Leptin receptor-expressing nucleus tractus solitarius neurons suppress food intake independently of GLP1 in mice.

Leptin receptor-expressing (LepRb-expressing) neurons of the nucleus tractus solitarius (NTS; LepRbNTS neurons) receive gut signals that synergize with leptin action to suppress food intake. NTS neurons that express preproglucagon (Ppg) (and that produce the food intake-suppressing PPG cleavage product glucagon-like peptide-1 [GLP1]) represent a subpopulation of mouse LepRbNTS cells. Using Leprcre, Ppgcre, and Ppgfl mouse lines, along with Designer Receptors Exclusively Activated by Designer Drugs (DREADDs), we examined roles for Ppg in GLP1NTS and LepRbNTS cells for the control of food intake and energy balance. We found that the cre-dependent ablation of NTS Ppgfl early in development or in adult mice failed to alter energy balance, suggesting the importance of pathways independent of NTS GLP1 for the long-term control of food intake. Consistently, while activating GLP1NTS cells decreased food intake, LepRbNTS cells elicited larger and more durable effects. Furthermore, while the ablation of NTS Ppgfl blunted the ability of GLP1NTS neurons to suppress food intake during activation, it did not impact the suppression of food intake by LepRbNTS cells. While Ppg/GLP1-mediated neurotransmission plays a central role in the modest appetite-suppressing effects of GLP1NTS cells, additional pathways engaged by LepRbNTS cells dominate for the suppression of food intake.

Calcitonin Receptor Neurons in the Mouse Nucleus Tractus Solitarius Control Energy Balance via the Non-aversive Suppression of Feeding.

To understand hindbrain pathways involved in the control of food intake, we examined roles for calcitonin receptor (CALCR)-containing neurons in the NTS. Ablation of NTS Calcr abrogated the long-term suppression of food intake, but not aversive responses, by CALCR agonists. Similarly, activating CalcrNTS neurons decreased food intake and body weight but (unlike neighboring CckNTS cells) failed to promote aversion, revealing that CalcrNTS neurons mediate a non-aversive suppression of food intake. While both CalcrNTS and CckNTS neurons decreased feeding via projections to the PBN, CckNTS cells activated aversive CGRPPBN cells while CalcrNTS cells activated distinct non-CGRP PBN cells. Hence, CalcrNTS cells suppress feeding via non-aversive, non-CGRP PBN targets. Additionally, silencing CalcrNTS cells blunted food intake suppression by gut peptides and nutrients, increasing food intake and promoting obesity. Hence, CalcrNTS neurons define a hindbrain system that participates in physiological energy balance and suppresses food intake without activating aversive systems.

Oea Signaling Pathways and the Metabolic Benefits of Vertical Sleeve Gastrectomy.

OBJECTIVE: The aim of this study was to determine whether downstream [peroxisome proliferator-activated-receptor alpha (PPARα) and the G-protein coupled receptor, GPR119] and upstream (a fatty acid translocase, CD36) signaling targets of N-oleoylethanolamide (OEA) were necessary for weight loss, metabolic improvements, and diet preference following vertical sleeve gastrectomy (VSG). SUMMARY BACKGROUND DATA: OEA is an anorectic N-acylethanolamine produced from dietary fats within the intestinal lumen that can modulate lipid metabolism, insulin secretion, and energy expenditure by activating targets such as PPARα and GPR119. METHODS: Diet-induced obese mice, including wild-type or whole body knockout (KO) of PPARα, GPR119, and CD36, were stratified to either VSG or sham surgery before body weight, body composition, diet preference, and glucose and lipid metabolic endpoints were assessed. RESULTS: We found increased duodenal production of OEA and expression of both GPR119 and CD36 were upregulated in wild-type mice after VSG. However, weight loss and glucose tolerance were improved in response to VSG in PPARαKO, GPR119KO, and CD36KO mice. In fact, VSG corrected hepatic triglyceride dysregulation in CD36KO mice, and circulating triglyceride and cholesterol levels in PPARαKO mice. Lastly, we found PPARα-mediated signaling contributes to macronutrient preference independent of VSG, while removal of CD36 signaling blunts the VSG-induced shift toward carbohydrate preference. CONCLUSIONS: In the search for more effective and less invasive therapies to help reverse the global acceleration of obesity and obesity-related disease OEA is a promising candidate; however, our data indicate that it is not an underlying mechanism of the effectiveness of VSG.

