Fu Liang Ng

Expression and function of the K+ channel KCNQ genes in human arteries

BACKGROUND AND PURPOSE KCNQ‐encoded voltage‐gated potassium channels (Kv7) have recently been identified as important anti‐constrictor elements in rodent blood vessels but the role of these channels and the effects of their modulation in human arteries remain unknown. Here, we have assessed KCNQ gene expression and function in human arteries ex vivo.

Functional analyses of coronary artery disease associated variation on chromosome 9p21 in vascular smooth muscle cells

Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genomic region contains the CDKN2A and CDKN2B genes which encode the cell cycle regulators p16(INK4a), p14(ARF) and p15(INK4b) and the ANRIL gene which encodes a non-coding RNA. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis which causes CAD. We ascertained whether 9p21 genotype had an influence on CDKN2A/CDKN2B/ANRIL expression levels in VSMCs, VSMC proliferation and VSMC content in atherosclerotic plaques. Immunohistochemical examination showed that VSMCs in atherosclerotic lesions expressed p16(INK4a), p14(ARF) and p15(INK4b). Analyses of primary cultures of VSMCs showed that the 9p21 risk genotype was associated with reduced expression of p16(INK4a), p15(INK4b) and ANRIL (P = 1.2 × 10(-5), 1.4 × 10(-2) and 3.1 × 10(-9)) and with increased VSMC proliferation (P = 1.6 × 10(-2)). Immunohistochemical analyses of atherosclerotic plaques revealed an association of the risk genotype with reduced p15(INK4b) levels in VSMCs (P = 3.7 × 10(-2)) and higher VSMC content (P = 5.6 × 10(-4)) in plaques. The results of this study indicate that the 9p21 variation has an impact on CDKN2A and CDKN2B expression in VSMCs and influences VMSC proliferation, which likely represents an important mechanism for the association between this genetic locus and susceptibility to CAD.

ADAMTS7 Cleavage and Vascular Smooth Muscle Cell Migration Is Affected by a Coronary-Artery-Disease-Associated Variant

Recent genome-wide association studies have revealed an association between variation at the ADAMTS7 locus and susceptibility to coronary artery disease (CAD). Furthermore, in a population-based study cohort, we observed an inverse association between atherosclerosis prevalence and rs3825807, a nonsynonymous SNP (A to G) leading to a Ser-to-Pro substitution in the prodomain of the protease ADAMTS7. In light of these data, we sought a mechanistic explanation for this association. We found that ADAMTS7 accumulated in smooth muscle cells in coronary and carotid atherosclerotic plaques. Vascular smooth muscle cells (VSMCs) of the G/G genotype for rs3825807 had reduced migratory ability, and conditioned media of VSMCs of the G/G genotype contained less of the cleaved form of thrombospondin-5, an ADAMTS7 substrate that had been shown to be produced by VSMCs and inhibit VSMC migration. Furthermore, we found that there was a reduction in the amount of cleaved ADAMTS7 prodomain in media conditioned by VSMCs of the G/G genotype and that the Ser-to-Pro substitution affected ADAMTS7 prodomain cleavage. The results of our study indicate that rs3825807 has an effect on ADAMTS7 maturation, thrombospondin-5 cleavage, and VSMC migration, with the variant associated with protection from atherosclerosis and CAD rendering a reduction in ADAMTS7 function.

Pharmacogenetics as a tool for optimising drug therapy in solid-organ transplantation

Existing immunosuppressive therapies used for solid-organ transplantation have narrow therapeutic indices, whereby underdosing is associated with acute immunological rejection of the transplanted organ and overdosing is associated with infections and malignancy, as well as organ-specific toxicities. There is significant inter-individual variation in the pharmacokinetics and pharmacodynamics of these drugs, an issue that has been addressed, in part, by therapeutic drug monitoring. Genetic polymorphisms in drug metabolising enzymes, drug efflux pumps and drug targets which may underly this heterogeneity have been identified and may provide a tool to guide prescribing. There are a number of associations between genotype and pharmacology, but as of now, only thiopurine-S-methyltransferase and cytochrome P450 3A5 have a sufficiently large influence to have potential in guiding therapy. Recent studies have also identified that donor genotype may play a significant role in immunosuppressive drug pharmacokinetics and pharmacodynamics.

