- Email: [email protected]
54
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS rate of phagocytosis by peritoneal macrophages from newborn mice with NEC was significantly enhanced as compared with control animals (111⫾20% increase over control, p⬍0.05), suggesting a mechanism whereby the effects of hypoxia on macrophage function could contribute to the development of this disease. Conclusion: These data indicate that hypoxia enhances phagocytosis by macrophages in a p38MAPKdependent manner, and suggest the possibility that enhanced macrophage priming within the peritoneal cavity may contribute to the pathogenesis of NEC.
53. INTERLEUKIN-1 BETA REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN THE INTESTINAL EPITHELIUM. B. Lugo, J. Upperman, P. Lockerbie, X. Zhang, J. Wang, A. Grishin, H. Ford; Children’s Hospital Los Angeles, Los Angeles, CA Introduction: Necrotizing Enterocolitis (NEC) is a lethal surgical emergency affecting the gastrointestinal tract of the premature infant. It is characterized by an unchecked inflammatory response leading to the accumulation of toxic nitric oxide (NO) metabolites and epithelial injury. The early molecular signaling pathways that govern this exuberant inflammatory response remain unclear. We hypothesize that interleukin-1beta (Il-1), a cytokine up regulated early in NEC, may be responsible for the upregulation of inducible NO synthase (iNOS), the enzyme responsible for excess NO production in the intestine. Methods: Newborn rats were either subjected to 10 min. of hypoxia (5% O2, t.i.d.) and fed a conventional formula by gavage (NEC) or were breast-fed without hypoxia (BF). Rats were sacrificed on day 0 thru 4, and the distal ilea were harvested for molecular and morphological studies. Il-1 mRNA levels in tissue were measured using RT-PCR. In order to test the role of Il-1 in enterocyte iNOS production, we used the rat intestinal epithelial cell line (IEC-6). SDS-PAGE and Western Blot were used to measure protein levels of iNOS, inhibitory subunit of nuclear regulatory factor-kappaB (NF-B), transcription factor-kappaB (IB), and activation of mitogen-activated protein kinases (MAPK) after stimulation with inflammatory cytokines and lipopolysaccharide (LPS). Specific pharmacologic inhibitors were used to inhibit NF-B, MAPK, and Janus kinase (JAK). Electromobility shift assay (EMSA) was used to measure activation of transcription factors NF-kappaB and STAT-1. Production of NO was measured using Greiss assay. Data expressed mean ⫾ SEM, ANOVA, p⬍.05. Results: Il-1 RNA levels were elevated in rat NEC in a time-dependent fashion compared to BF controls. In Vitro, Il-1 (1ng/ml) stimulated a concentrationdependent and time-dependent increase in NO production and iNOS expression in IEC-6 cells, whereas tumor necrosis factor alpha (TNF␣), interferon gamma (IFN␥), or LPS did not (Figure 1). IB levels decreased upon Il-1 stimulation, and Il-1 induced expression of iNOS was blocked by the NF-B inhibitors Bay11-8026 and MG-132. ERK inhibitors PD98059 and U0126 decreased, whereas p38 MAPK inhibitor SB202190 increased the Il-1 induced NO production and iNOS expression. In addition, Il-1 activated both ERK and p38.
173
SP600125 and Tyrphostin AG-490, inhibitors of JNK and JAK protein kinases, had no effect on Il-1 induced iNOS expression or NO production. EMSA demonstrated that Il-1, TNF, and LPS activated NF-B but not signal transduction activator of trascription-1 (STAT1). Pharmacologic inhibitors of ERK and p38 did not affect Il-1 induced activation of NFB. Conclusions: NF-B is necessary, but not sufficient for Il-1 induced expression of iNOS in enterocytes. ERK and p38 modulate Il-1 induced expression of iNOS in a manner independent of NF-B. JNK and JAK-STAT pathways have no role in Il-1 induced expression of iNOS. Understanding iNOS regulation during NEC and discovering potential therapeutic targets will require further study of molecular interplay between MAPK and NF-B pathways.
