An endogenous inhibitor of angiogenesis inversely correlates with side population phenotype and function in human lung cancer cells.

The side population (SP) in human lung cancer cell lines and tumors is enriched with cancer stem cells. An endogenous inhibitor of angiogenesis known as tissue inhibitor of matrix metalloproteinase-2 (TIMP-2), characterized for its ability to inhibit matrix metalloproteinases (MMPs), has been shown by several laboratories to impede tumor progression through MMP-dependent or -independent mechanisms. We recently reported that forced expression of TIMP-2, as well as the modified form Ala+TIMP-2 (that lacks MMP inhibitory activity) significantly blocks growth of A549 human lung cancer cells in vivo. However, the mechanisms underlying TIMP-2 antitumor effects are not fully characterized. Here, we examine the hypothesis that the TIMP-2 antitumor activity may involve regulation of the SP in human lung cancer cells. Indeed, using Hoechst dye efflux assay and flow cytometry, as well as quantitative reverse transcriptase-PCR analysis, we found that endogenous TIMP-2 mRNA levels showed a significant inverse correlation with SP fraction size in six non-small cell lung cancer cell lines. In A549 cells expressing increased levels of TIMP-2, a significant decrease in SP was observed, and this decrease was associated with lowered gene expression of ABCG2, ABCB1 and AKR1C1. Functional analysis of A549 cells showed that TIMP-2 overexpression increased chemosensitivity to cytotoxic drugs. The SP isolated from TIMP-2-overexpressing A549 cells also demonstrated impaired migratory capacity compared with the SP from empty vector control. More importantly, our data provide strong evidence that these TIMP-2 functions occur independent of MMP inhibition, as A549 cells overexpressing Ala+TIMP-2 exhibited identical behavior to those overexpressing TIMP-2 alone. Our findings provide the first indication that TIMP-2 modulates SP phenotype and function, and suggests that TIMP-2 may act as an endogenous suppressor of the SP in human lung cancer cells.

TIMP-2 upregulates RECK expression via dephosphorylation of paxillin tyrosine residues 31 and 118.

We previously demonstrated that TIMP-2 increases the association of Crk with C3G and via subsequent activation of Rap1 enhances the expression of RECK, a membrane-anchored MMP inhibitor. In the present study, we investigate the mechanism of how the TIMP-2 signal is transduced from the alpha3beta1 integrin receptor to the Crk-C3G-Rap1 molecular complex. TIMP-2 treatment of human microvascular endothelial cells (hMVECs) increased the phosphorylation levels of Src at Tyr-527, the negative regulatory site, through enhanced association of Src with Csk. This results in the reduction of Src kinase activity and dephosphorylation of paxillin at Tyr-31/118, the target sites for Src kinase phosphorylation and also the binding sites for the downstream effector Crk. Such TIMP-2 effects accompany the disassembly of paxillin-Crk-DOCK180 molecular complex and, in turn, Rac1 inactivation. On the contrary, levels of paxillin-Crk-C3G complex formation are not reduced, rather slightly increased, which is consistent with our previous finding. Therefore, TIMP-2-mediated inhibition of Src kinase activity leads to the signaling switch from Rac1 to Rap1, thereby leading to enhanced RECK expression.

Quantification of tissue inhibitor of metalloproteinases 2 in plasma from healthy donors and cancer patients.

Tissue inhibitor of metalloproteinases (TIMP)-2 is a highly conserved molecule, which binds both active and latent matrix metalloproteinase (MMP)-2. TIMP-2 is also involved in the activation of MMP-2 on the cell surface. A quantitative enzyme-linked immunosorbent assay (ELISA) was established and optimized for measurement of TIMP-2 in plasma. The capturing antibody in the ELISA was a monoclonal, while the detecting antibody was a chicken polyclonal antibody recognizing the native form of human TIMP-2. The levels of TIMP-2 were measured in ethylenediaminetetraacetic acid (EDTA) and citrate plasma from healthy donors. The median values were determined as 163 ng/ml (n = 186) with a range of 109-253 ng/ml for EDTA plasma and 139 ng/ml (n = 77) with a range of 95-223 ng/ml for citrate plasma. The TIMP-2 concentration in citrate plasma from 15 patients with advanced, stage IV breast cancer had a median value of 160 ng/ml, only slightly higher but statistically distinguishable from the level found in citrate plasma from the healthy donors. In addition, the TIMP-2 concentration in EDTA plasma from colorectal cancer patients revealed a significantly higher level in plasma from patients with Dukes stage A (P = 0.01) compared with patients with more advanced Dukes stages.