The role of GIP and pancreatic GLP-1 in the glucoregulatory effect of DPP-4 inhibition in mice.

AIMS/HYPOTHESIS: Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are two peptides that function to promote insulin secretion. Dipeptidyl peptidase-4 (DPP-4) inhibitors increase the bioavailability of both GLP-1 and GIP but the dogma continues to be that it is the increase in GLP-1 that contributes to the improved glucose homeostasis. We have previously demonstrated that pancreatic rather than intestinal GLP-1 is necessary for improvements in glucose homeostasis in mice. Therefore, we hypothesise that a combination of pancreatic GLP-1 and GIP is necessary for the full effect of DPP-4 inhibitors on glucose homeostasis. METHODS: We have genetically engineered mouse lines in which the preproglucagon gene (Gcg) is absent in the entire body (GcgRAΔNull) or is expressed exclusively in the intestine (GcgRAΔVilCre) or pancreas and duodenum (GcgRAΔPDX1Cre). These mice were used to examine oral glucose tolerance and GLP-1 and GIP responses to a DPP-4 inhibitor alone, or in combination with incretin receptor antagonists. RESULTS: Administration of the DPP-4 inhibitor, linagliptin, improved glucose tolerance in GcgRAΔNull mice and control littermates and in GcgRAΔVilCre and GcgRAΔPDX1Cre mice. The potent GLP-1 receptor antagonist, exendin-[9-39] (Ex9), blunted improvements in glucose tolerance in linagliptin-treated control mice and in GcgRAΔPDX1Cre mice. Ex9 had no effect on glucose tolerance in linagliptin-treated GcgRAΔNull or in GcgRAΔVilCre mice. In addition to GLP-1, linagliptin also increased postprandial plasma levels of GIP to a similar degree in all genotypes. When linagliptin was co-administered with a GIP-antagonising antibody, the impact of linagliptin was partially blunted in wild-type mice and was fully blocked in GcgRAΔNull mice. CONCLUSIONS/INTERPRETATION: Taken together, these data suggest that increases in pancreatic GLP-1 and GIP are necessary for the full effect of DPP-4 inhibitors on glucose tolerance.

Vertical sleeve gastrectomy corrects metabolic perturbations in a low-exercise capacity rat model.

OBJECTIVE: Bariatric surgery is currently our most effective strategy at weight loss, yet the mechanisms for its success remain unknown. Low exercise capacity, in humans and rodents, predicts poor metabolic outcome. The objective of this manuscript was to determine if bariatric surgery could restore metabolic perturbations in rats with low intrinsic exercise capacity. METHODS: We performed vertical sleeve gastrectomy (VSG) or sham surgery in high fat-fed rats selectively bred for low running capacity. RESULTS: We found that VSG reduced body mass through a reduction in fat mass, caused early reductions in food intake, and shifted macronutrient preference away from fat and toward carbohydrates. VSG had no impact on basal glucose but did improve the return to baseline after an oral glucose load. As has been shown previously, VSG increased postprandial insulin, GLP-1, and bile acids. There was no significant impact of VSG on plasma triglycerides, hepatic triglycerides, or cholesterol. Interestingly, the brown adipose tissue to white adipose tissue ratio tended to be greater in VSG compared to sham surgery animals. While VSG positively impacted several aspects of metabolism, it did not enhance maximal oxygen capacity and seemed to lower metabolic efficiency as indicated by lower resting oxygen consumption and fat and carbohydrate oxidation. CONCLUSION: VSG can improve the metabolic status of animals with a low exercise capacity independently of exercise capacity.

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.

P-selectin inhibition therapeutically promotes thrombus resolution and prevents vein wall fibrosis better than enoxaparin and an inhibitor to von Willebrand factor.

