Electroencephalography-Guided Anesthesia and Delirium in Older Adults After Cardiac Surgery: The ENGAGES-Canada Randomized Clinical Trial.

Importance: Intraoperative electroencephalogram (EEG) waveform suppression, suggesting excessive general anesthesia, has been associated with postoperative delirium. Objective: To assess whether EEG-guided anesthesia decreases the incidence of delirium after cardiac surgery. Design, Setting, and Participants: Randomized, parallel-group clinical trial of 1140 adults 60 years or older undergoing cardiac surgery at 4 Canadian hospitals. Recruitment was from December 2016 to February 2022, with follow-up until February 2023. Interventions: Patients were randomized in a 1:1 ratio (stratified by hospital) to receive EEG-guided anesthesia (n = 567) or usual care (n = 573). Patients and those assessing outcomes were blinded to group assignment. Main Outcomes and Measures: The primary outcome was delirium during postoperative days 1 through 5. Intraoperative measures included anesthetic concentration and EEG suppression time. Secondary outcomes included intensive care and hospital length of stay. Serious adverse events included intraoperative awareness, medical complications, and 30-day mortality. Results: Of 1140 randomized patients (median [IQR] age, 70 [65-75] years; 282 [24.7%] women), 1131 (99.2%) were assessed for the primary outcome. Delirium during postoperative days 1 to 5 occurred in 102 of 562 patients (18.15%) in the EEG-guided group and 103 of 569 patients (18.10%) in the usual care group (difference, 0.05% [95% CI, -4.57% to 4.67%]). In the EEG-guided group compared with the usual care group, the median volatile anesthetic minimum alveolar concentration was 0.14 (95% CI, 0.15 to 0.13) lower (0.66 vs 0.80) and there was a 7.7-minute (95% CI, 10.6 to 4.7) decrease in the median total time spent with EEG suppression (4.0 vs 11.7 min). There were no significant differences between groups in median length of intensive care unit (difference, 0 days [95% CI, -0.31 to 0.31]) or hospital stay (difference, 0 days [95% CI, -0.94 to 0.94]). No patients reported intraoperative awareness. Medical complications occurred in 64 of 567 patients (11.3%) in the EEG-guided group and 73 of 573 (12.7%) in the usual care group. Thirty-day mortality occurred in 8 of 567 patients (1.4%) in the EEG-guided group and 13 of 573 (2.3%) in the usual care group. Conclusions and Relevance: Among older adults undergoing cardiac surgery, EEG-guided anesthetic administration to minimize EEG suppression, compared with usual care, did not decrease the incidence of postoperative delirium. This finding does not support EEG-guided anesthesia for this indication. Trial Registration: ClinicalTrials.gov Identifier: NCT02692300.

Real-Time Use of SGLT2i Verified in Pre-dialysis: The RSVP Cross-sectional Study.

BACKGROUND: The use of sodium-glucose cotransporter 2 inhibitors (SGLT2i) in nephrology practice is increasingly becoming standard of care in patients with diabetes or those with proteinuria. OBJECTIVES: The primary outcome was to identify the proportion of pre-dialysis patients with chronic kidney disease (CKD) G3a, G3b, or G4 prescribed an SGLT2i and describe their characteristics. METHODS: This was a retrospective, multicentric, cross-sectional study of patients with CKD followed at 4 pre-dialysis clinics in the province of Quebec, Canada. We collected data of multiple covariates associated with prescribing SGLT2i in patients over 18 years of age with CKD G3a, G3b, or G4. We then performed a multivariate logistic regression to assess their associations. RESULTS: Of the 874 patients included, 22.7% were prescribed an SGLT2i. Factors most strongly associated included male sex (odds ratio [OR] = 4.88, 95% CI = 2.38-10.03), being prescribed metformin (OR = 4.30, 95% CI = 2.23-8.31), having type 2 diabetes (OR = 4.00, 95% CI = 1.86-8.62), or having an albumin-to-creatinine ratio greater than 300 mg/g (OR = 1.84, 95% CI = 1.08-3.14). The majority of patients (60.4%) had their SGLT2i initiated by the pre-dialysis clinic and the most frequent adverse event was an initial increase in serum creatinine 1 week after starting treatment (33.9%). CONCLUSION AND RELEVANCE: An increasing number of patients with CKD are being prescribed SGLT2i. Nonetheless, significant disparities in sex, severity of disease, and comorbidities remain. We suggest that specific strategies be put in place to promote prescribing of SGLT2i in women and other at-risk populations, in particular among nephrology teams, to improve patient care.

