Raymond G. Makhoul

Developmental anomalies at the thoracic outlet: An analysis of 200 consecutive cases***

Anatomic observations were made during 200 consecutive transaxillary surgical procedures performed in 175 patients because of unremitting signs and symptoms of nerve or vascular compression at the thoracic outlet. There were 160 cases of brachial plexus compression and 40 cases of arterial or venous occlusion. In 132 (66%) of these cases, single or multiple abnormalities were recognized that represented developmental variations previously described in anatomic dissections or in embryologic studies. There were 17 cases of cervical rib or first thoracic rib abnormalities and 20 supernumerary scalene muscles. Developmental variations were identified in 86 scalene and 39 subclavius muscles or their insertions. Comparisons revealed a higher percentage of developmental anomalies in this group of patients than in consecutive anatomic investigations reported in unselected populations. This information was interpreted in light of recent embryologic studies and histochemical and morphometric ultrastructural studies of scalene muscle. The preponderance of evidence suggests that neurovascular compression in the region of the thoracic outlet derives from a combination of these factors: predisposing morphologic variations, structural modifications conditioned by functional requirements, and changes in fiber type or myosin isoform consequent to trauma. The correlation of clinical syndrome with morphologic characteristics alone was significant only for the Paget-Schroetter syndrome.

L-arginine improves endothelium-dependent vasorelaxation and reduces intimal hyperplasia after balloon angioplasty.

Reductions in nitric oxide (NO) activity persist after arterial intimal injury and may be a factor in the development of intimal hyperplasia. NO inhibits in vitro platelet aggregation, leukocyte adhesion, and smooth muscle cell growth, all of which are key components in the process of intimal hyperplasia. We hypothesized that long-term supplementation with L-arginine, the precursor of NO, would increase NO production and thereby improve endothelium-dependent vasorelaxation and simultaneously reduce intimal hyperplasia. Twenty-six New Zealand White male rabbits were fed standard rabbit chow either with or without 2.25% L-arginine in their drinking water for 3 weeks. Then the animals underwent unilateral iliac artery angioplasty and were continued on their respective diets. Four weeks after angioplasty, the iliac arteries were harvested for functional and morphometric studies. The iliac arteries from several animals from each group were processed for study by electron microscopy. Maximal endothelium-dependent vasorelaxation in injured arteries was significantly greater in L-arginine-supplemented animals (mean +/- SEM, 71.8 +/- 4.1%; n = 6) than controls (51.4 +/- 4.0%, n = 7; P < .05). Furthermore, the intimal area in injured arteries was significantly reduced in L-arginine-supplemented animals (0.22 +/- 0.03 mm2, n = 5) compared with controls (0.34 +/- 0.03 mm2, n = 6; P < .05). These data suggest that L-arginine supplementation enhances NO production at sites of vascular healing and may reduce intimal hyperplasia.

Heparin-associated thrombocytopenia and thrombosis: A serious clinical problem and potential solution

Heparin-associated thrombocytopenia and thrombosis (HATT) is an unusual but serious complication of heparin therapy. Twenty-five patients (13 men and 12 women) had thrombocytopenia and arterial or venous thrombosis 1 to 10 days (mean, 6.3 days) after the start of heparin administration. The vessels in the affected extremity had been entered for catheterization, arteriography, or passage of a balloon counterpulsation device in 19 of the 25 patients. In vitro platelet aggregation with heparin was seen in all patients. Additional studies were performed to see whether other lots or sources of heparin also produced in vitro aggregation. Four separate lots of beef lung heparin, commercial heparin from porcine intestinal mucosa, and two types of low molecular weight heparin were all highly stimulatory in this system. However, Org 10172, a heparinoid, did not induce aggregation in any of 13 patient plasmas tested. Inhibition of platelet aggregation by aspirin was also examined. Aspirin abolished in vitro aggregation in 9 of 16 cases and decreased the degree of aggregation from 85% to 55% (p = 0.02) in the remaining seven cases. We conclude that in patients with HATT platelet aggregation is equally induced by beef lung, porcine intestinal, and some forms of low molecular weight heparin. Org 10172 does not stimulate platelet aggregation in plasma from these patients in vitro. Finally, there may be a role for aspirin in treating patients with HATT.

Management of Patients with Heparin-Associated Thrombocytopenia and Thrombosis Requiring Cardiac Surgery

Heparin-associated thrombocytopenia and thrombosis (HATT) is a serious complication of heparin therapy that results in intravascular platelet aggregation with arterial or venous thrombosis. Platelet aggregation to heparin in vitro is used to confirm the diagnosis. Cessation of heparin therapy with avoidance of reexposure to heparin is an important principle in the management of HATT. However, certain patients with HATT may require reexposure to heparin for emergency cardiac operations requiring cardiopulmonary bypass while still demonstrating positive in vitro platelet aggregation with heparin. The present report describes the management of 2 such patients. In each patient aspirin was shown to inhibit platelet aggregation to heparin in vitro; therefore, aspirin and dipyridamole were administered to each patient before heparin reexposure and continued throughout the perioperative period. Cardiopulmonary bypass with full heparinization was achieved without thrombotic or hemorrhagic complications in both patients. Despite the presence of a heparin-dependent platelet-aggregating factor in the plasma of these 2 patients, inhibition of platelet aggregation with aspirin and dipyridamole allowed uneventful reexposure to heparin.

