Hepatocyte ß-catenin loss is compensated by Insulin-mTORC1 activation to promote liver regeneration.

BACKGROUND AND AIMS: Liver regeneration (LR) following partial hepatectomy (PH) occurs via activation of various signaling pathways. Disruption of a single pathway can be compensated by activation of another pathway to continue LR. The Wnt-ß-catenin pathway is activated early during LR and conditional hepatocyte loss of ß-catenin delays LR. Here, we study mechanism of LR in the absence of hepatocyte-ß-catenin. APPROACH AND RESULTS: Eight-week-old hepatocyte-specific Ctnnb1 knockout mice (ß-catenin ΔHC ) were subjected to PH. These animals exhibited decreased hepatocyte proliferation at 40-120 h and decreased cumulative 14-day BrdU labeling of <40%, but all mice survived, suggesting compensation. Insulin-mediated mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) activation was uniquely identified in the ß-catenin ΔHC mice at 72-96 h after PH. Deletion of hepatocyte regulatory-associated protein of mTOR (Raptor), a critical mTORC1 partner, in the ß-catenin ΔHC mice led to progressive hepatic injury and mortality by 30 dys. PH on early stage nonmorbid Raptor ΔHC -ß-catenin ΔHC mice led to lethality by 12 h. Raptor ΔHC mice showed progressive hepatic injury and spontaneous LR with ß-catenin activation but died by 40 days. PH on early stage nonmorbid Raptor ΔHC mice was lethal by 48 h. Temporal inhibition of insulin receptor and mTORC1 in ß-catenin ΔHC or controls after PH was achieved by administration of linsitinib at 48 h or rapamycin at 60 h post-PH and completely prevented LR leading to lethality by 12-14 days. CONCLUSIONS: Insulin-mTORC1 activation compensates for ß-catenin loss to enable LR after PH. mTORC1 signaling in hepatocytes itself is critical to both homeostasis and LR and is only partially compensated by ß-catenin activation. Dual inhibition of ß-catenin and mTOR may have notable untoward hepatotoxic side effects.

SOD2 V16A amplifies vascular dysfunction in sickle cell patients by curtailing mitochondria complex IV activity.

Superoxide dismutase 2 (SOD2) catalyzes the dismutation of superoxide to hydrogen peroxide in mitochondria, limiting mitochondrial damage. The SOD2 amino acid valine-to-alanine substitution at position 16 (V16A) in the mitochondrial leader sequence is a common genetic variant among patients with sickle cell disease (SCD). However, little is known about the cardiovascular consequences of SOD2V16A in SCD patients or its impact on endothelial cell function. Here, we show SOD2V16A associates with increased tricuspid regurgitant velocity (TRV), systolic blood pressure, right ventricle area at systole, and declined 6-minute walk distance in 410 SCD patients. Plasma lactate dehydrogenase, a marker of oxidative stress and hemolysis, significantly associated with higher TRV. To define the impact of SOD2V16A in the endothelium, we introduced the SOD2V16A variant into endothelial cells. SOD2V16A increases hydrogen peroxide and mitochondrial reactive oxygen species (ROS) production compared with controls. Unexpectedly, the increased ROS was not due to SOD2V16A mislocalization but was associated with mitochondrial complex IV and a concomitant decrease in basal respiration and complex IV activity. In sum, SOD2V16A is a novel clinical biomarker of cardiovascular dysfunction in SCD patients through its ability to decrease mitochondrial complex IV activity and amplify ROS production in the endothelium.

A Novel Method of Mouse RPE Explant Culture and Effective Introduction of Transgenes Using Adenoviral Transduction for In Vitro Studies in AMD.

