Tumor-induced myeloid-derived suppressor cells (MDSC) promote immune suppression and mediate tumor progression. However, the molecular basis for the generation of MDSC, which in mice co-express the CD11b+ and Gr-1+ cell surface markers remains unclear. Because CD11b+Gr-1+ cells expand during progressive tumor growth, this suggests that tumor-induced events alter signaling pathways that affect normal myeloid cell development. Interferon regulatory factor-8 (IRF-8), a member of the IFN-γ regulatory factor family, is essential for normal myelopoiesis. We therefore examined whether IRF-8 modulated tumor-induced CD11b+Gr-1+ cell development or accumulation using both implantable (4T1) and transgenic (MMTV-PyMT) mouse models of mammary tumor growth. In the 4T1 model, both splenic and bone marrow-derived CD11b+Gr-1+ cells of tumor-bearing mice displayed a marked reduction in IRF-8 expression compared to control populations. A causal link between IRF-8 expression and the emergence of tumor-induced CD11b+Gr-1+ cells was explored in vivo using a double transgenic (dTg) mouse model designed to express transgenes for both IRF-8 and mammary carcinoma development. Despite the fact that tumor growth was unaffected, splenomegaly, as well as the frequencies and absolute numbers of CD11b+Gr-1+ cells were significantly lower in dTg mice when compared with single transgenic tumor-bearing mice. Overall, these data reveal that IRF-8 plays an important role in tumor-induced development and/or accumulation of CD11b+Gr-1+ cells, and establishes a molecular basis for the potential manipulation of these myeloid populations for cancer therapy.
IRF-8; myeloid-derived suppressor cells; tumor progression; hematopoiesis
The histone deacetylase (HDAC) inhibitor trichostatin A (TSA) has recently been shown to inhibit deleterious effects of cytokines on β-cells, but it is unable to protect β-cells from death due to its own cytotoxicity. Herein, we investigated novel HDAC inhibitors for their cytoprotective effects against IL-1β-induced damage to isolated β-cells. We report that three novel compounds (THS-73–44, THS-72–5 and THS-78–5) significantly inhibited HDAC activity and increased the acetylation of histone H4 in isolated β-cells. Further, these compounds exerted no toxic effects on metabolic cell viability in these cells. However, among the three compounds tested, only THS-78–5 protected against IL-1β-mediated loss in β-cell viability. THS-78–5 was also able to attenuate IL-1β-induced inducible nitric oxide synthase expression and subsequent NO release. Our data also indicate that the cytoprotective properties of THS-78–5 against IL-1β-mediated effects may, in part, be due to inhibition of IL-1β-induced transactivation of nuclear factor κB (NF-κB) in these cells. Together, we provide evidence for a novel HDAC inhibitor with a significant potential to prevent IL-1β-mediated effects on isolated β-cells. Potential implications of these findings in the development of novel therapeutics to prevent deleterious effects of cytokines and the onset of autoimmune diabetes are discussed.
HDAC inhibitors; pancreatic β-cell; inducible nitric oxide synthase; NO release; NF-κB
Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) affects cross-talk between the individual cell types of the neurovascular unit, which then contributes to disruption of the blood–brain barrier (BBB) and the development of neurological dysfunctions. While the toxicity of HIV-1 on neurons, astrocytes, and brain endothelial cells has been widely studied, there are no reports addressing the influence of HIV-1 on pericytes. Therefore, the purpose of this study was to evaluate whether pericytes can be infected with HIV-1 and how such an infection affects the barrier function of brain endothelial cells. Our results indicate that human brain pericytes express the major HIV-1 receptor CD4 and coreceptors CXCR4 and CCR5. We also determined that HIV-1 can replicate, though at a low level, in human brain pericytes as detected by HIV-1 p24 ELISA. Pericytes were susceptible to infection with both the X4-tropic NL4-3 and R5-tropic JR-CSF HIV-1 strains. Moreover, HIV-1 infection of pericytes resulted in compromised integrity of an in vitro model of the BBB. These findings indicate that human brain pericytes can be infected with HIV-1 and suggest that infected pericytes are involved in the progression of HIV-1-induced CNS damage.