Ventricular mapping during atrial and ventricular pacing. Relationship of multipotential electrograms to ventricular tachycardia reentry circuits after myocardial infarction.

AIMS: Conduction through separated myocyte bundles causes multipotential electrograms and reentrant ventricular tachycardia. We hypothesized that without initiating tachycardia, the reentry region could be detected by analysing the change in multipotential electrograms during two different activation sequences. METHODS AND RESULTS: During catheter mapping and ablation in 16 patients with ventricular tachycardia late after infarction ventricular electrograms were recorded from 1072 sites during atrial and right ventricular paced ventricular activation. Multipotential electrograms were present during both activation sequences at 285 (27%) sites, during atrial pacing only at 159 (15%) sites and during right ventricular pacing only at 152 (14%) sites. Sites with multipotential electrograms during both activation sequences were more often related to a ventricular tachycardia circuit isthmus (43%) as compared to sites where such electrograms were present during one activation sequence (20%). Multipotential electrograms with >2 low amplitude deflections and a >100 ms difference in duration between the two activation sequences were infrequent but highly predictive of the reentry circuit. CONCLUSION: Regions with fixed multipotentials consistent with conduction block might be useful guides for ablation approaches that target large regions of the infarct, but are not sufficiently specific to be the sole guide for focal ablation approaches.

Atrial fibrillation after cardiac surgery.

PURPOSE: To review the epidemiology, mechanisms, complications, predictors, prevention, and treatment of atrial fibrillation following cardiac surgery. DATA SOURCES: MEDLINE search of English-language reports published between 1966 and 2000 and a search of references of relevant papers. STUDY SELECTION: Clinical and basic research studies on atrial fibrillation after cardiac surgery. DATA EXTRACTION: Relevant clinical information was extracted from selected articles. DATA SYNTHESIS: Atrial fibrillation occurs in 10% to 65% of patients after cardiac surgery, usually on the second or third postoperative day. Postoperative atrial fibrillation is associated with increased morbidity and mortality and longer, more expensive hospital stays. Prophylactic use of beta-adrenergic blockers reduces the incidence of postoperative atrial fibrillation and should be administered before and after cardiac surgery to all patients without contraindication. Prophylactic amiodarone and atrial overdrive pacing should be considered in patients at high risk for postoperative atrial fibrillation (for example, patients with previous atrial fibrillation or mitral valve surgery). For patients who develop atrial fibrillation after cardiac surgery, a strategy of rhythm management or rate management should be selected. For patients who are hemodynamically unstable or highly symptomatic or who have a contraindication to anticoagulation, rhythm management with electrical cardioversion, amiodarone, or both is preferred. Treatment of the remaining patients should focus on rate control because most will spontaneously revert to sinus rhythm within 6 weeks after discharge. All patients with atrial fibrillation persisting for more than 24 to 48 hours and without contraindication should receive anticoagulation. CONCLUSIONS: Atrial fibrillation frequently complicates cardiac surgery. Many cases can be prevented with appropriate prophylactic therapy. A strategy of rhythm management for symptomatic patients and rate management for all other patients usually results in reversion to sinus rhythm within 6 weeks of discharge.

Implantable cardioverter defibrillators in heart failure.

Implantable cardioverter defibrillators provide effective and reliable treatment of spontaneous VT and VF. These devices can be expected to decrease the risk for arrhythmic death in patients with heart failure but do not improve overall survival when death from severe pump dysfunction is imminent. Appropriate patient selection is a major aspect of arrhythmia management. Future devices will incorporate features that have the potential to reduce atrial arrhythmias, improve ventricular function, monitor hemodynamics, and prevent sudden arrhythmic death.

Epidermal growth factor receptor signaling and the invasive phenotype of ovarian carcinoma cells.

