Phenotypic changes in EPCs predict outcome following lower extremity revascularization

Phenotypic changes in EPCs predict outcome following lower extremity revascularization

VASCULAR SURGERY II modulate target genes associated with vascular remodeling after exposure to IL-1. PARP-1 deficiency augments intimal hyperplasia ...

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VASCULAR SURGERY II modulate target genes associated with vascular remodeling after exposure to IL-1.

PARP-1 deficiency augments intimal hyperplasia in a mouse model of arterial injury Chandler A Long MD, Hassan Albadawi MD, Hyung-Jin Yoo BSc, Shirling Tsai MD, Rahmi Oklu MD, PhD, Mitchell Goldman MD, FACS, Michael T Watkins MD, FACS Massachusetts General Hospital/Harvard Medical School, Boston, MA, and University of Tennessee Medical Center/Graduate School of Medicine, Knoxville, TN

METHODS: Cultured vSMCs and NIvSMCs were infected with control virus (LacZ) or KLF-2 virus with or without IL-1 stimulation. RNA was harvested and gene expression of Thrombospondin-1 (THBS-1) and Plasminogen Activator Inhibitor-1 (PAI-1) for each treatment was evaluated using real-time PCR. Data were subsequently analyzed using a two-way ANOVA.

INTRODUCTION: Poly ADP polymerase (PARP) inhibition has been shown to limit experimental tissue injury and atherosclerotic lesion formation in mice. In contrast, PARP inhibition is known to decrease tumor cell proliferation and increase tumor sensitivity when combined with chemotherapeutic agents in cancer patients. These experiments were designed to determine whether genetic depletion of PARP-1 might modulate the proliferative response to arterial injury. METHODS: PARP-1 mutants (PARP-1⫺/⫺, n⫽7) and wild type mice (Control, n⫽12) underwent common carotid artery ligation proximal to the carotid bifurcation and were sacrificed 28 days later. Carotid arteries were harvested, embedded and submitted to axial sectioning. Proliferating smooth muscle cells were identified by alpha-Actin and PCNA using immunohistochemistry. Verhoeff’s van Gieson staining illustrated the medial elastic laminas for morphometric analysis (intimal to medial area ratio, I/M). Statistical analysis was done by a student t-test. RESULTS: PARP-1⫺/⫺ demonstrated significantly higher I/M ratios compared to Control (PARP1⫺/⫺: 1.47⫾0.32 vs. 0.24⫾0.06, p⫽0.0002). The increased I/M ratio correlated with more extensive alpha-Actin staining detected in the media and the developing subintimal hyperplasia. In addition, there was evidence of increased neointimal PCNA staining in the PARP-1⫺/⫺ compared to Control. CONCLUSIONS: These data demonstrate that genetic depletion of PARP-1 results in an exuberatant hyperplastic response in injured blood vessels. It remains to be determined whether pharmacologic inhibition of PARP activity has a similar impact on intimal injury. This observation may limit the use of PARP inhibitors for treatment of ischemia reperfusion injury in the setting of concomitant arterial injury. Kruppel-like factor 2 does not suppress interleukin-1 signaling in vascular smooth muscle cells Khayree Butler MD, David Zhou, Kerri O’Malley PhD, Angela Cuenca BSc, Scott Berceli MD, PhD, FACS University of Florida, Gainesville, FL INTRODUCTION: Kruppel-like factor 2 (KLF-2) has been shown to be a modulator of target genes associated with vascular remodeling in response to pro-inflammatory stimuli and shear stress in vascular endothelial cells. Interleukin-1 (IL-1) is a pro-inflammatory mediator which has been shown to be negatively modulated by KLF-2 in vascular endothelial cells. The regulatory effect of KLF-2 on vascular smooth muscle cells exposed to IL-1 is not currently known. Our hypothesis is that in murine vascular smooth muscle cells (vSMC) and neointimal vascular smooth muscle cells (NIvSMC) KLF-2 will

© 2011 by the American College of Surgeons Published by Elsevier Inc.

