Local effects of human PCSK9 on the atherosclerotic lesion.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes atherosclerosis by increasing low-density lipoprotein (LDL) cholesterol levels through degradation of hepatic LDL receptor (LDLR). Studies have described the systemic effects of PCSK9 on atherosclerosis, but whether PCSK9 has local and direct effects on the plaque is unknown. To study the local effect of human PCSK9 (hPCSK9) on atherosclerotic lesion composition, independently of changes in serum cholesterol levels, we generated chimeric mice expressing hPCSK9 exclusively from macrophages, using marrow from hPCSK9 transgenic (hPCSK9tg) mice transplanted into apoE(-/-) and LDLR(-/-) mice, which were then placed on a high-fat diet (HFD) for 8 weeks. We further characterized the effect of hPCSK9 expression on the inflammatory responses in the spleen and by mouse peritoneal macrophages (MPM) in vitro. We found that MPMs from transgenic mice express both murine (m) Pcsk9 and hPCSK9 and that the latter reduces macrophage LDLR and LRP1 surface levels. We detected hPCSK9 in the serum of mice transplanted with hPCSK9tg marrow, but did not influence lipid levels or atherosclerotic lesion size. However, marrow-derived PCSK9 progressively accumulated in lesions of apoE(-/-) recipient mice, while increasing the infiltration of Ly6C(hi) inflammatory monocytes by 32% compared with controls. Expression of hPCSK9 also increased CD11b- and Ly6C(hi) -positive cell numbers in spleens of apoE(-/-) mice. In vitro, expression of hPCSK9 in LPS-stimulated macrophages increased mRNA levels of the pro-inflammatory markers Tnf and Il1b (40% and 45%, respectively) and suppressed those of the anti-inflammatory markers Il10 and Arg1 (30% and 44%, respectively). All PCSK9 effects were LDLR-dependent, as PCSK9 protein was not detected in lesions of LDLR(-/-) recipient mice and did not affect macrophage or splenocyte inflammation. In conclusion, PCSK9 directly increases atherosclerotic lesion inflammation in an LDLR-dependent but cholesterol-independent mechanism, suggesting that therapeutic PCSK9 inhibition may have vascular benefits secondary to LDL reduction.

HMOX1 gene promoter alleles and high HO-1 levels are associated with severe malaria in Gambian children.

Heme oxygenase 1 (HO-1) is an essential enzyme induced by heme and multiple stimuli associated with critical illness. In humans, polymorphisms in the HMOX1 gene promoter may influence the magnitude of HO-1 expression. In many diseases including murine malaria, HO-1 induction produces protective anti-inflammatory effects, but observations from patients suggest these may be limited to a narrow range of HO-1 induction, prompting us to investigate the role of HO-1 in malaria infection. In 307 Gambian children with either severe or uncomplicated P. falciparum malaria, we characterized the associations of HMOX1 promoter polymorphisms, HMOX1 mRNA inducibility, HO-1 protein levels in leucocytes (flow cytometry), and plasma (ELISA) with disease severity. The (GT)(n) repeat polymorphism in the HMOX1 promoter was associated with HMOX1 mRNA expression in white blood cells in vitro, and with severe disease and death, while high HO-1 levels were associated with severe disease. Neutrophils were the main HO-1-expressing cells in peripheral blood, and HMOX1 mRNA expression was upregulated by heme-moieties of lysed erythrocytes. We provide mechanistic evidence that induction of HMOX1 expression in neutrophils potentiates the respiratory burst, and propose this may be part of the causal pathway explaining the association between short (GT)(n) repeats and increased disease severity in malaria and other critical illnesses. Our findings suggest a genetic predisposition to higher levels of HO-1 is associated with severe illness, and enhances the neutrophil burst leading to oxidative damage of endothelial cells. These add important information to the discussion about possible therapeutic manipulation of HO-1 in critically ill patients.

PTX3 genetic variation and dizygotic twinning in the Gambia: could pleiotropy with innate immunity explain common dizygotic twinning in Africa?

