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 effect of 17-beta-estradiol on smooth muscle cell and aortic explant MMP2.

OBJECTIVE: The present investigation tested the hypothesis that intrinsic gender-related differences exist in rat aortic smooth muscle cell matrix metalloproteinase 2 (MMP2). METHODS: This investigation comprised 3 sets of experiments. Experiment I: Adult male and female rat aortic smooth muscle cells (RASMCs) at passages 4-8 were stimulated in serum-free media for 48 h with interleukin(IL)1beta at doses encountered in human abdominal aortic aneurysms (2 ng/mL). Messenger RNA was extracted from the RASMCs, and gene expression of MMP2 and tissue inhibitor of metalloproteinase 2 (TIMP2), a major MMP2 inhibitor, was measured by real-time polymerase chain reaction. MMP2 protein levels in conditioned media were measured by Western blotting, and MMP2 and TIMP2 activity quantified by standard and reverse gelatin zymography. Experiment II: Male and female RASMCs were incubated for 48 h in Dulbecco's modified Eagler's medium containing IL-1beta and 17-beta-estradiol at doses from 1x10(-10) to 1x10(-6) molar. MMP2 activity in the conditioned media was then determined. Experiment III: Male rats underwent sustained 17-beta-estradiol exposure for 21 d using extended-release, subcutaneously implanted pellets prior to sacrifice and aortic explantation. Aortas from males, females, and estradiol-treated males were stimulated with IL-1beta for 48-h, and MMP2 activity in the conditioned media was determined. RESULTS: Experiment I: MMP2 gene expression was 3-fold higher in male compared with female IL-1beta stimulated RASMCs (P<0.0001). MMP2:TIMP2 gene expression ratio was 7.5-fold greater in male versus female RASMCs. MMP2 protein levels were 3-fold higher (2.68 versus 0.96 o.d./mg total protein, P=0.003) in male versus female RASMCs. Gelatinolytic activity was more than 6-fold higher (15,010 versus 2,472 o.d./mg total protein, P=0.002) in male versus female RASMCs. Experiment II: MMP2 activity in male and female RASMCs was not altered by a wide range of 17-beta-estradiol concentrations. Experiment III: When pretreated with 17-beta-estradiol, MMP2 activity in the media of male rat whole-aortic explants decreased 2-fold (P=0.002). This post-17-beta-estradiol treatment male level was not different than baseline female aortic explant MMP2 levels. CONCLUSIONS: MMP2 is higher in male RASMCs compared to female RASMCs. Exogenous 17-beta-estradiol did not alter MMP2 activity in vitro, but in vivo 17-beta-estradiol exposure greatly decreased male aortic MMP2 production to levels seen in the female aorta. Gender differences in MMP2 are speculated to be associated with phenotypic differences in human abdominal aortic aneurysm formation.

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.

Tamoxifen up-regulates catalase production, inhibits vessel wall neutrophil infiltration, and attenuates development of experimental abdominal aortic aneurysms.

BACKGROUND: Selective estrogen receptor modulators (SERMs), similar to estrogens, possess vasoprotective effects by reducing release of reactive oxygen species. Little is known about the potential effects of SERMs on the pathogenesis of abdominal aortic aneurysms (AAAs). This study's objective was to investigate the growth of experimental AAAs in the setting of the SERM tamoxifen. METHODS: In the first set of experiments, adult male rats underwent subcutaneous tamoxifen pellet (delivering 10 mg/kg/day) implantation (n = 14) or sham operation (n = 16). Seven days later, all animals underwent pancreatic elastase perfusion of the abdominal aorta. Aortic diameters were determined at that time, and aortas were harvested 7 and 14 days after elastase perfusion for immunohistochemistry, real-time polymerase chain reaction, Western blot analysis, and zymography. In the second set of experiments, a direct irreversible catalase inhibitor, 3-amino-1,2,4-triazole (AT), was administered intraperitoneally (1 mg/kg) daily to tamoxifen-treated (n = 6) and control rats (n = 6), starting on day 7 after elastase perfusion. Aortic diameters were measured on day 14. In a third set of experiments, rats were perfused with catalase (150 mg/kg) after the elastase (n = 5), followed by daily intravenous injections of catalase (150 mg/kg/day) administered for 10 days. A control group of rats (n = 7) received 0.9% NaCl instead of catalase. RESULTS: Mean AAA diameters were approximately 50% smaller in tamoxifen-treated rats compared with sham rats 14 days after elastase perfusion (P = .002). The tamoxifen-treated group's aortas had a five-fold increase in catalase mRNA expression (P = .02) on day 7 and an eight-fold increase in catalase protein on day 14 (P = .04). Matrix metalloprotroteinase-9 activity was 2.4-fold higher (P = .01) on day 7 in the aortas of the controls compared to the tamoxifen-treated group's aortas. Tamoxifen-treated rats had approximately 40% fewer aortic polymorphonuclear neutrophils compared to controls on day 7 (P = .05). Administration of the direct catalase inhibitor AT to tamoxifen-treated rats partially reversed the aneurysm inhibitory effect of tamoxifen by nearly 30% (P = .02). In contrast, catalase administration inhibited AAA formation by 44% (P = .002). CONCLUSIONS: The selective estrogen receptor modulator tamoxifen inhibits the development of AAAs in male rats in association with an up-regulation of catalase and inhibition of aortic wall neutrophil infiltration.

Computerized microfluidic cell culture using elastomeric channels and Braille displays.

Computer-controlled microfluidics would advance many types of cellular assays and microscale tissue engineering studies wherever spatiotemporal changes in fluidics need to be defined. However, this goal has been elusive because of the limited availability of integrated, programmable pumps and valves. This paper demonstrates how a refreshable Braille display, with its grid of 320 vertically moving pins, can power integrated pumps and valves through localized deformations of channel networks within elastic silicone rubber. The resulting computerized fluidic control is able to switch among: (i) rapid and efficient mixing between streams, (ii) multiple laminar flows with minimal mixing between streams, and (iii) segmented plug-flow of immiscible fluids within the same channel architecture. The same control method is used to precisely seed cells, compartmentalize them into distinct subpopulations through channel reconfiguration, and culture each cell subpopulation for up to 3 weeks under perfusion. These reliable microscale cell cultures showed gradients of cellular behavior from C2C12 myoblasts along channel lengths, as well as differences in cell density of undifferentiated myoblasts and differentiation patterns, both programmable through different flow rates of serum-containing media. This technology will allow future microscale tissue or cell studies to be more accessible, especially for high-throughput, complex, and long-term experiments. The microfluidic actuation method described is versatile and computer programmable, yet simple, well packaged, and portable enough for personal use.

Passively driven integrated microfluidic system for separation of motile sperm.

This paper describes a self-contained integrated microfluidic system that can separate motile sperm from small samples that are difficult to handle using conventional sperm-sorting techniques. The device isolates motile sperm from nonmotile sperm and other cellular debris, based on the ability of motile sperm to cross streamlines in a laminar fluid stream. The device is small, simple, and disposable yet is an integrated system complete with sample inlets, outlets, sorting channel, and a novel passively driven pumping system that provides a steady flow of liquid; it requires no external power source or controls. The device fulfills a need in clinical settings where small amounts of sperm need to be sorted. It also opens the way for convenient bioassays based on sperm motility including at-home motile sperm tests.