Ricky R. Lareu

Suberoylanilide hydroxamic acid: a potential epigenetic therapeutic agent for lung fibrosis?

Pulmonary fibrosis represents a fatal stage of interstitial lung diseases of known and idiopathic aetiology. No effective therapy is currently available. Based on an indication-discovery approach we present novel in vitro evidence that the histone deacetylases inhibitor suberoylanilide hydroxamic acid (SAHA), an FDA approved anti-cancer drug, has antifibrotic and anti-inflammatory potential. Human lung fibroblasts (fetal, adult and idiopathic adult pulmonary fibrosis) were treated with transforming growth factor (TGF)-β1 with or without SAHA. Collagen deposition, α-smooth muscle actin (α-SMA) expression, matrix metalloproteinase (MMP)1 activity, tissue inhibitor of MMP (TIMP)1 production, apoptosis and cell proliferation were assessed. Pro-inflammatory cytokines relevant to pulmonary fibrosis were assayed in SAHA-treated human peripheral blood mononuclear cells (PBMC) and its subpopulations. SAHA abrogated TGF-β1 effects on all the fibroblast lines by preventing their transdifferentiation into α-SMA positive myofibroblasts and increased collagen deposition without inducing apoptosis. However, MMP1 activity and TIMP1 production was modulated without a clear fibrolytic effect. SAHA also inhibited serum-induced proliferation of the fibroblast lines and caused hyperacetylation of α-tubulin and histone. Cytokine secretion was inhibited from PBMC and lymphocytes at nonapoptotic concentrations. Taken together, these data demonstrate combined antifibrotic and anti-inflammatory properties of SAHA, suggesting its therapeutic potential for pulmonary fibrosis.

Role of DDC-4/sFRP-4, a secreted Frizzled-related protein, at the onset of apoptosis in mammary involution

AbstractUsing differential display, we isolated DDC-4, a secreted frizzled-related protein (sFRP), which is induced in the physiological apoptosis of hormonally regulated, reproductive tissues such as mammary gland, prostate, corpus luteum and uterus. The role of this gene in apoptosis was studied in animals overexpressing ectopic DDC-4/sFRP-4. Transgenic mice bearing the DDC-4/sFRP-4 cDNA under the control of the MMTV-LTR promoter showed lactational insufficiency and many apoptotic cells in the alveoli between day 19 of pregnancy and day 4 of lactation as demonstrated by TUNEL reaction and the presence of activated caspase-3. We performed a PKB/Akt kinase assay and studied several of its substrates using phosphorylation-specific antibodies to show reduced phosphorylation in PKB/Akt itself, as well as in glycogen synthetase kinase-3β (GSK-3β), BAD, and Forkhead. Taken together, our results show a role for DDC-4/sFRP-4 in abrogating an epithelial cell survival pathway at the onset of mammary gland involution.

Protein markers for Alzheimer disease in the frontal cortex and cerebellum

Objective: To compare proteins related to Alzheimer disease (AD) in the frontal cortex and cerebellum of subjects with early-onset AD (EOAD) with or without presenilin 1 (PS1) mutations with sporadic late-onset AD (LOAD) and nondemented control subjects. Methods: Immunohistochemistry, immunoblot analysis, and ELISA were used to detect and assess protein levels in brain. Results: In EOAD and to a lesser extent in LOAD, there was increased amyloid beta (Aβ) deposition (by immunohistochemistry), increased soluble Aβ (by immunoblot analysis), and specific increases in Aβ40 and Aβ42 (by ELISA) in the frontal cortex and, in some cases, in the cerebellum. Surprisingly, immunoblot analysis revealed reduced levels of PS1 in many of the subjects with EOAD with or without PS1 mutations. In those PS1 mutation-bearing subjects with the highest Aβ, PS1 was barely, if at all, detectable. This decrease in PS1 was specific and not attributable solely to neuronal loss because amyloid precursor protein (APP) and the PS1-interacting protein β-catenin levels were unchanged. Conclusions: This study shows that in the frontal cortex and cerebellum from Alzheimer disease patients harboring certain presenilin 1 mutations, high levels of amyloid beta are associated with low levels of presenilin 1. The study provides the premise for further investigation of mechanisms underlying the downregulation of presenilin 1, which may have considerable pathogenic and therapeutic relevance.