Development and Evaluation of a Height-Based Tobramycin Initial Dosing Nomogram for the Treatment of Adult Cystic Fibrosis Pulmonary Exacerbations.

ABSTRACT: Tobramycin is widely used to treat pulmonary exacerbations of cystic fibrosis. Height has been previously found to be significantly more predictive of tobramycin pharmacokinetics than body weight. This study aimed to develop a height-based initial dosing nomogram and evaluate its performance in peak concentration (Cmax) precision relative to standard and fixed dosing. Monte Carlo simulations were performed to develop a nomogram representing the doses required to reach Cmax targets at different heights. Cmax data observed at 2 clinical centers [McGill University Health Centre (MUHC) and Institut universitaire de cardiologie et pneumologie de Québec (IUCPQ-UL)] were compared with population-predicted Cmax using the doses derived from the nomogram alongside a fixed dose. Height-based dosing resulted in significantly less variable-predicted Cmax values [coefficient of variation (CV) MUHC = 15.7% and IUCPQ-UL = 10.8%] than the Cmax values observed in clinical practice (CV MUHC = 30.0% and CV IUCPQ-UL = 26.9%) and predicted Cmax values obtained from a fixed dose (CV MUHC = 21.2% and CV IUCPQ-UL = 16.3%). An initial dosing nomogram was developed to help reduce pharmacokinetic variability in the observed Cmax. More precise dosing would allow for better clinical outcomes in adult patients with cystic fibrosis.

Consideration of height-based tobramycin dosing regimens for the treatment of adult cystic fibrosis pulmonary exacerbations.

AIMS: Some population pharmacokinetic models have been developed using height to explain some of the interindividual variability in tobramycin pharmacokinetics in cystic fibrosis patients. However, their predictive performance when extrapolated to other clinical centres is unclear. Therefore, the aim of this study was to externally evaluate the predictability of tobramycin population pharmacokinetic models with an independent dataset and perform simulations using previously recommended height-based dosing regimens. METHODS: A literature search was conducted through the PubMed database to identify relevant population pharmacokinetic models. Tobramycin plasma concentration data from April 2014 to November 2019 were retrospectively collected from the Institut universitaire de cardiologie et de pneumologie de Québec, Canada. External evaluations were performed using NONMEM® v7.5 and RStudio® v1.3.1073. Monte Carlo simulations were performed to evaluate the probability of target attainment of Cmax /MIC ratios for several dosing regimens. RESULTS: The validation dataset included 27 patients and 143 concentration samples. Three models were evaluated. Only the ones by Crass et al. and Alghanem et al. performed satisfactorily in terms of prediction-based diagnostics with MDPE values of -3.4% and 29.3% and MDAPE values of 19.0 and 29.5%, respectively. In simulation-based evaluations, both pcVPC and NPDE showed no evidence of model misspecification. Our simulations suggest that patients treated with a once-daily dose of 3.4 mg/cm should produce peak and trough levels consistent with current guidelines. CONCLUSION: Our results show that the models by Crass et al. and Alghanem et al. are appropriate for simulation-based applications to aid individualized dosing in our population and that height-based dosing regimens could be considered in cystic fibrosis patients.

Effect of Detecting and Isolating Clostridium difficile Carriers at Hospital Admission on the Incidence of C difficile Infections: A Quasi-Experimental Controlled Study.

