Development of a clinically relevant ex vivo model of cardiac ablation for testing of ablation catheters.

INTRODUCTION: Reliable ex vivo cardiac ablation models have the potential to increase catheter testing throughput while minimizing animal usage. The goal of this work was to develop a physiologically relevant ex vivo swine model of cardiac ablation displaying minimal variability and high repeatability and identify and optimize key parameters involved in ablation outcomes. METHODS AND RESULTS: A root cause analysis was conducted to identify variables affecting ablation outcomes. Parameters associated with the tissue, bath media, and impedance were identified. Variables were defined experimentally and/or from literature sources to best mimic the clinical cardiac ablation setting. The model was validated by performing three independent replicates of ex vivo myocardial ablation and a direct comparison of lesion outcomes of the ex vivo swine myocardial and in vivo canine thigh preparation (TP) models. Replicate experiments on the ex vivo model demonstrated low variance in ablation depth (6.5 ± 0.6, 6.3 ± 0.6, 6.2 ± 0.4 mm) and width (10.4 ± 1.1, 9.7 ± 1.0, 9.9 ± 0.9 mm) and no significant differences between replicates. In a direct comparison of the two models, the ex vivo model demonstrated ablation depths similar to the canine TP model at 35 W (6.9 ± 1.0, and 7.0 ± 0.9 mm) and 50 W (8.0 ± 0.7, and 8.4 ± 0.7 mm), as well as similar power to depth ratios (15% and 19% for the ex vivo cardiac and in vivo TP models, respectively). CONCLUSION: The ex vivo model exhibited strong lesion reproducibility and power-to-depth ratios comparable to the in vivo TP model. The optimized ex vivo model minimizes animal usage with increased throughput, lesion characteristics similar to the in vivo TP model, and ability to discriminate minor variations between different catheter designs.

Device safety assessment of bronchoscopic microwave ablation of normal swine peripheral lung using robotic-assisted bronchoscopy.

INTRODUCTION: The aim of this study was to assess the safety of bronchoscopic microwave ablation (MWA) of peripheral lung parenchyma using the NEUWAVE™ FLEX Microwave Ablation System, and robotic-assisted bronchoscopy (RAB) using the MONARCH™ Platform in a swine model. METHODS: Computed tomography (CT)-guided RAB MWA was performed in the peripheral lung parenchyma of 17 Yorkshire swine (40-50 kg) and procedural adverse events (AEs) documented. The acute group (day 0, n = 5) received 4 MWAs at 100 W for 1, 3, 5, and 10 min in 4 different lung lobes. Subacute and chronic groups (days 3 and 30, n = 6 each) received one MWA (100 W, 10 min) per animal. RESULTS: The study was completed without major procedural complications. No postprocedural AEs including death, pneumothorax, bronchopleural fistula, hemothorax, or pleural effusions were observed. No gross or histological findings suggestive of thromboembolism were found in any organ. One 3-Day and one 30-Day swine exhibited coughing that required no medication (minor AEs), and one 30-Day animal required antibiotic medication (major AE) for a suspected lower respiratory tract infection that subsided after two weeks. CT-based volumetric estimates of ablation zones in the acute group increased in an ablation time-dependent (1-10 min) manner, whereas macroscopy-based estimates showed an increasing trend in ablation zone size. CONCLUSION: The NEUWAVE FLEX and MONARCH devices were safely used to perform single or multiple RAB MWAs. The preclinical procedural safety profile of RAB MWA supports clinical research of both devices to investigate efficacy in select patients with oligometastatic disease or primary NSCLC.

Hemostatic efficacy of two topical adjunctive hemostats in a porcine spleen biopsy punch model of moderate bleeding.

