Activated intestinal muscle cells promote preadipocyte migration: a novel mechanism for creeping fat formation in Crohn's disease.

OBJECTIVE: Creeping fat, the wrapping of mesenteric fat around the bowel wall, is a typical feature of Crohn's disease, and is associated with stricture formation and bowel obstruction. How creeping fat forms is unknown, and we interrogated potential mechanisms using novel intestinal tissue and cell interaction systems. DESIGN: Tissues from normal, UC, non-strictured and strictured Crohn's disease intestinal specimens were obtained. The muscularis propria matrisome was determined via proteomics. Mesenteric fat explants, primary human preadipocytes and adipocytes were used in multiple ex vivo and in vitro cell migration systems on muscularis propria muscle cell derived or native extracellular matrix. Functional experiments included integrin characterisation via flow cytometry and their inhibition with specific blocking antibodies and chemicals. RESULTS: Crohn's disease muscularis propria cells produced an extracellular matrix scaffold which is in direct spatial and functional contact with the immediately overlaid creeping fat. The scaffold contained multiple proteins, but only fibronectin production was singularly upregulated by transforming growth factor-ß1. The muscle cell-derived matrix triggered migration of preadipocytes out of mesenteric fat, fibronectin being the dominant factor responsible for their migration. Blockade of α5ß1 on the preadipocyte surface inhibited their migration out of mesenteric fat and on 3D decellularised intestinal tissue extracellular matrix. CONCLUSION: Crohn's disease creeping fat appears to result from the migration of preadipocytes out of mesenteric fat and differentiation into adipocytes in response to an increased production of fibronectin by activated muscularis propria cells. These new mechanistic insights may lead to novel approaches for prevention of creeping fat-associated stricture formation.

Telomerase Reverse Transcriptase Deficiency Prevents Neointima Formation Through Chromatin Silencing of E2F1 Target Genes.

OBJECTIVE: Aberrant proliferation of smooth muscle cells (SMC) in response to injury induces pathological vascular remodeling during atherosclerosis and neointima formation. Telomerase is rate limiting for tissue renewal and cell replication; however, the physiological role of telomerase in vascular diseases remains to be determined. The goal of the present study was to determine whether telomerase reverse transcriptase (TERT) affects proliferative vascular remodeling and to define the molecular mechanism by which TERT supports SMC proliferation. APPROACH AND RESULTS: We first demonstrate high levels of TERT expression in replicating SMC of atherosclerotic and neointimal lesions. Using a model of guidewire-induced arterial injury, we demonstrate decreased neointima formation in TERT-deficient mice. Studies in SMC isolated from TERT-deficient and TERT overexpressing mice with normal telomere length established that TERT is necessary and sufficient for cell proliferation. TERT deficiency did not induce a senescent phenotype but resulted in G1 arrest albeit hyperphosphorylation of the retinoblastoma protein. This proliferative arrest was associated with stable silencing of the E2F1-dependent S-phase gene expression program and not reversed by ectopic overexpression of E2F1. Finally, chromatin immunoprecipitation and accessibility assays revealed that TERT is recruited to E2F1 target sites and promotes chromatin accessibility for E2F1 by facilitating the acquisition of permissive histone modifications. CONCLUSIONS: These data indicate a previously unrecognized role for TERT in neointima formation through epigenetic regulation of proliferative gene expression in SMC.

Vascular smooth muscle cell dysfunction in diabetes: nuclear receptors channel to relaxation.

Endothelial dysfunction and impaired vascular relaxation represent a common cause of microvascular disease in patients with diabetes. Although multiple mechanisms underlying altered endothelial cell function in diabetes have been described, there is currently no specific and approved pharmacological treatment. In this edition of Clinical Science, Morales-Cano et al. characterize voltage-dependent K(+) (Kv) channels as genes regulated by pharmacological activation of peroxisome proliferator-activated receptor-b/d (PPARb/d). Diabetes altered Kv channel function leading to impaired coronary artery relaxation, which was prevented by pharmacological activation of PPARb/d. These studies highlight an important mechanism of vascular dysfunction in diabetes and point to a potential approach for therapy, particularly considering that PPARb/d ligands have been developed and tested in small clinical trials.

A germ-free humanized mouse model shows the contribution of resident microbiota to human-specific pathogen infection.

