Michael J. Ashby

Matrix Metalloproteinase-9 (MMP-9), MMP-2, and Serum Elastase Activity Are Associated With Systolic Hypertension and Arterial Stiffness

Background— Arterial stiffness is an independent determinant of cardiovascular risk, and arterial stiffening is the predominant abnormality in systolic hypertension. Elastin is the main elastic component of the arterial wall and can be degraded by a number of enzymes, including matrix metalloproteinase-9 (MMP-9) and MMP-2. We hypothesized that elastase activity would be related to arterial stiffness and tested this using isolated systolic hypertension (ISH) as a model of stiffening and separately in a large cohort of healthy individuals. Methods and Results— A total of 116 subjects with ISH and 114 matched controls, as well as 447 individuals free from cardiovascular disease were studied. Aortic and brachial pulse wave velocity (PWV) and augmentation index were determined. Blood pressure, lipids, C-reactive protein, MMP-9, MMP-2, serum elastase activity (SEA), and tissue-specific inhibitor 2 of metalloproteinases were measured. Aortic and brachial PWV, MMP-9, MMP-2, and SEA levels were increased in ISH subjects compared with controls (P=0.001). MMP-9 levels correlated linearly and significantly with aortic (r=0.45; P=0.001) and brachial PWV (r=0.22; P=0.002), even after adjustments for confounding variables. In the younger, healthy subjects, MMP-9 and SEA were also independently associated with aortic PWV. Conclusions— Aortic stiffness is related to MMP-9 levels and SEA, not only in ISH, but also in younger, apparently healthy individuals. This suggests that elastases including MMP-9 may be involved in the process of arterial stiffening and development of ISH.

Variation in the Human Matrix Metalloproteinase-9 Gene Is Associated With Arterial Stiffness in Healthy Individuals

Background—Arterial stiffness is an important determinant of cardiovascular risk. Elastin is the main elastic component of the arterial wall and can be degraded by a number of enzymes including serine proteases and matrix metalloproteinases (MMPs). Serum MMP-9 levels correlate with arterial stiffness and predict cardiovascular risk. Polymorphisms in the MMP-9 gene are also associated with large artery function in subjects with coronary artery disease. Therefore, we investigated the influence of known MMP-9 (−1562C>T, R279Q) polymorphisms on arterial stiffness in a large cohort of healthy individuals (n=865). Methods and Results—Aortic pulse wave velocity (PWV) and augmentation index were assessed. Supine blood pressure, biochemical markers, MMP-9 levels, and serum elastase activity (SEA) were also determined. Genomic DNA was extracted and genotyping performed. Aortic PWV, serum MMP-9, and SEA were higher in carriers of the rare alleles for the −1562C>T and R279Q polymorphisms. These polymorphisms were also associated with aortic PWV after correction for other confounding factors. Stepwise regression models with known or likely determinants of arterial stiffness revealed that ≈60% of the variability in aortic PWV was attributable to age, mean arterial pressure, and genetic variants (P<0.001). Conclusions—We have demonstrated for the first time that aortic stiffness and elastase activity are influenced by MMP-9 gene polymorphisms. This suggests that the genetic variation in this protein may be involved in the process of large artery stiffening.

Positive Association of Tyrosine Hydroxylase Microsatellite Marker to Essential Hypertension