Topical hemostatic agents have become essential tools to aid in preventing excessive bleeding in surgical or emergency settings and to mitigate the associated risks of serious complications. In the present study, we compared the hemostatic efficacy of SURGIFLO® Hemostatic Matrix Kit with Thrombin (Surgiflo-flowable gelatin matrix plus human thrombin) to HEMOBLAST™ Bellows Hemostatic Agent (Hemoblast-a combination product consisting of collagen, chondroitin sulfate, and human thrombin). Surgiflo and Hemoblast were randomly tested in experimentally induced bleeding lesions on the spleens of four pigs. Primary endpoints included hemostatic efficacy measured by absolute time to hemostasis (TTH) within 5 min. Secondary endpoints included the number of product applications and the percent of product needed from each device to achieve hemostasis. Surgiflo demonstrated significantly higher hemostatic efficacy and lower TTH (p < 0.01) than Hemoblast. Surgiflo-treated lesion sites achieved hemostasis in 77.4% of cases following a single product application vs. 3.3% of Hemoblast-treated sites. On average, Surgiflo-treated sites required 63% less product applications than Hemoblast-treated sites (1.26 ± 0.0.51 vs. 3.37 ± 1.16). Surgiflo provided more effective and faster hemostasis than Hemoblast. Since both products contain thrombin to activate endogenous fibrinogen and accelerate clot formation, the superior hemostatic efficacy of Surgiflo in the porcine spleen punch biopsy model seems to be due to Surgiflo's property as a malleable barrier able to adjust to defect topography and to provide an environment for platelets to adhere and aggregate. Surgiflo combines a flowable gelatin matrix and a delivery system well-suited for precise application to bleeding sites where other methods of hemostasis may be impractical or ineffective.

Potential Role of Axonal Chemorepellent Slit2 in Modulating Adventitial Inflammation in a Rat Carotid Artery Balloon Injury Model.

Leukocyte infiltration of adventitial and perivascular tissues is an early event in the development of vascular remodeling after injury. We investigated whether Slit/Robo-an axonal chemorepellent system in vertebrate and invertebrate development-is activated during the inflammatory phase that follows endothelial denudation. Using the rat carotid artery model of angioplasty, we conducted a time course analysis of mRNAs encoding Slit ligands (Slit2 and Slit3) and Robo receptors (Robo1, Robo2, and Robo4), as well as proinflammatory cell adhesion molecule (CAM) genes. Adventitial inflammatory cells were counted in immunostained arterial sections. E-selectin, vascular CAM-1, and intercellular CAM-1 were upregulated 2-3 hours after injury, followed by infiltration of neutrophils and monocytes as evidenced by real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry. Slit2, Slit3, and Robo genes exhibited no expression changes at 3 hours; however, they were markedly upregulated 1 day after angioplasty. Intercellular CAM-1 expression was reduced by 50%, and the number of adventitial neutrophils decreased by >75% 1 day after angioplasty. Slit2 has been shown to be a potent chemorepelent of leukocytes, endothelial cells, and smooth muscle cells. Thus, we decided to further investigate the localization of Slit2 in injured vessels. Immunohistochemical stainings revealed the presence of Slit2 within the vessel wall and in the perivascular vasa vasorum of naive and injured arteries. Double immunohistochemical analyses showed that infiltrating monocytes expressed Slit2 in the perivascular and adventitial tissues of injured arteries 1 and 3 days postangioplasty. In addition, recombinant full-length Slit2 and Slit2-N/1118, an N-terminal fragment of Slit2, inhibited stromal cell-derived factor 1-mediated migration of circulating rat peripheral blood mononuclear cells. In summary, adventitial activation of CAM genes and neutrophil infiltration preceded upregulation of Slit/Robo genes. Sli2 expression colocalized with infiltrating inflammatory cells in the adventitial layer. This temporospatial association suggests that leukocyte chemorepellent Slit2 may be involved in halting the adventitial accumulation of inflammatory cells in injured vessels.

A vascular biology network model focused on inflammatory processes to investigate atherogenesis and plaque instability.

BACKGROUND: Numerous inflammation-related pathways have been shown to play important roles in atherogenesis. Rapid and efficient assessment of the relative influence of each of those pathways is a challenge in the era of "omics" data generation. The aim of the present work was to develop a network model of inflammation-related molecular pathways underlying vascular disease to assess the degree of translatability of preclinical molecular data to the human clinical setting. METHODS: We constructed and evaluated the Vascular Inflammatory Processes Network (V-IPN), a model representing a collection of vascular processes modulated by inflammatory stimuli that lead to the development of atherosclerosis. RESULTS: Utilizing the V-IPN as a platform for biological discovery, we have identified key vascular processes and mechanisms captured by gene expression profiling data from four independent datasets from human endothelial cells (ECs) and human and murine intact vessels. Primary ECs in culture from multiple donors revealed a richer mapping of mechanisms identified by the V-IPN compared to an immortalized EC line. Furthermore, an evaluation of gene expression datasets from aortas of old ApoE-/- mice (78 weeks) and human coronary arteries with advanced atherosclerotic lesions identified significant commonalities in the two species, as well as several mechanisms specific to human arteries that are consistent with the development of unstable atherosclerotic plaques. CONCLUSIONS: We have generated a new biological network model of atherogenic processes that demonstrates the power of network analysis to advance integrative, systems biology-based knowledge of cross-species translatability, plaque development and potential mechanisms leading to plaque instability.

