Strain and model dependent differences in inflammatory cell recruitment in mice.

OBJECTIVE AND DESIGN: The objective of this study was to determine genetic differences in inflammation in these distinct inbred mouse strains. METHODS: Peritoneal leukocyte recruitment, matrix metalloproteinases and cytokines were quantified in A/J, 129/svJ, C57BL/6J, using thioglycollate or biomaterial implants as inflammatory stimuli. RESULTS: In response to thioglycollate, A/J had significant decreases compared to C57BL/6J in both neutrophil (86 %) and macrophage (62 %) recruitment, and 129/svJ had a significant (43 %) decrease compared to C57BL/6J in macrophage recruitment. The reduced leukocyte recruitment corresponded to reduced matrix metalloproteinase-9. In the bioimplant model, 129/svJ had a 2-fold increase in neutrophil and macrophage recruitment compared to C57BL/6J, and the increased leukocyte recruitment corresponded to elevated cytokines, monocyte inhibitory protein-2 and monocyte chemoattractant protein-1, in the lavage compared to the values for C57BL/6J. CONCLUSION: Not only was leukocyte recruitment strain dependent, but the three strains had marked differences in metalloproteinases and cytokine response. In addition, there were model specific differences in the metalloproteinase and cytokine response to the two inflammatory stimuli. Thus, inflammatory cell recruitment is genetically determined and stimulus specific and may determine the susceptibility to complex diseases.

A central role for plasminogen in the inflammatory response to biomaterials.

The inflammatory response to implanted biomaterials severely limits their deployment in patients. Plasminogen has been shown to play a central role in cell migration, and therefore could regulate this inflammatory response. We sought to determine if plasminogen influences recruitment of inflammatory cells to a biomaterial implanted into plasminogen-deficient (Plg(-/-)) mice. Small disks of polyethylene terephthalate, a material used in vascular grafts, were surgically implanted into the peritoneum of wild-type and Plg(-/-) mice. Recruitment of neutrophils and monocytes/macrophages into the peritoneum and onto the disks was measured, primarily at 18 h. Monocyte/macrophage recruitment was markedly blunted in Plg(-/-) mice compared with wild-type mice. Unexpectedly, neutrophil recruitment was also markedly decreased in the Plg(-/-) mice. While recruitment of leukocytes into the peritoneum was plasminogen-dependent, the adhesion of the emigrating cells to the implants was not. In contrast, adhesion but not recruitment was reduced in fibrinogen-deficient mice. Reconstitution of Plg(-/-) mice with intravenous or intraperitoneal plasminogen differentially restored monocyte/macrophage and neutrophil recruitment. Tranexamic acid, an inhibitor of the lysine binding sites of plasminogen, suppressed leukocyte recruitment in wild-type mice, but aprotinin, a plasmin inhibitor, did not. Plasminogen exerts a marked influence on both neutrophil and monocyte/macrophage recruitment to implanted biomaterials. This role is distinct from that of fibrinogen, and the two inflammatory cell types use plasminogen in different ways. Plasminogen represents a therapeutic target for controlling the inflammatory response to implanted materials.

Diagnosis and management of aortoenteric fistulas.

Development of an aortoenteric fistula (AEF) is a devastating and life-threatening condition, which is as difficult to diagnose as it is to treat. Fortunately, it is rare, most commonly seen as a delayed complication of aortic reconstruction. Two types are recognized: primary and secondary. Primary fistulas occur de novo between the aorta and bowel, most commonly duodenum. Secondary fistulas occur between an aortic graft and segment of bowel. Diagnosis of AEF requires a high index of suspicion in patients who present with either signs of infection or gastrointestinal hemorrhage. Early diagnosis is essential for a successful outcome because of the lethal nature of AEF. Symptomatology can be varied but most often includes signs of infection and of gastrointestinal bleeding. Esophagogastroduodenoscopy (EGD) and computed tomography (CT) scans are the most useful tests to diagnose AEF. Treatment almost always requires excision of the infected graft and revascularization. Placement of an extra anatomic bypass, followed by graft excision, has been the usual treatment. Recent experience with in situ revascularization has shown that a variety of materials can be use for in situ reconstruction with good results. Morbidity and mortality rates still are high even in contemporary series. The mortality rate still is approximately 33%, but amputation rates have been reduced to less then 10%. Care of patients with AEF requires timely control of bleeding and infection followed by vascular reconstruction performed in a manor to minimize physiological stress.

Inhibition of endothelial cell migration by cigarette smoke condensate.

Cigarette smoking is among the leading risk factors in the etiology of atherosclerotic vascular disease. The mechanism, however, that links cigarette smoking to an increased incidence of atherosclerosis is poorly understood. Endothelial cell (EC) integrity is critical in preventing vascular lesion formation, and after a loss of EC integrity reendothelialization must be rapid and complete. We therefore investigated whether cigarette smoke affected the ECs ability to migrate or altered the intracellular signals generated during migration. The DMSO-soluble fraction of cigarette smoke condensate (CSC), derived from the standard research cigarette, was tested on cultured ECs (HUVEC) derived from human umbilical vein. The addition of CSC caused a dose-dependent decrease in the ability of EC to migrate as measured over a 24-h time period. Nicotine and cadmium sulfate, two constituents of cigarette smoke, individually or in combination, had no effect on migration. Examination of the tyrosine phosphorylation state of various intracellular proteins by Western blot analysis showed that CSC caused the hyperphosphorylation of a 130-kDa protein. In addition, other intracellular proteins showed changes in their phosphorylation states after CSC addition. These results support the hypothesis that CSC is detrimental to normal EC function in maintaining vascular integrity and suggest that smokers are more likely to develop complications of vascular disease due to delayed or incomplete reendothelialization as a consequence of decreased EC migration.