Despite advances in the understanding of monogenic hypertensive disorders, the genetic contribution to essential hypertension has yet to be elucidated. The position of tyrosine hydroxylase (TH) as the rate-limiting enzyme in catecholamine biosynthesis renders it a candidate gene for the etiology of hypertension. The TH gene contains an internal, informative microsatellite marker (TCAT)9. We undertook (1) an association study in a group of well-characterized hypertensive subjects (HT) and control subjects (NT) and (2) an affected sibling pair (ASP) study using sibships from our local family practices. Two hundred twenty-seven hypertensive patients (pretreatment systolic/diastolic blood pressure [BP] range, 139/94 to 237/133 mm Hg; age range [SD], 30 to 71 [8.5] years) were age- and gender-matched with 206 control subjects (BP range, 96/62 to 153/86 mm Hg; age range, 40 to 70 [7.6] years). One hundred thirty-six affected sibling pairs were recruited for our linkage study; 73 young borderline hypertensive subjects (YHT) (pretreatment BP range, 123/76 to 197/107 mm Hg; age range, 20 to 51 [9.4] years) were also recruited in whom recent pretreatment norepinephrine and epinephrine levels were available. All subjects were white. The TH short tandem repeat (STR) was amplified using specific polymerase chain reaction cycling conditions in all subjects, and products were run on an ABI 373A sequencer. TH alleles were assigned using Genescan and Genotyper software. Five TH alleles were present and designated A through E. Allele frequencies in the NT population (A, B, C, D, and E: 0.24, 0.17, 0.13, 0.20, and 0.26, respectively) were significantly different from the HT cohort (A, B, C, D, and E: 0.24, 0.19, 0.11, 0.11, and 0.35, respectively), P<0. 0005 (Pearsons test chi2=19.94; 4 df). The E allele appears overrepresented in the HT group, whereas the D allele appears to be overrepresented in the NT group. TH genotype frequencies were also significantly different between cases and controls (P<0.001; chi2=36. 57; 14 df). Both groups were in Hardy-Weinberg proportion. There was a trend (NS) for the D allele to be associated with a lower BP when BP was analyzed as a quantitative trait. ASP linkage data was analyzed using Splink, a nonparametric program. Expected values for sharing 0, 1, and 2 alleles (Z0, Z1, and Z2, respectively) may be expected to be 25%, 50%, and 25%, respectively, by chance (assuming identity by descent). These probabilities were calculated by Splink as 34, 68, and 34, respectively, and compared with observed values of 36.8, 67.9, and 31.3, respectively; thus, there was no excess sharing of TH alleles among affected sibling pairs (P=0.59; logarithm of odds ratio score, 0.0). TH allele frequencies in our YHT group (A, B, C, D, and E: 0.24, 0.20, 0.12, 0.15, and 0.29, respectively) were similar to those of our NT cohort (P>0.05). There was a trend for lower pretreatment plasma norepinephrine levels with the D allele in this YHT cohort. A common and potentially functional variant at codon 81(Val-->Met) within exon 2 of the TH gene (which we show to be in linkage disequilibrium with TH-STR) was also typed in our YHT but did not associate with catecholamine levels and is therefore unlikely to account for our findings with D and E TH-STR. In conclusion, the TH locus strongly associates with essential hypertension in a case-control model using well-characterized hypertensive and control groups. An ASP linkage model was negative, presumably because of lack of power. This study suggests that the TH gene, or a nearby gene, may be involved in the etiology of essential hypertension.

The Spironolactone, Amiloride, Losartan, and Thiazide (SALT) Double-Blind Crossover Trial in Patients With Low-Renin Hypertension and Elevated Aldosterone-Renin Ratio