OBJECTIVE: Aptamers are oligonucleotides targeting protein-protein interactions with pharmacokinetic profiles and activity reversal options. Although P-selectin and von Willebrand factor (vWF) have been implicated in the development of venous thrombosis (VT), no studies have directly compared aptamer efficacy with standard of care in VT. In this study, ARC5692, an anti-P-selectin aptamer, and ARC15105, an anti-vWF aptamer, were compared with low-molecular-weight heparin, enoxaparin, to test the efficacy of P-selectin or vWF inhibition in promoting thrombus resolution and preventing vein wall fibrosis, in a baboon model of VT. APPROACH AND RESULTS: Groups were as follows: treatment arm: animals received P-selectin or vWF aptamer inhibitors or enoxaparin (n=3 per group). Controls received no treatment (n=3). Prophylactic arm: animals received P-selectin inhibitor (n=4) or vWF inhibitor (n=3). Treatment arm: P-selectin-inhibitor demonstrated a significant improvement in vein recanalization by magnetic resonance venography (73% at day 21), and significantly decreased vein wall collagen, compared with all groups. Anti-P-selectin equaled enoxaparin in maintaining valve competency by ultrasound. All control animals had compromised valve competency post thrombosis. Prophylactic arm: animals receiving P-selectin and vWF inhibitors demonstrated improved vein recanalization by magnetic resonance venography versus controls (80% and 85%, respectively, at day 21). Anti-P-selectin protected iliac valve function better than anti-vWF, and both improved valve function versus controls. No adverse bleeding events were observed. CONCLUSIONS: The P-selectin inhibitor aptamer promoted iliac vein recanalization, preserved valve competency, and decreased vein wall fibrosis. The results of this work suggest that P-selectin inhibition maybe an ideal target in the treatment and prophylaxis of deep VT, warranting clinical trials.

Low-molecular-weight heparin modulates vein wall fibrotic response in a plasminogen activator inhibitor 1-dependent manner.

BACKGROUND: Treatment with low-molecular-weight heparin (LMWH) favorably alters the vein wall response to deep venous thrombosis (DVT), although the mechanisms remain unclear. Previous studies have suggested that LMWH alters the levels of circulating plasminogen activator inhibitor 1 (PAI-1), a known mediator of fibrosis, and may improve endogenous fibrinolysis. We hypothesized that LMWH favorably alters the vein wall response by binding of PAI-1 and acceleration of fibrinolysis. METHODS: Wild-type and PAI-1 -/- mice underwent treatment with LMWH after induction of occlusive DVT. Vein wall and plasma were harvested and analyzed by enzyme-linked immunosorbent assay, zymography, real-time polymerase chain reaction, and immunohistochemistry. RESULTS: Wild-type mice treated with LMWH exhibited diminished vein wall fibrosis (0.6 ± 0.6 vs 1.4 ± 0.2; P < .01; n = 5) and elevation of circulating PAI-1 (1776 ± 342 vs 567 ± 104 ρg/mL; P < .01; n = 5) compared with untreated controls after occlusive DVT. PAI-1-/- mice treated with LMWH were not similarly protected from fibrosis, despite improved thrombus resolution. Treatment with LMWH was associated with decreased intrathrombus interleukin-lß (68.6 ± 31.0 vs 223.4 ± 28.9 ρg/mg total protein; P < .01; n = 5) but did not alter inflammatory cell recruitment to the vein wall. PAI-1 -/- mice exhibited significantly elevated intrathrombus (257.2 ± 51.5 vs 4.3 ± 3.8 ρg/mg total protein; n = 5) and vein wall interleukin-13 (187.2 ± 57.6 vs 9.9 ± 1.1 ρg/mg total protein; P < .05; n = 5) as well as vein wall F4/80 positively staining monocytes (53 ± 11 vs 16 ± 2 cells/5 high-power fields; P < .05; n = 4). CONCLUSIONS: LMWH did not accelerate venous thrombosis resolution but did protect against vein wall fibrosis in a PAI-1-dependent manner in an occlusive DVT model. Lack of PAI-1 correlated with accelerated venous thrombosis resolution but no protection from fibrosis. PAI-1 inhibition as a treatment strategy for DVT is likely to accelerate clearance of the thrombus but may come at the expense of increased vein wall fibrosis. CLINICAL RELEVANCE: The pathophysiologic mechanism of post-thrombotic syndrome is not well understood clinically or experimentally. In this study, we evaluated the effect of the prominent fibrinolytic mechanism, plasminogen activator inhibitor 1 (PAI-1), and low-molecular-weight heparin (LMWH) on vein wall injury after thrombosis. We show here that LMWH is protective from vein wall fibrosis, but this is abrogated in PAI-1-deleted mice. This is also correlated with monocyte vein wall influx. These data support the clinical observation that LMWH may be protective from post-thrombotic vein wall injury in a PAI-1-dependent manner.

