Human C-reactive protein : genetic and hormonal regulation and role in atherogenesis
Author: Kovacs, Alexander
Date: 2007-04-13
Location: Leksellsalen, Medicinhistoriska Museet, Solna
Time: 09.00
Department: Institutionen för medicin / Department of Medicine
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thesis.pdf (4.511Mb)
Abstract
Although in decline in the developed world CHD is still the number one
killer. The decline is thought to be due to better risk prevention and
treatment. C-reactive protein (CRP) has emerged as an marker of CHD. It
is associated with several of the major risk factors of CHD. In vitro
studies have indicated that CRP is not merely a marker but involved in
several stages of disease progression, such as lipid oxidation,
thrombosis and tissue damage. Acute phase CRP expression is stimulated by
foremost IL-6, TNFalpha and IL1 acting in synergy with IL6. Less is known
about factors controlling basal levels but studies have suggested
hereditability as one factor. Several polymorphisms have been found in
and around the CRP gene. However, none of these have showed to mutate the
protein. Here, we report on new polymorphisms in the promoter region of
the CRP gene (paper I) and possible hormonal regulation of unstimulated
CRP levels (paper II). We also characterize a mouse model, developing
atherosclerosis (paper III) and investigate the effects of CRP on
atherogenesis In vitro (paper IV).
In short, 30 healthy individuals were screened for polymorphisms in the first 1.6 kb of the CRP 5' promoter using dHPLC. Five novel SNPs were found whereof two were frequent (-717 and -286). Four SNPs (-717, 286, +1059, +1444) were tested for associations with unstimulated (n=740) and stimulated (MI) (n=208) circulating CRP levels. One of the polymorphisms (-286 C>T>A) showed a significant association with unstimulated CRP levels. The A allele was associated to highest, T allele to intermediate and C allele to lowest CRP levels. 100 consecutive prostate cancer patients that were randomized to estrogen (n=53) or intervention by orchidectomy (n=47) were sampled for blood before and 6 month after treatment. IL-1, IL-6 and CRP were analysed. Univariate analysis showed that orchidectomy tended to decrease circulating CRP concentrations whereas estrogen treatment tended to increase CRP. Treatment with estrogen resulted in higher circulating CRP levels than orchidectomy. Multivariate analysis including treatment, CRP concentrations before treatment and IL-6 levels after treatment showed that the difference in CRP concentration after treatment was highly significant indicating a role for estrogen in CRP expression.
When characterising the mouse model, atherosclerosis progression develops slowly at first, expands rapidly after transformation of fatty streaks into plaques, and plateaus after advanced lesions form. This development was paralleled by the activity of 1259 genes forming four expression clusters. Genetic lowering of plasma cholesterol in mice with early lesions produced a distinct transcriptional response and prevented atherosclerosis development at 40 weeks. 37 cholesterol-responsive genes were identified. In THP-1 macrophages, inhibiting six of these genes with siRNA affected foam cell formation.
Human CRP was studied in relation to atherogenesis in atherosclerosis-prone mice. Lesion development was studied at 15, 30, 40 and 50 weeks of age. At 40 and 50 weeks, atherosclerotic lesions of CRP transgenic mice were smaller and at 50 weeks had larger collagen deposits compared to littermate controls. Analysis of gene expression profiles from lesions of 40 weeks-old CRP transgenic and littermate controls revealed differentially expressed genes related to the proteasome.
In conclusion, the results show that genetic variation and estrogen are intimately involved in the regulation of unstimulated circulating CRP concentrations and that transgenic expression of CRP in our mouse model results in less atherosclerosis. Furthermore, some of the cholesterol-sensitive genes may be considered as future targets for atherosclerosis treatment.
In short, 30 healthy individuals were screened for polymorphisms in the first 1.6 kb of the CRP 5' promoter using dHPLC. Five novel SNPs were found whereof two were frequent (-717 and -286). Four SNPs (-717, 286, +1059, +1444) were tested for associations with unstimulated (n=740) and stimulated (MI) (n=208) circulating CRP levels. One of the polymorphisms (-286 C>T>A) showed a significant association with unstimulated CRP levels. The A allele was associated to highest, T allele to intermediate and C allele to lowest CRP levels. 100 consecutive prostate cancer patients that were randomized to estrogen (n=53) or intervention by orchidectomy (n=47) were sampled for blood before and 6 month after treatment. IL-1, IL-6 and CRP were analysed. Univariate analysis showed that orchidectomy tended to decrease circulating CRP concentrations whereas estrogen treatment tended to increase CRP. Treatment with estrogen resulted in higher circulating CRP levels than orchidectomy. Multivariate analysis including treatment, CRP concentrations before treatment and IL-6 levels after treatment showed that the difference in CRP concentration after treatment was highly significant indicating a role for estrogen in CRP expression.