HIV-1; pericytes; blood–brain barrier; neurovascular unit
Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-βRI indicative of altered TGF-β signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.
scleroderma; fibroblast; hTERT; fibrosis; collagen; TGF-β; Smad1
Previous studies have shown that the transforming growth factor (TGF)β/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFβ. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFβ requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFβ-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFβ/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.
scleroderma; caveolin-1; Smad1; CCN2
Combined morphological, immunocytochemical, biochemical and molecular genetic studies were performed on skeletal muscle, heart muscle and liver tissue of a 16-months boy with fatal liver failure. The pathological characterization of the tissues revealed a severe depletion of mtDNA (mitochondrial DNA) that was most pronounced in liver, followed by a less severe, but still significant depletion in skeletal muscle and the heart. The primary cause of the disease was linked to compound heterozygous mutations in the polymerase γ (POLG) gene (DNA polymerase γ; A467T, K1191N). We present evidence, that compound heterozygous POLG mutations lead to tissue selective impairment of mtDNA replication and thus to a mosaic defect pattern even in the severely affected liver. A variable defect pattern was found in liver, muscle and heart tissue as revealed by biochemical, cytochemical, immunocytochemical and in situ hybridization analysis. Functionally, a severe deficiency of cytochrome-c-oxidase (cox) activity was seen in the liver. Although mtDNA depletion was detected in heart and skeletal muscle, there was no cox deficiency in these tissues. Depletion of mtDNA and microdissection of cox-positive or negative areas correlated with the histological pattern in the liver. Interestingly, the mosaic pattern detected for cox-activity and mtDNA copy number fully aligned with the immunohistologically revealed defect pattern using Pol γ, mtSSB- and mtTFA-antibodies, thus substantiating the hypothesis that nuclear encoded proteins located within mitochondria become unstable and are degraded when they are not actively bound to mtDNA. Their disappearance could also aggravate the mtDNA depletion and contribute to the non-homogenous defect pattern.
depletion of mtDNA; polymerase γ; mitochondrial single stranded binding protein(mtSSB); mitochondrial transcription factor A (mtTFA); liver failure; in situ hybridization
PV1 is an endothelial specific protein with structural roles in the formation of diaphragms in endothelial cells of normal vessels. PV1 is also highly expressed on endothelial cells of many solid tumors. Based on in vitro data, PV1 is thought to actively participate in angiogenesis. In order to test whether PV1 has a function in tumor angiogenesis and in tumor growth in vivo, we have treated pancreatic tumor bearing mice by single dose intratumoral delivery of lentiviruses encoding for two different shRNAs targeting murine PV1. We find that PV1 downregulation by shRNAs inhibits the growth of established tumors derived from two different human pancreatic adenocarcinoma cell lines (AsPC-1 and BxPC-3). The effect observed is due to downregulation of PV1 in the tumor endothelial cells of host origin, PV1 being specifically expressed in tumor vascular endothelial cells and not in cancer or other stromal cells. There are no differences in vascular density of tumors treated or not with PV1 shRNA and gain and loss of function of PV1 in endothelial cells does not modify either their proliferation or migration, suggesting that tumor angiogenesis is not impaired. Together, our data argue that down regulation of PV1 in tumor endothelial cells results in the inhibition of tumor growth via a mechanism different from inhibiting angiogenesis.