IMPORTANCE: Clostridium difficile infection (CDI) is a major cause of health care-associated infection worldwide, and new preventive strategies are urgently needed. Current control measures do not target asymptomatic carriers, despite evidence that they can contaminate the hospital environment and health care workers' hands and potentially transmit C difficile to other patients. OBJECTIVE: To investigate the effect of detecting and isolating C difficile asymptomatic carriers at hospital admission on the incidence of health care-associated CDI (HA-CDI). DESIGN, SETTING, AND PARTICIPANTS: We performed a controlled quasi-experimental study between November 19, 2013, and March 7, 2015, in a Canadian acute care facility. Admission screening was conducted by detecting the tcdB gene by polymerase chain reaction on a rectal swab. Carriers were placed under contact isolation precautions during their hospitalization. MAIN OUTCOMES AND MEASURES: Changes in HA-CDI incidence level and trend during the intervention period (17 periods of 4 weeks each) were compared with the preintervention control period (120 periods of 4 weeks each) by segmented regression analysis and autoregressive integrated moving average (ARIMA) modeling. Concomitant changes in the aggregated HA-CDI incidence at other institutions in Québec City, Québec (n = 6) and the province of Québec (n = 94) were also examined. RESULTS: Overall, 7599 of 8218 (92.5%) eligible patients were screened, among whom 368 (4.8%) were identified as C difficile carriers. During the intervention, 38 patients (3.0 per 10 000 patient-days) developed an HA-CDI compared with 416 patients (6.9 per 10 000 patient-days) during the preintervention control period (P < .001). There was no immediate change in the level of HA-CDIs on implementation (P = .92), but there was a significant decrease in trend over time of 7% per 4-week period (rate ratio, 0.93; 95% CI, 0.87-0.99 per period; P = .02). ARIMA modeling also detected a significant effect of the intervention, represented by a gradual progressive decrease in the HA-CDI time series by an overall magnitude of 7.2 HA-CDIs per 10 000 patient-days. We estimated that the intervention had prevented 63 of the 101 (62.4%) expected cases. By contrast, no significant decrease in HA-CDI rates occurred in the control groups. CONCLUSIONS AND RELEVANCE: Detecting and isolating C difficile carriers was associated with a significant decrease in the incidence of HA-CDI. If confirmed in subsequent studies, this strategy could help prevent HA-CDI.

Estradiol inhibits vascular endothelial cells pro-inflammatory activation induced by C-reactive protein.

In addition of being an important inflammatory biomarker and a risk factor for cardiovascular disease, much evidence indicates that the C-reactive protein (CRP) contributes to the atherosclerosis development process. This plasmatic protein synthesized by hepatocytes in response to inflammation and tissue injury induces pro-inflammatory molecules' expression by endothelial cells (ECs). Previous studies showed that the 17ß-estradiol (E2) has beneficial effects on vascular cells by reducing in vitro pro-inflammatory molecules expressions in EC. Therefore, we hypothesize that E2 blocks or reduces CRP-mediated inflammatory responses by modulating endogenous production of CRP in EC and/or activation mechanisms. Using human aortic ECs (HAECs), we first evaluated CRP production by vascular EC and second demonstrated its self-induction. Indeed, recombinant human CRP stimulation induces a fivefold increase of CRP expression. A 1-h pre-treatment of E2 at a physiologic dose (10(-9 )M) leads to an important decrease of CRP production suggesting a partial blockage of its amplification loop mechanism. Furthermore, in HAEC, E2 reduces the secretion of the most potent agonist of CRP induction, the IL-6, by 21 %. E2 pre-treatment also decreased the expression of pro-inflammatory molecules IL-8, VCAM-1, and ICAM-1 induced by CRP and involved in leukocytes recruitment. In addition, we demonstrated that E2 could restore vascular endothelial growth factor-mediated EC migration response impaired by CRP suggesting another pro-angiogenic property of this hormone. These findings suggest that E2 can interfere with CRP pro-inflammatory effects via activation signals using its rapid, non-genomic pathway that may provide a new mechanism to improve vascular repair.

Tuning the properties and functions of 17ß-estradiol-polysaccharide conjugates in thin films: impact of sample history.