RESULTS: NIvSMCs have increased PAI-1 and THBS-1 expression compared to vSMCs following IL-1 stimulation (p ⫽ 0.005, p ⫽ 0.021 respectively). The response to KLF-2 transfection after IL-1 exposure was not significantly different in target genes. CONCLUSIONS: Neointimal vascular smooth muscle cells are more responsive to IL-1 than vascular smooth muscle cells but this effect is not influenced by KLF-2. This is in contrast to vascular endothelial cells where KLF-2 is a known modulator of target genes associated with vascular remodeling in response to IL-1.

Phenotypic changes in EPCs predict outcome following lower extremity revascularization Christian David Restrepo BSc, Kerri O’Malley PhD, Michael Hong MD, Khayree Butler MD, Lyle Moldawer PhD, Scott Berceli MD, FACS, Peter R Nelson MD, FACS Malcolm Randall VA Medical Center, Gainesville, FL INTRODUCTION: Endothelial progenitor cells (EPCs) circulate in the peripheral blood of adults and function to replenish aging and damaged endothelial cells (EC) that line blood vessels. Studies suggest that EPCs home to sites of vascular injury leading to reendothelialization and vessel repair. We hypothesized that differences in EPC phenotype may predict success versus failure in patients undergoing lower extremity vein bypass grafting for critical limb ischemia. METHODS: Peripheral blood was collected from patients (n⫽39) preoperatively and then following vein bypass at 2 hours (2H), 1 day (1D), 1 week (1W), 1 month (1M), 6 months (6M), and one year (1Y). Samples underwent staining for markers CD45, CD34, CD133, VEGF, and CD146 followed by red blood cell lysis, fixation, and analysis using an LSRII flow cytometer. Data at each time point were then analyzed using a Student’s t-test to compare outcome groups with p ⬍0 .05 considered significant. RESULTS: 18.5% of the CD45⫺/CD133⫹/CD34⫺ EPC population expressed VEGF preoperatively in failure patients when compared to 43% in success patients (p⫽0.005). Failure patients also had significantly less CD146 expression in the CD45⫺/CD133⫹/ CD34⫺ EPC population when compared to success patients (p⫽0.022). There were no significant differences at later time points (2H, 1D, 1W, 1M, and 6M, and 1Y) that differentiated success or failure outcomes. CONCLUSIONS: Pre-operative differences in EPC phenotype were predictive of outcome following lower extremity bypass. EPCs in failure patients demonstrated phenotypic changes consistent with impaired homing (VEGF) and endothelial regeneration (CD146).

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ISSN 1072-7515/11/$36.00 doi:10.1016/j.jamcollsurg.2011.06.390

Vol. 213, No. 3S, September 2011

Post-operative temporal changes in EPC phenotype require further study.

EphB4 activates endothelial nitric oxide synthase to limit vein graft thickening Michael J Collins MD, Akihito Muto MD, PhD, Amanda Feigel MD, Matthew Goodwin, Kenneth Ziegler MD, Clinton Protack MD, Lynn Model MD, Alan Dardik MD, PhD, FACS Yale University, New Haven, CT INTRODUCTION: EphB4, a receptor tyrosine kinase, is a marker of venous identity in adult veins that inhibits vein graft thickening. Since the role of endothelial nitric oxide synthase (eNOS) in vein graft remodeling is not well understood, we evaluated whether eNOS is a potential downstream mechanism that mediates EphB4 signaling and control of vein graft thickness. METHODS: Intrathoracic inferior vena cava was harvested from wild-type (WT) or eNOS knockout (KO) mice and placed as an interposition vein graft into the infrarenal aorta of WT mice. Vein grafts were harvested and analyzed after 3 weeks. EphB4 was activated in mouse lung endothelial cells (EC) in vitro using the bivalent ligand Ephrin-B2/Fc. In some experiments EphB4 was clustered by pretreatment with anti-Fc. eNOS activation was assessed by eNOS phosphorylation on Western blot analysis. RESULTS: Vein grafts derived from eNOS-KO mice showed significantly less wall thickening compared with WT vein grafts (n⫽4, p⫽0.04). Eph-B4 increased phosphorylation of eNOS in a bimodal distribution, with maximal increases in eNOS phosphorylation at both 1 and 60 minutes (n⫽5; p⬍0.005). Clustering of Eph receptors altered the kinetics of the second, but not the first, peak of eNOS phosphorylation, suggesting a unique EphB4-eNOS signal pathway.

