Interleukin-31 and oncostatin-M mediate distinct signaling reactions and response patterns in lung epithelial cells.

Lung epithelial cells are primary targets of oncostatin M (OSM) and, to a lower degree, of interleukin (IL)-6 and IL-31, all members of the IL-6 cytokine family. The OSM receptor (OSMR) signals through activation of STAT and mitogen-activated protein kinase pathways to induce genes encoding differentiated cell functions, reduce cell-cell interaction, and suppress cell proliferation. IL-31 functions through the heteromeric IL-31 receptor, which shares with OSMR the OSMRbeta subunit, but does not engage gp130, the common subunit of all other IL-6 cytokine receptors. Because the response of epithelial cells to IL-31 is unknown, the action of IL-31 was characterized in the human alveolar epithelial cell line A549 in which the expression of the ligand-binding IL-31Ralpha subunit was increased. IL-31 initiated signaling that differed from other IL-6 cytokines by the particularly strong recruitment of the STAT3, ERK, JNK, and Akt pathways. IL-31 was highly effective in suppressing proliferation by altering expression of cell cycle proteins, including up-regulation of p27(Kip1) and down-regulation of cyclin B1, CDC2, CDK6, MCM4, and retinoblastoma. A single STAT3 recruitment site (Tyr-721) in the cytoplasmic domain of IL-31Ralpha exerts a dominant function in the entire receptor complex and is critical for gene induction, morphological changes, and growth inhibition. The data suggest that inflammatory and immune reactions involving activated T-cells regulate functions of epithelial cells by IL-6 cytokines through receptor-defined signaling reactions.

Endovascular treatment of aneurysms: gene expression of neointimal cells recruited on the embolic agent and evolution with recurrence in an experimental model.

PURPOSE: The authors attempted to identify genes associated with healing or recurrence after embolization in an aneurysm model in which neointima formation at the neck varies according to flow zones. A better understanding of the relationship between blood flow, molecular events, and healing or recurrence may provide future avenues to improve results of endovascular treatment of aneurysms. METHODS: Bilateral carotid venous pouch aneurysms were constructed in 36 dogs and embolized with gelatin sponges. Angiography and pathological studies were performed at T0 and/or 3 weeks (n=22). Angiographic results and neointima formation were scored using a qualitative index applied to the distal (inflow) and proximal (outflow) zones of the neck. In 14 animals, mRNA expression 1 to 14 days after embolization at the proximal or distal segment of the sponge was analyzed by RT-PCR, attempting to correlate flow zones, gene expression, and neointima formation. RESULTS: Aneurysms recurred at 3 weeks, as shown by significantly worse angiographic scores as compared to T0 (P<.01). Neointimal scores differed at pathology, with a more complete neointima at the proximal as compared to the distal aspect of the sponge at 3 weeks (P=.027). Embolization was followed by migration of CD31+, CD14+, smooth muscle alpha-actin+ (SMA+) cells that progressively expressed metalloproteinases (MMP-9,-12,-14), but stable or lesser, retarded expression of inhibitors (TIMP1-4). Growth factors (PDGF-BB, TGF-beta1, TNF-alpha, MCP-1 and Ang-1) were expressed at increasing levels, maximal at 7 to 14 days. Differences between distal and proximal zones were limited to increased expression of MMP-2 proximally (P<.035). CONCLUSION: Gene expression after embolization is compatible with patterns associated with neointima formation. The authors have not identified key factors involved in recurrence.

Recanalization of arterial thrombus, and inhibition with beta-radiation in a new murine carotid occlusion model: MRNA expression of angiopoietins, metalloproteinases, and their inhibitors.

