John W. Ford

Neutrophil Depletion Inhibits Experimental Abdominal Aortic Aneurysm Formation

Background—Neutrophils may be an important source of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), two matrix-degrading enzymes thought to be critical in the formation of an abdominal aortic aneurysm (AAA). The purpose of this investigation was to test the hypothesis that neutrophil depletion would limit experimental AAA formation by altering one or both of these enzymes. Methods and Results—Control, rabbit serum–treated (RS; n=27) or anti-neutrophil-antibody–treated (anti-PMN; n=25) C57BL/6 mice underwent aortic elastase perfusion to induce experimental aneurysms. Anti-PMN–treated mice became neutropenic (mean, 349 cells/&mgr;L), experiencing an 84% decrease in the circulating absolute neutrophil count (P<0.001) before elastase perfusion. Fourteen days after elastase perfusion, control mice exhibited a mean aortic diameter (AD) increase of 104±14% (P<0.0001), and 67% developed AAAs, whereas anti-PMN–treated mice exhibited a mean AD increase of 42±33%, with 8% developing AAAs. The control group also had increased tissue neutrophils (20.3 versus 8.6 cells per 5 high-powered fields [HPFs]; P=0.02) and macrophages (6.1 versus 2.1 cells per 5 HPFs, P=0.005) as compared with anti-PMN–treated mice. There were no differences in monocyte chemotactic protein-1 or macrophage inflammatory protein-1&agr; chemokine levels between groups by enzyme-linked immunosorbent assay. Neutrophil collagenase (MMP-8) expression was detected only in the 14-day control mice, with increased MMP-8 protein levels by Western blotting (P=0.017), and MMP-8–positive neutrophils were seen almost exclusively in this group. Conversely, there were no statistical differences in MMP-2 or MMP-9 mRNA expression, protein levels, enzyme activity, or immunostaining patterns between groups. When C57BL/6 wild-type (n=15) and MMP-8–deficient mice (n=17) were subjected to elastase perfusion, however, ADs at 14 days were no different in size (134±7.9% versus 154±9.9%; P=0.603), which suggests that MMP-8 serves only as a marker for the presence of neutrophils and is not critical for AAA formation. Conclusions—Circulating neutrophils are an important initial component of experimental AAA formation. Neutrophil depletion inhibits AAA development through a non–MMP-2/9–mediated mechanism associated with attenuated inflammatory cell recruitment.

New Drugs Targeting the Cardiac Ultra-Rapid Delayed-Rectifier Current (IKur): Rationale, Pharmacology and Evidence for Potential Therapeutic Value

There is a clear unmet medical need for new pharmacologic therapies for the treatment of atrial fibrillation (AF) with improved efficacy and safety. This article reviews the development of new and novel Kv1.5/ultra-rapid delayed-rectifier current (IKur) inhibitors and presents evidence that Kv1.5 modulation provides an atrial-selective mechanism for treating AF. Academia and industry have invested heavily in Kv1.5 (>500 scientific publications and >50 patents published since 1993); however, to realize the full value of this therapeutic drug target, clinical efficacy and safety data are required for a selective Kv1.5 modulator. The reward for demonstrating clinical efficacy and safety in a pivotal Phase 3 trial, on regulatory approval, is “first in class” status.

Transforming growth factor-β1 increases lysyl oxidase enzyme activity and mRNA in rat aortic smooth muscle cells

Abstract Purpose: This investigation was designed to test the hypothesis that transforming growth factor-β 1 (TGF-β 1 ) regulates lysyl oxidase secretion from vascular smooth muscle cells. Lysyl oxidase is an enzyme that catalyzes an essential step in collagen and elastin cross-linking in the extracellular matrix, and TGF-β 1 has been implicated in the pathogenesis of restenosis after vascular injury. The effect of TGF-β 1 on lysyl oxidase in vascular smooth muscle cells has not been previously defined. Methods: Rat aortic smooth muscle cells were grown in culture to confluence. Cells in passage 2 to 6 were incubated for 24 hours in media containing 0.1, 0.5, 1.0, or 10.0 ng/ml of TGF-β 1 . Lysyl oxidase activity in the media was quantitated with a tritium-release bioassay against an insoluble 3 H-labeled aortic elastin substrate. Northern blot analyses were performed to determine steady-state levels of lysyl oxidase mRNA in the smooth muscle cells. Results: Lysyl oxidase activity in the media increased 1.5-fold above control levels after exposure to 10 ng/ml of TGF-β 1 ( p 1 , respectively ( p 1 was also time-dependent over the 24-hour experimental period. Conclusions: TGF-β 1 appears to regulate lysyl oxidase in cultured rat aortic smooth muscle cells. Increases in lysyl oxidase activity may be one of the mechanisms by which TGF-β 1 contributes to arterial restenosis after vascular injury. (J Vasc Surg 1997;25:446-52.)