angiogenesis; fenestrae; vesiculo-vacuolar organelles; caveolae; pancreatic cancer; transendothelial channels; tumor microenvironment
Study of physiologic angiogenesis and associated signaling mechanisms in adult heart has been limited by the lack of a robust animal model. We investigated thyroid hormone induced sprouting angiogenesis and the underlying mechanism. Hypothyroidism was induced in C57BL/6J mice by feeding with propylthiouracil (PTU). One year of PTU treatment induced heart failure. Both 12 weeks- (young) and 1 year-PTU (middle age) treatment caused a remarkable capillary rarefaction observed in capillary density. Three-days Triiodothyronine (T3) treatment significantly induced cardiac capillary growth in hypothyroid mice. In cultured left ventricle (LV) tissues from PTU treated mice, T3 also induced robust sprouting angiogenesis where pericyte-wrapped endothelial cells formed tubes. The in vitro T3 angiogenic response was similar in mice pretreated with PTU for periods ranging from 1.5–12 months. Besides bFGF and VEGF164, PDGF-BB was the most robust angiogenic growth factor, which stimulated notable sprouting angiogenesis in cultured hypothyroid LV tissues with increasing potency but had little effect on tissues from euthyroid mice. T3 treatment significantly increased PDGF receptor beta (PDGFR-β) protein levels in hypothyroid heart. PDGFR inhibitors blocked the action of T3 both on sprouting angiogenesis in cultured LV tissue and on capillary growth in vivo. Additionally, activation of Akt signaling mediated in T3-induced angiogenesis was blocked by PDGFR inhibitor and neutralizing antibody. Our results suggest that hypothyroidism leads to cardiac microvascular impairment and rarefaction with increased sensitivity to angiogenic growth factors. T3 induced cardiac sprouting angiogenesis in adult hypothyroid mice was associated with PDGF-BB, PDGFR-β, and downstream activation of Akt.
thyroid hormone; sprouting angiogenesis; pericyte; PDGF; Akt
Renal medullary hypoxia-inducible factor (HIF)-1α and its target genes, such as heme oxygenase and nitric oxide synthase, have been indicated to play an important role in the regulation of sodium excretion and blood pressure. HIF prolyl hydroxylase domain-containing proteins (PHDs) are major enzymes to promote the degradation of HIF-1α. We recently reported that high salt intake suppressed the renal medullary PHD2 expression and thereby activated HIF-1α-mediated gene regulation in the renal medulla in response to high salt. To further define the functional role of renal medullary PHD2 in the regulation of renal adaptation to high salt intake and the longer-term control of blood pressure, we transfected PHD2 expression plasmids into the renal medulla in uninephrectomized rats and determined its effects on pressure natriuresis, sodium excretion after salt overloading and the long-term control of arterial pressure after high salt challenge. It was shown that overexpression of PHD2 transgene increased PHD2 levels and decreased HIF-1α levels in the renal medulla, which blunted pressure natriuresis, attenuated sodium excretion, promoted sodium retention and produced salt sensitive hypertension after high salt challenge compared with rats treated with control plasmids. There was no blood pressure change in PHD2-treated rats that were maintained in low salt diet. These results suggested that renal medullary PHD2 is an important regulator in renal adaptation to high salt intake and a deficiency in PHD2-mediated molecular adaptation in response to high salt intake in the renal medulla may represent a pathogenic mechanism producing salt sensitive hypertension.
pressure natriuresis; hypoxia inducible factor-1α; transcription factor; gene transfection; sodium excretion
Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction, and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1-AC10), the enzymes that generate cyclic AMP (cAMP). Our lab recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any maneuver causing reduction of free [Ca2+] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this “store-operated” pathway requires the ER Ca2+ sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here we used sensitive FRET-based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store-operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca2+ entry via the plasma membrane Ca2+ channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca2+/cAMP crosstalk system.
Ubiquitin-dependent proteolysis is a major mechanism that downregulates misfolded proteins or those that have finished a programmed task. In the last two decades, neddylation has emerged as a major regulatory pathway for ubiquitination. Central to the neddylation pathway is the amyloid precursor protein (APP)-binding protein APP-BP1, which together with Uba3, plays an analogous role to the ubiquitin-activating enzyme E1 in nedd8 activation. Activated nedd8 covalently modifies and activates a major class of ubiquitin ligases called Cullin-RING ligases (CRLs). New evidence suggests that neddylation also modifies Type-1 transmembrane receptors such as APP. Here we review the functions of neddylation and summarize evidence suggesting that dysfunction of neddylation is involved in Alzheimer’s disease.