Background— There is continuing variation in diagnosis and estimated prevalence of primary hyperaldosteronism. The higher estimates encourage search for adrenal adenomas in patients with elevated ratios of plasma aldosterone to renin. However, it is more likely that patients with normal plasma K+ and aldosterone belong to the polygenic spectrum of low-renin hypertension rather than have the same monogenic syndrome as classic Conn’s. Our primary hypothesis was that in low-renin patients with normal plasma K+ and aldosterone, a thiazide diuretic, bendroflumethiazide, would be as effective as spironolactone in overcoming the Na+ retention and lowering blood pressure. Secondary objectives were to compare the dose response for each diuretic and to evaluate amiloride as an alternative to spironolactone. Methods and Results— Fifty-seven patients entered and 51 patients completed a placebo-controlled, double-blind, randomized crossover trial. Entry criteria included low plasma renin, normal K+, elevated aldosterone-renin ratio, and a previous systolic blood pressure response to spironolactone of ≥20 mm Hg. Two doses each of spironolactone and bendroflumethiazide were compared. The crossover also included amiloride and losartan. Outcome measures were blood pressure, plasma renin, and other biochemical markers of diuretic action. Spironolactone 100 mg and bendroflumethiazide 5 mg caused similar falls in systolic blood pressure, whereas bendroflumethiazide 2.5 mg was 5/2 mm Hg less effective in reducing blood pressure than either bendroflumethiazide 5 mg or spironolactone 50 mg (P<0.005). Amiloride 40 mg was as effective as the other diuretics. Biochemical indices of natriuresis showed bendroflumethiazide to be less effective than either spironolactone or amiloride; plasma renin rose 4-fold on spironolactone but only 2-fold on bendroflumethiazide (P=0.003). Conclusions— In hypertensive patients with a low plasma renin but normal K+, bendroflumethiazide 5 mg was as effective as spironolactone 100 mg in lowering blood pressure, despite patients being selected for a previous large fall in blood pressure on spironolactone. Because this result differs from that expected in primary hyperaldosteronism, our finding argues against low-renin hypertension including a large, undiagnosed pool of primary hyperaldosteronism. However, spironolactone was the more effective natriuretic agent, suggesting that inappropriate aldosterone release or response may still contribute to the Na+ retention of low-renin hypertension.

Inducible nitric oxide synthase activity is increased in patients with rheumatoid arthritis and contributes to endothelial dysfunction

BACKGROUND Recent in vitro studies suggest that inducible nitric oxide synthase (iNOS) activity mediates endothelial dysfunction. Rheumatoid arthritis (RA) is a chronic inflammatory condition and is associated with endothelial dysfunction and increased risk of cardiovascular disease. The aim of the study was to establish the contribution of iNOS to endothelial function. METHODS Forearm blood flow (FBF) was measured during intra-arterial infusions of acetylcholine (ACh), sodium nitroprusside (SNP), N(G)-monomethyl-l-arginine (l-NMMA) and aminoguanidine (AG) in 12 RA patients and 13 healthy control subjects. Levels of C-reactive protein (CRP) and myeloperoxidase (MPO) were assessed. FBF data are presented as mean percentage changes in the ratio (infused/control arm) of FBF + or - SEM. RESULTS FBF response to ACh was reduced in patients with RA compared to controls (179 + or - 29 v. 384 + or - 72%, respectively; P=0.01), but SNP response was not (P=0.5). FBF response to AG differed between patients and controls (-15 + or - 2% v. 13 + or - 4%, respectively; P<0.001), whereas the response to l-NMMA did not (P=0.4). In a multiple regression model log CRP, AG response and LDL were found to be independent predictors of endothelial function (R(2)=0.617, P<0.001). CONCLUSION RA patients have endothelial dysfunction and increased iNOS activity in comparison to controls. Furthermore, CRP and iNOS activity were independently associated with endothelial function. Our data demonstrates that inflammation is a key mediator in a process of endothelial dysfunction possibly via activation of iNOS and increased production of MPO.

Characterization of Endothelin Isoforms in Human Heart: Endothelin-2 Demonstrated

In humans, three endothelin (ET) isoforms are predicted to exist by analysis of genomic DNA. However, evidence for the presence of all three mature ET peptides and their precursors remains unclear. Our aim was to identify the ET isoforms present in human heart, using radioimmunoassay (RIA) and reverse-phase high-performance liquid chromatography (RP-HPLC). Antisera raised against the ET-1[15-21] terminal sequence were specific for mature ETs, showing no cross-reactivity with their precursor pro-ETs. Antisera raised against the pro-ET-1[31-38] terminal sequence was specific for pro-ET-1, showing no cross-reactivity with other ET peptides. In extracts of human cardiac tissues, the concentrations of immunoreactive (IR) mature ET and pro-ET-1 were found to be as follows: left atrium (n = 3): 282.3 +/- 113.0, 21.9 +/- 11.0, respectively; right atrium (n = 5): 308.3 +/- 95.4, 43.1 +/- 12.8, respectively; left ventricle (n = 6): 218.5 +/- 64.6, 47.9 +/- 11.9, respectively; right ventricle (n = 4): 215.1 +/- 79.8, 53.9 +/- 13.0, respectively (fmol/g wet weight, mean +/- SEM, for total IR mature ET and pro-ET-1, respectively). RP-HPLC showed peaks of immunoreactivity that coeluted with authentic ET-1 in all extracts of human left atria and ventricle tested. In addition, peaks were also present corresponding to ET-2, ET-3, and pro-ET-1. These results suggest that in addition to ET-1 and pro-ET-1, ET-2 and ET-3 are present in the human heart.