Endoluminal arterial injury in plasminogen-deficient mice.

BACKGROUND: Vascular remodeling following arterial injury is characterized by an initial inflammatory reaction. Prior experiments using peritoneal inflammatory models have shown that the plasminogen system plays a role in the intensity of the inflammatory response. This study was undertaken to test the hypothesis that an absence of plasminogen would lead to a decrease in vascular remodeling. METHODS: A left carotid artery injury was created with a flexible guidewire in both wild-type [Plg(+/+)] and plasminogen deficient [Plg(-/-)] mice. The right carotid artery was uninjured and used as a control. Three weeks postinjury, the mice were sacrificed and perfusion fixed, and the bilateral carotid arteries were sectioned for histological examination and collection of morphometric data. RESULTS: After arterial injury, electron microscopy of the acutely injured artery revealed that the endothelium was denuded, that there were breaks in the internal elastic membrane, and that there was disruption of the medial layer of smooth muscle cells. The intimal and medial areas were significantly increased between the uninjured and injured carotid arteries of both Plg(+/+) (+80% intimal, +41% medial, P < 0. 05) and Plg(-/-) [+48% intimal, +24% medial, P < 0.05) mice. However, although there was a significant increase in the adventitial area of Plg(+/+) mice (+18%, P < 0.05), there was no difference in Plg(-/-) mice (-6%). Interestingly, even after 3 weeks, four of six injured arteries in Plg(-/-) mice had persistent thrombus within the medial layer, whereas this was not found in any of the nine Plg(+/+) mouse arteries. DISCUSSION: Plasminogen deficiency inhibited the increase in adventitial area seen after injury in Plg(+/+) mice, but not the increase in intimal or medial areas. Not surprisingly, plasminogen-deficient mice also demonstrated a severe alteration in intramural thrombus clearance. Thus, specific aspects of the vascular remodeling response are dependent on plasminogen.

Carotid duplex overestimation of stenosis due to severe contralateral disease.

BACKGROUND: In the quest to use carotid duplex to assess carotid occlusive disease, it has been reported that the current velocity criteria to calculate stenosis tends to overestimate the severity when there is a contralateral highly stenotic or occluded carotid artery. METHODS: Patient records were reviewed for 592 consecutive carotid endarterectomies performed from 1987 to 1994. Preoperative and postoperative duplex scan results were compared in a subset of patients in whom duplex overestimated the degree of stenosis, as compared to preoperative angiography. RESULTS: A total of 146 patients were identified in whom duplex overestimated the degree of stenosis contralateral to a high grade stenosis or an occlusion. Of 76 arteries, 18 (23.7%) contralateral to an occluded artery were overestimated by duplex, and 128 (27.0%) of 474 arteries contralateral to a high grade stenosis were overestimated. Following endarterectomy 44 (51.8%) of 128 nonoperated contralateral stenoses decreased by at least one duplex category. The average peak systolic frequency (PSF) decreased by 1175 Hz (P = 0.0018), and the average end diastolic frequency (EDF) decreased by 475 Hz (P = 0.011). CONCLUSIONS: Patients with high grade stenosis have a significant decrease in PSF and EDF in the unoperated carotid after endarterectomy, supporting a compensatory flow phenomenon. This often results in a decrease in the postoperative duplex defined stenosis by at least one category. The clinical significance of these findings is of increasing importance as carotid surgery is being performed more frequently without angiography.

Antisense suppresssion of protein kinase C-alpha and -delta in vascular smooth muscle.

Understanding and being able to manipulate intracellular signaling pathways which control VSMC gene expression and proliferation will be important in efforts to control neointimal hyperplastic vascular diseases. Activation of the protein kinase C (PKC) family of enzymes is a central event in growth factor-stimulated cells. PKC activation results in the activation of downstream protein kinases including mitogen activated protein kinase (MAPK). PKC isozymes alpha (alpha) and delta (delta) predominate in cultured rat aortic VSMC and both isozymes are completely downregulated upon prolonged (16-24 hr) stimulation with the PKC activator, phorbol 12,13 dibutyrate (PDBu). At these low levels of PKC, MAPK activation in response to PDBu is nearly abolished. To assess the role of specific PKC isozymes in regulating MAPK, isozyme-specific antisense oligodeoxynucleotides (ODNs) were used to inhibit reexpression of PKC in downregulated cells. ODNs were phosphorothioated to increase stability and contained C-5 propynyl modified pyrimidines which are reported to have increased binding affinity. ODNs were administered in low concentration (400 nM) with a cationic liposome carrier (Lipofectin; GibcoBRL). Optical imaging of cells treated with FITC-labeled ODNs confirmed that virtually all cells took up the ODNs within 2 hr. With this technique, PKCalpha-specific antisense ODNs selectively inhibited PKCalpha recovery compared to cells treated with an equal length nonsense ODN (76 +/- 3.9, P < 0.001), with no effect on recovery of PKCdelta. However, activation of MAPK by PDBu was not significantly inhibited in these PKCalpha downregulated cells. This suggests that only a small amount of the total PKCalpha is required for PDBu induced activation of MAPK and/or that PKCdelta can mediate the response. Manipulation of PKC isozymes using this model system should allow assessment of the roles of specific isozymes in controlling diverse downstream effectors and events related to VSMC growth and proliferation.