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Kidneys have one of the most complex three-dimensional cellular organizations in the body, but the spatial molecular principles of kidney health and disease are poorly understood. Here we generate high-quality single cell (sc), single nuclear (sn), spatial (sp) RNA expression and sn open chromatin datasets for 73 samples, capturing half a million cells from healthy, diabetic, and hypertensive diseased human kidneys. Combining the sn/sc and sp RNA information, we identify > 100 cell types and states and successfully map them back to their spatial locations. Computational deconvolution of spRNA-seq identifies glomerular/vascular, tubular, immune, and fibrotic spatial microenvironments (FMEs). Although injured proximal tubule cells appear to be the nidus of fibrosis, we reveal the complex, heterogenous cellular and spatial organization of human FMEs, including the highly intricate and organized immune environment. We demonstrate the clinical utility of the FME spatial gene signature for the classification of a large number of human kidneys for disease severity and prognosis. We provide a comprehensive spatially-resolved molecular roadmap for the human kidney and the fibrotic process and demonstrate the clinical utility of spatial transcriptomics.

Daprodustat (GSK1278863) is a hypoxia-inducible factor (HIF)-prolyl hydroxylase (PHD) inhibitor in development for treatment of anemia of chronic kidney disease. Daprodustat’s biological activity simulates components of the natural response to hypoxia; inhibition of PHDs results in HIF stabilization and modulation of HIF-controlled gene products, including erythropoietin. The carcinogenic potential of daprodustat was evaluated in 2-year carcinogenicity studies in Sprague-Dawley rats and CD-1 mice, where once-daily doses were administered. The mouse study also included evaluation of daprodustat’s 3 major circulating human metabolites. There were no neoplastic findings that were considered treatment related in either study. Exaggerated pharmacology resulted in significantly increased red cell mass and subsequent multiorgan congestion and secondary non-neoplastic effects in both species, similar to those observed in chronic toxicity studies. In rats, these included aortic thrombosis and an exacerbation of spontaneous rodent cardiomyopathy, which contributed to a statistically significant decrease in survival in high-dose males (group terminated in week 94). Survival was not impacted in mice at any dose. Systemic exposures (area under the plasma concentration–time curve) to daprodustat at the high doses in rats and mice exceed predicted maximal human clinical exposure by ≥143-fold. These results suggest that daprodustat and metabolites do not pose a carcinogenic risk at clinical doses.

Most kidney diseases begin with abnormalities in glomerular podocytes, motivating the need for podocyte models to study pathophysiological mechanisms and new treatment options. However, podocytes cultured in vitro face a limited ability to maintain appreciable extents of differentiation hallmarks, raising concerns over the relevance of study results. Many key properties such as nephrin expression and morphology reach plateaus that are far from the in vivo levels. Here, we demonstrate that a biomimetic topography, consisting of microhemispheres arrayed over the cell culture substrate, promotes podocyte differentiation in vitro. We define new methods for fabricating microscale curvature on various substrates, including a thin porous membrane. By growing podocytes on our topographic substrates, we found that these biophysical cues augmented nephrin gene expression, supported full-size nephrin protein expression, encouraged structural arrangement of F-actin and nephrin within the cell, and promoted process formation and even interdigitation compared to the flat substrates. Furthermore, the topography facilitated nephrin localization on curved structures while nuclei lay in the valleys between them. The improved differentiation was also evidenced by tracking barrier function to albumin over time using our custom topomembranes. Overall, our work presents accessible methods for incorporating microcurvature on various common substrates, and demonstrates the importance of biophysical stimulation in supporting higher-fidelity podocyte cultivation in vitro.

Supplemental Material, gales_supplemental_Table_1 for Carcinogenicity Assessment of Daprodustat (GSK1278863), a Hypoxia-Inducible Factor (HIF)-Prolyl Hydroxylase Inhibitor by David F. Adams, Mark S. Watkins, Luc Durette, Josée Laliberté, Félix Goulet, Elaine Debien, Kendall S. Frazier, Nacéra Mellal, Liangfu Chen, Wei Shi, Roberta Thomas and Erding Hu in Toxicologic Pathology

Hypoxia-inducible factor- [Formula: see text] ([Formula: see text]) is a major regulator of cellular adaptation to hypoxia and oxidative stress, and recent advances of prolyl-4-hydroxylase (P4H) inhibitors have produced powerful tools to stabilize [Formula: see text] for clinical applications. However, whether [Formula: see text] provokes or resists neonatal hypoxic-ischemic (HI) brain injury has not been established in previous studies. We hypothesize that systemic and brain-targeted [Formula: see text] stabilization may have divergent effects. To test this notion, herein we compared the effects of GSK360A, a potent P4H inhibitor, in in-vitro oxygen-glucose deprivation (OGD) and in in-vivo neonatal HI via intracerebroventricular (ICV), intraperitoneal (IP), and intranasal (IN) drug-application routes. We found that GSK360A increased the erythropoietin (EPO), heme oxygenase-1 (HO1) and glucose transporter 1 (Glut1) transcripts, all [Formula: see text] target-genes, and promoted the survival of neurons and oligodendrocytes after OGD. Neonatal HI insult stabilized [Formula: see text] in the ipsilateral hemisphere for up to 24 h, and either ICV or IN delivery of GSK360A after HI increased the [Formula: see text] target-gene transcripts and decreased brain damage. In contrast, IP-injection of GSK360A failed to reduce HI brain damage, but elevated the risk of mortality at high doses, which may relate to an increase of the kidney and plasma EPO, leukocytosis, and abundant vascular endothelial growth factor (VEGF) mRNAs in the brain. These results suggest that brain-targeted [Formula: see text]-stabilization is a potential treatment of neonatal HI brain injury, while systemic P4H-inhibition may provoke unwanted adverse effects.

In vitro studies of cardiac physiology and drug response have traditionally been performed on individual isolated cardiomyocytes or isotropic monolayers of cells that may not mimic desired physiological traits of the laminar adult myocardium. Recent studies have reported a number of advances to Heart-on-a-Chip platforms for the fabrication of more sophisticated engineered myocardium, but cardiomyocyte immaturity remains a challenge. In the anisotropic musculature of the heart, interactions between cardiac myocytes, the extracellular matrix (ECM), and neighboring cells give rise to changes in cell shape and tissue architecture that have been implicated in both development and disease. We hypothesized that engineered myocardium fabricated from cardiac myocytes cultured in vitro could mimic the physiological characteristics and gene expression profile of adult heart muscle. To test this hypothesis, we fabricated engineered myocardium comprised of neonatal rat ventricular myocytes with laminar architectures reminiscent of that observed in the mature heart and compared their sarcomere organization, contractile performance characteristics, and cardiac gene expression profile to that of isolated adult rat ventricular muscle strips. We found that anisotropic engineered myocardium demonstrated a similar degree of global sarcomere alignment, contractile stress output, and inotropic concentration–response to the β-adrenergic agonist isoproterenol. Moreover, the anisotropic engineered myocardium exhibited comparable myofibril related gene expression to muscle strips isolated from adult rat ventricular tissue. These results suggest that tissue architecture serves an important developmental cue for building in vitro model systems of the myocardium that could potentially recapitulate the physiological characteristics of the adult heart. Impact statement With the recent focus on developing in vitro Organ-on-Chip platforms that recapitulate tissue and organ-level physiology using immature cells derived from stem cell sources, there is a strong need to assess the ability of these engineered tissues to adopt a mature phenotype. In the present study, we compared and contrasted engineered tissues fabricated from neonatal rat ventricular myocytes in a Heart-on-a-Chip platform to ventricular muscle strips isolated from adult rats. The results of this study support the notion that engineered tissues fabricated from immature cells have the potential to mimic mature tissues in an Organ-on-Chip platform.