Degeneration of retinal pigment epithelium (RPE) is one of the most critical phenotypic changes of age-related macular degeneration (AMD), the leading cause of vision loss in the elderly. While cultured polarized RPE cells with original properties are valuable in in vitro models to study RPE biology and the consequences of genetic and/or pharmacological manipulations, the procedure to establish mouse primary PRE cell culture or pluripotent stem cell-derived RPE cells is time-consuming and yields a limited number of cells. Thus, establishing a mouse in situ RPE culture system is highly desirable. Here we describe a novel and efficient method for RPE explant culture that allows for obtaining biologically relevant RPE cells in situ. These RPE explants (herein referred to as RPE flatmounts) are viable in culture for at least 7 days, can be efficiently transduced with adenoviral constructs, and/or treated with a variety of drugs/chemicals followed by downstream analysis of the signaling pathways/biological processes of interest, such as assessment of the autophagy flux, inflammatory response, and receptor tyrosine kinases stimulation. This method of RPE explant culture is highly beneficial for pharmacological and mechanistic studies in the field of RPE biology and AMD research.

Compensatory increases of select proteostasis networks after Hsp70 inhibition in cancer cells.

Cancer cells thrive when challenged with proteotoxic stress by inducing components of the protein folding, proteasome, autophagy and unfolded protein response (UPR) pathways. Consequently, specific molecular chaperones have been validated as targets for anti-cancer therapies. For example, inhibition of Hsp70 family proteins (hereafter Hsp70) in rhabdomyosarcoma triggers UPR induction and apoptosis. To define how these cancer cells respond to compromised proteostasis, we compared rhabdomyosarcoma cells that were sensitive (RMS13) or resistant (RMS13-R) to the Hsp70 inhibitor MAL3-101. We discovered that endoplasmic reticulum-associated degradation (ERAD) and autophagy were activated in RMS13-R cells, suggesting that resistant cells overcome Hsp70 ablation by increasing misfolded protein degradation. Indeed, RMS13-R cells degraded ERAD substrates more rapidly than RMS cells and induced the autophagy pathway. Surprisingly, inhibition of the proteasome or ERAD had no effect on RMS13-R cell survival, but silencing of select autophagy components or treatment with autophagy inhibitors restored MAL3-101 sensitivity and led to apoptosis. These data indicate a route through which cancer cells overcome a chaperone-based therapy, define how cells can adapt to Hsp70 inhibition, and demonstrate the value of combined chaperone and autophagy-based therapies.This article has an associated First Person interview with the first author of the paper.

Graft-infiltrating PD-L1hi cross-dressed dendritic cells regulate antidonor T cell responses in mouse liver transplant tolerance.

Although a key role of cross-dressing has been established in immunity to viral infection and more recently in the instigation of transplant rejection, its role in tolerance is unclear. We investigated the role of intragraft dendritic cells (DCs) and cross-dressing in mouse major histocompatibility complex (MHC)-mismatched liver transplant tolerance that occurs without therapeutic immunosuppression. Although donor interstitial DCs diminished rapidly after transplantation, they were replaced in the liver by host DCs that peaked on postoperative day (POD) 7 and persisted indefinitely. Approximately 60% of these recipient DCs displayed donor MHC class I, indicating cross-dressing. By contrast, only a very minor fraction (0%-2%) of cross-dressed DCs (CD-DCs) was evident in the spleen. CD-DCs sorted from liver grafts expressed much higher levels of T cell inhibitory programed death ligand 1 (PD-L1) and high levels of interleukin-10 compared with non-CD-DCs (nCD-DCs) isolated from the graft. Concomitantly, high incidences of programed death protein 1 (PD-1)hi T cell immunoglobulin and mucin domain containing 3 (TIM-3)+ exhausted graft-infiltrating CD8+ T cells were observed. Unlike nCD-DCs, the CD-DCs failed to stimulate proliferation of allogeneic T cells but markedly suppressed antidonor host T cell proliferation. CD-DCs were much less evident in allografts from DNAX-activating protein of 12 kDa (DAP12)-/- donors that were rejected acutely. CONCLUSION: These findings suggest that graft-infiltrating PD-L1hi CD-DCs may play a key role in the regulation of alloimmunity and in the induction of liver transplant tolerance. (Hepatology 2018;67:1499-1515).

Support of bone mineral deposition by regulation of pH.

