John R. Tippins

Ischemia Modified Albumin Is a Sensitive Marker of Myocardial Ischemia After Percutaneous Coronary Intervention

Background—Ischemia modified albumin (IMA; Ischemia Technologies, Inc) blood levels rise in patients who develop ischemia during percutaneous coronary intervention (PCI). It is not known whether IMA elevations correlate with increases in other markers of oxidative stress, ie, 8-iso prostaglandin F2-A (iP). Methods and Results—We compared IMA versus iP plasma levels in 19 patients (mean age 62.8±11.9 years) undergoing PCI and 11 patients (mean age 64±13.6 years) undergoing diagnostic angiography (controls). In the PCI patients, blood samples for IMA and iP were taken from the guide catheter before PCI and after balloon inflations, and from the femoral sheath 30 minutes after PCI. IMA was measured by the albumin cobalt binding (ACB) test and plasma iP by enzyme immunoassay. During PCI, all 19 patients had chest pain and 18 had transient ischemic ST segment changes. IMA was elevated from baseline in 18 of the 19 patients after PCI. Median IMA levels were higher after PCI (101.4 U/mL, 95%CI 82 to 116) compared with baseline (72.8 U/mL, CI 55 to 93;P <0.0001). Levels remained elevated at 30 minutes (87.9 U/mL, CI 78 to 99;P <0.0001) and returned to baseline at 12 hours (70.3 U/mL, CI 65 to 87;P =0.65). iP levels were raised after PCI in 9 of the 19 patients. However, median iP levels were not significantly different immediately (P =0.6) or 30 minutes after PCI (P =0.1). In the control group, IMA and iP levels remained unchanged before and after angiography (P =0.2 and 0.16, respectively). Conclusions—IMA is a more consistent marker of ischemia than iP in patients who develop chest pain and ST segment changes during PCI.

The myotropic and plasma-calcium modulating effects of calcitonin gene-related peptide (CGRP).

Human and rat calcitonin gene-related peptides cause a dose-related contraction of guinea pig ileum, which is antagonised by an anti-histamine, mepyramine, and an anticholinergic compound, hyoscine. Both peptides also cause a positive inotropic and a positive chronotropic effect in the rat isolated auricle and these responses are antagonised by propranolol, a B adrenoceptor blocker. Further, the peptides lower plasma calcium levels in both rats and rabbits in a dose-related manner resembling calcitonin; in the rabbit, but not in the rat, the initial calcium lowering effect is succeeded by hypercalcaemia at higher doses, while in the chick, only the parathyroid hormone-like calcium-raising effect is seen.

Reactive oxygen species mediate endothelium-dependent relaxations in tetrahydrobiopterin-deficient mice.

(6 R)-5,6,7,8-Tetrahydro-biopterin (H4B) is essential for the catalytic activity of all NO synthases. The hyperphenylalaninemic mouse mutant (hph-1) displays 90% deficiency of the GTP cyclohydrolase I, the rate-limiting enzyme in H4B synthesis. A relative shortage of H4B may shift the balance between endothelial NO synthase (eNOS)-catalyzed generation of NO and reactive oxygen species. Therefore, the hph-1 mouse represents a unique model to assess the effect of chronic H4B deficiency on endothelial function. Aortas from 8-week-old hph-1 and wild-type mice (C57BL×CBA) were compared. H4B levels were determined by high-performance liquid chromatography and NO synthase activity by [3H]citrulline assay in homogenized tissue. Superoxide production by the chemiluminescence method was measured. Isometric tension was continuously recorded. The intracellular levels of H4B as well as constitutive NO synthase activity were significantly lower in hph-1 compared with wild-type mice. Systolic blood pressure was increased in hph-1 mice. However, endothelium-dependent relaxations to acetylcholine were present in both groups and abolished by inhibition of NO synthase with NG-nitro-l-arginine methyl ester as well. Only in hph-1 mice were the relaxations inhibited by catalase and enhanced by superoxide dismutase. After incubation with exogenous H4B, the differences between the 2 groups disappeared. Our findings demonstrate that H4B deficiency leads to eNOS dysfunction with the formation of reactive oxygen species, which become mediators of endothelium-dependent relaxations. A decreased availability of H4B may favor an impaired activity of eNOS and thus contribute to the development of vascular diseases.