54. THE FORKHEAD BOX (FOX) TRANSCRIPTION FACTOR FOXF1 ATTENUATES LIVER INJURY DURING BILIARY OBSTRUCTION IN MICE. M. Chen, G. Zheng, M. Wang, A. Holterman; U Illinois at Chicago, Chicago, IL Introduction: The Forkhead Box (Fox)f1 is a mesodermal transcription factor which regulates epithelial-mesenchymal interaction during organ development and function. In the liver, the hepatic stellate cells and periportal fibroblasts are derived from the mesodermal cells and are implicated in the pathogenesis of biliary fibrosis through their production of collagen. This study tests the hypothesis that functional activation of Foxf1 in the liver through the replacement of the Foxf1 DNA binding domain with the nuclear localizing LacZ gene can attenuate liver injury in a mouse model of biliary fibrosis by bile duct ligation (BDL). Methods: Foxf1 expression in the liver was assessed by b-galactosidase staining. Foxf1 mutant mice (⫹/-) and wild type littermates (WT) underwent sham operation or BDL and sacrificed at 3 and 9 days (n⫽6) for evaluation of hepatocyte proliferation by BrdU labeling. Cytokine, collagen production and stellate cell activation (alpha-smooth muscle actin upregulation) were monitored by RNAse protection assay. Histological damage was assessed by Trichrome, TUNEL and Caspase-3 immunostaining. Results: Similarly to the stellate cells, Foxf1 expression was also found to be inactivated in the periportal, perivenous and capsular fibroblasts in Foxf1 ⫹/- mice. Despite functional inactivation of Foxf1 in these fibrogenic cells, markers of fibrosis such as TGFb, collagen and alpha-smooth muscle actin upregulation following BDL were comparable between the groups. Although no differences in the expression of proinflammatory and profibrotic hepatic cytokines TNF-a or IL-6 was seen, hepatic mRNA expression of HGF was diminished in the Foxf1 ⫹/- mice (70% of WT level; p⬍ 0.05), leading to extensive biliary infarcts, hepatic apoptosis, and a 2.5-fold reduction in hepatocyte proliferation (p⫽0.05) at day 3 of BDL. In support of HGF as a Foxf1-regulated gene, Foxf1 transcription factor has binding sites to the HGF promoter region. Conclusion: Foxf1 does not play an important role in the early fibrogenic response to biliary obstruction. In the Foxf1 ⫹/- mice, abnormal HGF expression is however associated with increased hepatic damage and impaired hepatocyte proliferation, suggesting a protective role for Foxf1
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ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
against parenchymal liver injury during biliary fibrosis through its regulation of hepatic HGF production.
VASCULAR I: VASCULAR TONE AND REMODELING 55. CHLOROTYROSINE INDUCES ENDOTHELIAL DYSFUNCTION IN HUMAN CORONARY ARTERY ENDOTHELIAL CELLS AND PORCINE CORONARY ARTERIES. H. Chai, I. Mohiuddin, P. Lin, A. Lumsden, Q. Yao, C. J. Chen; Baylor College of Medicine, Houston, TX. Introduction: The heme-containing enzyme myeloperoxidase (MPO) play a critical role in many inflammatory conditions including atherosclerosis. Hypochlorous acid (HOCl), a major product of MPO, converts tyrosine into the 3-chlorotyrosine (CT). Recently, high CT levels have been associated with clinical progression of atherosclerosis. However, the molecular functions of CT in the vascular cells remain unclear. In this study, we investigated the effects of CT on coronary endothelial cell functions. Methods: Human coronary artery endothelial cells (HCAECs) were treated with CT (0, 10 -7, 10 -6 or 10 -5 ⌴) ° 6, 24 ° 48 . Tyrosine (10 -5 ⌴) was used as a control. The mRNA levels of endothelial nitric oxide synthase (eNOS) were determined by real-time PCR. Superoxide anion (O 2 -) production was determined by fluorescence dye DHE staining and flow cytometry analysis. Activation status of MAPKs and NF-B was studied using BioPlex immunoassay. In addition, porcine coronary artery rings were cultured with CT (0, 10 -8, 10 -7, 10 -6 or 10 -5 ⌴) for 24 hrs. Endothelium-dependent vasorelaxation was studied using a myograph tension system. The eNOS protein level and O 2 - production were studied using immunohistochemistry and chemiluminescence assay, respectively. Antioxidant seleno-L-methionine (SeMet) was included. Results: When HCAECs were cultured with CT (10 -6 or 10 -5 ⌴) for 24 hrs, the eNOS mRNA levels were significantly decreased by 