Niren Angle

A role for VEGF as a negative regulator of pericyte function and vessel maturation

Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively. PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators. Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rβ signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rβ and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rβ/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively. PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators. Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rbeta signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rbeta and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rbeta/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.

Hypertonic Saline Resuscitation Decreases Susceptibility to Sepsis after Hemorrhagic Shock

BACKGROUND We hypothesized that improvements in cellular immune function after hypertonic saline (HTS) resuscitation will alter the outcome of sepsis after hemorrhage. METHODS To test this hypothesis, a two-hit model was used. Hemorrhage was induced in BALB/c mice by catheterizing the femoral artery and bleeding until a mean arterial pressure = 35 mm Hg was reached and maintained for 1 hour. Resuscitation was performed with HTS (NaCl 7.5%, 4 mL/kg) or lactated Ringers (LR, twice the shed blood volume), plus the shed blood. Cecal ligation and puncture (CLP) was performed 24 hours after hemorrhage. Mortality was assessed for 72 hours, comparing HTS (n = 14) and LR (n = 13) resuscitation. Another set of animals (n = 10 in each group at each time point) were killed at 2 and 24 hours after blood collection. Liver and blood were cultured for the presence of bacteria, and lung and liver samples were scored on a scale from 0 (normal) to 4 (most severe) in a blind fashion by a pathologist. RESULTS Mortality 72 hours after CLP was 14.3% in HTS and 76.9% in LR treated animals (p < 0.002). At 24 hours after CLP, 44% of HTS, but 77% of LR treated animals had > 1,000 colony forming units/mL of blood. Positive liver cultures (> 100,000 colony forming units/g) also showed the same trend (HTS = 30%, LR = 60%). Autopsies revealed a better containment of the infection (abscess formation) in the HTS group. At 2 hours, lung scores were 1.2 +/- 0.25 and 2.6 +/- 0.31 for HTS and LR, respectively (p < 0.002). At 24 hours, HTS treated animals showed marked improvement of lung injury, while the scores in the LR group remained high. A significant difference was also observed regarding liver injury. At 2 hours, scores were 0.4 +/- 0.22 and 2.3 +/- 0.16 for HTS and LR, respectively (p < 0.002). At 24 hours, HTS treated animals showed normal hepatic architecture, although mild injury was still observed in the LR group. CONCLUSION HTS resuscitation leads to increased survival after hemorrhage and CLP. Marked improvements were observed in lung and liver injury compared with isotonic resuscitation. The better containment of the infection observed with HTS resuscitation corresponds to a marked decreased in bacteremia. HTS resuscitation stands as an alternative resuscitation regimen with immunomodulatory potential.

Hypertonic saline resuscitation diminishes lung injury by suppressing neutrophil activation after hemorrhagic shock.

Hypertonic saline (HS) resuscitation after hemorrhage and sepsis has been shown to markedly reduce the development of lung injury in animals, compared with traditional resuscitation with lactated Ringers (LR). These experiments examined the effect of HS on lung injury after hemorrhage without sepsis. The effects of HS and LR resuscitation on neutrophil trafficking, neutrophil adhesion, and neutrophil oxidative burst were studied. Methods: BALB/c mice were hemorrhaged to a mean arterial pressure of 40 torr for 1 h. Animals were resuscitated with shed blood and either 4 mL/kg of 7.5% HS or LR in twice the volume of the shed blood. Lung histology was examined 24 h after hemorrhage. Lung myeloperoxidase content and bronchoalveolar lavage fluid neutrophil counts were obtained. Peripheral blood smears were obtained to determine the neutrophil percentage. Peripheral blood neutrophil CD11b expression and neutrophil H2O2 production were assayed by flow cytometry. Results: HS animals had less lung injury than LR animals. The mean myeloperoxidase activity in HS versus LR animals was 1.79 ± 1.33 U/100 mg versus 3.0 ± 1.33 U/100 mg, respectively. The percentage of neutrophils in the bronchoalveolar lavage fluid of HS animals (3.8% ± .8) was significantly less than that of LR animals (10.8% ± 2.1). This corresponded to a significantly higher peripheral blood neutrophil count in HS animals compared with LR animals, 41% vs. 20%, respectively. There was no difference in neutrophil expression of the CD11b integrin between the HS and LR groups. The neutrophils of LR animals had basal H2O2 production that was 107% greater than that of controls; HS suppressed this hemorrhage-induced activation by > 60%. HS resuscitation after hemorrhagic shock protects against the development of lung injury. This protection is due, in part, to suppression of the hemorrhage-induced neutrophil oxidative burst. HS resuscitation offers immunomodulatory potential after hemorrhagic shock.