MACROMOLECULAR CROWDING IN BIOLOGICAL SYSTEMS: DYNAMIC LIGHT SCATTERING (DLS) TO QUANTIFY THE EXCLUDED VOLUME EFFECT (EVE)

Macromolecules crowd defined spaces, thereby excluding other like-sized molecules from the volume they occupy. These excluded-volume effect(s) (EVE) are well characterized for intracellular and partially for extracellular compartments such as blood plasma. We showed that EVE in fibroblast culture leads to faster enzymatic procollagen conversion and matrix deposition. Apparently, EVE can be applied to emulate in vivo conditions in an in vitro setting. Thus, we attempted to quantitatively capture the crowding potential of various macromolecules using dynamic light scattering under physiological conditions. We found that charged macromolecules like dextran sulfate (negative, 500 kDa) have a hydrodynamic radii of 46.4 ± 0.3 nm i.e. ~4 fold larger than that of neutral macromolecules like Ficoll (neutral, 400 kDa) and thus show greater EVE potential. At biologically effective concentrations viscosity was not increased. Unexpectedly, we observed a dramatic drop of hydrodynamic radii of all macromolecules tested ...

Role of apoptosis in functional luteolysis in the pregnant rabbit corpus luteum: Evidence of a role for placental-derived factors in promoting luteal cell survival

Corpora lutea (CL) were isolated from one rabbit ovary on days 4, 8, 16 (peak luteal function), 28 (functional regression) and 30 of pregnancy and processed for biochemical analysis of DNA integrity. Analysis of DNA integrity revealed the presence of oligonucleosomal fragments in day 28 and day 30 CL but not in day 16 CL. The extent of low molecular weight (<15 kb) DNA labeling was 6.6 ± 0.84 fold higher in day 30 as compared to day 16 CL (mean ± SEM; n = 4, P < 0.01). In a second series of experiments, healthy CL collected from day 16 pregnant rabbits were incubated for 2 h in the absence or presence of 250 μg/ml of placental extract (PE) obtained from day 16 and/or day 30 placentas. Analysis of DNA integrity revealed that extensive apoptosis occurred in CL incubated in medium alone and in medium containing day 30 PE. In contrast, day 16 PE significantly suppressed apoptosis vs control (70 ± 4%). In a third series of experiments, expression of mRNA for bcl-x and bax was measured by Northern analysis of CL treated without and with day 16 PE using cRNA probes for bcl-x and bax developed in our laboratory by RT-PCR. Treatment with PE significantly reduced bax mRNA levels but did not change bcl-x mRNA levels. These studies provide evidence that functional luteolysis in the pregnant rabbit CL is correlated with the occurrence of apoptosis. The data suggest that a factor(s) derived from the placenta may be responsible for the prolongation of CL life span during pregnancy by its ability to alter the bax:bcl-x rheostat and suppress apoptosis.

Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence

Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands.

Regulated Expression of Inhibitor of Apoptosis Protein 3 in the Rat Corpus Luteum

Abstract We sought to investigate the role inhibitor of apoptosis proteins (IAPs) play in the life cycle of the corpus luteum (CL) of the rat. We isolated two clones with amino acid homology to rat IAP2 (BIRC 3) and three to rat IAP3 (rIAP3; BIRC 4). The expression of rIAP3 mRNA was examined in the rat CL during and after pregnancy, in Day 8 pregnant rats after 24-h treatment of gonadotropin-releasing hormone-agonist (GnRH-Ag), and in a CL organ culture model of spontaneous apoptosis in the absence of tropic support with and without superoxide dismutase. We used real-time RT-PCR to quantitate rIAP3 mRNA expression. Interestingly, a significant reduction in rIAP3 levels was seen at the time of CL regression in the course of natural pregnancy and the GnRH-Ag model. Surprisingly, rIAP3 mRNA levels in the CL organ culture model of spontaneous apoptosis failed to show significant changes, although TUNEL (terminal deoxynucleotide transferase-mediated dUTP nick end-labeling) reaction showed 30%–40% of the cells undergoing DNA fragmentation after 2 h in culture. In situ hybridization revealed that rIAP3 expression was localized to the cytoplasm of luteal and granulosa cells. These data clearly demonstrate both the presence of IAPs in the rat CL and the regulation of rIAP3 during in vivo apoptotic cell death, indicating a role for IAPs in the maintenance of CL function and demise.

