Purkinje cell development. An essential relationship between rKirre and Purkinje cells was implied by the co-localization of rKirre and NF-200 on the cell bodies of Purkinje cells. These results suggest that rKirre may play a potential role in postnatal developing rat cerebellum.

A method to isolate morphologically distinct clones of smooth muscle cells from human saphenous vein

The monoclonal theory of atherosclerosis postulates that a certain subpopulation of vascular smooth muscle cells (VSMC) is selectively expanded in response to pathological stimuli thereby contributing to the formation of atherosclerotic plaques. VSMC cloning experiments will be important in characterizing the phenotypic composition of VSMC in atherosclerotic plaques. However, the difficulty in cloning human VSMC is well recognized. Here a technique is described that produced multiple clones from human saphenous vein. The clones could be divided into two categories based on their distinctly different morphology: (1) spindle-shaped; and, (2) epithelioid-shaped. Each clone expressed smooth muscle-a-actin and calponin, two smooth muscle-specific differentiation markers. The clonal study presented here reports for the first time that phenotypically heterogeneous smooth muscle cells coexist within human saphenous veins.