Hypertonic saline resuscitation reduces neutrophil margination by suppressing neutrophil L selectin expression.

UNLABELLED Hypertonic saline (HS) reduces hemorrhage-induced lung injury by suppressing the neutrophil oxidative burst and reducing lung neutrophil influx. This study investigated whether this is caused by the effects of HS on endothelial adhesion molecule expression, the production of chemoattractants in the lung, or a direct effect of HS on neutrophil selectin expression. METHODS BALB/c mice were made to hemorrhage to 40 mm Hg for 1 hour and resuscitated with shed blood and either 4 mL/kg 7.5% HS or two times the shed blood volume of lactated Ringers solution (LRS). Neutrophil L selectin expression was determined by flow cytometry, total neutrophil counts were obtained by differential staining, and pulmonary endothelial P and E selectin expression was evaluated by immunohistochemistry. Chemoattractants in lung lavages were determined with a modified Boyden chamber migration assay. RESULTS Chemotactic activity of lavage fluid of HS-treated animals was not significantly different from that of LRS-treated animals, and endothelial P and E selectin expression was not altered by HS resuscitation. Neutrophils of HS-treated animals, however, expressed significantly less L selectin than those of LRS-treated mice. Concomitantly, circulating neutrophil counts of LRS-treated animals were significantly decreased compared with those of HS-treated mice. CONCLUSION HS had little effect on endothelial selectin expression and chemoattractant production in the lung. HS significantly decreased neutrophil L selectin expression, however. This suggests that HS resuscitation may reduce lung injury by preventing neutrophil L selectin expression and endothelial adhesion.

hypertonic Saline Infusion : can It Regulate Human Neutrophil Function?

The down-regulation of neutrophil adhesion molecule expression after hemorrhagic shock may reduce neutrophil-mediated organ injury. Hypertonic saline (HS) blocks neutrophil activation, and HS infusion in animals reduces organ injury. In this study, we investigated whether HS infusion in healthy human volunteers can affect neutrophil function. Healthy human volunteers were administered either 4 mL/kg of a 7.5% HS (n = 6) or normal saline (NS, 0.9%; n = 5) over 15 min. Mean arterial pressure (MAP) and plasma sodium levels were measured. Blood samples were obtained before and 1 h after fluid administration. Cells were stimulated with fMLP or left untreated. Neutrophil phagocytosis and expression of CD11b and L-selectin was determined with flow cytometry. HS infusion caused a 7 +/- 2 mM rise in plasma Na+ levels that was sustained at 6 +/- 1 mM for 60 min. MAP was affected only in one subject. HS and NS infusion had little effect on neutrophil phagocytosis. After HS infusion, CD1lb expression of unstimulated neutrophils was 26 +/- 6% lower than before HS infusion, and that of fMLP-stimulated cells was 12 +/- 2% lower compared to pre-infusion values. NS infusion had no significant effects on neutrophil CD11b expression. L-selectin expression of unstimulated cells after HS infusion was 9 +/- 3% higher than in the pre-infusion samples. These data suggest that HS infusion could indeed affect human neutrophils by suppressing CD11b expression. Although modest in healthy subjects, this effect may be more pronounced in trauma patients where reduced neutrophil-endothelial cell interactions might lessen neutrophil-mediated tissue damage.