In addition to its role in the regulation of sex-related processes, 17ß-estradiol (E2) participates in the prevention and treatment of cardiovascular diseases via nongenomic pathways mediated by estrogen receptors (ER-α) located in the cell membrane. To achieve specific nongenomic activity of E2, we linked E2 (4.4 mol %) to chitosan-phosphorylcholine (CH-PC) (20 mol % PC). Injections of ER-α solutions (5 to 100 nmol L(-1)) over rehydrated CH-PC-E2 thin films led to permanent adsorption of ER-α to the film surface, as detected by quartz crystal microbalance with dissipation (QCM-D). However, ER-α did not bind onto CH-PC-E2 films formed in situ and never dried. X-ray photoelectron spectroscopy (XPS) analysis of spin-cast CH-PC-E2 films revealed significant E2 enrichment of the topmost section of the film, attributed to the preferential migration of E2 toward the film/air interface upon drying. Mechanical analysis of CH-PC-E2 films in the frequency domain probed by QCM-D indicated that rehydrated films behave as an entangled network with junction points formed by self-assembly of hydrophobic E2 moieties and by ion pairing among PC groups, whereas films formed in situ are entangled polymer solutions with temporary junctions. The structural analysis presented offers useful guidelines for the study of amphiphilic biomacromolecules designed for therapeutic use as thin films.

Vascular smooth muscle cell alterations triggered by mice adipocytes: role of high-fat diet.

AIM: Inherent mechanisms leading to vascular smooth muscle cells (VSMC) alterations in obesitylinked type 2 diabetes (T2D) situation remain to be clarified. This study evaluates the impact of supernatant of adipocytes extracted from mice fed high-fat-diets (HFD) on the proliferation and apoptosis of VSMC. METHODS: Adipocytes were extracted from visceral white fat pads of male and female C57Bl6 mice showing different stages of metabolic alterations after 20 weeks of vegetal or animal HFD feeding. These cells were stimulated or not with insulin or glucose to condition VSMC media. After 24h of stimulation with adipocyte supernatants (AdS), VSMC proliferation and sustainability were assessed in the absence and presence of AdS. CD36 and insulin receptor mRNA levels were also evaluated. RESULTS: Proliferation and viability of VSMC were significantly modulated by the nature of the AdS used and the gender of mice from which adipocytes have been extracted. The most extensive effects on VSMC were triggered by adipocytes from males fed animal HFD and females fed vegetal HFD. These effects were concurrent with increased leptin concentration and decreased adiponectin levels in AdS. In addition, adipocytes of HFD-fed mice increased caspase-3 activity and apoptosis in VSMC. Significant up-regulation of CD36 mRNA was also found in these cells. CONCLUSION: Adipocytes of HFD-fed mice induce VSMC alterations. These changes involved mouse gender, most probably correlated to the diet-induced adipocyte secretion profile. Greater sensitivity to AdS effects in VSMC raises concerns about the more frequent cardiovascular events associated with obesity in the presence of T2D, which impairs adipocyte activity.

Key fatty acid combinations define vascular smooth muscle cell proliferation and viability.

High plasma concentrations of free fatty acids (FFA) and insulin are common features in atherosclerotic patients with type 2 diabetes. FFA, according to their nature, can have various effects on vascular smooth muscle cells (VSMC). These cells play important roles throughout atherosclerosis pathogenesis, from plaque development to plaque instability. Thus, this study aims to assess the impact of two FFA combinations and insulin on murine VSMC viability. The two combinations contain the same FFA but at different ratios, one being richer in saturated fatty acids (SFA) and the other having a higher proportion of monounsaturated fatty acids (MUFA). Both combinations inhibited VSMC proliferation due to their pro-apoptotic potential, with SFA being the major inducers of apoptosis. However, the presence of oleic acid (OLA) attenuated this impact in a dose-dependent manner. OLA had also the capacity to reduce apoptosis rates more strongly when combined with a SFA than when used alone in VSMC treatments. This effect was significant only for specific proportions of these FFA and was even more effective in presence of insulin. These results highlight the presence of a competition between pro-apoptotic and anti-apoptotic mechanisms in VSMC that is dependent on FFA ratios (saturated vs. monounsaturated) and on insulinemia. They also underline the importance of the presence of MUFA such as OLA in diets containing high proportions of SFA.

