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74. ADRENOMEDULLIN AND ADRENOMEDULLIN BINDING PROTEIN-1 IMPROVE GUT PERMEABILITY AND ATTENUATE TISSUE INJURY AFTER GUT ISCHEMIA/ REPERFUSION. S. Higuchi, R. Wu, M. Zhou, C. P. Marini, T. S. Ravikumar, P. Wang; Department of Surgery, North Shore University Hospital and Long Island Jewish Medical Center, Manhasset, NY Introduction: Ischemia bowel remains a critical problem resulting in up to 80% mortality. Previous studies have shown that combined treatment of adrenomedullin (AM) and AM binding protein-1 (AMBP-1) reduce systemic inflammatory responses. Our recent study has also shown that AM/AMBP-1 downregulates inflammatory cytokines and attenuates acute lung injury after gut ischemiareperfusion (I/R). We hypothesized that administration of AM/ AMBP-1 after gut I/R can ameliorate functional and structural damages to the intestinal mucosa, which play an important role in producing reperfusion organ injury. Methods: Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery (SMA) for 90 min min in male adult rats. After release of the SMA clamp, AM (12 ␮g/kg BW) and AMBP-1 (40 ␮g/kg BW) in combination or vehicle (1 ml normal saline) was administered intravenously over a period of 30 min. Small intestinal mucosal barrier function was assessed in an isolated everted ileum sac using fluorescein-isothiocyanate dextran (FD4) at 4 h after AM/AMBP-1 treatment. FD4 clearance was used as a measure of gut permeability. In additional groups of animals, blood and small intestine samples were collected at 4 h after the treatment. Small intestine morphological changes were assessed by H-E staining. The gene expression and protein levels of TNF-␣ in the small intestine were determined by RT-PCR and ELISA, respectively. Serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (T-Bil), and direct bilirubin (D-Bil) were also assessed. Results: The results (means ⫾ SE, n⫽6/group) are as follows. The above results showed that AM/AMBP-1 treatment significantly inhibited the development of intestinal mucosal hyperpermeability during the reperfusion. Histopathological findings showed that AM/AMBP-1 treatment dramatically improved I/R induced intestinal mucosal damages. Moreover, treatment with AM/AMBP-1 downregulated gene expression and protein levels of ⌻⌵F-␣ in the small intestine, and attenuated liver injury. Conclusion: AM/ AMBP-1 attenuates structural and functional damages to the intestinal mucosa, and appears to be a novel treatment for reperfusion injury after gut ischemia.

Gut permeability Tissue (Small Intestine) TNF-␣

Plasma Data

FD4 Clearance (nL/min/cm2) TNF-␣/G3PDH Tissue TNF-␣ (ng/g protein) AST (IU/L) ALT (IU/L) T-Bil (mg/dL) D-Bil (mg/dL)

Sham

I/RVehicle

I/R-AM/ AMBP1

38⫾2.7

93⫾28.4*

52⫾7.9#

0.16⫾0.11

0.45⫾0.13*

0.11⫾0.03#

2.1⫾0.2 23⫾2.0 15⫾1.2 3.1⫾0.7 0.3⫾0.2

5.7⫾1.2* 88⫾17.4* 46⫾4.5* 9.8⫾2.7* 3.2⫾0.5*

3.3⫾0.2# 56⫾4.2# 33⫾2.3*# 5.3⫾1.0# 2.1⫾0.5

(ANOVA and SNK test: *P⬍.05 vs. Sham; #P⬍.05 vs. I/R-Vehicle)

75. STEM CELL CONDITIONED MEDIA ATTENUATES HYPOXIC PULMONARY VASOCONSTRICTION. K. Patel, P. Crisostomo, T. Markel, M. Wang, K. D. Lillemoe, D. R. Meldrum; Indiana University, Indianapolis, IN Background: Hypoxic pulmonary vasoconstriction (HPV) may be an adaptive mechanism to correct ventilation-perfusion mismatch. It has been shown that, during chronic hypoxia, mesenchymal stem cells (MSCs) may contribute to pulmonary vascular remodeling, anti-