Dizygotic (DZ) twinning has a genetic component and is common among sub-Saharan Africans; in The Gambia its frequency is up to 3% of live births. Variation in PTX3, encoding Pentraxin 3, a soluble pattern recognition receptor that plays an important role both in innate immunity and in female fertility, has been associated with resistance to Mycobacterium tuberculosis pulmonary disease and to Pseudomonas aeruginosa infection in cystic fibrosis patients. We tested whether PTX3 variants in Gambian women associate with DZ twinning, by genotyping five PTX3 single nucleotide polymorphisms (SNPs) in 130 sister pairs (96 full sibs and 34 half sibs) who had DZ twins. Two, three and five SNP haplotypes differed in frequency between twinning mothers and those without a history of twinning (from P = 0.006 to 3.03e-06 for two SNP and three SNP haplotypes, respectively). Twinning mothers and West African tuberculosis-controls from a previous study shared several frequent haplotypes. Most importantly, our data are consistent with an independently reported association of PTX3 and female fertility in a sample from Ghana. Taken together, these results indicate that selective pressure on PTX3 variants that affect the innate immune response to infectious agents, could also produce the observed high incidence of DZ twinning in Gambians.

Population differences in allele frequencies at the OLR1 locus may suggest geographic disparities in cardiovascular risk events.

BACKGROUND: Several studies have demonstrated a link between cardiovascular disease (CVD) susceptibility and the genetic background of populations. Endothelial activation and dysfunction induced by oxidized low-density lipoprotein (ox-LDL) is one of the key steps in the initiation of atherosclerosis. The oxidized low density lipoprotein (lectin-like) receptor 1 (OLR1) gene is the main receptor of ox-LDL. We have previously characterized two polymorphisms (rs3736235 and rs11053646) associated with the risk for coronary artery disease (CAD) and acute myocardial infarction (AMI). AIM: Given their clinical significance, it is of interest to know the distribution of these variants in populations from different continents. SUBJECTS AND METHODS: A total of 1229 individuals from 17 different African, Asian and European populations was genotyped for the two considered markers. RESULTS: The high frequencies of ancestral alleles in South-Saharan populations is concordant with the African origin of our species. The results highlight that African populations are closer to Asians, and clearly separated from the Europeans. CONCLUSION: The results confirm significant genetic structuring among populations and suggest a possible basis for varying susceptibility to CVD among groups correlated with the geographical location of populations linked with the migrations out of Africa, or with different lifestyle.

Human PCSK9 promotes hepatic lipogenesis and atherosclerosis development via apoE- and LDLR-mediated mechanisms.

AIMS: Proprotein convertase subtilisin/kexin type 9 (PCSK9) promotes the degradation of hepatic low-density lipoprotein (LDL) receptors (LDLR), thereby, decreasing hepatocyte LDL-cholesterol (LDL-C) uptake. However, it is unknown whether PCSK9 has effects on atherogenesis that are independent of lipid changes. The present study investigated the effect of human (h) PCSK9 on plasma lipids, hepatic lipogenesis, and atherosclerotic lesion size and composition in transgenic mice expressing hPCSK9 (hPCSK9tg) on wild-type (WT), LDLR⁻/⁻, or apoE⁻/⁻ background. METHODS AND RESULTS: hPCSK9 expression significantly increased plasma cholesterol (+91%), triglycerides (+18%), and apoB (+57%) levels only in WT mice. The increase in plasma lipids was a consequence of both decreased hepatic LDLR and increased hepatic lipid production, mediated transcriptionally and post-transcriptionally by PCSK9 and dependent on both LDLR and apoE. Despite the lack of changes in plasma lipids in mice expressing hPCSK9 and lacking LDLR (the main target for PCSK9) or apoE (a canonical ligand for the LDLR), hPCSK9 expression increased aortic lesion size in the absence of apoE (268 655 ± 97 972 µm² in hPCSK9tg/apoE⁻/⁻ vs. 189 423 ± 65 700 µm(2) in apoE⁻/⁻) but not in the absence of LDLR. Additionally, hPCSK9 accumulated in the atheroma and increased lesion Ly6C(hi) monocytes (by 21%) in apoE⁻/⁻ mice, but not in LDLR⁻/⁻ mice. CONCLUSIONS: PCSK9 increases hepatic lipid and lipoprotein production via apoE- and LDLR-dependent mechanisms. However, hPCSK9 also accumulate in the artery wall and directly affects atherosclerosis lesion size and composition independently of such plasma lipid and lipoprotein changes. These effects of hPCSK9 are dependent on LDLR but are independent of apoE.

