C. Makena Hightower

Integration of cardiovascular regulation by the blood/ endothelium cell-free layer

The cell‐free layer (CFL) width separating red blood cells in flowing blood from the endothelial cell membrane is shown to be a regulator of the balance between nitric oxide (NO) production by the endothelium and NO scavenging by blood hemoglobin. The CFL width is determined by hematocrit (Hct) and the vessel wall flow velocity gradient. These factors and blood and plasma viscosity determine vessel wall shear stress which regulates the production of NO in the vascular wall. Mathematical modeling and experimental findings show that vessel wall NO concentration is a strong nonlinear function of Hct and that small Hct variations have comparatively large effects on blood pressure regulation. Furthermore, NO concentration is a regulator of inflammation and oxygen metabolism. Therefore, small, sustained perturbations of Hct may have long‐term effects that can promote pro‐hypertensive and pro‐inflammatory conditions. In this context, Hct and its variability are directly related to vascular tone, peripheral vascular resistance, oxygen transport and delivery, and inflammation. These effects are relevant to the analysis and understanding of blood pressure regulation, as NO bioavailability regulates the contractile state of blood vessels. Furthermore, regulation of the CFL is a direct function of blood composition therefore understanding of its physiology relates to the design and management of fluid resuscitation fluids. From a medical perspective, these studies propose that it should be of clinical interest to note small variations in patients Hct levels given their importance in modulating the CFL width and therefore NO bioavailability. WIREs Syst Biol Med 2011 3 458–470 DOI: 10.1002/wsbm.150

Neuropeptide Y (NPY): genetic variation in the human promoter alters glucocorticoid signaling, yielding increased NPY secretion and stress responses

OBJECTIVES This study sought to understand whether genetic variation at the Neuropeptide Y (NPY) locus governs secretion and stress responses in vivo as well as NPY gene expression in sympathochromaffin cells. BACKGROUND The NPY is a potent pressor peptide co-released with catecholamines during stress by sympathetic axons. Genome-wide linkage on NPY secretion identified a LOD (logarithm of the odds ratio) peak spanning the NPY locus on chromosome 7p15. METHODS Our approach began with genomics (linkage and polymorphism determination), extended into NPY genetic control of heritable stress traits in twin pairs, established transcriptional mechanisms in transfected chromaffin cells, and concluded with observations on blood pressure (BP) in the population. RESULTS Systematic polymorphism tabulation at NPY (by re-sequencing across the locus: promoter, 4 exons, exon/intron borders, and untranslated regions; on 2n = 160 chromosomes of diverse biogeographic ancestries) identified 16 variants, of which 5 were common. We then studied healthy twin/sibling pairs (n = 399 individuals), typing 6 polymorphisms spanning the locus. Haplotype and single nucleotide polymorphism analyses indicated that proximal promoter variant ∇-880Δ (2-bp TG/-, Ins/Del, rs3037354) minor/Δ allele was associated with several heritable (h(2)) stress traits: higher NPY secretion (h(2) = 73 ± 4%) as well as greater BP response to environmental (cold) stress, and higher basal systemic vascular resistance. Association of ∇-880Δ and plasma NPY was replicated in an independent sample of 361 healthy young men, with consistent allelic effects; genetic variation at NPY also associated with plasma NPY in another independent series of 2,212 individuals derived from Australia twin pairs. Effects of allele -880Δ to increase NPY expression were directionally coordinate in vivo (on human traits) and in cells (transfected NPY promoter/luciferase reporter activity). Promoter -880Δ interrupts a novel glucocorticoid response element motif, an effect confirmed in chromaffin cells by site-directed mutagenesis on the transfected promoter, with differential glucocorticoid stimulation of the motif as well as alterations in electrophoretic mobility shifts. The same -880Δ allele also conferred risk for hypertension and accounted for approximately 4.5/approximately 2.1 mm Hg systolic BP/diastolic BP in a population sample from BP extremes. CONCLUSIONS We conclude that common genetic variation at the NPY locus, especially in proximal promoter ∇-880Δ, disrupts glucocorticoid signaling to influence NPY transcription and secretion, raising systemic vascular resistance and early heritable responses to environmental stress, eventuating in elevated resting BP in the population. The results point to new molecular strategies for probing autonomic control of the human circulation and ultimately susceptibility to and pathogenesis of cardiovascular and neuropsychiatric disease states.