Coronary artery constriction by the isoprostane 8-epi prostaglandin F2α

1 This study was undertaken to compare the effects of 8‐epi prostaglandin F2α (8‐epi PGF2α) to those of prostaglandin F2α (PGF2α) and U46619, a thromboxane mimetic, on ovine, bovine and porcine coronary arteries. 2 8‐epi PGF2α constricted porcine and bovine coronary arteries in a concentration‐dependent manner with EC50 values of 689.0 ± 229.3 and 1361.0 ± 272.3 nM, respectively, but had no effect on ovine coronary arteries. 3 U46619 was a potent vasoconstrictor of porcine, ovine and bovine coronary arteries with EC50 values of 33.0 ± 23.5, 373.3 ± 69.7 and 254.1 ± 134.3 nM, respectively. Emax values were significantly greater than those obtained with 8‐epi PGF2α. 4 PGF2α constricted porcine and bovine coronary arteries in a concentration‐dependent manner with EC50 values of 1631.0 ± 207.6 and 3644.0 ± 344.8 nM, respectively, but had no effect on ovine coronary arteries. 5 Concentration‐dependent constriction to U46619 in porcine coronary arteries was competitively inhibited by SQ29548 (10−8 m to 10−7 m) and BM13505 (10−8 m to 10−6 m) with no decrease in maximal responses. 6 Concentration‐dependent constriction to 8‐epi PGF2α in porcine coronary arteries was inhibited in a concentration‐dependent manner by SQ29548 (10−8 m to 10−7 m) and BM13505 (10−8 m to 10−6 m). However, the inhibition was associated with a decrease in maximal response. 7 Maximal responses of porcine coronary artery to U46619 (1 μm) and 8‐epi PGF2α (30 μm) were inhibited in a concentration‐dependent manner by SQ29548 with IC50 values 99 ± 12.36 nM and 46.5 ± 18.67 nM, respectively. 8 Although ovine coronary arteries did not constrict to 8‐epi PGF2α, pre‐incubation of these vessels with 8‐epi PGF2α caused a rightward shift of the U46619 response curve in a concentration‐dependent manner. 9 Pre‐incubation of porcine coronary arteries with 8‐epi PGF2aL competitively inhibited responses to U46619 with a Schild slope of 0.99 and a pA2 of 6.13. 10 We conclude that 8‐epi PGF2α is a vasoconstrictor within porcine and bovine coronary arteries, with a potency approximately twice that of PGF2α but 5–20 times lower than U46619. The data suggest that 8‐epi PGF2α is acting as a partial agonist on the TP‐receptor in the coronary vasculature.

Investigation of the structure/activity relationship of human calcitonin gene-related peptide (CGRP).

The biological activities of calcitonin gene-related peptide (CGRP) enzymic digest fragments, chemically modified products and beta-CGRP have been compared to that of intact alpha-CGRP on rat isolated paired atria. Tryptic and chymotryptic digests both produced inactive fragments. Acetylation of the N-terminal amino acid (Alanine) or either of Lys 24 or Lys 35, resulted in reduced, but measurable, biological activity. Destruction of the disulphide bridge between Cys 2 and Cys 7 abolished biological activity. Substitution of several amino acids, Asp 3, Val 22 and Asn 25, with Asn, Met and Ser respectively (beta-CGRP), produced a peptide with similar biological activity to alpha-CGRP.

