Florin Niculescu

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.

Immunohistochemical localization of the terminal C5b-9 complement complex in human aortic fibrous plaque

The terminal C5b-9 complex of the complement system was localized in 26 aortic, 3 iliac and 4 femoral human fibrous plaques using indirect immunofluorescence and immunoperoxidase. IgG, IgA, IgM, Clq, C3c, C4, C9 and fibrinogen were investigated simultaneously. All the fibrous plaques presented C5b-9 deposits appearing like thin threads in the fibrous cap and masses and spots in the amorphous areas. The extent and intensity were in agreement with the size of the fibrous plaques. The intimal thickenings presented less intense deposits which were absent in atherosclerosis-free samples. The C5b-9 deposits were frequently associated with immunoglobulins and complement components in the same areas. Whereas the demonstration of complement components reflected only a nonspecific trapping, the presence of assembled C5b-9 in the damaged tissues is more indicative of the involvement of complement activation in the tissue injury. The absence of C5b-9 in the atherosclerosis-free intima and its presence at lower intensity in the intimal thickenings than in the fibrous plaques suggest a pathogenic involvement in the chronic progression of the atherosclerotic lesion.

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.

Immunoglobulins and complement components in human aortic atherosclerotic intima

Concentration and preferential retention of immunoglobulins and complement components were studied in comparison with other plasma proteins in 42 human aortae with atherosclerosis. Saline and acid extracted IgG, IgA, IgM, C1q, C3c, C4, C9, C3A, C-reactive protein, alpha 1-antitrypsin, alpha 2-macroglobulin, albumin, transferrin and fibrinogen were quantitatively determined using the radial immunodiffusion. The fibrous plaques and their adjacent areas contained higher levels of each protein than intima with only fatty streaks. No significant differences were found between the fibrous plaques and their adjacent areas presenting intimal thickenings. Saline eluted IgG and IgA were significantly higher in the fibrous plaque intima than in intimal samples with fatty streaks and were the only proteins detected in the acid eluates. The complement components were present in all saline eluates, while C-reactive protein was found in 23 samples. Crossed immunoelectrophoretic studies showed the activation of saline C3 and C4. In 8 cases serum levels of the studied proteins were compared with their concentration in saline eluates obtained from intima and media. The immunoglobulins and complement components presented higher intima/serum and lower media/intima retention ratios than the other studied proteins suggesting their preferential retention in the intima. The presence of immune related proteins in the atherosclerotic intima and their preferential retention might be explained not only by an altered permeability but also in relation to their function.

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.

Immunohistochemical localization of C5b-9, S-protein, C3d and apolipoprotein B in human arterial tissues with atherosclerosis

The terminal C5b-9 neoantigens of the complement complex, S-protein (Vitronectin), C3c, C3d and apolipoprotein B were localized on 16 aortic fibrous plaques, 8 aortic intimal thickenings, 4 fatty streaks intimae, 12 coronary fibrous plaques, 3 coronary intimal thickenings, 6 femoral and 5 basilar fibrous plaques, using an indirect and double-staining immunoperoxidase method. The granular specific deposits were localized in the fibrous cap and deeper parts of the plaque or in the deeper intima and inner-third media of intimal thickenings and fatty streaks intimae, in relation to the degree of atherosclerotic involvement. The different localization of C5b-9 and S-protein demonstrated by the double-staining technique is more suggestive for the assembly of the complex into the arterial wall and not for its preformed passage from circulation. The relation of these immune deposits to the degree of fibrosis and necrosis and their presence from the initial stages through to the advanced lesions could ascribe a role to the complement system in atherosclerosis.

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.