Peter Shamamian

Activation of progelatinase A (MMP-2) by neutrophil elastase, cathepsin G, and proteinase-3: a role for inflammatory cells in tumor invasion and angiogenesis.

Gelatinase A (MMP‐2), a matrix metalloproteinase (MMP) involved in tumor invasion and angiogenesis, is secreted as an inactive zymogen (proMMP‐2) and activated by proteolytic cleavage. Here we report that polymorphonuclear neutrophil (PMN)‐derived elastase, cathepsin G, and proteinase‐3 activate proMMP‐2 through a mechanism that requires membrane‐type 1 matrix metalloproteinase (MT1‐MMP) expression. Immunoprecipitation of human PMN‐conditioned medium with a mixture of antibodies to elastase, cathepsin G, and proteinase‐3 abolished proMMP‐2 activation, whereas individual antibodies were ineffective. Incubation of HT1080 cells with either purified PMN elastase or cathepsin G or proteinase‐3 resulted in dose‐and time‐dependent proMMP‐2 activation. Addition of PMN‐conditioned medium to MT1‐MMP expressing cells resulted in increased proMMP‐2 activation and in vitro invasion of extracellular matrix (ECM), but had no effect with cells that express no MT1‐MMP. MMP‐2 activation by PMN‐conditioned medium or purified elastase was blocked by the elastase inhibitor α1‐antitrypsin but not by Batimastat, an MMP inhibitor, showing that elastase activation of MMP‐2 is not mediated by MMP activities. The PMN‐conditioned medium‐induced increase in cell invasion was blocked by Batimastat as well as by α1‐antitrypsin, showing that PMN serine proteinases trigger a proteinase cascade that entails proMMP‐2 activation: this gelatinase is the downstream effector of the proinvasive activity of PMN proteinases. These findings indicate a novel role for PMN‐mediated inflammation in a variety of tissue remodeling processes including tumor invasion and angiogenesis.

Expression of von Willebrand factor, an endothelial cell marker, is up- regulated by angiogenesis factors: A potential method for objective assessment of tumor angiogenesis

von Willebrand factor (vWF), a glycoprotein produced uniquely by endothelial cells and megakaryocytes, is routinely used to identify vessels in tissue sections. Vessel density in tumor specimens, as determined by immuno‐histochemical staining for vWF or other endothelial cell markers, is a negative prognostic factor for many solid tumors. vWF is heterogeneously distributed throughout the vasculature, transcriptional control in response to the tissue microenvironment being responsible for local variations in endothelial cell levels of vWF. Here, we report that fibroblast growth factor‐2 and vascular endothelial growth factor, potent angiogenesis inducers expressed in a variety of tumors, up‐regulate expression of vWF mRNA and protein in cultured endothelial cells with a synergistic effect. Our data support the measurement of vWF mRNA in tumors to detect activated endothelium or angiogenesis. For this purpose, we developed a semi‐quantitative RT‐PCR for vWF mRNA. Preliminary results obtained with specimens from colon carcinoma and the corresponding normal colonic mucosa showed higher vWF mRNA levels in most tumors than in their normal counterparts. The differences in vWF mRNA levels were much larger than the differences in vessel counts between a tumor and the corresponding normal mucosa, indicating that high vWF mRNA levels in tumors may indeed be an early sign of activation of the endothelium. The rapidity, objectivity, sensitivity and specificity of this technique make it suitable for routine clinical application to identify aggressive, highly angiogenic tumors. Int. J. Cancer 85:281–288, 2000. ©2000 Wiley‐Liss, Inc.