Device-based Therapy for Hypertension.

Hypertension continues to be a major contributor to global morbidity and mortality, fuelled by an abundance of patients with uncontrolled blood pressure despite the multitude of pharmacological options available. This may occur as a consequence of true resistant hypertension, through an inability to tolerate current pharmacological therapies, or non-adherence to antihypertensive medication. In recent years, there has been a rapid expansion of device-based therapies proposed as novel non-pharmacological approaches to treating resistant hypertension. In this review, we discuss seven novel devices—renal nerve denervation, baroreflex activation therapy, carotid body ablation, central iliac arteriovenous anastomosis, deep brain stimulation, median nerve stimulation, and vagal nerve stimulation. We highlight how the devices differ, the varying degrees of evidence available to date and upcoming trials. This review also considers the possible factors that may enable appropriate device selection for different hypertension phenotypes.

Exploring hypertension genome-wide association studies findings and impact on pathophysiology, pathways, and pharmacogenetics.

Hypertension is a major risk factor for global mortality. Recent genome‐wide association studies (GWAS) have led to successful identification of many genetic loci influencing blood pressure, although these studies account for less than 5% of heritability. While genetic discovery efforts continue, it is timely to pause and reflect on what information has been gained to date from reported loci. Knowledge from GWAS findings inform our understanding of the pathways and pleiotropy underpinning hypertension and aid in the identification of potential druggable targets. By reviewing blood pressure loci we aim to determine how much potential the current observations have for future clinical utility. WIREs Syst Biol Med 2015, 7:73–90. doi: 10.1002/wsbm.1290

Black renal transplant recipients have poorer long-term graft survival than CYP3A5 expressers from other ethnic groups

BACKGROUND African American transplant recipients have poorer long-term outcomes than Caucasian Americans. This difference was not found in French patients, suggesting socialized medicine overcame this disparity. It has also been hypothesized that the difference relates to the higher prevalence of Black individuals who express the metabolic enzyme cytochrome P4503A5 (CYP3A5), with consequent altered handling of immunosuppressive drugs. METHODS Records of 555 (50 Black; 505 non-Black) sequential renal transplant recipients from a single UK centre were analysed. RESULTS Outcomes were significantly worse for Black patients: death-censored graft survival (5-year 66% versus 87%, P = 0.001); halving of year one estimated glomerular filtration rate (mean 8.8 versus 10.8 years, P = 0.008); first-year graft loss (12% versus 3.8%, P = 0.02); and death-censored graft survival in patients surviving the first year with functioning grafts (5-year 77% versus 94%, P = 0.02). Death-censored 5-year graft survival was poorer in Black CYP3A5 expressers than in non-Black CYP3A5 expressers (62% versus 93%, P = 0.002). Following multivariate analysis, the Black group demonstrated poorer graft survival as compared to the non-Black group (hazard ratio 0.46, 95% CI 0.25-0.85, P = 0.002). In a subgroup of genotyped transplant recipients, ethnicity (hazard ratio 0.31, 95% CI 0.15-0.64, P = 0.002), and not CYP3A5 expresser status, persists as an independent risk factor for graft survival following multivariate analysis. CONCLUSION In this cohort of patients with socialized medicine, Black recipients had poorer long-term outcomes than individuals from other ethnic groups. This was independent of CYP3A5 expresser status.