Chronic venous ulcer

Venous ulceration is the most severe and debilitating outcome of chronic venous insufficiency in the leg. It is a common problem in clinical practice, with an estimated prevalence of 1-1.3%.1 2 Such ulceration is part of the complex of chronic venous insufficiency and is associated with distal vein hypertension. Chronic venous insufficiency and its accompanying venous hypertension have been termed the post-thrombotic syndrome, but it is now known that primary valvar incompetence, not just prior thrombosis, is an important cause of venous leg ulcer.3 Dysfunction of the superficial or perforating veins of the legs, alone or in combination, may be the only finding in leg ulceration.4 Nevertheless, a risk factor for venous ulceration remains a previous episode of deep vein thrombosis, which may or may not destroy venous valve function.5 It is clear, however, that a venous ulcer in the leg occurs mainly from reflux and not usually from persistence of the original obstructive process.6 7 We relied largely on our own files to prepare this review. We accumulated these by weekly searches of Current Contents , which provides tables of contents of almost all medical publications published worldwide. Our files are arranged into categories, and the category of venous ulcer crosses many of these designations. Searches for evidence based information about venous disorders are often disappointing, but we used at least level 2 information whenever possible, tempered by our own experience. Diagnosis and treatment of venous disorders commands greater interest, and level 1 information is starting to appear, including treatment of venous thromboembolic disorders. This heralds better care of patients with venous problems. (Level 1 information means findings from large prospective randomised studies with definite conclusions that should be considered when managing any patient with the specified problem; level 2 information means findings from …

Vascular endothelial growth factor inhibits mitogen-induced vascular smooth muscle cell proliferation.

INTRODUCTION Delivery of vascular endothelial growth factor (VEGF) protein or gene transfer has been shown to accelerate re-endothelialization and attenuate neointimal hyperplasia in various arterial injury models, including balloon injury, stent implantation, and vein grafts. In addition to stimulating re-endothelialization, we hypothesize that VEGF has further vascular protective functions to prevent neointimal hyperplasia by directly inhibiting mitogen-induced proliferation of vascular smooth muscle cells (VSMCs) via the mitogen-activated protein kinase pathway. MATERIALS AND METHODS Human aortic VSMCs were seeded and serum starved for 24 h. The cells were then stimulated with a mitogen, recombinant human platelet derived growth factor at 20 ng/mL together with 0, 10, 20, 30, 40, 50 ng/mL recombinant human VEGF. A proliferation assay was used to quantitate bromodeoxyuridine uptake into newly synthesized DNA. Western immunoassay was used to quantify extracellular signal-regulated kinase (ERK) 2 protein and phosphorylation of retinoblastoma and ERK 1/2 protein. RESULTS VEGF inhibited bromodeoxyuridine incorporation into mitogen-induced VSMC in a dose-dependent manner, reaching statistical significance at concentrations of 30 (P < 0.05), 40 (P < 0.05), and 50 ng/mL (P < 0.01). Densitometry of western immunoblots revealed an inhibition of phosphorylation of retinoblastoma at VEGF concentrations of 40 and 50 ng/mL and ERK 1/2 phosphorylation at concentrations of 30, 40 and 50 ng/mL. CONCLUSION In addition to stimulating re-endothelialization, VEGF appears to have a vascular protective function by directly inhibiting VSMC proliferation. This effect occurs in the absence of endothelial cells and via the mitogen-activated protein kinase pathway. VEGF may serve as an important modulator of mitogen-induced VSMC proliferation after vascular injury.

