Eugene S. Lee

Molecular mechanisms of endothelial hyperpermeability: implications in inflammation

Endothelial hyperpermeability is a significant problem in vascular inflammation associated with trauma, ischaemia-reperfusion injury, sepsis, adult respiratory distress syndrome, diabetes, thrombosis and cancer. An important mechanism underlying this process is increased paracellular leakage of plasma fluid and protein. Inflammatory stimuli such as histamine, thrombin, vascular endothelial growth factor and activated neutrophils can cause dissociation of cell-cell junctions between endothelial cells as well as cytoskeleton contraction, leading to a widened intercellular space that facilitates transendothelial flux. Such structural changes initiate with agonist-receptor binding, followed by activation of intracellular signalling molecules including calcium, protein kinase C, tyrosine kinases, myosin light chain kinase, and small Rho-GTPases; these kinases and GTPases then phosphorylate or alter the conformation of different subcellular components that control cell-cell adhesion, resulting in paracellular hypermeability. Targeting key signalling molecules that mediate endothelial-junction-cytoskeleton dissociation demonstrates a therapeutic potential to improve vascular barrier function during inflammatory injury.

ADAM15 regulates endothelial permeability and neutrophil migration via Src/ERK1/2 signalling

AIMS Endothelial barrier dysfunction is a key event in the pathogenesis of vascular diseases associated with inflammation. ADAM (a disintegrin and metalloprotease) 15 has been shown to contribute to the development of vascular inflammation. However, its role in regulating endothelial barrier function is unknown. The aim of this study was to examine the effect of ADAM15 on endothelial permeability and its underlying mechanisms. METHODS AND RESULTS By measuring albumin transendothelial flux and transendothelial electric resistance in cultured human umbilical vein endothelial cell monolayers, we found that depletion of ADAM15 expression via siRNA decreased endothelial permeability and attenuated thrombin-induced barrier dysfunction. In contrast, endothelial cells overexpressing either wild-type or catalytically dead mutant ADAM15 displayed a higher basal permeability and augmented hyperpermeability in response to thrombin. In addition, ADAM15 knockdown inhibited whereas ADAM15 overexpression promoted neutrophil transendothelial migration. Further molecular assays revealed that ADAM15 did not cleave vascular endothelial-cadherin or cause its degradation. However, overexpression of ADAM15 promoted extracellular signal-regulated kinase (ERK)1/2 phosphorylation in both non-stimulated and thrombin-stimulated endothelial cells in a protease activity-independent manner. Pharmacological inhibition of Src kinase or ERK activation reversed ADAM15-induced hyperpermeability and neutrophil transmigration. CONCLUSION The data provide evidence for a novel function of ADAM15 in regulating endothelial barrier properties. The mechanisms of ADAM15-induced hyperpermeability involve Src/ERK1/2 signalling independent of junction molecule shedding.

Implementation of an aortic screening program in clinical practice: Implications for the Screen for Abdominal Aortic Aneurysms Very Efficiently (SAAAVE) Act

OBJECTIVE Screening for abdominal aortic aneurysms (AAA) significantly reduces aneurysm-related death. In January 2007, the Federal government enacted Medicare coverage guideline to screen persons at risk for the presence of an AAA, the Screen for Abdominal Aortic Aneurysms Very Efficiently (SAAAVE) Act. The purpose of this study is to evaluate the efficacy and costs of a large scale screening effort for identifying AAAs in patients in clinical practice. METHODS A regional veterans affairs mandate for screening for AAA was implemented in February 2007. Data were extracted through the Northern California Veterans Affairs (VA) Service Network to identify veteran males 65-75 years of age who ever smoked at least 100 cigarettes during their lifetime. An AAA was defined as an aortic diameter 3.0 cm or greater. A Decision Support Systems software (LumiData, Minneapolis, Minn) package tracked true costs of conducting a large AAA screening protocol in the Northern California VA Health Care System. RESULTS A total of 2918 patients (average age, 71 +/- 6 years) were screened for AAA over a 1-year period from February 2007 to February 2008. An AAA was diagnosed in 5.1% (148/2918) of patients. Two hundred ninety patients out of the 2918 (9.9%) were inappropriately screened. The aneurysm distribution was as follows: 83% (123/148) of the aneurysms were 3.0-4.4 cm, 13% (19/148) were 4.5-5.5 cm, and 4.1% (6/148) were greater than 5.5 cm. Incidental findings of isolated iliac artery aneurysms were found in 0.1% (3/2918) of patients. The cost of AAA screening per patient is

Wound infection after infrainguinal bypass operations: multivariate analysis of putative risk factors.

53. CONCLUSION The results of a large AAA screening effort in clinical practice reflect the results reported in the major clinical trials at a reasonable cost. The identification of large iliac artery aneurysms in the screening has not been previously reported.