Increased estrogen receptor alpha in experimental aortic aneurysms in females compared with males.

BACKGROUND: Estrogen receptor alpha (ERα) has been identified in the vessel wall, offering vasoprotective effects when upregulated. Estrogens are known to mediate the inflammatory milieu, and inflammation has long been associated with abdominal aortic aneurysm (AAA) formation. Therefore, it is theorized that increased estrogen receptor in females contributes to their relative resistance to AAAs. The objective of this study was to determine gender differences in ERα levels during experimental AAA formation. METHODS: Infrarenal aortas of male and female C57 mice (n = 18 and n = 16, respectively) were infused with 0.4% elastase. Diameters were measured at days 0 and 14. Aortic messenger RNA expression of ERα was determined on day 3 by reverse transcription-polymerase chain reaction, whereas ERα protein levels were measured via Western blot. Immunohistochemistry using rabbit antibody for ERα was performed on day 14 samples and quantified. Zymography was done for matrix metalloproteinases (MMP)2 and 9 activity levels. Samples of human AAAs were collected and Western blot performed. Data were compared for significance using a student t-test. RESULTS: Infrarenal aortic diameter increased in elastase-perfused males (ME) by 80% at 14 days after perfusion, whereas females (FE) increased by only 35% (P = 0.0012). FE had ×10 greater ERα messenger RNA expression compared with ME at day 3 (P = 0.003). Similarly, ERα protein levels were 100% higher in FE compared with those in ME on day 14 (P = 0.035). ERα protein levels were 80% higher in female human patients with AAA than those in their male counterparts (P = 0.029). ERα visualized via immunohistochemistry was 1.5 fold higher in FE than ME (P = 0.029). MMP2 and 9 activity levels were decreased in female compared with male aortas. CONCLUSIONS: This study demonstrates an increase in aortic wall ERα in females compared with males that correlates inversely with MMP activity and AAA formation. These findings, coupled with observations that exogenous estrogen inhibits AAA formation in males, further suggest that estrogen supplementation may be important to prevent AAA formation and growth.

Differential gender- and species-specific formation of aneurysms using a novel method of inducing abdominal aortic aneurysms.

BACKGROUND: The objective of this study was to test a novel model of inducing abdominal aortic aneurysms (AAAs) in different mouse strains and genders. MATERIALS AND METHODS: Male and female C57BL/6 and B6129 mice (n = 5 per group) underwent periaortic dissection and porcine pancreatic elastase (30 µL) or inactivated elastase application (5 min) to the aorta. Aortic measurements were taken on days 0 and 14. Aortic samples were analyzed for histology and zymography for matrix metalloproteinase (MMP) activity. Comparison statistics were performed using unpaired t-test. RESULTS: AAA phenotype (50% aortic increase) occurred in external elastase-treated males (100%) and females (90%). No control animals developed AAAs. The aortic diameter was larger in C57BL/6 and B6129 elastase-treated versus control males (P = 0.0028 and P < 0.0001, respectively) and females (P < 0.0001 and P = 0.0458, respectively). Histology verified phenotype via disrupted internal elastic laminae. Macrophage counts in elastase-treated animals were >6-fold higher than in controls (all groups significant). MMP9 activity was greater in elastase-treated males and females in C57BL/6 (P = 0.0031, P = 0.0004) and B6129 (P = 0.025, P = 0.2) mice; MMP2 activity was greater in C57BL/6 versus B6129 male elastase-treated mice. CONCLUSIONS: This rodent model produced AAAs in both genders and strains of mice. This model is simple, has little variability, and occurs in the infrarenal aorta, substantiating the external elastase model for future studies.