Background The amino acid response ( AAR ) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti‐inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl‐ tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure. Methods and Results Consistent with its ability to inhibit prolyl‐ tRNA synthetase, halofuginone elicited a general control nonderepressible 2–dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l ‐proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II /phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2–dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell–derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin‐1‐mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/ eIF 2α‐dependent manner. Conclusions Halofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

There is interest in pharmacologic preconditioning for end-organ protection by targeting the HIF system. This can be accomplished by inhibition of prolyl 4-hydroxylase (PHD). GSK360A is an orally active PHD inhibitor that has been previously shown to protect the failing heart. We hypothesized that PHD inhibition can also protect the brain from injuries and resulting behavioral deficits that can occur as a result of surgery. Thus, our goal was to investigate the effect of pre-stroke surgery brain protection using a verified GSK360A PHD inhibition paradigm on post-stroke surgery outcomes. Vehicle or an established protective dose (30 mg/kg, p.o.) of GSK360A was administered to male Sprague-Dawley rats. Initially, GSK360A pharmacokinetics and organ distribution were determined, and then PHD-HIF pharmacodynamic markers were measured (i.e., to validate the pharmacological effects of the GSK360A administration regimen). Results obtained using this validated PHD dose-regimen indicated significant improvement by GSK360A (30mg/kg); administered at 18 and 5 hours prior to transient middle cerebral artery occlusion (stroke). GSK360A exposure and plasma, kidney and brain HIF-PHD pharmacodynamics endpoints (e.g., erythropoietin; EPO and Vascular Endothelial Growth Factor; VEGF) were measured. GSK360A provided rapid exposure in plasma (7734 ng/ml), kidney (45-52% of plasma level) and brain (1-4% of plasma level), and increased kidney EPO mRNA (80-fold) and brain VEGF mRNA (2-fold). We also observed that GSK360A increased plasma EPO (300-fold) and VEGF (2-fold). Further assessments indicated that GSK360A reduced post-stroke surgery neurological deficits (47-64%), cognitive dysfunction (60-75%) and brain infarction (30%) 4 weeks later. Thus, PHD inhibition using GSK360A pretreatment produced long-term post-stroke brain protection and improved behavioral functioning. These data support PHD inhibition, specifically by GSK360A, as a potential strategy for pre-surgical use to reduce brain injury and functional decline due to surgery-related cerebral injury.

Hypoxia inducible factor (HIF) stabilization by HIF-prolyl hydroxylase (PHD) inhibitors may improve ischemic conditions such as peripheral artery disease (PAD). This multicenter, randomized, placebo-controlled study evaluated the safety and efficacy of GSK1278863 (an oral PHD inhibitor) in subjects with PAD. The study assessed two active treatment paradigms: single dosing and subchronic daily dosing (300 mg single dose and 15 mg daily for 14 days, respectively). Neither regimen improved exercise performance compared with placebo (change from baseline in the 6-minute walk test (6MWT; feet), (GSK1278863, placebo): single dose (–46, –44), p=0.96; repeat dose (9, 8), p=0.99; change in number of contractions to onset of claudication (goniometry): single dose (4, –1), p=0.053; repeat dose (–2, 1), p=0.08). A calf-muscle biopsy substudy showed no increases in mRNA or protein levels of HIF target genes. More subjects receiving GSK1278863 than placebo experienced adverse events, particularly following the 300 mg single dose. Thus, assessing the safety of GSK1278863 in this setting would require a larger population exposed to the agent for a longer duration. These data do not support a benefit of GSK1278863 in PAD using the regimens tested. ClinicalTrials.gov Identifier: NCT01673555

Cardiomyocytes derived from pluripotent stem cells can be applied in drug testing, disease modeling and cell-based therapy. However, without procardiogenic growth factors, the efficiency of cardiomyogenesis from pluripotent stem cells is usually low and the resulting cardiomyocyte population is heterogeneous. Here, we demonstrate that induced pluripotent stem cells (iPSCs) can be derived from murine ventricular myocytes (VMs), and consistent with other reports of iPSCs derived from various somatic cell types, VM-derived iPSCs (ViPSCs) exhibit a markedly higher propensity to spontaneously differentiate into beating cardiomyocytes as compared to genetically matched embryonic stem cells (ESCs) or iPSCs derived from tail-tip fibroblasts. Strikingly, the majority of ViPSC-derived cardiomyocytes display a ventricular phenotype. The enhanced ventricular myogenesis in ViPSCs is mediated via increased numbers of cardiovascular progenitors at early stages of differentiation. In order to investigate the mechanism of enhanced ventricular myogenesis from ViPSCs, we performed global gene expression and DNA methylation analysis, which revealed a distinct epigenetic signature that may be involved in specifying the VM fate in pluripotent stem cells.

Hypoxia inducible factors (HIFs) are transcription factors that are regulated by HIF-prolyl 4-hydroxylases (PHDs) in response to changes in oxygen tension. Once activated, HIFs play an important role in angiogenesis, erythropoiesis, proliferation, cell survival, inflammation, and energy metabolism. We hypothesized that GSK360A, a novel orally active HIF-PHD inhibitor, could facilitate local and systemic HIF-1 alpha signaling and protect the failing heart after myocardial infarction (MI).GSK360A is a potent (nanomolar) inhibitor of HIF-PHDs (PHD1PHD2 = PHD3) capable of activating the HIF-1 alpha pathway in a variety of cell types including neonatal rat ventricular myocytes and H9C2 cells. Male rats treated orally with GSK360A (30 mg x kg x d) had a sustained elevation in circulating levels of erythropoietin and hemoglobin and increased hemoxygenase-1 expression in the heart and skeletal muscle. In a rat model of established heart failure with systolic dysfunction induced by ligation of left anterior descending coronary artery, chronic treatment with GSK360A for 28 days prevented the progressive reduction in ejection fraction, ventricular dilation, and increased lung weight, which were observed in the vehicle-treated animals, for up to 3 months. In addition, the microvascular density in the periinfarct region was increased (2-fold) in GSK360A-treated animals. Treatment was well tolerated (survival was 89% in the GSK360A group vs. 82% in the placebo group).Chronic post-myocardial infarction treatment with a selective HIF PHD inhibitor (GSK360A) exerts systemic and local effects by stabilizing HIF-1 alpha signaling and improves long-term ventricular function, remodeling, and vascularity in a model of established ventricular dysfunction. These results suggest that HIF-PHD inhibitors may be suitable for the treatment of post-MI remodeling and heart failure.