A histological and micro-CT investigation in to the effect of NGF and EGF on the periodontal, alveolar bone, root and pulpal healing of replanted molars in a rat model - a pilot study

BackgroundThis study aims to investigate, utilising micro-computed tomography (micro-CT) and histology, whether the topical application of nerve growth factor (NGF) and/or epidermal growth factor (EGF) can enhance periodontal, alveolar bone, root and pulpal tissue regeneration while minimising the risk of pulpal necrosis, root resorption and ankylosis of replanted molars in a rat model.MethodsTwelve four-week-old male Sprague-Dawley rats were divided into four groups: sham, collagen, EGF and NGF. The maxillary right first molar was elevated and replanted with or without a collagen membrane impregnated with either the growth factors EGF or NGF, or a saline solution. Four weeks after replantation, the animals were sacrificed and the posterior maxilla was assessed using histological and micro-CT analysis. The maxillary left first molar served as the control for the corresponding right first molar.ResultsMicro-CT analysis revealed a tendency for all replanted molars to have reduced root length, root volume, alveolar bone height and inter-radicular alveolar bone volume. It appears that the use of the collagen membrane had a negative effect while no positive effect was noted with the incorporation of EGF or NGF. Histologically, the incorporation of the collagen membrane was found to negatively affect pulpal, root, periodontal and alveolar bone healing with pulpal inflammation and hard tissue formation, extensive root resorption and alveolar bone fragmentation. The incorporation of EGF and NGF did not improve root, periodontal or alveolar bone healing. However, EGF was found to improve pulp vascularisation while NGF-improved pulpal architecture and cell organisation, although not to the level of the control group.ConclusionsResults indicate a possible benefit on pulpal vascularisation and pulpal cell organisation following the incorporation of EGF and NGF, respectively, into the alveolar socket of replanted molars in the rat model. No potential benefit of EGF and NGF was detected in periodontal or root healing, while the use of a collagen membrane carrier was found to have a negative effect on the healing response.

Inhibition of nitric oxide synthesis potentiates apoptosis in the rabbit corpus luteum.

To determine if nitric oxide (NO) plays a role in corpus luteum (CL) physiology by affecting progesterone secretion or luteal apoptosis, an in-vitro pseudopregnant rabbit ovarian perfusion system was used to measure the effects of an inhibitor of NO synthesis, NG-nitro-L-arginine methyl ester (L-NAME), on progesterone secretion and corpus luteal apoptosis as measured by internucleosomal DNA breakdown. Pseudopregnant rabbit ovaries perfused in vitro with L-NAME did not demonstrate any significant differences compared with control ovaries in progesterone secretion. However, apoptosis, as measured by internucleosomal breakdown, was significantly increased in L-NAME-perfused CL compared with controls. While NO does not appear to directly affect progesterone secretion, there does appear to be a role for NO in CL maintenance, or a role for inhibition of NO production in CL regression.

Divergent LIN28-mRNA associations result in translational suppression upon the initiation of differentiation

LIN28 function is fundamental to the activity and behavior of human embryonic stem cells (hESCs) and induced pluripotent stem cells. Its main roles in these cell types are the regulation of translational efficiency and let-7 miRNA maturation. However, LIN28-associated mRNA cargo shifting and resultant regulation of translational efficiency upon the initiation of differentiation remain unknown. An RNA-immunoprecipitation and microarray analysis protocol, eRIP, that has high specificity and sensitivity was developed to test endogenous LIN28-associated mRNA cargo shifting. A combined eRIP and polysome analysis of early stage differentiation of hESCs with two distinct differentiation cues revealed close similarities between the dynamics of LIN28 association and translational modulation of genes involved in the Wnt signaling, cell cycle, RNA metabolism and proteasomal pathways. Our data demonstrate that change in translational efficiency is a major contributor to early stages of differentiation of hESCs, in which LIN28 plays a central role. This implies that eRIP analysis of LIN28-associated RNA cargoes may be used for rapid functional quality control of pluripotent stem cells under manufacture for therapeutic applications.