Osteoblasts secrete collagen and isolate bone matrix from extracellular space. In the matrix, alkaline phosphatase generates phosphate that combines with calcium to form mineral, liberating 8 H+ per 10 Ca+2 deposited. However, pH-dependent hydroxyapatite deposition on bone collagen had not been shown. We studied the dependency of hydroxyapatite deposition on type I collagen on pH and phosphate by surface plasmon resonance in 0-5 mM phosphate at pH 6.8-7.4. Mineral deposition saturated at <1 mM Ca2+ but was sensitive to phosphate. Mineral deposition was reversible, consistent with amorphous precipitation; stable deposition requiring EDTA removal appeared with time. At pH 6.8, little hydroxyapatite deposited on collagen; mineral accumulation increased 10-fold at pH 7.4. Previously, we showed high expression Na+/H+ exchanger (NHE) and ClC transporters in osteoblasts. We hypothesized that, in combination, these move protons across osteoblasts to the general extracellular space. We made osteoblast membrane vesicles by nitrogen cavitation and used acridine orange quenching to characterize proton transport. We found H+ transport dependent on gradients of chloride or sodium, consistent with apical osteoblast ClC family Cl-,H+ antiporters and basolateral osteoblast NHE family Na+/H+ exchangers. Little, if any, active H+ transport, supported by ATP, occurred. Major transporters include cariporide-sensitive NHE1 in basolateral membranes and ClC3 and ClC5 in apical osteoblast membranes. The mineralization inhibitor levamisole reduced bone formation and expression of alkaline phosphatase, NHE1, and ClC5. We conclude that mineral deposition in bone collagen is pH-dependent, in keeping with H+ removal by Cl-,H+ antiporters and Na+/H+-exchangers. Periodic orientation hydroxyapatite is organized on type I collagen-coiled coils.

Profilin-1 deficiency leads to SMAD3 upregulation and impaired 3D outgrowth of breast cancer cells.

BACKGROUND: Adhesion-mediated activation of FAK/ERK signalling pathway, enabled by the formation of filopodial protrusions (FLP), has been shown to be an important event for triggering of dormancy-to-proliferation switch and metastatic outgrowth of breast cancer cells (BCC). We studied the role of actin-binding protein profilin1 (Pfn1) in these processes. METHODS: Quantitative immunohistochemistry (IHC) of BC tissue microarray (TMA) and survival analyses of curated transcriptome datasets of BC patients were performed to examine Pfn1's association with certain clinicopathological features. FLP formation and single cell outgrowth of BCC were assessed using a 3D matrigel culture that accurately predicts dormant vs metastatic outgrowth phenotypes of BCC in certain microenvironment. Gene expression studies were performed to identify potential biological pathways that are perturbed under Pfn1-depleted condition. RESULTS: Lower Pfn1 expression is correlated with lower nuclear grade of breast tumours and longer relapse-free survival of BC patients. Pfn1 depletion leads to defects in FLP and outgrowth of BCC but without impairing either FAK or ERK activation. Guided by transcriptome analyses, we further showed that Pfn1 depletion is associated with prominent SMAD3 upregulation. Although knockdown and overexpression experiments revealed that SMAD3 has an inhibitory effect on the outgrowth of breast cancer cells, SMAD3 knockdown alone was not sufficient to enhance the outgrowth potential of Pfn1-depleted BCC suggesting that other proliferation-regulatory pathways in conjunction with SMAD3 upregulation may underlie the outgrowth-deficient phenotype of BCC cells upon depletion of Pfn1. CONCLUSION: Overall, these data suggest that Pfn1 may be a novel biomarker for BC recurrence and a possible target to reduce metastatic outgrowth of BCC.

Hierarchically aligned fibrous hydrogel films through microfluidic self-assembly of graphene and polysaccharides.

