Genes for familial hemiplegic migraine (FHM) and episodic ataxia type-2 (EA-2) have been mapped to chromosome 19p13. We characterized a brain-specific P/Q-type Ca2+ channel alpha1-subunit gene, CACNL1A4, covering 300 kb with 47 exons. Sequencing of all exons and their surroundings revealed polymorphic variations, including a (CA)n-repeat (D19S1150), a (CAG)n-repeat in the 3'-UTR, and different types of deleterious mutations in FHM and EA-2. In FHM, we found four different missense mutations in conserved functional domains. One mutation has occurred on two different haplotypes in unrelated FHM families. In EA-2, we found two mutations disrupting the reading frame. Thus, FHM and EA-2 can be considered as allelic channelopathies. A similar etiology may be involved in common types of migraine.

Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-κB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-κB activation in macrophages during atherogenesis, we used LDL receptor-deficient mice with a macrophage-restricted deletion of IκB kinase 2 (IKK2), which is essential for NF-κB activation by proinflammatory signals. These mice showed increased atherosclerosis as quantified by lesion area measurements. In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions. Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-κB activation, as compared with controls. In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar. Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages. Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10. Our data show that inhibition of the NF-κB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the proand anti-inflammatory balance that controls the development of atherosclerosis.

Inflammatory cytokines are well-recognized mediators of atherosclerosis. Depending on the pathological context, type I interferons (IFNs; IFNalpha and IFNbeta) exert either pro- or anti-inflammatory immune functions, but their exact role in atherogenesis has not been clarified. Here, we demonstrate that IFNbeta enhances macrophage-endothelial cell adhesion and promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner. Moreover, IFNbeta treatment accelerates lesion formation in two different mouse models of atherosclerosis and increases macrophage accumulation in the plaques. Concomitantly, absence of endogenous type I IFN signaling in myeloid cells inhibits lesion development, protects against lesional accumulation of macrophages, and prevents necrotic core formation. Finally, we show that type I IFN signaling is upregulated in ruptured human atherosclerotic plaques. Hereby, we identify type I IFNs as proatherosclerotic cytokines that may serve as additional targets for prevention or treatment.

Atherosclerosis is now generally accepted as a chronic inflammatory condition. The transcription factor NF-kappaB is a key regulator of inflammation, immune responses, cell survival, and cell proliferation. To investigate the role of NF-kappaB activation in macrophages during atherogenesis, we used LDL receptor-deficient mice with a macrophage-restricted deletion of IkappaB kinase 2 (IKK2), which is essential for NF-kappaB activation by proinflammatory signals. These mice showed increased atherosclerosis as quantified by lesion area measurements. In addition, the lesions were more advanced and showed more necrosis and increased cell number in early lesions. Southern blotting revealed that deletion of IKK2 was approximately 65% in macrophages, coinciding with a reduction of 50% in NF-kappaB activation, as compared with controls. In both groups, the expression of differentiation markers, uptake of bacteria, and endocytosis of modified LDL was similar. Upon stimulation with LPS, production of TNF was reduced by approximately 50% in IKK2-deleted macrophages. Interestingly, we also found a major reduction in the anti-inflammatory cytokine IL-10. Our data show that inhibition of the NF-kappaB pathway in macrophages leads to more severe atherosclerosis in mice, possibly by affecting the pro- and anti-inflammatory balance that controls the development of atherosclerosis.

Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-laden macrophages in the vessel wall. One of the major transcription factors in inflammation is nuclear factor B (NF-B), and we have studied its role in the development of atherosclerosis. Bone marrow from mice targeted in the NF-B1 gene encoding for the p50 subunit was used to reconstitute irradiated LDLR ؊/؊ mice as a model for atherosclerosis. After feeding the mice a highfat diet, those deficient in NF-B1 had a 41% lower rate of atherosclerosis than control mice, as judged by the sizes of the lesions. Furthermore, in the absence of NF-B1, the lesions were characterized by an inflammatory phenotype, contained increased numbers of small cells, and were almost devoid of normal foam cells. In vitro studies using bone marrow (BM)-derived macrophages showed that macrophages lacking p50 had a prolonged production of tumor necrosis factor (TNF) in response to lipopolysaccharide (LPS), and other cytokines were also affected. Interestingly, the uptake of oxidized lowdensity lipoprotein (LDL) was greatly reduced in activated p50-deficient macrophages, probably because of a reduction in the expression of scavenger receptor class A. The effects on atherosclerosis might have resulted from the changes in cytokine production and the uptake of modified lipoproteins, making p50 a pivotal regulator of atherogenesis. IntroductionAtherosclerosis is an inflammatory disease of the large arteries. An initiating event is the retention and subsequent modification of lipoproteins in the vessel wall. 1,2 This leads to the attraction of monocytes that migrate through the vessel wall and eventually differentiate into macrophages engorged with modified lipoproteins, turning them into foam cells. These processes mark the start of atherosclerosis, in which the immune system plays a key role. 3 Macrophages play a dual role in atherogenesis. First, they become foam cells upon uptake of modified lipoproteins in the vessel wall. Second, they are the major immune cells in the lesion, and they mediate the inflammatory response accompanying atherosclerotic plaque formation by producing cytokines, chemokines, and growth factors. These inflammatory mediators contribute to the attraction of more monocytes and other cells of the immune system and can influence other cells in the vicinity, such as smooth muscle cells and endothelial cells. Furthermore, they can produce factors that affect plaque development, morphology, and stability. Hence, macrophages have essential functions in all phases of plaque development, from fatty streaks to advanced plaques and eventually to plaque rupture.The transcription factor nuclear factor B (NF-B) plays a major role in inflammation and immune regulation. NF-B complexes consist of heterodimers or homodimers that are kept in the cytoplasm in an inactive form. On activation, NF-B translocates to the nucleus, mediating the transcription of a wide range of genes, including cytokines, growth factors, and antiapoptotic proteins. 4 ...

Familial hemiplegic migraine (FHM) is an autosomal dominant subtype of migraine with aura, with half of the families being assigned to chromosome 19p13. We identified missense mutations in a brain-specific calcium channel alpha1A-subunit (CACNA1A) gene on 19p13 segregating with FHM and truncating mutations in families with episodic ataxia type 2 (EA-2). Expansions of an intragenic CAG repeat have been shown in autosomal dominant cerebellar ataxia (SCA6). Hence, FHM, EA-2, and SCA6 are allelic ion channel disorders. We analyzed the phenotype-genotype relation in three unrelated FHM families with the calcium channel alpha1A-subunit gene mutations I1811L (two families) and V714A (one family). We found mutations in all but three patients with FHM (i.e., three phenocopies). In addition, the I1811L mutation occurred in two patients with "nonhemiplegic" migraine and in one subject without migraine. Cerebellar ataxia was found in both families with the I1811L mutation but not in the family with the V714A mutation. We failed to find expansions of the intragenic CAG repeat in FHM patients with cerebellar ataxia. We conclude that the I1811L mutation causes both FHM and cerebellar ataxia independent of the number of CAG repeats. The I1811L mutation may also occur in "normal" migraine patients, supporting the hypothesis that FHM is part of the migraine spectrum.

The increased allele sharing in the CACNA1A gene region on 19p13 is consistent with an important involvement of this region in migraine, especially migraine with aura.

Secreted phospholipase A2 group X (sPLA 2 -X) is one of the most potent enzymes of the phospholipase A 2 lipolytic enzyme superfamily. Its high catalytic activity toward phosphatidylcholine (PC), the major phospholipid of cell membranes and lowdensity lipoproteins (LDL), has implicated sPLA 2 -X in chronic inflammatory conditions such as atherogenesis. We studied the role of sPLA 2 -X enzyme activity in vitro and in vivo, by generating sPLA 2 -X-overexpressing macrophages and transgenic macrophage-specific sPLA 2 -X mice. Our results show that sPLA 2 -X expression inhibits macrophage activation and inflammatory responses upon stimulation, characterized by reduced cell adhesion and nitric oxide production, a decrease in tumor necrosis factor (TNF), and an increase in interleukin (IL)-10. These effects were mediated by an increase in IL-6, and enhanced production of prostaglandin E 2 (PGE 2 ) and 15-deoxy-⌬12,14-prostaglandin J 2 (PGJ 2 ). Moreover, we found that overexpression of active sPLA 2 -X in macrophages strongly increases foam cell formation upon incubation with native LDL but also oxidized LDL (oxLDL), which is mediated by enhanced expression of scavenger receptor CD36. Transgenic sPLA 2 -X mice died neonatally because of severe lung pathology characterized by interstitial pneumonia with massive granulocyte and surfactant-laden macrophage infiltration. We conclude that overexpression of the active sPLA 2 -X enzyme results in enhanced foam cell formation but reduced activation and inflammatory responses in macrophages in vitro. Interestingly, enhanced sPLA 2 -X activity in macrophages in vivo leads to fatal pulmonary defects, suggesting a crucial role for sPLA 2 -X in inflammatory lung disease.