Germ-free (GF) mice, which are depleted of their resident microbiota, are the gold standard for exploring the role of the microbiome in health and disease; however, they are of limited value in the study of human-specific pathogens because they do not support their replication. Here, we develop GF mice systemically reconstituted with human immune cells and use them to evaluate the role of the resident microbiome in the acquisition, replication and pathogenesis of two human-specific pathogens, Epstein-Barr virus (EBV) and human immunodeficiency virus (HIV). Comparison with conventional (CV) humanized mice showed that resident microbiota enhance the establishment of EBV infection and EBV-induced tumorigenesis and increase mucosal HIV acquisition and replication. HIV RNA levels were higher in plasma and tissues of CV humanized mice compared with GF humanized mice. The frequency of CCR5+ CD4+ T cells throughout the intestine was also higher in CV humanized mice, indicating that resident microbiota govern levels of HIV target cells. Thus, resident microbiota promote the acquisition and pathogenesis of two clinically relevant human-specific pathogens.

Inhibitors of histone deacetylases: correlation between isoform specificity and reactivation of HIV type 1 (HIV-1) from latently infected cells.

Deacetylation of histone proteins at the HIV type 1 (HIV-1) long terminal repeat (LTR) by histone deactylases (HDACs) can promote transcriptional repression and virus latency. As such, HDAC inhibitors (HDACI) could be used to deplete reservoirs of persistent, quiescent HIV-1 proviral infection. However, the development of HDACI to purge latent HIV-1 requires knowledge of the HDAC isoforms contributing to viral latency and the development of inhibitors specific to these isoforms. In this study, we identify the HDACs responsible for HIV-1 latency in Jurkat J89GFP cells using a chemical approach that correlates HDACI isoform specificity with their ability to reactivate latent HIV-1 expression. We demonstrate that potent inhibition or knockdown of HDAC1, an HDAC isoform reported to drive HIV-1 into latency, was not sufficient to de-repress the viral LTR. Instead, we found that inhibition of HDAC3 was necessary to activate latent HIV-1. Consistent with this finding, we identified HDAC3 at the HIV-1 LTR by chromatin immunoprecipitation. Interestingly, we show that valproic acid is a weak inhibitor of HDAC3 (IC(50) = 5.5 mm) relative to HDAC1 (IC(50) = 170 µm). Because the total therapeutic concentration of valproic acid ranges from 275 to 700 µm in adults, these data may explain why this inhibitor has no effect on the decay of latent HIV reservoirs in patients. Taken together, our study suggests an important role for HDAC3 in HIV-1 latency and, importantly, describes a chemical approach that can readily be used to identify the HDAC isoforms that contribute to HIV-1 latency in other cell types.

Nuclear receptor co-repressor is required to maintain proliferation of normal intestinal epithelial cells in culture and down-modulates the expression of pigment epithelium-derived factor.

Stem cells of the gut epithelium constantly produce precursors that progressively undergo a succession of molecular changes resulting in growth arrest and commitment to a specific differentiation program. Few transcriptional repressors have been identified that maintain the normal intestinal epithelial cell (IEC) proliferation state. Herein, we show that the nuclear receptor co-repressor (NCoR1) is differentially expressed during the proliferation-to-differentiation IEC transition. Silencing of NCoR1 expression in proliferating cells of crypt origin resulted in a rapid growth arrest without associated cell death. A genechip profiling analysis identified several candidate genes to be up-regulated in NCoR1-deficient IEC. Pigment epithelium-derived factor (PEDF, also known as serpinf1), a suspected tumor suppressor gene that plays a key role in the inhibition of epithelial tissue growth, was significantly up-regulated in these cells. Chromatin immunoprecipitation experiments showed that the PEDF gene promoter was occupied by NCoR1 in proliferating epithelial cells. Multiple retinoid X receptor (RXR) heterodimers interacting sites of the PEDF promoter were confirmed to interact with RXR and retinoid acid receptor (RAR). Cotransfection assays showed that RXR and RAR were able to transactivate the PEDF promoter and that NCoR1 was repressing this effect. Finally, forced expression of PEDF in IEC resulted in a slower rate of proliferation. These observations suggest that NCoR1 expression is required to maintain IEC in a proliferative state and identify PEDF as a novel transcriptional target for NCoR1 repressive action.

Selective deletion of MyD88 signaling in α-SMA positive cells ameliorates experimental intestinal fibrosis via post-transcriptional regulation.