BACKGROUND: Recanalization is an important physiologic phenomenon because it can efficiently reestablish circulation after thrombosis. We attempted to characterize molecular events related to recanalization or organization of arterial thrombus in a new murine model by studying genes reported to be involved in angiogenesis or neointima formation. METHODS: Platinum coils, radioactive phosphorus 32 coils or not, were implanted in the carotid artery in mice to cause thrombotic occlusion. The outcome of the occlusion was followed up with transmyocardial angiography and pathologic analysis at 2, 6, or 15 days. Angiographic results were compared with the Pearson chi2 test. Messenger RNA expression of von Willebrand factor (vWF); smooth muscle alpha-actin (SMA+); platelet endothelial cell adhesion molecule-1 (PECAM-1); vascular endothelium cadherin (VE-Cad); endothelial nitric oxide synthase (eNOS); vascular cell adhesion molecule-1 (VCAM-1); tumor necrosis factor alpha (TNF-alpha); matrix metalloproteinase (MMP-9, MMP-12, and MMP-14), and tissue inhibitors of MMPs (TIMPs: TIMP-1, TIMP-2, TIMP-3, TIMP-4); angiopoietins (Ang-1, Ang-2); and receptors Tie-1 and Tie-2, were analyzed with reverse transcriptase polymerase chain reaction 2, 6, and 15 days after surgery. Levels of mRNA expression were compared with analysis of variance and the Student t test. RESULTS: Carotid arteries implanted with nonradioactive 0.015-caliber coils were occluded in 84% of arteries on day 2, but in only 57% of arteries on day 15, which confirms that recanalization occurred in this model. Arteries implanted with 0.015-caliber 32P coils did not become recanalized, and 100% were occluded on day 15 (n = 13; P = .006). Recanalization was associated with endothelial-like cell-lined channels, whereas persistent occlusion was caused by complete filling of the lumen with conjunctive tissue. Coil occlusion, with or without recanalization, was followed by decreased expression of vWf, VE-Cad, eNOS, VCAM-1, MMP-2, TIMP-1, and TIMP-2; stable expression of PECAM-1, SMA+, and TIMP-3; and overexpression of Ang-1 and Ang-2, MMP-9, MMP-14, and TIMP-4. Statistically significant differences when arteries were implanted with 32P coils included decreased expression of TIMP-4 (P = .011) and increased expression of MMP-9 (P = .02). CONCLUSION: Recanalization and organization of arterial thrombus is associated with expression of genes involved in angiogenesis and neointima formation. Recanalization can be prevented with beta-radiation, but molecular mechanisms remain to be refined. CLINICAL RELEVANCE: A better understanding of molecular mechanisms involved in angiogenesis has permitted its regulation as a new option in treatment of various diseases. Inhibition of angiogenesis may help control diseases such as cancer, arthritis, or diabetes retinopathy. On the other hand, stimulation of angiogenesis may palliate conditions associated with insufficient blood supply, such as ischemic heart disease or critical limb ischemia. Yet little is known regarding recanalization (to be differentiated from thrombolysis), a cellular process that occurs concurrently with thrombus "organization." Recanalization is an important physiologic phenomenon because it can efficiently reestablish antegrade circulation after thrombosis both in veins and in arteries, and could be modulated for therapeutic purposes. Thus our efforts at better understanding of mechanisms involved in recanalization could be used, in addition to its promotion to recover flow after thrombotic occlusions, to prevent its occurrence after endovascular interventions designed to permanently occlude aneurysms.

Lingual artery bifurcation aneurysms for training and evaluation of neurovascular devices.

We present a canine lingual artery bifurcation aneurysm and assess its value for training in endovascular techniques and testing new embolic agents. The experimental aneurysm described herein mirrors human bifurcation aneurysms, and with this model, we sought to reproduce endovascular technical difficulties. However, the lesions created in this canine model did not show angiographic or histologic evidence of aneurysmal recurrence. We conclude that this model may be useful for training in endovascular techniques, but because of the lack of sufficient aneurysmal recurrence, it is not suitable for evaluating new embolic agents.

Role of the endothelial lining in recurrences after coil embolization: prevention of recanalization by endothelial denudation.

BACKGROUND AND PURPOSE: Endovascular treatment can improve the outcome of patients treated for ruptured intracranial aneurysms as compared with surgical clipping, but angiographic recurrences are frequent. Endothelial denudation before coil embolization may prevent recanalization and improve results of endovascular treatment. METHODS: We compared angiographic and pathological results 3 months after coil occlusion of paired canine arteries (n=16), with or without previous denudation of the endothelial lining using an endovascular device. The technique was then used to denude the neck of carotid venous pouch bifurcation aneurysms before coil embolization in 8 dogs, and the angiographic evolution at 12 weeks was compared with 7 control aneurysms treated by coiling only. Qualitative scoring systems were used to compare angiographic results with time and neointimal coverage at the neck of aneurysm after necropsy. The evolution of angiographic scores was analyzed using Wilcoxon signed rank tests whereas angiographic and neointimal scores of the 2 groups were compared using the Mann-Whitney test. RESULTS: All arteries embolized with platinum coils recanalized, whereas most arteries (12/16 or 75%) denuded before coil embolization remained occluded at 3 and 12 weeks (P<0.001). Aneurysms treated with coils without previous denudation tended to recur, with angiographic scores significantly worse at 12 weeks as compared with T(0) (P=0.015). Median angiographic and neointimal scores were significantly better at 12 weeks with endothelial denudation (P=0.011 and 0.026, respectively). CONCLUSIONS: Endothelial denudation can prevent recanalization after coil embolization.

ICAM-1 isoforms: specific activity and sensitivity to cleavage by leukocyte elastase and cathepsin G.