Is Isolated Low High-Density Lipoprotein Cholesterol a Cardiovascular Disease Risk Factor? New Insights From the Framingham Offspring Study.

BACKGROUND: Although the inverse association between high-density lipoprotein cholesterol (HDL-C) and risk of cardiovascular disease (CVD) has been long established, it remains unclear whether low HDL-C remains a CVD risk factor when levels of low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) are not elevated. This is a timely issue because recent studies have questioned whether HDL-C is truly an independent predictor of CVD. METHODS AND RESULTS: 3590 men and women from the Framingham Heart Study offspring cohort without known CVD were followed between 1987 and 2011. Low HDL-C (<40 mg/dL in men and <50 mg/dL in women) was defined as isolated if TG and LDL-C were both low (<100 mg/dL). We also examined higher thresholds for TG (150 mg/dL) and LDL-C (130 mg/dL) and compared low versus high HDL-C phenotypes using logistic regression analysis to assess association with CVD. Compared with isolated low HDL-C, CVD risks were higher when low HDL-C was accompanied by LDL-C ≥100 mg/dL and TG <100 mg/dL (odds ratio 1.3 [1.0, 1.6]), TG ≥100 mg/dL and LDL-C <100 mg/dL (odds ratio 1.3 [1.1, 1.5]), or TG and LDL-C ≥100 mg/dL (odds ratio 1.6, [1.2, 2.2]), after adjustment for covariates. When low HDL-C was analyzed with higher thresholds for TG (≥150 mg/dL) and LDL-C (≥130 mg/dL), results were essentially the same. In contrast, compared with isolated low HDL-C, high HDL-C was associated with 20% to 40% lower CVD risk except when TG and LDL-C were elevated. CONCLUSIONS: CVD risk as a function of HDL-C phenotypes is modulated by other components of the lipid panel.

Sex-Specific Parental Effects on Offspring Lipid Levels.

BACKGROUND: Plasma lipid levels are highly heritable traits, but known genetic loci can only explain a small portion of their heritability. METHODS AND RESULTS: In this study, we analyzed the role of parental levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TGs) in explaining the values of the corresponding traits in adult offspring. We also evaluated the contribution of nongenetic factors that influence lipid traits (age, body mass index, smoking, medications, and menopause) alone and in combination with variability at the genetic loci known to associate with TC, LDL-C, HDL-C, and TG levels. We performed comparisons among different sex-specific regression models in 416 families from the Framingham Heart Study and 304 from the SardiNIA cohort. Models including parental lipid levels explain significantly more of the trait variation than models without these measures, explaining up to ≈39% of the total trait variation. Of this variation, the parent-of-origin effect explains as much as ≈15% and it does so in a sex-specific way. This observation is not owing to shared environment, given that spouse-pair correlations were negligible (<1.5% explained variation in all cases) and is distinct from previous genetic and acquired factors that are known to influence serum lipid levels. CONCLUSIONS: These findings support the concept that unknown genetic and epigenetic contributors are responsible for most of the heritable component of the plasma lipid phenotype, and that, at present, the clinical utility of knowing age-matched parental lipid levels in assessing risk of dyslipidemia supersedes individual locus effects. Our results support the clinical utility of knowing parental lipid levels in assessing future risk of dyslipidemia.

G6PD A- deficiency and severe malaria in The Gambia: heterozygote advantage and possible homozygote disadvantage.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is frequent in Africa, because it confers resistance to Plasmodium falciparum malaria; however, the nature of the protection and the genotypes associated with it have been controversial. In 1972, Bienzle and others described protection from malaria in West African females heterozygous for G6PD A-. They determined that G6PD A- heterozygotes had lower parasite counts than A- homozygotes, hemizygous males, and normal individuals. However, other studies have reached different conclusions about the protective genotypes. DNA samples from 135 children with severe malaria and 146 children with mild malaria from The Gambia were genotyped for the G6PD A- mutation that is most frequent among Gambians (G6PD 968 T->C); there was a marked deficiency of heterozygotes and an excess of homozygotes with severe malaria, producing a strong deviation from Hardy-Weinberg equilibrium. Our results support the protective effect in G6PD A- heterozygous females and suggest that homozygotes might be more susceptible to severe malaria attacks.