23% and 46%, respectively, as compared to controls (P⬍0.05). An increase production in O 2 - after CT (10 -6 M) treatment was observed. Significant decreases in the catalase and superoxide dismutase enzyme activities were also noted. Antioxidant SeMet reversed CT-induced eNOS mRNA decrease. JNK, but not p38 and ERK1/2, was activated at 15 min after CT (10 -6 M) treatment. NF-B activation was also observed 45-60 min after CT (10 -6 M) treatment. In porcine coronary artery ring cultures, CT (10 -6 or 10 -5 ⌴) treatment induced a significant decrease in endothelium-dependent vasorelaxation by 19% and 32%, respectively, as compared to controls (P⬍0.05). The eNOS immunoreactivity was decreased in the CTtreated vessels, while O 2 - production was significantly increased. Conclusions: These data demonstrate that clinically relevant concentrations of CT significantly reduce eNOS expression, while increase O 2 - production in both HCAECs and porcine coronary arteries. In addition, CT also activates JNK and NF-B pathways. Antioxidant SeMet effectively blocks the CT-mediated action in vascular cells. These results suggest CT may contribute to the vascular lesion formation through endothelial dysfunction. 56. IN VITRO GENERATION OF HUMAN MICROVESSEL NETWORKS. W. B. Carter 1, M. Sekharem 1, J. B. Hoying 2; 1H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, 2 University of Arizona, Tucson, AZ Introduction: Successful incorporation of bioengineered tissues requires vascularization. Attempts at de novo vascular generation or angiogenic induction have had little success in improving tissue perfusion. An alternate approach to neovascularization is to form a prevascularized construct that is capable of rapid inosculation and rapid cellular perfusion. Human parathyroid (PTH) tissue is uniquely capable of a rapid angiogenic induction, with restoration of calcium homeostasis within 6 weeks of PTH autografting. We hy-
pothesized that human PTH cells could induce human microvessel fragments to form networks of capillary microvessels in vitro. Because PTH cells release VEGF and Angiopoietin-2 (Ang-2) after devascularization, we further hypothesized that PTH angiogenic induction is driven by PTH release of VEGF and Ang-2. Methods: Human subcutaneous fat was partially digested with collagenase to yield microvessel fragments. After sequential filtering, 30-350 micron fragments were embedded in DMEM ⫹ 10% FBS-containing collagen I matrix in 3 dimensional culture. PTH cells were cocultured on the matrix surface to induce an angiogenic response. Other matrix cultures were treated with VEGF (50 ng/ml) or VEGF ⫹ Ang-2 (50 ng/ml) and microvessel responses noted. Because Stat3 phosphorylation has been identified with endothelial cell (EC) angiogenic responses, we also tested for Stat3 activation in human dermal microvessel EC (HDMEC) after treatment with VEGF or VEGF ⫹ Ang-2. Results: Human microvessel fragments in culture are completely quiescent. Coculture with PTH cells induces the rapid development of neovascular networks. Treatment of the human microvessels in vitro with VEGF failed to induce an angiogenic event. VEGF ⫹ Ang-2 induced EC migration but no capillary formation. Both VEGF and Ang-2 induced Stat3 phosphorylation in HDMEC, but not in iliac vein EC. Conclusions: Human PTH cells release or induce release of all necessary angiogenic factors for capillary network angiogenic induction of human microvessels in vitro. The mechanism of angiogenic induction is more complex than VEGF stimulation alone. Stat 3 activity is upregulated in microvessel EC after VEGF or Ang-2 stimulation, but not in large vessel EC, suggesting a possible role in microvessel angiogenic processes. PTH cells have the angiogenic potential to induce capillary network development in vitro. This system may be capable of providing vascularization of bioengineered tissues for therapeutic intervention. 