The extracellular moiety of ICAM-1 consists of five Ig-like domains, the first and third domains mediating adhesion to integrin ligands. The ICAM-1 gene, however, gives rise to the expression of five alternative splice variants containing two, three, or four Ig-like domains. In this work, we have investigated whether the rearrangement of the architecture of ICAM-1 affects its structural properties and function. We showed that, in contrast to the common form, all alternative isoforms of ICAM-1 were susceptible to cleavage by leukocyte elastase and cathepsin G. We found that the length of an isoform did not influence the susceptibility to proteolysis. The molecular diversity provided by the skipping of entire Ig domains and the level of expression on the APC, however, significantly influenced their ability to potentiate the proliferation of T cells. Finally, we found that the expression of minor ICAM-1 isoforms encoding the third Ig-like domains was sufficient to sustain neutrophil infiltration in the liver and confer exon-5-targeted ICAM-1-deficient mice susceptibility to LPS-induced septic shock. These findings not only demonstrate that ICAM-1 isoforms are fully functional, but support the concept that alternative RNA splicing in the Ig superfamily may fulfill distinct roles during the development of the immune response.

Evidence for the role of promoter methylation in the regulation of MMP-9 gene expression.

Several studies have reported that elevated MMP-9 expression in lymphoma tissues correlated with tumor stage, grade, or prognosis. Because the DNA methylation pattern is critical for gene expression, detailed methylation analysis using genomic bisulfite sequencing was performed on a series of lymphoma cell lines. We found an inverse correlation between level of methylation of the MMP-9 promoter and the level of MMP-9 expression. Treating lymphoma cells with a DNA methylation inhibitor decreased MMP-9 promoter methylation and increased MMP-9 messenger RNA and protein secretion. This increased expression was potentiated by PMA, a known stimulus of MMP-9 in lymphoma cells. Finally, experiments using in vitro methylated MMP-9 promoter constructs confirmed the fact that DNA methylation exerts suppression on transcriptional activity. The results thus indicate that methylation may contribute to the transcriptional activity of the MMP-9 promoter.

Induced expression of MMP-9 in C6 glioma cells is inhibited by PDGF via a PI 3-kinase-dependent pathway.

The involvement of phosphatidylinositol 3 (PI 3)-kinase in the signalling pathways leading to MMP-9 expression in glioma cells remains unclear. Here, we report that PI 3-kinase inhibits MMP-9 expression induced by either IL-1 or TNF-alpha in rat C6 glioma cells. Using zymography and semi-quantitative RT-PCR analysis, we showed that treatment of C6 cells with wortmannin, an inhibitor of PI 3-kinase activity, potentiated the expression of MMP-9 induced by both cytokines. In contrast, platelet-derived growth factor (PDGF), an inducer of PI 3-kinase activity in C6 cells, inhibited IL-1- or TNF-alpha-induced MMP-9 secretion. Accordingly, this inhibition by PDGF was prevented by wortmannin. Furthermore, stable C6 clones over-expressing the dominant-negative form the regulatory subunit of PI 3-kinase potentiated the expression of MMP-9 induced by IL-1 or TNF-alpha. Taken together, these results suggest that PI 3-kinase may act as a negative regulator of MMP-9 expression in C6 glioma cells.

Protein kinase C-zeta regulates transcription of the matrix metalloproteinase-9 gene induced by IL-1 and TNF-alpha in glioma cells via NF-kappa B.

The regulation of matrix metalloproteinase-9 (MMP-9) expression in glioma cells is one of the key processes in tumor invasion through the brain extracellular matrix. Although some studies have demonstrated the implication of classic protein kinase C (PKC) isoforms in the regulation of MMP-9 production by phorbol esters or lipopolysaccharide, the involvement of specific PKC isoforms in the signaling pathways leading to MMP-9 expression by inflammatory cytokines remains unclear. Here we report that the atypical PKC-zeta isoform participates in the induction of MMP-9 expression by interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) in rat C6 glioma cells. Indeed, zymography and semi-quantitative reverse transcriptase-PCR analysis showed that pretreatment of C6 cells with PKC-zeta pseudosubstrate abolished MMP-9 activity and gene expression induced by IL-1 or TNF-alpha. Accordingly, IL-1 and TNF-alpha were able to induce PKC-zeta activity, as demonstrated by in vitro kinase assay using immunoprecipitated PKC-zeta. Furthermore, stable C6 clones overexpressing PKC-zeta, but not PKC-epsilon, displayed an up-regulation of MMP-9 constitutive expression as well as an increase of mmp-9 promoter activity. These processes were inhibited by an NF-kappaB-blocking peptide and completely prevented by NF-kappaB-binding site mutation in the mmp-9 promoter. Taken together, these results indicate that PKC-zeta plays a key role in the regulation of MMP-9 expression in C6 glioma cells through NF-kappaB.