Type I interferon signaling genes in recurrent major depression: increased expression detected by whole-blood RNA sequencing.

A study of genome-wide gene expression in major depressive disorder (MDD) was undertaken in a large population-based sample to determine whether altered expression levels of genes and pathways could provide insights into biological mechanisms that are relevant to this disorder. Gene expression studies have the potential to detect changes that may be because of differences in common or rare genomic sequence variation, environmental factors or their interaction. We recruited a European ancestry sample of 463 individuals with recurrent MDD and 459 controls, obtained self-report and semi-structured interview data about psychiatric and medical history and other environmental variables, sequenced RNA from whole blood and genotyped a genome-wide panel of common single-nucleotide polymorphisms. We used analytical methods to identify MDD-related genes and pathways using all of these sources of information. In analyses of association between MDD and expression levels of 13 857 single autosomal genes, accounting for multiple technical, physiological and environmental covariates, a significant excess of low P-values was observed, but there was no significant single-gene association after genome-wide correction. Pathway-based analyses of expression data detected significant association of MDD with increased expression of genes in the interferon α/ß signaling pathway. This finding could not be explained by potentially confounding diseases and medications (including antidepressants) or by computationally estimated proportions of white blood cell types. Although cause-effect relationships cannot be determined from these data, the results support the hypothesis that altered immune signaling has a role in the pathogenesis, manifestation, and/or the persistence and progression of MDD.

Statistical analysis of MPSS measurements: application to the study of LPS-activated macrophage gene expression.

Massively Parallel Signature Sequencing (MPSS), a recently developed high-throughput transcription profiling technology, has the ability to profile almost every transcript in a sample without requiring prior knowledge of the sequence of the transcribed genes. As is the case with DNA microarrays, effective data analysis depends crucially on understanding how noise affects measurements. We analyze the sources of noise in MPSS and present a quantitative model describing the variability between replicate MPSS assays. We use this model to construct statistical hypotheses that test whether an observed change in gene expression in a pair-wise comparison is significant. This analysis is then extended to the determination of the significance of changes in expression levels measured over the course of a time series of measurements. We apply these analytic techniques to the study of a time series of MPSS gene expression measurements on LPS-stimulated macrophages. To evaluate our statistical significance metrics, we compare our results with published data on macrophage activation measured by using Affymetrix GeneChips.

Local delivery of c-myc neutrally charged antisense oligonucleotides with transport catheter inhibits myointimal hyperplasia and positively affects vascular remodeling in the rabbit balloon injury model.

Myointimal hyperplasia after percutaneous transluminal coronary angioplasty (PTCA) is a key component of the process of restenosis. The c-myc is a critical cell-cycle division protein involved in the formation of neointima. We evaluated the long-term impact of local delivery of c-myc neutrally charged antisense oligonucleotides (Resten-NG) on myointimal hyperplasia after PTCA in a rabbit model. PTCA was performed in the iliac arteries of 25 New Zealand white rabbits, using a Transport catheter at 8 atm for 30 sec, three times; 500 microg Resten-NG (n = 11) or saline (n = 14) was delivered to the PTCA site at 2 atm with the outer balloon for 2 min. The diet was supplemented with 0.25% cholesterol for 10 days before and 60 days after PTCA. Angiography was performed at harvest, and vessels were fixed in formalin, processed, and stained with hematoxylin and eosin (H&E) and Movat. Quantitative angiography showed that local delivery of antisense c-myc at PTCA reduced late luminal loss from 1.8 +/- 0.30 mm in control animals to 0.90 +/- 0.30 mm in the treatment group (P = 0.001). Histological analysis by planimetry showed that intimal areas were 1.67 +/- 0.44 mm(2) and 0.82 +/- 0.32 mm(2) in the control and antisense delivery groups, respectively (P < 0.05). We conclude that local delivery of Resten-NG inhibited myointimal hyperplasia after PTCA in cholesterol-fed rabbits for up to 60 days.

Cortactin potentiates bone metastasis of breast cancer cells.