Nature of fatty acids in high fat diets differentially delineates obesity-linked metabolic syndrome components in male and female C57BL/6J mice.

BACKGROUND: Adverse effects of high-fat diets (HFD) on metabolic homeostasis are linked to adipose tissue dysfunction. The goal of this study was to examine the effect of the HFD nature on adipose tissue activity, metabolic disturbances and glucose homeostasis alterations in male mice compared with female mice. METHODS: C57BL/6J mice were fed either a chow diet or HFD including vegetal (VD) or animal (AD) fat. Body weight, plasmatic parameters and adipose tissue mRNA expression levels of key genes were evaluated after 20 weeks of HFD feeding. RESULTS: HFD-fed mice were significantly heavier than control at the end of the protocol. Greater abdominal visceral fat accumulation was observed in mice fed with AD compared to those fed a chow diet or VD. Correlated with weight gain, leptin levels in systemic circulation were increased in HFD-fed mice in both sexes with a significant higher level in AD group compared to VD group. Circulating adiponectin levels as well as adipose tissue mRNA expression levels were significantly decreased in HFD-fed male mice. Although its plasma levels remained unchanged in females, adiponectin mRNA levels were significantly reduced in adipose tissue of both HFD-fed groups with a more marked decrease in AD group compared to VD group. Only HFD-fed male mice were diabetic with increased fasting glycaemia. On the other hand, insulin levels were only increased in AD-fed group in both sexes associated with increased resistin levels. VD did not induce any apparent metabolic alteration in females despite the increased weight gain. Peroxisome Proliferator-Activated Receptors gamma-2 (PPARγ2) and estrogen receptor alpha (ERα) mRNA expression levels in adipose tissue were decreased up to 70% in HFD-fed mice but were more markedly reduced in male mice as compared with female mice. CONCLUSIONS: The nature of dietary fat determines the extent of metabolic alterations reflected in adipocytes through modifications in the pattern of adipokines secretion and modulation of key genes mRNA expression. Compared with males, female mice demonstrate higher capacity in controlling glucose homeostasis in response to 20 weeks HFD feeding. Our data suggest gender specific interactions between the diet's fatty acid source, the adipocyte-secreted proteins and metabolic disorders.

A phosphorylcholine-modified chitosan polymer as an endothelial progenitor cell supporting matrix.

The aim of the present study was to develop a new biopolymer to increase endothelial progenitor cells (EPC) survival and amplification. As a cell culture platform, bone marrow-derived cells (BMDC) were used to investigate the biocompatibility of chitosan-phosphorylcholine (CH-PC). On CH-PC, BMDC were found in colonies with a mortality rate similar to that of fibronectin (FN), the control matrix. Adhesion/proliferation assays demonstrated a greater number of BMDC on CH-PC after 7 days with an amplification phase occurring during the second week. Difference in adhesion mechanisms between (CH-PC) and the control FN matrix suggest distinctive cell retention ability. Confocal microscopy analyses confirmed that (CH-PC) supported the survival/differentiation of endothelial cells. Moreover, flow cytometry analyses demonstrated that, (CH-PC) increased the percentage of progenitor cells (CD117(+)CD34(+)) (7.1 ± 0.8%, FN: 4.1 ± 0.8%) and EPC (CD117(+)CD34(+)VEGFR-2(+)CD31(+)) (2.33 ± 0.6%, FN: 0.25 ± 0.1%), while the mesenchymal stem cell fraction (CD44(+)CD106(+)CD90(+)) was decreased (0.07 ± 0.01%, FN: 0.55 ± 0.22%). Polymeric substrate CH-PC might provide a suitable surface to promote the amplification of EPC for future vascular therapeutic applications.

Group V secreted phospholipase A2 contributes to LPS-induced leukocyte recruitment.