Lessons learned from the institution of the Surgical Care Improvement Project at a teaching medical center

BACKGROUND The Surgical Care Improvement Project (SCIP) was designed to reduce perioperative complications. We describe our institutional experience in 6 major areas: surgical site infection, venous thromboembolism prevention, use of perioperative beta-blockade, serum glucose level greater than 200 mg/dL, normothermia, and the use of electric razors for hair removal. METHODS This was a retrospective review of surgical cases. Evidence-based training and standardization of system and process were undertaken. Compliance with SCIP guidelines was determined. RESULTS Overall SCIP compliance improved from 80% to 94% over a 2-year period. Standardized antibiotic dosing times improved compliance to more than 90%. Appropriate preoperative antibiotic choice improved to 100%. Cessation of antibiotics postoperatively within 24 hours remains a difficult task. Venous thromboembolism prophylaxis has been difficult to achieve because of postoperative bleeding concerns. Administration of beta-blockers has remained one of the most difficult problems to correct because of the multiplicity of avenues by which a patient may arrive to the operating suite. CONCLUSIONS Achievement of the SCIP goals is a formidable, but achievable, process requiring individual, cultural, systems, and institutional changes to achieve success.

Direct imaging and quantification of carotid plaque calcification

Carotid plaque calcification normally appears as a signal void with clinical MR sequences. Here, we describe the use of an adiabatic inversion recovery prepared two‐dimensional ultrashort echo time sequence to image and characterize carotid plaque calcification using a clinical 3‐T scanner. T1, T  2* , and free water content were measured for seven carotid samples, and the results were compared with micro‐CT imaging. Conventional gradient echo and fast spin echo images were also acquired for comparison. Correlations between T1, T  2* , free water concentration, and mineral density were performed. There was a close correspondence between inversion recovery prepared two‐dimensional ultrashort echo time morphologic and micro‐CT appearances. Carotid plaque calcification varied significantly from sample to sample, with T1s ranging from 94 ± 19 to 328 ± 21 msec, T  2* s ranging from 0.31 ± 0.12 to 2.15 ± 0.25 msec, and free water concentration ranging from 5.7 ± 2.3% to 16.8 ± 3.4%. There was a significant positive correlation between T1 (R = 0.709; P < 0.074), T  2* (R = 0.816; P < 0.025), and free water concentration, a negative correlation between T1 (R = 0.773; P < 0.042), T  2* (R = 0.948; P < 0.001) and CT measured mineral density, and a negative correlation between free water concentration (R = 0.936; P < 0.002) and mineral density. Magn Reson Med, 2010.

Surgical Bypass of Symptomatic Central Venous Obstruction for Arteriovenous Fistula Salvage in Hemodialysis Patients

Venous hypertension due to proximal central venous outflow obstruction coexisting with a functioning arteriovenous fistula in the ipsilateral arm presents with a complex management problem in hemodialysis patients. Ligation of the arteriovenous communication is the simplest procedure to relieve symptoms; however, this sacrifices the patients hemodialysis access, which may be the only available access in that patient. Surgical bypass of the occlusion is a potential option as it obviates the symptoms of venous hypertension while preserving dialysis access. Our objective was to evaluate our experience and outcome with dialysis patients undergoing surgical bypass for symptomatic central venous obstruction and dialysis access salvage. There were three hemodialysis patients with severe venous hypertension secondary to subclavian vein obstruction who had functioning ipsilateral arteriovenous fistulae. All underwent cephalic vein (n = 2) or axillary vein (n = 1) to internal jugular vein bypass of the obstructed subclavian segment via an 8-mm polytetrafluoroethylene bridge graft. All patients had unsuccessful percutaneous transluminal angioplasty (PTA) attempts prior to surgical bypass. In two patients, a wire could not be passed through the occlusion; in the third, PTA was only transiently successful despite four repeated procedures. All patients had complete resolution of symptoms without operative mortality. The bypass grafts remained patent, allowing the arteriovenous fistulae to provide functional access for the entire duration of follow-up after surgery (3-8 months). Surgical bypass of a central vein obstruction relieves the symptoms of venous hypertension and prolongs the use of the existing hemodialysis access. This surgical option should be well recognized within the dialysis community.