Pharmacogenetics in cardiovascular disorders: an update on the principal drugs.

In the coming years, genomics will impact clinical practice in multiple ways. However, one of the most important applications will be in the determination of the best treatments in personalized medicine. This is, in fact, one of the fields in which genetic variants have already been most successful and useful to clinicians. Here, we briefly review the current state of the art on pharmacogenomics and its applications to modern cardiovascular medicine.

Putting pleiotropy and selection into context defines a new paradigm for interpreting genetic data.

BACKGROUND: Natural selection shapes many human genes, including some related to complex diseases. Understanding how selection affects genes, especially pleiotropic ones, may be important in evaluating disease associations and the role played by environmental variation. This may be of particular interest for genes with antagonistic roles that cause divergent patterns of selection. The lectin-like low-density lipoprotein 1 receptor, encoded by OLR1, is exemplary. It has antagonistic functions in the cardiovascular and immune systems because the same protein domain binds oxidized low-density lipoprotein and bacterial cell wall proteins, the former contributing to atherosclerosis and the latter presumably protecting from infection. We studied patterns of selection in this gene, in humans and nonhuman primates, to determine whether variable selection can lead to conflicting results in cardiovascular disease association studies. METHODS AND RESULTS: We analyzed sequences from 11 nonhuman primate species, as well as single-nucleotide polymorphisms and sequence data from multiple human populations. Results indicate that the derived allele is favored across primate lineages (probably because of recent positive selection). However, both the derived and ancestral alleles were maintained in human populations, especially European ones (possibly because of balancing selection derived from dual roles of LOX-1). Balancing selection likely reflects response to diverse environmental pressures among humans. CONCLUSIONS: These data indicate that differential selection patterns, within and between species, in OLR1 render association studies difficult to replicate even if the gene is etiologically connected to cardiovascular disease. Selection analyses can identify genes exhibiting gene-environment interactions critical for unraveling disease association.

APOE haplotypes are associated with human longevity in a Central Italy population: evidence for epistasis with HP 1/2 polymorphism.

BACKGROUND: Apolipoprotein E (APOE) functional haplotypes determined by rs429358 and rs7412 SNPs have been extensively studied and found to be one of the most consistent association in human longevity studies. However, the search for longevity-determining genes in human has largely neglected the operation of genetic interactions. METHODS: APOE haplotypes have been determined for 1072 unrelated healthy individuals from Central Italy, 18-106 years old, divided into three gender-specific age classes defined according to demographic information and accounting for the different survival between sexes. The epistasis between APOE haplotypes and Haptoglobin (HP) 1/2 polymorphism was tested according to three-way contingency table analysis by a log-linear model. RESULTS: APOE genotype and haplotype distributions differ significantly along the age classes (Genotype: p=0.014; Haplotype: p=0.005) with APOE*ε4 genotype status and haplotype displaying negative association (Genotype: O.R.=0.377, p=0.002, Haplotype: O.R.=0.447, p=0.005). A significant interaction between APOE*ε4 genotype status, HP 1/2 genotype and age classes is reported (p=0.006). CONCLUSION: APOE haplotypes are significantly associated with longevity in our population. Of note, HP*1/*1 genotype seems to protects APOE*ε4 carriers from age-related negative selection. Collectively, these results also suggest and claim for further investigations on APOE/HP interaction in other age-related diseases such as Alzheimer's disease, atherosclerosis and Parkinson's disease.

Role of genomics in cardiovascular medicine.

As all branches of science grow and new experimental techniques become readily accessible, our knowledge of medicine is likely to increase exponentially in the coming years. Recently developed technologies have revolutionized our analytical capacities, leading to vast knowledge of many genes or genomic regions involved in the pathogenesis of congenital heart diseases, which are often associated with other genetic syndromes, coronary artery disease and non-ischemic cardiomyopathies and channelopathies. The knowledge-base of the genesis of cardiovascular diseases is likely going to be further revolutionized in this new era of genomic medicine. Here, we review the advances that have been made over the last several years in this field and discuss different genetic mechanisms that have been shown to underlie a variety of cardiovascular diseases.