Phase II randomized trial of radiation therapy, cetuximab, and pemetrexed with or without bevacizumab in patients with locally advanced head and neck cancer.

BACKGROUND: We previously reported the safety of concurrent cetuximab, an antibody against epidermal growth factor receptor (EGFR), pemetrexed, and radiation therapy (RT) in patients with locally advanced squamous cell carcinoma of the head and neck (SCCHN). In this non-comparative phase II randomized trial, we evaluated this non-platinum combination with or without bevacizumab, an inhibitor of vascular endothelial growth factor (VEGF). PATIENTS AND METHODS: Patients with previously untreated stage III-IVB SCCHN were randomized to receive: conventionally fractionated radiation (70 Gy), concurrent cetuximab, and concurrent pemetrexed (arm A); or the identical regimen plus concurrent bevacizumab followed by bevacizumab maintenance for 24 weeks (arm B). The primary end point was 2-year progression-free survival (PFS), with each arm compared with historical control. Exploratory analyses included the relationship of established prognostic factors to PFS and quality of life (QoL). RESULTS: Seventy-eight patients were randomized: 66 oropharynx (42 HPV-positive, 15 HPV-negative, 9 unknown) and 12 larynx; 38 (49%) had heavy tobacco exposure. Two-year PFS was 79% [90% confidence interval (CI) 0.69-0.92; P < 0.0001] for arm A and 75% (90% CI 0.64-0.88; P < 0.0001) for arm B, both higher than historical control. No differences in PFS were observed for stage, tobacco history, HPV status, or type of center (community versus academic). A significantly increased rate of hemorrhage occurred in arm B. SCCHN-specific QoL declined acutely, with marked improvement but residual symptom burden 1 year post-treatment. CONCLUSIONS: RT with a concurrent non-platinum regimen of cetuximab and pemetrexed is feasible in academic and community settings, demonstrating expected toxicities and promising efficacy. Adding bevacizumab increased toxicity without apparent improvement in efficacy, countering the hypothesis that dual EGFR-VEGF targeting would overcome radiation resistance, and enhance clinical benefit. Further development of cetuximab, pemetrexed, and RT will require additional prospective study in defined, high-risk populations where treatment intensification is justified.

Selective neutrophil desensitization to chemotactic factors.

In the presence of extracellular calcium and magnesium, a series of chemotactic oligopeptides and C5a caused aggregation of human polymorphonuclear neutrophils (PMNs). This cellular response developed rapidly and began to reverse 2 min after exposure to the chemotactin. In the absence of the bivalent cations, none of the chemotactins stimulated the aggregation response. If cells were first exposed to a chemotactin and then treated with calcium and magnesium, aggregation was detected only after addition of the cations, and the magnitude of the response fell sharply as the interval between the addition of chemotactin and addition of cations was lengthened: when this interval exceeded 2 min, aggregation was barely detectable. This loss of reactivity persisted even when cells were re-exposed to fresh chemotactic factor and washed between the first and second exposures. In all instances, however, loss of cellular reactivity was highly selective: cells preincubated with any chemotactic oligopeptide were hyporesponsive to subsequent stimulation with an oligopeptide but remained fully responsive to C5a; cells preincubated with C5A were hyporesponsive to C5a but retained their responsitivity to the oligopeptides. Because this selectivity parallels the known specificities of these chemotactic factors for their receptors in or on the neutrophil, desensitization may reflect functional loss of receptors after stimulation. Alternatively, this selectivity may indicate that morphologically identical neutrophils contain subpopulations of cells with varying reactivities to receptor-bound chemotactic factors. In either event, desensitization may be useful in functionally defining chemotactic factors and their respective receptors. The rapidity of development of desensitization suggests that it may operate to limit or moderate various in vitro and in vivo neutrophil responses to chemotactic factors.

Recombinant superoxide dismutase reduces oxygen free radical concentrations in reperfused myocardium.

