Indranil Sinha

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.

Testosterone supplementation reverses sarcopenia in aging through regulation of myostatin, c-Jun NH2-terminal kinase, Notch, and Akt signaling pathways.

Aging in rodents and humans is characterized by loss of muscle mass (sarcopenia). Testosterone supplementation increases muscle mass in healthy older men. Here, using a mouse model, we investigated the molecular mechanisms by which testosterone prevents sarcopenia and promotes muscle growth in aging. Aged mice of 22 months of age received a single sc injection of GnRH antagonist every 2 wk to suppress endogenous testosterone production and were implanted subdermally under anesthesia with 0.5 or 1.0 cm testosterone-filled implants for 2 months (n = 15/group). Young and old mice (n = 15/group), of 2 and 22 months of age, respectively, received empty implants and were used as controls. Compared with young animals, a significant (P < 0.05) increase in muscle cell apoptosis coupled with a decrease in gastrocnemius muscles weight (by 16.7%) and muscle fiber cross-sectional area, of both fast and slow fiber types, was noted in old mice. Importantly, such age-related changes were fully reversed by higher dose (1 cm) of testosterone treatment. Testosterone treatment effectively suppressed age-specific increases in oxidative stress, processed myostatin levels, activation of c-Jun NH(2)-terminal kinase, and cyclin-dependent kinase inhibitor p21 in aged muscles. Furthermore, it restored age-related decreases in glucose-6-phosphate dehydrogenase levels, phospho-Akt, and Notch signaling. These alterations were associated with satellite cell proliferation and differentiation. Collectively these results suggest involvement of multiple signal transduction pathways in sarcopenia. Testosterone reverses sarcopenia through stimulation of cellular metabolism and survival pathway together with inhibition of death pathway.

Comparison of morbidity, functional outcome, and satisfaction following bilateral TRAM versus bilateral DIEP flap breast reconstruction.

Background: The potential for donor-site morbidity associated with bilateral pedicled transverse rectus abdominis myocutaneous (TRAM) flap breast reconstruction has led to the popularization of deep inferior epigastric artery perforator (DIEP) flap reconstruction. This study compares postoperative morbidity and satisfaction following bilateral pedicled TRAM and DIEP flap reconstruction. Methods: One hundred five women with bilateral pedicled TRAM flaps were compared with 58 women with bilateral DIEP flap reconstruction. Medical records were reviewed for complications and demographic data. Postoperative follow-up data were obtained through Short Form-36, Functional Assessment of Cancer Therapy-Breast, Michigan Breast Satisfaction, and Qualitative Assessment of Back Pain surveys. Results: The mean follow-up interval was 6.2 years in the bilateral TRAM group and 2.3 years in the bilateral DIEP group (p < 0.001). Demographic data were otherwise similar. Abdominal hernias occurred in three TRAM patients (2.9 percent) and in no DIEP patients, whereas abdominal bulges occurred in three TRAM patients (2.9 percent) and four DIEP patients (6.9 percent); these differences were not statistically significant. Fat necrosis occurred less frequently in the TRAM group (p = 0.04). Postoperative survey results revealed no significant difference in patient satisfaction, incidence of back pain, or physical function. The TRAM group scored higher in the Medical Outcome Study Short Form-36 subjective energy category (p = 0.01) and mean Functional Assessment of Cancer Therapy-Breast score (p = 0.01). Conclusions: This study suggests no significant differences in donor-site morbidity, survey-based functional outcome, or patient satisfaction between bilateral TRAM and DIEP flap breast reconstruction. Although perforator flaps represent an important technological advancement, bilateral pedicled TRAM flap reconstruction still represents a good option for autologous breast reconstruction.

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.

Outcomes and patient satisfaction following breast reconstruction with bilateral pedicled TRAM flaps in 105 consecutive patients.

Background: Breast reconstruction using pedicled transverse rectus abdominis musculocutaneous (TRAM) flaps has come under increasing scrutiny secondary to presumed abdominal wall morbidity. This study analyzes morbidity and patient satisfaction in a consecutive series of breast reconstructions performed using bilateral pedicled TRAM flaps. Methods: Between 1991 and 2007, 105 women underwent bilateral pedicled TRAM flap breast reconstruction performed by the senior author (J.J.P.). Charts were reviewed for postoperative complications and demographic data. Minimum 11-month follow-up survey data were obtained through the Michigan Breast Satisfaction Questionnaire, the Qualitative Assessment of Back Pain Questionnaire, and the Short Form-36 questionnaire. Results: The mean follow-up interval was 6 years. Abdominal wall complications included three abdominal hernias (2.9 percent), three abdominal wall bulges (2.9 percent), and four partial donor-site skin dehiscences (3.8 percent). Fat necrosis was the most common flap complication, present in 24 flaps (11.4 percent). The survey response rate was 61.9 percent. Short Form-36 data demonstrated no significant difference from a mean age-matched female population in general well-being, although patients with a body mass index greater than or equal to 30 reported significantly lower emotional well-being (p = 0.01), social functioning (p = 0.03), and overall energy scores (p = 0.03) in comparison with patients with a body mass index less than 30. Thirteen of the 65 patients who completed the survey (20 percent) complained of postoperative back pain, although most of these patients reported their symptoms to be mild in nature. Conclusions: Low complication rates were demonstrated and patients were generally satisfied in this series of 105 consecutive bilateral pedicled TRAM flaps. This suggests that the bilateral pedicled TRAM flap remains a viable option for breast reconstruction.