A blood pressure-associated variant of the SLC39A8 gene influences cellular cadmium accumulation and toxicity

Genome-wide association studies have revealed a relationship between inter-individual variation in blood pressure and the single nucleotide polymorphism rs13107325 in the SLC39A8 gene. This gene encodes the ZIP8 protein which co-transports divalent metal cations, including heavy metal cadmium, the accumulation of which has been associated with increased blood pressure. The polymorphism results in two variants of ZIP8 with either an alanine (Ala) or a threonine (Thr) at residue 391. We investigated the functional impact of this variant on protein conformation, cadmium transport, activation of signalling pathways and cell viability in relation to blood pressure regulation. Following incubation with cadmium, higher intracellular cadmium was detected in cultured human embryonic kidney cells (HEK293) expressing heterologous ZIP8-Ala391, compared with HEK293 cells expressing heterologous ZIP8-Thr391. This Ala391-associated cadmium accumulation also increased the phosphorylation of the signal transduction molecule ERK2, activation of the transcription factor NFκB, and reduced cell viability. Similarly, vascular endothelial cells with the Ala/Ala genotype had higher intracellular cadmium concentration and lower cell viability than their Ala/Thr counterpart following cadmium exposure. These results indicate that the ZIP8 Ala391-to-Thr391 substitution has an effect on intracellular cadmium accumulation and cell toxicity, providing a potential mechanistic explanation for the association of this genetic variant with blood pressure.

Coronary-Heart-Disease-Associated Genetic Variant at the COL4A1/COL4A2 Locus Affects COL4A1/COL4A2 Expression, Vascular Cell Survival, Atherosclerotic Plaque Stability and Risk of Myocardial Infarction

Genome-wide association studies have revealed an association between coronary heart disease (CHD) and genetic variation on chromosome 13q34, with the lead single nucleotide polymorphism rs4773144 residing in the COL4A2 gene in this genomic region. We investigated the functional effects of this genetic variant. Analyses of primary cultures of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) from different individuals showed a difference between rs4773144 genotypes in COL4A2 and COL4A1 expression levels, being lowest in the G/G genotype, intermediate in A/G and highest in A/A. Chromatin immunoprecipitation followed by allelic imbalance assays of primary cultures of SMCs and ECs that were of the A/G genotype revealed that the G allele had lower transcriptional activity than the A allele. Electrophoretic mobility shift assays and luciferase reporter gene assays showed that a short DNA sequence encompassing the rs4773144 site interacted with a nuclear protein, with lower efficiency for the G allele, and that the G allele sequence had lower activity in driving reporter gene expression. Analyses of cultured SMCs from different individuals demonstrated that cells of the G/G genotype had higher apoptosis rates. Immunohistochemical and histological examinations of ex vivo atherosclerotic coronary arteries from different individuals disclosed that atherosclerotic plaques with the G/G genotype had lower collagen IV abundance and thinner fibrous cap, a hallmark of unstable, rupture-prone plaques. A study of a cohort of patients with angiographically documented coronary artery disease showed that patients of the G/G genotype had higher rates of myocardial infarction, a phenotype often caused by plaque rupture. These results indicate that the CHD-related genetic variant at the COL4A2 locus affects COL4A2/COL4A1 expression, SMC survival, and atherosclerotic plaque stability, providing a mechanistic explanation for the association between the genetic variant and CHD risk.

Increased NBCn1 expression, Na+/HCO3− co-transport and intracellular pH in human vascular smooth muscle cells with a risk allele for hypertension

Abstract Genome‐wide association studies have revealed an association between variation at the SLC4A7 locus and blood pressure. SLC4A7 encodes the electroneutral Na+/HCO3− co‐transporter NBCn1 which regulates intracellular pH (pHi). We conducted a functional study of variants at this locus in primary cultures of vascular smooth muscle and endothelial cells. In both cell types, we found genotype‐dependent differences for rs13082711 in DNA‐nuclear protein interactions, where the risk allele is associated with increased SLC4A7 expression level, NBCn1 availability and function as reflected in elevated steady‐state pHi and accelerated recovery from intracellular acidosis. However, in the presence of Na+/H+ exchange activity, the SLC4A7 genotypic effect on net base uptake and steady‐state pHi persisted only in vascular smooth muscle cells but not endothelial cells. We found no discernable effect of the missense polymorphism resulting in the amino acid substitution Glu326Lys. The finding of a genotypic influence on SLC4A7 expression and pHi regulation in vascular smooth muscle cells provides an insight into the molecular mechanism underlying the association of variation at the SLC4A7 locus with blood pressure.