We use molecular dynamics simulations to characterize the influence of cholesterol (Chol) on the interaction between the anticancer drug doxorubicin (DOX) and a dipalmitoyl phosphatidylcholine/Chol ...lipid bilayer. We calculate the potential of mean force, which gives us an estimate of the free energy barrier for DOX translocation across the membrane. We find free energy barriers of 23.1 ± 3.1 kBT, 36.8 ± 5.1 kBT, and 54.5 ± 4.7 kBT for systems composed of 0%, 15%, and 30% Chol, respectively. Our predictions agree with Arrhenius activation energies from experiments using phospholipid membranes, including 20 kBT for 0% Chol and 37.2 kBT for 20% Chol. The location of the free energy barrier for translocation across the bilayer is dependent on composition. As Chol concentration increases, this barrier changes from the release of DOX into the water to flip-flop over the membrane center. The drug greatly affects local membrane structure by attracting dipalmitoyl phosphatidylcholine headgroups, curving the membrane, and allowing water penetration. Despite its hydrophobicity, DOX facilitates water transport via its polar groups.
The authors use molecular dynamics simulations to investigate viral peptide interactions as the cause of pH-dependent fusion in liposomal drug delivery. Viral peptides (LEFN) are composed of a linker ...peptide (LELELELE) connected to a synthetic viral peptide (DRGWGNGCGLFGKGSI). Rather than being anchored in a lipid bilayer, the viral peptides are anchored to a neutral surface by the amino termini of the linker peptide (anchor atoms are mobile in the xy-plane). Atomistic-level peptide pair arrangement on a surface depends on pH; however, the overall propensity to cluster is independent of pH, indicating that pH-sensitive liposome fusion is not due to peptide clustering. To further investigate a molecular cause of pH-sensitive fusion, the authors treat the linker peptides as ectodomains, with the assumption that the viral peptides are already inserted into a target membrane. In these simulations, the linker peptides are elongated to encourage them to bundle. At both high and low pH, the peptides readily bundle. At high pH, however, bundling was constrained by long-range order induced by sodium ions bridging negatively charged glutamic acid residues on neighboring peptides. The authors hypothesize that this constraint hinders the ability of the linker peptides to support viral peptide insertion, resulting in decreased levels of fusion observed experimentally.
Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar ...macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.
Abstract only
Rationale
Fibrotic interstitial lung disease (ILD) is among the top indications for patients undergoing lung transplantation worldwide. While our group and others have demonstrated a ...role for monocyte‐derived alveolar macrophages in the development of lung fibrosis in murine models, it is unknown if human alveolar macrophages play a similar role in the development of fibrotic ILD. We hypothesized that transcriptional profiling of AMs isolated from lung transplant donors and recipients as well as from murine alveolar macrophages in a bleomycin‐induced lung fibrosis model would demonstrate homology of involved genes.
Methods
Fluorescence‐activated cell sorting (FACS) was used to isolate AMs (CD45
+
CD169
+
HLA‐DR
+
) from samples of 18 donor lungs and 22 explanted lungs from patients at the time of lung transplantation and to isolate separately tissue‐resident and monocyte‐derived AMs from mice at fourteen and nineteen days following the administration of intratracheal bleomycin. RNA extraction from sorted cells was performed with poly(A) enrichment, followed by single‐end RNA‐seq. Exploratory data analysis using principle component analysis (PCA) and K‐mean clustering were performed. Differentially expressed genes were estimated between donors and patients with interstitial lung disease, and between murine tissue‐resident and monocyte‐derived AMs during bleomycin‐induced fibrosis.
Results
The median age of the 22 lung transplant recipients was 56 (IQR 13–64). Among recipients, there were eleven patients with the diagnosis of fibrotic ILD (five with scleroderma‐associated ILD, three with myositis‐ILD, two with idiopathic pulmonary fibrosis, and one with mixed connective tissue disease ILD). PCA of sorted populations showed grouping by diagnosis. 61 genes were identified that were differentially expressed (adjusted p < 0.05) in AMs between donors and patients with fibrotic ILD, and that were also homologs of genes highly expressed in murine monocyte‐derived AMs relative to tissue‐resident AMs during bleomycin‐induced lung fibrosis. Hierarchical clustering of samples according to expression of these homologous genes showed just one ILD sample clustering among donors, and of 61 total differentially expressed homologous genes, 51 were upregulated in AMs from patients with fibrotic ILD and only 10 were downregulated.
Conclusions
Transcriptional profiling of AMs collected at the time of lung transplantation identifies homology between human fibrotic interstitial lung disease and a murine model of bleomycin‐induced lung fibrosis. By focusing on key lung cellular populations such as alveolar macrophages, future transcriptional studies of lung disease in humans may identify new pathways and new potential targets for therapy for patients with lung disease.