APP; NAE1; Alzheimer’s disease; Parkinson’s disease; neurodegeneration; proteasome; neurogenesis; ubiquitination; neddylation; differentiation
Mice with spontaneous mutations in the Sharpin gene develop chronic proliferative dermatitis that is characterized by eosinophilic inflammation of the skin and other organs with increased expression of type 2 cytokines and dysregulated development of lymphoid tissues. The mutant mice share phenotypic features with human hypereosinophilic syndromes. The biological function of SHARPIN and how its absence leads to such a complex inflammatory phenotype in mice are poorly understood. However, recent studies identified SHARPIN as a novel modulator of immune and inflammatory responses. The emerging mechanistic model suggests that SHARPIN functions as an important adaptor component of the linear ubiquitin chain assembly complex that modulates activation of NF-κB signaling pathway, thereby regulating cell survival and apoptosis, cytokine production and development of lymphoid tissues. In this review, we will summarize the current understanding of the ubiquitin-dependent regulatory mechanisms involved in NF-κB signaling, and incorporate the recently obtained molecular insights of SHARPIN into this pathway. Recent studies identified SHARPIN as an inhibitor of β1-integrin activation and signaling, and this may be another mechanism by which SHARPIN regulates inflammation. Furthermore, the disrupted lymphoid organogenesis in SHARPIN-deficient mice suggests that SHARPIN-mediated NF-κB regulation is important for de novo development of lymphoid tissues.
Sharpin; immune system; inflammation; chronic dermatitis; NF-κB; ubiquitination; lymphoid organogenesis; eosinophilic dermatitis; scaly skin disease
Recent work identified L-asparaginase (L-ASP) as a putative therapeutic target for ovarian cancer. We hypothesized L-ASP, a dysregulator of glycosylation, would interrupt the local microenvironment, affecting the ovarian cancer cell—endothelial cell interaction and thus angiogenesis without cytotoxic effects. Ovarian cancer cell lines and human microvascular endothelial cells (HMVEC) were exposed to L-ASP at physiologically attainable concentrations and subjected to analyses of endothelial tube formation, invasion, adhesion, and the assessment of sialylated proteins involved in matrix-associated and heterotypic cell adhesion. Marked reduction in HMVEC tube formation in vitro, HMVEC and ovarian cancer cell invasion, and heterotypic cell-cell and cell-matrix adhesion was observed (p<0.05 – 0.0001). These effects were associated with reduced binding to ß1integrin, activation of FAK, and cell surface sialyl LewisX (sLex) expression. No reduction in HMVEC E-selectin expression was seen consistent with the unidirectional inhibitory actions observed. L-ASP concentrations were non-toxic to either ovarian cancer or HMVEC lines in the time frame of the assays. However, early changes of autophagy were observed in both cell types with induction of ATG12, beclin-1, and cleavage of LC-3, indicating cell injury did occur. These data and the known mechanism of action of L-ASP on glycosylation of nascent proteins suggest that L-ASP reduction of ovarian cancer dissemination and progression through modification of its microenvironment. The reduction of ovarian cancer cell surface sLex inhibits interaction with HMVEC and thus HMVEC differentiation into tubes, inhibits interaction with the local matrix reducing invasive behavior, and causes cell injury initiating autophagy in tumor and vascular cells.
asparaginase; ovarian cancer; sialyl Lewis X; angiogenesis; autophagy
Despite mounting preclinical and clinical evidence of the beneficial effects of cell-based therapy, optimal cell dosing and delivery approaches have not been identified. Cardiospheres are self-assembling 3D microtissues formed by cardiac stem cells and supporting cell types. The ability of cardiospheres to augment cardiac function has been demonstrated in animal models of ischemic cardiomyopathy. In the present study, we studied the dose-dependence of the benefits of human cardiospheres, delivered via intramyocardial injection, upon cardiac function and ventricular remodeling in SCID mice with acute myocardial infarction. Four doses of cardiospheres were used: 1×104, 5×104, 1×105, and 5×105 (expressed as number of plated cardiosphere-forming cells). Acute (24 hour) cell retention rates in all groups were similar. Functional assessment and quantitative heart morphometry indicated benefit from higher cell doses (≥5×104) in terms of ejection fraction, infarct size and capillary density. Histological analysis indicated that the dose-dependent benefit was primarily due to indirect effects of transplanted cells. The results provide scalable data on cardiosphere dosing for intramyocardial injection.