A model-based approach to investigating the pathophysiological mechanisms of hypertension and response to antihypertensive therapies: extending the Guyton model.

Reproducibly differential responses to different classes of antihypertensive agents are observed among hypertensive patients and may be due to interindividual differences in hypertension pathology. Computational models provide a tool for investigating the impact of underlying disease mechanisms on the response to antihypertensive therapies with different mechanisms of action. We present the development, calibration, validation, and application of an extension of the Guyton/Karaaslan model of blood pressure regulation. The model incorporates a detailed submodel of the renin-angiotensin-aldosterone system (RAAS), allowing therapies that target different parts of this pathway to be distinguished. Literature data on RAAS biomarker and blood pressure responses to different classes of therapies were used to refine the physiological actions of ANG II and aldosterone on renin secretion, renal vascular resistance, and sodium reabsorption. The calibrated model was able to accurately reproduce the RAAS biomarker and blood pressure responses to combinations of dual-RAAS agents, as well as RAAS therapies in combination with diuretics or calcium channel blockers. The final model was used to explore the impact of underlying mechanisms of hypertension on the blood pressure response to different classes of antihypertensive agents. Simulations indicate that the underlying etiology of hypertension can impact the magnitude of response to a given class of therapy, making a patient more sensitive to one class and less sensitive others. Given that hypertension is usually the result of multiple mechanisms, rather than a single factor, these findings yield insight into why combination therapy is often required to adequately control blood pressure.

Theoretical and Computational Analysis of a Wurtzite-AlGaN DUV-LED to Mitigate Quantum-Confined Stark Effect with a Zincblende Comparison Considering Mg- and Be-Doping.

In this work, an AlGaN-based Deep-Ultraviolet Light-Emitting Diode structure has been designed and simulated for the zincblende and wurtzite approaches, where the polarization effect is included. DFT analysis was performed to determine the band gap direct-to-indirect cross-point limit, AlN carrier mobility, and activation energies for p-type dopants. The multiple quantum wells analysis describes the emission in the deep-ultraviolet range without exceeding the direct-to-indirect bandgap cross-point limit of around 77% of Al content. Moreover, the quantum-confined Stark effect on wavefunctions overlapping has been studied, where Al-graded quantum wells reduce it. Both zincblende and wurtzite have improved electrical and optical characteristics by including a thin AlGaN with low Al content. Mg and Be acceptor activation energies have been calculated at 260 meV and 380 meV for Be and Mg acceptor energy, respectively. The device series resistance has been decreased by using Be instead of Mg as the p-type dopant from 3 kΩ to 0.7 kΩ.

Evaluation of the Hemostatic Efficacy of Two Powdered Topical Absorbable Hemostats Using a Porcine Liver Abrasion Model of Mild to Moderate Bleeding.

