Horea Rus

Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall

Interleukin 6 (IL-6) and interleukin 8 (IL-8) are present in the human arterial atherosclerotic wall as cellular and extracellular deposits in the connective tissue matrix. Quantitative determinations of IL-6 by ELISA showed mean values of 27.6 +/- 3.3 ng/100 mg protein in normal intima, 37.3 +/- 2.1 ng/100 mg protein in fibrous plaque and 25.7 +/- 4.3 ng/100 mg total extracted protein in media. IL-8 levels were 3.5 +/- 0.6 ng/100 mg protein in normal intima, 11.3 +/- 2.1 ng/100 mg protein in fibrous plaque and 8.5 +/- 1.4 ng/100 mg total extracted protein in media. Fibrous plaques presented statistically significant higher levels of both IL-6 and IL-8. IL-6 and IL-8 gene transcripts were present in human iliac fibrous plaque and media prelevated at surgery indicating that a local production by the cells of the arterial wall participate to their accumulation. We also tested the role of complement activation in induction of IL-6 and IL-8 protein synthesis as well as the subsequent activation of endothelial cells. Only IL-8 was induced by complement activation and this may contribute to increased IL-8 levels found in the atherosclerotic wall. When exposed to terminal complement complexes, endothelial cells in culture also showed an increase of both DNA-synthesis and p70 S6 kinase activity indicating that complement is able to induce not only IL-8 synthesis but also cell activation. The presence of IL-6 and IL-8 in the arterial wall where complement activation also occurred, clearly show the involvement of inflammatory events in initiation and progression of atherosclerosis.

Membrane attack by complement: the assembly and biology of terminal complement complexes

Complement system activation plays an important role in both innate and acquired immunity. Activation of the complement and the subsequent formation of C5b-9 channels (the membrane attack complex) on the cell membranes lead to cell death. However, when the number of channels assembled on the surface of nucleated cells is limited, sublytic C5b-9 can induce cell cycle progression by activating signal transduction pathways and transcription factors and inhibiting apoptosis. This induction by C5b-9 is dependent upon the activation of the phosphatidylinositol 3-kinase/Akt/FOXO1 and ERK1 pathways in a Gi protein-dependent manner. C5b-9 induces sequential activation of CDK4 and CDK2, enabling the G1/S-phase transition and cellular proliferation. In addition, it induces RGC-32, a novel gene that plays a role in cell cycle activation by interacting with Akt and the cyclin B1-CDC2 complex. C5b-9 also inhibits apoptosis by inducing the phosphorylation of Bad and blocking the activation of FLIP, caspase-8, and Bid cleavage. Thus, sublytic C5b-9 plays an important role in cell activation, proliferation, and differentiation, thereby contributing to the maintenance of cell and tissue homeostasis.

The role of the complement system in innate immunity.

Complement is a major component of innate immune system involved in defending against all the foreign pathogens through complement fragments that participate in opsonization, chemotaxis, and activation of leukocytes and through cytolysis by C5b-9 membrane attack complex. Bacterias and viruses have adapted in various ways to escape the complement activation, and they take advantage of the complement system by using the host complement receptors to infect various cells. Complement activation also participates in clearance of apoptotic cells and immune, complexes. Moreover at sublytic dose, C5b-9 was shown to promote cell survival. Recently it was also recognized that complement plays a key role in adaptive immunity by modulating and modifying the T cell responses. All these data suggest that complement activation constitutes a critical link between the innate and acquired immune responses.

Role of the C5b-9 complement complex in cell cycle and apoptosis

Assembly of C5b‐9 on cell membranes results in transmembrane channels and causes cell death. When the number of C5b‐9 molecules is limited, nucleated cells are able to escape cell death by endocytosis and by shedding of membranes bearing C5b‐9. Sublytic C5b‐9 induces proto‐oncogenes, activates the cell cycle, and enhances cell survival. In addition, C5b‐9 reverses the differentiated phenotype of post‐mitotic cells, such as oligodendrocytes and skeletal muscle cells. The signal transduction pathways responsible for cell cycle activation by C5b‐9 include Gi‐mediated activation of extracellular signal‐regulated kinase 1 and phosphatidylinositol 3‐kinase (PI3‐K). Cell survival enhanced by C5b‐9 is mediated by the PI3‐K/Akt pathway, which inhibits apoptosis through regulation of BAD. These findings indicate that complement activation and membrane assembly of sublytic C5b‐9 play an important role in inflammation by promoting cell proliferation and by rescuing apoptotic cells.