Adenosine A2A receptor activation promotes wound neovascularization by stimulating angiogenesis and vasculogenesis

Recent reports indicate that circulating endothelial progenitor cells (EPCs) may be recruited to sites of neovascularization where they differentiate into endothelial cells (EC). As we have previously demonstrated that adenosine A(2A) agonists promote neovascularization in wounds, we sought to determine whether adenosine A(2A) receptor agonist-augmented wound healing involves vessel sprouting (angiogenesis) or EPC recruitment (vasculogenesis) or both. Four weeks after bone marrow reconstitution from donor FVB/N Tie2GFP transgenic mice, two full-thickness excisional wounds were performed on the dorsum of FVB/N wild-type mice and treated with either an A(2A) receptor agonist (CGS-21680) or vehicle alone. Vessel density, as measured by CD31 staining, and density of EPC-derived vessels, as measured by GFP expression, were quantified in a blinded fashion using two-color fluorescence microscopy. We observed nearly a threefold increase in CD31-positive vessels and a more than 10-fold increase in GFP-positive cells in A(2A) agonist-treated 3-day old wounds, but by 6 days after wounding the differences between A(2A) agonist-treated and vehicle-treated wounds were no longer statistically significant. In conclusion, this is the first evidence that an exogenous agent such as an adenosine A(2A) receptor agonist increases neovascularization in the early stages of wound repair by increasing both EPC recruitment (vasculogenesis) and local vessel sprouting (angiogenesis).

Diagnostic standards for acute pancreatitis

An accurate history and thorough physical examination will often raise clinical suspicion of acute pancreatitis in the differential diagnosis of a patient presenting with acute abdominal pain. An accurate diagnosis is needed to eliminate etiologies of acute abdominal pain and to appropriately direct therapy. Confirmation of the diagnosis is most often made by evaluation of serum amylase and lipase levels. Although hyperamylasemia is found in the majority of patients with acute pancreatitis, other nonpancreatic acute abdominal conditions may be present with hyperamylasemia. CT scanning provides an accurate confirmation of clinical and laboratory findings and offers excellent anatomic and morphologic representation of the pancreas and peripancreatic tissue. The following article, written by the late John H.C. Ranson, presents a discussion of the modalities available for diagnosing acute pancreatitis.RésuméLa pancréatitc aiguë est une maladie relativement fréquente dont l’incidence est en hausse. Son diagnostic est difficile. Dans une étude présentée par Bockus à partir de 94 admissions pour pancréatite aiguë, le diagnostic était inexact chez 43 % des cas. La pancréatite a été prise le plus souvent pour une cholécystitc aiguë (20%), une perforation de viscère creux (7%) ou une occlusion intestinale (5%). Chez le patient ayant une pancréatite aiguë fatale, le diagnostic correct n’a pas été fait avant l’autopsie dans 41.6% des cas. Il n’existe aucun critère clinique ou biologique qui permette le diagnostic certain de pancréatite aiguë à tous les coups. Les données opératoires ou autopsiques ne sont disponibles que chez un faible pourcentage des patients. Les données de la tomodensitométrie sont parfois diagnostiques, mais cet examen peut être normal chez le patient ayant une forme mineure de la maladie. Les critères diagnostiques de la pancréatite aiguë sont passés en revue. Il faut souligner que le diagnostic initial dépend principalement d’un interrogatoire précis ainsi que d’un examen physique soigneux.ResumenLa pancreatitis aguda es una entidad común, con frecuencia de dificil diagnóstico, cuya incidencia es creciente. En un estudio realizado por Bockus en 94 pacientes hospitalizados por pancreatitis aguda, el diagnósti co inicial resultó incorrecto en 43% de los casos. Comúnmente se confundio la pancreatitis aguda con colecistitis aguda (20%), víscera perforada (7%) u obstructión intestinal (5%). En los casos de pancreatitis aguda letal, el diagnóstico correcto solo es establecido en el momento de la autopsia, lo cual ocurre en 41.6% de los casos. Desafortunadamente no existen criterios clínicos o de laboratorio que permitan un diagnóstico certero de pancreatitis aguda en la totalidad de los pacientes. Los hallazgos operatorios o de necropsia están disponibles sólo en una pequena minoría de los casos. Los hallazgos radiológicos en tomografia computadorizada pueden ser diagnósticos de la entidad, pero el estudio puede aparecer normal en los pacientes con enfermedad leve. Es por ello que en el presente artìculo se revisan las características diagnósticas. Debe hacerse énfasis en que el diagnóstico inicial depende, primordialmente, de una bien orientada historia clìnica y de un meticuloso examen fisico.