BACKGROUND: Most (70%-100%) ovarian carcinomas express high levels of the epidermal growth factor receptor (EGFR). To examine the relationship between EGFR and the invasive phenotype, we assessed integrin expression, adhesion, matrix metalloproteinase (MMP) activity, and migration in ovarian cancer cells in which EGFR expression was modified. METHODS: NIH:OVCAR-8 human ovarian carcinoma cells were transfected with an expression vector containing the human EGFR complementary DNA in an antisense orientation (EGFR-antisense cells) or the vector alone (vector control cells). We compared vector control and EGFR-antisense cells for cell morphology and adhesion by light microscopy, expression of alpha(6)- and alpha(3)-integrin subunits by flow cytometry, MMP and tissue inhibitor of MMP (TIMP) activity by zymography, and migration by a wound migration assay. In some experiments, EGFR kinase activity in parental cells was inhibited by treatment with PD153035. All statistical tests were two-sided. RESULTS: EGFR-antisense cells were morphologically distinct from vector control cells and had a selective decrease in adhesion to laminin-1 that was not observed with vector control cells (P = .008) or on other extracellular matrix substrates. Compared with vector control cells, cell surface alpha(6)-integrin expression decreased by approximately 80% (difference = 78.7%; 95% confidence interval [CI] = 77.8% to 79.6), MMP-9 activity decreased by approximately 50%, and TIMP activity increased by approximately 50% in EGFR-antisense cells. Vector control cells were highly motile (5.51 arbitrary distance unit; 95% CI = 4.98 to 6.04), whereas the EGFR-antisense cells were not (0.99 arbitrary distance units; 95% CI = 0.38 to 1.60). The morphology and integrin profile of NIH:OVCAR-8 parental cells treated with PD153035 were similar to those of the EGFR-antisense cells. CONCLUSIONS: Reduced EGFR expression in ovarian carcinoma cells decreased their adhesion to laminin-1, expression of the alpha(6)-integrin subunit (a well-characterized laminin-1 receptor), and MMP-9 activity. These data support the hypothesis that EGFR overexpression in ovarian cancer cells results in multiple phenotypic changes that enhance the invasive phenotype.

Catheter ablation in patients with multiple and unstable ventricular tachycardias after myocardial infarction: short ablation lines guided by reentry circuit isthmuses and sinus rhythm mapping.

BACKGROUND: Extensive lines of radiofrequency (RF) lesions through infarct (MI) can ablate multiple and unstable ventricular tachycardias (VTs). Methods for guiding ablation that minimize unnecessary RF applications are needed. This study assesses the feasibility of guiding RF line placement by mapping to identify a reentry circuit isthmus. METHODS AND RESULTS: Catheter mapping and ablation were performed in 40 patients (MI location: inferior, 28; anterior, 7; and both, 5) with an electroanatomic mapping system to measure the infarct region and ablation lines. The initial line was placed in the MI region either through a circuit isthmus identified from entrainment mapping or a target identified from pace mapping. A total of 143 VTs (42 stable, 101 unstable) were induced. An isthmus was identified in 25 patients (63%; 5 with only stable VTs, 5 with only unstable VTs, and 15 with both VTs). Inducible VTs were abolished or modified in 100% of patients when the RF line included an isthmus compared with 53% when RF had to be guided by pace mapping (P=0.0002); those with an isthmus identified received shorter ablation lines (4.9+/-2.4 versus 7.4+/-4.3 cm total length, P=0.02). During follow-up, spontaneous VT decreased markedly regardless of whether an isthmus was identified. VT stability and number of morphologies did not influence outcome. CONCLUSIONS: A 4- to 5-cm line of RF lesions abolishes all inducible VTs in more than 50% of patients. Less ablation is required if a reentry circuit isthmus is identified even when multiple and unstable VTs are present.

Recalls and safety alerts involving pacemakers and implantable cardioverter-defibrillator generators.

