Sean P. Allen

Differential Leukotriene Constrictor Responses in Human Atherosclerotic Coronary Arteries

BACKGROUND Leukotrienes are a class of biologically active lipids that have potent effects on the heart. To assess their role in coronary artery disease, we compared the contractile responses of leukotriene C4 (LTC4) and leukotriene D4 (LTD4) and their binding activity in both atherosclerotic and nonatherosclerotic human coronary arteries. We also studied expression of the enzymes that control their formation to understand how the 5-lipoxygenase (5-LO) pathway is activated in the coronary arteries. METHODS AND RESULTS The capacity of leukotrienes to affect coronary vessel tone and the influence of atherosclerosis was tested in organ baths. Leukotriene receptors were examined by autoradiography, and antibody binding to the various enzymes responsible for their formation was assessed by use of immunocytochemistry. Nonatherosclerotic coronary artery ring segments were unresponsive to LTC4 and LTD4. In contrast, LTC4 and LTD4 induced concentration-dependent contractions in atherosclerotic coronary arteries. Specific [3H]-LTC4 but not LTD4 binding to atherosclerotic coronary artery was evident, with no evidence of specific binding of [3H]-leukotrienes to nonatherosclerotic coronary artery. High-resolution autoradiography identified specific [3H]-LTC4 binding sites to smooth muscle cell and to regions of intimal proliferation and plaque. Cells showing positive antibody binding to 5-LO, FLAP (5-lipoxygenase activating protein), and leukotriene A4 hydrolase were also present in the coronary arteries and had a similar distribution to macrophages. CONCLUSIONS Atherosclerosis is associated with a specific leukotriene receptor(s) capable of inducing hyperreactivity of human epicardial coronary arteries in response to LTC4 and LTD4.

Endothelin-1 stimulates proliferation of human coronary smooth muscle cells via the ETA receptor and is co-mitogenic with growth factors

We investigated the effects of endothelin-1 (ET-1) on growth of cultured human coronary artery smooth muscle cells (cSMC). ET-1 alone stimulated DNA synthesis in growth-arrested cSMC as measured by [3H]thymidine incorporation, with a maximum 63 +/- 23% increase above control by 10(-7) M (P < 0.05). ET-1 (10(-7) M) also stimulated increases in cyclin D1 protein levels after 24 h, and in absolute cell number after 4 days. Furthermore, ET-1 stimulated protein synthesis (maximum 73 +/- 32% increase in [3H]leucine incorporation by 10(-7) M (P < 0.05)), as well as triggering intracellular calcium transients in human cSMC, as visualised under fura-2 fluorescence microscopy. The selective ET(A) receptor antagonist BQ123 inhibited the increases in DNA synthesis, cell number, protein synthesis and intracellular calcium concentration in response to ET-1, whereas the ET(B) receptor antagonist BQ788 had no such effects. Furthermore, the ET(B) agonist sarafotoxin 6c had no effect on cSMC DNA synthesis. In addition, co-incubation of ET-1 with threshold concentrations of the growth factors, platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), resulted in pronounced synergistic increases in DNA synthesis over that observed with the factors alone. In conclusion, we have shown that ET-1 stimulates proliferation of human cSMC via the ET(A) receptor and is also a co-mitogen with the growth factors tested. These findings indicate a role for ET-1 in the development of coronary intimal hyperplasia in man.

Expression of adhesion molecules by Lp(a): a potential novel mechanism for its atherogenicity