57. DELETION OF CCR2 BUT NOT CCR5 INHIBITS AORTIC ANEURYSM FORMATION. J. N. Mactaggart, W. Xiong, R. Knispel, B. T. Baxter; University of Nebraska Medical Center, Omaha, NE Introduction: Microscopic analysis of abdominal aortic aneurysms (AAA) demonstrates an abundance of infiltrating leukocytes. The mechanism underlying the recruitment of these leukocytes to the abdominal aorta during aneurysm formation is unknown. Chemokines are cytokines that attract leukocytes to areas of inflammation. RANTES and MCP-1 are chemokines previously shown to be upregulated in experimental aneurysm formation. CCR5 and CCR2 are chemokine receptors present on leukocytes that bind RANTES and MCP-1 respectively. We hypothesized that the genetic deletions of CCR5 (CCR5-/-) and CCR2 (CCR2-/-) would limit leukocyte infiltration and subsequent aneurysm formation in a mouse model of AAA. Methods: CCR2-/-, CCR5-/-, and control mice of matching genetic backgrounds, all obtained from Jackson Laboratories, were subject to periaortic application of calcium chloride. This model has been described in several publications (Nature Medicine and JCI). Aortic diameter was measured with video microscopy before aneurysm induction and at harvest 6 weeks later. The mean diameter and diameter change of each treatment group were compared with concurrent controls using the Mann-Whitney test. Aortas were perfusion fixed with formalin and stained with H&E and trichrome for histologic analysis. Results: There was no inhibition of aneurysm formation in the CCR5-/- mice. This was corroborated by the tissue histology which showed inflammatory cell infiltration and matrix degradation. Aneurysm formation was attenuated in the CCR2-/- mice. The final mean diameter in the CCR2-/- mice was significantly less than the controls (*P⫽0.0001) and the CCR5-/- mice (P⬍0.05). The inflammatory infiltration was decreased compared to controls and the normal lamellar architecture of the aorta was preserved. Conclusions: Deletion of the CCR2 gene attenuates experimental aneurysm formation, while the deletion of the CCR5 gene has no effect. The CCR2 receptor and its chemokine ligands should be investigated further and may provide a therapeutic target in the development of pharmacologic inhibitors of AAA expansion.
53. INTERLEUKIN-1 BETA REGULATION OF INDUCIBLE NITRIC OXIDE SYNTHASE IN THE INTESTINAL EPITHELIUM. B. Lugo, J. Upperman, P. Lockerbie, X. Zhang, J. Wang, A. Grishin, H. Ford; Children’s Hospital Los Angeles, Los Angeles, CA Introduction: Necrotizing Enterocolitis (NEC) is a lethal surgical emergency affecting the gastrointestinal tract of the premature infant. It is characterized by an unchecked inflammatory response leading to the accumulation of toxic nitric oxide (NO) metabolites and epithelial injury. The early molecular signaling pathways that govern this exuberant inflammatory response remain unclear. We hypothesize that interleukin-1beta (Il-1), a cytokine up regulated early in NEC, may be responsible for the upregulation of inducible NO synthase (iNOS), the enzyme responsible for excess NO production in the intestine. Methods: Newborn rats were either subjected to 10 min. of hypoxia (5% O2, t.i.d.) and fed a conventional formula by gavage (NEC) or were breast-fed without hypoxia (BF). Rats were sacrificed on day 0 thru 4, and the distal ilea were harvested for molecular and morphological studies. Il-1 mRNA levels in tissue were measured using RT-PCR. In order to test the role of Il-1 in enterocyte iNOS production, we used the rat intestinal epithelial cell line (IEC-6). SDS-PAGE and Western Blot were used to measure protein levels of iNOS, inhibitory subunit of nuclear regulatory factor-kappaB (NF-B), transcription factor-kappaB (IB), and activation of mitogen-activated protein kinases (MAPK) after stimulation with inflammatory cytokines and lipopolysaccharide (LPS). Specific pharmacologic inhibitors were used to inhibit NF-B, MAPK, and Janus kinase (JAK). Electromobility shift assay (EMSA) was used to measure activation of transcription factors NF-kappaB and STAT-1. Production of NO was measured using Greiss assay. Data expressed mean ⫾ SEM, ANOVA, p⬍.05. Results: Il-1 RNA levels were elevated in rat NEC in a time-dependent fashion compared to BF controls. In Vitro, Il-1 (1ng/ml) stimulated a concentrationdependent and time-dependent increase in NO production and iNOS expression in IEC-6 cells, whereas tumor necrosis factor alpha (TNF␣), interferon gamma (IFN␥), or LPS did not (Figure 1). IB levels decreased upon Il-1 stimulation, and Il-1 induced expression of iNOS was blocked by the NF-B inhibitors Bay11-8026 and MG-132. ERK inhibitors PD98059 and U0126 decreased, whereas p38 MAPK inhibitor SB202190 increased the Il-1 induced NO production and iNOS expression. In addition, Il-1 activated both ERK and p38.