CONTEXT: Unanticipated pacemaker and implantable cardioverter-defibrillator (ICD) generator malfunctions sometimes warrant recall by the US Food and Drug Administration (FDA). Despite increasingly frequent device implantation, pacemaker and ICD recalls and safety alerts (advisories) remain poorly characterized. OBJECTIVES: To determine pacemaker and ICD generator advisory rates in the United States, to identify trends in these rates, and to examine their clinical and financial implications. DESIGN AND SETTING: Analysis of weekly FDA Enforcement Reports issued between January 1990 and December 2000 to identify all advisories involving pacemaker or ICD generators in the United States. Recalls and safety alerts involving lead malfunctions were not included. MAIN OUTCOME MEASURES: Number of pacemakers and ICD generators in the United States subject to FDA recall or safety alert in 1990-2000; annual pacemaker and ICD advisory rates in the United States in 1990-2000; and estimated cost of device advisories. RESULTS: During the study period, 52 advisories (median [25th and 75th percentiles], 4 [4 and 7] per year) involving 408 500 pacemakers and 114 645 ICDs (523 145 total devices) were issued. Hardware malfunctions (35 advisories affecting 280 641 devices) and computer errors (10 advisories affecting 216 533 devices) accounted for 95% of device recalls. Implantable cardioverter-defibrillators were recalled more frequently than pacemakers (mean [SD], 16.4 [1.6] vs 6.7 [0.8] advisories per 100 person-years; P<.001). Between 1995 and 2000, the annual advisory rate increased for both pacemakers (P for trend <.001) and ICDs (P for trend =.02). An estimated 1.3 million device checks and analyses and 36 187 device replacements resulted from the advisories and cost approximately $870 million. CONCLUSIONS: Pacemaker and ICD recalls and safety alerts occur frequently, affect many patients, and appear to be increasing in number and rate. With the growing number of device implants and expanding indications for device therapy, the number of patients affected by device advisories will likely continue to increase.

Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1-matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma.

Vasculogenic mimicry describes a process where aggressive tumor cells in three-dimensional matrices mimic embryonic vasculogenesis by forming extracellular matrix (ECM)-rich, patterned tubular networks. Microarray gene chip analyses revealed significant increases in the expression of laminin 5 (Ln-5, gamma2 chain) and matrix metalloproteinases (MMP)-1, -2, -9, and MT1-MMP (MMP-14) in aggressive compared with poorly aggressive melanoma cells. These components colocalized with developing patterned networks and antisense oligonucleotides to the Ln-5 gamma2 chain (but not sense oligonucleotides), and antibodies to MMP-2 or MT1-MMP (but not MMP-9) inhibited the formation of these networks. Cultures which did not receive antibodies to either MMPs-2 or -14 contained the Ln-5 gamma2 chain promigratory cleavage fragments. Poorly aggressive melanoma cells seeded on collagen I matrices preconditioned by the aggressive cells formed tubular networks along the Ln-5 gamma2 chain-enriched tracks deposited by the aggressive cells. These results suggest that increased expression of MMP-2 and MT1-MMP, along with matrix deposition of the Ln-5 gamma2 chain and/or its cleavage fragments, are required for vasculogenic mimicry by aggressive melanoma cells. Furthermore, the apparent recapitulation of laminin-rich, patterned networks observed in aggressive melanoma patients' tissue sections by aggressive melanoma tumor cells in three-dimensional culture may also serve as a model to help identify specific molecular targets which could function as templates for the coordinated migration of aggressive tumor cells and their proteolytic remodeling of the ECM and may have profound implications for the development of novel therapies directed at the ECM to alter tumor progression.

Single catheter determination of local electrogram prematurity using simultaneous unipolar and bipolar recordings to replace the surface ECG as a timing reference.

Bipolar recordings eliminate much of the far-field signal, while minimally filtered unipolar recordings contain substantial far-field signal components. These properties may allow the onset of the unipolar recording to serve as a timing reference for the bipolar recording obtained from the same electrode catheter during mapping of focal atrial or ventricular tachycardias. Mapping and RF ablation were performed in 26 patients with focal ventricular tachycardia and 14 patients with focal atrial tachycardia. At 205 mapping sites, simultaneous recordings of (1) minimally filtered unipolar electrograms (0.5-500 Hz), (2) high pass filtered unipolar electrograms (100 Hz), and (3) filtered bipolar recordings (30-500 Hz) were analyzed. The interval between the onset of the minimally filtered unipolar electrogram and the first peak of the bipolar electrogram (UniOn-BiP) correlated closely with the timing of the local electrogram referenced to the surface ECG (r = 0.85, P < 0.001). Of 53 sites where RF ablation was performed, UniOn-BiP was shorter at successful compared to unsuccessful sites (3.8 +/- 3.5 vs 9.2 +/- 5.2 ms, P < 0.001) and was < 15 ms at all successful sites. In conclusion, the comparison of simultaneous unipolar and bipolar electrograms from a single catheter allows assessment of the prematurity of local electrograms from a focal source without the use of the P wave or QRS onset as a timing reference.

Multi atrial maco-re-entry circuits in adults with repaired congenital heart disease: entrainment mapping combined with three-dimensional electroanatomic mapping.