Gene amplification of the chromosome 11q13 in breast cancer and squamous carcinomas in the head and neck results in frequent overexpression of cortactin, a prominent substrate of Src-related tyrosine kinases in the cell cortical areas. To investigate the role of cortactin in tumor progression, we analyzed MDA-MB-231 breast cancer cells overexpressing green fluorescent protein-tagged murine cortactin (GFP-cortactin) and a cortactin mutant deficient in tyrosine phosphorylation under the control of a retroviral vector. Injection of MDA-MB-231 cells overexpressing GFP-cortactin into nude mice through cardiac ventricles caused bone osteolysis at a frequency approximately 85% higher than that of cells expressing the vector alone, whereas injection of cells overexpressing the mutant deficient in tyrosine phosphorylation induced 74% fewer osteolytic metastases as compared with the control group. Interestingly, the cells expressing either GFP-cortactin or the mutant did not show significant differences in growth in vitro or when injected m.f.p. in vivo. On the other hand, the cells overexpressing GFP-cortactin but not the mutant acquired a >60% enhanced capability for transendothelial invasion and endothelial cell adhesion. These data suggest that cortactin contributes to tumor metastasis by enhancing the interaction of tumor cells with endothelial cells and the invasion of tumor cells into bone tissues.

Overexpression of cyclooxygenase-2 is sufficient to induce tumorigenesis in transgenic mice.

The cyclooxygenase (COX)-2 gene encodes an inducible prostaglandin synthase enzyme that is overexpressed in adenocarcinomas and other tumors. Deletion of the murine Cox-2 gene in Min mice reduced the incidence of intestinal tumors, suggesting that it is required for tumorigenesis. However, it is not known if overexpression of Cox-2 is sufficient to induce tumorigenic transformation. We have derived transgenic mice that overexpress the human COX-2 gene in the mammary glands using the murine mammary tumor virus promoter. The human Cox-2 mRNA and protein are expressed in mammary glands of female transgenic mice and were strongly induced during pregnancy and lactation. Female virgin Cox-2 transgenic mice showed precocious lobuloalveolar differentiation and enhanced expression of the beta-casein gene, which was inhibited by the Cox inhibitor indomethacin. Mammary gland involution was delayed in Cox-2 transgenic mice with a decrease in apoptotic index of mammary epithelial cells. Multiparous but not virgin females exhibited a greatly exaggerated incidence of focal mammary gland hyperplasia, dysplasia, and transformation into metastatic tumors. Cox-2-induced tumor tissue expressed reduced levels of the proapoptotic proteins Bax and Bcl-x(L) and an increase in the anti-apoptotic protein Bcl-2, suggesting that decreased apoptosis of mammary epithelial cells contributes to tumorigenesis. These data indicate that enhanced Cox-2 expression is sufficient to induce mammary gland tumorigenesis. Therefore, inhibition of Cox-2 may represent a mechanism-based chemopreventive approach for carcinogenesis.

Low-energy laser irradiation reduces formation of scar tissue after myocardial infarction in rats and dogs.

BACKGROUND: Low-energy laser irradiation (LELI) has been found to attenuate various biological processes in tissue culture and experimental animal models. The aim of the present study was to investigate the effect of LELI on the formation of scar tissue in experimentally induced chronic infarct in rats and dogs. METHODS AND RESULTS: Myocardial infarction (MI) was induced in 50 dogs and 26 rats by ligation of the left anterior descending coronary artery. After induction of MI, the laser-irradiated (LI) group received laser irradiation (infrared laser, 803-nm wavelength) epicardially. Control MI-induced non-laser irradiated (NLI) dogs were sham-operated, and laser was not applied. All dogs were euthanized at 5 to 6 weeks after MI. Infarct size was determined by TTC staining and histology. The laser treatment (P:<0.05) lowered mortality significantly, from 30% to 6.5%, after induction of MI. The infarct size in the LI dogs was reduced significantly (P:<0.0001) (52%) compared with NLI dogs. Histological observation of the infarct revealed a typical scar tissue in NLI dogs and cellularity in most of the LI dogs. Only 14+/-3% of the mitochondria in the cardiomyocytes in the ischemic zone (4 hours after MI) of LI MI-induced rats were severely damaged, compared with 36+/-1% in NLI rats. Accordingly, ATP content in that zone was 7.6-fold (significantly) higher in LI than in NLI rats. CONCLUSIONS: Our observations indicate that epicardial LELI of rat and dog hearts after chronic MI caused a marked reduction in infarct size, probably due to a cardioprotective effect of the LELI.

Endothelium and atherogenesis: endothelial therapy revisited.