Genetic Implication of a Novel Thiamine Transporter in Human Hypertension

OBJECTIVES This study coupled 2 strategies-trait extremes and genome-wide pooling-to discover a novel blood pressure (BP) locus that encodes a previously uncharacterized thiamine transporter. BACKGROUND Hypertension is a heritable trait that remains the most potent and widespread cardiovascular risk factor, although details of its genetic determination are poorly understood. METHODS Representative genomic deoxyribonucleic acid (DNA) pools were created from male and female subjects in the highest- and lowest-fifth percentiles of BP in a primary care population of >50,000 patients. The peak associated single-nucleotide polymorphisms were typed in individual DNA samples, as well as in twins/siblings phenotyped for cardiovascular and autonomic traits. Biochemical properties of the associated transporter were evaluated in cellular assays. RESULTS After chip hybridization and calculation of relative allele scores, the peak associations were typed in individual samples, revealing an association between hypertension, systolic BP, and diastolic BP and the previously uncharacterized solute carrier SLC35F3. The BP genetic association at SLC35F3 was validated by meta-analysis in an independent sample from the original source population, as well as the International Consortium for Blood Pressure Genome-Wide Association Studies (across North America and western Europe). Sequence homology to a putative yeast thiamine (vitamin B1) transporter prompted us to express human SLC35F3 in Escherichia coli, which catalyzed [(3)H]-thiamine uptake. SLC35F3 risk-allele homozygotes (T/T) displayed decreased erythrocyte thiamine content on microbiological assay. In twin pairs, the SLC35F3 risk allele predicted heritable cardiovascular traits previously associated with thiamine deficiency, including elevated cardiac stroke volume with decreased vascular resistance, and elevated pressor responses to environmental (cold) stress. Allelic expression imbalance confirmed that cis variation at the human SLC35F3 locus influenced expression of that gene, and the allelic expression imbalance peak coincided with the hypertension peak. CONCLUSIONS Novel strategies were coupled to position a new hypertension-susceptibility locus, uncovering a previously unsuspected thiamine transporter whose genetic variants predicted several disturbances in cardiac and autonomic function. The results have implications for the pathogenesis and treatment of systemic hypertension.

Chromogranin B: intra- and extra-cellular mechanisms to regulate catecholamine storage and release, in catecholaminergic cells and organisms

Chromogranin B (CHGB) is the major matrix protein in human catecholamine storage vesicles. CHGB genetic variation alters catecholamine secretion and blood pressure. Here, effective Chgb protein under‐expression was achieved by siRNA in PC12 cells, resulting in ~ 48% fewer secretory granules on electron microscopy, diminished capacity for catecholamine uptake (by ~ 79%), and a ~ 73% decline in stores available for nicotinic cholinergic‐stimulated secretion. In vivo, loss of Chgb in knockout mice resulted in a ~ 35% decline in chromaffin granule abundance and ~ 44% decline in granule diameter, accompanied by unregulated catecholamine release into plasma. Over‐expression of CHGB was achieved by transduction of a CHGB‐expressing lentivirus, resulting in ~ 127% elevation in CHGB protein, with ~ 122% greater abundance of secretory granules, but only ~ 14% increased uptake of catecholamines, and no effect on nicotinic‐triggered secretion. Human CHGB protein and its proteolytic fragments inhibited nicotinic‐stimulated catecholamine release by ~ 72%. One conserved‐region CHGB peptide inhibited nicotinic‐triggered secretion by up to ~ 41%, with partial blockade of cationic signal transduction. We conclude that bi‐directional quantitative derangements in CHGB abundance result in profound changes in vesicular storage and release of catecholamines. When processed and released extra‐cellularly, CHGB proteolytic fragments exert a feedback effect to inhibit catecholamine secretion, especially during nicotinic cholinergic stimulation.

Clinical ResearchGenetics and GenomicsNeuropeptide Y (NPY): Genetic Variation in the Human Promoter Alters Glucocorticoid Signaling, Yielding Increased NPY Secretion and Stress Responses

