Haloom Rafehi

Vascular histone deacetylation by pharmacological HDAC inhibition

HDAC inhibitors can regulate gene expression by post-translational modification of histone as well as nonhistone proteins. Often studied at single loci, increased histone acetylation is the paradigmatic mechanism of action. However, little is known of the extent of genome-wide changes in cells stimulated by the hydroxamic acids, TSA and SAHA. In this article, we map vascular chromatin modifications including histone H3 acetylation of lysine 9 and 14 (H3K9/14ac) using chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq). Since acetylation-mediated gene expression is often associated with modification of other lysine residues, we also examined H3K4me3 and H3K9me3 as well as changes in CpG methylation (CpG-seq). RNA sequencing indicates the differential expression of ∼30% of genes, with almost equal numbers being up- and down-regulated. We observed broad deacetylation and gene expression changes conferred by TSA and SAHA mediated by the loss of EP300/CREBBP binding at multiple gene promoters. This study provides an important framework for HDAC inhibitor function in vascular biology and a comprehensive description of genome-wide deacetylation by pharmacological HDAC inhibition.

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 1956. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811). Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

Genetic and epigenetic events in diabetic wound healing

The prevalence of the chronic metabolic disorder, diabetes mellitus, is expected to increase in the coming years and worldwide pandemic levels are predicted. Inevitably, this will be accompanied by an increase in the prevalence of diabetic complications, including diabetic foot ulcers. At present, treatment options for diabetic foot ulcers are in many cases insufficient, and progression of the condition results in the requirement for limb amputation in a proportion of patients. To improve therapy, an increase in our understanding of the pathobiology of diabetic complications such as impaired wound healing is necessary. In this review, recent advances in molecular aspects of normal and impaired diabetic wound healing are discussed. Furthermore, investigations of the role of epigenetic processes in the pathogenesis of impaired diabetic wound healing are now emerging. Indeed, epigenetic changes have already been identified as key factors in diabetes and related complications and these are overviewed in this review.

HDAC inhibition attenuates cardiac hypertrophy by acetylation and deacetylation of target genes

Pharmacological histone deacetylase (HDAC) inhibitors attenuate pathological cardiac remodeling and hypertrophic gene expression; yet, the direct histone targets remain poorly characterized. Since the inhibition of HDAC activity is associated with suppressing hypertrophy, we hypothesized histone acetylation would target genes implicated in cardiac remodeling. Trichostatin A (TSA) regulates cardiac gene expression and attenuates transverse aortic constriction (TAC) induced hypertrophy. We used chromatin immunoprecipitation (ChIP) coupled with massive parallel sequencing (ChIP-seq) to map, for the first time, genome-wide histone acetylation changes in a preclinical model of pathological cardiac hypertrophy and attenuation of pathogenesis with TSA. Pressure overload-induced cardiac hypertrophy was associated with histone acetylation of genes implicated in cardiac contraction, collagen deposition, inflammation, and extracellular matrix identified by ChIP-seq. Gene set enrichment analysis identified NF-kappa B (NF-κB) transcription factor activation with load induced hypertrophy. Increased histone acetylation was observed on the promoters of NFκB target genes (Icam1, Vcam1, Il21r, Il6ra, Ticam2, Cxcl10) consistent with gene activation in the hypertrophied heart. Surprisingly, TSA attenuated pressure overload-induced cardiac hypertrophy and the suppression of NFκB target genes by broad histone deacetylation. Our results suggest a mechanism for cardioprotection subject to histone deacetylation as a previously unknown target, implicating the importance of inflammation by pharmacological HDAC inhibition. The results of this study provides a framework for HDAC inhibitor function in the heart and argues the long held views of acetylation is subject to more flexibility than previously thought.

Controversies surrounding the clinical potential of cinnamon for the management of diabetes.

Obesity levels have increased significantly in the past five decades and are predicted to continue rising, resulting in important health implications. In particular, this has translated to an increase in the occurrence of type II diabetes mellitus (T2D). To alleviate associated problems, certain nutraceuticals have been considered as potential adjuncts or alternatives to conventional prescription drugs. Cinnamon, a commonly consumed spice originating from South East Asia, is currently being investigated as a potential preventative supplement and treatment for insulin resistance, metabolic syndrome and T2D. Extensive in vitro evidence has shown that cinnamon may improve insulin resistance by preventing and reversing impairments in insulin signalling in skeletal muscle. In adipose tissue, it has been shown that cinnamon increases the expression of peroxisome proliferator‐activated receptors including, PPARγ. This is comparable to the action of commonly used thiazolinediones, which are PPAR agonists. Studies have also shown that cinnamon has potent anti‐inflammatory properties. However, numerous human clinical trials with cinnamon have been conducted with varying findings. While some studies have showed no beneficial effect, others have indicated improvements in cholesterol levels, systolic blood pressure, insulin sensitivity and postprandial glucose levels with cinnamon. However, the only measurement consistently improved by cinnamon consumption is fasting glucose levels. While it is still premature to suggest the use of cinnamon supplementation based on the evidence, further investigation into mechanisms of action is warranted. Apart from further characterization of genetic and epigenetic changes in model systems, systematic large‐scale clinical trials are required. In this study, we discuss the mechanisms of action of cinnamon in the context of T2D and we highlight some of the associated controversies.