173
SP600125 and Tyrphostin AG-490, inhibitors of JNK and JAK protein kinases, had no effect on Il-1 induced iNOS expression or NO production. EMSA demonstrated that Il-1, TNF, and LPS activated NF-B but not signal transduction activator of trascription-1 (STAT1). Pharmacologic inhibitors of ERK and p38 did not affect Il-1 induced activation of NFB. Conclusions: NF-B is necessary, but not sufficient for Il-1 induced expression of iNOS in enterocytes. ERK and p38 modulate Il-1 induced expression of iNOS in a manner independent of NF-B. JNK and JAK-STAT pathways have no role in Il-1 induced expression of iNOS. Understanding iNOS regulation during NEC and discovering potential therapeutic targets will require further study of molecular interplay between MAPK and NF-B pathways.
54. THE FORKHEAD BOX (FOX) TRANSCRIPTION FACTOR FOXF1 ATTENUATES LIVER INJURY DURING BILIARY OBSTRUCTION IN MICE. M. Chen, G. Zheng, M. Wang, A. Holterman; U Illinois at Chicago, Chicago, IL Introduction: The Forkhead Box (Fox)f1 is a mesodermal transcription factor which regulates epithelial-mesenchymal interaction during organ development and function. In the liver, the hepatic stellate cells and periportal fibroblasts are derived from the mesodermal cells and are implicated in the pathogenesis of biliary fibrosis through their production of collagen. This study tests the hypothesis that functional activation of Foxf1 in the liver through the replacement of the Foxf1 DNA binding domain with the nuclear localizing LacZ gene can attenuate liver injury in a mouse model of biliary fibrosis by bile duct ligation (BDL). Methods: Foxf1 expression in the liver was assessed by b-galactosidase staining. Foxf1 mutant mice (⫹/-) and wild type littermates (WT) underwent sham operation or BDL and sacrificed at 3 and 9 days (n⫽6) for evaluation of hepatocyte proliferation by BrdU labeling. Cytokine, collagen production and stellate cell activation (alpha-smooth muscle actin upregulation) were monitored by RNAse protection assay. Histological damage was assessed by Trichrome, TUNEL and Caspase-3 immunostaining. Results: Similarly to the stellate cells, Foxf1 expression was also found to be inactivated in the periportal, perivenous and capsular fibroblasts in Foxf1 ⫹/- mice. Despite functional inactivation of Foxf1 in these fibrogenic cells, markers of fibrosis such as TGFb, collagen and alpha-smooth muscle actin upregulation following BDL were comparable between the groups. Although no differences in the expression of proinflammatory and profibrotic hepatic cytokines TNF-a or IL-6 was seen, hepatic mRNA expression of HGF was diminished in the Foxf1 ⫹/- mice (70% of WT level; p⬍ 0.05), leading to extensive biliary infarcts, hepatic apoptosis, and a 2.5-fold reduction in hepatocyte proliferation (p⫽0.05) at day 3 of BDL. In support of HGF as a Foxf1-regulated gene, Foxf1 transcription factor has binding sites to the HGF promoter region. Conclusion: Foxf1 does not play an important role in the early fibrogenic response to biliary obstruction. In the Foxf1 ⫹/- mice, abnormal HGF expression is however associated with increased hepatic damage and impaired hepatocyte proliferation, suggesting a protective role for Foxf1
174
ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS
against parenchymal liver injury during biliary fibrosis through its regulation of hepatic HGF production.