Despite significant interest in developing extracellular matrix (ECM)-inspired biomaterials to recreate native cell-instructive microenvironments, the major challenge in the biomaterial field is to recapitulate the complex structural and biophysical features of native ECM. These biophysical features include multiscale hierarchy, electrical conductivity, optimum wettability, and mechanical properties. These features are critical to the design of cell-instructive biomaterials for bioengineering applications such as skeletal muscle tissue engineering. In this study, we used a custom-designed film fabrication assembly, which consists of a microfluidic chamber to allow electrostatic charge-based self-assembly of oppositely charged polymer solutions forming a hydrogel fiber and eventually, a nanocomposite fibrous hydrogel film. The film recapitulates unidirectional hierarchical fibrous structure along with the conductive properties to guide initial alignment and myotube formation from cultured myoblasts. We combined high conductivity, and charge carrier mobility of graphene with biocompatibility of polysaccharides to develop graphene-polysaccharide nanocomposite fibrous hydrogel films. The incorporation of graphene in fibrous hydrogel films enhanced their wettability, electrical conductivity, tensile strength, and toughness without significantly altering their elastic properties (Young's modulus). In a proof-of-concept study, the mouse myoblast cells (C2C12) seeded on these nanocomposite fibrous hydrogel films showed improved spreading and enhanced myogenesis as evident by the formation of multinucleated myotubes, an early indicator of myogenesis. Overall, graphene-polysaccharide nanocomposite fibrous hydrogel films provide a potential biomaterial to promote skeletal muscle tissue regeneration.

Peroxisome Proliferator-activated Receptor-γ Coactivator 1-α (PGC1α) Protects against Experimental Murine Colitis.

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is the primary regulator of mitochondrial biogenesis and was recently found to be highly expressed within the intestinal epithelium. PGC1α is decreased in the intestinal epithelium of patients with inflammatory bowel disease, but its role in pathogenesis is uncertain. We now hypothesize that PGC1α protects against the development of colitis and helps to maintain the integrity of the intestinal barrier. We selectively deleted PGC1α from the intestinal epithelium of mice by breeding a PGC1α(loxP/loxP) mouse with a villin-cre mouse. Their progeny (PGC1α(ΔIEC) mice) were subjected to 2% dextran sodium sulfate (DSS) colitis for 7 days. The SIRT1 agonist SRT1720 was used to enhance PGC1α activation in wild-type mice during DSS exposure. Mice lacking PGC1α within the intestinal epithelium were more susceptible to DSS colitis than their wild-type littermates. Pharmacologic activation of PGC1α successfully ameliorated disease and restored mitochondrial integrity. These findings suggest that a depletion of PGC1α in the intestinal epithelium contributes to inflammatory changes through a failure of mitochondrial structure and function as well as a breakdown of the intestinal barrier, which leads to increased bacterial translocation. PGC1α induction helps to maintain mitochondrial integrity, enhance intestinal barrier function, and decrease inflammation.

Unwrapping the origins and roles of the renal endothelium.

The renal vasculature, like all vessels, is lined by a thin layer of simple squamous epithelial cells called an endothelium. These endothelial-lined vessels can be subdivided into four major compartments: arteries, veins, capillaries and lymphatics. The renal vasculature is a highly integrated network that forms through the active processes of angiogenesis and vasculogenesis. Determination of the precise contribution of these two processes and of the molecular signaling that governs the differentiation, specification and maturation of these critical cell populations is the focus of an actively evolving field of research. Although much of the focus has concentrated on the origin of the glomerular capillaries, in this review we extend the investigation to the origins of the endothelial cells throughout the entire kidney and the signaling events that cause their distinct functional and molecular profiles. A thorough understanding of endothelial cell biology may play a critical role in a better understanding of renal vascular diseases.

Mechanism of transfer of functional microRNAs between mouse dendritic cells via exosomes.

Dendritic cells (DCs) are the most potent APCs. Whereas immature DCs down-regulate T-cell responses to induce/maintain immunologic tolerance, mature DCs promote immunity. To amplify their functions, DCs communicate with neighboring DCs through soluble mediators, cell-to-cell contact, and vesicle exchange. Transfer of nanovesicles (< 100 nm) derived from the endocytic pathway (termed exosomes) represents a novel mechanism of DC-to-DC communication. The facts that exosomes contain exosome-shuttle miRNAs and DC functions can be regulated by exogenous miRNAs, suggest that DC-to-DC interactions could be mediated through exosome-shuttle miRNAs, a hypothesis that remains to be tested. Importantly, the mechanism of transfer of exosome-shuttle miRNAs from the exosome lumen to the cytosol of target cells is unknown. Here, we demonstrate that DCs release exosomes with different miRNAs depending on the maturation of the DCs. By visualizing spontaneous transfer of exosomes between DCs, we demonstrate that exosomes fused with the target DCs, the latter followed by release of the exosome content into the DC cytosol. Importantly, exosome-shuttle miRNAs are functional, because they repress target mRNAs of acceptor DCs. Our findings unveil a mechanism of transfer of exosome-shuttle miRNAs between DCs and its role as a means of communication and posttranscriptional regulation between DCs.