It has been proposed that oxygen free radicals mediate damage that occurs during postischemic reperfusion. Recombinant human superoxide dismutase (r-h-SOD) has been shown to be effective at reducing reperfusion injury, but it is not known if this infused enzyme actually reduces oxygen free radical concentrations in the myocardial tissue. Electron paramagnetic resonance spectroscopy was used to directly measure the effect of r-h-SOD on free radical concentrations in the postischemic heart. Hearts were freeze clamped at 77 degrees K after 10 min of normothermic global ischemia followed by 10 s of reflow with control perfusate (n = 7) or perfusate containing 60,000 U r-h-SOD (n = 7). The spectra of these hearts exhibited three different signals: signal A isotropic, g = 2.004, identical to the carbon-centered ubiquinone free radical; signal B anisotropic with axial symmetry, g parallel = 2.033, g perpendicular = 2.005, identical to the oxygen-centered alkyl peroxyl free radical; and the signal C an isotropic triplet, g parallel = 2.000, an = 24 G, similar to a nitrogen-centered free radical such as a peroxyl amine. With r-h-SOD administration the concentration of the oxygen free radical, signal B, was reduced 49% from 6.8 +/- 0.3 microM to 3.5 +/- 0.3 microM (P less than 0.01) and the concentration of the nitrogen free radical, signal C, was reduced 38% from 3.4 +/- 0.3 to 2.1 +/- 0.3 microM (P less than 0.01). The concentration of the carbon-centered free radical, signal A, however, was increased 51% from 3.3 +/- 0.2 to 5.0 +/- 0.2 microM (P less than 0.01). Identical reperfusion with peroxide-inactivated r-h-SOD did not alter the concentrations of free radicals indicating that the specific enzymatic activity of r-h-SOD is required to decrease the concentrations of reactive oxygen free radicals. Additional measurements performed varying the duration of reflow demonstrate a burst of oxygen free radical generation peaking at 10 s of reperfusion. r-h-SOD entirely abolished this burst. These studies demonstrate that superoxide-derived free radicals are generated during postischemic reperfusion and suggest that the beneficial effect of r-h-SOD is due to its specific enzymatic scavenging of superoxide free radicals.

Oxygen radicals generated at reflow induce peroxidation of membrane lipids in reperfused hearts.

To test whether generation of oxygen radicals during postischemic reperfusion might promote peroxidation of cardiac membrane lipids, four groups of Langendorff-perfused rabbit hearts were processed at the end of (a) control perfusion, (b) 30 min of total global ischemia at 37 degrees C without reperfusion, (c) 30 min of ischemia followed by reperfusion with standard perfusate, (d) 30 min of ischemia followed by reperfusion with the oxygen radical scavenger human recombinant superoxide dismutase (h-SOD). The left ventricle was homogenized and tissue content of malonyldialdehyde (MDA), an end product of lipid peroxidation, was measured on the whole homogenate as well as on various subcellular fractions. Reperfusion was accompanied by a significant increase in MDA content of the whole homogenate and of the fraction enriched in mitochondria and lysosomes. This phenomenon was not observed in hearts subjected to ischemia but not reperfused, and was similarly absent in those hearts which received h-SOD at reflow. Reperfused hearts also had significantly greater levels of conjugated dienes (another marker of lipid peroxidation) in the mitochondrial-lysosomal fraction. Again, this phenomenon did not occur in ischemic hearts or in reperfused hearts treated with h-SOD. Unlike the effect on tissue MDA and conjugated dienes, reperfusion did not significantly stimulate release of MDA in the cardiac effluent. Treatment with h-SOD was also associated with significant improvement in the recovery of cardiac function. In conclusion, these data directly demonstrate that postischemic reperfusion results in enhanced lipid peroxidation of cardiac membranes, which can be blocked by h-SOD, and therefore is most likely secondary to oxygen radical generation at reflow.

Neutropenia induced by systemic infusion of 5,12-dihydroxy-6,8,10,14-eicosatetraenoic acid: correlation with its in vitro effects upon neutrophils.

5(S), 12(S)-Dihydroxy-cis-14,trans-6,8,10-eicosatetraenoate (compound I), 5(S),12(R)-dihydroxy-cis-14,trans-6,8,10-eicosatetraenoate (compound II), and 5(S),12(R)-dihydroxy-cis-6,14,trans-8,10-eicosatetraenoate (compound III) were prepared from rabbit peritoneal neutrophils challenged with arachidonic acid plus ionophore A23187. Each arachidonate metabolite caused rabbit neutrophils to aggregate and, in cells treated with cytochalasin B, release granule-bound enzymes. Compound III was 10- to 100-fold more potent than compounds II and I. When intravenously infused into rabbits at doses of 100--1,000 ng/kg, compound III induced abrupt, profound, transient neutropenia associated with a rapidly reversing accumulation of neutrophils in the pulmonary circulation. This in vivo action correlated closely with the ability of the fatty acid to activate neutrophils in vitro: neutropenia, aggregation, and degranulation occurred at similar doses of stimulus and the rapid, reversing kinetics of the neutropenic response paralleled the equally rapid, reversing formation of aggregates. The fatty acid did not alter the circulating levels of lymphocytes or platelets and did not aggregate platelets in vitro. At comparable doses (i.e., 100--1,000 ng/kg), compounds I and II did not cause neutropenia. Thus, compound III possesses a high degree of structural and target-cell specificity in stimulating neutrophils in vitro and in vivo. Clinical and experimental syndromes associating neutropenia with increased levels of circulating arachidonate metabolites may involve compound III as a mediator of neutrophil sequestration in lung.