Summary. Background: Endothelial microparticles (EMPs) are irregularly shaped membrane fragments shed into the circulation in patients with vascular diseases, and may themselves act to enhance the endothelial response to inflammation. On the basis of the importance of p38 mitogen-activated protein kinase (MAPK) in endothelial responses to inflammatory stimuli, we sought to define the role of p38 in EMP generation and function. Methods: Microparticle generation from cultures of human aortic endothelial cells (hAECs) treated with tumor necrosis factor-α (TNF-α) and p38 inhibition was quantified via multiple modalities. The response of target endothelial cells was assessed by treatment of cells with EMPs generated under various conditions. Results: Inhibition of p38 in hAECs, using pharmacologic agents, resulted in a 50% reduction of TNF-α-induced EMPs. Importantly, suppression of microparticles was specific to p38 MAPK pathways. EMPs triggered by TNF-α activation induced an approximately four-fold increase in soluble intercellular adhesion molecule-1 (sICAM-1) release from targeted cells. However, inhibition of p38 MAPK in the targeted cell prior to EMP treatment did not alter the sICAM1 response. Conclusions: Our findings implicate p38 MAPK signaling as significant and selective in the formation and maturation of EMPs. EMPs elicited a proinflammatory response from targeted hAECs that was dependent on the conditions under which EMPs were generated. However, our results imply a unidirectional model in which p38 MAPK is critical at the source of microparticle formation, but not the target cell response to EMPs. These findings indicate a novel mechanism by which p38 inhibition may offer therapeutic benefit in vivo via direct inhibition of EMP formation.

SIRT1, the founding member of the mammalian family of seven NAD+-dependent sirtuins, is composed of 747 amino acids forming a catalytic domain and extended N- and C-terminal regions. We report the design and characterization of an engineered human SIRT1 construct (mini-hSIRT1) containing the minimal structural elements required for lysine deacetylation and catalytic activation by small molecule sirtuin-activating compounds (STACs). Using this construct, we solved the crystal structure of a mini-hSIRT1-STAC complex, which revealed the STAC-binding site within the N-terminal domain of hSIRT1. Together with hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis using full-length hSIRT1, these data establish a specific STAC-binding site and identify key intermolecular interactions with hSIRT1. The determination of the interface governing the binding of STACs with human SIRT1 facilitates greater understanding of STAC activation of this enzyme, which holds significant promise as a therapeutic target for multiple human diseases., Sirtuins are NAD+-dependent deacylases implicated in the regulation of stress responses, bioenergetics and epigenetic control. Here the authors describe the crystal structure of a sirtuin-activating compounds (STAC)-sirtuin complex and begin to elucidate the mechanism of sirtuins activation by STACs.

Rho kinase (ROCK1) mediates vascular smooth muscle contraction and is a potential target for the treatment of hypertension and related disorders. Indazole amide 3 was identified as a potent and selective ROCK1 inhibitor but possessed poor oral bioavailability. Optimization of this lead resulted in the discovery of a series of dihydropyridones, exemplified by 13, with improved pharmacokinetic parameters relative to the initial lead. Indazole substitution played a critical role in decreasing clearance and improving oral bioavailability.

Rho protein represents a family of small GTP binding proteins that are involved in many important cellular functions including cell proliferation, migration and cytoskeletal reorganization. Rho protein is activated by GTP binding and is inactivated by hydrolyzing GTP to GDP. This process is influenced by variety of physiological and pathophysiological stimuli including growth factors, many vasoactive substances, smoking and mechanic stress or injury. Recent evidence suggest that targeting Rho protein per se or its downstream effector proteins such as Rho kinase or LIM kinase may have therapeutic potential in diseases such as hypertension, angina, myocardiac infarction (MI), atherosclerosis, tumor metastasis and spinal cord injury. Several recent patents have described modalities that regulate the activity of Rho, Rho kinase and LIM kinase as potential therapeutics. In this article, we will review the current knowledge on the cellular functions of Rho signaling pathway and strategies in targeting different components in Rho signaling pathway for human diseases with an emphasis on cardiovascular indications.

Increased Rho kinase (ROCK) activity contributes to smooth muscle contraction and regulates blood pressure homeostasis. We hypothesized that potent and selective ROCK inhibitors with novel structural motifs would help elucidate the functional role of ROCK and further explore the therapeutic potential of ROCK inhibition for hypertension. In this article, we characterized two aminofurazan-based inhibitors, GSK269962A [N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1H-imidazo[4, 5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]-oxy}benzamide] and SB-7720770-B [4-(7-{[(3S)-3-amino-1-pyrrolidinyl]carbonyl}-1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,2,5-oxadiazol-3-amine], as members of a novel class of compounds that potently inhibit ROCK enzymatic activity. GSK269962A and SB-772077-B have IC50 values of 1.6 and 5.6 nM toward recombinant human ROCK1, respectively. GSK269962A also exhibited more than 30-fold selectivity against a panel of serine/threonine kinases. In lipopolysaccharide-stimulated monocytes, these inhibitors blocked the generation of inflammatory cytokines, such as interleukin-6 and tumor necrosis factor-alpha. Furthermore, both SB-772077-B and GSK269962A induced vasorelaxation in preconstricted rat aorta with an IC50 of 39 and 35 nM, respectively. Oral administration of either GSK269962A or SB-772077-B produced a profound dose-dependent reduction of systemic blood pressure in spontaneously hypertensive rats. At doses of 1, 3, and 30 mg/kg, both compounds induced a reduction in blood pressure of approximately 10, 20, and 50 mm Hg. In addition, administration of SB-772077-B also dramatically lowered blood pressure in DOCA salt-induced hypertensive rats. SB-772077-B and GSK269962A represent a novel class of ROCK inhibitors that have profound effects in the vasculature and may enable us to further evaluate the potential beneficial effects of ROCK inhibition in animal models of cardiovascular as well as other chronic diseases.

Rho Kinase I (ROCK I) is a serine/threonine kinase that is involved in diverse cellular signaling. To further understand the physiological role of ROCK I and to identify and develop potent and selective inhibitors of ROCK I, we have overexpressed and purified a constitutively active dimeric human ROCK I (3-543) kinase domain using the Sf9- baculovirus expression system. In addition, using a limited proteolysis technique, we have identified a minimal functional subdomain of ROCK I that can be used in crystallization studies. The availability of multimilligram amounts of purified and well characterized functional human ROCK I kinase domains will be useful in screening and structural studies.