To determine in the baboon model the identities and functional characteristics of endothelial progenitor cells mobilized in response to artery ligation, we collected peripheral blood mononuclear cells before and 3 days after a segment of femoral artery was removed. Our goal was to find EPC subpopulations with highly regenerative capacity. We identified 12 subpopulations of putative EPCs that were altered >1.75 fold; two subpopulations (CD146+/CD54-/CD45- at 6.63 fold, and CD146+/UEA-1-/CD45- at 12.21 fold) were dramatically elevated. To investigate the regenerative capacity of putative EPCs, we devised a new assay that maximally resembled their in vivo scenario, we purified CD34+ and CD146+ cells and co-cultured them with basal and mobilized peripheral blood mononuclear cells; both cell types took up Dil-LDL, but purified CD146+ cells exhibited accelerated differentiation by increasing expression of CD31 and CD144, and by exhibiting more active cord-like structure formation by comparison to the CD34+ subpopulation in a co-culture with mobilized PBMNCs. We demonstrate that ischemia due to vascular ligation mobilizes multiple types of cells with distinct roles. Baboon CD146+ cells exhibit higher reparative capacity than CD34+ cells, and thus are a potential source for therapeutic application.
EPC mobilization; nonhuman primate model; ischemia
The present study tested the hypothesis that homocysteine (Hcys)-induced ceramide production stimulates lipid rafts (LRs) clustering on the membrane of glomerular endothelial cells (GECs) to form redox signalling platforms by aggregation and activation of NADPH oxidase subunits and thereby enhances superoxide (O2•−) production, leading to glomerular endothelial dysfunction and ultimate injury or sclerosis. Using confocal microscopy, we first demonstrated a co-localization of LR clusters with NADPH oxidase subunits, gp91phox and p47phox in the GECs membrane upon Hcys stimulation. Immunoblot analysis of floated detergent-resistant membrane fractions found that in LR fractions NADPH oxidase subunits gp91phox and p47phox are enriched and that the activity of this enzyme dramatically increased. We also examined the effect of elevated Hcys on the cell monolayer permeability in GECs. It was found that Hcys significantly increased GEC permeability, which was blocked by inhibition of LR redox signalling platform formation. Finally, we found that Hcys-induced enhancement of GEC permeability is associated with the regulation of microtubule stability through these LR-redox platforms. It is concluded that the early injurious effect of Hcys on the glomerular endothelium is associated with the formation of redox signalling platforms via LR clustering, which may lead to increases in glomerular permeability by disruption of microtubule network in GECs.