OBJECTIVES This study sought to understand whether genetic variation at the Neuropeptide Y (NPY) locus governs secretion and stress responses in vivo as well as NPY gene expression in sympathochromaffin cells. BACKGROUND The NPY is a potent pressor peptide co-released with catecholamines during stress by sympathetic axons. Genome-wide linkage on NPY secretion identified a LOD (logarithm of the odds ratio) peak spanning the NPY locus on chromosome 7p15. METHODS Our approach began with genomics (linkage and polymorphism determination), extended into NPY genetic control of heritable stress traits in twin pairs, established transcriptional mechanisms in transfected chromaffin cells, and concluded with observations on blood pressure (BP) in the population. RESULTS Systematic polymorphism tabulation at NPY (by re-sequencing across the locus: promoter, 4 exons, exon/intron borders, and untranslated regions; on 2n = 160 chromosomes of diverse biogeographic ancestries) identified 16 variants, of which 5 were common. We then studied healthy twin/sibling pairs (n = 399 individuals), typing 6 polymorphisms spanning the locus. Haplotype and single nucleotide polymorphism analyses indicated that proximal promoter variant ∇-880Δ (2-bp TG/-, Ins/Del, rs3037354) minor/Δ allele was associated with several heritable (h(2)) stress traits: higher NPY secretion (h(2) = 73 ± 4%) as well as greater BP response to environmental (cold) stress, and higher basal systemic vascular resistance. Association of ∇-880Δ and plasma NPY was replicated in an independent sample of 361 healthy young men, with consistent allelic effects; genetic variation at NPY also associated with plasma NPY in another independent series of 2,212 individuals derived from Australia twin pairs. Effects of allele -880Δ to increase NPY expression were directionally coordinate in vivo (on human traits) and in cells (transfected NPY promoter/luciferase reporter activity). Promoter -880Δ interrupts a novel glucocorticoid response element motif, an effect confirmed in chromaffin cells by site-directed mutagenesis on the transfected promoter, with differential glucocorticoid stimulation of the motif as well as alterations in electrophoretic mobility shifts. The same -880Δ allele also conferred risk for hypertension and accounted for approximately 4.5/approximately 2.1 mm Hg systolic BP/diastolic BP in a population sample from BP extremes. CONCLUSIONS We conclude that common genetic variation at the NPY locus, especially in proximal promoter ∇-880Δ, disrupts glucocorticoid signaling to influence NPY transcription and secretion, raising systemic vascular resistance and early heritable responses to environmental stress, eventuating in elevated resting BP in the population. The results point to new molecular strategies for probing autonomic control of the human circulation and ultimately susceptibility to and pathogenesis of cardiovascular and neuropsychiatric disease states.

Polymorphisms at the F12 and KLKB1 loci have significant trait association with activation of the renin-angiotensin system

BackgroundPlasma coagulation Factor XIIa (Hageman factor; encoded by F12) and kallikrein (KAL or Fletcher factor; encoded by KLKB1) are proteases of the kallikerin-kinin system involved in converting the inactive circulating prorenin to renin. Renin is a key enzyme in the formation of angiotensin II, which regulates blood pressure, fluid and electrolyte balance and is a biomarker for cardiovascular, metabolic and renal function. The renin-angiotensin system is implicated in extinction learning in posttraumatic stress disorder.Methods & ResultsActive plasma renin was measured from two independent cohorts- civilian twins and siblings, as well as U.S. Marines, for a total of 1,180 subjects. Genotyping these subjects revealed that the carriers of the minor alleles at the two loci- F12 and KLKB1 had a significant association with reduced levels of active plasma renin. Meta-analyses confirmed the association across cohorts. In vitro studies verified digestion of human recombinant pro-renin by kallikrein (KAL) to generate active renin. Subsequently, the active renin was able to digest the synthetic substrate angiotensinogen to angiotensin-I. Examination of mouse juxtaglomerular cell line and mouse kidney sections showed co-localization of KAL with renin. Expression of either REN or KLKB1 was regulated in cell line and rodent models of hypertension in response to oxidative stress, interleukin or arterial blood pressure changes.ConclusionsThe functional variants of KLKB1 (rs3733402) and F12 (rs1801020) disrupted the cascade of enzymatic events, resulting in diminished formation of active renin. Using genetic, cellular and molecular approaches we found that conversion of zymogen prorenin to renin was influenced by these polymorphisms. The study suggests that the variant version of protease factor XIIa due to the amino acid substitution had reduced ability to activate prekallikrein to KAL. As a result KAL has reduced efficacy in converting prorenin to renin and this step of the pathway leading to activation of renin affords a potential therapeutic target.