Atherosclerosis, a chronic systemic disease of the vasculature with an inflammatory component, is the primary cause of cardiovascular morbidity and mortality in industrialized countries. Impairment of vascular endothelial cell function in atherosclerosis and in conditions associated with increased cardiovascular risk are important determinants of disease progression. Reduced endothelium-dependent relaxation in the coronary and systemic circulation due to decreased bioavailability of nitric oxide (NO) and increased release of oxygen-derived free radicals promotes the adhesion of leukocytes, thrombosis, inflammation, cell proliferation, and increases in vascular tone. In addition to decreases in bioactive NO, enhanced production of the 21-amino acid peptide endothelin-1 contributes to the progression of atherosclerosis. This paper discusses mechanisms and therapeutic approaches to improving endothelial pathways in atherosclerosis. Restoration of endothelium-derived NO bioactivity through inhibition of the renin-angiotensin system, the endothelin system, or statin therapy improves vascular function in experimental hypercholesterolemia, hypertension and heart failure. These treatments may also have therapeutic benefit for patients at risk or with overt atherosclerosis, and are likely to reduce vascular and myocardial complications of this disease.

Endoluminal reconstruction of the arterial wall with endothelial cell/glue matrix reduces restenosis in an atherosclerotic rabbit.

OBJECTIVES: The objectives of this study were 1) to improve the attachment of reimplanted endothelial cells (EC) using a fibrin glue, and 2) to assess the impact of endothelial reseeding on restenosis eight weeks after balloon angioplasty. BACKGROUND: A possible mechanism contributing to restenosis after balloon angioplasty is the loss of the EC lining. Previous attempts to reseed EC had little effect due to rapid loss of the seeded cells. METHODS: Twelve atherosclerotic rabbits were subjected to angioplasty of iliac arteries and reseeding procedure. One iliac artery was subjected to EC/glue reconstruction and a contralateral site to EC seeding without glue. The animals were sacrificed after 4 h. In another series 12 rabbits were treated in the same fashion and were restudied at eight weeks. Additionally, in 10 animals one iliac was subjected to glue treatment, and another served as control. RESULTS: Histological examination demonstrated the ability of this method to reattach the EC/glue matrix circumferentially to 68.0 +/- 6.7% of the arterial wall in comparison with 13.5 +/- 3.9% reattachment after EC seeding. Morphometry at eight weeks showed that the lumen area was significantly greater in the EC/glue group (1.23 +/- 0.35 mm2) than in the EC seeding alone (0.65 +/- 0.02 mm2) and 0.72 +/- 0.41 mm2 in the glue group. This was principally accounted for by the statistically significant differences in the intimal area (0.76 +/- 0.18 mm vs. 1.25 +/-0.26 mm2 and 1.01 +/- 0.53 mm2, respectively). CONCLUSIONS: The attachment of EC after angioplasty can be greatly improved with fibrin glue matrix. The near 70% endothelial coverage achieved by this method resulted in a significant reduction of restenosis in atherosclerotic rabbit.

Role of CD154 in the secondary immune response: the reduction of pre-existing splenic germinal centers and anti-factor VIII inhibitor titer.

Using the murine model of hemophilia A, we have examined the role of CD154 in the secondary immune response to factor VIII (FVIII). We previously reported that repeated i.v. injection of FVIII in hemophilia A mice induces a T cell-dependent anti-FVIII antibody formation. Herein, blocking of CD154 by a monoclonal antibody in FVIII-primed hemophilia A mice resulted in the disappearance of pre-existing spleen germinal centers (GC) in the white pulp within 24 h of treatment. Moreover, further expansion of GC in response to FVIII challenge was completely inhibited. In parallel, anti-FVIII antibody titers were markedly reduced and T cell responses to FVIII were abolished. The rapid disappearance of the GC after anti-CD154 treatment was not accompanied by increased B cell apoptosis; instead B cells accumulated in the peripheral zone of the splenic white pulp. Interestingly, repeated exposure to FVIII with anti-CD154 antibody administration blocked anti-FVIII antibody formation but failed to induce long-lasting unresponsiveness. Our data demonstrate that the CD40-CD154 interaction is critical for B cell homeostasis and the secondary immune response to FVIII. For potential clinical application, the data also suggest that therapies targeting the CD154 molecule may be useful for the treatment of high titer FVIII inhibitors in hemophilia A.

Role of hepatic and lipoprotein lipase in lipoprotein metabolism and atherosclerosis: studies in transgenic and knockout animal models and somatic gene transfer.