Characterization of [125I]-PD164333, an ETA selective non-peptide radiolabelled antagonist, in normal and diseased human tissues

We have synthesized a new low molecular weight, non‐peptide radioligand, [125I]‐PD164333, an analogue of the orally active butenolide antagonists of the endothelin ETA receptor. Analysis of saturation binding assays demonstrated that [125I]‐PD164333 bound with high affinity to a single population of receptors (n3 individuals ±s.e.mean) in human aorta (KD=0.26±0.08 nM; Bmax=8.8±3.95 fmol mg‐1 protein), left ventricle from the heart (KD=0.16±0.02 nM; Bmax=34.2± 3.02 fmol mg‐1 protein) and kidney (KD=1.24±0.16 nM; Bmax=125.3±35.07 fmol mg‐1 protein). In each case Hill slopes were close to unity. In kinetic experiments, the binding of [125I]‐PD164333 to ETA receptors in sections of heart was time‐dependent and rapid at 23°C. The data were fitted to a one site model, with an association rate constant (K1 of 2.66±0.213×108 M‐1 min‐1, and a half‐time for association of 11 min. The binding was reversible at 23°C: analysis of the data indicated [125I]‐PD164333 dissociated from a single site, with a dissociation rate constant of 0.0031±0.0004 min‐1, a half‐time for dissociation of 216 min and a KD calculated from these kinetic data of 0.01 nM. Unlabelled PD164333 inhibited the binding of [125I]‐ET‐1 to left ventricle (which expresses both subtypes) in a biphasic manner with a KDETA of 0.99±0.32 nM and KDETB of 2.41±0.22 μM, giving a selectivity of 2500 fold. ETA‐selective ligands competed monophasically for [125I]‐PD164333 binding in left ventricle, a one site fit was preferred to a two site model giving similar nanomolar affinities: BQ123, KD=3.93 ±0.18 nM; FR139317 KD=3.53±0.69 nM. In contrast, the ETB selective agonists, BQ3020 and sarafotoxin S6c (1 μM) did not inhibit binding. In human isolated saphenous vein, unlabelled PD164333 was a functional antagonist, producing parallel rightward shifts of the endothelin‐1 (ET‐1) concentration‐response curve (pA2=8.84) and a slope of unity. In the human brain, autoradiography revealed high levels of [125I]‐PD164333 binding to the pial arteries of the cerebral cortex and to the numerous smaller intercerebral vessels penetrating the underlying grey and white matter. Conduit and resistance vessels contributing to the control of blood pressure from the heart, kidney, lungs and adrenal also displayed high densities of binding. In diseased vessels, binding of [125I]‐PD164333 was confined to the medial layer of both coronary arteries with advanced atherosclerotic lesions or occluded saphenous vein grafts. In contrast, little or no binding was detected in the proliferated smooth muscle of the intimal layer or occluded lesion. These results show [125I]‐PD164333 is a specific, high affinity, reversible non‐peptide radioligand for human ETA receptors, which will facilitate the further characterization of this subtype, in vitro and in vivo.

AT2 receptor stimulation may halt progression of pheochromocytoma.

Abstract:  Two patients with pheochromocytoma are described in whom treatment with a high dose of an angiotensin receptor blocker was associated with cessation of tumor growth as assessed by serial CT scanning and plasma norepinephrine estimation. If the association with drug treatment is causal, the mechanism may be through stimulation of the AT2 receptor consequent upon the elevation of plasma angiotensin II levels induced by AT1 receptor blockade. Similar observations in further patients should lead to a placebo‐controlled study in patients with malignant or familial pheochromocytoma, or other tumors found to express the AT2 receptor.