inflammation, and vascular stability. Also, MSCs have been shown to release growth factors when stressed by hypoxia. We hypothesize that media conditioned from MSCs challenged by hypoxia will acutely affect the vasoreactivity phenomenon of HPV. Methods: Adult male (250-350g) Sprague-Dawley pulmonary arteries (n⫽10) were isolated. Conditioned media was prepared by stressing mesenchymal stem cells with hypoxia for 60 mins using oil immersion technique. Conditioned media was added 20 minutes prior to precontraction with phenylephrine and then hypoxia was initiated. We measured force displacement of isolated pulmonary artery rings in the presence of MSC-conditioned media and vehicle (MSCBM) for the 60 minute duration of hypoxia. All data are reported as mean ⫾ SEM and were analyzed with two-way analysis of variance (ANOVA) with post-hoc Bonferroni test. A two tailed probability value of ⬍0.05 was considered statistically significant. Results: Pulmonary arteries exposed to MSC-conditioned media experienced an augmented vasodilatory phase as compared to vehicle (MSCBM). Maximum vasodilation was 53.58 ⫾ 6.42% vs. 39.76 ⫾ 4.05% for vehicle (P⬍0.001). In addition, delayed, phase II vasoconstriction was significantly attenuated as compared to vehicle. Maximum phase II vasoconstriction was 28.51 ⫾ 12.42 vs. 86.29 ⫾ 15.99% for vehicle (P⬍0.001). Conclusions: We conclude that mesenchymal stem cells may indirectly play a role in pulmonary artery vasoreactivity in acute hypoxia. In addition to directly contributing to vessel remodeling in chronic hypoxia, mesenchymal stem cells produce factors that significantly alter pulmonary vasoreactivity in acute hypoxia presumably by altering the inflammatory state of the vasculature.

76. LENTIVIRAL-MEDIATED OVEREXPRESSION OF STROMAL DERIVED GROWTH FACTOR-1〈 IMPROVES IMPAIRED DIABETIC WOUND HEALING. A. Badillo, S. Chung, P. Zoltick, K. Liechty; Children’s Hospital of Philadelphia, Philadelphia, PA Background: Chronic wound related morbidity continues to be a major clinical problem and novel therapeutic approaches are needed. Stromal derived growth factor -1␣ (SDF-1␣) is a chemokine important in the recruitment of progenitor cells and more recently has also been shown to be down-regulated in response to inflammation in dermal wound healing. Previous work from our laboratory has demonstrated that improved cutaneous wound healing with the local application of fetal murine MSC in diabetic mice was due in part to increased production of SDF-1␣. We hypothesized that lentiviral mediated increased production of SDF-1␣ in the wound environment could improve the wound healing impairment seen in diabetic wounds. Methods: 8mm full thickness excisional wounds were created in Db-/Db- and Db⫹/Db- heterozygous mice. At 7 days post wounding, wounds were harvested for molecular and histological analysis. Total wound RNA was isolated and RT-PCR for SDF was performed to determine baseline SDF mRNA expression in wounds. Densitometry for individual sample message level was performed and standardized to GAPDH. Additional Db-/Db- mice were wounded and immediately treated with 10 8 or 10 9 plaque-forming units of a lentiviral construct containing GFP-SDF-1␣ or GFP alone. Digital images were taken of the wounds daily using an in picture ruler for scale. The surface area of the wound was measured using Sigma Scan image analysis software. Results: SDF-1␣ mRNA expression was 33% higher in wounded nondiabetic Db⫹/Db- compared to diabetic Db-/Db- animals (p ⬍ 0.05). When Db-/Db- wounds were treated with 10 8 SDF, there was a decrease in wound surface area from 54⫾12 mm 2 in vehicle treated wounds to 35⫾2 mm 2 at 7 days (mean ⫾ SD, p⬍0.003). Morphologically SDF treated wounds exhibited greater dermal cellularity with increased granulation tissue volume compared to controls (3.4 ⫾ 0.46 mm 2 vs 1.5 ⫾ 0.06 mm 2, p⬍0.05). When treated with 10 9 SDF, wounds were not significantly smaller. At 14d, all SDF treated wounds were fully epithelialized (n⫽6) compared to only 1 of 6 controls. Conclusions: SDF-1␣ mRNA

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183

expression is deficient in diabetic wounds suggesting impaired progenitor cell recruitment may be a contributing factor in diabetes related wound healing impairment. Lentiviral-mediated overproduction of SDF-1␣ is sufficient to correct the pathophysiologic abnormalities in diabetic wound healing resulting in complete epithelialization at 2 weeks. SDF-1␣ mediated improvement in diabetic wound healing has significant implications for the development of novel therapeutic strategies to facilitate wound closure which target progenitor cell mobilization and recruitment.