Temporal expression and activation of matrix metalloproteinases-2, -9, and membrane type 1-matrix metalloproteinase following acute hindlimb ischemia.

OBJECTIVE Matrix metalloproteinase (MMP) activity is essential for remodeling of ischemic tissue. The murine hindlimb ischemia model exhibits tissue remodeling including revascularization in part due to angiogenesis. MMP-2 and -9 are type IV collagenases necessary for basement membrane degradation as a part of extracellular matrix remodeling and angiogenesis. Polymorphonuclear leukocytes (PMNs) contain MMP-9, and in the presence of membrane type 1 (MT1)-MMP, are able to activate proMMP-2 in vitro. Activation of MMP-2 and -9 may be essential in ischemic limbs both for tissue remodeling and revascularization via angiogenesis. We hypothesized that MMP-2 and -9 would be activated following acute hindlimb ischemia (HI), and this activation would be temporally related to PMN infiltration. DESIGN OF STUDY HI was achieved by unilateral femoral artery ligation in 20 FVB/N mice. Five mice underwent sham operation without hindlimb ischemia. Gastrocnemius muscle was harvested from both hindlimbs at 1, 3, 14, and 30 days following ligation and assayed for MMP-2, -9 (gelatin zymography), and MT1-MMP (Western blotting). MMP-2 and -9 expression and activation were analyzed by gelatin zymography and quantified by densitometry with NIH Image Analysis software. Neutrophils per high power field were counted. The results were expressed as a ratio of ischemic to nonischemic limbs and compared at each time point using ANOVA. RESULTS Zymographic analysis revealed a 212% increase in active MMP-2 3 days postligation (P <.05). Active MMP-9 reached its maximum level (800% over baseline) on postoperative day 3 and continued to be elevated on day 14 (737% over baseline) (P <.05). The increase in active MMP-2 and -9 levels paralleled PMN infiltration that also peaked 3 days postligation (1184% over baseline) (P <.05). PMN count, MMP-2, and -9 all returned to baseline levels by postoperative Day 30. MT1-MMP was present in tissue samples from all time points as confirmed by Western blot. CONCLUSIONS Limb ischemia causes an early activation of MMP-2 and -9 in temporal relation to PMN infiltration. HI may prime PMNs, leading to their sequestration in ischemic tissue. Primed PMNs, along with constitutively expressed MT1-MMP, may activate MMPs-2 and -9 and enable tissue remodeling essential for limb revascularization and angiogenesis.

Polymorphonuclear Neutrophils Promote rFGF-2-Induced Angiogenesis in Vivo

BACKGROUND The role of neutrophils in angiogenesis remains largely unknown. Recent evidence has shown that polymorphonuclear neutrophils (PMNs) produce several proangiogenic cytokines, including VEGF, TNF-alpha, IL-1, IL-6, and IL-8. In addition, PMN-derived proteinases promote endothelial cell migration. We hypothesized that PMNs may facilitate angiogenesis and that reducing circulating PMNs might alter the host angiogenic response. MATERIALS AND METHODS We utilized a corneal pocket assay to compare rFGF-2-induced vessel formation in the corneas of mice with normal levels of circulating neutrophils to those in a neutropenic state. Circulating PMNs were reduced using serial intraperitoneal injections of monoclonal antibody to Gr-1. Slow release rFGF2 pellets were implanted into the corneas of neutropenic mice and controls. Corneal neovascularization, measured as vessel length and area of vessel in-growth, was quantified using slit-lamp microscopy on day 7. RESULTS The average number of circulating PMNs was significantly reduced in the experimental group compared to the control group on days 1-7 (P < 0.05). No statistical differences in circulating monocytes or lymphocytes were observed from days 0 to 6. Mice in the experimental group had a vascular area of 2.58 +/- 0.2 mm(2) compared to 3.55 +/- 0.3 mm(2) in the control group (P < 0.05). CONCLUSIONS Corneal neovascularization in response to rFGF-2 is diminished by PMN depletion. PMNs play an important role in facilitating rFGF-2-induced angiogenesis.