Secreted phospholipases A(2) (sPLA(2)s) are well known for their contribution in the biosynthesis of inflammatory eicosanoids. These enzymes also participate in the inflammatory process by regulating chemokine production and protein expression of adhesion molecules. The majority of sPLA(2) isoforms are up-regulated by proinflammatory stimuli such as bacterial lipopolysaccharide (LPS), which predominantly increases the expression of group V sPLA(2) (sPLA(2)-V). Furthermore, it has recently been shown that sPLA(2)-V is a critical messenger in the regulation of cell migration during allergic airway responsiveness. Herein, we investigated the effect of sPLA(2)-V on LPS-mediated leukocyte recruitment and its capacity to modulate adhesion molecule expression. We conducted our study in the murine air pouch model, using sPLA(2)-V null mice (sPLA(2)-V(-/-)) and control wild-type (WT) littermates. We observed that LPS (1 microg/ml)-mediated leukocyte emigration in sPLA(2)-V(-/-) was attenuated by 52% and 86% upon 6 and 12 h of treatment respectively, as compared to WT mice. In WT mice, treatment with the cell-permeable sPLA(2) inhibitor (12-epi-scalaradial; SLD) reduced LPS-mediated leukocyte recruitment by 67%, but had no additional inhibitory effect in sPLA(2)-V(-/-) mice. Protein analyses from the air pouch skin were carried out upon LPS-challenge, and the expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were both significantly reduced in sPLA(2)-V(-/-) mice as compared to control WT mice. Together, our data demonstrate the role of sPLA(2)-V in LPS-induced ICAM-1 and VCAM-1 protein overexpression and leukocyte recruitment, supporting the contribution of sPLA(2)-V in the development of inflammatory innate immune responses.

Biophysical studies of the interactions between the phage varphiKZ gp144 lytic transglycosylase and model membranes.

The use of naturally occurring lytic bacteriophage proteins as specific antibacterial agents is a promising way to treat bacterial infections caused by antibiotic-resistant pathogens. The opportunity to develop bacterial resistance to these agents is minimized by their broad mechanism of action on bacterial membranes and peptidoglycan integrity. In the present study, we have investigated lipid interactions of the gp144 lytic transglycosylase from the Pseudomonas aeruginosa phage varphiKZ. Interactions with zwitterionic lipids characteristic of eukaryotic cells and with anionic lipids characteristic of bacterial cells were studied using fluorescence, solid-state nuclear magnetic resonance, Fourier transform infrared, circular dichroism, Langmuir monolayers, and Brewster angle microscopy (BAM). Gp144 interacted preferentially with anionic lipids, and the presence of gp144 in anionic model systems induced membrane disruption and lysis. Lipid domain formation in anionic membranes was observed by BAM. Gp144 did not induce disruption of zwitterionic membranes but caused an increase in rigidity of the lipid polar head group. However, gp144 interacted with zwitterionic and anionic lipids in a model membrane system containing both lipids. Finally, the gp144 secondary structure was not significantly modified upon lipid binding.

Menstrual cycle influences endothelial progenitor cell regulation: a link to gender differences in vascular protection?

BACKGROUND: The potential role of endothelial progenitor cells (EPCs) in the beneficial effects of estrogen on women's cardiovascular health is of great interest. We thus evaluated if menstrual cycle influences circulating levels of EPC subpopulations in normally menstruating women and if this could underline gender differences. METHODS AND RESULTS: Ten women and ten men were recruited for this study. Peripheral blood samples were collected at each menstrual cycle phase in women and, three times over a one month period in men. Flow cytometry analysis revealed that, in women, the number of CD133+/CD34-, CD133+/CD34+ progenitor cells (PCs) and CD133+/CD34+/VEGF-R2+ EPCs per ml of blood fluctuated significantly throughout the cycle in synchronization with the level of circulating 17beta-estradiol (17betaE). Maturation of CD133+/VEGF-R2+ and CD133+/CD34-/VEGF-R2+ EPCs towards respective CD144+ advanced EPC (aEPC) subpopulations was reduced at mid-luteal phase. Greater mean global number of CD133+/CD34+ PC, CD133+/VEGF-R2+ and CD133+/CD34-/VEGF-R2+ EPC subpopulations was found in women and 17betaE was identified as a predictive factor for the gender differences perceived. Finally, maturation of CD133+ and CD133+/CD34- towards respective EPCs or aEPCs was increased in women compared to men. CONCLUSIONS: Our results suggest a physiological regulation of the availability of PC and EPC subpopulations in premenopausal women throughout menstrual cycle and reveal gender differences in the level and maturity of specific PC, EPC and aEPC subpopulations.This cyclic regulation in premenopausal women, may in part explain the lower prevalence of cardiovascular events at middle age compared with men or the timing of such events during the menstrual cycle.