Isolation of progenitors that exhibit myogenic/osteogenic bipotency in vitro by fluorescence-activated cell sorting from human fetal muscle.

Summary Fluorescence-activated cell sorting (FACS) strategies to purify distinct cell types from the pool of fetal human myofiber-associated (hMFA) cells were developed. We demonstrate that cells expressing the satellite cell marker PAX7 are highly enriched within the subset of CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells. These CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi cells lack adipogenic capacity but exhibit robust, bipotent myogenic and osteogenic activity in vitro and engraft myofibers when transplanted into mouse muscle. In contrast, CD45−CD11b−GlyA−CD31−CD34+ fetal hMFA cells represent stromal constituents of muscle that do not express PAX7, lack myogenic function, and exhibit adipogenic and osteogenic capacity in vitro. Adult muscle likewise contains PAX7+ CD45−CD11b−GlyA−CD31−CD34−CD56intITGA7hi hMFA cells with in vitro myogenic and osteogenic activity, although these cells are present at lower frequency in comparison to their fetal counterparts. The ability to directly isolate functionally distinct progenitor cells from human muscle will enable novel insights into muscle lineage specification and homeostasis.

Comic books can educate children about burn safety in developing countries.

Burns in developing countries account for significant morbidity and many occur within the pediatric population. This study investigates whether a comic book can increase burn awareness in primary school age children, both domestically and abroad. Based on demographic data regarding pediatric burns in developing nations, a comic book was developed to educate primary school age children on key risk factors regarding burn safety, including teaching children to not touch active stoves, not to light fireworks without supervision, and to “stop, drop, and roll” after burn injury. Students, aged 5 to 7 years, in both West Virginia, United States (N = 74), and West Bengal, India (N = 39), answered a three-question survey regarding these issues both before and after reading the comic book. Groups were compared using Fishers exact test and significance was defined as P < .05. Initially, students answered 67.8 and 66.9% of the questionnaire correctly overall in West Virginia and West Bengal, respectively. These scores improved to 81.6 and 99.1% (P < .01 for each group), respectively, after reading the comic as a class. Specifically, there were significant increases in both groups for the questions regarding avoiding hot stoves (P < .01) and fireworks (P < .01). The lesson required 30 minutes total per class. The teachers reported that students enjoyed reading the comic and were engaged during the sessions. This study demonstrates that a comic book has value in teaching children about burn awareness. Comic books may be a cost-effective method as an outreach tool for children.

Attenuation of Experimental Aortic Aneurysm Formation in P-Selectin Knockout Mice

Abstract:  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 Fishers 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.

Sox Factors Transcriptionally Regulate ROBO4 Gene Expression in Developing Vasculature in Zebrafish

Despite their importance as members of the Roundabout (Robo) family in the control of axonal and vascular patterning, the transcriptional regulation of these genes is poorly understood. In this study, we show that members of the Sry-related high mobility box (Sox) transcription factor family as being transcriptional regulators of roundabout4 (robo4), a Robo gene family member that participates in sprouting angiogenesis in vivo, in zebrafish. Double whole mount in situ hybridization analysis in zebrafish embryos revealed co-localization of the vascular relevant Sox factors sox7 or sox18 mRNA with robo4 transcripts in developing intersomitic vessels. A 3-kb human ROBO4 promoter element was able to drive reporter expression in zebrafish to recapitulate the endogenous temporal intersomitic vessel expression pattern of robo4. EMSA analysis confirmed binding of Sox18 to a canonical Sox binding site (from −1170 bp to −1176 bp) in the ROBO4 promoter (3 kb), and mutation analysis indicated that this site was partially responsible for ROBO4 promoter activity in ECs. A combination of gain- and loss-of-function analysis identified Sox7 and Sox18 co-regulation of robo4 but not fli1a transcripts in zebrafish. Finally, Sox-mediated robo4 transcriptional regulation is conserved across evolution. These studies imply Sox-mediated transcriptional regulation of Robo4 in the developing embryonic vasculature.