Aortic anatomic severity grade correlates with resource utilization.

BACKGROUND: Potential cost effectiveness of endovascular aneurysm repair (EVAR) compared with open aortic repair (OAR) is offset by the use of intraoperative adjuncts (components) or late reinterventions. Anatomic severity grade (ASG) can be used preoperatively to assess abdominal aortic aneurysms, and provide a quantitative measure of anatomic complexity. The hypothesis of this study is that ASG is directly related to the use of intraoperative adjuncts and cost of aortic repair. METHODS: Patients who undergo elective OAR and EVAR for abdominal aortic aneurysms were identified over a consecutive 3-year period. ASG scores were calculated manually using three-dimensional reconstruction software by two blinded reviewers. Statistical analysis of cost data was performed using a log transformation. Regression analyses, with a continuous or dichotomous outcome, used a generalized estimating equations approach with the sandwich estimator, being robust with respect to deviations from model assumptions. RESULTS: One hundred forty patients were identified for analysis, n = 33 OAR and n = 107 EVAR. The mean total cost (± standard deviation) for OAR was per thousand (k) $38.3 ± 49.3, length of stay (LOS) 13.5 ± 14.2 days, ASG score 18.13 ± 3.78; for EVAR, mean total cost was k $24.7 ± 13.0 (P = .016), LOS 3.0 ± 4.4 days (P = .012), ASG score 15.9 ± 4.13 (P = .010). In patients who underwent EVAR, 25.2% required intraoperative adjuncts, and analysis of this group revealed a mean total cost of k $31.5 ± 15.9, ASG score 18.48 ± 3.72, and LOS 3.9 ± 4.5, which were significantly greater compared with cases without adjunctive procedures. An ASG score of ≥15 correlated with an increased propensity for requirement of intraoperative adjuncts; odds ratio, 5.75 (95% confidence interval, 1.82-18.19). ASG >15 was also associated with chronic kidney disease, end stage renal disease, hypertension, female sex, increased cost, and use of adjunctive procedures. CONCLUSIONS: Complex aneurysm anatomy correlates with increased total cost and need for adjunctive procedures during EVAR. Preoperative assessment with ASG scores can delineate patients at greater risk for increased resource use. Patient comorbid factors are associated with anatomic complexity defined according to ASG. A critical examination of the relationship between anatomic complexity and finances is required within the context of aggressive endovascular treatment strategies and shifts toward value-based reimbursement.

Differential expression of Hedgehog/Notch and transforming growth factor-ß in human abdominal aortic aneurysms.

OBJECTIVE: The molecular mechanisms leading to the development of abdominal aortic aneurysms (AAAs) remain poorly understood. The aim of this study was to determine the expression of Sonic Hedgehog (SHh), transforming growth factor ß (TGF-ß), and Notch signaling components in human aneurysmal and nonaneurysmal aorta in vivo. METHODS: Paired tissue samples were obtained from aneurysmal and nonaneurysmal (control) segments of the aortic wall of eight patients with suitable anatomy undergoing open repair of infrarenal AAAs. Protein and messenger RNA (mRNA) expression levels were determined by Western blot and quantitative real-time polymerase chain reaction analysis. RESULTS: Aneurysm development resulted in a significant reduction in vascular smooth muscle (vSMC) differentiation genes α-actin and SMC22α at both mRNA and protein levels. In parallel experiments, an 80.0% ± 15% reduction in SHh protein expression was observed in aneurysmal tissue compared with control. SHh and Ptc-1 mRNA levels were also significantly decreased, by 82.0% ± 10% and 75.0% ± 5%, respectively, in aneurysmal tissue compared with nonaneurysmal control tissue. Similarly, there was a 50.0% ± 9% and 60.0% ± 4% reduction in Notch receptor 1 intracellular domain and Hrt-2 protein expression, respectively, in addition to significant reductions in Notch 1, Notch ligand Delta like 4, and Hrt-2 mRNA expression in aneurysmal tissue compared with nonaneurysmal tissue. There was no change in Hrt-1 expression observed in aneurysmal tissue compared with control. In parallel experiments, we found a 2.2 ± 0.2-fold and a 5.6 ± 2.2-fold increase in TGF-ß mRNA and protein expression, respectively, in aneurysmal tissue compared with nonaneurysmal tissue. In vitro, Hedgehog signaling inhibition with cyclopamine in human aortic SMCs resulted in decreased Hedgehog/Notch signaling component and vSMC differentiation gene expression. Moreover, cyclopamine significantly increased TGF-ß1 mRNA expression by 2.6 ± 0.9-fold. CONCLUSIONS: These results suggest that SHh/Notch and TGF-ß signaling are differentially regulated in aneurysmal tissue compared with nonaneurysmal tissue. Changes in these signaling pathways and the resulting changes in vSMC content may play a causative role in the development of AAAs.