Postischemic extremities exhibit immediate release of tumor necrosis factor

PURPOSE Although reperfusion of acutely ischemic extremities can cause cardiopulmonary collapse and death, the humoral agent(s) released from limbs promoting this distant organ injury are not well characterized. We hypothesized that tumor necrosis factor-alpha (TNF-alpha), a cytokine that causes cardiopulmonary dysfunction in septic shock, may be released from postischemic extremities. METHODS Isolated rat hindlimbs were perfused at constant pressure with a nonrecirculating crystalloid-based buffer. After 60 or 120 minutes of normothermic ischemia, TNF activity was measured in sequential samples of venous effluent by L929 bioassay. Associated limb injury was assessed by the extent of no-reflow after reperfusion, changes in endothelial permeability to iodine 125-labeled albumin and skeletal muscle injury by uptake of technetium 99 pyrophosphate. RESULTS After 120 minutes of normothermic ischemia (n = 10), a 15-fold increase in TNF-alpha activity in venous effluent occurred, with peak activity at 1.5 minutes of reperfusion (30.6 +/- 8.7 U/ml) falling to near control levels by 5 minutes. This group had a 3.3-fold increase in vascular permeability, a 2.2-fold increase in the muscle injury index and a 71.2% decline in reperfusion flow (all p < 0.05 vs control). Pretreatment of extremities with an anti-TNF-alpha antibody in a second group of limbs undergoing 120 minutes of ischemia (n = 10) decreased TNF activity to levels not significantly different from the nonischemic control group (n = 12). The extent of no-reflow in limbs treated with antibodies was significantly attenuated (flow 44.9% of control vs 29.8% [untreated], p < 0.05). Antibody treatment affected neither muscle injury nor vascular permeability. CONCLUSIONS Postischemic extremities exhibited a transient, early burst of TNF release on reperfusion, which likely represented washout of TNF produced during ischemia. Suppression of TNF activity with an antibody to TNF-alpha resulted in a salutary increase in postischemic flow rates, suggesting that TNF may play a role in the no-reflow phenomenon.

Stabilization and partial characterization of endothelium-derived relaxing factor from cultured bovine aortic endothelial cells

The extreme lability of the chemically undefined vasoregulatory mediator, endothelium-derived relaxing factor (EDRF), has been overcome. The activity of EDRF, from cultured bovine aortic endothelial cells stimulated with A23187, was stabilized by acidification. An additional action of EDRF, platelet disaggregation was used as a sensitive and convenient bioassay to monitor purification. EDRF appears to be a hydrophilic molecule, rapidly inactivated under alkaline conditions. However, activity is restored upon reacidification suggesting that this instability results from a readily reversible chemical process. The stabilization and partial purification of EDRF sets the stage for its further biochemical and chemical characterization.

Heparin prevents postischemic endothelial cell dysfunction by a mechanism independent of its anticoagulant activity

PURPOSE Heparin may have protective effects on postischemic vascular endothelial cell function that are distinct from its anticoagulant, antiplatelet, or anticomplement activity. We tested this hypothesis in isolated rat hindlimbs. METHODS Isolated rat hindlimbs underwent 60 minutes of normothermic ischemia and 10 minutes of reperfusion. Potential heparin interaction with plasma-based proteins or cells was eliminated by perfusion of the hindlimbs with a nonrecirculated albumin-enriched crystalloid buffer. Endothelial function was assessed by measurement of endothelium-derived relaxing factor (EDRF) activity. Limbs perfused at constant pressure were subjected to increasing log dose infusions of acetylcholine and nitroprusside to measure endothelial-dependent (EDRF-mediated) and endothelial-independent vasoreactivity, respectively. Fifty limbs were divided into seven groups: two nonischemic groups (one with heparin) and five ischemia/reperfusion groups treated with increasing doses of heparin (0 to 1.0 U/ml perfusate). RESULTS The nontreated ischemia/reperfusion group (n = 12) had a 46.2% reduction in endothelial-dependent vasodilation of the rat hindlimb when compared with the nonischemic control (n = 7, p < 0.05). Treatment with heparin 0.5 U/ml (n = 6) nearly abolished this attenuation of endothelial-dependent vasodilation (4.3% reduction, p = not significant vs nonischemic control). The endothelial protective effect of heparin was dose-dependent: groups treated with 0.25 U/ml (n = 6) and 0.1 U/ml heparin (n = 7) showed progressive impairment in postischemic EDRF-mediated vasodilation. Endothelial-independent vasodilation induced by nitroprusside was unchanged by ischemia/reperfusion or heparin treatment, which confirmed that the postischemic damage and its protection by heparin were specific to the endothelium. CONCLUSIONS Heparin prevented postischemic endothelial cell dysfunction by a mechanism independent of its interactions with plasma-based proteins or cells. This nonanticoagulant protective effect may contribute to the salutary effects of heparinization during acute ischemic events.