Support or Funding Information
5T32HL076139‐13, AG049665, HL071643, ES013995, The Veterans Administration, DOD W81XWH‐15‐1‐0215
In Chapter 2, we use molecular dynamics simulations to characterize the influence of cholesterol (Chol) on the interaction between the anticancer drug doxorubicin (DOX) and a dipalmitoyl ...phosphatidylcholine/Chol lipid bilayer. We calculate the potential of mean force, which gives us an estimate of the free energy barrier for DOX translocation across the membrane. We find free energy barriers of 23.1 ± 3.1 kT, 36.8 ± 5.1 kT, and 54.5 ± 4.7 kT for systems composed of 0%, 15%, and 30% Chol, respectively. Our predictions agree with Arrhenius activation energies from experiments using phospholipid membranes, including 20 kT for 0% Chol and 37.2 kT for 20% Chol. The location of the free energy barrier for translocation across the bilayer is dependent on composition. As Chol concentration increases, this barrier changes from the release of DOX into the water to flip-flop over the membrane center. The drug greatly affects local membrane structure by attracting dipalmitoyl phosphatidylcholine headgroups, curving the membrane, and allowing water penetration. Despite its hydrophobicity, DOX facilitates water transport via its polar groups. In Chapter 3, we design a pH-sensitive synthetic flavivirus-type peptide and measure lipid mixing due to liposome fusion with Forster Resonance Electron Transfer (FRET). The viral peptide comprises a pH-sensitive linker peptide and a synthetic fusion pep- tide attached to two 16-carbon lipid tails. In the absence of PEG, we find fusion to be pH-dependent, but not dependent on cholesterol (in host liposomes) or viral peptide concentration. A physical model is created, describing pre-experimental hemifusion and fusion between viral liposomes. We propose a new measure of liposome fusion. Using a traditional two-step kinetic model, we assert that the ratio of the parameters beta (overall level of lipid mixing) to k1, the rate of outer leaflet mixing, indicates the size of viral particles due to pre-experimental interactions. beta increases as viral liposome aggregates fuse, as there is more surface area exposed for host binding. k1 decreases as the particles fuse, due to decreased curvature and increased competitive binding, resulting in longer time needed to saturate the viral particles with host liposomes. A strong correlation was found between beta/ k1 and k2, the rate of inner leaflet mixing (and traditional measure of fusion measured with FRET). When 5% PEG is added to viral liposomes, this correlation is lost, as PEG prevents the pre-experimental interactions between the viral liposomes. Fusion is found to be dependent on both pH and peptide concentration in the case of PEG. In Chapter 4, we investigate molecular interactions between viral peptides as the cause for the pH-dependent fusion found in Chapter3. The same peptide from Chapter3 is used, but without the lipid tails. Rather than being anchored in a lipid bilayer, the viral peptides are anchored to a neutral surface. A potential of mean force was calculated between two peptides, with the distance between anchor atoms as the reaction coordinate. The range of peptide interactions is 3.5 nm, and the energy needed to separate peptides is approximately 9 ± 2 kT. Peptide clustering was found to be independent of pH, indicating that the pH-sensitive liposome fusion observed in Chapter3 is not due to peptide clustering, as was first hypothesized. To further investigate a molecular cause of pH-sensitive fusion, we treated the linker peptides as ectodomains, with the assumption that the viral peptides had already inserted into a target membrane. In these simulations, the linker peptides were elongated to encourage them to bundle. At both high and low pH, the peptides bundled. At high pH, however, bundling was constrained by long-range order induced by sodium ions bridging negatively-charged glutamic acid residues on neighboring peptides. We hypothesize that this constraint hinders the ability of the linker peptides to support viral peptide insertion, resulting in decreased levels of fusion observed in Chapter 3 at pH 7.4.
Background
Hemodialysis (HD) access failure is a common cause of increased morbidity and healthcare cost in patients with end stage renal disease (ESRD). Percutaneous balloon angioplasty has been ...used to treat hemodialysis access stenosis but is complicated by a high rate of restenosis. Percutaneous cutting balloon (PCB) angioplasty is an alternative approach that has shown to reduce restenosis.
Objectives
The aim of the study is to assess the safety and efficacy of PCB angioplasty in comparison with conventional and high‐pressure balloon angioplasty in the treatment of hemodialysis access site stenosis.
Methods
We searched PubMed, EMBASE and the Cochrane Central register of controlled trials (CENTRAL) databases through August 2014 and selected studies using the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses (PRISMA) checklist. We included all randomized clinical trials with a head‐to‐head comparison between PCB and conventional or high‐pressure balloon angioplasty
Results
Three studies with 1034 participants (age 60.7 (±12.9) years and 50.1% males) with 525 in PCB and 509 in control arm were included in the analysis. The immediate procedural success rate was not significantly different in the PCB angioplasty and control arm respectively, (87.2% vs. 83.7% RD −0.02; 95%CI −0.06 to 0.01; P = 0.38). The six‐month target lesion patency was significantly higher in the PCB angioplasty arm (67.2% vs. 55.6% RD 0.12; 95%CI 0.05–0.19; P < 0.05) with number needed to treat (NNT) of 9. The device related complications were not statistically significant between groups (RD 0.03; 95%CI −0.02 to 0.07; P = 0.26).
Conclusions
PCB angioplasty is effective in treatment of hemodialysis access stenosis, with significantly higher six‐month patency compared to balloon angioplasty. (J Interven Cardiol 2015;28:288–295)
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