Mechanisms of Action of Phenolic Compounds in Olive

ABSTRACT Olive oil, an oil rich in monounsaturated fatty acids (MUFCs) and minor constituents including phenolic compounds, is a major component of the Mediterranean diet. The potential health benefits of the Mediterranean diet were highlighted by the seminal Seven Countries Study, and more contemporary research has identified olive oil as a major element responsible for these effects. It is emerging that the phenolic compounds are the most likely candidates accounting for the cardioprotective and cancer preventative effects of extra virgin olive oil (EVOO). In particular, the phenolic compound, hydroxytyrosol has been identified as one of the most potent antioxidants found in olive oil. This review will briefly consider historical aspects of olive oil research and the biological properties of phenolic compounds in olive oil will be discussed. The focus of the discussion will be related to the mechanisms of action of hydroxytyrosol. Studies have demonstrated that hydroxytyrosol induces apoptosis and cell cycle arrest in cancer cells. Further, research has shown that hydroxytyrosol can prevent cardiovascular disease by reducing the expression of adhesion molecules on endothelial cells and preventing the oxidation of low-density lipoprotein (LDL). The molecular mechanisms accounting for these effects are reviewed.

Multicellular transcriptional analysis of mammalian heart regeneration

Background: The inability of the adult mammalian heart to regenerate following injury represents a major barrier in cardiovascular medicine. In contrast, the neonatal mammalian heart retains a transient capacity for regeneration, which is lost shortly after birth. Defining the molecular mechanisms that govern regenerative capacity in the neonatal period remains a central goal in cardiac biology. Here, we assemble a transcriptomic framework of multiple cardiac cell populations during postnatal development and following injury, which enables comparative analyses of the regenerative (neonatal) versus nonregenerative (adult) state for the first time. Methods: Cardiomyocytes, fibroblasts, leukocytes, and endothelial cells from infarcted and noninfarcted neonatal (P1) and adult (P56) mouse hearts were isolated by enzymatic dissociation and fluorescence-activated cell sorting at day 3 following surgery. RNA sequencing was performed on these cell populations to generate the transcriptome of the major cardiac cell populations during cardiac development, repair, and regeneration. To complement our transcriptomic data, we also surveyed the epigenetic landscape of cardiomyocytes during postnatal maturation by performing deep sequencing of accessible chromatin regions by using the Assay for Transposase-Accessible Chromatin from purified mouse cardiomyocyte nuclei (P1, P14, and P56). Results: Profiling of cardiomyocyte and nonmyocyte transcriptional programs uncovered several injury-responsive genes across regenerative and nonregenerative time points. However, the majority of transcriptional changes in all cardiac cell types resulted from developmental maturation from neonatal stages to adulthood rather than activation of a distinct regeneration-specific gene program. Furthermore, adult leukocytes and fibroblasts were characterized by the expression of a proliferative gene expression network following infarction, which mirrored the neonatal state. In contrast, cardiomyocytes failed to reactivate the neonatal proliferative network following infarction, which was associated with loss of chromatin accessibility around cell cycle genes during postnatal maturation. Conclusions: This work provides a comprehensive framework and transcriptional resource of multiple cardiac cell populations during cardiac development, repair, and regeneration. Our findings define a regulatory program underpinning the neonatal regenerative state and identify alterations in the chromatin landscape that could limit reinduction of the regenerative program in adult cardiomyocytes.

Epigenetic mechanisms in the pathogenesis of diabetic foot ulcers

The incidence of diabetes mellitus, a chronic metabolic disease associated with both predisposing genetic and environmental factors, is increasing globally. As a result, it is expected that there will also be an increasing incidence of diabetic complications which arise as a result of poor glycemic control. Complications include cardiovascular diseases, nephropathy, retinopathy and diabetic foot ulcers. The findings of several major clinical trials have identified that diabetic complications may arise even after many years of proper glycemic control. This has led to the concept of persistent epigenetic changes. Various epigenetic mechanisms have been identified as important contributors to the pathogenesis of diabetes and diabetic complications. The aim of this review is to provide an overview of the pathobiology of type 2 diabetes with an emphasis on complications, particularly diabetic foot ulcers. An overview of epigenetic mechanisms is provided and the focus is on the emerging evidence for aberrant epigenetic mechanisms in diabetic foot ulcers.

Metabolism and chromatin dynamics in health and disease.

The regulation of gene expression in response to environmental and behavioural cues is critical for many biological processes. Histone tail modifications are dynamic and, as such, can regulate gene expression in response to extracellular conditions. Many of the enzymes involved in adding and removing these modifications require cofactors that are products of intermediary metabolism pathways, thus linking cellular metabolism to the regulation of gene expression. Furthermore, the expression and activity of such enzymes are influenced by the cellular concentrations of metabolic products. Under- and over-nutrition can induce epigenetic changes that influence chromatin structure and define a metabolic program. Importantly, recent studies have demonstrated that such changes during the pre- and peri-natal periods can be long-lasting, influencing the disease risk later in life and could be transmitted to subsequent generations. Moreover, damaging gene expression patterns observed in metabolic diseases such as diabetes are driven by persistent changes in chromatin structure, raising the possibility of targeting epigenetic pathways for the treatment of disease.

NET silencing by let-7i in postural tachycardia syndrome

While strongly implicated in postural tachycardia syndrome (POTS), considerable controversy exists regarding norepinephrine transporter (NET) loss of function. POTS is characterized by the clinical symptoms of orthostatic intolerance, lightheadedness, tachycardia, and syncope or near syncope with upright posture. Abnormal sympathetic nervous system activity is typical, of a type which suggests dysfunction of the NET, with evidence that the gene responsible is under tight epigenetic control. Using RNA of isolated chromatin combined with massive parallel sequencing (RICh-seq) we show that let-7i miRNA suppresses NET by methyl-CpG-binding protein 2 (MeCP2). Vorinostat restores epigenetic control and NET expression in leukocytes derived from POTS participants.