Effects of intra-abdominal sepsis on atherosclerosis in mice.

INTRODUCTION: Sepsis and other infections are associated with late cardiovascular events. Although persistent inflammation is implicated, a causal relationship has not been established. We tested whether sepsis causes vascular inflammation and accelerates atherosclerosis. METHODS: We performed prospective, randomized animal studies at a university research laboratory involving adult male ApoE-deficient (ApoE-/-) and young C57B/L6 wild-type (WT) mice. In the primary study conducted to determine whether sepsis accelerates atherosclerosis, we fed ApoE-/- mice (N = 46) an atherogenic diet for 4 months and then performed cecal ligation and puncture (CLP), followed by antibiotic therapy and fluid resuscitation or a sham operation. We followed mice for up to an additional 5 months and assessed atheroma in the descending aorta and root of the aorta. We also exposed 32 young WT mice to CLP or sham operation and followed them for 5 days to determine the effects of sepsis on vascular inflammation. RESULTS: ApoE-/- mice that underwent CLP had reduced activity during the first 14 days (38% reduction compared to sham; P < 0.001) and sustained weight loss compared to the sham-operated mice (-6% versus +9% change in weight after CLP or sham surgery to 5 months; P < 0.001). Despite their weight loss, CLP mice had increased atheroma (46% by 3 months and 41% increase in aortic surface area by 5 months; P = 0.03 and P = 0.004, respectively) with increased macrophage infiltration into atheroma as assessed by immunofluorescence microscopy (0.52 relative fluorescence units (rfu) versus 0.97 rfu; P = 0.04). At 5 months, peritoneal cultures were negative; however, CLP mice had elevated serum levels of interleukin 6 (IL-6) and IL-10 (each at P < 0.05). WT mice that underwent CLP had increased expression of intercellular adhesion molecule 1 in the aortic lumen versus sham at 24 hours (P = 0.01) that persisted at 120 hours (P = 0.006). Inflammatory and adhesion genes (tumor necrosis factor α, chemokine (C-C motif) ligand 2 and vascular cell adhesion molecule 1) and the adhesion assay, a functional measure of endothelial activation, were elevated at 72 hours and 120 hours in mice that underwent CLP versus sham-operations (all at P <0.05). CONCLUSIONS: Using a combination of existing murine models for atherosclerosis and sepsis, we found that CLP, a model of intra-abdominal sepsis, accelerates atheroma development. Accelerated atheroma burden was associated with prolonged systemic, endothelial and intimal inflammation and was not explained by ongoing infection. These findings support observations in humans and demonstrate the feasibility of a long-term follow-up murine model of sepsis.

DLK1: a novel target for immunotherapeutic remodeling of the tumor blood vasculature.