Estrogen-induced gene expression in bone marrow c-kit+ stem cells and stromal cells: identification of specific biological processes involved in the functional organization of the stem cell niche.

The recent interest in the role of bone marrow (BM)-derived endothelial progenitor cells (EPCs) and the benefits of estrogen on cardiovascular health brought us to evaluate if estrogen could affect cardiac repair more broadly by regulating biological processes involved in the functional organization of the BM stem cell (SC) niche. To assess such possibility, we evaluated gene expression profiles of BM c-kit+ SCs and CD44+ stromal cells (StroCs) after exposure to a physiological concentration of 17beta-estradiol (17betaE). Data analysis showed that 17betaE altered the expression (>1.5 fold) of 509 and 682 gene probes in c-kit+ SCs and CD44+ StroCs, respectively. Among them, 199 genes in c-kit+ SCs and 283 in CD44+ StroCs were associated to biological process categories of the Gene Ontology classification. Within processes highly regulated by 17betaE, we identified key factors involved in adhesion, migration, proteolysis, and signaling by which 17betaE influences physiological regulation of the functional organization of the SC niche. Together, our results demonstrate that estrogen benefits on cardiovascular health could involve other BM-derived cells than EPCs and that this capacity of estrogen to influence the physiology of the BM SC niche deserves to be investigated clinically.

Local delivery of 17-beta-estradiol modulates collagen content in coronary porcine arteries after PTCA and stent implantation.

BACKGROUND: Percutaneous transluminal coronary angioplasty (PTCA) and stent implantation are associated with intimal hyperplasia and extracellular matrix (ECM) accumulation, resulting in restenosis. We showed that local delivery of 17-beta-estradiol (17betaE) reduced restenosis following PTCA and stent implantation by 47 and 23%, respectively. Because estrogens decreased type I and type III collagen synthesis in vitro, we hypothesized that local delivery of 17betaE may influence intimal hyperplasia formation by modulating ECM expression. METHODS: Porcine coronary arteries underwent PTCA or stenting and were randomly assigned to 17betaE or placebo. After 28 days, animals were sacrificed for histology and collagen type I and III content analysis. RESULTS: Both collagen subtypes increased in the media by 1.7 to 2.6-fold after PTCA and by 15.7 to 16.1-fold after stenting, as compared to PTCA segments. In the neointima, the ratio of collagen type III to type I was 2.7 in stented arteries and only 0.3 in PTCA arteries. In the neointima of 17betaE-treated animals, collagen type I (but not type III) content upregulation was limited by 53% after PTCA and by 74% after stenting. CONCLUSION: Local delivery of 17betaE reduces restenosis, in part by decreasing the density of collagen type I in the neointima in PTCA and stented arteries.

In situ conformation of spider silk proteins in the intact major ampullate gland and in solution.

To understand the spinning process of dragline silk by spiders, the protein conformation before spinning has to be determined. Raman confocal spectromicroscopy has been used to study the conformation of the proteins in situ in the intact abdominal major ampullate gland of Nephila clavipes and Araneus diadematus spiders. The spectra obtained are typical of natively unfolded proteins and are very similar to that of a mixture of recombinant silk proteins. Vibrational circular dichroism reveals that the conformation is composed of random and polyproline II (PPII) segments with some alpha-helices. The alpha-helices seem to be located in the C-terminal part whereas the repetitive sequence is unfolded. The PPII structure can significantly contribute to the efficiency of the spinning process in nature.

Peptidoglycan lytic activity of the Pseudomonas aeruginosa phage phiKZ gp144 lytic transglycosylase.