Analysis of endogenous human endothelin peptides by high-performance liquid chromatography and mass spectrometry

Summary: The three isoforms of both endothelin (ET) and big ET are not readily distinguishable by radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) alone. We have previously used high-performance liquid chromatography (HPLC) linked with RIA to determine ET isoforms in various tissue extracts, and cell supernatants. Our aim was to confirm that the isoforms of ET secreted by human umbilical vein endothelial cells (HUVECs) are ET-1 and big ET-1, using HPLC linked to electrospray mass spectrometry (ESI-MS). Authentic synthetic peptide standards were used to obtain the specific ions for ET isoforms and to calculate their retention times on the HPLC system. ET-1, [Met7]-sulfoxy ET-1, ET-2, ET-3, and big ET-1 could all be separated by HPLC and by specific ions on ESI-MS. Pooled supernatant (500 ml) from 50 cultures of HUVECs was applied first to an anti-C-terminal ET-1 immunoaffinity column and then to an anti-C-terminal big ET-1 immunoaffinity column. The eluates were solid phase-extracted and then injected onto an HPLC system linked to ESI-MS. Peaks of mass intensity at the specific ions and retention times corresponding to ET-1, [Met7]-sulfoxy ET-1, and big ET-1 were found in these eluates. However, no signal was present for ET-2 or ET-3. These data confirm that HUVECs in culture produce ET-1, [Met7]-sulfoxy ET-1, and big ET-1.

Response to Letter Regarding Article, “The Spironolactone, Amiloride, Losartan, and Thiazide (SALT) Double-Blind Crossover Trial in Patients With Low-Renin Hypertension and Elevated Aldosterone-Renin Ratio”

BACKGROUND There is continuing variation in diagnosis and estimated prevalence of primary hyperaldosteronism. The higher estimates encourage search for adrenal adenomas in patients with elevated ratios of plasma aldosterone to renin. However, it is more likely that patients with normal plasma K+ and aldosterone belong to the polygenic spectrum of low-renin hypertension rather than have the same monogenic syndrome as classic Conns. Our primary hypothesis was that in low-renin patients with normal plasma K+ and aldosterone, a thiazide diuretic, bendroflumethiazide, would be as effective as spironolactone in overcoming the Na+ retention and lowering blood pressure. Secondary objectives were to compare the dose response for each diuretic and to evaluate amiloride as an alternative to spironolactone. METHODS AND RESULTS Fifty-seven patients entered and 51 patients completed a placebo-controlled, double-blind, randomized crossover trial. Entry criteria included low plasma renin, normal K+, elevated aldosterone-renin ratio, and a previous systolic blood pressure response to spironolactone of > or = 20 mm Hg. Two doses each of spironolactone and bendroflumethiazide were compared. The crossover also included amiloride and losartan. Outcome measures were blood pressure, plasma renin, and other biochemical markers of diuretic action. Spironolactone 100 mg and bendroflumethiazide 5 mg caused similar falls in systolic blood pressure, whereas bendroflumethiazide 2.5 mg was 5/2 mm Hg less effective in reducing blood pressure than either bendroflumethiazide 5 mg or spironolactone 50 mg (P<0.005). Amiloride 40 mg was as effective as the other diuretics. Biochemical indices of natriuresis showed bendroflumethiazide to be less effective than either spironolactone or amiloride; plasma renin rose 4-fold on spironolactone but only 2-fold on bendroflumethiazide (P=0.003). CONCLUSIONS In hypertensive patients with a low plasma renin but normal K+, bendroflumethiazide 5 mg was as effective as spironolactone 100 mg in lowering blood pressure, despite patients being selected for a previous large fall in blood pressure on spironolactone. Because this result differs from that expected in primary hyperaldosteronism, our finding argues against low-renin hypertension including a large, undiagnosed pool of primary hyperaldosteronism. However, spironolactone was the more effective natriuretic agent, suggesting that inappropriate aldosterone release or response may still contribute to the Na+ retention of low-renin hypertension.