lipid microdomains; hyperhomocysteinemia; glomerular sclerosis; hyperfiltration
Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent intracellular Ca2+ signaling second messenger, but the mechanism of NAADP-induced Ca2+ release is still poorly understood. The present study tested the hypothesis that NAADP induces Ca2+ release from the lysosomal store via a TRP-ML1 (transient receptor potential-mucolipin 1)-mediated Ca2+ release channel in coronary arterial myocytes (CAMs). RT-PCR and Western blot analyses demonstrated that TRP-ML1 was present in CAMs, and fluorescence resonance energy transfer (FRET) detection revealed that the TRP-ML1 was closely associated with some lysosomal proteins in these CAMs. ET-1, a well-known NAADP stimulator, was found to induce a local Ca2+ burst from lysosomes followed by a global Ca2+ release. This lysosome-associated Ca2+ release was significantly inhibited in the TRP-ML1 siRNA pre-treated CAMs by 46.8 ± 12.6 % in the local Ca2+ burst and 73.3 ± 14.9 % in the global Ca2+ wave. In the reconstituted lysosomal channels from CAMs, NAADP activated Ca2+ release channels at concentrations of 1–1000 nM, but neither activators (1 μM IP3, 5 μM Rya) nor blockers (100 μM 2-APB, 50 μM Rya) of sarcoplasmic reticulum (SR) Ca2+ release channels had effect on the channel activity. Moreover, TRP-ML1 gene silencing reduced this NAADP-sensitive Ca2+ release channel activity in lysosomes by 71.5 ± 18.5%. Immunoprecipitation or blockade of TRP-ML1 by anti-TRP-ML1 antibodies almost abolished NAADP-induced activation of lysosomal Ca2+ channels (to 14.0 ± 4.4% of control). These results for the first time provide direct evidence that an NAADP-sensitive Ca2+ release channel is characteristic of TRP-ML1 channels.
Transient receptor potential channel; lysosomal Ca2+ store; Ca2+ mobilization; organelles; coronary circulation
CD38 is a multifunctional protein involving in a number of signaling pathways. Given that the lack of CD38 is considered as a dedifferentiation marker of lymphocytes and other cells, we hypothesized that CD38 and its signaling pathway may participate in the epithelial-to-mesenchymal transition (EMT) process of podocytes and thereby regulates the integrity of glomerular structure and function. Western blot analysis and RT-PCR demonstrated that renal tissue CD38 expression was lacking in CD38−/− mice or substantially reduced in renal CD38 shRNA-transfected WT (CD38-shRNA) mice compared to CD38+/+ littermates. Confocal fluorescent microscopy demonstrated the reduced expression of epithelial markers (P-Cadherin, ZO-1 and podocin) and increased expression of mesenchymal markers (FSP-1, α-SMA and desmin) in the glomeruli of CD38−/− and CD38-shRNA mice compared to CD38+/+ mice. Morphological examinations showed profound injury in the glomeruli of CD38−/− or CD38-shRNA mice compared to CD38+/+ mice. This enhanced glomerular injury in CD38−/− or CD38-shRNA mice was accompanied by increased albuminuria and proteinuria. DOCA/high salt treatment further decreased the expression of epithelial markers and increased the abundance of mesenchymal markers, which were accompanied by more increased glomerular damage index and mean arterial pressure in CD38−/− and CD38-shRNA mice than CD38+/+ mice. In vitro studies showed that inhibition of CD38 enhances the EMT in podocytes. In conclusion, our observations reveal that the normal expression of CD38 importantly contributes to the differentiation and function of podocytes and the defect of this gene expression may be a critical mechanism inducing EMT and consequently resulting in glomerular injury and sclerosis.
CD38; glomerulosclerosis; ADP–ribosylcyclase; end-stage renal disease; podocyte transdifferentiation
Previously, we have shown that CCR5 transcription is regulated by CREB-1. However, the ubiquitous pattern of CREB-1 expression suggests the involvement of an additional level of transcriptional control in the cell type-specific expression of CCR5. In this study we show that epigenetic changes (i.e. DNA methylation and histone modifications) within the context of the CCR5 P1 promoter region correlate with transcript levels of CCR5 in healthy and in malignant CD4+ T lymphocytes as well as in CD14+ monocytes. In normal naïve T cells and CD14+ monocytes the CCR5 P1 promoter resembles a bivalent chromatin state, with both repressive and permissive histone methylation and acetylation marks. The CCR5 expressing CD14+ monocytes however show much higher levels of acetylated histone H3 (AcH3) compared to the non–CCR5-expressing naïve T cells. Combined with a highly methylated promoter in CD14+ monocytes, this indicates a dominant role for AcH3 in CCR5 transcription.
