Cell migration in response to the amino-terminal fragment of urokinase requires epidermal growth factor receptor activation through an ADAM-mediated mechanism

BackgroundCell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of amino-terminal fragment (ATF) of urokinase on a pivotal cross-talk receptor, epidermal growth factor receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. We hypothesize that A Disintegrin and Metalloproteinase Domains (ADAM) allows the transactivation of EGFR by ATF.ObjectiveTo determine the role of ADAM in EGFR transactivation by ATF in human vascular smooth muscle cells (VSMC) during cell migration.MethodsHuman coronary VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to ATF (10 nM) in the presence and absence of the matrix metalloprotease (MMP) inhibitor GM6001, the ADAM inhibitors TAPI-0 and TAPI-1, heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, epidermal growth factor (EGF) inhibitory antibodies, and the EGFR inhibitor AG1478. The small interference ribonucleic acid (siRNA) against EGFR and ADAM-9, ADAM-10, ADAM-12, and adenoviral delivered Gbg inhibitor, βARKCT were also used.ResultsATF produced concentration-dependent VSMC migration (by wound assay and Boyden chamber), which was inhibited by increasing concentrations of AG1478. ATF was shown to induce time-dependent EGFR phosphorylation, which peaked at fourfold greater than control. Pre-incubation with the Gβγ inhibitor βARKCT inhibited EGFR activation by ATF. This migratory and EGFR response was inhibited by AG1478 in a concentration-dependent manner. Incubation with siRNA against EGFR blocked the ATF-mediated migratory and EGFR responses. EGFR phosphorylation by ATF was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of MMP, ADAMs, or HB-EGF. Direct blockade of the EGFR prevented activation by both ATF and EGF. Incubation with siRNA to ADAM-9 and -10 significantly reduced HB-EGF release from VSMC and EGFR activation in response to ATF. The siRNA against ADAM-12 had no effect.ConclusionATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR is an attractive molecular target to inhibit cell migration.Clinical RelevanceCell migration is an integral component of intimal hyperplasia development and proteases are pivotal in the process. Understanding the role of urokinase signaling within the cells of vasculature remains poorly defined. The study examines the role of ATF of urokinase on a pivotal cross-talk receptor, EGFR. EGFR is transactivated by both G-protein-coupled receptors and receptor tyrosine kinases and is key to many of their responses. ATF can induce transactivation of EGFR by an ADAM-mediated, HB-EGF-dependent process. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target

Radiation arteritis: A contraindication to carotid stenting?

BackgroundCarotid artery stenting (CAS) for high-risk anatomic lesions is accepted practice. Neck irradiation and radiotherapy-induced arteritis are common indications. The clinical outcomes of CAS for radiation arteritis have been poorly defined.MethodsA prospective database of patients undergoing CAS at a tertiary referral academic medical center was maintained from 1999 to 2006. Patients undergoing primary carotid artery stenting for significant atherosclerotic (ASOD) and radiotherapy (XRT)-induced occlusive disease were analyzed. Life-table analyses were performed to assess time-dependent outcomes. Cox proportional hazard analysis or Fisher’s exact test was performed to identify factors associated with outcomes. Data are presented as the mean ± SEM unless otherwise indicated.ResultsDuring the study period, 150 patients underwent primary CAS, 75% with embolic protection. Fifty-eight percent were symptomatic. One hundred twenty-seven (85%) were treated for ASOD, and 23 (15%) had XRT. The 30-day all-cause mortality rate was 1% for ASOD and 0% for XRT (P = NS); overall survival at 3 years was equivalent. There was no significant difference in major adverse event rates as defined by the Stenting and Angioplasty with Protection in Patients at High Risk for Endarterectomy (SAPPHIRE) trial between the groups. The 3-year neurologic event-free rate was 85% for ASOD and 87% for XRT (P = NS). Late asymptomatic occlusions were seen only in XRT patients. The 3-year freedom from restenosis rate was significantly worse for the XRT group, at 20%, vs 74% for the ASOD group (P < .05). Likewise, the 3-year patency rate was also worse for the XRT group, at 91%, vs 100% for ASOD by Kaplan-Meier analysis (P < .05). No factor was predictive of occlusion or stenosis by Cox proportional hazards analysis.ConclusionCAS for radiation arteritis has poor long-term anatomic outcome and can present with late asymptomatic occlusions. These findings suggest that these patients require closer postoperative surveillance and raise the question of whether CAS is appropriate for carotid occlusive lesions caused by radiation arteritis