Intestinal fibrosis leading to strictures remains a significant clinical problem in inflammatory bowel diseases (IBD). The role of bacterial components in activating intestinal mesenchymal cells and driving fibrogenesis is largely unexplored. Tamoxifen-inducible α-SMA promoter Cre mice crossed with floxed MyD88 mice were subjected to chronic dextran sodium sulfate colitis. MyD88 was deleted prior to or after induction of colitis. Human intestinal myofibroblasts (HIMF) were exposed to various bacterial components and assessed for fibronectin (FN) and collagen I (Col1) production. RNA sequencing was performed. Post-transcriptional regulation was assessed by polysome profiling assay. Selective deletion of MyD88 in α-SMA-positive cells prior to, but not after induction of, experimental colitis decreased the degree of intestinal fibrosis. HIMF selectively responded to flagellin with enhanced FN or Col1 protein production in a MyD88-dependent manner. RNA sequencing suggested minimal transcriptional changes induced by flagellin in HIMF. Polysome profiling revealed higher proportions of FN and Col1 mRNA in the actively translated fractions of flagellin exposed HIMF, which was mediated by eIF2 alpha and 4EBP1. In conclusion, selectivity of flagellin-induced ECM secretion in HIMF is post-transcriptionally regulated. The results may represent a novel and targetable link between the gut microbiota and intestinal fibrogenesis.

Telomerase Deficiency Predisposes to Heart Failure and Ischemia-Reperfusion Injury.

Introduction: Elevated levels of mitochondrial reactive oxygen species (ROS) contribute to the development of numerous cardiovascular diseases. TERT, the catalytic subunit of telomerase, has been shown to translocate to mitochondria to suppress ROS while promoting ATP production. Acute overexpression of TERT increases survival and decreases infarct size in a mouse model of myocardial infarct, while decreased telomerase activity predisposes to mitochondrial defects and heart failure. In the present study, we examined the role of TERT on cardiac structure and function under basal conditions and conditions of acute or prolonged stress in a novel rat model of TERT deficiency. Methods: Cardiac structure and function were evaluated via transthoracic echocardiogram. Langendorff preparations were used to test the effects of acute global ischemia reperfusion injury on cardiac function and infarction. Coronary flow and left ventricular pressure were measured during and after ischemia/reperfusion (I/R). Mitochondrial DNA integrity was measured by PCR and mitochondrial respiration was assessed in isolated mitochondria using an Oxygraph. Angiotensin II infusion was used as an established model of systemic stress. Results: No structural changes (echocardiogram) or coronary flow/left ventricle pressure (isolated hearts) were observed in TERT-/- rats at baseline; however, after I/R, coronary flow was significantly reduced in TERT-/- compared to wild type (WT) rats, while diastolic Left Ventricle Pressure was significantly elevated (n = 6 in each group; p < 0.05) in the TERT-/-. Interestingly, infarct size was less in TERT-/- rats compared to WT rats, while mitochondrial respiratory control index decreased and mitochondrial DNA lesions increased in TERT-/- compared to WT. Angiotensin II treatment did not alter cardiac structure or function; however, it augmented the infarct size significantly more in TERT-/- compared to the WT. Conclusion: Absence of TERT activity increases susceptibility to stress like cardiac injury. These results suggest a critical role of telomerase in chronic heart disease.

Loss of cathepsin L activity promotes claudin-1 overexpression and intestinal neoplasia.

Intestinal epithelial integrity and polarity are maintained by cohesive interactions between cells via the formation of tight junctions. Irregularities in tight junctions have only recently been found to be associated with the initiation and progression of intestinal neoplasia. The claudin family of proteins is integral to the structure and function of the tight junction but little is known of the molecular events that regulate the expression of these components. The present report identifies cathepsin L, classically a lysosomal cysteine protease, as being induced during intestinal epithelial cell polarization and differentiation. Inhibition of intracellular cathepsin L activity results in the accumulation of disorganized cell layers and a decline in the expression of differentiation markers in cultured intestinal epithelial cells. This coincides with a rapid up-regulation of claudin-1 protein accumulation. Mutant mice defective in cathepsin L activity (furless) display an elevated level of intestinal claudin-1 and claudin-2 expression. Loss of cathepsin L activity leads to a marked increase in tumor multiplicity in the intestine of Apc(Min) mice. Given the traditionally viewed biological role of cathepsin L in the processing of lysosomal content as well as in pathological extracellular matrix remodeling, the results here demonstrate an as yet unsuspected intracellular role for this protease in normal intestinal epithelial polarization and initiation of neoplasia.

Discovery of a small molecule agonist of phosphatidylinositol 3-kinase p110α that reactivates latent HIV-1.