VASCULAR I: VASCULAR TONE AND REMODELING 55. CHLOROTYROSINE INDUCES ENDOTHELIAL DYSFUNCTION IN HUMAN CORONARY ARTERY ENDOTHELIAL CELLS AND PORCINE CORONARY ARTERIES. H. Chai, I. Mohiuddin, P. Lin, A. Lumsden, Q. Yao, C. J. Chen; Baylor College of Medicine, Houston, TX. Introduction: The heme-containing enzyme myeloperoxidase (MPO) play a critical role in many inflammatory conditions including atherosclerosis. Hypochlorous acid (HOCl), a major product of MPO, converts tyrosine into the 3-chlorotyrosine (CT). Recently, high CT levels have been associated with clinical progression of atherosclerosis. However, the molecular functions of CT in the vascular cells remain unclear. In this study, we investigated the effects of CT on coronary endothelial cell functions. Methods: Human coronary artery endothelial cells (HCAECs) were treated with CT (0, 10 -7, 10 -6 or 10 -5 ⌴) ° 6, 24 ° 48 . Tyrosine (10 -5 ⌴) was used as a control. The mRNA levels of endothelial nitric oxide synthase (eNOS) were determined by real-time PCR. Superoxide anion (O 2 -) production was determined by fluorescence dye DHE staining and flow cytometry analysis. Activation status of MAPKs and NF-B was studied using BioPlex immunoassay. In addition, porcine coronary artery rings were cultured with CT (0, 10 -8, 10 -7, 10 -6 or 10 -5 ⌴) for 24 hrs. Endothelium-dependent vasorelaxation was studied using a myograph tension system. The eNOS protein level and O 2 - production were studied using immunohistochemistry and chemiluminescence assay, respectively. Antioxidant seleno-L-methionine (SeMet) was included. Results: When HCAECs were cultured with CT (10 -6 or 10 -5 ⌴) for 24 hrs, the eNOS mRNA levels were significantly decreased by 23% and 46%, respectively, as compared to controls (P⬍0.05). An increase production in O 2 - after CT (10 -6 M) treatment was observed. Significant decreases in the catalase and superoxide dismutase enzyme activities were also noted. Antioxidant SeMet reversed CT-induced eNOS mRNA decrease. JNK, but not p38 and ERK1/2, was activated at 15 min after CT (10 -6 M) treatment. NF-B activation was also observed 45-60 min after CT (10 -6 M) treatment. In porcine coronary artery ring cultures, CT (10 -6 or 10 -5 ⌴) treatment induced a significant decrease in endothelium-dependent vasorelaxation by 19% and 32%, respectively, as compared to controls (P⬍0.05). The eNOS immunoreactivity was decreased in the CTtreated vessels, while O 2 - production was significantly increased. Conclusions: These data demonstrate that clinically relevant concentrations of CT significantly reduce eNOS expression, while increase O 2 - production in both HCAECs and porcine coronary arteries. In addition, CT also activates JNK and NF-B pathways. Antioxidant SeMet effectively blocks the CT-mediated action in vascular cells. These results suggest CT may contribute to the vascular lesion formation through endothelial dysfunction. 56. IN VITRO GENERATION OF HUMAN MICROVESSEL NETWORKS. W. B. Carter 1, M. Sekharem 1, J. B. Hoying 2; 1H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL, 2 University of Arizona, Tucson, AZ Introduction: Successful incorporation of bioengineered tissues requires vascularization. Attempts at de novo vascular generation or angiogenic induction have had little success in improving tissue perfusion. An alternate approach to neovascularization is to form a prevascularized construct that is capable of rapid inosculation and rapid cellular perfusion. Human parathyroid (PTH) tissue is uniquely capable of a rapid angiogenic induction, with restoration of calcium homeostasis within 6 weeks of PTH autografting. We hy-