Binding and metabolism of platelet-activating factor by human neutrophils.

Human polymorphonuclear neutrophils rapidly incorporated radiolabeled platelet-activating factor, 1-O-[hexadecyl-9, 10-3H2]-2-acetyl-sn-glycero-3-phosphocholine ([3H]PAF), and then metabolized it into its sn-2-fatty acyl derivative. Fractionation of radiolabel-pretreated cells over Percoll gradients revealed that virtually all of the intact [3H]PAF was located in nongranule membranes that were enriched with alkaline phosphatase and cell surface glycoproteins. While still membrane associated, the ligand was rapidly converted to its acyl derivative and then more slowly transferred to specific granules and, to a lesser extent, azurophilic granules. In contrast, neutrophils did not metabolize [3H]PAF at 4 degrees C but rather gradually accumulated it in their alkaline phosphatase-enriched membrane subfractions. These same subfractions contained receptors for the ligand, as determined by their capacity to bind [3H]PAF specifically. Binding was readily saturated, partially reversible, and fit a two receptor model; dissociation constant (Kd) values for high and low affinity sites were 0.2 and 500 nM, respectively. Receptors with similar affinities were detected in whole cells. Furthermore, the potencies of several structural analogues in inhibiting binding of [3H]PAF to membranes correlated closely with their respective potencies in stimulating degranulation responses. Finally, quantitative studies suggested all or most of the cell's receptors were membrane associated. We conclude that PAF rapidly enters cellular membranes to bind with specific receptors that trigger function. The intramembranous ligand is also deacetylated, acylated, and then transferred to granules. This metabolism may be sufficiently rapid to limit ligand-receptor binding and distort quantitative analyses of receptors.

IL6 is associated with response to dasatinib and cetuximab: Phase II clinical trial with mechanistic correlatives in cetuximab-resistant head and neck cancer.

OBJECTIVE: Src family kinase (SFK) activation circumvents epidermal growth factor receptor (EGFR) targeting in head and neck squamous cell carcinoma (HNSCC); dual SFK-EGFR targeting could overcome cetuximab resistance. PATIENTS AND METHODS: We conducted a Simon two-stage, phase II trial of the SFK inhibitor, dasatinib, and cetuximab in biomarker-unselected patients with cetuximab-resistant, recurrent/metastatic HNSCC. Pre- and post-treatment serum levels of interleukin-6 (IL6) were measured by ELISA. HNSCC cell lines were assessed for viability and effects of IL6 modulation following dasatinib-cetuximab treatment. RESULTS: In the first stage, 13 patients were evaluable for response: 7 had progressive and 6 had stable disease (SD). Enrollment was halted for futility, and biomarker analysis initiated. Low serum IL6 levels were associated with SD (raw p=0.028, adjusted p=0.14) and improved overall survival (p=0.010). The IL6 classifier was validated in a separate trial of the same combination, but was unable to segregate survival risk in a clinical trial of cetuximab and bevacizumab suggesting serum IL6 may be specific for the dasatinib-cetuximab combination. Enhanced in vitro HNSCC cell death was observed with dasatinib-cetuximab versus single agent treatment; addition of IL6-containing media abrogated this effect. CONCLUSION: Clinical benefit and overall survival from the dasatinib-cetuximab combination were improved among patients with low serum IL6. Preclinical studies support IL6 as a modifier of dasatinib-cetuximab response. In the setting of clinical cetuximab resistance, serum IL6 is a candidate predictive marker specific for combined dasatinib-cetuximab. The trial was modified and redesigned as a biomarker-enriched Phase II study enrolling patients with undetectable IL6.

Chemical and biological characterization of oxo-eicosatetraenoic acids.