Increased PAI-1 in females compared with males is protective for abdominal aortic aneurysm formation in a rodent model.

The serine proteases, along with their inhibitor plasmin activator inhibitor-1 (PAI-1), have been shown to play a role in abdominal aortic aneurysm (AAA) formation. The aim of this study is to determine if PAI-1 may be a protective factor for AAA formation and partially responsible for the gender difference observed in AAAs. Male and female wild-type (WT) C57BL/6 and PAI-1(-/-) mice 8-12 wk of age underwent aortic perfusion with porcine pancreatic elastase. Animals were harvested 14 days following perfusion and analyzed for phenotype, PAI-1 protein levels, and matrix metalloproteinase (MMP)-9 and -2 activity. WT males had an average increase in aortic diameter of 80%, whereas females only increased 32% (P < 0.001). PAI-1(-/-) males increased 204% and females 161%, significantly more than their WT counterparts (P < 0.001). Western blot revealed 61% higher PAI-1 protein levels in the WT females compared with the WT males (P = 0.01). Zymography revealed higher levels of pro-MMP-2 and active MMP-2 in the PAI-1(-/-) males and females compared with their WT counterparts. PAI-1(-/-) females had significantly higher serum plasmin levels compared with WT females (P = 0.003). In conclusion, WT female mice are protected from aneurysm formation and have higher levels of PAI-1 compared with males during experimental aneurysm formation. Additionally, both male and female PAI-1(-/-) animals develop significantly larger aneurysms than WT animals, correlating with higher pro- and active MMP-2 levels. These findings suggest that PAI-1 is protective for aneurysm formation in the elastase model of AAA and plays a role in the gender differences seen in AAA formation.

Increased JNK in males compared with females in a rodent model of abdominal aortic aneurysm.

BACKGROUND: In humans, there is a 4:1 male:female ratio in the incidence of abdominal aortic aneurysms (AAAs). c-Jun-N-terminal kinase (JNK) is an important upstream regulator of several enzymes involved in AAA formation, including the matrix metalloproteinases (MMPs). The purpose of this study was to determine if there is a gender difference between males and females in JNK during AAA formation. MATERIALS AND METHODS: Male and female C57/B6 mice underwent aortic perfusion with elastase or heat inactivated elastase with aortas harvested at d 3 and 14 for phenotype determination, RT-PCR, Western blot, and zymography. Additionally, in vitro experiments using siRNA were conducted to define JNK regulation of matrix metalloproteinases (MMPs). A t-test was used to compare between groups. RESULTS: Males formed larger AAAs at d 14 compared with females (P < 0.001), with significantly higher levels of JNK1 protein, proMMP9, proMMP2, and active MMP2. At d 3, males had more JNK1 mRNA, protein, and MMP activity. Knockdown of JNK 1 or 2 in vitro decreased MMP activity, while knockdown of JNK 1 and 2 together blocked all MMP activity. CONCLUSION: Alterations in JNK between genders is partially responsible for the differential rates of experimental AAA formation, likely through differential regulation of MMPs.

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.

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 Masson's 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.

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.

Differential regulation of the superoxide dismutase family in experimental aortic aneurysms and rat aortic explants.

