Karen J. Roelofs

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.

Gender Differences in Experimental Aortic Aneurysm Formation

Objective—It is hypothesized that a male predominance, similar to that in humans, persists in a rodent model of experimental abdominal aortic aneurysm (AAA) via alterations in matrix metalloproteinases (MMPs). Methods and Results—Group I experiments were as follows: elastase perfusion of the infrarenal aorta was performed in male (M) and female (F) rats. At 14 days, aortas were harvested for immunohistochemistry, real-time polymerase chain reaction (PCR), and zymography. Group II experiments were the following: abdominal aorta was transplanted from F or M donors into F or M recipients. At 14 days, rodents that had undergone transplantation underwent elastase perfusion. In group III, male rats were given estradiol or sham 5 days before elastase perfusion. In group I, M rats had larger AAAs with higher frequency than did F rats. M rat aortas had more significant macrophage infiltrates and increased matrix metalloproteinase (MMP)-9 production and activity. In group II, M-to-M aortic transplants uniformly developed aneurysms after elastase perfusion, whereas F-to-F aortic transplants remained resistant to aneurysm formation. F aortas transplanted into M recipients, however, lost aneurysm resistance. In group III, estradiol-treated rats demonstrated smaller aneurysms and less macrophage infiltrate and MMP-9 compared with M controls after elastase. Conclusions—These data provide evidence of gender-related differences in AAA development, which may reflect an estrogen-mediated reduction in macrophage MMP-9 production.

L-Selectin-Mediated Neutrophil Recruitment in Experimental Rodent Aneurysm Formation

Background—This investigation tested the hypothesis that L-selectin is important in experimental abdominal aortic aneurysm (AAA) formation in rodents. Methods and Results—Rat abdominal aortas were perfused with saline (control) or porcine pancreatic elastase and studied on postperfusion days 1, 2, 4, 7, and 14 (n=5 per treatment group per day). Neutrophil (polymorphonucleur leukocyte, PMN) and macrophage counts per high-powered field (HPF) were performed on fixed sections. L-selectin expression and protein levels in aortic tissue were determined by polymerase chain reaction and Western blot, respectively. Elastase-perfused aortic diameters were significantly increased compared with control aortas at all time points except day 1 (P<0.05). PMN counts significantly increased in elastase-perfused aortas compared with control aortas at days 1, 2, and 4, reaching maximum levels at day 7 (40.8 versus 0.3 PMNs/HPF, P=0.001). L-selectin mRNA expression in elastase-perfused aortas was 18 (P=0.018), 17 (P<0.001), and 8 times (P=0.02) times greater than control aortas at days 1, 2, and 4, respectively. Western blot demonstrated a significant 69% increase in L-selectin protein at day 7 in elastase- as compared with saline-perfused aortas (P=0.005). Subsequent experiments involved similar studies on postperfusion days 4, 7, and 14 of aortas from C57BL/6 wild-type (WT) mice (n=21) and L-selectin–knockout (LKO) mice (n=19). LKO mice had significantly smaller aortic diameters at day 14 as compared with WT mice (88% versus 123%, P=0.02). PMN counts were significantly greater in elastase-perfused WT mouse aortas as compared with LKO mouse aortas at day 4 after perfusion (12.8 versus 4.8 PMNs/HPF, P=0.02). Macrophage counts were significantly greater at all time points after perfusion in elastase-perfused WT mouse aortas compared with elastase-perfused LKO mouse aortas, with a maximum difference at day 7 after perfusion (13.3 versus 0.5 macrophages/HPF, P<0.001). Conclusion—L-selectin–mediated neutrophil recruitment may be a critical early step in AAA formation.

Female Gender Attenuates Cytokine and Chemokine Expression and Leukocyte Recruitment in Experimental Rodent Abdominal Aortic Aneurysms

Abstract:  Female gender appears to be protective in the development of abdominal aortic aneurysms (AAAs). This study sought to identify gender differences in cytokine and chemokine expression in an experimental rodent AAA model. Male and female rodent aortas were perfused with either saline (control) or elastase to induce AAA formation. Aortic diameter was determined and aortic tissue was harvested on postperfusion days 4 and 7. Cytokine and chemokine gene expression was examined using focused gene arrays. Immunohistochemistry was used to quantify aortic leukocyte infiltration. Data were analyzed by Students t‐tests and ANOVA. Elastase‐perfused female rodents developed significantly smaller aneurysms compared to males by day 7 (93 ± 10% vs. 201 ± 25%, P= 0.003). Elastase‐perfused female aortas exhibited a fivefold decrease in expression of the BMP family and ligands of the TNF superfamily compared to males. In addition, the expression of members of the TGF β and VEGF families were three to fourfold lower in female elastase‐perfused aortas compared to males. Multiple members of the interleukin, CC chemokine receptor, and CC ligand families were detectable in only the male elastase‐perfused aortas. Female elastase‐perfused aortas demonstrated a corollary twofold lower neutrophil count (females: 17.5 ± 1.1 PMN/HPF; males: 41 ± 5.2 neutrophils/HPF, P= 0.01) and a 1.5‐fold lower macrophage count (females: 12 ± 1.1 macrophages/HPF; males: 17.5 ± 1.1 macrophages/HPF, P= 0.003) compared to male elastase‐perfused aortas. This study documents decreased expression of multiple cytokines and chemokines and diminished leukocyte trafficking in female rat aortas compared to male aortas following elastase perfusion. These genes may contribute to the gender disparity seen in AAA formation.

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.