Tumor blood vessels are frequently inefficient in their design and function, leading to high interstitial fluid pressure, hypoxia, and acidosis in the tumor microenvironment (TME), rendering tumors refractory to the delivery of chemotherapeutic agents and immune effector cells. Here we identified the NOTCH antagonist delta-like 1 homologue (DLK1) as a vascular pericyte-associated antigen expressed in renal cell carcinomas (RCC), but not in normal kidney tissues in mice and humans. Vaccination of mice bearing established RCC against DLK1 led to immune-mediated elimination of DLK1(+) pericytes and to blood vessel normalization (i.e., decreased vascular permeability and intratumoral hypoxia) in the TME, in association with tumor growth suppression. After therapeutic vaccination, tumors displayed increased prevalence of activated VCAM1(+)CD31(+) vascular endothelial cells (VECs) and CXCL10, a type-1 T cell recruiting chemokine, in concert with increased levels of type-1 CD8(+) tumor-infiltrating lymphocytes (TIL). Vaccination against DLK1 also yielded (i) dramatic reductions in Jarid1B(+), CD133(+), and CD44(+) (hypoxia-responsive) stromal cell populations, (ii) enhanced tumor cell apoptosis, and (iii) increased NOTCH signaling in the TME. Coadministration of a γ-secretase inhibitor (N-[N-(3,5-Difluorophenacetyl-l-alanyl)]-(S)-phenylglycine t-butyl ester (DAPT)) that interferes with canonical NOTCH signaling resulted in the partial loss of therapeutic benefits associated with lentivirus encoding full-length murine (lvDLK1)-based vaccination.

Disabled-2 protein facilitates assembly polypeptide-2-independent recruitment of cystic fibrosis transmembrane conductance regulator to endocytic vesicles in polarized human airway epithelial cells.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl(-) channel expressed in the apical plasma membrane of fluid-transporting epithelia, where the plasma membrane abundance of CFTR is in part controlled by clathrin-mediated endocytosis. The protein networks that control CFTR endocytosis in epithelial cells have only been partially explored. The assembly polypeptide-2 complex (AP-2) is the prototypical endocytic adaptor critical for optimal clathrin coat formation. AP-2 is essential for recruitment of cargo proteins bearing the YXXΦ motif. Although AP-2 interacts directly with CFTR in vitro and facilitates CFTR endocytosis in some cell types, it remains unknown whether it is critical for CFTR uptake into clathrin-coated vesicles (CCVs). Disabled-2 (Dab2) is a clathrin-associated sorting protein (CLASP) that contributes to clathrin recruitment, vesicle formation, and cargo selection. In intestinal epithelial cells Dab2 was not found to play a direct role in CFTR endocytosis. By contrast, AP-2 and Dab2 were shown to facilitate CFTR endocytosis in human airway epithelial cells, although the specific mechanism remains unknown. Our data demonstrate that Dab2 mediates AP-2 independent recruitment of CFTR to CCVs in polarized human airway epithelial cells. As a result, it facilitates CFTR endocytosis and reduces CFTR abundance and stability in the plasma membrane. These effects are mediated by the DAB homology domain. Moreover, we show that in human airway epithelial cells AP-2 is not essential for CFTR recruitment to CCVs.

Metallothionein-induced zinc partitioning exacerbates hyperoxic acute lung injury.

Hypozincemia, with hepatic zinc accumulation at the expense of other organs, occurs in infection, inflammation, and aseptic lung injury. Mechanisms underlying zinc partitioning or its impact on extrahepatic organs are unclear. Here we show that the major zinc-binding protein, metallothionein (MT), is critical for zinc transmigration from lung to liver during hyperoxia and preservation of intrapulmonary zinc during hyperoxia is associated with an injury-resistant phenotype in MT-null mice. Particularly, lung-to-liver zinc ratios decreased in wild-type (WT) and increased significantly in MT-null mice breathing 95% oxygen for 72 h. Compared with female adult WT mice, MT-null mice were significantly protected against hyperoxic lung injury indicated by reduced inflammation and interstitial edema, fewer necrotic changes to distal airway epithelium, and sustained lung function at 72 h hyperoxia. Lungs of MT-null mice showed decreased levels of immunoreactive LC3, an autophagy marker, compared with WT mice. Analysis of superoxide dismutase (SOD) activity in the lungs revealed similar levels of manganese-SOD activity between strains under normoxia and hyperoxia. Lung extracellular SOD activity decreased significantly in both strains at 72 h of hyperoxia, although there was no difference between strains. Copper-zinc-SOD activity was ~4× higher under normoxic conditions in MT-null compared with WT mice but was not affected in either group by hyperoxia. Collectively the data suggest that genetic deletion of MT-I/II in mice is associated with compensatory increase in copper-zinc-SOD activity, prevention of hyperoxia-induced zinc transmigration from lung to liver, and hyperoxia-resistant phenotype strongly associated with differences in zinc homeostasis during hyperoxic acute lung injury.