Eicosatetraenoates (ETEs) with 5-oxo residues are known to induce human neutrophil (PMN) Ca2+ transients and chemotaxis. We find that 5-oxoETE, 5-oxo-8-trans-ETE, 5-oxo-15-hydroxy-ETE, 5-hydroxy-ETE, 5-hydroxy-15-oxoETE, 5,15-dioxoETE, and 5,15-dihydroxy-ETE have respective relative potencies of 10, 5, 3, 1, 0.2, 0.1, and 0.02 in: a) causing PMN to mobilize Ca2+, aggregate, and release small amounts of granule enzymes and b) promoting large degranulation and oxidative burst responses in PMN co-challenged with platelet-activating factor, tumor necrosis factor-alpha, or ATP. Contrastingly, 12(R)-hydroxy-ETE, 12(S)-hydroxy-ETE, and 12-oxoETE induced PMN Ca2+ transients and aggregation [respective potencies (5-hydroxy-ETE = 1) of 0.1, 0.01, and 0.003] but did not effect degranulation, and 15-hydroxy-ETE, 15-oxoETE, and 15-oxo-11-trans-ETE were inactive in all assays. Finally, 5-oxo/hydroxy-ETEs desensitized PMN to themselves but not to 12-oxo/hydroxy-ETEs or leukotriene (LT)B4; 12-oxo/hydroxy-ETEs and LTB4 desensitized PMN to themselves and each other but not to 5-oxo/hydroxy-ETEs; 15-oxo/hydroxy-ETEs did not desensitize PMN; and a LTB4 receptor antagonist blocked responses to LTB4 and 12-oxo/hydroxy-ETEs but not to 5-oxo/hydroxy-ETEs. Thus, 5-oxo/hydroxy-ETEs act by a common, LTB4 receptor-independent mechanism that recognizes 5- but not 12- or 15-oxo/hydroxy-ETEs and prefers oxo over hydroxy residues at C5 whereas 12-oxo/hydroxy-ETEs act via a LTB4 receptor mechanism that recognizes 12- but not 5- or 15-oxo/hydroxy-ETEs and prefers hydroxy over oxo residues at C12.

Comparison of 1-O-alkyl-, 1-O-alk-1'-enyl-, and 1-O-acyl-2-acetyl-sn-glycero-3-phosphoethanolamines and -3-phosphocholines as agonists of the platelet-activating factor family.

Four naturally occurring platelet-activating factor (PAF) analogs, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, 1-octadecanoyl-2-acetyl-sn-glycero-3-phosphocholine, and 1-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine, stimulated human neutrophils (PMN) to mobilize Ca2+, degranulate, and produce superoxide anion. They were, respectively, 5-, 300-, 500-, and 4000-fold weaker than PAF in each assay; inhibited PMN-binding of [3H]PAF at concentrations paralleling their biological potencies; and showed sensitivity to the inhibitory effects of PAF antagonists. PAF and the analogs, moreover, desensitized PMN responses to each other but not to leukotriene B4 and actually increased (or primed) PMN responses to N-formyl-MET-LEU-PHE. Finally, 5-hydroxyeicosatetraenoate-enhanced PMN responses to PAF and the analogs without enhancing the actions of other stimuli. It stereospecifically raised each analog's potency by as much as 100-fold and converted a fifth natural analog, 1-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine from inactive to a weak stimulator of PMN. PAF and its analogs thus represent a structurally diverse family of cell-derived phospholipids which can activate, prime, and desensitize neutrophils by using a common, apparently PAF receptor-dependent mechanism.

Evidence for protein-catalyzed transfer of platelet activating factor by macrophage cytosol.

Platelet activating factor (PAF) is a potent, proinflammatory lipid. PAF is synthesized in response to stimuli and is rapidly destroyed by specific acetylhydrolases. In order to express its biological activity, PAF and its metabolites are transported among subcellular membranes by as yet unexplained mechanisms. We report here an assay system using methylcarbamyl-PAF (CPAF, 1-O-hexadecyl-2-O-(N-methylcarbamyl)-sn-glycero-3-phosphocholine) and a vesicle-extrusion technique for examining protein-catalyzed intermembrane transfer of CPAF, and demonstrate the presence of proteins catalyzing the separate transfer of CPAF and diacyl phosphatidylcholine in macrophage cytosol. The CPAF transfer activity is heat- and trypsin-sensitive and elutes from gel-filtration columns well separated from proteins catalyzing the transfer of phosphatidylcholine.