Hepatic lipase (HL) and lipoprotein lipase (LPL) are the two major lipolytic enzymes responsible for the hydrolysis of triglycerides and phospholipids present in circulating plasma lipoproteins. Both lipases are attached to the vascular endothelium via cell surface proteoglycans. HL is primarily involved in the metabolism of chylomicron remnants, intermediate density lipoproteins and high-density lipoproteins whereas LPL catalyzes the hydrolysis of triglycerides from chylomicrons and very low-density lipoproteins. In addition to their traditional function as lipolytic enzymes, HL and LPL appear to serve as ligands that mediate the interaction of lipoproteins to cell surface receptors and/or proteoglycans. Over the past several years significant advances have been made in our understanding of new, alternative mechanisms by which HL and LPL modulate lipoprotein metabolism and the development of atherosclerosis in vivo. This review will summarize some of the new insights generated from the study of transgenic and knockout HL and LPL animal models as well as somatic gene transfer of these two lipases.

Complete genomic sequence of the human ABCA1 gene: analysis of the human and mouse ATP-binding cassette A promoter.

The ABCA1 gene, a member of the ATP-binding cassette A (ABCA1) transporter superfamily, encodes a membrane protein that facilitates the cellular efflux of cholesterol and phospholipids. Mutations in ABCA1 lead to familial high density lipoprotein deficiency and Tangier disease. We report the complete human ABCA1 gene sequence, including 1,453 bp of the promoter, 146,581 bp of introns and exons, and 1 kb of the 3' flanking region. The ABCA1 gene spans 149 kb and comprises 50 exons. Sixty-two repetitive Alu sequences were identified in introns 1-49. The transcription start site is 315 bp upstream of a newly identified initiation methionine codon and encodes an ORF of 6,783 bp. Thus, the ABCA1 protein is comprised of 2,261 aa. Analysis of the 1,453 bp 5' upstream of the transcriptional start site reveals multiple binding sites for transcription factors with roles in lipid metabolism. Comparative analysis of the mouse and human ABCA1 promoter sequences identified specific regulatory elements, which are evolutionarily conserved. The human ABCA1 promoter fragment -200 to -80 bp that contains binding motifs for SP1, SP3, E-box, and AP1 modulates cellular cholesterol and cAMP regulation of ABCA1 gene expression. These combined findings provide insights into ABCA1-mediated regulation of cellular cholesterol metabolism and will facilitate the identification of new pharmacologic agents for the treatment of atherosclerosis in humans.

Functional expression of regiospecific cytochrome P450 limonene hydroxylases from mint (Mentha spp.) in Escherichia coli and saccharomyces cerevisiae.

The oxygenation pattern of the essential oil monoterpenes of commercial mint (Mentha) species is determined by regiospecific cytochrome P450-catalyzed hydroxylation of the common olefinic precursor (-)-4S-limonene. In spearmint (M. spicata), C6-allylic hydroxylation leads to (-)-trans-carveol and thence (-)-carvone, whereas in peppermint (M. x piperita), C3-allylic hydroxylation leads to (-)-trans-isopiperitenol and ultimately (-)-menthol. cDNAs encoding the C6-hydroxylase and C3-hydroxylase from spearmint and peppermint, respectively, were isolated by a combination of reverse genetic and homology-based cloning methods (S. Lupien, F. Karp, M. Wildung, and R. Croteau, Arch. Biochem. Biophys. 368, 181-192, 1999). Although both hydroxylase genes were confirmed by functional expression using the baculovirus-Spodoptera system, too little protein was available by this approach to permit detailed study of the structure-function relationships of these catalysts, especially the substrate binding determinants that underlie the regiochemistry and stereochemistry of the reactions. Therefore, heterologous overexpression systems based on Escherichia coli and Saccharomyces cerevisiae were developed to produce several N-terminally modified versions of the recombinant hydroxylases. Ancillary methods for the solubilization, purification, and reconstitution (with recombinant spearmint cytochrome P450 reductase) of the limonene hydroxylases were also devised, with which substrate binding behavior and precise regiochemistry and stereochemistry of product formation were determined.

The Swiss database on quality projects in medical institutions.

Since the early 1990s it is important for every medical institution to report on activities in the field of quality improvement and quality assessment because there is a certain pressure from the market and from health insurance laws in various countries. Nevertheless, researchers as well as clinicians or administrators are rarely informed on ongoing projects. To register projects in quality research, an Internet-based information system was established to register projects on quality of medical institutions. Among others, hospitals, private doctors' offices, medical specialty societies and cost-payers are regarded as institutions in this effort. An interactive database provides information on the institution performing a project and on what is being/has been performed in a certain place during a certain period of time. At present medical institutions are invited to report on their projects, but this initiative can only succeed if it provides information from as many different institutions as possible: for data skills and help your colleagues! Place your knowledge, activities and information at disposal to the public and profit from your colleagues.