Endoscopic biliary drainage before pancreaticoduodenectomy for periampullary malignancies

Despite decreased operative mortality, pancreaticoduodenectomy (PD) remains a formidable operation with substantial morbidity. We have evaluated the influence of preoperative endoscopic biliary drainage (EBD) on morbidity after PD for malignant biliary obstruction by retrospectively reviewing the medical records of 182 patients undergoing PD between April 1985 and August 1996. Of 52 study patients with malignant obstructive jaundice, 22 underwent preoperative EBD, and 30 were not drained. Eighty-three patients were excluded for bilirubin levels less than 5 mg/dl, 43 had other biliary drainage, and 4 had jaundice with benign pathology. Preoperative, intraoperative, and postoperative factors were compared. The two groups were well matched for clinical presentation and operative characteristics except for lower preoperative values of liver chemistries in patients undergoing EBD. Length of postoperative hospitalization for patients undergoing EBD was 13.5 days, compared with 19 days for patients who were not drained (p = 0.02). Patients who were not drained tended to have more overall complications (p = 0.054). Multivariate analysis revealed time to regular diet (p < 0.0001) and no preoperative drainage (p = 0.04) to be independent factors significantly increasing the length of hospitalization. Endoscopic biliary drainage before PD significantly reduced the length of postoperative hospitalization and was associated with less postoperative morbidity. Further studies, including cost analysis, are warranted.

Topical Hepatic Hypothermia Attenuates Pulmonary Injury after Hepatic Ischemia and Reperfusion

BACKGROUND Prolonged periods of hepatic ischemia are associated with hepatocellular injury and distant organ dysfunction in experimental models. Neutrophils (PMN) and tumor necrosis factor (TNF)-alpha have been implicated, mostly because of their local deleterious effects on the hepatocyte after hepatic ischemia and reperfusion (I/R) injury. We hypothesize that topical hepatic hypothermia (THH) reduces ischemia and reperfusion-induced hepatic necrosis, PMN infiltration, TNF-alpha release, and consequent acute pulmonary injury. STUDY DESIGN Sprague-Dawley rats (250 to 300g) were evenly divided into three groups: 90 minutes of normothermic (37 degrees C) partial hepatic ischemia (normothermic I/R), 90 minutes of hypothermic (25 degrees C) partial hepatic ischemia (hypothermic I/R), and sham laparotomy (without ischemia). There were six animals in each experimental group per time point unless otherwise specified. Hepatic necrosis and PMN infiltration were evaluated and scored on hematoxylin and eosin-stained liver specimens 12 hours after reperfusion. Serum TNF-alpha levels were determined by ELISA at 0 minutes, 15 minutes, 30 minutes, 1 hour, and 12 hours postreperfusion. Pulmonary PMN infiltration and vascular permeability were measured by myeloperoxidase activity and Evans blue dye extravasation, respectively, to quantitate pulmonary injury 12 hours after reperfusion. RESULTS Normothermic I/R results in a significant increase in TNF-alpha at 15 and 30 minutes (p < 0.005), PMN infiltration (p < 0.001), and hepatic necrosis (p < 0.001), compared with sham. Institution of THH reduced peak serum TNF-alpha levels by 54% at 15 minutes (p < 0.005) and by 73% at 30 minutes (p < 0.001) postreperfusion compared with normothermic I/R. Similarly, hepatic PMN infiltration and necrosis at 12 hours were reduced by 60% (p < 0.05) and 47% (p < 0.05), respectively. Myeloperoxidase activity and Evans blue extravasation (measures of acute lung injury) were reduced by 42% and 39%, respectively, with institution of THH compared with animals undergoing normothermic I/R (p < 0.001). CONCLUSIONS These results demonstrate that THH protects the liver from ischemia and reperfusion-induced necrosis and PMN infiltration. In addition, THH reduces the serum levels of TNF-alpha and associated pulmonary injury. These data suggest that the ischemic liver is a potential source of inflammatory mediators associated with hepatic ischemia and reperfusion-induced pulmonary injury.

Inhibition of matrix metalloproteinases reduces local and distant organ injury following experimental acute pancreatitis.