INTRODUCTION: Topical hemostatic agents, used alone or in combination, have become common adjuncts to manage tissue and organ bleeding resulting from trauma and surgical procedures. Oxidized regenerated cellulose (ORC) is one of the most commonly used adjunctive hemostatic agents. The aim of the present study was to compare the hemostatic efficacy of a novel ORC-based product, SURGICEL® Powder Absorbable Hemostat (Surgicel-P) to that of HEMOBLAST™ Bellows (Hemoblast-B), a collagen-based combination powder. METHODS: Using an established porcine liver abrasion model, we randomly tested Surgicel-P and Hemoblast-B in 60 experimental lesion sites (30 per product tested). Primary endpoints included hemostatic efficacy measured by absolute time to hemostasis (TTH) within 5 minutes. We also examined number of applications required to achieve hemostasis, and sustained hemostasis following saline irrigation of test sites that achieved hemostasis. RESULTS: Surgicel-P demonstrated significantly higher hemostatic efficacy and lower TTH (p < 0.01) than Hemoblast-B. Surgicel-P-treated lesion sites achieved hemostasis in 73.3% of cases following one product application vs. 3.3% of Hemoblast-B-treated sites. Of all sites that were assessed, hemostasis was achieved and sustained following irrigation at 93.3% of Surgicel-P-treated sites vs. 50.0% of Hemoblast-B-treated sites. The average number of Surgicel-P applications per site was 51% lower than the average number of applications used for Hemoblast-B. CONCLUSION: Surgicel-P provided more effective and sustained hemostasis and faster TTH than Hemoblast-B. Surgicel-P represents a novel clinical alternative to provide adjunctive control of diffuse mild and moderate bleeding. Surgicel-P combines an ORC powder formulation and a delivery system in a device that is particularly useful for application on large surfaces and difficult-to-access anatomical locations where application of other forms of topical hemostats may be impractical.

Laparoscopic versus open total mesorectal excision: a nonrandomized comparative prospective trial in a tertiary center in Mexico City.

Because definitive long-term results are not yet available, the oncologic safety of laparoscopic surgery in rectal cancer remains controversial. Laparoscopic total mesorectal excision (LTME) for rectal cancer has been proposed to have several short-term advantages in comparison with open total mesorectal excision (OTME). However, few prospective randomized studies have been performed. The main purpose of our study was to evaluate whether relevant differences in safety and efficacy exist after elective LTME for the treatment of rectal cancer compared with OTME in a tertiary referral medical center. This comparative nonrandomized prospective study analyzes data in 56 patients with middle and lower rectal cancer treated with low anterior resection or abdominoperineal resection from November 2005 to November 2007. Follow-up was determined through office charts or direct patient contact. Statistical analysis was performed using chi2 test and Student's t test. Twenty-eight patients underwent LTME and 28 patients were in the OTME group. No conversion was required in the LTME group. Mean operating time was shorter in the laparoscopic group (LTME) (181.3 vs 206.1 min, P < 0.002). Less intraoperative blood loss and fewer postoperative complications were seen in the LTME group. Return of bowel motility was observed earlier after laparoscopic surgery. There was no 30-day mortality and the overall morbidity was 17 per cent in the LTME group versus 32 per cent in the OTME group. The mean number of harvested lymph nodes was greater in the laparoscopic group than in the OTME group (12.1 +/- 2 vs 9.3 +/- 3). Mean follow-up time was 12 months (range 9-24 months). No local recurrence was found. LTME is a feasible procedure with acceptable postoperative morbidity and low mortality, however it is technically demanding. This series confirms its safety, although oncologic results are at present comparable with the OTME published series with the limitation of a short followup period. Further randomized studies are necessary to evaluate long-term clinical outcome.

Oxygenation strategies for encapsulated islet and beta cell transplants.

Human allogeneic islet transplantation (ITx) is emerging as a promising treatment option for qualified patients with type 1 diabetes. However, widespread clinical application of allogeneic ITx is hindered by two critical barriers: the need for systemic immunosuppression and the limited supply of human islet tissue. Biocompatible, retrievable immunoisolation devices containing glucose-responsive insulin-secreting tissue may address both critical barriers by enabling the more effective and efficient use of allogeneic islets without immunosuppression in the near-term, and ultimately the use of a cell source with a virtually unlimited supply, such as human stem cell-derived ß-cells or xenogeneic (porcine) islets with minimal or no immunosuppression. However, even though encapsulation methods have been developed and immunoprotection has been successfully tested in small and large animal models and to a limited extent in proof-of-concept clinical studies, the effective use of encapsulation approaches to convincingly and consistently treat diabetes in humans has yet to be demonstrated. There is increasing consensus that inadequate oxygen supply is a major factor limiting their clinical translation and routine implementation. Poor oxygenation negatively affects cell viability and ß-cell function, and the problem is exacerbated with the high-density seeding required for reasonably-sized clinical encapsulation devices. Approaches for enhanced oxygen delivery to encapsulated tissues in implantable devices are therefore being actively developed and tested. This review summarizes fundamental aspects of islet microarchitecture and ß-cell physiology as well as encapsulation approaches highlighting the need for adequate oxygenation; it also evaluates existing and emerging approaches for enhanced oxygen delivery to encapsulation devices, particularly with the advent of ß-cell sources from stem cells that may enable the large-scale application of this approach.