Sublytic C5b-9 induces proliferation of human aortic smooth muscle cells Role of mitogen activated protein kinase and phosphatidylinositol 3-kinase

Proliferation of vascular smooth muscle cells contributes to initimal hyperplasia during atherogenesis, but the factors regulating their proliferation are not well known. In the present study we report that sublytic C5b-9 assembly induced proliferation of differentiated human aortic smooth muscle cells (ASMC) in culture. Cell cycle re-entry occurred through activation of cdk4, cdk2 kinase and the reduction of p21 cell cycle inhibitor. We also investigated if C5b-9 cell cycle induction is mediated through activation of mitogen activated protein kinase (MAPK) pathways. Extracellular signal regulated kinase (ERK) 1 activity was significantly increased, while c-jun NH2-terminal kinase (JNK) 1 and p38 MAPK activity were only transiently increased. Pretreatment with wortmannin inhibits ERK1 activation by C5b-9, suggesting the involvement of phosphatidylinositol 3-kinase (PI 3-kinase). Both PI 3-kinase and p70 S6 kinase were activated by C5b-9 but not by C5b6. C5b-9 induced DNA synthesis was abolished by pretreatment with inhibitors of ERK1 and PI 3-kinase, but not by p38 MAPK. These data indicated that ERK1 and PI 3-kinase play a major role in C5b-9 induced ASMC proliferation.

Complement activation and atherosclerosis.

Atherosclerosis is an inflammatory disease mediated through the action of monocyte/macrophages, complement and T-lymphocytes. C5a and monocyte chemotactic factor released during complement activation in the arterial wall may participate in the initial monocyte recruitment. Assembly of C5b-9 on cells of the arterial wall may also induce cell lysis. On the other hand, sublytic assembly of C5b-9 on smooth muscle cells (SMC) and endothelial cells (EC) induces cell activation and proliferation. Analysis of mitogen activated protein kinases (MAPK) pathways induced by C5b-9 in aortic SMC revealed that extracellular signal regulated kinase (ERK) 1, c-jun NH2-terminal kinase (JNK) 1, and p38 MAPK are all activated by C5b-9. ERK1 activity was inhibited by wortmannin suggesting that ERK1 pathway is activated through phosphatidyl inositol -3 (PI 3-) kinase. Sublytic C5b-9 assembly on the plasma membrane was also able to activate Janus kinase (JAK) 1, signal transducer and activator (STAT) 3 and STAT4 in EC. JAK1 but not STAT3 activation induced by C5b-9 is dependent on Gi protein activation. New evidence accumulated during the last decade support the role of complement activation in both initiation and progression of the atherosclerotic lesions. Complement system activation is a major component of the chronic inflammatory process associated with atherosclerosis.

The role of complement activation in atherosclerosis

Atherosclerosis is a chronic inflammatory disease in which dyslipidemia, inflammation, and the immune system play an important pathogenetic role. A role in atherogenesis was demonstrated for monocyte/macrophages, complement system, and T-lymphocytes. Complement activation and C5b-9 deposition occurs both in human and experimental atherosclerosis. Complement C6 deficiency has a protective effect on diet-induced atherosclerosis, indicating that C5b-9 assembly is required for the progression of atherosclerotic lesions. The maturation of atherosclerotic lesions beyond the foam cell stage was shown to be strongly dependent on an intact complement system. C5b-9 may be responsible for cell lysis, and sublytic assembly of C5b-9 induces smooth muscle cell (SMC) and endothelial cell (EC) activation and proliferation. All these data suggest that activation of the complement system plays an important role in atherogenesis.