Heritable Influence of DBH on Adrenergic and Renal Function: Twin and Disease Studies

Background Elevated sympathetic activity is associated with kidney dysfunction. Here we used twin pairs to probe heritability of GFR and its genetic covariance with other traits. Methods We evaluated renal and adrenergic phenotypes in twins. GFR was estimated by CKD-EPI algorithm. Heritability and genetic covariance of eGFR and associated risk traits were estimated by variance-components. Meta-analysis probed reproducibility of DBH genetic effects. Effect of DBH genetic variation on renal disease was tested in the NIDDK-AASK cohort. Results Norepinephrine secretion rose across eGFR tertiles while eGFR fell (p<0.0001). eGFR was heritable, at h2 = 67.3±4.7% (p = 3.0E-18), as were secretion of norepinephrine (h2 = 66.5±5.0%, p = 3.2E-16) and dopamine (h2 = 56.5±5.6%, p = 1.8E-13), and eGFR displayed genetic co-determination (covariance) with norepinephrine (ρG = −0.557±0.088, p = 1.11E-08) as well as dopamine (ρG = −0.223±0.101, p = 2.3E-02). Since dopamine β-hydroxylase (DBH) catalyzes conversion of dopamine to norepinephrine, we studied functional variation at DBH; DBH promoter haplotypes predicted transcriptional activity (p<0.001), plasma DBH (p<0.0001) and norepinephrine (p = 0.0297) secretion; transcriptional activity was inversely (p<0.0001) associated with basal eGFR. Meta-analysis validated DBH haplotype effects on eGFR across 3 samples. In NIDDK-AASK, we established a role for DBH promoter variation in long-term renal decline rate (GFR slope, p = 0.003). Conclusions The heritable GFR trait shares genetic determination with catecholamines, suggesting new pathophysiologic, diagnostic and therapeutic approaches towards disorders of GFR as well as CKD. Adrenergic activity may play a role in progressive renal decline, and genetic variation at DBH may assist in profiling subjects for rational preventive treatment.

Seasonal hematocrit variation and health risks in the adult population of Kinshasa, Democratic Republic of Congo.

Hematocrit (Hct) as an indicator of blood viscosity and mean arterial blood pressure (MAP) were assessed according to the season in adult participants of health screenings conducted throughout Kinshasa, Democratic Republic of Congo. Data was collected at the end of summer (April) and the end of winter (August) and identified by gender. Male Hcts in August were significantly higher (P < 0.0001) than in April (48.3% ± 4.2% and 45.7% ± 2.3%, respectively) while male MAP (85.0 ± 8.4 mm Hg) was identical to that recorded in April (85.4 ± 7.7 mm Hg). August female Hcts (41.4% ± 3.1%) were statistically higher than those recorded in April (39.6% ± 1.9%, P = 0.001), MAP being 82.3 ± 7.3 vs 87.9 ± 6.6 mm Hg, respectively (P = 0.0001). Systolic and diastolic blood pressures, heart rate, body mass indices, ages, and personal and familial medical histories of the August and April groups were not significantly different. This study offers further support for the assertion that the relationship between blood viscosity and pressure of a healthy population shows that increased Hct, and therefore increased blood viscosity is associated with lowered MAP, and presumably peripheral vascular resistance.

Plasma expander and blood storage effects on capillary perfusion in transfusion after hemorrhage

BACKGROUND: Treating hemorrhage with blood transfusions in subjects previously hemodiluted with different colloidal plasma expanders, using fresh autologous blood or blood that has been stored for 2 weeks, allows identifying the interaction between type of plasma expander and differences in blood storage.

Effect of plasma expander viscosity on the cell free layer

The effect of low and high viscosity hemodilution with plasma expanders on the extent of the cell free layer (CFL) width was analyzed in the microcirculation of the exteriorized cremaster muscle preparation of Sprague-Dawley male rats. Anesthetized animals were subjected to 40% hemodilution by blood volume, using 5% human serum albumin (HSA) or 6% Hetastarch (hydroxyethyl starch 670 kDa). Arterioles (n=5 for each treatment) were investigated. Mean arterial pressure, heart rate, vessel flow velocity and CFL width were measured at baseline and 5, 20 and 40 min post-exchange transfusion. Blood and plasma viscosity was determined from terminal blood collections. CFL width and pseudoshear rate, diameter and flow, normalized to baseline, were significantly elevated at all post-exchange assessments. Peripheral vascular resistance decreased. The increase of the CFL width was greater with HSA by comparison with Hetastarch hemodilution (p<0.05). Hetastarch blood and plasma viscosities increased significantly compared to those of HSA (p<0.05). This study shows that CFL widths are influenced by plasma expander viscosity, a phenomenon proportional to the increase in molecular weight of the colloids in solution.