Neuronal chemorepellent Slit2 inhibits vascular smooth muscle cell migration by suppressing small GTPase Rac1 activation.

The Slits are secreted proteins with roles in axonal guidance and leukocyte migration. On binding to Robo receptors, Slit2 repels developing axons and inhibits leukocyte chemotaxis. Slit2 is cleaved into Slit2-N, a protein tightly binding to cell membranes, and Slit2-C, a diffusible fragment. In the present study, we characterized the functional role of Slit2-N in vascular smooth muscle cells (VSMCs) and the cell association properties of 2 truncated versions of Slit2-N. Here, we document for the first time that Slit2-N is a chemorepellent of VSMCs. Intact blood vessels expressed Slit2 and Robo receptors as demonstrated by immunohistochemistry and quantitative real time PCR. Recombinant Slit2-N prevented the platelet-derived growth factor (PDGF)-stimulated migration of VSMCs. Slit2-N also abrogated PDGF-mediated activation of small guanosine triphosphatase (GTPase) Rac1, a member of the Rho GTPase superfamily of proteins involved in regulating the actin cytoskeleton. Furthermore, Slit2-N inhibited the PDGF-induced formation of lamellipodia, a crucial cytoskeletal reorganization event for cell motility. Slit2-N had no effect on the PDGF-mediated increase in DNA synthesis determined by [3H]thymidine uptake, suggesting that VSMC growth is unaffected by Slit2. Analysis of 2 engineered Slit2-N fragments (Slit2-N/1118 and Slit2-N/1121) indicated that 3 amino acids upstream of the putative cleavage site (Arg1121, Thr1122) are involved in the association of Slit2-N to the cell membrane. Our data assign a novel functional role to Slit2 in vascular function and show that cell guidance mechanisms that operate in the developing central nervous system are conserved in VSMCs.

Laparoscopic vs. open total mesorectal excision for treatment of rectal cancer.

INTRODUCTION: Because definitive long-term results are not yet available, the oncologic safety of laparoscopic surgery in rectal cancer remains controversial. Laparoscopic total mesorectal excision (LTME) for rectal cancer has been proposed to have several short-term advantages in comparison with open total mesorectal excision (OTME). However, few prospective randomized studies have been performed. OBJECTIVES: The main purpose was to evaluate whether there are relevant differences in safety and efficacy after elective LTME for the treatment of rectal cancer compared with OTME in a tertiary academic medical center. MATERIAL AND METHODS: This comparative non-randomized prospective study analyzes data of 20 patients with middle and low rectal cancer treated with low anterior resection (LAR) or abdomino perineal resection (APR) from November 2005 to April 2006. Follow-up was determined through office charts or direct patient contact. Statistical analysis was performed using chi2 test and Student's t-test. RESULTS: Ten patients underwent LTME and 10 patients underwent OTME. No conversion was required in the LTME group. Mean operating time was shorter in the laparoscopic group (LTME) (186.7 vs. 204.4 min, p < 0.007). Less intraoperative blood loss and fewer postoperative complications were seen in the LTME group. An earlier return of bowel motility was achieved after laparoscopic surgery. There was no 30-day mortality and the overall morbidity was 20% in the LTME group vs. 40% in the OTME group. The mean number of harvested lymph nodes was greater in the laparoscopic group than in OTME group (10.2 +/- 2.5 vs. 8.3 +/- 3). Mean follow-up time was 12 months (range 9-15 months). No local recurrence was found. CONCLUSION: LTME is a feasible procedure with acceptable postoperative morbidity and low mortality, however it is technically demanding. This series confirms its safety, while oncologic results are at present comparable to the OTME published series, with limitation of a short follow-up period though. Further randomized studies are necessary to evaluate long-term clinical outcome.

Rad GTPase attenuates vascular lesion formation by inhibition of vascular smooth muscle cell migration.