C5b-9 Terminal Complement Complex Protects Oligodendrocytes from Death by Regulating Bad Through Phosphatidylinositol 3-Kinase/Akt Pathway

Apoptosis of oligodendrocytes is induced by serum growth factor deprivation. We showed that oligodendrocytes and progenitor cells respond to serum withdrawal by a rapid decline of Bcl-2 mRNA expression and caspase-3-dependent apoptotic death. Sublytic assembly of membrane-inserted terminal complement complexes consisting of C5b, C6, C7, C8, and C9 proteins (C5b-9) inhibits caspase-3 activation and apoptotic death of oligodendrocytes. In this study, we examined an involvement of the mitochondria in oligodendrocyte apoptosis and the role of C5b-9 on this process. Decreased phosphatidylinositol 3-kinase and Akt activities occurred in association with cytochrome c release and caspase-9 activation when cells were placed in defined medium. C5b-9 inhibited the mitochondrial pathway of apoptosis in oligodendrocytes, as shown by decreased cytochrome c release and inhibition of caspase-9 activation. Phosphatidylinositol 3-phosphate kinase and Akt activities were also induced by C5b-9, and the phosphatidylinositol 3-phosphate kinase inhibitor LY294002 reversed the protective effect of C5b-9. Phosphatidylinositol 3-phosphate kinase activity was also responsible for the phosphorylation of Bad at Ser112 and Ser136. This phosphorylation resulted in dissociation of Bad from the Bad/Bcl-xL complex in a Giα-dependent manner. The mitochondrial pathway of oligodendrocyte apoptosis is, therefore, inhibited by C5b-9 through post-translational regulation of Bad. This mechanism may be involved in the promotion of oligodendrocyte survival in inflammatory demyelinating disorders affecting the CNS.

Cell Cycle-dependent Phosphorylation of the RUNX2 Transcription Factor by cdc2 Regulates Endothelial Cell Proliferation

RUNX2 is a member of the runt family of DNA-binding transcription factors. RUNX2 mediates endothelial cell migration and invasion during tumor angiogenesis and is expressed in metastatic breast and prostate tumors. Our published studies showed that RUNX2 DNA-binding activity is low during growth arrest, but elevated in proliferating endothelial cells. To investigate its role in cell proliferation and cell cycle regulation, RUNX2 was depleted in human bone marrow endothelial cells using RNA interference. Specific RUNX2 depletion inhibited DNA-binding activity as measured by electrophoretic mobility shift assay resulting in inhibition of cell proliferation. Cells were synchronized at the G1/S boundary with excess thymidine or in mitosis (M phase) with nocodazole. Endogenous or ectopic RUNX2 activity was maximal at late G2 and during M phase. Inhibition of RUNX2 expression by RNA interference delayed entry into and exit out of the G2/M phases of the cell cycle. RUNX2 was coimmunoprecipitated with cyclin B1 in mitotic cells, which further supported a role for RUNX2 in cell cycle progression. Moreover, in vitro kinase assays using recombinant cdc2 kinase showed that RUNX2 was phosphorylated at Ser451. The cdc2 inhibitor roscovitine dose dependently inhibited in vivo RUNX2 DNA-binding activity during mitosis and the RUNX2 mutant S451A exhibited lower DNA-binding activity and reduced stimulation of anchorage-independent growth relative to wild type RUNX2. These results suggest for the first time that RUNX2 phosphorylation by cdc2 may facilitate cell cycle progression possibly through regulation of G2 and M phases, thus promoting endothelial cell proliferation required for tumor angiogenesis.

Molecular Cloning and Characterization of RGC-32, a Novel Gene Induced by Complement Activation in Oligodendrocytes

Sublytic complement activation on oligodendrocytes (OLG) down-regulates expression of myelin genes and induces cell cycle in culture. Differential display (DD) was used to search for new genes whose expression is altered in response to complement and that may be involved in cell cycle activation. DD bands showing either increased or decreased mRNA expression in response to complement were identified and designated ResponseGenes to Complement (RGC) 1–32.RGC-1 is identical with heat shock protein 105,RGC-2 with poly(ADP-ribose) polymerase, andRGC-10 with IP-10. A new gene, RGC-32, that encodes a protein of 137 amino acids was cloned. RGC-32 has no homology with other known proteins, and contains no motif that would indicate its function. In OLG, the mRNA expression was increased by complement activation and by terminal complement complex assembly. RGC-32 protein was localized in the cytoplasm and co-immunoprecipitated with cdc2 kinase. Overexpression of RGC-32 increased DNA synthesis in OLGxC6 glioma cell hybrids. These results suggest thatRGC-32 may play a role in cell cycle activation.