In vivo evidence for both lipolytic and nonlipolytic function of hepatic lipase in the metabolism of HDL.

To investigate the in vivo role that hepatic lipase (HL) plays in HDL metabolism independently of its lipolytic function, recombinant adenovirus (rAdV) expressing native HL, catalytically inactive HL (HL-145G), and luciferase control was injected in HL-deficient mice. At day 4 after infusion of 2 x 10(8) plaque-forming units of rHL-AdV and rHL-145G-AdV, similar plasma concentrations were detected in postheparin plasma (HL=8.4+/-0.8 microg/mL and HL-145G=8.3+/-0.8 microg/mL). Mice expressing HL had significant reductions of cholesterol (-76%), phospholipids (PL; -68%), HDL cholesterol (-79%), apolipoprotein (apo) A-I (-45%), and apoA-II (-59%; P<0.05 for all), whereas mice expressing HL-145G decreased their cholesterol (-49%), PL (-40%), HDL cholesterol (-42%), and apoA-II (-89%; P<0.005 for all) but had no changes in apoA-I. The plasma kinetics of (125)I-labeled apoA-I HDL, (131)I-labeled apoA-II HDL, and [(3)H]cholesteryl ester (CE) HDL revealed that compared with mice expressing luciferase control (fractional catabolic rate [FCR] in d(-1): apoA-I HDL=1.3+/-0.1; apoA-II HDL=2.1+/-0; CE HDL=4.1+/-0.7), both HL and HL-145G enhanced the plasma clearance of CEs and apoA-II present in HDL (apoA-II HDL=5.6+/-0.5 and 4.4+/-0.2; CE HDL=9.3+/-0. 0 and 8.3+/-1.1, respectively), whereas the clearance of apoA-I HDL was enhanced in mice expressing HL (FCR=4.6+/-0.3) but not HL-145G (FCR=1.4+/-0.4). These combined findings demonstrate that both lipolytic and nonlipolytic functions of HL are important for HDL metabolism in vivo. Our study provides, for the first time, in vivo evidence for a role of HL in HDL metabolism independent of lipolysis and provides new insights into the role of HL in facilitating distinct metabolic pathways involved in the catabolism of apoA-I- versus apoA-II-containing HDL.

Obesity is associated with tissue-specific activation of renal angiotensin-converting enzyme in vivo: evidence for a regulatory role of endothelin.

In the C57BL/6J mice model, we investigated whether obesity affects the function or expression of components of the tissue renin-angiotensin system and whether endothelin (ET)-1 contributes to these changes. ACE activity (nmol. L His-Leu. mg protein(-1)) was measured in lung, kidney, and liver in control (receiving standard chow) and obese animals treated for 30 weeks with a high-fat, low cholesterol diet alone or in combination with LU135252, an orally active ET(A) receptor antagonist. ACE mRNA expression was measured in the kidney, and the effects of LU135252 on purified human ACE were determined. Aortic and renal tissue ET-1 protein content was measured, and the vascular contractility to angiotensin II was assessed. Obesity was associated with a tissue-specific increase in ACE activity in the kidney (55+/-4 versus 33+/-3 nmol/L) but not in the lung (34+/-2 versus 32+/-2 nmol/L). Long-term LU135252 treatment completely prevented this activation (13.3+/-0.3 versus 55+/-4 nmol/L, P<0.05) independent of ACE mRNA expression, body weight, or renal ET-1 protein but did not affect pulmonary or hepatic ACE activity. Obesity potentiated contractions in response to angiotensin II in the aorta (from 6+/-2% to 33+/-5% KCl) but not in the carotid artery (4+/-1% to 3.6+/-1% KCl), an effect that was completely prevented with LU135252 treatment (6+/-0.4% versus 33+/-5% KCl). No effect of LU135252 on purified ACE was observed. Thus, obesity is associated with the activation of renal ACE in vivo independent of its mRNA expression and enhanced vascular contractility to angiotensin II. These effects are regulated by ET in an organ-specific manner, providing novel mechanisms by which ET antagonists may exert organ protection.

Ligation of CD28 in vivo induces CD40 ligand expression and promotes B cell survival.