Human neutrophils incorporate arachidonic acid and saturated fatty acids into separate molecular species of phospholipids.

The incorporation of radiolabeled arachidonic acid and saturated fatty acids into choline-linked phosphoglycerides (PC) of rabbit and human neutrophils was investigated by resolving the individual molecular species by reversed-phase high performance liquid chromatography. PC from neutrophils incubated with a mixture of [3H]arachidonic acid and [14C]stearic or [14C]palmitic acid contains both radiolabels; however, double labeling of individual molecular species is minimal. After labeling for 2 h, the [3H]arachidonate is distributed almost equally between diacyl and 1-O-alkyl-2-acyl species, but it is incorporated into diacyl species containing unlabeled stearate or palmitate at the sn-1 position. In contrast, labeled saturated fatty acids are incorporated only into diacyl species and contain predominantly oleate and linoleate at the sn-2 position. Labeled linoleate is not incorporated into ether-linked species, but is found in the same species as labeled stearate. The findings suggest that mechanisms exist in neutrophils for specific shunting of exogenous arachidonic acid into certain phospholipid molecular species and support the concept that the 1-O-alkyl-2-arachidonoyl species may be a functionally segregated pool of arachidonic acid within the PC of neutrophils.

Comparison of alkylacylglycerol vs. diacylglycerol as activators of mitogen-activated protein kinase and cytosolic phospholipase A2 in human neutrophil priming.

In human neutrophils, the choline-containing phosphoglycerides contain almost equal amounts of alkylacyl- and diacyl-linked subclasses. In contrast to phosphatidylinositol hydrolysis which yields diacylglycerol, hydrolysis of choline-containing phosphoglycerides by phospholipase D coupled with phosphohydrolase yields both alkylacyl- and diacylglycerol. While diacylglycerol activates protein kinase C, alkylacylglycerol does not, and its role is unclear. Yet previous studies have shown that exogenous alkylacyl- and diacylglycerols can prime for the release of radiolabeled arachidonic acid (AA) in intact neutrophils stimulated by formyl-methionyl-leucyl-phenylalanine. We have now examined the effects of both diacylglycerol (1-oleoyl-2-acetylglycerol; OAG) and alkylacylglycerol (1-O-hexadecyl-2-acetylglycerol; EAG) on the activation of mitogen-activated protein (MAP) kinase and the 85-kDa cytosolic phospholipase A2 (cPLA2) in human neutrophils. We observed that while OAG could effectively activate p42 and p44 MAP kinases along with cPLA2 in a time- and concentration-dependent manner, EAG could not. A novel p40 MAP kinase isoform is also present and activated in response to OAG treatment; the behavior of this MAP kinase isoform is discussed. The activation of cPLA2 and MAP kinase by 20 microM OAG could be inhibited by pretreatment with 1 microM GF-109203X, a selective inhibitor of protein kinase C. Although only OAG activated cPLA2, both OAG and EAG primed for the release of AA mass as determined by gas chromatography/mass spectrometry. The priming of AA release by OAG may be explained by the phosphorylation of cPLA2 through the activation of protein kinase C linked to MAP kinase. However, priming by EAG appears to involve a separate mechanism that is dependent on a different PLA2. Our results support a role for phospholipase D-derived products modulating the activation of cPLA2, further supporting the idea of cross-talk among various phospholipases.

Role of Ca2+ and Mg2+ in neutrophil hexose transport.

The influence of extracellular Ca2+ and Mg2+ on the transport of 2-deoxy-[3H]glucose into human polymorphonuclear neutrophils was studied. Omission of these cations from the cell suspensions had little effect on resting hexose uptake. Furthermore, the addition of the bivalent cation chelator, EDTA, depressed uptake only slightly. Similarly, neither cation was essential for the enhanced 2-deoxy-D-[3H]glucose uptake stimulated by two chemotactic factors (C5a and N-formylmethionylleucylphenylalanine) and arachidonic acid: enhanced uptake was only partially depressed by the omission of Ca2+ and Mg2+ from the suspensions and was still prominent in the presence of EDTA. Two other neutrophil stimulants, the ionophores, A23187 and ionomycin, also enhanced hexose uptake but their actions were heavily dependent upon extracellular bivalent cations and were totally abrogated by EDTA. In all instances, extracellular Ca2+, but not Mg2+, supported optimal enhanced hexose transport induced by stimuli. Activation of 2-deoxy-D-[3H]glucose uptake by each of the five stimuli was totally blocked by cytochalasin B (a blocker of carrier-mediated hexose transport) and D-glucose but not by L-glucose. The data indicate, therefore, that a variety of neutrophil stimulants activate carrier-mediated hexose transport. Although this transport can be triggered by the movement of extracellular Ca2+ into the cell (as exemplified by the action of the two ionophores), such Ca2+ movement is not required for the actions of chemotactic factors or arachidonic acid. Other mechanisms, such as a rearrangement of intracellular Ca2+, may be involved in mediating the activation of hexose transport induced by the latter stimuli.