A mouse model of accelerated liver aging caused by a defect in DNA repair.

UNLABELLED: The liver changes with age, leading to an impaired ability to respond to hepatic insults and increased incidence of liver disease in the elderly. Therefore, there is critical need for rapid model systems to study aging-related liver changes. One potential opportunity is murine models of human progerias or diseases of accelerated aging. Ercc1(-/Δ) mice model a rare human progeroid syndrome caused by inherited defects in DNA repair. To determine whether hepatic changes that occur with normal aging occur prematurely in Ercc1(-/Δ) mice, we systematically compared liver from 5-month-old progeroid Ercc1(-/Δ) mice to old (24-36-month-old) wild-type (WT) mice. Both displayed areas of necrosis, foci of hepatocellular degeneration, and acute inflammation. Loss of hepatic architecture, fibrosis, steatosis, pseudocapillarization, and anisokaryosis were more dramatic in Ercc1(-/Δ) mice than in old WT mice. Liver enzymes were significantly elevated in serum of Ercc1(-/Δ) mice and old WT mice, whereas albumin was reduced, demonstrating liver damage and dysfunction. The regenerative capacity of Ercc1(-/Δ) liver after partial hepatectomy was significantly reduced. There was evidence of increased oxidative damage in Ercc1(-/Δ) and old WT liver, including lipofuscin, lipid hydroperoxides and acrolein, as well as increased hepatocellular senescence. There was a highly significant correlation in genome-wide transcriptional changes between old WT and 16-week-old, but not 5-week-old, Ercc1(-/Δ) mice, emphasizing that the Ercc1(-/Δ) mice acquire an aging profile in early adulthood. CONCLUSION: There are strong functional, regulatory, and histopathological parallels between accelerated aging driven by a DNA repair defect and normal aging. This supports a role for DNA damage in driving aging and validates a murine model for rapidly testing hypotheses about causes and treatment for aging-related hepatic changes.

PEG-farnesylthiosalicylate conjugate as a nanomicellar carrier for delivery of paclitaxel.

S-trans, trans-farnesylthiosalicylic acid (FTS) is a synthetic small molecule that acts as a potent and especially nontoxic Ras antagonist. It inhibits both oncogenically activated Ras and growth factor receptor-mediated Ras activation, resulting in the inhibition of Ras-dependent tumor growth. In this work, an FTS conjugate with poly(ethylene glycol) (PEG) through a labile ester linkage, PEG5K-FTS2(L), was developed. PEG5K-FTS2 conjugate readily forms micelles in aqueous solutions with a critical micelle concentration of 0.68 µM, and hydrophobic drugs such as paclitaxel (PTX) could be effectively loaded into these particles. Both drug-free and PTX-loaded micelles were spherical in shape with a uniform size of 20-30 nm. The release of PTX from PTX-loaded PEG5K-FTS2 micelles was significantly slower than that from Taxol formulation. In vitro cytotoxicity studies with several tumor cell lines showed that PEG5K-FTS2(L) was comparable to FTS in antitumor activity. Western immunoblotting showed that total Ras levels were downregulated in several cancer cell lines treated with FTS or PEG5K-FTS2(L). The micellar formulation of PTX exhibited more in vitro cytotoxic activity against several tumor cell lines compared with free PTX, suggesting a possible synergistic effect between the carrier and the codelivered drug. The antitumor activity of the PTX loaded PEG5K-FTS2(L) micelles in a syngeneic murine breast cancer model was found to be significantly higher than that of Taxol, which may be attributed to their preferential tumor accumulation and a possible synergistic effect between PEG5K-FTS2 carrier and loaded PTX.

Morphological and mechanical examination of the atrial 'intima'.