(1) The involvement of Rho-kinase (ROCK) in the contractile mechanisms mediating smooth muscle contraction of the rat urinary bladder was investigated using expression studies and the ROCK inhibitor Y-27632. (2) Both isoforms of ROCK (ROCK I and ROCK II) were detected in high levels in rat urinary bladder. (3) Y-27632 (10 micro M) significantly attenuated contractions of rat urinary bladder strips evoked by the G-protein coupled receptor agonists carbachol (58.1+/-10.5% at 0.3 micro M) and neurokinin A (68.6+/-12.7% at 1 micro M) without affecting contractions to potassium chloride (10-100 mM). In addition, basal tone was reduced by 47.8+/-2.0% by 10 micro M Y-27632 in the absence of stimulation. (4) Contractions of urinary bladder strips evoked by the P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-mATP; 10 micro M) were also attenuated by Y-27632 (30.0+/-7.2% at 10 micro M). (5) Y-27632 (10 micro M) significantly attenuated contractions evoked by electrical field stimulation (2-16 Hz). The effect of Y-27632 on the tonic portion of the neurogenic response (4-16 Hz) was not significantly different from the effect of atropine (1 micro M) alone. (6) While the mechanism underlying the ability of Y-27632 to inhibit alpha,beta-mATP-evoked contractions remains undetermined, the results of the present study clearly demonstrate a role for ROCK in the regulation of rat urinary bladder smooth muscle contraction and tone.

We report the identification and characterization of a novel cytokine-like gene family using structure-based methods to search for novel four-helix-bundle cytokines in genomics databases. There are four genes in this family, FAM3A, FAM3B, FAM3C, and FAM3D, each encoding a protein (224–235 amino acids) with a hydrophobic leader sequence. Northern analysis indicates that FAM3B is highly expressed in pancreas, FAM3D in placenta, and FAM3A and FAM3C in almost all tissues. Immunohistochemistry showed that FAM3A is expressed prominently in the vascular endothelium, particularly capillaries. We found that FAM3A and FAM3B protein were both localized to the islets of Langerhans of the endocrine pancreas. Recombinant FAM3B protein has delayed effects on β-cell function, inhibiting basal insulin secretion from a β-cell line in a dose-dependent manner.

Histone acetylation alters chromatin state by modifying lysines on histone and plays an important role in modulating gene transcription. A dynamic balance of histone acetylation/deacetylation is maintained by histone acetyltransferases and histone deacetylases. Emerging evidence suggests that a family of histone deacetylases may exist to regulate diverse cellular functions, including chromatin structure, gene expression, cell cycle progression, and oncogenesis. We describe here a novel human histone deacetylase, named HDAC8, cloned from human kidney. HDAC8 encodes 377 amino acid residues and shares extensive homology to several known HDACs, in particular a histone deacetylase from Arabidopsis thaliana. Northern blot analyses revealed that HDAC8 expression pattern for HDAC8 is distinct from that for HDAC1 and HDAC3, and expression of HDAC8 mRNA occurs in multiple organs including heart, lung, kidney, and pancreas. HDAC8 mRNA was also observed in several cell lines derived from cancerous tissues. When expressed in HEK293 cells, HDAC8 exhibited deacetylase activity toward acetylated histone, indicating that this protein is a bona fide histone deacetylase. Its histone deacetylase activity was inhibited by trichostatin and other known histone deacetylase inhibitors. Furthermore, active recombinant HDAC8 was expressed and purified from Escherichia coli. When ectopically expressed in cells, HDAC8 was found to be localized to the nucleus. Co-transfection experiments demonstrated that expression of HDAC8 repressed a viral SV40 early promoter activity. These results indicate that HDAC8 is a novel member of the histone deacetylase family, which may play a role in the development of a broad range of tissues and potentially in the etiology of cancer.

The adipose cell is now known to play a complex role in energy homeostasis, storing energy and signaling to other tissues concerning the state of energy balance. The past several years have seen an explosive increase in our knowledge of the transcriptional basis of adipocyte differentiation. This review describes the role of peroxisome proliferator-activated receptor gamma in this process, and describes how other transcription factors may affect adipogenesis by modulating the amount or activity of peroxisome proliferator-activated receptor gamma. Furthermore, peroxisome proliferator-activated receptor gamma and other adipogenic transcription factors provide a focus for beginning to understand how various hormones and metabolites influence the development of this tissue in vivo.

A splice form of IGF-1, IGF-1Eb, is upregulated after exercise or injury. Physiological responses have been ascribed to the 24-amino acid COOH-terminal peptide that is cleaved from the NH3-terminal 70-amino acid mature IGF-1 protein. This COOH-terminal peptide was termed “mechano-growth factor” (MGF). Activities claimed for the MGF peptide included enhancing muscle satellite cell proliferation and delaying myoblast fusion. As such, MGF could represent a promising strategy to improve muscle regeneration. Thus, at our two pharmaceutical companies, we attempted to reproduce the claimed effect of MGF peptides on human and mouse muscle myoblast proliferation and differentiation in vitro. Concentrations of peptide up to 500 ng/ml failed to increase the proliferation of C2C12 cells or primary human skeletal muscle myoblasts. In contrast, all cell types exhibited a proliferative response to mature IGF-1 or full-length IGF-1Eb. MGF also failed to inhibit the differentiation of myoblasts into myotubes. To address whether the response to MGF was lost in these tissue culture lines, we measured proliferation and differentiation of primary mouse skeletal muscle stem cells exposed to MGF. This, too, failed to demonstrate a significant effect. Finally, we tested whether MGF could alter a separate documented in vitro effect of the peptide, activation of p-ERK, but not p-Akt, in cardiac myocytes. Although a robust response to IGF-1 was observed, there were no demonstrated activating responses from the native or a stabilized MGF peptide. These results call in to question whether there is a physiological role for MGF.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) have been recently derived and are used for basic research, cardiotoxicity assessment, and phenotypic screening. However, the hiPS-CM phenotype is dependent on their derivation, age, and culture conditions, and there is disagreement as to what constitutes a functional hiPS-CM. The aim of the present study is to characterize the temporal changes in hiPS-CM phenotype by examining five determinants of cardiomyocyte function: gene expression, ion channel functionality, calcium cycling, metabolic activity, and responsiveness to cardioactive compounds. Based on both gene expression and electrophysiological properties, at day 30 of differentiation, hiPS-CMs are immature cells that, with time in culture, progressively develop a more mature phenotype without signs of dedifferentiation. This phenotype is characterized by adult-like gene expression patterns, action potentials exhibiting ventricular atrial and nodal properties, coordinated calcium cycling and beating, suggesting the formation of a functional syncytium. Pharmacological responses to pathological (endothelin-1), physiological (IGF-1), and autonomic (isoproterenol) stimuli similar to those characteristic of isolated adult cardiac myocytes are present in maturing hiPS-CMs. In addition, thyroid hormone treatment of hiPS-CMs attenuated the fetal gene expression in favor of a more adult-like pattern. Overall, hiPS-CMs progressively acquire functionality when maintained in culture for a prolonged period of time. The description of this evolving phenotype helps to identify optimal use of hiPS-CMs for a range of research applications.

Adipose differentiation is accompanied by changes in cellular morphology, a dramatic accumulation of intracellular lipid and activation of a specific program of gene expression. Using an mRNA differential display technique, we have isolated a novel adipose cDNA, termed adipoQ. The adipoQ cDNA encodes a polypeptide of 247 amino acids with a secretory signal sequence at the amino terminus, a collagenous region (Gly-X-Y repeats), and a globular domain. The globular domain of adipoQ shares significant homology with subunits of complement factor C1q, collagen alpha 1(X), and the brain-specific factor cerebellin. The expression of adipoQ is highly specific to adipose tissue in both mouse and rat. Expression of adipoQ is observed exclusively in mature fat cells as the stromal-vascular fraction of fat tissue does not contain adipoQ mRNA. In cultured 3T3-F442A and 3T3-L1 preadipocytes, hormone-induced differentiation dramatically increases the level of expression for adipoQ. Furthermore, the expression of adipoQ mRNA is significantly reduced in the adipose tissues from obese mice and humans. Whereas the biological function of this polypeptide is presently unknown, the tissue-specific expression of a putative secreted protein suggests that this factor may function as a novel signaling molecule for adipose tissue.