Regional Variations in Endothelin-1 and its Receptor Subtypes in Human Coronary Vasculature: Pathophysiological Implications in Coronary Disease

Endothelin-1 is a potent vasoconstrictor peptide and mitogen for vascular smooth muscle cells. Increased plasma or tissue levels of endothelin-1 have been described after myocardial infarction and in atherosclerosis, suggesting that this peptide may play a pathophysiological role in various coronary syndromes. Here, we have studied regional variations in ET-1 and its receptors in control and atherosclerotic human coronary vasculature using standard immunohistochemistry and in vitro autoradiography. ET-1 immunoreactivity was associated with luminal endothelial cells and smooth muscle cells at regions of atherosclerosis. ET(A) receptors were present on smooth muscle cells of coronary arteries and on cardiac myocytes. Medial ET(B) receptor binding at the proximal region of coronary arteries was weak, but increased significantly towards distal regions of this vessel (p<0.005 in control and p<0.0005 in ischaemic heart disease). Microvascular endothelial cells in the adventitia of coronary arteries, myocardial microvessels and the endocardial endothelium expressed the ET(B) receptor exclusively. The receptor variations revealed in this study provide supporting evidence that ET-1 is associated with (1) vascular smooth muscle and endothelial cell proliferation, including areas of intimal hyperplasia and regions of neovascularization (2) increased ET-1-induced reactivity of distal portions of the human coronary artery, (3) ET-1-mediated constriction of myocardial microvessels. These results provide new insights into different potential roles for this peptide in healthy and diseased human coronary vasculature.

The vasoconstrictor effect of 8‐epi prostaglandin F2α in the hypoxic rat heart

8‐epi prostaglandin (PG) F2α, a vasoconstrictor isoprostane, is synthesized under conditions of oxidative stress. This study was undertaken to investigate the vasoconstrictor effect of 8‐epi PGF2α in the coronary circulation before and after a period of oxidative stress. The effects of the isoprostane 8‐epi PGF2α and the thromboxane mimetic U46619 were compared in the isolated rat heart perfused in the Langendorff mode at a constant pressure of 80u2003mmHg. In normal hearts U46619 caused a dose‐related reduction in coronary flow (ED50 4.7±2.2u2003nmol). In contrast, 8‐epi PGF2α had no effect. After reducing perfusion pressure to 20u2003mmHg for 30u2003min and reperfusing at 80u2003mmHg, the dose‐response curve to U46619 was unaffected. In contrast, 8‐epi PGF2α caused a dose‐dependent drop in coronary flow (ED50 52.6±12.7u2003nmol), producing a similar maximal reduction to U46619. Similarly, after perfusion with xanthine and xanthine oxidase for either 15 or 30u2003min there was little change in the response to U46619 in comparison to control hearts. In contrast, 8‐epi PGF2α caused a reduction in coronary flow similar to that produced by U46619, the magnitude of the response being related to the length of xanthine/xanthine oxidase perfusion. Responses to both U46619 and 8‐epi PGF2α after xanthine/xanthine oxidase perfusion were blocked by the selective thromboxane receptor antagonist SQ29548 10−7u2003M. These results show that oxidative stress in the isolated perfused rat heart reveals a potent vasoconstrictor effect of the isoprostane 8‐epi PGF2α by an action on the thromboxane receptor. The data also suggest that, since 8‐epi PGF2α is a partial agonist at the thromboxane receptor, thromboxane receptor reserve is increased by oxidative stress.

7-Ketocholesterol and 5,6-secosterol induce human endothelial cell dysfunction by differential mechanisms.

7-Ketocholesterol and 5,6-secosterol are cholesterol autoxidation products generated under oxidative stress by two distinct mechanisms. They are present in atherosclerotic plaques and are candidate players in the disease initiation and progression. While 7-ketocholesterol affects at cellular level, in particular apoptosis, are well known and reported on diverse cell lines, 5,6-secosterol is a recently discovered oxysterol with relatively few reports on the potential to affect endothelial cell functions. Endothelial cells have a central role in cardiovascular disease as they provide the barrier between blood and the vessel wall where atherosclerosis starts and progresses. Insults to endothelial cells provoke their dysfunction favoring pro-atherogenic and pro-thrombotic effects. In the present work, we tested 7-ketocholesterol and 5,6-secosterol on endothelial cells - focusing on apoptosis and the associated mitochondrial/lysosome alterations - and on endothelial function using the in vitro model of arterial relaxation of aortic rings. Our data provide evidence that 7-ketocholesterol and 5,6-secosterol are efficient instigators of apoptosis, which for 5,6-secosterol is associated to PKC and p53 up-regulation. In addition 5,6-secosterol is a potent inhibitor of endothelial-dependent arterial relaxation through PKC-dependent mechanisms. This may contribute to pro-atherogenic and pro-thrombotic mechanisms of 5,6-secosterol and highlights the role of cholesterol autoxidation in cardiovascular disease.