Pericardial Patch Angioplasty Heals via an Ephrin-B2 and CD34 Positive Cell Mediated Mechanism

Pericardial patches are commonly used in vascular surgery to close arteriotomies. The mechanism of early healing after patch implantation is still not well defined. We used a rat aortic patch model to assess pericardial patch healing and examined Ephrin-B2, a marker of arterial identity, expression within the post-implantation patch. We also determined whether endothelial progenitor cells (EPC) are associated with early patch healing in the arterial environment.Wistar rats (200-250 grams) underwent infrarenal aortic arteriotomy and then closure via bovine or porcine pericardial patch angioplasty. Control groups included subcutaneously implanted patches. Patches were harvested at 0-30 days and analyzed by histology, immunohistochemistry, immunofluorescence and Western blot as well as quantitative PCR.Prior to implantation, pericardial patches are largely composed of collagen and are acellular. Following arterial implantation, increasing numbers of CD68-positive cells as well as Ephrin-B2 and CD34 dual-positive cells are found within both bovine and porcine pericardial patches, whereas the infiltrating cells are negative for vWF and α-actin. Porcine patches have a luminal monolayer of cells at day 7, compared to bovine patches that have fewer luminal cells. Subcutaneously implanted patches do not attract Ephrin-B2/CD34-positive cells. By day 30, both bovine and porcine pericardial patches develop a neointima that contains Ephrin-B2, CD34, and VEGFR2-positive cells.Both CD68-positive and Ephrin-B2 and CD34 dual-positive cells infiltrate the pericardial patch early after implantation. Arteriotomy closure via pericardial patch angioplasty shows patch adaptation to the arterial environment that may involve a foreign body response as well as localization of EPC. Arterial remodeling of pericardial patches support endothelialization and may represent a paradigm of healing of scaffolds used for tissue engineering

Long-term outcomes of catheter directed thrombolysis for lower extremity deep venous thrombosis without prophylactic inferior vena cava filter placement

BackgroundWhile the predominant treatment of lower extremity deep venous thrombosis (DVT) remains systemic anticoagulation, there is a growing consensus that more aggressive percutaneous catheter directed thrombolysis (CDT) carries both short-term and long-term benefits. There remains controversy as to whether an inferior vena cava (IVC) filter is always required during CDT.ObjectiveTo define the short- and long-term outcomes of CDT with and without prophylactic IVC filter placement for lower extremity DVT.MethodsA database of patients treated by CDT from 1996 to 2006 was compiled. Results were standardized to current Society for Vascular Surgery criteria. Average follow-up was 2.1 years, range of 1-8 years. Kaplan-Meier survival analyses were performed to assess time-dependent outcomes. Factor analyses were performed using a Cox proportional hazard model for time dependent variables. Data are presented as mean ± SD where appropriate.ResultsSixty-nine patients (39% male, average age 48 ± 17 years) underwent CDT: (27 received pharmacological thrombolysis, 12 received mechanical thrombolysis, and 30 received mechanical and pharmacological thrombolysis). Fourteen patients (20%) had IVC filter placement prior to or during CDT. Twenty-one had a hypercoagulable state. Technical success with grade III lysis of clot burden was achieved in 63%. Fifty-one patients required an adjuvant stent. Overall, 90-day all-cause mortality was 4% and peri-procedural morbidity was 4%. No patients developed a pulmonary embolus (PE) during therapy. By Kaplan-Meier analysis 83%, 83%, and 75% of patients were free of recurrent DVT at 1, 2, and 3 years, respectively. Hypercoagulability was associated with DVT recurrence by Cox proportional hazards analysis. No analyzed factor was predictive of PE.ConclusionCatheter directed thrombolysis without universal prophylactic IVC filter placement is safe and effective in treating acute DVT. Pulmonary embolization did not occur during CDT. Selective rather than routine IVC filter placement is a safe and appropriate approach