The Role of Pancreatic Preproglucagon in Glucose Homeostasis in Mice

Glucagon-like peptide 1 (GLP-1) is necessary for normal gluco-regulation, and it has been widely presumed that this function reflects the actions of GLP-1 released from enteroendocrine L cells. To test the relative importance of intestinal versus pancreatic sources of GLP-1 for physiological regulation of glucose, we administered a GLP-1R antagonist, exendin-[9-39] (Ex9), to mice with tissue-specific reactivation of the preproglucagon gene (Gcg). Ex9 impaired glucose tolerance in wild-type mice but had no impact on Gcg-null or GLP-1R KO mice, suggesting that Ex9 is a true and specific GLP-1R antagonist. Unexpectedly, Ex-9 had no effect on blood glucose in mice with restoration of intestinal Gcg. In contrast, pancreatic reactivation of Gcg fully restored the effect of Ex9 to impair both oral and i.p. glucose tolerance. These findings suggest an alternative model whereby islet GLP-1 also plays an important role in regulating glucose homeostasis.

Differential Regulation of Aortic Growth in Male and Female Rodents Is Associated With AAA Development

BACKGROUND The objective was to examine effects of gonadal hormone manipulation on aortic diameter and macrophage infiltration in rodents during abdominal aortic aneurysm (AAA) formation. METHODS Experiment 1: 17-beta estradiol and testosterone pellets were implanted in male (ME) and female (FT) rats. No pellet was implanted in shams (MES, FTS). Experiment 2: Testes and ovaries were removed from males (MO) and females (FO), respectively. No organs were removed from shams (MOS, FOS). Experiment 3: Male and female rats were orchiectomized and oophorectomized, respectively. Four weeks post-castration, testosterone (MOT) and 17-beta estradiol (FOE) pellets were implanted. Shams underwent castration, but no pellet was implanted (MOTS, FOES). All rats underwent infrarenal aortic infusion with elastase postimplantation/postcastration. Diameters were measured on postoperative d 14. Tissue was stained for macrophages by immunohistochemistry. RESULTS Diameter (P = 0.046) and macrophage counts (P = 0.014) decreased in ME compared with shams, but not in females treated with testosterone (FT). Diameter (P = 0.019) and macrophage infiltration (P = 0.024) decreased in MO compared with shams, but not in FO. Diameter increased in MOT compared with MOTS (P = 0.033), but decreased in FOE compared with FOES (P = 0.002). Macrophages decreased in FOE compared with FOES (P = 0.002). CONCLUSION This study documents a decrease in AAA diameter in males treated with estrogen or undergoing orchiectomy, but no changes in females treated with testosterone or undergoing oophorectomy; and an increase in diameter in MOT and a decrease in FOE. These data suggest that gonadal hormones differentially regulate AAA growth in association with changes in macrophages.

Gender-Dependent Differential Phosphorylation in the ERK Signaling Pathway is Associated with Increased MMP2 Activity in Rat Aortic Smooth Muscle Cells

BACKGROUND The present experiments were conducted to explore the role of mitogen-activated protein kinase (MAPK) pathways, potential upstream regulators of MMPs, in abdominal aortic aneurysms (AAAs). METHODS Rat aortic smooth muscle cells (RASMCs) from males and females were treated with media containing interleukin (IL)-1beta (2 ng/mL), a concentration known to be present in AAAs. Levels of both total and phosphorylated (activated) extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38 were examined by Western blotting at various time intervals up to 60 min. Similar experiments were conducted following exposure of RASMCs to elastase (6 U/mL), a concentration known to induce AAA formation in rodents. Finally, media was assayed for MMP activity by zymography. RESULTS Total ERK (t-ERK) was consistently no different in females compared with males prior to or following IL-1beta exposure. In contrast, levels of phosphorylated ERK (p-ERK) were significantly higher in males than females throughout the postexposure period (P < 0.0001). Levels of t-p38, p-p38, and t-JNK were not altered in a gender-dependent manner. The lack of p-JNK levels detected in both male and female RASMCs did not allow for conclusions to be drawn regarding gender disparities in this pathway. Results were similar following RASMC elastase exposure, although t-ERK levels were consistently higher in females than males (P < 0.0001). Pro-MMP2 levels were significantly higher (P = 0.0035) in males than females at each time point following elastase exposure. CONCLUSIONS These data provide evidence implicating alterations in p-ERK signaling via the up-regulation of MMPs as a potential explanation for gender-related discrepancies in AAA formation.

Ultrasound measurement of aortic diameters in rodent models of aneurysm disease.

BACKGROUND This investigation was undertaken to evaluate transabdominal ultrasound (US) measurements of aortic diameters in rats and mice as a complementary method to video microscopy (VM), the current standard for assessing the diameter of rodent aortas. METHODS Aortic diameters were measured in 64 rats (n = 132 sets) and 12 mice (n = 36 sets) following experimental induction of aortic aneurysms. Diameters were measured at the renal vein, midinfrarenal aorta, and aortic bifurcation. RESULTS In the rat, anteroposterior (AP) US measurements were closely correlated with transverse VM measurements, with correlation coefficients ranging from 0.66 to 0.77 (P < 0.0001) for axial US images and 0.58 to 0.63 (P < 0.0001) for sagittal US images. In the mouse, significant correlation coefficients were 0.57 (P < 0.001) near the renal vein and 0.44 (P = 0.007) at the midinfrarenal aorta. Aortic diameters increased significantly with increasing animal age and weight (R = 0.40, P = 0.003 at the renal vein, R = 0.29, P = 0.04 in the midinfrarenal aorta, and R = 0.39, P = 0.004 at the aortic bifurcation), suggesting that weight matched rodents must be used to define aortic dimensions in treatment groups as opposed to repeated comparisons with baseline measurements in a growing rat. CONCLUSION Noninvasive aortic US measurements throughout the course of a rodent study of aneurysmal disease provide a practical alternative to VM for the repeated determinations of aortic diameters.