Outcomes of an abdominal aortic aneurysm screening program

Reported wound infection rates for infrainguinal bypass operations range from 17% to 44%, but there is limited appreciation of which characteristics of patients or operations are reliable markers of increased wound infection risk. The purpose of the present study was to analyze all wound infections observed after infrainguinal bypass operations during 20 years of practice in a large teaching institution. Independent risk factors for wound infection development were identified. During the 20-year period ending 31 December, 1997, 978 male patients underwent infrainguinal bypass operations at the Minneapolis Department of Veterans Affairs Medical Center. Wound infections complicated the recovery of 129 of these patients during a 30-day postoperative surveillance interval. Multivariate logistic regression analysis was used to test the association between wound infection occurrence and putative risk factors that were either features of patients or characteristics of the operations. The following variables were examined: obesity, prosthetic graft placement, diabetes mellitus, steroid use, anticoagulation use, length of preoperative hospital stay, development of incisional hematoma, duration of operation, and the preoperative presence of a non-healing wound in the extremity being revascularized. The overall wound infection rate was 13.2% (129/978). In a final logistic regression model, obesity was a significant and independent predictor of wound infection (Relative Risk 2.6, 95% confidence interval, 1.35-4.90), as was development of a post-operative incisional hematoma (Relative Risk 6.44, 95% confidence interval, 2.95-14.08). No other explanatory variable was significantly associated with wound infection development.

A Disintegrin and Metalloproteinase 15 Contributes to Atherosclerosis by Mediating Endothelial Barrier Dysfunction via Src Family Kinase Activity

OBJECTIVE In 2007, Medicare guidelines were established to identify persons at risk for the presence of an abdominal aortic aneurysm (AAA). The purpose of this study is to evaluate the 5-year outcomes of an AAA screening program in a regional Veterans Affairs (VA) health care system. METHODS Data were extracted from a regional VA health care network identifying all veteran males 65 to 75 years of age who smoked at least 100 cigarettes during their lifetime. In 2007, an AAA screening mandate was implemented allowing patients meeting screening criteria to be evaluated for AAA as part of the patients health maintenance. AAA is identified as an aortic diameter size of 3.0 cm or greater. Clinician adherence to screening protocols and referral to a vascular surgeon for aneurysms >5.5 cm were also evaluated. RESULTS A total of 9751 patients (71.5 ± 5.6 standard deviation years of age) were screened for an AAA over a 5-year period from January 1, 2007 to December 31, 2011. A total of 698 aneurysms (7.1%) were found. Referrals to a vascular surgeon were made on 45 patients with aneurysms >5.5 cm. Over a 5-year period, a total of 2754 patients (28.2%) were inappropriately screened: 416 patients were under 65 years old, 2243 patients were over 75 years old, 36 patients were women, and 123 patients without aneurysms had multiple screenings. In 2007, during the first year of implementation, 39.2% of patients were inappropriately screened. Over the next 4 years, inappropriate screenings decreased with 33.7% in 2008, 28.6% in 2009, 17.7% in 2010, and 14.3% in 2011. CONCLUSIONS A large AAA screening program at the VA detects more aneurysms, but at smaller diameters than that published in clinical trials. Over time, the number of inappropriate AAA screenings has continued to decrease, demonstrating greater awareness and application of the AAA screening guidelines by primary care providers. Developing surveillance guidelines for small and medium aneurysms is a potential area for future research.

Tissue Inhibitor of Metalloproteinase-2 Regulates Matrix Metalloproteinase-2–Mediated Endothelial Barrier Dysfunction and Breast Cancer Cell Transmigration through Lung Microvascular Endothelial Cells

Objective—Endothelium dysfunction is an initiating factor in atherosclerosis. A disintegrin and metalloproteinase 15 (ADAM 15) is a multidomain metalloprotease recently identified as a regulator of endothelial permeability. However, whether and how ADAM15 contributes to atherosclerosis remains unknown. Methods and Results—Genetic ablation of ADAM15 in apolipoprotein E–deficient mice led to a significant reduction in aortic atherosclerotic lesion size (by 52%), plaque macrophage infiltration (by 69%), and smooth muscle cell deposition (by 82%). In vitro studies implicated endothelial-derived ADAM15 in barrier dysfunction and monocyte transmigration across mouse aortic and human umbilical vein endothelial cell monolayers. This role of ADAM15 depended on intact functioning of the cytoplasmic domain, as evidenced in experiments with site-directed mutagenesis targeting the metalloprotease active site (E349A), the disintegrin domain (Arginine-Glycine-Aspartic acid→Threonine-Aspartic acid-Aspartic acid), or the cytoplasmic tail. Further investigations revealed that ADAM15-induced barrier dysfunction was concomitant with dissociation of endothelial adherens junctions (vascular endothelial [VE]-cadherin/&ggr;-catenin), an effect that was sensitive to Src family kinase inhibition. Through small interfering RNA-mediated knockdown of distinct Src family kinase members, c-Src and c-Yes were identified as important mediators of these junctional effects of ADAM15. Conclusion—These results suggest that endothelial cell-derived ADAM15, signaling through c-Src and c-Yes, contributes to atherosclerotic lesion development by disrupting adherens junction integrity and promoting monocyte transmigration.