pothesized that human PTH cells could induce human microvessel fragments to form networks of capillary microvessels in vitro. Because PTH cells release VEGF and Angiopoietin-2 (Ang-2) after devascularization, we further hypothesized that PTH angiogenic induction is driven by PTH release of VEGF and Ang-2. Methods: Human subcutaneous fat was partially digested with collagenase to yield microvessel fragments. After sequential filtering, 30-350 micron fragments were embedded in DMEM ⫹ 10% FBS-containing collagen I matrix in 3 dimensional culture. PTH cells were cocultured on the matrix surface to induce an angiogenic response. Other matrix cultures were treated with VEGF (50 ng/ml) or VEGF ⫹ Ang-2 (50 ng/ml) and microvessel responses noted. Because Stat3 phosphorylation has been identified with endothelial cell (EC) angiogenic responses, we also tested for Stat3 activation in human dermal microvessel EC (HDMEC) after treatment with VEGF or VEGF ⫹ Ang-2. Results: Human microvessel fragments in culture are completely quiescent. Coculture with PTH cells induces the rapid development of neovascular networks. Treatment of the human microvessels in vitro with VEGF failed to induce an angiogenic event. VEGF ⫹ Ang-2 induced EC migration but no capillary formation. Both VEGF and Ang-2 induced Stat3 phosphorylation in HDMEC, but not in iliac vein EC. Conclusions: Human PTH cells release or induce release of all necessary angiogenic factors for capillary network angiogenic induction of human microvessels in vitro. The mechanism of angiogenic induction is more complex than VEGF stimulation alone. Stat 3 activity is upregulated in microvessel EC after VEGF or Ang-2 stimulation, but not in large vessel EC, suggesting a possible role in microvessel angiogenic processes. PTH cells have the angiogenic potential to induce capillary network development in vitro. This system may be capable of providing vascularization of bioengineered tissues for therapeutic intervention. 57. DELETION OF CCR2 BUT NOT CCR5 INHIBITS AORTIC ANEURYSM FORMATION. J. N. Mactaggart, W. Xiong, R. Knispel, B. T. Baxter; University of Nebraska Medical Center, Omaha, NE Introduction: Microscopic analysis of abdominal aortic aneurysms (AAA) demonstrates an abundance of infiltrating leukocytes. The mechanism underlying the recruitment of these leukocytes to the abdominal aorta during aneurysm formation is unknown. Chemokines are cytokines that attract leukocytes to areas of inflammation. RANTES and MCP-1 are chemokines previously shown to be upregulated in experimental aneurysm formation. CCR5 and CCR2 are chemokine receptors present on leukocytes that bind RANTES and MCP-1 respectively. We hypothesized that the genetic deletions of CCR5 (CCR5-/-) and CCR2 (CCR2-/-) would limit leukocyte infiltration and subsequent aneurysm formation in a mouse model of AAA. Methods: CCR2-/-, CCR5-/-, and control mice of matching genetic backgrounds, all obtained from Jackson Laboratories, were subject to periaortic application of calcium chloride. This model has been described in several publications (Nature Medicine and JCI). Aortic diameter was measured with video microscopy before aneurysm induction and at harvest 6 weeks later. The mean diameter and diameter change of each treatment group were compared with concurrent controls using the Mann-Whitney test. Aortas were perfusion fixed with formalin and stained with H&E and trichrome for histologic analysis. Results: There was no inhibition of aneurysm formation in the CCR5-/- mice. This was corroborated by the tissue histology which showed inflammatory cell infiltration and matrix degradation. Aneurysm formation was attenuated in the CCR2-/- mice. The final mean diameter in the CCR2-/- mice was significantly less than the controls (*P⫽0.0001) and the CCR5-/- mice (P⬍0.05). The inflammatory infiltration was decreased compared to controls and the normal lamellar architecture of the aorta was preserved. Conclusions: Deletion of the CCR2 gene attenuates experimental aneurysm formation, while the deletion of the CCR5 gene has no effect. The CCR2 receptor and its chemokine ligands should be investigated further and may provide a therapeutic target in the development of pharmacologic inhibitors of AAA expansion.