BACKGROUND: Rad (Ras associated with diabetes) GTPase is a prototypic member of a new subfamily of Ras-related GTPases with unique structural features, although its physiological role remains largely unknown. In the present study, we characterized the Rad function in vascular smooth muscle cells (VSMCs) and the influence of adenovirus-mediated Rad (Ad-Rad) gene delivery on vascular remodeling after experimental angioplasty. METHODS AND RESULTS: We documented for the first time that neointimal formation using balloon-injured rat carotid arteries was associated with a significant increase in Rad expression as determined by immunohistochemistry and quantitative real-time reverse-transcriptase polymerase chain reaction. The levels of Rad expression in VSMCs were highly induced by platelet-derived growth factor and tumor necrosis factor-alpha. Morphometric analyses 14 days after injury revealed significantly diminished neointimal formation in the Ad-Rad-treated carotid arteries compared with Ad-GFP or PBS controls, whereas the mutated form of Rad GTPase, which can bind GDP but not GTP, increased neointimal formation. Overexpression of Rad significantly inhibited the attachment and migration of VSMCs. In addition, Rad expression dramatically reduced the formation of focal contacts and stress fibers in VSMCs by blocking the Rho/ROK signaling pathway. CONCLUSIONS: Our data clearly identified Rad GTPase as a novel and critical mediator that inhibits vascular lesion formation. Manipulation of the Rad signaling pathway may provide new therapeutic approaches that will limit vascular pathological remodeling.

[Mortality-associated factors in major surgery: retrospective analysis in a referral center]. / Factores asociados a mortalidad en cirugia mayor: análisis retrospectivo en un centro de referencia.

BACKGROUND: There are very few studies that analyze surgical morbidity and mortality in the general population and the factors associated with those events. OBJECTIVE: To determine factors associated to mortality in surgical procedures performed in a tertiary referral center in Mexico City. METHODS: We retrospectively analyze surgical mortality in 4,157 consecutive surgical procedures performed in a one-year period from 1/1/2000 through 12/31/2000. Categorical variables were analyzed with the chi-square test and continuous variables with the t-Student test. Significance was defined as p < 0.05. RESULTS: During the study period there were 76 postoperative deaths, representing a mortality rate of 1.82%. Mean patient's age in the entire cohort was 48.7 +/- 17.6 years and for the patients who died in the postoperative period 57.8 +/- 17.8 years (p < 0.05). Sixty-six percent of deaths were attributed to the primary or surgical disease. In 33.8% of postoperative deaths an adverse event was identified as responsible for the outcome. In 23% of cases there was a potentially preventable event, representing 0.3% of surgical procedures. Most patients (96.9%) had at least one comorbid condition and 61.5% had two or more. Almost 80% of surgical deaths occurred in patients with ASA score III of IV and albumin levels below 3.5 g/dL. Most common cause of death was sepsis, reported in 35% of patients who died in the postoperative period. CONCLUSION: Surgical mortality in our series is low. In 0.3% of procedures it was detected a potentially preventable event. Postoperative deaths occurred in older patients with low albumin levels.

Systems toxicology-based assessment of the candidate modified risk tobacco product THS2.2 for the adhesion of monocytic cells to human coronary arterial endothelial cells.

Alterations of endothelial adhesive properties by cigarette smoke (CS) can progressively favor the development of atherosclerosis which may cause cardiovascular disorders. Modified risk tobacco products (MRTPs) are tobacco products developed to reduce smoking-related risks. A systems biology/toxicology approach combined with a functional in vitro adhesion assay was used to assess the impact of a candidate heat-not-burn technology-based MRTP, Tobacco Heating System (THS) 2.2, on the adhesion of monocytic cells to human coronary arterial endothelial cells (HCAECs) compared with a reference cigarette (3R4F). HCAECs were treated for 4h with conditioned media of human monocytic Mono Mac 6 (MM6) cells preincubated with low or high concentrations of aqueous extracts from THS2.2 aerosol or 3R4F smoke for 2h (indirect treatment), unconditioned media (direct treatment), or fresh aqueous aerosol/smoke extracts (fresh direct treatment). Functional and molecular investigations revealed that aqueous 3R4F smoke extract promoted the adhesion of MM6 cells to HCAECs via distinct direct and indirect concentration-dependent mechanisms. Using the same approach, we identified significantly reduced effects of aqueous THS2.2 aerosol extract on MM6 cell-HCAEC adhesion, and reduced molecular changes in endothelial and monocytic cells. Ten- and 20-fold increased concentrations of aqueous THS2.2 aerosol extract were necessary to elicit similar effects to those measured with 3R4F in both fresh direct and indirect exposure modalities, respectively. Our systems toxicology study demonstrated reduced effects of an aqueous aerosol extract from the candidate MRTP, THS2.2, using the adhesion of monocytic cells to human coronary endothelial cells as a surrogate pathophysiologically relevant event in atherogenesis.