Peroxisome proliferator activated receptor (PPAR) γ is an orphan member of the nuclear hormone receptor superfamily and is expressed at high levels specifically in adipose tissue. Recent data suggest that this factor is a central regulator of adipocyte gene expression and differentiation. Fibroblastic cell lines that express PPARγ ectopically can be induced to differentiate into fat cells by a variety of lipids and lipid-like activators of PPARs, suggesting that this protein may function to link adipogenesis with systemic lipid metabolism.

Phosphoenolpyruvate carboxykinase (PEPCK) is expressed at high levels in liver, kidney, and adipose tissue. This enzyme catalyzes the rate-limiting step in hepatic and renal gluconeogenesis and adipose glyceroneogenesis. The regulatory factors important for adipose expression of the PEPCK gene are not well defined. Previous studies with transgenic mice established that the region between bp -2086 and -888 is required for expression in adipose tissue but not for expression in liver or kidney tissue. We show here that a DNA fragment containing this region can function as an enhancer and direct differentiation-dependent expression of a chloramphenicol acetyltransferase gene from a heterologous promoter in cultured 3T3-F442A preadipocytes and adipocytes. We further demonstrate that the adipocyte-specific transcription factor PPAR gamma 2, previously identified as a regulator of the adipocyte P2 enhancer, binds in a heterodimeric complex with RXR alpha to the PEPCK 5'-flanking region at two sites, termed PCK1 (bp -451 to -439) and PCK2 (bp -999 to -987). Forced expression of PPAR gamma 2 and RXR alpha activates the PEPCK enhancer in non-adipose cells. This activation is potentiated by peroxisome proliferators and fatty acids but not by 9-cis retinoic acid. Mutation of the PPAR gamma 2 binding site (PCK2) abolishes both the activity of the enhancer in adipocytes and its ability to be activated by PPAR gamma 2 and RXR alpha. These results establish a role for PPAR gamma 2 in the adipose expression of the PEPCK gene and suggest that this factor functions as a coordinate regulator of multiple adipocyte-specific genes.

Background: Hypoxia inducible factors (HIFs) are transcription factors that are upregulated by decreased oxygen tension and ischemic preconditioning. HIFs increase angiogenesis, erythropoiesis, cell proliferation/survival and energy metabolism and decrease inflammation. GSK360A is a novel, orally active prolyl 4-hydroxylase (PHD) inhibitor that upregulates HIF-1α signaling and can protect the failing heart. Since GSK360A upregulates systems involved in ischemic tolerance, its administration prior to surgery/ischemia could be beneficial. GSK360A effects in cerebral ischemia have not been explored. Method: Male Sprague Dawley rats received 2 hours transient middle cerebral artery occlusion (tMCAO) followed by 22 hours of reperfusion. GSK dose selection was based on previous pharmacodynamic data on HIF system upregulation that was verified in these studies. Vehicle (1% Methyl cellulose) or GSK360A (30 mg/kg) was administered by oral gavage at 18 and 5 hr prior to tMCAO. Measurements were made of kidney erythropoietin (EPO) mRNA and protein over 1 day, modified neurological severity score (mNSS) over 5 and 24 hrs, cognitive performance (Active Place Avoidance; APA) and motor activity at 3-4 weeks and terminal brain infarction were made. Different groups of rats (N=5-10 per group) were studied over 1 day to 4 weeks. Results: GSK360A increased kidney EPO mRNA and protein up to 80-fold (p Conclusions: This is the first demonstration of GSK360A brain protection and long-term improvement in post-stroke neurological outcome, including beneficial effects to reduce stroke-induced loss in cognitive function. These data suggest that pre-stroke administration of GSK360A can produce PHD inhibition that stimulates HIF signaling useful for high-stroke-risk situations. In addition, its use prior to surgery might reduce the probability of post-surgical cognitive decline.

Previously, we identified a novel transcription factor, ARF6, as a key regulator of the tissue-specific adipocyte P2 (aP2) enhancer. In order to identify the proteins which comprise the adipocyte ARF6 complex, we have purified this DNA binding activity from a cultured adipocyte cell line. We have developed a system for growth and differentiation of HIB-1B brown adipocytes in suspension culture that facilitates the production of large quantities of adipocyte nuclear extract. ARF6 was purified from HIB-1B nuclear extract by a combination of conventional and sequence-specific DNA affinity chromotography. Chemical sequencing and mass spectral analysis of tryptic peptides derived from the purified polypeptides identifies the ARF6 complex as a heterodimer of the retinoid X receptor alpha (RXR alpha) and the murine peroxisome proliferator activated receptor gamma (PPAR gamma). Of the known PPAR gamma isoforms, PPAR gamma is the predominant form expressed in adipose tissue. These results suggest that PPAR gamma 2 serves a unique function among PPAR family members as an important regulator of adipocyte-specific gene expression.

Diabetes mellitus is associated with accelerated atherosclerosis, leading to a two to three fold increase in the incidence of cardiovascular disease (1). Quantitative methods to distinguish cardiovascular disease risk remain elusive in the diabetic population. Current approaches rely on assessing endothelial function, including invasive and non-invasive methods (2), and the association of circulating plasma levels of soluble proteins such as high sensitivity C reactive protein (CRP) (3). Many of these approaches are either complicated, operator dependant or nonspecific (2). For example, the precise cellular source of soluble proteins is often unknown and can confound interpretation. Accurate, convenient and biologically informative markers and associated methods are necessary to assist in the early detection and treatment of vascular complications in diabetic patients. In this regard, cell derived MPs and progenitor cells (PCs), defined as CD34 positive cells, and pro-angiogenic cells (PACs), also CD34 positive along with having endothelial surface markers, have emerged as surrogate markers for cellular damage and repair respectively. Both have been associated independently with a range of vascular diseases including diabetes (4–7). MPs are membrane encapsulated vesicles of approximately 1µm or less in diameter, derived from surface membranes under conditions of cellular activation or apoptosis (8). These submicron vesicles are released into circulation, carrying with them an array of surface markers, used to identify their cellular source. Exposed membrane phosphatidylserine (PS) and tissue factor, along with a plethora of other surface molecules and cytoplasmic components, including nuclear material (9), enable MPs to impact on a variety of biological functions, including coagulation, thrombosis and angiogenesis (10). Direct receptor-ligand interactions (11) and fusion and delivery of intracellular contents (9,12) are mechanisms employed by MP’s to target and impact host cells. Increases in endothelial MPs (EMPs) (13), platelet MPs (PMPs)(14) and monocyte MPs (MMPs) (15) have all been implicated in vascular pathology, including myocardial infarction. Circulating EMPs, generated from the surface of the endothelium have received considerable attention as a surrogate marker for endothelial dysfunction (4,16,17). Due to disease specific changes and associated biological activity, these vesicles are considered active paracrine agents that aggravate further vascular dysfunction(18) and promote inflammation (19). To date, studies assessing MPs have relied on flow cytometry methods performed in specialized laboratories. The utility of a plate based method has not been tested in comparison to flow cytometry assay. PACs (formerly given the designation of endothelial progenitor cells) play a significant role in vascular homeostasis, and in the development and resolution of cardiovascular pathologies. For example, the number and function of PACs are altered immediately after a myocardial ischemic event, and in individuals at high risk of pathologic cardiovascular events (7). In addition, MPs are lowered and PACs are increased after treatment with HMG CoA reductase inhibitors (statins) (20,21). A study of hypercholesterolemic patients with higher CD31+/CD42− EMPs and lower PACs than normocholesterolemic subjects supports this hypothesis (22). We hypothesize that the ratio of MPs to PCs and/or PACs provides an indicator of cellular damage, including the integrity of the endothelium and a loss of endothelial repair capacity that could be relevant in type 2 diabetes. This ratio was directly associated with aortic pulse wave velocity (aPWV), providing a functional link between plasma cholesterol levels, MPs, PACs, endothelial injury and arterial stiffness. To test this hypothesis we evaluated the vascular health of two diabetic subpopulations using MPs and PCs/PACs as surrogate markers. As the risk of vascular events increases with the duration of diabetes (23), we recruited individuals recently diagnosed and those diagnosed for five years or more with diabetes to provide separation of these two populations.