Fibrinolytic activity of endothelial cells from different venous beds.

BACKGROUND: Little is known about the molecular biology of endothelial cells from different venous vascular beds. As a result, our treatment of deep vein thrombosis and pulmonary artery embolism remain identical. As an initial step in understanding venous thromboembolic disease in the trauma and surgical patients, this study sought to investigate the balance between coagulation and fibrinolysis in the pulmonary and deep venous vascular beds and how trauma might influence this balance. MATERIALS AND METHODS: Confluent human iliac vein endothelial cells (HIVECs) and human pulmonary artery endothelial cells (HPAECs), were cultured in the absence or presence of tumor necrosis factor (TNFα; 10 ng/mL) for 24 h. The expression of mediators of coagulation and fibrinolysis were determined by Western blot analysis, and plasminogen activator activity was determined by a fibrin clot degradation assay. RESULTS: After TNFα stimulation, there was decreased expression of endothelial protein C receptor and thrombomodulin in both HIVECs and HPAECs. TNFα stimulation increased urokinase plasminogen activator expression in both HIVECs and HPAECs. There was an increase in the expression of tissue plasminogen activator and plasminogen activator inhibitor-1 in response to TNFα in HPAECs, but not in HIVECs. There was significantly greater clot degradation in the presence of both the conditioned media and cell extracts from HIVECs, when compared with HPAECs. CONCLUSIONS: HPAECs and HIVECs react differently in terms of fibrinolytic potential when challenged with a cytokine associated with inflammation. These findings suggest that endothelial cells from distinct venous vascular beds may differentially regulate the fibrinolytic pathway.

Inhibition of patched-1 prevents injury-induced neointimal hyperplasia.

OBJECTIVE: To determine the role of patched receptor (Ptc)-1 in mediating pulsatile flow-induced changes in vascular smooth muscle cell growth and vascular remodeling. APPROACH AND RESULTS: In vitro, human coronary arterial smooth muscle cells were exposed to normal or pathological low pulsatile flow conditions for 24 hours using a perfused transcapillary flow system. Low pulsatile flow increased vascular smooth muscle cell proliferation when compared with normal flow conditions. Inhibition of Ptc-1 by cyclopamine attenuated low flow-induced increases in Notch expression while concomitantly decreasing human coronary arterial smooth muscle cell growth to that similar under normal flow conditions. In vivo, ligation injury-induced low flow increased vascular smooth muscle cell growth and vascular remodeling, while increasing Ptc-1/Notch expression. Perivascular delivery of Ptc-1 small interfering RNA by pluronic gel inhibited the pathological low flow-induced increases in Ptc-1/Notch expression and markedly reduced the subsequent vascular remodeling. CONCLUSIONS: These results suggest that pathological low flow stimulates smooth muscle cell growth in vitro and vascular remodeling in vivo via Ptc-1 regulation of Notch signaling.

Mapping the distribution of health equity research and practice across a university: a network analysis.