BACKGROUND Pulmonary complications from pancreatitis involve parenchymal destruction via proteolytic enzymes. Matrix metalloproteinases (MMPs) may play an important role in pulmonary injury following acute severe pancreatitis. We hypothesized that local and distant organ injury would be decreased by the presence of an MMP inhibitor (Batimistat; BB-94) following severe acute pancreatitis (AP). METHODS Eighteen male rats were randomized into two groups: BB-94 (AP + 40 mg/kg/24 h BB-94 ip x three doses) or control (AP + 20 ml/kg/24 h normal saline ip x three doses). Necrotizing AP was induced by retrograde infusion of 5% sodium taurocholate (1.5 ml/kg) into the pancreatic duct. Twenty additional animals were randomized into BB-94 and control groups for the survival study. Serum was evaluated for amylase and MMP activity. Pancreatic sections were graded for edema, necrosis, neutrophil infiltrate, and hemorrhage. Myloperoxidase (MPO) activity was used to determine PMN infiltration in the lung. Evans Blue dye extravasation was used to quantify vascular permeability. RESULTS Animals in the BB-94 group had decreased amylase levels (1086.0 +/- 61.7 U/L vs 2232.7 +/- 309.9 U/L; P < 0.05), decreased cellular infiltrate (1.4 +/- 0.2 vs 2.3 +/- 0.2; P < 0.02), and decreased necrosis (4.1 +/- 0.3 vs 6.1 +/- 0.4; P < 0.005) compared to the control group. Lung tissue following pancreatitis in the BB-94 group demonstrated decreased MPO activity (41.5 +/- 2.4 units vs 57.3 +/- 2.9 units; P < 0.05) and decreased vascular permeability (18.3 +/- 2.8 mg/100 g vs 30.1 +/- 4.6 mg/100 g; P < 0.05). Animals treated with BB-94 had 100% survival compared to 50% survival in control at 72 h. CONCLUSIONS Pancreatitis results in increased local and distant MMP activity. Pulmonary and pancreatic injury following AP can be abrogated by treatment with an MMP inhibitor (Batimistat; BB-94) which may result in decreased morbidity and mortality.

Increased membrane type 1 matrix metalloproteinase expression from adenoma to colon cancer: a possible mechanism of neoplastic progression.

AbstractPURPOSE: Membrane type 1 matrix metalloproteinase is a membrane-associated matrix metalloproteinase central to the degradation of basement membrane components via the activation of matrix metalloproteinase-2. Although membrane type 1 matrix metalloproteinase is overexpressed in invasive colon cancer, its expression in colonic polyps and carcinoma in situ has not been defined. In addition, the association of membrane type 1 matrix metalloproteinase expression by a primary tumor and recurrence of colon cancers has not been examined. METHODS: Immunoperoxidase staining was performed on randomly selected specimens containing adenoma (n = 17), carcinoma in situ (n = 9), or metastatic colon carcinoma (n = 8) with mouse monoclonal antibody to human membrane type 1 matrix metalloproteinase. Similar staining was also performed on randomly selected node-negative colon cancers that recurred within five years of resection (n = 17), matched for age, gender, stage, grade, and vascular, lymphatic, and perineural invasion, and node-negative colon cancers that did not recur within five years of resection (n = 17). Staining for membrane type 1 matrix metalloproteinase was graded. Mean scores for the groups were compared by Wilcoxon test. RESULTS: We found a progressive and significant increase in the mean score of membrane type 1 matrix metalloproteinase from normal mucosa to adenoma (P < 0.001), carcinoma in situ (P < 0.006), and invasive cancer (P < 0.009). However, there was no difference in membrane type 1 matrix metalloproteinase expression between the recurrent and nonrecurrent groups of node-negative colon cancer (P = not significant). CONCLUSIONS: These data suggest that membrane type 1 matrix metalloproteinase expression increases with progression from normal mucosa to invasive adenocarcinoma; however, it cannot be used as a prognostic indicator on which adjuvant therapy is based in node-negative colon cancer because of its failure to predict recurrence in this patient group.