Complementary DNAs encoding the beta subunit of casein kinase II (CKII beta) from the nematode Caenorhabditis elegans were cloned and sequenced. The predicted beta subunit polypeptide comprises 234 amino acid residues and has a Mr of 26,452. CKII beta is not homologous with other types of proteins. In synchronously developing C. elegans the abundance of the 1.3-kilobase mRNA for CKII beta varies in parallel with the level of mRNA encoding the catalytic subunit (alpha) of CKII. Thus, the developmental expression of CKII subunits is controlled coordinately and pretranslationally. CKII beta and CKII alpha mRNAs are enriched 5-10-fold in C. elegans embryos relative to their concentrations at several other stages of nematode development. A 3.8-kilobase pair segment of C. elegans DNA that contains the CKII beta gene and an extensive 5'-flanking region was cloned and sequenced. The CKII beta gene is divided into 6 exons by introns ranging from 49 to 533 base pairs in length. The first exon encodes 88 nucleotides of 5'-untranslated mRNA. Exon 2 (72 base pairs) contains the initiator Met codon and only 5 additional codons. Exons 3-6 encode 52, 63, 64, and 49 amino acid residues, respectively. The 5' terminus of CKII beta mRNA is modified post-transcriptionally by trans-splicing with a leader sequence of 22 nucleotides. The CKII beta gene was mapped to a position on C. elegans chromosome 2 that is in close proximity to the lin-11 gene.

Diabetes Mellitus (DM) is strongly associated with a high incidence of cardiovascular disease (CVD) although often individuals show no symptoms or signs of disease. Therefore, quantitative measures are needed to distinguish those diabetic patients at higher risk for CV events. Cell derived microparticles (MPs), submicron membrane vesicles released from activated cells, and endothelial progenitor cells (EPCs) are mediators of cellular damage and endothelial reparative capacity respectively. Both are emerging biomarkers of vascular health and have been assessed individually in diabetic populations. To extend on this, we examined whether the relationship of MPs to EPCs could be used as a potential index of vascular pathology. Plasma samples were collected from patients with early-stage (ES, Diagnosis5yrs, n=21, 64±3yrs, 7.1±0.3% HbA1c) with type 2 DM and compared to age related healthy volunteers (H, n=18, 57±3yrs, 5.3±0.7% HbA1c). EPCs positive for CD133 + /CD34 + and MP subtypes, AnnexinV + (AnnV + ), platelets (CD41 + ), monocytes (CD14 + ) and endothelial (CD144 + ) were measured by flow cytometry. Pro-coagulant microparticles and quantification of soluble proteins was measured using ELISA based methods. A significant relationship was found between MPs and EPCs which correlated with disease duration (H vs. ES vs. LT). Significant changes of pro-coagulant MPs ( P =0.02) and AnnV + MPs ( P =0.02) were noted. Of particular interest, the ratio of CD144 + MPs/EPC increased dramatically ( P =0.009) followed by ratios of AnnV + ( P =0.01), CD41 + ( P =0.01) and CD14 + ( P =0.03) MPs/EPCs. These indices of compromised vascular function were highest in the LT group despite intensive statin therapy (LDL mg/dL, 109±8 in H, vs. 80±7 in LT, P =0.01). Of note, the ratio of endothelial MPs/endothelial progenitor cells (CD144 + MPs/EPCs) was more informative than CRP and a range of inflammatory mediators. This is the first report of a relationship of MPs and EPCs in DM. This ratio provides a quantitative measurement of both pro-coagulant and endothelial injury and can identify DM patients with potentially increased risk of CV disease.

Recent studies using known Rho-associated kinase isoform 1 (ROCK1) inhibitors along with cellular and molecular biology data have revealed a pivotal role of this enzyme in many aspects of cardiovascular function. Here we report a series of ROCK1 inhibitors which were originally derived from a dihydropyrimidinone core 1. Our efforts focused on the optimization of dihydropyrimidine 2, which resulted in the identification of a series of dihydropyrimidines with improved pharmacokinetics and P450 properties.

Heme oxygenase-1 (HO) metabolizes heme to form the vasodilator carbon monoxide and antioxidant biliverdin. Upregulation of HO-1 by hemin, which is also a substrate attenuates thrombosis in rodent models, however, whether protection is due to HO-1 upregulation or to increased substrate availability is unknown. This study tested the hypothesis that treatment of mice with cobalt protoporphyrin (CoPP), a non-substrate HO-1 inducer, would protect the endothelium from laser injury. C57Bl/J6 mice were treated with vehicle, CoPP (20 mg/kg), CoPP plus the HO-1 inhibitor tin protoporphyrin (SnPP; 20 mg/kg) or SnPP alone for 18 h. Intravital microscopy was used to quantitate thrombus formation in cremaster arterioles in response to laser ablation of the endothelium. CoPP treatment inhibited thrombosis by 43% compared to vehicle (P0.05). SnPP co-treatment negated the inhibitory effect of CoPP while SnPP alone potentiated thrombosis compared to vehicle. In CoPP-treated animals, cremaster HO-1 mRNA expression was increased 59+/-17-fold over vehicle (P0.001). Co-treatment with CoPP+SnPP attenuated this effect by 36%, however the increase in HO-1 protein induced by CoPP was unaffected by SnPP. Induction of HO-1 by the non-substrate inducer CoPP protects against laser induced endothelial injury without the need for increased substrate. Small molecule, substrate-independent upregulation of HO-1 expression represents a feasible approach to ameliorate endothelial dysfunction in cardiovascular disease.