OBJECTIVE: Oxidative stress has been implicated in abdominal aortic aneurysm pathogenesis. This study sought to characterize the relevance of superoxide dismutases (SOD), a family of reactive oxygen catalyzing metalloenzymes, including manganese SOD (MnSOD), copper-zinc SOD (CuZnSOD), and extracellular SOD (EcSOD), in a rodent aortic aneurysm model. METHODS: Male rat infrarenal abdominal aortas were perfused with either saline (control) or porcine pancreatic elastase (6 U/mL). Aortic diameter was measured and aortas harvested on post-operation days 1, 2, and 7 (N=5-6 per treatment group per day). MnSOD, CuZnSOD, EcSOD, catalase, MMP-2, MMP-9, and beta-actin expression in aortic tissue was determined by quantitative real-time PCR. MnSOD protein levels were measured using western immunoblotting and immunohistochemistry. In subsequent experiments, aimed at understanding the mechanism by which SOD is involved in AAA pathogenesis, rat aortic explants (RAEs) were incubated in media for 24 h in the presence of interleukin-1beta (IL-1beta, 2 ng/mL) and TEMPOL (SOD mimetic), catalase, or a combined SOD and catalase mimetic. Media MMP-2 and MMP-9 activity was determined by zymography. Data were analyzed by Student's t-tests and ANOVA. RESULTS: Elastase-perfused aortic diameters were significantly increased compared to control aortas by post-perfusion day 7 (P=0.016). MnSOD mRNA levels in elastase perfused aortas were 6.0 (P=0.05) and 7.5 times (P<0.01) greater than control aortas at post-perfusion days 1 and 2, respectively. EcSOD, CuZnSOD, catalase, and MMP-2 mRNA expression did not statistically vary between the two groups. MMP-9 expression was 3.5-fold higher in the elastase group on post-perfusion day 2 (P=0.04). Western immunoblotting confirmed MnSOD protein was up-regulated on day 4 in the elastase-perfused group compared to controls (P=0.02). Immunohistrochemistry demonstrated increased MnSOD staining in the elastase group on day 4. In RAE experiments, TEMPOL increased both MMP-9 and MMP-2 activity 2 (P=0.09) and 3-fold (P=0.05), respectively, whereas catalase and the combined SOD/catalase mimetic failed to increase MMP-2 or MMP-9 activity. CONCLUSION: Experimental abdominal aortic aneurysm formation is associated with early increases in MnSOD expression and an increase in MMP-9 activity. Strategies aimed at inhibiting oxidative stress during AAA formation should focus on MnSOD.

Diffusion of Alexa Fluor 488-conjugated dendrimers in rat aortic tissue.

In this study, the distribution of labeled dendrimers in native and aneurysmal rat aortic tissue was examined. Adult male rats underwent infrarenal aorta perfusion with generation 5 (G5) acetylated Alexa Fluor 488-conjugated dendrimers for varying lengths of time. In a second set of experiments, rats underwent aortic elastase perfusion followed by aortic dendrimer perfusion 7 days later. Aortic diameters were measured prior to and postelastase perfusion, and again on the day of harvest. Aortas were harvested 0, 12, or 24 h postperfusion, fixed, and mounted. Native aortas were harvested and viewed as negative controls. Aortic cross-sections were viewed and imaged using confocal microscopy. Dendrimers were quantified (counts/high-powered field). Results were evaluated by repeated measures ANOVA and Student's t-test. We found that in native aortas, dendrimers penetrated the aortic wall in all groups. For all perfusion times, fewer dendrimers were present as time between dendrimer perfusion and aortic harvest increased. Longer perfusion times resulted in increased diffusion of dendrimers throughout the aortic wall. By 24 h, the majority of the dendrimers were through the wall. Dendrimers in aneurysmal aortas, on day 0 postdendrimer perfusion, diffused farther into the aortic wall than controls. In conclusion, this study documents labeled dendrimers delivered intra-arterially to native rat aortas in vivo, and the temporal diffusion of these molecules within the aortic wall. Increasing perfusion time and length of time prior to harvest resulted in continued dendrimer diffusion into the aortic wall. These preliminary data provide a novel mechanism whereby local inhibitory therapy may be delivered locally to aortic tissue.

Attenuation of experimental aortic aneurysm formation in P-selectin knockout mice.