AIMS: To examine morphology and mechanical properties of the atrial 'intima', which we defined as the tissue interposed between atrial endocardium and myocardium, in patients without known cardiovascular disease. METHODS AND RESULTS: Post-mortem right and left atrial tissue was obtained from male infants (<1 year, n = 4), children (10-19 years, n = 4), and adults (58-69 years, n = 7). Using light microscopy and an ocular micrometer, atrial intimal (AIT) thickness was measured. Intimal collagen bundle thickness was measured using electron microscopy. Passive atrial wall stiffness was measured using a planar biaxial testing device. Among infants, left AIT (0.2 ± 0.2 mm) and right (0.2 ± 0.1 mm) AIT were not significantly different (P = 0.84). Among children, left AIT (0.6 ± 0.2 mm) was significantly greater than right (0.2 ± 0.1 mm) AIT (P = 0.03), and left AIT was marginally greater than in infants (P = 0.07). Among adults, with the exception of the appendage region, left AIT (1.0 ± 0.2 mm) was markedly greater than right AIT (0.3 ± 0.1 mm; P < 0.05), and left AIT was significantly greater than that in other age groups (P < 0.05). There were no differences in right AIT among age groups. Left intimal collagen bundle thickness was greater in adults (0.0512 ± 0.0056 µm) than infants (0.0432 ± 0.0071 µm) or children (0.0435 ± 0.0013 µm), and bundles were less organized. Wall stiffness was attributable primarily to the intima (1245 ± 132, vs. 260 ± 45 N/m(2) for the remaining atrial wall). CONCLUSION: The left atrial intima, but not the right, thickens with age, becomes more disorganized ultrastructurally, and is responsible for the majority of atrial wall stiffness.

IL-33 expands suppressive CD11b+ Gr-1(int) and regulatory T cells, including ST2L+ Foxp3+ cells, and mediates regulatory T cell-dependent promotion of cardiac allograft survival.

IL-33 administration is associated with facilitation of Th2 responses and cardioprotective properties in rodent models. However, in heart transplantation, the mechanism by which IL-33, signaling through ST2L (the membrane-bound form of ST2), promotes transplant survival is unclear. We report that IL-33 administration, while facilitating Th2 responses, also increases immunoregulatory myeloid cells and CD4(+) Foxp3(+) regulatory T cells (Tregs) in mice. IL-33 expands functional myeloid-derived suppressor cells, CD11b(+) cells that exhibit intermediate (int) levels of Gr-1 and potent T cell suppressive function. Furthermore, IL-33 administration causes an St2-dependent expansion of suppressive CD4(+) Foxp3(+) Tregs, including an ST2L(+) population. IL-33 monotherapy after fully allogeneic mouse heart transplantation resulted in significant graft prolongation associated with increased Th2-type responses and decreased systemic CD8(+) IFN-γ(+) cells. Also, despite reducing overall CD3(+) cell infiltration of the graft, IL-33 administration markedly increased intragraft Foxp3(+) cells. Whereas control graft recipients displayed increases in systemic CD11b(+) Gr-1(hi) cells, IL-33-treated recipients exhibited increased CD11b(+) Gr-1(int) cells. Enhanced ST2 expression was observed in the myocardium and endothelium of rejecting allografts, however the therapeutic effect of IL-33 required recipient St2 expression and was dependent on Tregs. These findings reveal a new immunoregulatory property of IL-33. Specifically, in addition to supporting Th2 responses, IL-33 facilitates regulatory cells, particularly functional CD4(+) Foxp3(+) Tregs that underlie IL-33-mediated cardiac allograft survival.

RTD-1mimic containing γPNA scaffold exhibits broad-spectrum antibacterial activities.

Macrocyclic peptides with multiple disulfide cross-linkages, such as those produced by plants and those found in nonhuman primates, as components of the innate immunity, hold great promise for molecular therapy because of their broad biological activities and high chemical, thermal, and enzymatic stability. However, for some, because of their intricate spatial arrangement and elaborate interstrand cross-linkages, they are difficult to prepare de novo in large quantities and high purity, due to the nonselective nature of disulfide-bond formation. We show that the disulfide bridges of RTD-1, a member of the θ-defensin subfamily, could be replaced with noncovalent Watson-Crick hydrogen bonds without significantly affecting its biological activities. The work provides a general strategy for engineering conformationally rigid, cyclic peptides without the need for disulfide-bond reinforcement.