Direct measurement of myocardial free radical generation in an in vivo model: effects of postischemic reperfusion and treatment with human recombinant superoxide dismutase.

OBJECTIVES: The purpose of this study was to determine whether postischemic reperfusion of the heart in living rabbits induces a burst of oxygen free radical generation that can be attenuated by recombinant human superoxide dismutase administered at the moment of reflow. BACKGROUND: This phenomenon was previously demonstrated in crystalloid perfused, globally ischemic rabbit hearts. METHODS: Thirty-two open chest rabbits were assigned to one of four groups of eight animals each: Group I (control animals), no coronary artery occlusion; Group II, 30 min of circumflex marginal coronary artery occlusion without reperfusion; Group III, 30 min of coronary occlusion followed by 60 s of reperfusion, and Group IV, 30 min of coronary occlusion followed by treatment with recombinant human superoxide dismutase (a 20-mg/kg body weight bolus 90 s before reperfusion and a 0.17-mg/kg infusion during 60 s of reperfusion). Full thickness biopsy specimens taken from the ischemic region were then rapidly freeze clamped and electron paramagnetic resonance spectroscopy was performed at 77 degrees K. RESULTS: Three radical signals similar to those previously identified in the isolated, crystalloid perfused rabbit heart were observed: an isotropic signal with g = 2.004 suggestive of a semiquinone, an anisotropic signal with g parallel = 2.033 and g perpendicular = 2.005 suggestive of an oxygen-centered alkyl peroxy radical, and a triplet with g = 2.000 and aN = 24 G suggestive of a nitrogen-centered radical. In addition, a fourth signal consistent with an iron-sulfur center was seen. The oxygen-centered free radical concentration during normal perfusion (Group I) was 1.8 +/- 0.8 mumol compared with 4.4 +/- 0.9 mumol after 30 min of regional ischemia without reperfusion (Group II) and 13.0 +/- 2.5 mumol after 60 s of reperfusion (Group III) (p < 0.05 among all three groups). In contrast, superoxide dismutase treated-rabbits (Group IV) demonstrated a peak oxygen radical concentration of only 5.9 +/- 1.2 mumol (p < 0.05 vs. Group III). CONCLUSIONS: This study demonstrates that reperfusion after regional myocardial ischemia in the intact rabbit is associated with a burst of oxygen-centered free radicals. The magnitude of this burst is greater than that seen after a comparable duration of global ischemia in the isolated, buffer-perfused rabbit heart preparation and is significantly reduced by superoxide dismutase administration begun just before reflow.

Immediate improvement of dysfunctional myocardial segments after coronary revascularization: detection by intraoperative transesophageal echocardiography.

To ascertain the immediate effects of coronary artery bypass grafting on regional myocardial function, intraoperative transesophageal two-dimensional echocardiograms were obtained in 20 patients using a 3.5 MHz phased array transducer at the tip of a flexible gastroscope. Cross-sectional images of the left ventricle were obtained at multiple levels before skin incision and were repeated serially before and immediately after cardiopulmonary bypass. Using a computer-aided contouring system, percent systolic wall thickening was determined for eight anatomic segments in each patient at similar loading conditions (four each at mitral and papillary muscle levels). Of the 152 segments analyzed, systolic wall thickening improved from a prerevascularization mean value (+/- SEM) of 42.7 +/- 2.9% to a postrevascularization mean value of 51.6 +/- 2.6% (p less than 0.001). Thickening improved most in those segments with the worst preoperative function (p less than 0.001). Chest wall echocardiograms obtained 8.4 +/- 2.3 days after operation showed no deterioration or further improvement in segmental motion compared with transesophageal echocardiograms obtained after revascularization. Thus: regional myocardial function frequently improves immediately after bypass grafting, with increases in regional thickening being most marked in those segments demonstrating the most severe preoperative dysfunction, and this improvement appears to be sustained; and in some patients, chronic subclinical ischemic dysfunction is present which can be improved by revascularization.