The gp144 endolysin gene from the Pseudomonas aeruginosa phage phiKZ was cloned and studies of gp144 expression into Escherichia coli showed host cell lysis. The gp144 protein was purified directly from the culture supernatant and from the bacterial cell pellet and showed in vitro antibacterial lytic activity against P. aeruginosa bacteria and degraded purified peptidoglycan of Gram-negative bacteria. MS analysis identified the gp144 peptidoglycan cleavage site and confirmed a lytic transglycosylase enzyme. Studies of gp144 expression in the presence of sodium azide (NaN(3)), an inhibitor of the protein export machinery, and into an E. coli MM52 secA(ts) mutant at permissive and restrictive temperatures showed that gp144 was secreted independently of the Sec system. The solution conformation of purified gp144 analyzed by circular dichroism spectroscopy was 61% in alpha-helical content, and showed a 72% decrease when interacting with dimyristoylphosphatidylglycerol (DMPG), one of the major components of bacterial membranes and less than 10% with dimyristoylphosphatidylcholine (DMPC) found in eukaryotic membranes. Membrane vesicles of DMPG anionic lipids containing calcein indicated that gp144 caused a rapid release of fluorescent calcein when interacting with synthetic membranes. These results indicated that gp144 from phiKZ is a lytic transglycosylase capable of interacting with and disorganizing bacterial membranes and has potential as an antipseudomonal in phage therapy.

Stent implantation in coronary porcine arteries is associated with early activation of TNFalpha and TNFalpha receptor II expression.

Inflammation present in restenosis after angioplasty is associated with production of cytokines such as tumor necrosis factor (TNFalpha). However, limited data exist on the possible increase in TNFalpha and TNFalpha receptor expression induced during the chronic phase after stenting. To this end, swine underwent balloon denudation (PTCA) and stent implantation in coronary arteries. At day 1, 7 or 28 post-procedure, sections from injured and reference vessel segments were evaluated for extent of pathology and expression of TNFalpha and TNFalpha receptors (RI and RII). Restenosis assessed at days 7 and 28 showed, respectively, two- and six-fold more neointimal (NI) area in stented than in PTCA segments. Unlike reference segments, TNFalpha-positive cells were detected in both the media and the NI of injured segments, with a significant increase over the 28-day time frame. Stenting was associated with an eight-fold enhancement in TNFalpha expression over PTCA. TNFalpha expression and NI area tended to correlate in injured segments. Furthermore, the pattern of expression of TNFalpha-RII, but not TNFalpha-RI, resembled that of TNFalpha itself. These results implicate TNFalpha and TNFalpha-RII as important actors in both the acute and the chronic phases of inflammation following stent implantation.

Estradiol blocks the induction of CD40 and CD40L expression on endothelial cells and prevents neutrophil adhesion: an ERalpha-mediated pathway.

OBJECTIVE: Interferon gamma (IFN-gamma) was shown to induce CD40 and CD40L expression on endothelial cells (ECs) and consequently to promote neutrophil adhesion. The pro- and anti-inflammatory effects of estrogens are well recognized but their role on the regulation of CD40 and CD40L expression on ECs remains undefined. METHODS AND RESULTS: Treatment of porcine aortic endothelial cells (PAEC) with IFN-gamma for 24 h enhanced CD40 and CD40L expression by 97% and 78%, respectively. Pretreatment of PAEC with 17-beta-estradiol (17betaE) for 24 h prevented the latter expression of CD40/CD40L. Treatment of PAEC with antisense oligomers targeting ERalpha mRNA attenuated the ability of 17betaE to inhibit the IFN-gamma-induced CD40 and CD40L protein expression. The IFN-gamma activation pathway of CD40 is known to involve the phosphorylation of the Janus activated kinase (JAK) and the signal transducer and activator of transcription 1 (Stat1). 17betaE, acting via the estrogen receptor alpha (ERalpha), abrogated IFN-gamma-mediated effects on Stat1 but failed to inhibit Jak1 and Jak2 phosphorylation. Furthermore, 17betaE prevented neutrophil adhesion induced by IFN-gamma. CONCLUSION: In summary, 17betaE binding to ERalpha blocked IFN-gamma-induced Stat1 phosphorylation, CD40 and CD40L protein expression, and neutrophil adhesion onto ECs.