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before manipulation (unmanipulated, UM). Additional HSV was collected after back-table manipulation, including hand-held syringe distention and marking for orientation (after manipulation, AM). Paired UM and AM segments were obtained. SV was sectioned into 1-mm rings. Baseline histology was obtained. Rings were maintained in RPMI 1640 medium with 30% FBS, 1% L-glutamine, and 1% penicillin/streptomycin for 14 days at 37°C/5% CO2. The experiments were conducted in duplicate for every vein segment and both rings were used for histological evaluation. Four-quadrant measurements were made of intimal and medial thickness. RESULTS: After two weeks in organ culture, UM-SV had a mean increase in intimal thickness of 22.8⫾18.8 ␮m, and a mean increase in intimal/medial ratio of 22.3⫾26.9%. AM-SV had a mean increase in intimal thickness of 38.5⫾26.5 ␮m, and a mean increase in intimal/medial ratio of 49.6⫾37.3%. Compared with UM-SV, AM-SV had a 68.9% greater increase in intimal thickness (p⫽0.043), and a 122.3% greater increase in intimal/medial ratio (p⫽0.015). CONCLUSIONS: Back-table preparation causes injury which promotes development of intimal hyperplasia. These results argue for less injurious means of preparing HSV prior to autologous transplantation into the arterial circulation.

Localized gene silencing in a rat model of intimal hyperplasia Julia D Glaser, Christoph S Nabzdyk MD, Mauricio Contreras MD, Leena Pradhan PhD, Frank W LoGerfo MD, FACS Beth Israel Deaconess Medical Center, Boston, MA

CONCLUSIONS: Eph-B4 stimulates eNOS phosphorylation in adult venous cells via a novel intracellular signaling pathway. Our data suggests that eNOS is critical for normal vein graft adaptation to the arterial circulation. Methods that promote eNOS activation may also promote vein graft adaptation and reduce vein graft failure.

INTRODUCTION: Myristoylated, alanine-rich C-kinase substrate (MARCKS) is upregulated in intimal hyperplasia (IH) in vein grafts. Silencing of MARCKS is known to reduce cell proliferation and migration of vascular smooth muscle, but not endothelial cells, making it a desirable target for gene therapy. Previously, this lab demonstrated that the intraluminal route facilitates delivery of unmodified siRNA into the wall of human saphenous veins in an ex-vivo model. This study evaluates intraluminal siRNA delivery in a rat model of carotid artery balloon injury.

“Back-table” manipulation of human saphenous vein promotes intimal hyperplasia Michael J Osgood MD, Kyle M Hocking BE, Kevin W Sexton MD, Padmini Komalavilas PhD, Joyce Cheung-Flynn PhD, Colleen Brophy MD, FACS Vanderbilt University Medical Center, Nashville, TN and Tennessee Valley Veterans Affairs Medical Center, Nashville, TN

METHODS: Several MARCKS siRNA sequences were evaluated in rat aortic smooth muscle cells. Rat carotid arteries were injured using a 2F balloon catheter. A 15-minute intraluminal injection was used to transfect the vessels with siRNA solutions. Fluorophore-conjugated siRNA was used to examine transmural delivery. Samples were harvested at 24 hours, 5 and 21 days. Gene silencing was evaluated by Q-RT-PCR. IH was assessed morphometrically.

INTRODUCTION: Saphenous vein (SV) is the most widely used arterial bypass conduit despite a high rate of intimal hyperplasia (IH). IH is thought to evolve as a response to vascular injury. We investigated whether injury from back-table surgical preparation promotes IH in an organ culture model.

RESULTS: In vitro, MARCKS siRNA complexed with transfection reagent RNAiMax reduced mRNA levels by 67% and 52% compared to no treatment or control siRNA. In vivo, fluorophore-tagged siRNA was visible throughout the vessel wall. Unmodified MARCKS siRNA only reduced mRNA levels by 26% and 47% compared to saline or control siRNA. Preliminary data reveals no differences in IH between groups at 21 days.

METHODS: SV segments were collected from patients undergoing arterial bypass (n⫽11). SV was collected after surgical removal but