Sequential studies of healing in endothelial seeded vascular prostheses histologic and ultrastructure characteristics of graft incorporation

Abstract Chronological events leading to incorporation of endothelial cell seeded prosthetic vascular grafts were documented in this investigation. Forty-one adult dogs underwent thoracoabdominal bypass using double-velour Dacron grafts. Experimental grafts were preclotted with blood containing enzymatically derived endothelium immediately after derivation, or after 14 days of cultivation. Control grafts were preclotted without addition of endothelial cells. Grafts were studied grossly as well as by light, scanning electron, transmission electron, and fluorescence microscopy, 1 to 28 days postimplantation. Control graft healing proceeded from pannus and perigraft ingrowth. Experimental grafts healed from seeded cells as well. Platelets covered all grafts by Days 1 and 2. Thrombus accumulations on control grafts, first evident on Day 4, became maximal by Day 14. Seeded grafts appeared relatively thrombus free with patches of endothelial cells noted by 4 days. These cells were initially separated by gaps, often containing leukocytes. Endothelium became densely packed with cellular migration and proliferation. Subendothelial tissues were composed of fibrin and smooth muscle. Control and experimental grafts were approximately 10 and 80% endothelialized, respectively, by Day 28. Smooth muscle dominated subintimal tissue in experimental grafts. These cells initially appeared fibroblastoid. Endothelial seeding enhances both pseudointimal development and rapid graft incorporation.

Regulation of Lysyl Oxidase by Interferon-γ in Rat Aortic Smooth Muscle Cells

Abstract —Lysyl oxidase is an essential catalyst for the cross-linking of extracellular collagen and elastin. Abnormalities in lysyl oxidase activity may contribute to the pathogenesis of arterial diseases characterized by abnormal matrix remodeling. This study tested the hypothesis that interferon (IFN)-γ, a proinflammatory cytokine present in aortic aneurysm and arteriosclerotic plaque rupture, downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells. Steady-state lysyl oxidase mRNA levels decreased in a concentration- and time-dependent manner to 30% of control levels after 24 hours of treatment with IFN-γ. Cell layer lysyl oxidase activity decreased in parallel with the observed changes in steady-state mRNA. Nuclear runoff studies suggested that transcriptional regulation was responsible for at least 40% of the observed downregulation. mRNA decay studies suggested that IFN-γ also decreased lysyl oxidase mRNA half-life from 9 to 6 hours. Downregulation of lysyl oxidase by IFN-γ did not appear to require new protein synthesis. This study documents that IFN-γ downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells by transcriptional and posttranscriptional mechanisms. If similar regulation occurs in vivo, it is possible that IFN-γ–mediated changes in lysyl oxidase may contribute to arterial diseases characterized by abnormal extracellular matrix.

Platelet reactivity in vivo in dogs with arterial prostheses seeded with endothelial cells.

This study was designed to assess platelet activity in vivo with vascular prostheses seeded with endothelial cells to determine the time course for development of thromboresistance and to test the ability of prostheses to produce prostacyclin. Sixteen dogs were randomly allocated to receive seeded (experimental group) or unseeded (control group) velour Dacron aortic prostheses. Serial measurements of platelet survival were performed to assess platelet interaction with prostheses in vivo, and platelet serotonin was monitored as an index of platelet release in vivo. After placement of prostheses, dogs in the experimental group had rapid normalization of platelet survival, with most having normal platelet survival at 4 to 8 weeks after surgery. In contrast, most control animals had reduced platelet survival throughout the 12 week period of study. Significant differences between groups in mean platelet survival were noted at 8 weeks after surgery (p less than .005) and in mean platelet serotonin at 12 weeks after surgery (p less than .05). Luminal surface production of 6-keto-PGF1 alpha from seeded prostheses was similar to aortic production and significantly greater (p less than .05) than that of control prostheses. Gross thrombus was present on 6.0 +/- 3.4% of the prosthetic surface in experimental animals in comparison to 26.6 +/- 19.2% in controls (p less than .005). The results of these studies document accelerated nonreactivity with platelets of seeded prostheses due to rapid coverage with endothelium possessing a normal ability to produce prostacyclin.