Contrast echocardiographic mapping of collateralized myocardium in humans before and after coronary angioplasty.

Conventional coronary arteriography is able to demonstrate the presence of coronary collateral vessels but cannot delineate the specific region of myocardium to which they supply blood. To test the hypothesis that contrast echocardiography can specifically identify collateralized myocardium, contrast echocardiographic perfusion "maps" were compared in patients with (n = 12) and without (n = 12) angiographic evidence of coronary collateral flow, both before and after coronary angioplasty. Contrast echocardiographic images of the mid-left ventricle in the short-axis view at end-diastole were obtained after separate injections of a sonicated contrast agent into both the right and the left coronary arteries. A computer-based contouring system was used to determine the individual areas of myocardium perfused by each of the two coronary arteries and then to superimpose the images of the two perfusion beds. The resulting area of overlapping perfusion represented myocardium receiving blood flow from both coronary systems and was defined as collateralized myocardium. To normalize for heart size, overlap area was expressed as a percent of total myocardial area, which was the area between endocardium and epicardium in the short-axis view. To adjust for differences in vascular distribution, overlap area was expressed as a percent of the perfusion area of the recipient vessel. In patients with angiographic collateral flow, the recipient vessel was that vessel receiving the collateral flow. In patients without angiographic collateral flow, the right coronary artery was considered the recipient vessel. Overlap area was 1.3 +/- 0.4% of total myocardial area and 6.6 +/- 1.7% of recipient vessel area in patients without angiographic evidence of collateral flow compared with 30.6 +/- 2.5% and 89.2 +/- 6.4%, respectively, in patients with angiographic collateral flow (p less than 0.001 for both). In four patients in whom angiographic collateral flow was abolished by angioplasty, overlap area decreased from 30.3 +/- 5.3% to 6.8 +/- 2.7% of total myocardial area and from 100% to 18.5 +/- 5.4% of recipient vessel area (p less than 0.05 for both). Thus, contrast echocardiography is able to map the specific myocardial territory perfused by coronary collateral flow and document an immediate reduction in perfusion in this territory when collateral flow is abolished by angioplasty.

Results of a randomized prospective trial of intraaortic balloon counterpulsation and intravenous nitroglycerin in patients with acute myocardial infarction.

A randomized prospective clinical trial compared combined treatment with intraaortic balloon pumping and intravenous nitroglycerin for 4 to 5 days with routine clinical management in 20 patients with extensive myocardium at risk for infarction as evidenced by a thallium defect score of 7.0 units or greater. No significant differences in mortality or clinical outcome were observed between the 10 patients receiving the combined treatment and the 10 receiving routine management. In 14 patients two-dimensional echocardiograms obtained 6 to 24 hours after the onset of symptoms and at follow-up 6 to 16 days later (after completion of combined intraaortic balloon pumping plus nitroglycerin therapy) were analyzed to determine whether infarct segment or noninfarct segment lengths were affected by therapy. Among these 14 patients, 5 (3 receiving the combined therapy and 2 receiving routine management) demonstrated an increase in infarct segment length of greater than 1.0 cm. Mean infarct segment length increased 0.30 +/- 0.44 cm in patients receiving the combined therapy and 0.29 +/- 0.36 cm in patients on routine management (p = NS). In contrast, noninfarct segment length increased greater than 1.0 cm (mean increase 1.20 +/- 0.39) in five of seven patients on routine management but in none of 7 patients receiving intraaortic balloon pumping plus nitroglycerin therapy (mean decrease 0.22 +/- 0.20 cm) (p less than 0.05). No significant differences were noted in left ventricular ejection fraction, as measured by gated blood pool scintigraphy, or thallium perfusion defect score in a comparison of day 1 (pretreatment) with day 4 thallium or day 7 to 14 gated blood pool scintigrams. Thus, in patients with extensive myocardium at risk, it is unlikely that a reduction in mortality or a significant improvement in myocardial perfusion or ventricular function can be obtained by early intervention with intraaortic balloon pumping in combination with nitroglycerin. Although this combined therapy failed to prevent infarct segment lengthening (infarct expansion), the combined afterload-lowering effects of intraaortic balloon pumping and nitroglycerin did appear to prevent dilation or remodeling of noninfarcted segments during the first 2 weeks after acute myocardial infarction.