Association of polymorphonuclear leukocytes with sites of aortic catheter-induced injury in rabbits

The kinetics of the association of polymorphonuclear leukocytes (PMNs) with arterial balloon catheter-induced injury have been examined. An average of 6 X 10(7) PMNs were isolated from 20 ml of blood and labelled with 111In-oxine for reinfusion into the donor rabbit. The cells remained viable as demonstrated by both in vitro and in vivo tests of cell function. The abdominal aorta of rabbits was denuded of endothelium and immediately, 24 h, or 5 weeks later, exposed to autologous radiolabelled PMNs for 1 h. The presence of PMNs at sites of denudation was demonstrated by detection of the radioactive label and was confirmed by light and electron microscopy after 24 h, but not at 5 weeks. Immediately following denudation radioactivity was 2.44 +/- 0.33 times control (P = 0.006); 2.52 +/- 0.18 at 24 h (P = 0.005); and 1.88 +/- 0.32 times control at 5 weeks (P = 0.045). The presence of PMNs, or their products, 5 weeks after denudation suggests a more complex role of PMNs and possibly a direct involvement in the long term changes resulting from arterial balloon catheter injury.

The role of aspirin and dipyridamole on vascular DNA synthesis and intimal hyperplasia following deendothelialization

Platelets are implicated both in acute thrombotic events and, through platelet-derived growth factor, in the development of intimal hyperplasia. We have investigated, in vivo, the influence of aspirin and dipyridamole on vascular smooth muscle cell proliferation and DNA synthesis following balloon catheter injury. Fifty-eight male, New Zealand white rabbits were divided equally into two groups; the test group was fed aspirin (14 mg/kg/day) and dipyridamole (9 mg/kg/day) from 2 days prior to surgery until sacrifice at 1, 2, 3, 4, 7, 14, or 28 days after injury. All animals were sacrificed 1 h after injection of [3H]thymidine and the smooth muscle cell DNA specific activity and total kinetic activity were determined. Intimal hyperplasia was measured by light microscopy and intimal nuclear proliferation was determined by counting nuclei per millimeter of internal elastic lamina. Nuclear proliferation was maximal at 14 days (25 +/- 1.2) but intimal hyperplasia was still increasing at 28 days. DNA specific activity after 24 hr (test: 4 +/- 2 dpm/micrograms DNA; control: 3.3 +/- 3 dpm/micrograms DNA) was similar to basal levels in uninjured rabbits. DNA synthesis peaked in both groups between the second and third day (test: 177 +/- 27 dpm/micrograms DNA; control: 185 +/- 39 dpm/micrograms DNA) and then declined slowly toward baseline values. There was no significant difference between treated and normal rabbits in either [3H]thymidine incorporation, nuclear proliferation, or development of intimal hyperplasia despite 90% inhibition of platelet aggregation and a significant reduction (78%) in [14C]serotonin release following collagen challenge (6 micrograms/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Inducible nitric oxide synthase expression in human vein grafts

BACKGROUND The patency of vascular reconstructive procedures is limited by the development of intimal hyperplasia (IH). Nitric oxide (NO) seems to be beneficial in abrogating this process. Currently, there is little information concerning inducible nitric oxide synthase (iNOS), the enzyme responsible for NO synthesis, and human vein grafts. The purpose of this study was to examine iNOS gene expression in human aortocoronary vein grafts (ACVG) and infrainguinal vein bypass grafts (IVG). METHODS Nonthrombosed sections from ACVG (n = 5), IVG (n = 5), and control saphenous vein (SV; n = 4) were harvested and processed for RNA isolation. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was performed on samples using 32P radioactively end labeled primers. Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was the internal control, and results were expressed as iNOS pmol/GAPDH pmol. RESULTS There was a significant increase in the iNOS gene expression in the ACVG (0.049 +/- 0.01) when compared with IVG (0.019 +/- 0.001) or normal SV (0.011 +/- 0.002; P < or = 0.05). There was no significant difference between normal vein and the infrainguinal grafts. Sequencing of a fragment of the amplified 428 bp gene product confirmed 84% homology with the available gene bank human sequence. CONCLUSIONS This study proves that iNOS is expressed in human vein bypass grafts. Additionally, there is a significant elevation of iNOS message in human ACVGs compared with IVG or normal SV. This difference may be the result of the unique vascular beds supplied by these grafts. Ultimately, manipulation of iNOS expression may lead to therapies to alleviate IH in these grafts.