The aim of this study was to determine the role of P-selectin, an adhesion molecule found on the surface of activated platelets and endothelial cells during experimental aortic aneurysm formation. Infrarenal abdominal aortas of C57 black wild-type (WT) mice and P-selectin knockout (PKO) mice were measured in situ and then perfused with porcine pancreatic elastase (0.332 U/mL). Whole blood was drawn from the tail artery on day 2 pre-perfusion to determine total and differential white blood cell (WBC) counts. On day 14 postperfusion, aortic diameters (AD) of WT mice (N = 19) and PKO mice (N = 9) were measured. An aortic aneurysm was defined as a 100% or greater increase in AD from pre-perfusion measurement. Immunohistochemistry, including H&E, trichrome and von Gieson staining, was performed on harvested aortic tissue. Statistical analysis was performed by t-test and Fisher's exact test. There were no significant differences in peripheral leukocyte counts at baseline between the two groups. WT mice had significantly larger AD compared to PKO mice at day 14 postperfusion (116 % vs. 38 %, P < 0.001). Aortic aneurysm penetrance was 52% in WT mice, while 0% (P = 0.01) of PKO mice formed aneurysms. On histologic examination, WT mouse aortas were associated with a significant inflammatory response and degradation of elastin and collagen fibers, while PKO mouse aortas lacked signs of inflammation or vessel wall injury. P-selectin deficiency attenuates aneurysm formation in the elastase aortic perfusion model. This was associated with a blunting of the inflammatory response and preserved vessel wall intergrity following elastase perfusion in the P-selectin knockout mice. Further investigation to elucidate the independent contributions of endothelial cell and platelet P-selectin in experimental aortic aneurysm formation is required.

Female gender attenuates cytokine and chemokine expression and leukocyte recruitment in experimental rodent abdominal aortic aneurysms.

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 Student's 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 beta 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.

A biologic basis for asymmetric growth in descending thoracic aortic aneurysms: a role for matrix metalloproteinase 9 and 2.

OBJECTIVE: This study was undertaken to define matrix metalloproteinase (MMP) expression in the anterior and posterior wall of descending thoracic aortic aneurysms (TAAs) and correlate it with specific computed tomography (CT) image sites within the descending thoracic aorta. METHODS: Serial CT images of patients with TAAs were compared with age- and gender-matched normal descending thoracic aortas at levels T4-T12. The mean circumference of the TAAs was 153 mm (n = 12) and 148 mm (n = 11) at T8 and T10, respectively, compared with 75 mm (n = 12) and 75 mm (n = 10) in controls (P < .001). Aortic tissue was collected from a separate set of eight patients undergoing descending TAA resection (processed < or =12 hours of excision) and six cadavers (processed < or =24 hours of death). Tissue collected between the intercostals arteries was defined as posterior wall, and directly opposite was the anterior wall. MMP-9 and MMP-2 messenger RNA (mRNA) extracted from aortic tissue was analyzed by quantitative real time polymerase chain reaction (PCR) and normalized to beta-actin. Immunohistochemistry was performed for MMP-9 and MMP-2. CT aortic measurements and MMP expression were compared by t tests and analysis of variance, respectively. RESULTS: The ratio of arc distance between the intercostals on the posterior wall to total aortic circumference was 0.14 in healthy controls compared with 0.08 in TAAs at vertebral level T8 (P = .001). At T10, the ratio was 0.15 in healthy controls compared with 0.11 in TAAs (P = .001). MMP-9 expression in TAAs was 4.3-fold higher in the anterior wall compared with the posterior wall (P = .03). Conversely, MMP-2 expression in TAAs was 3.2-fold higher in the posterior wall compared with the anterior wall (P = .008). MMP expression was not detected in control cadaver aortas. CONCLUSION: Anterior walls of expanding TAAs grow at a greater rate than the posterior wall, as determined from the lower ratio of intercostal arc distance to total circumference in TAAs. Differential MMP expression appears to be a biologic marker for asymmetric growth in the TAA wall. CLINICAL RELEVANCE: The pathogenesis of thoracic aortic aneurysms (TAAs) is poorly understood. Multiple lines of evidence suggest that matrix metalloproteinases (MMPs), a family of enzymes, are important in aneurysm development. Earlier experiments documented a regional variation of MMP-9 in stimulated rodent aortas, with production greater in the abdominal aorta compared with the thoracic aorta. The present study extends that observation and documents asymmetric aneurysm development in the TAA wall, with increased anterior wall growth in correlation to increased MMP-9 production. An improved understanding of the mechanisms by which MMP production is regulated is critical.