Regional wall motion improvement after coronary thrombolysis with recombinant tissue plasminogen activator: importance of coronary angioplasty.

To evaluate functional recovery in 20 consecutive patients with acute myocardial infarction who received recombinant tissue-type plasminogen activator, serial two-dimensional echocardiograms were performed before and immediately after tissue plasminogen activator administration and at 1 and 10 days postinfarction. Tissue plasminogen activator was administered intravenously (17 patients) or by intracoronary infusion (3 patients) after angiographic confirmation of total occlusion. Reperfusion, documented by angiography, occurred in 13 of the 20 patients. The mean time from onset of chest pain to thrombolysis was 5.1 +/- 1.1 hours. Echocardiograms were evaluated for regional function with a visual semiquantitative scoring system by two independent observers who had no knowledge of patient identity, temporal sequence, therapy or effect of therapy. There was no immediate or 24 hour improvement in wall motion. At day 10 compared with pretreatment, 28 of 33 reperfused infarct zone segments versus 6 of 20 nonreperfused infarct segments demonstrated improved wall motion (p = 0.01). This improvement did not relate to time from onset of chest pain to successful thrombolysis. Of reperfused infarct zone segments in the distribution of coronary artery balloon dilation, 19 of 23 segments exhibited improvement versus 7 of 17 (reperfused, no angioplasty) and 6 of 20 (nonreperfused, no angioplasty) segments (p = 0.001). Infarct zone segments reperfused at the time of ongoing chest pain demonstrated functional recovery compared with segments reperfused in the absence of chest pain (18 of 23 versus 10 of 20, respectively; p = 0.05). Thus, in this uncontrolled series, there was echocardiographically detectable improvement in function of reperfused infarct segments 10 days after coronary thrombolysis with recombinant tissue plasminogen activator.

Hypertension following coronary artery bypass surgery. Role of preoperative propranolol therapy.

Over a 9-month period, the incidence and characteristics of hypertension following coronary artery bypass surgery were studied in a group of 52 patients. Hypertension occurred in 61% of the patients and was characterized by an increase in arterial blood pressure of 35 +/- 2 mm Hg mean +/- SEM during the early postoperative period. Preoperative blood pressures and hemodynamic variables were similar in those who developed hypertension of those who remained normotensive. Ninety-four percent of those who developed hypertension as compared to only 40% of those who remained normotensive received propranolol during the 24 hours preceding surgery (x2 = 15.4; p less than 0.001). Maximal blood pressures during the first 5 hours following the termination of cardiopulmonary bypass were significantly positively correlated with preoperative propranolol dosage (p less than 0.01). Hypertension was not associated with significant changes in plasma renin activity or angiotensin II levels, but concomitant plasma catecholamine concentrations were elevated significantly (p less than 0.005). However, a similar rise in plasma catecholamine concentrations was found in those who remained normotensive. Hypertension was associated with an increase in systemic vascular resistance (p less than 0.001) and left ventricular stroke work index (p less than 0.05), and a fall in stroke volume (p less than 0.005) and cardiac index (p less than 0.001). These studies suggest that hypertension following coronary artery bypass surgery is common, results from an increase in systemic vascular resistance, is not renin-angiotensin mediated, and may, in part, be related to preoperative propranolol administration.

Reperfusion injury.

Several lines of evidence point to a major role of oxygen free radicals in the pathogenesis of cell death or dysfunction in a variety of disease processes. Recent studies from this as well as other laboratories have demonstrated that oxygen free radicals play a major role in the pathogenesis of post-ischemic reperfusion injury in the heart. We have recently developed methods for direct measurement of radical species and/or specific byproducts of radical injury. Timely administration of oxygen radical scavengers reduced the quantity of free radicals generated following reperfusion and in addition improved recovery of post-ischemic ventricular function and metabolism. In a regionally ischemic model the free radical scavenger recombinant human superoxide dismutase also administered at the time of reflow was shown to limit infarct size. In this article we review the biophysical and molecular mechanisms of oxygen free radical generation that are viewed as contributing to post-ischemic reperfusion injury. We also discuss the mechanisms by which the body defends against free radical attack and the interrelationships of free radical injury to other mechanisms of tissue injury.