Functional activation of T cells requires ligation of Ag receptors with specific peptides presented by MHC molecules on APCs concurrent with appropriate contacts of cell surface accessory molecules. Among these accessory molecules, interactions between CD28/CTLA-4 with B7 family members (CD80 and CD86) and CD40 with CD40 ligand (CD40L) play a decisive role in regulating the progression of balanced immune responses. However, most information regarding the role of accessory molecules in immune responses has been derived in the context of signals from the TCRs. Little understanding has been achieved regarding the consequence of ligation of costimulation molecules in absence of signals from the TCR. By employing an in vivo murine system, we show, herein, that ligation of CD28 alone with anti-CD28 Abs leads to a dramatic enlargement of the peripheral lymphoid organs characterized primarily by the expansion of B cells. B cells from anti-CD28-treated mice are resistant to spontaneous and anti-IgM-induced apoptosis. These cells are also unsusceptible to FasL-mediated apoptosis. Interestingly, this in vivo effect of CD28 on B cells is largely mediated by inducing the expression of CD40L, since coadministration of a blocking Ab against CD40L inhibited CD28-mediated B cell survival and expansion. Therefore, CD28-mediated expression of CD40L may play an important role in the regulation of lymphocyte homeostasis.

Studies on the histogenesis of myxomatous tissue of human coronary lesions.

Myxomatous tissue is a characteristic component of human coronary artery lesions, found more often in restenotic lesions. It represents a bulky accumulation of stellate-shaped cells of unknown histogenesis that are embedded in a loose stroma. We analyzed 64 atherectomy specimens containing substantial amounts of myxomatous tissue by using immunohistochemistry, in situ hybridization, and electron microscopy techniques. Stellate cells represented a heterogeneous population, sharing features of smooth muscle cells (SMCs), macrophages, as well as antigen-presenting dendritic cells. Like quiescent medial SMCs, the stellate cells in all specimens expressed high levels of SM alpha-actin message and protein and showed heterogeneity with respect to heavy-chain myosin, SM22, desmin, and vimentin. Ultrastructurally, stellate cells resembled SMCs, with some peculiarities that distinguish them from both differentiated and dedifferentiated SMCs. In contrast to quiescent SMCs, the stellate cells expressed high levels of acidic fibroblast growth factor mRNA and protein similar to cells of monocyte/macrophage lineage. However, stellate cells did not express the marker of mature macrophages, HAM56, and were heterogeneous with respect to CD68. Moreover, unlike SMCs, the stellate cells bore some of the major phenotypic markers of dendritic cells: they were S100-positive and showed various reactivity with respect to CD1a and human leukocyte antigen (HLA)-DR. Invasion of myxomatous tissue with CD45RO-positive T lymphocytes was correlated with strong expression of CD1a in these specimens. Stellate cells also expressed a pericyte marker, high-molecular-weight melanoma-associated antigen. We conclude that stellate cells of myxomatous tissue represent a specific phenotype of mesenchymal cells (possibly pericytes), which is activated to express some markers of antigen-presenting cells. These findings suggest involvement of the stellate cells in a local immune response.

Feasibility and safety of percutaneous laser revascularization using the Biosense system in porcine hearts.

BACKGROUND: Direct myocardial revascularization (DMR) is being explored to improve symptoms in patients with refractory ischemic coronary syndromes. This study assessed the safety and feasibility of percutaneous DMR using the Biosense non-fluoroscopic navigation system (incorporating the holmium : yttrium aluminium garnet laser) in pig hearts. METHODS AND RESULTS: Twenty pigs underwent left ventricular mapping using the Biosense system, followed by percutaneous DMR with a holmium : yttrium aluminium garnet laser at 2 J x 1 pulse, and were sacrificed acutely (n = 5), at 24 h (n = 5), and at 3 weeks (n = 10) after the DMR procedure. The hearts were examined grossly and microscopically. Serial creatine kinase-MB level was measured up to 24 h. There were no procedural complications. Animals received 13 +/- 2 channels in 19 +/- 7 min. The catheter was able to reach all regions, including the septum. There was no increase in creatine kinase-MB level up to 24 h. One animal died within an hour of the procedure. There was one sealed perforation into the subepicardial fat as a result of same site laser activation. Histologic evaluation in the acute and 24 h groups revealed laser channels which were 3.6 +/- 2.2 mm long, 1.5 +/- 0.7 mm wide, with an entry angle of 73 +/- 12 degrees. Channels were filled with platelet thrombi acutely and were surrounded by well-defined rims of thermal coagulation and an 'impact zone' of viable myocardium; at 3 weeks, no channels remained patent and they had been replaced by well-healed granulation tissue. CONCLUSIONS: It is feasible and safe to perform percutaneous DMR with the Biosense system and the channels created with the chosen laser parameters are rapidly sealed with platelet thrombi and at 3 weeks are replaced by well-healed granulation tissue.