Hypothalamic Hypertensive Factor: An Inhibitor of Nitric Oxide Synthase Activity

Human and rat plasma and rat hypothalamus contain a cytochemically detectable substance, the concentration of which rises with an increase in salt intake. The plasma concentration of this material is also raised in essential hypertension and in the spontaneously hypertensive rat (SHR), the Milan hypertensive rat, and the reduced renal mass (RRM) hypertensive rat. In the normal rat, the greatest concentration is found in the hypothalamus of the SHR and the RRM hypertensive rat. The physicochemical characteristics of this cytochemically detectable hypothalamic hypertensive factor (HHF), including chromatographic behavior and molecular weight range, suggest that it may share features common to a substituted guanidine that is present in established nitric oxide synthase (NOS) inhibitors. It was therefore decided to determine the effect on NOS activity of the HHF obtained from mature SHR. The ability of HHF to inhibit NOS activity was studied on (1) NOS extracted from bovine aorta, rat brain, and human platelets by measuring the conversion of radiolabeled L-arginine to L-citrulline and (2) rat liver NOS measured indirectly with a cytochemical technique based on the stimulation of soluble guanylate cyclase activity in hepatocytes by NO. HHF showed a biphasic inhibitory action on platelet NOS activity that was greater with HHF obtained from SHR than from Wistar-Kyoto rats. HHF also had a biphasic inhibitory effect on hepatocyte NOS activity that was more potent when obtained from SHR. It is proposed that the increase in HHF, a novel form of NOS inhibitor that is elevated in SHR, may be involved in the rise in arterial pressure.

Regulation of Thromboxane Receptor Signaling at Multiple Levels by Oxidative Stress-Induced Stabilization, Relocation and Enhanced Responsiveness

Background Thromboxane A2 (TxA2) is a major, unstable arachidonic acid metabolite, and plays a key role in normal physiology and control of vascular tone. The human thromboxane receptor (TPβ), expressed in COS-7 cells, is located predominantly in the endoplasmic reticulum (ER). Brief hydrogen peroxide exposure increases the efficiency of translocation of TPβ from the ER into the Golgi complex, inducing maturation and stabilization of TPβ. However, the ultimate fate of this post-ER TPβ pool is not known, nor is its capacity to initiate signal transduction. Here we specifically assessed if functional TPβ was transported to the plasma membrane following H2O2 exposure. Results We demonstrate, by biotinylation and confocal microscopy, that exposure to H2O2 results in rapid delivery of a cohort of TPβ to the cell surface, which is stable for at least eight hours. Surface delivery is brefeldin A-sensitive, indicating that translocation of this receptor cohort is from internal pools and via the Golgi complex. H2O2 treatment results in potentiation of the increase to intracellular calcium concentrations in response to TPβ agonists U46619 and 8-iso PGF2α and also in the loss of ligand-dependent receptor internalization. Further there is increased responsiveness to a second application of the agonist. Finally we demonstrate that the effect of H2O2 on stimulating surface delivery is shared with the FP prostanoid receptor but not the EP3 or EP4 receptors. Conclusions/Significance In summary, brief exposure to H2O2 results in an immediate and sustained increase in the surface pool of thromboxane receptor that is capable of mediating a persistent hyper-responsiveness of the cell and suggests a highly sophisticated mechanism for rapidly regulating thromboxane signaling.