A full-length Caenorhabditis elegans cDNA that encodes the alpha subunit of casein kinase II was inserted into the inducible bacterial expression vector pET3a to generate the plasmid pCK alpha. Escherichia coli DE21 lysozyme S that was transformed with pCK alpha expressed soluble, catalytically active casein kinase II alpha upon induction with isopropyl beta-D-thiogalactopyranoside. The expressed alpha subunit was purified to homogeneity with a 60% yield by chromatography on CM-Sephadex, P-11 phosphocellulose, and heparin-agarose. The Mr values estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 42,000) or calculated from hydrodynamic measurements (s20,w = 3.3 S, Stokes radius = 2.8 nm, Mr = 37,000) were similar, thereby indicating that the expressed enzyme is monomeric. The native holoenzyme and the expressed alpha subunit exhibited several similar properties including the utilization of both ATP and GTP as substrates and the susceptibility to inhibition of phosphotransferase activity by low concentrations of heparin. However, the kcat for E. coli-derived alpha was only 9% of the kcat for the native holoenzyme, and catalytic activity was not stimulated by polyamines. Recombinant casein kinase II alpha aggregates at low ionic strength, and the aggregation is partially reversible. A mutant alpha subunit in which Lys74 and Lys75 were substituted by glutamic acid residues was constructed by site-directed mutagenesis. The mutant enzyme was not inhibited by typically effective concentrations of heparin (e.g. IC50 = 0.3 micrograms/ml) because the affinity of modified recombinant casein kinase II Glu-74Glu-75 for heparin decreased approximately 70-fold. Thus, Lys74 and Lys75 are implicated in the heparin binding, inhibitory domain. The successful expression of casein kinase II alpha in E. coli will facilitate the analysis of the structural basis for functional domains in this enzyme.

The nematode Caenorhabditis elegans provides a model system for investigating the structure, function, and regulation of casein kinase II. Cytosols from C. elegans embryos and gravid adults, which contain fertilized eggs and embryos, are enriched in casein kinase II activity; cytosols from newly hatched larva, four subsequent larval stages, and immature adults exhibit casein kinase II levels that are 3-10-fold lower than those observed in embryo cytosol. C. elegans casein kinase II contains alpha (Mr = 42,000) and beta (Mr = 29,000) subunits and has a Stokes radius of 50 nm. The enzyme utilizes ATP and GTP as substrates, is potently inhibited by heparin and undergoes autophosphorylation. Sequence analyses of cloned cDNAs corresponding to the 1.7-kilobase mRNA encoding the alpha (catalytic) subunit of casein kinase II indicate that the alpha polypeptide contains 359 amino acid residues. Variations in the abundance of casein kinase II alpha mRNA are coordinated with changes in enzyme activity during C. elegans development, indicating that alpha subunit expression is controlled at a pretranslational level. However, the magnitude of the developmentally controlled changes in phosphotransferase activity exceeded the corresponding increments in alpha subunit mRNA content. This suggests that translational and/or post-translational mechanisms also play an important role in the developmental regulation of C. elegans casein kinase II activity. The 2.9-kilobase casein kinase II alpha gene is divided into eight exons by intervening sequences ranging from 48 to 457 base pairs in length. The alpha gene promoter contains a TATA box, and a unique transcription start site has been identified. The intron/exon organization of the casein kinase II alpha gene differs markedly from the gene structure of the catalytic subunit of murine cAMP-dependent protein kinase (Chrivia, J. C., Uhler, M. D., and McKnight, G. S. (1988) J. Biol. Chem. 263, 5739-5744).

The discovery, proposed binding mode, and optimization of a novel class of Rho-kinase inhibitors are presented. Appropriate substitution on the 6-position of the azabenzimidazole core provided subnanomolar enzyme potency in vitro while dramatically improving selectivity over a panel of other kinases. Pharmacokinetic data was obtained for the most potent and selective examples and one (6n) has been shown to lower blood pressure in a rat model of hypertension.

Rho kinase (ROCK) belongs to a family of Ser/Thr protein kinases that are activated via interaction with the small GTP-binding protein RhoA. Growing evidence suggests that RhoA and ROCK participate in a variety of important physiological functions in vasculature including smooth muscle contraction, cell proliferation, cell adhesion and migration, and many aspects of inflammatory responses. As these processes mediate the onset and progression of cardiovascular disease, modulation of the Rho/ROCK signalling pathway is a potential strategy for targeting an array of cardiovascular indications. Two widely employed ROCK inhibitors, fasudil and Y-27632, have provided preliminary but compelling evidence supporting the potential cardiovascular benefits of ROCK inhibition in preclinical animal disease models and in the clinic. This review summarises the molecular biology of ROCK and its biological functions in smooth muscle, endothelium and other vascular tissues. In addition, there will be a focus on recent progress demonstrating the benefits of ROCK inhibition in several animal models of cardiovascular diseases. Finally, recent progress in the identification of novel ROCK inhibitors and challenges associated with their development for clinical use will be discussed.

RhoA and Rho-kinase (ROCK) participate in diverse cellular signaling functions such as smooth muscle contraction, cytoskeleton rearrangement, and cell migration and proliferation. In smooth muscle, ROCK plays an important role in calcium sensitization, an event that controls vascular vessel tone. Recent studies using ROCK inhibitors along with cellular and molecular biology techniques have revealed a pivotal role of this enzyme in many other aspects of cardiovascular function. This review will focus on the current understanding of Rho/ROCK signaling pathways and discuss the use of ROCK inhibitors as therapeutic agents for cardiovascular diseases ranging from hypertension to atherosclerosis.

Histone deacetylases (HDACs) represent an expanding family of protein modifying-enzymes that play important roles in cell proliferation, chromosome remodeling, and gene transcription. We have previously shown that recombinant human HDAC8 can be expressed in bacteria and retain its catalytic activity. To further explore the catalytic activity of HDACs, we expressed two additional human class I HDACs, HDAC1 and HDAC3, in baculovirus. Recombinant HDAC1 and HDAC3 fusion proteins remained soluble and catalytically active and were purified to near homogeneity. Interestingly, trichostatin (TSA) was found to be a potent inhibitor for all three HDACs (IC50 value of approximately 0.1-0.3 microM), whereas another HDAC inhibitor MS-27-275 (N-(2-aminophenyl)-4-[N-(pyridin-3-methyloxycarbonyl)-aminomethyl]benzamide) preferentially inhibited HDAC1 (IC50 value of approximately 0.3 microM) versus HDAC3 (IC50 value of approximately 8 microM) and had no inhibitory activity toward HDAC8 (IC50 value >100 microM). MS-27-275 as well as TSA increased histone H4 acetylation, induced apoptosis in the human colon cancer cell line SW620, and activated the simian virus 40 early promoter. HDAC1 protein was more abundantly expressed in SW620 cells compared with that of HDAC3 and HDAC8. Using purified recombinant HDAC proteins, we identified several novel HDAC inhibitors that preferentially inhibit HDAC1 or HDAC8. These inhibitors displayed distinct properties in inducing histone acetylation and reporter gene expression. These results suggest selective HDAC inhibitors could be identified using recombinantly expressed HDACs and that HDAC1 may be a promising therapeutic target for designing HDAC inhibitors for proliferative diseases such as cancer.