Lp(a) is a major inherited risk factor for premature atherosclerosis. The mechanism of Lp(a) atherogenicity has not been elucidated, but likely involves both its ability to interfere with plasminogen activation and its atherogenic potential as a lipoprotein particle after receptor‐mediated uptake. We demonstrate that Lp(a) stimulates production of vascular cell adhesion molecule 1 (VCAM‐1) and E‐selectin in cultured human coronary artery endothelial cells (HCAEC). This effect resulted from a rise in intracellular free calcium induced by Lp(a) and could be inhibited by the intracellular calcium chelator, BAPTA/AM. The involvement of the LDL and VLDL receptors in Lp(a) activation of HCAEC were ruled out since Lp(a) induction of adhesion molecules was not prevented by an antibody (IgGC7) to the LDL receptor or by receptor‐activating protein, an antagonist of ligand binding to the VLDL receptor. Addition of α2‐macroglobulin as well as treatment with heparinase, chondroitinase ABC, and sodium chlorate did not decrease levels of VCAM‐1 and E‐selectin stimulated by Lp(a), suggesting that neither the low density lipoprotein receptor‐related protein nor cell‐surface proteoglycans are involved in Lp(a)‐induced adhesion molecule production. Neither does the binding site on HCAEC responsible for adhesion molecule production by Lp(a) appear to involve plasminogen receptors, as levels of VCAM‐1 and E‐selectin were not significantly decreased by the addition of glu‐plasminogen, the lysine analog ε‐aminocaproic acid, or by trans‐4‐(aminomethyl)‐cyclohexanecarboxymethylic acid (tranexamic acid), which acts by binding to the lysine binding sites carried on the kringle structures in plasminogen. In contrast, recombinant apolipoprotein (a) [r‐apo(a)] competed with Lp(a) and attenuated the expression of VCAM‐1 and E‐selectin. In summary, we have identified a calcium‐dependent interaction of Lp(a) with HCAEC capable of inducing potent surface expression of VCAM‐1 and E‐selectin that does not appear to involve any of the known potential Lp(a) binding sites. Because leukocyte recruitment to the vessel wall appears to represent one of the important early events in atherogenesis, this newly described endothelial cell‐activating effect of Lp(a) places it at a crucial juncture in the initiation of atherogenic disease and may lead to a better understanding of the role of Lp(a) in the vascular biology of atherosclerosis.—Allen, S., Khan, S., Tam, S.‐P., Koschinsky, M., Taylor, P., Yacoub, M. Expression of adhesion molecules by Lp(a): a potential novel mechanism for its atherogenicity. FASEB J. 12, 1765–1776 (1998)

Expression of functional angiotensin-converting enzyme and AT1 receptors in cultured human cardiac fibroblasts

BACKGROUND Angiotensin II (Ang II) has been implicated in the development of cardiac fibrosis. The aims of the present study were to examine expression and activity of ACE and of angiotensin receptors in human cardiac fibroblasts cultured from dilated cardiomyopathic and ischemic hearts. The effects of Ang II on fibroblasts were also investigated. METHODS AND RESULTS Human cardiac fibroblasts were cultured from ventricular and atrial myocardium and characterized immunohistochemically. Expression of ACE and the angiotensin AT1 receptor was demonstrated in cardiac fibroblasts by reverse transcriptase-polymerase chain reaction and radioligand binding. Functional ACE activity, measured by radiolabeled substrate conversion assay, was detected in both ventricular (Vmax. Km-1. mg-1, 0.031+/-0.010; n=13) and atrial (0. 034+/-0.012; n=6) fibroblasts. Fibroblast ACE activity was increased after 48 hours of treatment with basic fibroblast growth factor, dexamethasone, and phorbol ester. Ang II did not affect DNA synthesis but stimulated [3H]proline incorporation in cardiac fibroblasts (20.0+/-4.0% increase above control by 10 micromol/L; P<0.05, n=7), which was abolished by losartan 10 micromol/L but not PD123319 1 micromol/L. Ang II also stimulated a rise in intracellular calcium (basal, 56+/-1 nmol/L; Ang II, 355+/-24 nmol/L) via the AT1 receptor, as shown by complete inhibition with losartan. CONCLUSIONS We have demonstrated expression and activity of ACE and AT1 receptor in cultured human cardiac fibroblasts. In addition, cardiac fibroblasts respond to Ang II with AT1 receptor-mediated collagen synthesis. The presence of local ACE and AT1 receptors in human fibroblasts suggests their involvement in the development of cardiac fibrosis.

Enhanced excretion of urinary leukotriene E4 in coronary artery disease and after coronary artery bypass surgery.

Background:Excretion of leukotriene (LT) E4, the major urinary metabolite of cysteinyl leukotrienes in humans, is increased in patients with unstable angina and myocardial infarction, suggesting that cysteinyl leukotrienes are released into the circulation during episodes of myocardial ischaemia. Furthermore, leukotrienes are known to induce potent vasoconstrictive effects in human atherosclerotic coronary arteries and the saphenous vein. Accordingly, we measured urinary excretion of LTE4 in patients with stable coronary artery disease both before and after coronary artery bypass surgery, and in age-matched healthy controls, to study the relation between the systemic synthesis of cysteinyl leukotrienes and stable coronary artery disease, as well as the possible changes after bypass surgery. Methods:LTE4 was isolated from urine samples by solid-phase extraction, purified by reverse-phase high-performance liquid chromatography, and subsequently quantified by radioimmunoassay. Results:In patients with coronary artery disease, preoperative urinary LTE4 levels were normally distributed on a log10 scale, with a geometric mean of 115 pmol/mmol creatinine (95% confidence interval 67–196) compared with 63.0 pmol/mmol creatinine (95% confidence interval 47.9–82.7) in healthy subjects (P<0.05). Urinary LTE4 levels increased further in patients after coronary artery bypass surgery with levels peaking on the second day after surgery (266.2 pmol/mmol creatinine, 95% confidence interval 167.2–423.9) at significantly higher than preoperative levels (P<0.02), and then decreasing by day 3. Conclusions:Levels of cysteinyl leukotrienes are raised in coronary artery disease patients both before and after coronary artery bypass surgery. As these mediators are capable of inducing potent vasoconstrictive effects on atherosclerotic coronary arteries and the saphenous vein, our results could have important clinical and possibly therapeutic implications.