OBJECTIVES: We sought to characterize re-entry circuits causing intra-atrial re-entrant tachycardias (IARTs) late after the repair of congenital heart disease (CHD) and to define an approach for mapping and ablation, combining anatomy, activation sequence data and entrainment mapping. BACKGROUND: The development of IARTs after repair of CHD is difficult to manage and ablate due to complex anatomy, variable re-entry circuit locations and the frequent co-existence of multiple circuits. METHODS: Forty-seven re-entry circuits were mapped in 20 patients with recurrent IARTs refractory to medical therapy. In the first group (n = 7), ablation was guided by entrainment mapping. In the second group (n = 13), entrainment mapping was combined with a three-dimensional electroanatomic mapping system to precisely localize the scar-related boundaries of re-entry circuits and to reconstruct the activation pattern. RESULTS: Three types of right atrial macro-re-entrant circuits were identified: those related to a lateral right atriotomy scar (19 IARTs), the Eustachian isthmus (18 IARTs) or an atrial septal patch (8 IARTs). Two IARTs originated in the left atrium. Radiofrequency (RF) lesions were applied to transect critical isthmuses in the right atrium. In three patients, the combined mapping approach identified a narrow isthmuses in the lateral atrium, where the first RF lesion interrupted the circuit; the remaining circuits were interrupted by a series of RF lesions across a broader path. Overall, 38 (81%) of 47 IARTs were successfully ablated. During follow-up ranging from 3 to 46 months, 16 (80%) of 20 patients remained free of recurrence. Success was similar in the first 7 (group 1) and last 13 patients (group 2), but fluoroscopy time decreased from 60 +/- 30 to 24 +/- 9 min/procedure, probably related to the increasing experience and ability to monitor catheter position non-fluoroscopically. CONCLUSIONS: Entrainment mapping combined with three-dimensional electroanatomic mapping allows delineation of complex re-entry circuits and critical isthmuses as targets for ablation. Radiofrequency catheter ablation is a reasonable option for treatment of IARTs related to repair of CHD.

The role of matrix metalloproteinases in tumor invasion, metastasis, and angiogenesis.

Tumor invasion and metastasis formation are the hallmarks of malignant cancer. Metastatic spread of cancer cells is a result of a complex cascade of cellular events. This article discusses the matrix metalloproteinase family, the regulation of matrix metalloproteinase activity, and the functions of matrix metalloproteinases in tumor progression.

Tissue inhibitor of metalloproteinase-1 alters the tumorigenicity of Burkitt's lymphoma via divergent effects on tumor growth and angiogenesis.

Epstein-Barr virus (EBV)-positive Burkitt's lymphoma cells and EBV-infected B cells elicit humoral factors that inhibit tumor-induced angiogenesis, resulting in tumor necrosis and regression. Of the chemokine factors identified in association with this growth behavior, none have induced complete tumor regression. We have previously identified tissue inhibitors of metalloproteinase (TIMP)-1 in various B cell lymphoma cell lines. Here we show that induction of TIMP-1 expression in an EBV-negative Burkitt's lymphoma cell line results in a biphasic, in vivo tumor growth pattern in the nude mouse that is essentially identical to EBV-positive Burkitt's lymphoma cell lines. The initial effect of TIMP-1 is to enhance tumor growth, consistent with the reported anti-apoptotic effect of TIMP-1 on B cell growth. Tumor necrosis and regression then follow the initial period of rapid, increased tumor growth. Only microscopic foci of residual, proliferating tumor cells are observed on biopsy of the tumor site. This latter effect is mediated by TIMP-1 inhibition of an angiogenic response within the developing tumor mass, as demonstrated by immunostaining and microvessel counts. These findings suggest that TIMP-1 is an important mediator of the in vivo growth properties of EBV-positive Burkitt's lymphoma.

Proteases in invasion: matrix metalloproteinases.

The role of proteases in general, and the matrix metalloproteinases in particular, in tumor invasion and metastasis is well established. However, the classic view that these enzymes simply provide a mechanism for the breakdown of connective tissue barriers has been challenged. This overview summarizes recent evidence to support the changing view of the role of matrix metalloproteinases in cancer progression. First we briefly review the central role of cell invasion in cancer progression and also the matrix metalloproteinase family members. We then focus on the emerging roles for these enzymes in cancer progression, including the role of matrix metalloproteinases in cell proliferation and release of growth factors, cell migration and in modification of the extracellular matrix to reveal cryptic sites that alter cell behaviour.