Serum metalloproteinases MMP-2, MMP-9, and metalloproteinase tissue inhibitors in patients are associated with arteriovenous fistula maturation

Matrix metalloproteinases (MMP) have been implicated in multiple stages of cancer metastasis. Tissue inhibitor of metalloproteinase-2 (TIMP-2) plays an important role in regulating MMP-2 activity. By forming a ternary complex with pro-MMP-2 and its activator MMP-14 on the cell surface, TIMP-2 can either initiate or restrain the cleavage and subsequent activation of MMP-2. Our recent work has shown that breast cancer cell adhesion to vascular endothelial cells activates endothelial MMP-2, promoting tumor cell transendothelial migration (TEME). However, the mechanism of MMP-2 regulation during TEME remains unclear. In the current study, we present evidence that MMP-14 is expressed in both invasive breast cancer cells (MDA-MB-231 and MDA-MB-436) and lung microvascular endothelial cells (HBMVEC-L), whereas TIMP-2 is exclusively expressed and released from the cancer cells. The tumor cell–derived TIMP-2 was further identified as a major determinant of endothelial MMP-2 activity during tumor cell transmigration in the presence of MMP-14. This response was associated with endothelial barrier dysfunction because coculture of MDA-MB-231 or MDA-MB-436 with HBMVEC-L caused a significant decrease in transendothelial electrical resistance concomitantly with endothelial cell-cell junction disruption and tumor cell transmigration. Knockdown of TIMP-2 or inhibition of TIMP-2/MMP-14 attenuated MMP-2–dependent transendothelial electrical resistance response and TEME. These findings suggest a novel interactive role of breast cancer cells and vascular endothelial cells in regulating the TIMP-2/MMP-14/MMP-2 pathway during tumor metastasis. Mol Cancer Res; 8(7); 939–51. ©2010 AACR.

Surgical Resident Research Productivity Over 16 Years

OBJECTIVE Many vascular surgeons construct arteriovenous fistulas (AVFs) for hemodialysis access as the primary choice access. A significant number of AVFs fail to mature, however, leading to patient frustration and repeated operations. Metalloproteinase (MMP) activity, particularly MMP-2 and MMP-9, may be important for AVF maturation. We therefore sought to identify whether serum MMP levels could serve as a biomarker for predicting future successful AVF maturation. METHODS Blood was collected from patients with chronic renal insufficiency at the time of surgery for long-term hemodialysis access. Serum was separated from whole blood and ultracentrifuged at 1000g for 10 minutes. Serum aliquots were frozen at -80°C until used for analysis. Enzyme-linked immunosorbent assay was used to assay levels of MMP-2, MMP-9, and tissue inhibitor of metalloproteinase type 2 (TIMP-2), and TIMP type 4 (TIMP-4). Clinical end points were used to divide patients into failed and matured AVF groups. Successful maturation was considered in patients who had specific duplex findings or 1 month of successful two-needle cannulation hemodialysis. MMP/TIMP ratios were calculated as an index of the MMP axis activity because MMP activity parallels alterations in TIMP levels. RESULTS Of 20 enrolled patients, AVF maturation was successful in 13 and failed in 7. Serum levels of MMP-2/TIMP-2 were significantly higher in patients with matured AVFs vs levels in those that failed (P = .003). Similarly, a trend toward increased serum levels of MMP-9/TIMP-4 was found in patients with successful AVF (P = .06). CONCLUSIONS MMP-2 and TIMP-2 levels were different among patients whose AVF matured vs those who did not. Further follow-up studies to determine the predictability of AVF maturation using relative patient serum levels of MMP-2 and TIMP-2 should be performed.

Vein tissue expression of matrix metalloproteinase as biomarker for hemodialysis arteriovenous fistula maturation.

BACKGROUND General surgery training has changed over the past decade due to the 80-hour work week and increasing demands on the surgery faculty to generate clinical revenue with ever decreasing reimbursements. The purpose of this study was to evaluate surgery resident productivity over the years and the surgery residents contribution to clinical and basic research literature. METHOD A PubMed literature search of all graduating chief residents (n = 95) over a 16-y period from a single university-based general surgery program were evaluated. Number and types of publications (clinical paper versus basic science paper) were analyzed for each resident. A cohort of residents graduating from the years 1990 to 1996 (n = 42) were deemed the early group and a cohort of residents graduating from the years 1999 to 2005 (n = 41) were deemed the late group. Residents graduating in 1997 and 1998 were deemed the washout group. RESULTS From 1990 to 2005, there were 95 graduates with 204 published articles. Resident research time ranged from 0 to 2 y, with most residents spending 1 y of research time. In the early group, residents averaged 2.0 +/- 0.4 papers versus the late group where each resident published 2.6 +/- 0.5 papers (P = NS). In the early group, 24.4% of the papers were basic science in nature as opposed to the late group where 27.7% of the papers were with a basic science topic (P = NS, chi(2) analysis). CONCLUSIONS Resident research productivity at a single university-based program with an elective research time does not appear to be deteriorating over time. A majority of research performed by residents is clinically oriented; however. basic science research does not appear to be decreasing. Careful scrutiny to resident research productivity is needed to ensure productive future academic surgeons.