Introduction: Health equity research spans various disciplines, crossing formal organizational and departmental barriers and forming invisible communities. This study aimed to map the nomination network of scholars at the University of Rochester Medical Center who were active in racial and ethnic health equity research, education, and social/administrative activities, to identify the predictors of peer recognition. Methods: We conducted a snowball survey of faculty members with experience and/or interest in racial and ethnic health equity, nominating peers with relevant expertise. Results: Data from a total of 121 individuals (64% doing research on extent and outcomes of racial/ethnic disparities and racism, 48% research on interventions, 55% education, and 50% social/administrative activities) were gathered in six rounds of survey. The overlap between expertise categories was small with coincidence observed between education and social/administrative activities (kappa: 0.27; p < 0.001). Respondents were more likely to nominate someone if both were involved in research (OR: 3.1), if both were involved in education (OR: 1.7), and if both were affiliated with the same department (OR: 3.7). Being involved in health equity research significantly predicted the centrality of an individual in the nomination network, and the most central actors were involved in multiple expertise categories. Conclusions: Compared with equity researchers, those involved in racial equity social/administrative activities were less likely to be recognized by peers as equity experts.

Structural racism in healthcare and research: A community-led model of curriculum development and implementation.

Structural racism in the USA has roots that extend deep into healthcare and medical research, and it remains a key driver of illness and early death for Black, Indigenous, People of Color (BIPOC). Furthermore, the persistence of racism within academic medicine compels an interrogation of education and research within this context. In the spirit of this interrogation, this article highlights a unique model of community-engaged education that integrates cultural humility. As an individual and institutional stance, cultural humility denotes lifelong learning and self-critique, the mitigation of power imbalances, and accountability. The integration of cultural humility emphasizes that when space is created for BIPOC communities to lead the way, education regarding healthcare and research can be effectively reimagined. Demonstrating this effectiveness, six community partners led the development and implementation of a five-module Structural Racism in Healthcare and Research course. Using a cohort model approach, the pilot course enrolled 12 community members and 12 researchers. The curriculum covered topics such as history of racism in healthcare and research, and introduced participants to a cultural resilience framework. Evaluation results demonstrated a significant increase in participants' knowledge and ability to identify and take action to address inequities related to racism in healthcare and research.

How to overcome hesitancy for COVID-19 and other vaccines.

These evidence-based strategies (and list of do's and don'ts) can help you to increase the likelihood of vaccine uptake in hesitant patients.

Glycogen synthase kinase 3 beta positively regulates Notch signaling in vascular smooth muscle cells: role in cell proliferation and survival.

The role of glycogen synthase kinase 3 beta (GSK-3ß) in modulating Notch control of vascular smooth muscle cell (vSMC) growth (proliferation and apoptosis) was examined in vitro under varying conditions of cyclic strain and validated in vivo following changes in medial tension and stress. Modulation of GSK-3ß in vSMC following ectopic expression of constitutively active GSK-3ß, siRNA knockdown and pharmacological inhibition with SB-216763 demonstrated that GSK-3ß positively regulates Notch intracellular domain expression, CBF-1/RBP-Jκ transactivation and downstream target gene mRNA levels, while concomitantly promoting vSMC proliferation and inhibiting apoptosis. In contrast, inhibition of GSK-3ß attenuated Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to cyclic strain environments in vitro using both a Flexercell™ Tension system and a novel Sylgard™ phantom vessel following bare metal stent implantation revealed that cyclic strain inhibits GSK-3ß activity independent of p42/p44 MAPK and p38 activation concomitant with reduced Notch signaling and decreased vSMC proliferation and survival. Exposure of vSMC to changes in medial strain microenvironments in vivo following carotid artery ligation revealed that enhanced GSK-3ß activity was predominantly localized to medial and neointimal vSMC concomitant with increased Notch signaling, proliferating nuclear antigen and decreased Bax expression, respectively, as vascular remodeling progressed. GSK-3ß is an important modulator of Notch signaling leading to altered vSMC cell growth where low strain/tension microenvironments prevail.

Alcohol inhibits smooth muscle cell proliferation via regulation of the Notch signaling pathway.