An 8-Month Systems Toxicology Inhalation/Cessation Study in Apoe-/- Mice to Investigate Cardiovascular and Respiratory Exposure Effects of a Candidate Modified Risk Tobacco Product, THS 2.2, Compared With Conventional Cigarettes.

Smoking cigarettes is a major risk factor in the development and progression of cardiovascular disease (CVD) and chronic obstructive pulmonary disease (COPD). Modified risk tobacco products (MRTPs) are being developed to reduce smoking-related health risks. The goal of this study was to investigate hallmarks of COPD and CVD over an 8-month period in apolipoprotein E-deficient mice exposed to conventional cigarette smoke (CS) or to the aerosol of a candidate MRTP, tobacco heating system (THS) 2.2. In addition to chronic exposure, cessation or switching to THS2.2 after 2 months of CS exposure was assessed. Engaging a systems toxicology approach, exposure effects were investigated using physiology and histology combined with transcriptomics, lipidomics, and proteomics. CS induced nasal epithelial hyperplasia and metaplasia, lung inflammation, and emphysematous changes (impaired pulmonary function and alveolar damage). Atherogenic effects of CS exposure included altered lipid profiles and aortic plaque formation. Exposure to THS2.2 aerosol (nicotine concentration matched to CS, 29.9 mg/m(3)) neither induced lung inflammation or emphysema nor did it consistently change the lipid profile or enhance the plaque area. Cessation or switching to THS2.2 reversed the inflammatory responses and halted progression of initial emphysematous changes and the aortic plaque area. Biological processes, including senescence, inflammation, and proliferation, were significantly impacted by CS but not by THS2.2 aerosol. Both, cessation and switching to THS2.2 reduced these perturbations to almost sham exposure levels. In conclusion, in this mouse model cessation or switching to THS2.2 retarded the progression of CS-induced atherosclerotic and emphysematous changes, while THS2.2 aerosol alone had minimal adverse effects.

Aerosol from a candidate modified risk tobacco product has reduced effects on chemotaxis and transendothelial migration compared to combustion of conventional cigarettes.

Reduction of harmful constituents by heating rather than combusting tobacco is a promising new approach to reduce harmful effects associated with cigarette smoking. We investigated the effect from a new candidate modified risk tobacco product, the tobacco heating system (THS) 2.2, on the migratory behavior of monocytes in comparison with combustible 3R4F reference cigarettes. The monocytic cell line (THP-1) and human coronary arterial endothelial cells (HCAECs) were used to analyze chemotaxis and transendothelial migration (TEM). To assess the influence of aerosol extract from THS2.2 and smoke extract from 3R4F on toxicity and inflammation, flow cytometry and ELISA assays were performed. The results show that treatment of THP-1 cells with extract from 3R4F or THS2.2 induced concentration-dependent increases in cytotoxicity and inflammation. The inhibitory effects of THS2.2 extract for chemotaxis and TEM were ∼18 times less effective compared to 3R4F extract. Furthermore, extract from 3R4F or THS2.2 induced concentration-dependent decreases in the integrity of HCAEC monolayer. For all examined endpoints, the extract from 3R4F showed more than one order of magnitude stronger effects than that from THS2.2 extract. These data indicate the potential of a heat not burn tobacco product to reduce the risk for cardiovascular disease compared to combustible cigarettes.

Systems Biology Reveals Cigarette Smoke-Induced Concentration-Dependent Direct and Indirect Mechanisms That Promote Monocyte-Endothelial Cell Adhesion.