We also show that pharmacological interference in the epigenetic repressive mechanisms that account for the lack of CCR5 transcription in T leukemic cell lines results in an increase in CREB-1 association with CCR5 P1 chromatin. Furthermore RNA polymerase II was also recruited into CCR5 P1 chromatin resulting in CCR5 re-expression. Together, these data indicate that epigenetic modifications of DNA, and of histones, contribute to the control of CCR5 transcription in immune effector cells.
Chromatin remodeling; histone modifications; DNA methylation; bivalent chromatin; poised chromatin; CCR5; T Cells; Monocytes
In this study, we tested the hypothesis that the Angiopoietin 1 (Ang1)/Tie2 pathway mediates simvastatin-induced vascular integrity and migration of neuroblasts after stroke. Rats were subjected to 2 hrs of middle cerebral artery occlusion (MCAo) and treated, starting 1 day after stroke with or without simvastatin (1 mg/kg, daily) for 7 days. Simvastatin treatment significantly decreased blood–brain barrier (BBB) leakage and concomitantly, increased Ang1, Tie2 and Occludin expression in the ischaemic border (IBZ) compared to the MCAo control group. Simvastatin also significantly increased doublecortin (DCX, a marker of migrating neuroblasts) expression in the IBZ compared to control MCAo rats. DCX was highly expressed around vessels. To further investigate the signalling pathway of simvastatin-induced vascular stabilization and angiogenesis, rat brain microvascular endothelial cell (RBMEC) culture was employed. The data show that simvastatin treatment of RBMEC increased Ang1 and Tie2 gene and protein expression and promoted phosphorylated-Tie2 activity. Simvastatin significantly increased endothelial capillary tube formation, an index of angiogenesis, compared to non-treated control. Inhibition of Ang1 or knockdown of Tie2 gene expression in endothelial cells significantly attenuated simvastatin-induced capillary tube formation. In addition, simvastatin significantly increased subventricular zone (SVZ) explant cell migration compared to non-treatment control. Inhibition of Ang1 significantly attenuated simvastatin-induced SVZ cell migration. Simvastatin treatment of stroke increases Ang1/Tie2 expression and thereby reduces BBB leakage and promotes vascular stabilization. Ang1/Tie2 expression induced by simvastatin treatment promotes neuroblast microvascular coupling after stroke.
stroke; angiopoietin 1; Tie2; simvastatin; vascular stabilization
The ultrastructural changes in diabetic and idiopathic gastroparesis are not well studied and it is not known whether there are different defects in the two disorders. As part of the Gastroparesis Clinical Research Consortium, full thickness gastric body biopsies from 20 diabetic and 20 idiopathic gastroparetics were studied by light microscopy. Abnormalities were found in many (83%) but not all patients. Among the common defects were loss of interstitial cells of Cajal (ICC) and neural abnormalities. No distinguishing features were seen between diabetic and idiopathic gastroparesis. Our aim was to provide a detailed description of the ultrastructural abnormalities, compare findings between diabetic and idiopathic gastroparesis and determine if patients with apparently normal immunohistological features have ultrastructural abnormalities. Tissues from 40 gastroparetic patients and 24 age and sex matched controls were examined by transmission electron microscopy (TEM). ICC showing changes suggestive of injury, large and empty nerve endings, presence of lipofuscin and lamellar bodies in the smooth muscle cells were found in all patients. However, the ultrastructural changes in ICC and nerves differed between diabetic and idiopathic gastroparesis and were more severe in idiopathic gastroparesis. A thickened basal lamina around smooth muscle cells and nerves was characteristic of diabetic gastroparesis whereas idiopathic gastroparetics had fibrosis, especially around the nerves. In conclusion, in all the patients TEM showed abnormalities in ICC, nerves and smooth muscle consistent with the delay in gastric emptying. The significant differences found between diabetic and idiopathic gastroparesis offers insight into pathophysiology as well as into potential targeted therapies.
electron microscopy; smooth muscle; enteric nerves; interstitial cells of Cajal
Background and Aims
An obstacle to understanding motor pathologies of the GI tract is that the physiology of some of the cellular components of the gut wall is not understood. Morphologists identified fibroblast-like cells in the tunica muscularis many years ago, but little is know about these interstitial cells because of inadequate techniques to identify these cells. Recent findings have shown that fibroblast-like cells express PDGFRα in mice and antibodies for these receptors can be used to label the cells.