OBJECTIVE: To determine the role of Notch signaling in mediating alcohol's inhibition of smooth muscle cell (SMC) proliferation. METHODS AND RESULTS: Treatment of human coronary artery SMCs with ethanol (EtOH) decreased Notch 1 mRNA and Notch 1 intracellular domain protein levels, in the absence of any effect on Notch 3. EtOH treatment also decreased C-promoter binding factor-1 (CBF-1)/recombination signal-binding protein (RBP)-jk promoter activity and Notch target gene (hairy related transcription factor [HRT-1] or HRT-2) expression. These effects were concomitant with an inhibitory effect of EtOH on SMC proliferation. Overexpression of constitutively active Notch 1 intracellular domain or human hairy related transcription factor-1 (hHRT-1) prevented the EtOH-induced inhibition of SMC proliferation. In vivo, Notch 1 and HRT-1 mRNA expression was increased after ligation-induced carotid artery remodeling. The vessel remodeling response was inhibited in mice that received "moderate" amounts of alcohol by gavage daily; intimal-medial thickening was markedly reduced, and medial and neointimal SMC proliferating cell nuclear antigen expression was decreased. Moreover, Notch 1 and HRT-1 expression, induced after ligation injury, was inhibited by moderate alcohol consumption. CONCLUSIONS: EtOH inhibits Notch signaling and, subsequently, SMC proliferation, in vitro and in vivo. The modulation of Notch signaling in SMCs by EtOH may be relevant to the cardiovascular protective effects of moderate alcohol consumption purported by epidemiological studies.

Sonic Hedgehog induces Notch target gene expression in vascular smooth muscle cells via VEGF-A.

OBJECTIVE: Notch, VEGF, and components of the Hedgehog (Hh) signaling pathway have been implicated in vascular morphogenesis. The role of Notch in mediating hedgehog control of adult vascular smooth muscle cell (SMC) growth and survival remains unexplored. METHODS AND RESULTS: In cultured SMCs, activation of Hh signaling with recombinant rShh (3.5 mug/mL) or plasmid encoded Shh increased Ptc1 expression, enhanced SMC growth and survival and promoted Hairy-related transcription factor (Hrt) expression while concomitantly increasing VEGF-A levels. These effects were significantly reversed after Hh inhibition with cyclopamine. Shh-induced stimulation of Hrt-3 mRNA and SMC growth and survival was attenuated after inhibition of Notch-mediated CBF-1/RBP-Jk-dependent signaling with RPMS-1 while siRNA knockdown of Hrt-3 inhibited SMC growth and survival. Recombinant VEGF-A increased Hrt-3 mRNA levels while siRNA knockdown abolished rShh stimulated VEGF-A expression while concomitantly inhibiting Shh-induced increases in Hrt-3 mRNA levels, proliferating cell nuclear antigen (PCNA), and Notch 1 IC expression, respectively. Hedgehog components were expressed within intimal SMCs of murine carotid arteries after vascular injury concomitant with a significant increase in mRNA for Ptc1, Gli(2), VEGF-A, Notch 1, and Hrts. CONCLUSIONS: Hedgehog promotes a coordinate regulation of Notch target genes in adult SMCs via VEGF-A.

Video. Magnetic retraction for NOTES transvaginal cholecystectomy.

BACKGROUND: Natural orifice translumenal endoscopic surgery (NOTES) has the potential to decrease the burden of an operation on a patient. Limitations of the endoscopic platform require innovative solutions to provide retraction and create an operation comparable with the gold standard, laparoscopic cholecystectomy. METHODS: Four patients underwent transvaginal cholecystectomy. All procedures were performed under laparoscopic vision to ensure safety. The endoscope and a long articulating RealHand instrument were placed via a 15-mm vaginal trocar. A magnetic retraction system was used to retract the gallbladder safely. Laparoscopic clips were used to ligate the cystic duct and artery. All four gallbladders were successfully removed. No complications occurred. The mean operating time was 102 min. RESULTS: All four procedures were completed without complications. The four patients all were discharged shortly after surgery and reported normal sexual activity without pain. CONCLUSIONS: Transvaginal cholecystectomy can be completed safely using current technology. Further studies are needed to determine the safety of the procedure and to determine whether it confers any benefits other than cosmesis.

Natural orifice surgery: initial clinical experience.

