Monique N. Vergouwe

Inhibition of NF-kappaB activation in macrophages increases atherosclerosis in LDL receptor-deficient mice

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.

Involvement of the CACNA1A gene containing region on 19p13 in migraine with and without aura

Objective: To assess the involvement of the 19p13 familial hemiplegic migraine (FHM) locus in migraine with and without aura. Background: Migraine with and without aura are likely to be polygenetic multifactorial disorders. FHM is a rare dominantly inherited type of migraine with aura. In about 50% of families, FHM is caused by mutations in the P/Q-type calcium channel α1A-subunit (CACNA1A) gene on chromosome 19p13. The CACNA1A gene is thus a good candidate gene for “nonhemiplegic” migraine with or without aura. Methods: The authors performed an affected sibpair analysis using flanking and CACNA1A intragenic markers. The authors assessed the occurrence of shared parental marker alleles among 189 affected siblings from 36 extended families with typical migraine with or without aura. Results: Sibling pairs with any form of migraine had inherited the same 19p13 CACNA1A-containing region significantly more frequently than expected by chance (maximum multipoint lod score = 1.22). This result was almost exclusively dependent on the increased sharing found in sibling pairs with migraine with aura (maximum multipoint lod score = 1.41). The locus-specific relative risk for a sibling (λs) to suffer from migraine with aura, defined as the increase in risk of the trait attributable to the 19p13 locus, was λs = 1.56. When combining migraine with and without aura, λs was 1.22. Conclusions: 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.

5-HT1B Receptor Polymorphism and Clinical Response to Sumatriptan

The 5‐HT1 receptor agonist, sumatriptan, is highly effective in the treatment of migraine. Some patients, however, do not respond or experience recurrence of the headache. In addition, some patients report chest symptoms after sumatriptan. We investigated whether these different responses could be attributed to genetic diversity of the 5‐HT1B receptor, which most likely mediates the therapeutic action and the coronary side effects of sumatriptan. Allele frequencies of two polymorphisms in the 5‐HT1B receptor gene (G861C and T‐261G) were investigated in migraine patients with consistently good response to sumatriptan (n=14), with no response (n=12), with recurrence of the headache (n=12), with chest symptoms (n=13), and in patients without chest symptoms (n=27). Allele frequencies (G:0.74; C:0.26 at nt 861 and T:0.39; G:0.61 at nt ‐261) did not differ between patient groups, indicating that genetic diversity of the 5‐HT1B receptor does not seem to be involved in the different clinical responses to sumatriptan.

Lipopolysaccharide-induced gene expression in murine macrophages is enhanced by prior exposure to oxLDL

Uptake of modified lipoproteins by macrophages results in the formation of foam cells. We investigated how foam cell formation affects the inflammatory response of macrophages. Murine bone marrow-derived macrophages were treated with oxidized LDL (oxLDL) to induce foam cell formation. Subsequently, the foam cells were activated with lipopolysaccharide (LPS), and the expression of lipid metabolism and inflammatory genes was analyzed. Furthermore, gene expression profiles of foam cells were analyzed using a microarray. We found that prior exposure to oxLDL resulted in enhanced LPS-induced tumor necrosis factor (TNF) and interleukin-6 (IL-6) gene expression, whereas the expression of the anti-inflammatory cytokine IL-10 and interferon-β was decreased in foam cells. Also, LPS-induced cytokine secretion of TNF, IL-6, and IL-12 was enhanced, whereas secretion of IL-10 was strongly reduced after oxLDL preincubation. Microarray experiments showed that the overall inflammatory response induced by LPS was enhanced by oxLDL loading of the macrophages. Moreover, oxLDL loading was shown to result in increased nuclear factor-κB activation. In conclusion, our experiments show that the inflammatory response to LPS is enhanced by loading of macrophages with oxLDL. These data demonstrate that foam cell formation may augment the inflammatory response of macrophages during atherogenesis, possibly in an IL-10-dependent manner.

Involvement of a Ca 2+ Channel Gene in Familial Hemiplegic Migraine and Migraine With and Without Aura

A gene for familial hemiplegic migraine, a subtype of migraine with aura, was assigned to chromosome 19p13. In this region, we identified a brain‐specific P/Q‐type calcium channel α1a‐subunit gene, CACNA1A, with 47 exons covering 300 kb. Sequencing of all exons and their flanking surroundings revealed polymorphic variations, including a (CA)0‐repeat anti a (CAG)n‐repeat in the 34 untranslated region. In patients with familial hemiplegic migraine, we found four different missense mutations in conserved functional domains. One of the mutations has occurred on two different haplotypes in unrelated familial hemiplegic migraine families. Moreover, in episodic ataxia type 2, we found two mutations disrupting the reading frame. Thus, familial hemiplegic migraine and episodic ataxia type 2 can be considered as allelic channelopathies. Involvement of this familial hemiplegic migraine locus in migraine with and without aura was demonstrated by sib‐pair analysis. We showed an increase of shared marker alleles of locus D19S394, which is tightly linked to the gene. The association between the α1A calcium channel and familial hemiplegic migraine, and the increase of shared alleles in migraine‐affected sib‐pairs, have uncovered a new pathway for the pathophysiology of migraine. This finding may provide a rationale for the development of specific prophylactic therapy for migraine and other (paroxysmal) cerebral disorders.

A 3-Mb Region for the Familial Hemiplegie Migraine Locus on 19p13.1-p13.2: Exclusion of PRKCSH as a Candidate Gene

Familial hemiplegie migraine (FHM) is an autosomal dominant subtype of migraine with attacks, associated with transient episodes of hemiparesis. One of the genes for FHM has been assigned to chromosome 19p13. Detailed analysis of critical recombinants from two different chromosome 19-linked FHM families, using new markers indicated a 6-cM candidate region on 19p13.1–p13.2 flanked by loci D19S394 and D19S226. Another paroxysmal neurological disorder, episodic ataxia type 2 (EA-2), has also been linked to the same chromosomal region. Most of the interval was completely covered by YAC and cosmid contigs; the physical map yielded approximately 3 Mb encompassing several genes including the protein kinase substrate 80K-H (PRKCSH) gene. Since PRKCSH is involved in neuronal signal transduction, it was considered to be an FHM candidate gene. The genomic structure of this gene was established and mutation analysis for all exon and flanking intron sequences was performed in FHM- and EA-2-affected individuals. Five polymorphisms were identified, including a trinucleotide repeat length variation in the coding sequence. However, no potential disease causing mutation was found and therefore the PRKCSH gene can be excluded for both FHM and EA-2.