We used immunohistochemical techniques to study the phenotype and intercellular relationships of fibroblast-like cells in the human colon.
Fibroblast-like cells are labeled specifically with antibodies to PDGFRα and widely distributed through the tunica muscularis of human colon. These cells form discrete networks in the region of the myenteric plexus and within the circular and longitudinal muscle layers. PDGFRα+ cells are distinct from c-Kit+ interstitial cells of Cajal and closely associated with varicose processes of neurons expressing substance P (excitatory motor neurons) or nNOS (inhibitory motor neurons). PDGFRα+ cells express small conductance Ca2+-activated K+ channels (SK3), which are likely to mediate purinergic neural regulation of colonic muscles.
Our data suggest that PDGFRα+ cells may have an important role in transducing inputs from enteric motor neurons. This study identifies reagents and techniques that will allow investigation of this class of interstitial cells and help to develop an understanding of the role of PDGFRα+ cells in the human GI tract in health and disease.
interstitial cells of Cajal; fibroblast-like cell; enteric inhibitory neurotransmission; GI motility; purinergic neurotransmission
In this study, an injectable, biodegradable hydrogel composite of oligo(poly(ethylene glycol) fumarate) (OPF) was investigated as a carrier of mouse embryonic stem cells (mESCs) for the treatment of myocardial infarction (MI). 10K OPF hydrogels were used to encapsulate mESCs. The cell differentiation in vitro over 14 days was determined via immunohistochemical examination. Then, mESCs encapsulated in OPF hydrogels were injected into the left ventricular wall of a rat myocardial infarction model. Detailed histological analysis and echocardiography were used to determine the structural and functional consequences after 4 weeks of transplantation. With ascorbic acid induction, mESCs could differentiate into cardiomyocytes and other cell types in all three lineages in the OPF hydrogel. After transplantation, both the 24h-cell retention and 4-week graft size were significantly greater in the OPF + ESC group than that of the PBS + ESC group (p<0.01). Four weeks after transplantation, OPF hydrogel alone significantly reduced the infarct size and collagen deposition and improved the cardiac function. The heart function and revascularization improved significantly, while the infarct size and fibrotic area decreased significantly in the OPF + ESC group compared with that of the PBS +ESC group, OPF group and PBS group (p<0.01). All treatments had significantly reduced MMP2 and MMP9 protein levels compared to the PBS control group, and the OPF + ESC group decreased most by Western blotting. Transplanted mESCs expressed cardiovascular markers. This study suggest the potential of a method for heart regeneration involving oligo(poly(ethylene glycol) fumarate) hydrogels for stem cell encapsulation and transplantation.
cardiac tissue engineering; injectable hydrogels; cell encapsulation; embryonic stem cell; myocardial infarction
The low levels of CFTR gene expression and paucity of CFTR protein in human airway epithelial cells are not easily reconciled with the pivotal role of the lung in cystic fibrosis pathology. Previous data suggested that the regulatory mechanisms controlling CFTR gene expression might be different in airway epithelium in comparison to intestinal epithelium where CFTR mRNA and protein is much more abundant. Here we examine chromatin structure and modification across the CFTR locus in primary human tracheal (HTE) and bronchial (NHBE) epithelial cells and airway cell lines including 16HBE14o- and Calu3. We identify regions of open chromatin that appear selective for primary airway epithelial cells and show that several of these are enriched for a histone modification (H3K4me1) that is characteristic of enhancers. Consistent with these observations, three of these sites encompass elements that have cooperative enhancer function in reporter gene assays in 16HBE14o- cells. Finally, we use chromosome conformation capture (3C) to examine the three dimensional structure of nearly 800 kb of chromosome 7 encompassing CFTR and observe long range interactions between the CFTR promoter and regions far outside the locus in cell types that express high levels of CFTR.