Inability of plasma high-density lipoproteins to inhibit cell adhesion molecule expression in human coronary artery endothelial cells

High-density lipoproteins (HDL) have several antiatherogenic actions, including the ability to sequester cellular cholesterol, to protect low-density lipoproteins from oxidation and to inhibit platelet aggregation. An early event in atherogenesis is the adhesion and recruitment of blood monocytes, a process mediated by cell adhesion molecules (CAMs), including vascular cell adhesion molecule-1 (VCAM-1) which is rapidly synthesized by endothelial cells in response to cytokines. It has been reported that HDL limits CAM expression in cultured human umbilical vein endothelial cells (HUVECs), implying that HDL also protects at an early stage in lesion development. Here, we have studied HDL suppression of CAM induction in human coronary artery endothelial cells (HCAECs), a model directly relevant to blood vessels susceptible to atherosclerosis. Arterial endothelial cells were preincubated with increasing amounts of total HDL, or different subfractions, and then activated with the inflammatory cytokine, tumor necrosis factor-alpha (TNF-alpha). Flow cytometric analysis failed to detect any downregulation of VCAM-1 or E-selectin expression by HDL in this model of vascular endothelium. Moreover, we were unable to confirm that HDL could suppress CAM induction in well-characterized, low-passage HUVECs, even though positive controls, 17beta-estradiol or a nitric oxide donor, did cause downregulation and factors such as variability in donors and HDL preparation, or culture conditions, were excluded. We tentatively conclude that, as isolated HDL did not downregulate CAM expression in cultured HCAECs or HUVECs, attenuation of CAM induction in arterial endothelium is unlikely to contribute to HDL antiatherogenic actions in vivo.

GROWTH RESPONSE OF HUMAN CORONARY SMOOTH MUSCLE CELLS TO ANGIOTENSIN II AND INFLUENCE OF ANGIOTENSIN AT1 RECEPTOR BLOCKADE

BackgroundThe renin-angiotensin system has been implicated in the development of vascular wall thickening in cardiovascular disease, through the growth-promoting actions of the vasoconstrictive agent, angiotensin II, on vascular smooth muscle cells. ObjectiveTo investigate the effect of angiotensin II on growth of human coronary artery smooth muscle cells (cSMCs) in culture, and to identify the angiotensin receptor(s) mediating such a response. MethodsHuman cSMCs were isolated from coronary arteries of recipient hearts obtained during transplantation, and characterized by immunohistochemistry. The effect of angiotensin II on protein synthesis by cSMCs was measured by [3H]leucine incorporation and protein concentration assays. Human cSMC proliferation was assessed by [3H]thymidine incorporation assay and cell count. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect angiotensin receptor expression. Transient increases in intracellular calcium concentration in cSMCs in response to angiotensin II stimulation were visualized under fura-2 fluorescence microscopy. ResultsAngiotensin II (1 nmol/l-10 μmol/l) stimulated protein synthesis in cSMCs (maximum 24 ± 2% increase in incorporation of [3H]leucine over 48 h; n = 4, P < 0.01). An increase in cellular protein content was also measured. However, angiotensin II had no effect on proliferation of quiescent cSMCs. The increased protein synthesis was completely inhibited by pretreatment with the angiotensin AT1 receptor antagonist, losartan, but not the AT2 receptor antagonist, PD123319. Expression of the angiotensin AT1 receptor subtype was detected in cSMCs by RT-PCR. Angiotensin II stimulation of cells triggered transient increases in intracellular calcium concentration, which were abolished by losartan, but were insensitive to PD123319 and pertussis toxin. ConclusionsThe results of this study in human coronary VSMCs indicate that angiotensin II and the AT1 receptor may be involved in the development of coronary artery disease in man.