BACKGROUND: Natural orifice translumenal endoscopic surgery (NOTES) has moved quickly from preclinical investigation to clinical implementation. However, several major technical problems limit clinical NOTES including safe access, retraction and dissection of the gallbladder, and clipping of key structures. This study aimed to identify challenges and develop solutions for NOTES during the initial clinical experience. METHODS: Under an Institutional Review Board (IRB)-approved protocol, patients consented to a natural orifice operation for removal of either the gallbladder or the appendix via either the vagina or the stomach using a single umbilical trocar for safety and assistance. RESULTS: Nine transvaginal cholecystectomies, one transgastric appendectomy, and one transvaginal appendectomy have been completed to date. All but one patient were discharged on postoperative day 1 as per protocol. No complications occurred. CONCLUSION: The limited initial evidence from this study demonstrates that NOTES is feasible and safe. The addition of an umbilical trocar is a bridge allowing safe performance of NOTES procedures until better instruments become available. The addition of a flexible long grasper through the vagina and a flexible operating platform through the stomach has enabled the performance of NOTES in a safe and easily reproducible manner. The use of a uterine manipulator has facilitated visualization of the cul de sac in women with a uterus to allow for safe transvaginal access.

Video. NOTES: transvaginal cholecystectomy with assisting articulating instruments.

BACKGROUND: Transvaginal cholecystectomy has been performed at several institutions using hybrid natural orifice translumenal endoscopic surgery (NOTES) techniques. METHODS: A 42-year-old woman with symptomatic cholelithiasis was taken to the operating room for transvaginal cholecystectomy after giving informed consent. A single 5-mm laparoscope was placed at the umbilicus, followed by a 15-mm trocar through the vaginal conduit. The endoscope and a long flexible RealHand surgical instrument (Novare, Cupertino, CA) were placed via the vaginal trocar. The cystic duct and artery were identified and clipped using laparoscopic clips from the umbilical port. The long articulating laparoscopic instrument provided stable retraction. Hook cautery was used to dissect the gallbladder, which was removed via the vaginal trocar. The vaginal incision was closed using a single figure-of-eight absorbable suture under direct vision. The procedure lasted 96 min. RESULTS: The cholecystectomy was successfully performed without spillage of bile. The patient was kept overnight for observation only as a precaution. She reported no pain and did not require a discharge prescription for narcotics. CONCLUSIONS: The described technique for NOTES cholecystectomy results in a virtually scarless operation. The single 5-mm umbilical trocar allows for safe clipping of the cystic duct. Further work is needed to determine the efficacy of this approach.

Ethanol stimulates endothelial cell angiogenic activity via a Notch- and angiopoietin-1-dependent pathway.

AIMS: Our aims were to determine the effect of alcohol (EtOH) on endothelial angiogenic activity and to delineate the cell signalling mechanisms involved. METHODS AND RESULTS: Treatment of human umbilical vein endothelial cells (HUVECs) with EtOH (1-100 mM, 24 h) dose-dependently increased their network formation on Matrigel (an index of angiogenesis) with a maximum response (2.5- to 3-fold increase) at 25 mM. Ethanol also stimulated the proliferation (by cell count and proliferating cell nuclear antigen expression) and migration (by scratch wound assay) of HUVECs. In parallel cultures, EtOH stimulated Notch receptor (1 and 4) and Notch target gene (hrt-1, -2, and -3) mRNA and protein expression and enhanced CBF-1/RBP-Jk promoter activity. EtOH also stimulated, at the mRNA and protein level, the expression of angiopoietin-1 (Ang1) and its Tie2 receptor in these cells. Knockdown of Notch 1 or 4 by siRNA or inhibition of Notch-mediated, CBF-1/RBP-Jk-regulated gene expression by the Epstein-Barr virus-encoded protein RPMS-1 inhibited both ethanol-induced Ang1/Tie2 expression in HUVECs and their network formation on Matrigel. Moreover, knockdown of Ang1 or Tie2 by siRNA inhibited ethanol-induced endothelial network formation. CONCLUSION: These data demonstrate that ethanol, at levels consistent with moderate consumption, enhances endothelial angiogenic activity in vitro by stimulating a novel Notch/CBF-1/RBP-JK-Ang1/Tie2-dependent pathway. These actions of ethanol may be relevant to the cardiovascular effects of alcohol consumption purported by epidemiological studies.