Isolation of adult canine venous endothelium for tissue culture

SummaryIn order to provide autologous adult endothelial cells for the production of cell-lined artificial vascular prostheses, we have developed a method for harvesting large numbers of cells with minimal contamination by other cellular types. In this technique, the vein to be stripped is isolated, removed, and everted over a stainless steel rod. After washing, the vein is incubated in trypsin-EDTA solution followed by collagenase and the endothelial cells flushed off with a stream of culture medium. With care and appropriate timing, the endothelium can be selectively removed leaving the underlying basal lamina intact.

Increased MMP-9 expression and activity by aortic smooth muscle cells after nitric oxide synthase inhibition is associated with increased nuclear factor-κB and activator protein-1 activity

OBJECTIVE To determine the mechanism underlying increased expression and activity of matrix metalloproteinase 9 (MMP-9) by rat aortic smooth muscle cells (RA-SMC) after inhibition of inducible nitric oxide synthase (iNOS). METHODS AND RESULTS Treatment of interleukin-1beta-stimulated RA-SMC with aminoguanidine led to an increase of 96% in MMP-9 activity (P = 0.003) by gelatin zymography, a 40% increase in pro-MMP-9 protein (P = 0.018) by Western blot, and a 155% increase in MMP-9 mRNA (P = 0.06) by reverse transcription polymerase chain reaction. Aminoguanidine also caused a 26% decrease in cytosolic IkappaB levels (P = 0.014) by Western blot, as well as a 97% increase in nuclear factor-kappaB binding and a 216% increase in activator protein-1 binding as measured by electrophoretic mobility shift assay. No significant changes were noted in MMP-2 or TIMP-1 expression, protein levels, or activity after aminoguanidine administration. CONCLUSIONS MMP-9 expression and activity is increased in cytokine stimulated RA-SMCs after iNOS inhibition, coincident with activation of the nuclear factor-kappaB and activator protein-1 pathways. We speculate that local derangements in iNOS may favor MMP-9-dependent vessel wall damage in vivo via an inflammatory cascade mechanism.

Decreased Collagen and Increased Matrix Metalloproteinase-13 in Experimental Abdominal Aortic Aneurysms in Males Compared with Females

BACKGROUND This study examined differences in sex in collagen regulation during rodent experimental abdominal aortic aneurysm formation. METHODS Infrarenal aortas of male and female rats were perfused with elastase or saline (control). Aortic diameters were measured at baseline (day 0) and on postoperative days 7 and 14. Transforming growth factor-beta 1, collagen subtypes I and III, and matrix metalloproteinase-13 (MMP-13; collagenase-3) expression and/or protein levels from aortic tissue were determined by real-time reverse transcription polymerase chain reaction and Western blotting. Aortic tissue was stained for total collagen, neutrophils, and macrophages using immunohistochemistry on days 4 and 7. RESULTS At 7 and 14 days after perfusion, aortic diameter increased in elastase-perfused males compared with females (P < .001 for each). At 4 and 7 days postperfusion, significantly more neutrophils and macrophages were present in elastase-perfused males compared with females. By 7 days postperfusion, protein levels of transforming growth factor-beta 1 were less in males compared with females (P = .04). Type I collagen levels also decreased on days 7 (P < .001) and 14 (P = .002), and type III collagen levels decreased on days 7 (P < .001) and 14 (P < .001) in males compared with females. With Massons trichrome stain, less adventitial collagen was observed in the elastase-perfused males compared with females. MMP-13 expression (P < .001) and protein levels (P = .006) in elastase-perfused males were greater than females on day 14. CONCLUSION This study documents a decrease in types I and III collagen with a concurrent increase in MMP-13 after elastase perfusion in males compared with females. These data suggest that alterations in extracellular matrix collagen turnover may be responsible for altered abdominal aortic aneurysm formation between sexes.

Near‐Thorough QT Study as Part of a First‐In‐Man Study

Detailed electrocardiographic (ECG) support was provided to a first‐in‐man, single‐ascending‐dose study that included 6 cohorts of 8 male volunteers each. In each cohort, 6 and 2 subjects received active compound and placebo, respectively. Long‐term 12‐lead ECGs were obtained on baseline day −1, dosing day 1, and day 2. Automatic QT‐interval measurements were made at 63 time points (28 at baseline and 35 on treatment). Based on QT/RR distribution, 20% of measurements were visually verified. Baseline‐corrected time‐matched ΔQTc values were obtained at 35 postdose time points. Placebo subjects of all cohorts were pooled. When 2 cohorts of the lowest, middle, and highest doses were pooled (12 subjects per active treatment group), the spreads of placebo‐corrected ΔΔQTc values were within the regulatory requirements (single‐sided 95% confidence interval <1 0 milliseconds) at all time points. Thus, this ECG support of the first‐in‐man study provided data of regulatory acceptable accuracy at a small fraction of the cost of a full thorough QT study.