When characterising the mouse model, atherosclerosis progression develops slowly at first, expands rapidly after transformation of fatty streaks into plaques, and plateaus after advanced lesions form. This development was paralleled by the activity of 1259 genes forming four expression clusters. Genetic lowering of plasma cholesterol in mice with early lesions produced a distinct transcriptional response and prevented atherosclerosis development at 40 weeks. 37 cholesterol-responsive genes were identified. In THP-1 macrophages, inhibiting six of these genes with siRNA affected foam cell formation.
Human CRP was studied in relation to atherogenesis in atherosclerosis-prone mice. Lesion development was studied at 15, 30, 40 and 50 weeks of age. At 40 and 50 weeks, atherosclerotic lesions of CRP transgenic mice were smaller and at 50 weeks had larger collagen deposits compared to littermate controls. Analysis of gene expression profiles from lesions of 40 weeks-old CRP transgenic and littermate controls revealed differentially expressed genes related to the proteasome.
In conclusion, the results show that genetic variation and estrogen are intimately involved in the regulation of unstimulated circulating CRP concentrations and that transgenic expression of CRP in our mouse model results in less atherosclerosis. Furthermore, some of the cholesterol-sensitive genes may be considered as future targets for atherosclerosis treatment.
List of papers:
I. Kovacs A, Green F, Hansson LO, Lundman P, Samnegard A, Boquist S, Ericsson CG, Watkins H, Hamsten A, Tornvall P (2005). "A novel common single nucleotide polymorphism in the promoter region of the C-reactive protein gene associated with the plasma concentration of C-reactive protein. " Atherosclerosis 178(1): 193-8
Pubmed
II. Kovacs A, Henriksson P, Hamsten A, Wallen H, Bjorkegren J, Tornvall P (2005). "Hormonal regulation of circulating C-reactive protein in men." Clin Chem 51(5): 911-3
Pubmed
III. Skogsberg J, Kovacs A, Nilsson R, Noori P, Maleki S, Lundstrom J, Kohler M, Brinne B, Hamsten A, Tegner J, Bjorkegren J (2007). "Transcriptional profiling and genetic lowering of plasma cholesterol to identify cholesterol-responsive atherosclerosis target genes." (Manuscript)
IV. Kovacs A, Hamsten A, Nilsson R, Tegner J, Tornvall P, Bjorkegren J (2007). "Atherosclerosis development in human C-reactive protein transgenic apoB100-only low density receptor deficient mice." (Manuscript)
I. Kovacs A, Green F, Hansson LO, Lundman P, Samnegard A, Boquist S, Ericsson CG, Watkins H, Hamsten A, Tornvall P (2005). "A novel common single nucleotide polymorphism in the promoter region of the C-reactive protein gene associated with the plasma concentration of C-reactive protein. " Atherosclerosis 178(1): 193-8
Pubmed
II. Kovacs A, Henriksson P, Hamsten A, Wallen H, Bjorkegren J, Tornvall P (2005). "Hormonal regulation of circulating C-reactive protein in men." Clin Chem 51(5): 911-3
Pubmed
III. Skogsberg J, Kovacs A, Nilsson R, Noori P, Maleki S, Lundstrom J, Kohler M, Brinne B, Hamsten A, Tegner J, Bjorkegren J (2007). "Transcriptional profiling and genetic lowering of plasma cholesterol to identify cholesterol-responsive atherosclerosis target genes." (Manuscript)
IV. Kovacs A, Hamsten A, Nilsson R, Tegner J, Tornvall P, Bjorkegren J (2007). "Atherosclerosis development in human C-reactive protein transgenic apoB100-only low density receptor deficient mice." (Manuscript)
Issue date: 2007-03-23
Rights:
Publication year: 2007
ISBN: 978-91-7357-160-9
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