Designated Interpreters: A Model to Promote the Diversity and Inclusion of Deaf Professionals in Academic Medicine.

PROBLEM: Deaf professionals who use American Sign Language (ASL) are a growing population in academic medicine. Reasonable accommodations for this group include providing an ASL interpreter. Many institutions contract with external agencies to provide ad hoc interpreters, but this model has hidden costs for deaf professionals and institutions. APPROACH: The University of Rochester School of Medicine and Dentistry (URSMD) uses the designated interpreter model in which interpreters are on staff and embedded with deaf professionals so they can learn both the work environment and the deaf professionals' specialized science and medicine content. This model addresses many of the limitations of the external agency approach and better facilitates the inclusion of deaf professionals in the institution. OUTCOMES: This model has been in use at URSMD since 1990 but has seen exponential growth recently (increasing from 3 deaf professionals with designated interpreters in 2011 to a peak of 17 in 2016). Designated interpreters have worked in different research and clinical settings from dentistry and nursing to community and global health. This growth highlights the increasing number of deaf professionals in medicine and the need to train more designated interpreters. NEXT STEPS: In response to this growing demand, URSMD is developing an ASL Interpreting in Medicine and Science program, a master's degree-level program to train interpreters who are bilingual in ASL and English to be designated interpreters. The designated interpreter model is one step toward creating an environment that is fully inclusive of deaf professionals to the benefit of the whole institution.

Cyclic strain regulates the Notch/CBF-1 signaling pathway in endothelial cells: role in angiogenic activity.

OBJECTIVE: The purpose of this study was to determine the effect of cyclic strain on Notch signaling in endothelial cells. METHODS AND RESULTS: Exposure of human endothelial cells (ECs) to cyclic strain (10%) resulted in temporal upregulation of Notch receptors (1 and 4) at the mRNA and protein level. Cyclic strain significantly increased EC network formation on Matrigel (an index of angiogenesis); network AU=775+/-127 versus 3928+/-400 for static and strained ECs, respectively. In addition, Angiopoietin 1 (Ang1), Tie1, and Tie2 expression were increased and knockdown of Ang1/Tie1,2 by siRNAs decreased cyclic strain-induced network formation. Knockdown of Notch 1 and 4 by siRNA, or inhibition of Notch mediated CBF-1/RBP-Jk regulated gene expression by RPMS-1, caused a significant decrease in cyclic strain-induced network formation and in Tie1 and Tie2 mRNA expression. Notch 1 or Notch 4 siRNA, but not RPMS-1, inhibited cyclic strain-induced Ang1. Constitutive overexpression of Notch IC resulted in increased network formation, and Ang1 and Tie2 mRNA expression, under both static and strain conditions. CONCLUSIONS: These data suggest that cyclic strain-stimulated EC angiogenesis is mediated in part through a Notch-dependent, Ang1/Tie2 signaling pathway. This pathway may represent a novel therapeutic target for disease states in which hemodynamic force-induced angiogenesis occurs.

Ethanol inhibits pulse pressure-induced vascular smooth muscle cell migration by differentially modulating plasminogen activator inhibitor type 1, matrix metalloproteinase-2 and -9.

We investigated the effect of ethanol on the pulse pressure-induced expression of PAI-1 and MMP-2/9 in human smooth muscle cells (SMC). Human SMC were exposed to static or pulse pressure (25 mL/min; pulse pressure 106/50 mm Hg) conditions for 24 h in the absence or presence of ethanol (0.1-100 mM). SMC migration was then measured by Transwell migration assay. SMC exposed to pulse pressure demonstrated a significant increase in PAI-1 mRNA and protein expression (approximately 4-fold and approximately 3-fold) concomitant with a 3- and 8-fold increase in MMP-2 and MMP-9 protein, respectively. Ethanol dose-dependently inhibited the pulse pressure-induced SMC migration with complete inhibition observed at 20 mM. There was no effect of ethanol on basal PAI-1 or MMP-2/9 in SMC under static conditions. However, ethanol significantly enhanced the pulse pressure-induced PAI-1 mRNA and protein expression (2.2 +/- 0.52 fold and 2.5 +/- 0.27 fold, for 10 mM), respectively. In contrast, ethanol dose-dependently inhibited the pulse pressure-induced increases in MMP-9 protein and pro-MMP-9 activity and to a lesser extent MMP-2 mRNA and protein and pro-MMP-2 activity, with significant inhibition observed at 1 mM. These data provide a molecular mechanism mediating the inhibitory effect of ethanol on pulse-pressure-induced SMC migration and may be relevant to the cardioprotective effects of ethanol in vivo.