77. INHIBITION OF ANGIOCIDIN BY TWO NOVEL PEPTIDES DECREASES TUMOR BURDEN IN AN ORTHOTOPIC NUDE MOUSE MODEL. C. Liebig 1, N. Agarwal 1, G. Ayala 1, G. Verstovsek 1, G. P. Tuszynski 2, D. Albo 1; 1Baylor College of Medicine, Houston, TX, 2Temple University, Philadelphia, PA Introduction: We have recently identified angiocidin, a novel thrombospondin-1 receptor. We have previously shown that angiocidin promotes colon cancer tumor cell invasion in vitro. Two angiocidin inhibitory peptides recently developed in our laboratory show a potent inhibitory effect on colon cancer tumor cell invasion in vitro. In the present study we look at the expression patterns of angiocidin in human colon cancer specimens and evaluate angiocidin as a potential therapeutic target in colon cancer. We hypothesize that human colon cancer expresses angiocidin and that inhibition of angiocidin results in decreased tumor burden in vivo. Methods: We created a tissue array composed of 50 consecutive patients who underwent resection of primary colon cancers at our institution. We included normal colonic tissue, primary tumor, negative lymph nodes and when available, positive lymph nodes and metastases from each patient. Angiocidin expression was determined by immunohistochemistry. Staining intensity was evaluated by two blinded observers and graded on a scale of 1 (negative) to 4 (strongly positive). KM12L4 human colon cancer cells were injected into the spleens of nude mice and twenty-four hours after tumor cell injection, animals began QOD intra-peritoneal injections of either 10mg/kg of CSVTCG peptide, 10mg/kg of 25-mer peptide or 10ml/kg PBS (control). Animals were sacrificed at four weeks and assigned a health score (HS) based on activity level, nuchal fat and external evidence of primary tumor burden. At necropsy, the primary tumor was measured, liver metastases were counted and animals were assigned an internal disease score (IDS) based on extent of local tumor invasion. Using Western blots and computerized image analysis, angiocidin expression was measured in both the primary tumor and in liver specimens from each group. Results: Seventy-seven percent of primary tumor specimens in the tissue array expressed angiocidin, with 62% of those staining strongly positive. Normal colonic tissues were negative or only weakly positive in 92% of specimens. Normal lymph nodes were negative or weakly positive in 90% of specimens. Eighty-five percent of positive lymph nodes expressed angiocidin with 64% staining strongly positive. All liver metastases (6/6) stained strongly positive for angiocidin. Angiocidin expression in both the primary tumors and the livers of peptidetreated mice was 50% higher in those animals with combined HS and IDS greater than 3 when compared to peptide-treated mice with combined scores of 3 or less (p⬍0.05). Animals treated with CSVTCG peptide or 25-mer peptide showed a five-fold and sixteen-fold reduction, respectively, in primary tumor volume when compared to controls (p⬍0.001) (figure1). Animals in both peptide treatment groups showed at least 30% improvement in HS and IDS when compared to control animals (p⬍0.001). Conclusions: Angiocidin is expressed in human colon cancer specimens. Angiocidin expression correlates with tumor burden and disease severity in vivo. Treatment with angiocidin inhibitory peptides results in decreased primary tumor volume and decreased tumor burden in nude mice.

78. DO STANDARDIZED TEMPLATES IMPROVE DOCUMENTATION OF QUALITY OF CARE? J. Parikh 1, I. Yermilov 1, S. Jain 1, C. KO 1, M. Maggard 2; 1West Los Angeles Veterans Adminstration Hospital, Los Angeles, CA, 2University of California, Los Angeles, Los Angeles, CA Introduction: Data abstraction from patient charts is a common method for measuring quality of surgical care; however, it has inherent difficulties (i.e., lack of documentation of data in medical records). Recently there has been increased use of standardized templates and clinical pathways in many surgical fields. Whether or not the use of standardized templates is associated with better documentation of quality of care measures remains unknown. This study investigates adherence rates of documentation for intraoperative bariatric quality measures by comparing surgeons who use standardized operative dictation templates to those who do not. Methods: Two independent reviewers evaluated adherence to established intraoperative bariatric quality measures from 40 charts (20 charts from surgeons who used standard template operative reports, and 20 charts from those who did not). An abstraction tool, which included possible responses of yes, no, not reported, and not applicable was utilized. Operative reports for both laparoscopic and open gastric bypass were reviewed, which included cases from 4 surgeons who used a standardized template to dictate operative reports, and 8 surgeons who did not. Four quality measures were investigated: 1) exploration of the abdomen, 2) intra- or postoperative evaluation of the anastomosis for leak, 3) closure of the small bowel mesenteric defect, and 4) closure of the large bowel mesenteric defect or antecolic placement of Roux limb. Results: Comparing the total responses of “yes/no” for all 4 quality measures, operative reports more consistently contained quality assessment information for cases where a dictation template was used versus where it was not (84% vs. 55%, respectively, p⬍0.0001). The greatest discrepancies between surgeons that used a standardized operative report and those that did not were found in the “exploration of the abdomen” measure (100% vs. 35%, p⬍0.0001, respectively) and in the “evaluation of the anastomosis for leak” measure (95% vs. 75%, one-sided p⫽0.04 respectively). Rates for closure of mesenteric defects were similar in both groups. “Closure of small bowel mesenteric defect” adherence rate was similar between groups (80% for each). “Closure of the large bowel mesentery/antecolic placement of Roux limb” adherence rate was 80% for cases where a standardized operative report template was used, as compared to 65% where it was not used (p⫽0.14). Of