Unique Sensitivities to Cytokine Regulated Expression of Adhesion Molecules in Human Heart-Derived Endothelial Cells

The expression of adhesion molecules by endothelial cells is crucial in many inflammatory processes and plays an active role in the development of reperfusion injury, acute and chronic rejection. The expression of adhesion molecules in different parts of the coronary tree to cytokine stimulation is not known. We describe here a detailed study of the effects of the inflammatory cytokines TNFalpha and IL-1beta on the expression of adhesion molecules vascular cell adhesion molecule-1 (VCAM-1), E-selectin and intracellular cell adhesion molecule-1 (ICAM-1) on human aortic root (HAEC), coronary artery (HCAEC) and heart microvascular (HHMEC)) endothelial cells in culture, using flow cytometry. We found constitutive levels of both VCAM-1 and E-Selectin on HCAEC and HHMEC (approximately 20%) which were significantly higher compared to HAEC (approximately 3%). There was an extreme sensitivity of HCAEC and HHMEC to 0.002 ng/ml TNFalpha: (VCAM-1 approximately 40%, E-Selectin approximately 25%) respectively, compared to HAEC (VCAM-1 approximately 5%, E-selectin approximately 5%). IL-1beta showed a similar pattern of expression at low doses (5 U/ml), but was less potent. We also observed prolonged expression of these adhesion molecules, especially on the HHMEC (>48 hours) compared to HAEC. There was also increased binding of peripheral blood mononuclear cells (PBMC) to both non-stimulated and TNFalpha stimulated HCAEC and HHMEC compared to HAEC. This data suggest that endothelial cells in different regions of the coronary tree express different patterns of basal and cytokine-stimulated adhesion molecule expression.

Altered endothelial and smooth muscle cell reactivity caused by University of Wisconsin preservation solution in human saphenous vein

PURPOSE We have investigated the effect of University of Wisconsin (UW) solution at different temperatures on endothelial and smooth muscle cell function of the human saphenous vein to define the efficacy of UW solution as a preservation solution for saphenous vein conduits. METHODS Saphenous vein segments from 38 patients undergoing coronary artery bypass surgery were examined with an isolated organ bath technique to monitor changes in vessel reactivity. RESULTS Endothelial-dependent relaxations to acetylcholine were attenuated after incubation in UW solution at both 4 degrees C and 28 degrees C (p < 0.05, n = 10). In contrast, relaxations to sodium nitroprusside were unchanged after incubation in UW solution at both temperatures (n = 8). The responses to 90 mmol/L KCl were increased at both 4 degrees C and 28 degrees C, respectively. Tyrodes: 27.2% +/- 3.1% and 23.8% +/- 3.0%, UW: 64.7% +/- 8.0% and 73.1% +/- 11% (p < 0.001). In addition, the responses to 5-HT were enhanced at 4 degrees C and 28 degrees C (p < 0.05). In contrast, responses to noradrenaline were enhanced only at 28 degrees C compared with the responses after incubations in Tyrodes solution (p < 0.05, n = 6). Furthermore U46619 (0.3 nmol/L to 1 nmol/L) responses were augmented at 4 degrees C (p < 0.05, n = 7). The potency (pD2) values for each agonist were not significantly different after incubations in UW solution. CONCLUSIONS We conclude that UW solution produces attenuation of acetylcholine relaxations and temperature-dependent increased reactivity of smooth-muscle cell function in the isolated human saphenous vein. These studies document the complex interactions brought about by UW solution on the different components of the vascular wall that need to be elucidated further if this solution is to attain a place in vascular preservation.

Effect of aprotinin on vascular reactivity of coronary bypass grafts

OBJECTIVE Aprotinin reduces postoperative bleeding and the need for transfusion after cardiopulmonary bypass. The current clinical concern about aprotinin is that it may increase the incidence of postoperative graft thrombosis and thromboembolic phenomena. The fact that the mechanism of action of aprotinin is not completely elucidated and that its effects on the vascular reactivity of bypass conduits are unknown raise doubts regarding its safety. In an attempt to clarify these issues we investigated the vascular reactivity of the human saphenous vein and internal thoracic artery to a range of vasoconstrictor agents in the presence or absence of aprotinin. METHODS Human saphenous vein was obtained from 24 patients and internal thoracic artery from 7 patients undergoing coronary artery bypass. Vessels were set up in organ baths to record changes in vessel wall tension. RESULTS Endothelium-dependent relaxations to acetylcholine in saphenous vein rings were unaffected after aprotinin treatment. Contractions to the thromboxane analog U46619 were significantly attenuated after aprotinin treatment in the saphenous vein. Maximum responses were reduced from control values of 88 +/- 7.5 mN to 49.3 +/- 4.8 mN with 1 mumol/L doses of aprotinin (p < 0.05) and 36.6 +/- 4.8 mN with 10 mumol/L doses of 5-hyroxytryptamine or noradrenaline after aprotinin incubations. Furthermore, contractions to U46619 in the internal thoracic artery were unaffected by aprotinin. CONCLUSION Our data show that there is a preservation of endothelium-dependent responses to acetylcholine and a reduced U46619 vasoconstrictor action on the saphenous vein after aprotinin treatment. Thus the direct effect of aprotinin on the vessel wall could counteract the potential effect of its prothrombotic action on graft patency.