Cigarette smoke (CS) affects the adhesion of monocytes to endothelial cells, a critical step in atherogenesis. Using an in vitro adhesion assay together with innovative computational systems biology approaches to analyze omics data, our study aimed at investigating CS-induced mechanisms by which monocyte-endothelial cell adhesion is promoted. Primary human coronary artery endothelial cells (HCAECs) were treated for 4 h with (1) conditioned media of human monocytic Mono Mac-6 (MM6) cells preincubated with low or high concentrations of aqueous CS extract (sbPBS) from reference cigarette 3R4F for 2 h (indirect treatment, I), (2) unconditioned media similarly prepared without MM6 cells (direct treatment, D), or (3) freshly generated sbPBS (fresh direct treatment, FD). sbPBS promoted MM6 cells-HCAECs adhesion following I and FD, but not D. In I, the effect was mediated at a low concentration through activation of vascular inflammation processes promoted in HCAECs by a paracrine effect of the soluble mediators secreted by sbPBS-treated MM6 cells. Tumor necrosis factor α (TNFα), a major inducer, was actually shed by unstable CS compound-activated TNFα-converting enzyme. In FD, the effect was triggered at a high concentration that also induced some toxicity. This effect was mediated through an yet unknown mechanism associated with a stress damage response promoted in HCAECs by unstable CS compounds present in freshly generated sbPBS, which had decayed in D unconditioned media. Aqueous CS extract directly and indirectly promotes monocytic cell-endothelial cell adhesion in vitro via distinct concentration-dependent mechanisms.

Systems Biology Research into Cardiovascular Disease: Contributions of Lipidomics-based Approaches to Biomarker Discovery.

Atherosclerosis is a progressive inflammatory thickening of the arterial wall resulting from increased cellularity and the accumulation of lipids, cellular debris, and extracellular matrix. Conventional determinations of plasma lipoproteins have resulted in a wealth of clinical data documenting the correlation between low- and high-density lipoproteins (LDL and HDL) and cardiovascular disease (CVD) risk. Current mass spectrometry methodologies allow the detection and quantification of multiple molecular lipid species with various structural and functional roles. The opportunities provided by lipidomics for lipid-based biomarker discovery are prominent in disease diagnostics, monitoring of drug efficacy, and translational model development. For example, the analysis of human plasma samples assessing the effects of statins has shown correlative effects between the LDL/HDL ratio and sphingomyelin and phosphatidylcholine. Additionally, at the vascular tissue level, lipids from different classes are enriched in human plaques of coronary arteries and in the aortas of apolipoprotein E-deficient mice exposed to cigarette smoke, highlighting a set of lipid biomarkers for translational research. Molecular lipidomics will provide insights in which other lipids may play important roles in vascular disease progression and will serve as novel markers for preventive as well as therapeutic purposes.

A prototypic modified risk tobacco product exhibits reduced effects on chemotaxis and transendothelial migration of monocytes compared with a reference cigarette.

Monocyte adhesion and migration to the subendothelial space represent critical steps in atherogenesis. Here, we investigated whether extracts from the aerosol of a prototypic modified risk tobacco product (pMRTP), based on heating rather than combusting tobacco, exhibited differential effects on the migratory behavior of monocytes compared with that from the reference cigarette, 3R4F. THP-1 cells, a monocytic cell line, and human coronary arterial endothelial cells (HCAECs) were used to investigate chemotaxis and transendothelial migration (TEM) of monocytes in conventional and impedance-based systems. THP-1 cells migrated through a monolayer of HCAECs in response to C-X-C motif ligand 12 (CXCL12), a chemokine involved in diverse cellular functions including chemotaxis and survival of stem cells. Treatment of THP-1 cells with extracts from 3R4F or pMRTP induced concentration-dependent increases in cytotoxicity (7-aminoactinomycin D), and inflammation (IL-8 and TNF-α). CXCL12-mediated chemotaxis and TEM were decreased in extract-treated THP-1 cells. Extracts from 3R4F were ~21 times more potent than those from pMRTP in all examined endpoints. Extracts from 3R4F and pMRTP induced concentration-dependent responses in assays of inflammation, cytotoxicity, chemotaxis, and TEM. Furthermore, our findings indicate that extracts from a pMRTP are significantly less cytotoxic and induce less inflammation than those from the reference cigarette, 3R4F.