Pulsatile flow-induced angiogenesis: role of G(i) subunits.

OBJECTIVE: Angiogenesis plays a key role in the growth and function of normal and pathological tissues. We investigated the effect of pulsatile flow on endothelial cell (EC) in vitro angiogenic activity. METHODS AND RESULTS: Bovine aortic ECs were exposed to "static" or "flow" (1.2 to 67.0 mL/min, shear stress 1.4 to 19.2 dyne/cm2) conditions for 2 to 24 hours. After exposure, angiogenesis was measured as tubule formation on Matrigel, and EC migration was assessed by filter migration assay. Pulsatile flow increased angiogenesis and EC migration in a temporal and force-dependent manner, with a maximal effect at 16 hours (13.2 dyne/cm2). Pertussis toxin completely inhibited the effect of pulsatile flow on angiogenesis and migration. Transfection of ECs with inhibitory mutants of the alpha subunit of G(i)1 or G(i)3, but not G(i)2, inhibited the flow-induced angiogenic response by 61+/-2% and 32+/-6%, respectively, whereas transfection with constitutively activated mutants of the alpha subunit of G(i)1 or G(i)3, but not G(i)2, increased the flow-induced response by 202+/-23% and 70+/-4%, respectively. In contrast, inhibition of Gbetagamma by the carboxy terminal fragment of beta-adrenergic receptor kinase overexpression increased the flow-induced response by 82+/-8%. CONCLUSIONS: These results suggest that pulsatile flow stimulates angiogenesis and that this effect is mediated by activation of G(ialpha)1 or G(ialpha)3, but not Gbetagamma, subunits.

Cyclic strain-mediated regulation of endothelial matrix metalloproteinase-2 expression and activity.

OBJECTIVE: To investigate the role of cyclic strain in controlling matrix metalloproteinase-2 (MMP-2) expression and activity in endothelial cells (ECs) in vitro. METHODS: A Flexercell Tension Plus FX-4000T system was used to apply a physiological level of equibiaxial cyclic strain (0-10% strain, 60 cycles/min, 0-24 h, cardiac waveform) to bovine aortic endothelial cells (BAECs). Cells and conditioned media were harvested for analysis of MMP-2/9 expression and activity (pro and active) using reverse-transcriptase polymerase chain reaction (RT-PCR), Western blotting and zymography techniques. RESULTS: Cyclic strain significantly increased MMP-2 expression and activity force- and time-dependently. Pretreatment with Gialpha-protein inhibitors, pertussis toxin (PTX) and NF023, transient expression of inhibitory mutants of Gialpha-subunits, or pretreatment with RGD peptides to block RGD-dependent integrin signaling failed to attenuate strain-induced increases in MMP-2 expression in BAECs. In contrast, inhibition of Gbetagamma-signaling with betaArk-ct or tyrosine kinase blockade with genistein reduced strain-induced MMP-2 expression while concomitantly inhibiting strain-induced p38 and ERK activity in these cells. Pretreatment with PD169316 and PD98059 to selectively inhibit p38 and ERK activity, respectively, also resulted in a significant inhibition of the strain-induced MMP-2 response. Finally, inhibition of the adaptor protein, Shc, (via Shc-SH2 transfection) resulted in a significant decrease in strain-induced MMP-2 activity concomitant with a reduction in ERK activity in BAECs. CONCLUSION: Cyclic strain stimulates MMP-2 expression, in part, by stimulating both p38- and ERK-dependent pathways through activation of Gbetagamma and tyrosine kinase in BAECs.