VASCULAR SURGERY

Carbon monoxide treated macrophages release asoluble factor that inhibits smooth muscleproliferationJitesh Patel, MD,* Chase Gladstone, BS, Edith Tzeng, MD, FACSUniversity of Pittsburgh Medical Center, Pittsburgh, PA

INTRODUCTION: Carbon monoxide (CO) possesses antiprolifera-tive and anti-inflammatory properties. Short exposure to inhaled COdramatically inhibits intimal hyperplasia (IH) following vascular in-jury and CO induces an anti-inflammatory phenotype in macro-phages treated with LPS. We hypothesize that CO alters the secretoryprofile of macrophages which may mediate SMC antiproliferationand IH inhibition.

METHODS: Rats were exposed to inhaled CO (250ppm) for one hr.Control rats were treated with room air (RA). Peritoneal macro-phages (PMs) were isolated and cultured �/� LPS (100 ng/ml) for24hrs. Rat aortic SMC were treated with the conditioned medium(CM) from these macrophages. Proliferation was measured by 3H-thymidine incorporation. CM from RAW cells cultured in a conven-tional or CO incubator (250 ppm) �/� LPS were also tested.

RESULTS: CM from LPS treated PMs from control rats had noeffect on SMC proliferation vs CM from untreated PMs from con-trol rats (Table). However, CM from unstimulated PMs isolatedfrom CO treated rats significantly inhibited SMC proliferation. CMfrom LPS stimulated PMs from CO treated rats was similarly anti-proliferative. A greater antiproliferative effect was observed with CMfrom CO treated RAW cells �/� LPS.

CM sourceSMC proliferation

(% of control macrophages)

PM RAWControl 100 100Control � LPS 100.2 � 0.1 109.8 � 3.3CO treated 87.2 � 0.9� 34.6 � 11.8#CO treated � LPS 75.8 � 6.5� 37.5 � 9.6#

Mean � SEM, N, 3/treatment, experiments repeated twice; �p�0.05 vs.control and control � LPS CM; #p�0.01 vs. control and control � LPS CMby ANOVA.

CONCLUSIONS: CM from PMs from CO treated rats or CO-cultured RAW cells inhibit SMC proliferation vs RA controls, sug-gesting that CO treated macrophages secrete a soluble antiprolifera-tive factor(s) independent of LPS. Identifying the factor(s) willprovide a better understanding of the in vivo mechanisms of tissueprotection and inhibition of IH offered by CO.

Thermoreversible poloxamers and applications forvascular biologyEdward I Chang, MD, Cynthia D Hamou, MD,Michael G Galvez, BA, Michael T Longaker, MD, MBA, FACS,Geoffrey C Gurtner, MD, FACSStanford University, Stanford, CA

INTRODUCTION: Conventional suture techniques for vascularanastomoses are time consuming and prone to thrombosis and reste-

nosis due to intimal damage and inflammation. We propose a novelsutureless technique using thermoreversible poloxamers.

METHODS: Rheological studies determined the formulation ofP407/P188 to obtain a phase transition at 42°C. Anastomoses wereperformed on Fisher rat aortas using P407/P188 and bioadhesives(n�30) and 10-0 nylon sutures (n�30). CT angiograms, burststrength assays, histology, and scanning electron microscopy (SEM)were performed at designated timepoints. Tissue factor pathway in-hibitor (TFPI) ELISA was performed on media harvested fromHUVECs exposed to heparin and heparinized P407/P188.

RESULTS: A formulation of 17% P407 and 6% P188 achieved aphase transition at 42°C. Anastomoses performed using P407/P188were completed more efficiently than hand sewn anastomoses(10.0�4.2min vs. 47.3�5.0 min, p�0.05) with equivalent burststrengths (�1500mm Hg, p�0.05). Angiograms demonstratedequivalent patency, and flow through native aorta, hand-sewn anas-tomoses, and sutureless anastomoses were not significantly different(26mL/sec vs. 27mL/sec vs. 29mL/sec respectively). Histology andSEM demonstrated less fibrosis in the sutureless group comparedwith the traditional technique. Heparinized poloxamer induced asignificant percentage increase in secretion of TFPI compared withheparin administered directly to HUVECs (231.8%, p�0.05) witheffects lasting up to 24 hours (125.4%, p�0.05).

CONCLUSIONS: P407/P188 provides an efficient and sustainedmeans of delivering antithrombotic agents, and sutureless anastomo-ses achieve equivalent patency rates and burst strength with signifi-cantly less fibrosis. This technology has promising implications forthe fields of vascular, cardiothoracic, plastic, and transplant surgery.

HMGB-1 induces pro-angiogenic effects inendothelial cells: Implications for lower extremityischemiaUlka Sachdev, MD, Edith Tzeng, MD, FACSUniversity of Pittsburgh Medical Center, Pittsburgh, PA

INTRODUCTION: High-mobility group box 1 (HMGB-1), a nuclearfactor released by necrotic cells, is a mediator of sepsis and ischemia/reperfusion injury. We hypothesize that HMGB-1 may also be pro-angiogenic, promoting tube formation and survival of endothelial cells.

METHODS: Human dermal microvascular endothelial cells (HD-MVEC) were plated alone or on growth-factor reduced Matrigel in24-well plates at 30,000 cells/well. Cells were maintained in endo-thelial cell media with 1% fetal bovine serum containing either 1microgram/ml HMGB-1 or formulation buffer. After 24 hours ofincubation in 1% hypoxia or normoxic conditions, tube formationwas assessed by quantifying the number of boxes within endothelialtube networks. Four areas per well were analyzed at 10X magnifica-tion. Cells without Matrigel were trypsinized and counted. Condi-tions were performed in duplicate and replicated. Statistical differ-ences were determined using ANOVA.

RESULTS: Hypoxia alone increased complex endothelial tube for-mation compared to normoxia (Table). HMGB-1 significantly in-creased endothelial tube networks under both normoxic and hypoxic

S108© 2007 by the American College of Surgeons ISSN 1072-7515/07/$32.00Published by Elsevier Inc.