Combination antiretroviral therapy (cART) can effectively suppress HIV-1 replication, but the latent viral reservoir in resting memory CD4(+) T cells is impervious to cART and represents a major barrier to curing HIV-1 infection. Reactivation of latent HIV-1 represents a possible strategy for elimination of this reservoir. In this study we describe the discovery of 1,2,9,10-tetramethoxy-7H-dibenzo[de,g]quinolin-7-one (57704) which reactivates latent HIV-1 in several cell-line models of latency (J89GFP, U1 and ACH-2). 57704 also increased HIV-1 expression in 3 of 4 CD8(+)-depleted blood mononuclear cell preparations isolated from HIV-1-infected individuals on suppressive cART. In contrast, vorinostat increased HIV-1 expression in only 1 of the 4 donors tested. Importantly, 57704 does not induce global T cell activation. Mechanistic studies revealed that 57704 reactivates latent HIV-1 via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. 57704 was found to be an agonist of PI3K with specificity to the p110α isoform, but not the p110ß, δ or γ isoforms. Taken together, our work suggests that 57704 could serve as a scaffold for the development of more potent activators of latent HIV-1. Furthermore, it highlights the involvement of the PI3K/Akt pathway in the maintenance of HIV-1 latency.

Disulfiram reactivates latent HIV-1 expression through depletion of the phosphatase and tensin homolog.

OBJECTIVE: Disulfiram (DSF), an inhibitor of acetaldehyde dehydrogenase that is used for the treatment of alcoholism, was shown to reactivate latent HIV-1 expression in a primary cell model of virus latency and is currently being assessed in a clinical trial for its potential to deplete the latent HIV-1 reservoir in patients on combination antiretroviral therapy. The mechanism by which DSF reactivates latent HIV-1 expression, however, is not known and was the focus of this study. DESIGN/METHODS: The impact of DSF treatment on HIV-1 latency was assessed in the ACH2, J89GFP and U1 cell line models of HIV-1 latency and in resting CD4 T cells isolated from HIV-negative donors. RESULTS: DSF reactivated latent HIV-1 expression in the U1 cell line, but not in the J89GFP or ACH2 cell lines. Interestingly, we found that DSF significantly reduced phosphatase and tensin homolog (PTEN) protein levels in U1 cells and in resting CD4 T cells from HIV-negative donors. Decreased PTEN resulted in increased phosphorylation of protein kinase B (Akt) and activation of the Akt signaling pathway. Consistent with these finding, pharmacological inhibitors of Akt and nuclear factor-kappaB (NF-κB) block the latent HIV-1-reactivating activity of DSF. Furthermore, we show that HIV-1 expression in the U1 cell line could be activated by a small molecule inhibitor of PTEN or by siRNA knockdown of PTEN expression. Neither the J89GFP nor ACH2 cells express PTEN, explaining the lack of DSF effect on HIV-1 expression in both these cell lines. CONCLUSION: DSF reactivates latent HIV-1 expression via the Akt signaling pathway through depletion of PTEN.

IL-1 beta-dependent regulation of C/EBP delta transcriptional activity.

We have previously shown that the transcription factor C/EBP delta is involved in the intestinal inflammatory response. C/EBP delta regulates several inflammatory response genes, such as haptoglobin, in the rat intestinal epithelial cell line IEC-6 in response to IL-1. However, the different C/EBP delta domains involved in IL-1 beta-mediated transcriptional activation and the kinases implicated have not been properly defined. To address this, we determined the role of the p38 MAP kinase in the regulation of C/EBP delta transcriptional activity. The IL-1-dependent induction of the acute phase protein gene haptoglobin in IEC-6 cells was decreased in response to the p38 MAP kinase inhibitor SB203580, as determined by Northern blot. Transcriptional activity of C/EBP delta was repressed by the specific inhibitor of the p38 MAP kinase, as assessed by transient transfection assays. Mutagenesis studies and transient transfection assays revealed an important domain for transcriptional activation between amino acids 70 and 108. This domain overlapped with a docking site for the p38 MAP kinase, between amino acids 75 and 85, necessary to insure C/EBP delta phosphorylation. Deletion of this domain led to a decrease in basal transcriptional activity of C/EBP delta and in p300-dependent transactivation, as assessed by transient transfection assays, and in IL-1-dependent haptoglobin induction. This unusual arrangement of a kinase docking site within a transactivation domain may functionally be important for the regulation of C/EBP delta transcriptional activity.