(1) The involvement of Rho-kinase (ROCK) in the contractile mechanisms mediating smooth muscle contraction of the rat urinary bladder was investigated using expression studies and the ROCK inhibitor Y-27632. (2) Both isoforms of ROCK (ROCK I and ROCK II) were detected in high levels in rat urinary bladder. (3) Y-27632 (10 micro M) significantly attenuated contractions of rat urinary bladder strips evoked by the G-protein coupled receptor agonists carbachol (58.1+/-10.5% at 0.3 micro M) and neurokinin A (68.6+/-12.7% at 1 micro M) without affecting contractions to potassium chloride (10-100 mM). In addition, basal tone was reduced by 47.8+/-2.0% by 10 micro M Y-27632 in the absence of stimulation. (4) Contractions of urinary bladder strips evoked by the P2X receptor agonist alpha,beta-methylene ATP (alpha,beta-mATP; 10 micro M) were also attenuated by Y-27632 (30.0+/-7.2% at 10 micro M). (5) Y-27632 (10 micro M) significantly attenuated contractions evoked by electrical field stimulation (2-16 Hz). The effect of Y-27632 on the tonic portion of the neurogenic response (4-16 Hz) was not significantly different from the effect of atropine (1 micro M) alone. (6) While the mechanism underlying the ability of Y-27632 to inhibit alpha,beta-mATP-evoked contractions remains undetermined, the results of the present study clearly demonstrate a role for ROCK in the regulation of rat urinary bladder smooth muscle contraction and tone.

Histone deacetylase (HDAC) inhibitors such as trichostatin (TSA) and butyrate have been shown to inhibit cancer cell proliferation, induce apoptosis and regulate the expression of genes involved in cell cycle. Although the precise mechanism underlying HDAC inhibitor-induced cell growth arrest is not fully understood, induction of cell cycle related genes such as p21(cip/waf), is thought to be important. Here we showed that in the SW620 human colon cancer cell line, TSA and butyrate induced the growth arrest and DNA damage gene 45alpha (GADD45alpha) and GADD45beta. Furthermore, GADD45beta and p21(cip/waf) messenger RNA were induced in the absence of protein synthesis, indicating that both genes were immediate target genes for TSA. Cyclohexamide and TSA super-induced the expression of GADD45alpha and beta, but not p21(cip/waf). Interestingly while mitogen-activated kinase (MEK) inhibitor PD98059 and p38 kinase inhibitor SB242235 were unable to affect GADD45 induction, two serine/threonine protein kinase inhibitors (H7 and H8) as well as curcumin completely blocked the super-induction. Concomitant to the inhibition of GADD45 induction, H7 and H8 also blocked TSA-induced apoptosis. Taken together, these results suggest that GADD45 induction may play important role in TSA-induced cellular effects.

Profilin is a small actin-binding protein that is involved in diverse functions such as maintaining cell structure integrity, cell mobility, tumor cell metastasis, as well as growth factor signal transduction. In this paper, we describe the molecular cloning and characterization of a novel form of profilin, termed profilin-3, from rat kidney using a PCR-based procedure for isolating tissue-specific genes. The profilin-3 cDNA encoded 137 amino acids, and it shared extensive homology to profilin-1 and profilin-2 from mice and humans. More strikingly, the expression of profilin-3 was highly selective, and its mRNA was only found in the kidney and to a much lesser extent in the testis. The size of the mRNA for profilin-3 in the testis was 1.2 kb, while in kidney it was approximately 4.4–5 kb, suggesting the presence of tissue-specific transcription from different promoters. In addition, we also found that the expression of profilin-3 mRNA was significantly elevated in two types of renal diseases: diabetic nephropathy (db/db mice) and polycystic kidney diseases (cpk mice). Similar to profilin-1 and profilin-2, profilin-3 was localized in the cytoplasmic domain. Furthermore, it was capable of interacting with actin and poly-L-proline in an in vitro assay as well as in transfected cells. These results strongly suggest that, contrary to the ubiquitous presence of profilin-1 and profilin-2, there is a tissue-specific form of these cytoskeleton-regulatory proteins and that the kidney/testes-specific profilin-3 may play a unique role in renal and/or reproductive functions.

A systematic effort to isolate kidney-specific genes was performed using recently described PCR-select methodology. Using this technique, a kidney-specific mini-gene library was generated and a number of kidney-specific genes that share significant homology to previously characterized kidney genes from rats and other species were isolated. These included three renal-specific transporters (an ADH water channel, the anion transporters RST and ROAT1), a cell adhesion molecule (K-cadherin) and a kidney-specific protein upregulated in renal carcinoma (DD96). In addition, we isolated two novel genes from a rat kidney. One of the genes shares limited homology to rat profilin-1 while the other did not share any similarity to genes in the Genbank. Northern blot analysis revealed that the mRNA for each of these genes is expressed in a highly kidney-restricted fashion. Our results suggested that tissue-specific genes can be rapidly isolated and characterized using PCR-select techniques and this methodology may be generally applicable to isolate specific genes from a variety of tissues.

To gain further insights into the molecular mechanisms involved in acute renal failure, we have isolated a new gene from rat and human, named KSP32 (kidney-specific protein with a molecular mass of 32 kDa). KSP32 encodes a novel gene that shows little homology to other mammalian proteins. It, however, shares extensive homology with several proteins found in the nematode Caenorhabditis elegans and plants. The expression of KSP32 mRNA is highly restricted to kidney. In situ hybidization analysis revealed that the expression of KSP32 mRNA was prominent in the boundary of kidney cortex and outer medulla, exhibiting a raylike formation extending from the medulla into the cortex. Finally, KSP32 mRNA was dramatically downregulated in rat following induction of acute ischemic renal failure. Rapid loss of KSP32 mRNA expression was observed beginning at ∼5 h following renal injury and mRNA levels remained depressed for at least 96 h. Both KSP32 mRNA levels as well as renal function recovered 14 days after injury. Administration of an endothelin receptor antagonist (SB-209670), known to restore renal function, significantly increased KSP32 expression.

The Src homology 3 (SH3) motif is found in numerous signal transduction proteins involved in cellular growth and differentiation. We have purified and cloned a novel protein, DEF-1 (differentiation-enhancing factor), from bovine brain by using a Src SH3 affinity column. Ectopic expression of DEF-1 in fibroblasts resulted in the differentiation of a significant fraction of the culture into adipocytes. This phenotype appears to be related to the induction of the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma), since DEF-1 NIH 3T3 cells demonstrated augmented levels of PPARgamma mRNA and, when treated with activating PPARgamma ligands, efficient induction of differentiation. Further evidence for a role for DEF-1 in adipogenesis was provided by heightened expression of DEF-1 mRNA in adipose tissue isolated from obese and diabetes mice compared to that in tissue isolated from wild-type mice. However, DEF-1 mRNA was detected in multiple tissues, suggesting that the signal transduction pathway(s) in which DEF-1 is involved is not limited to adipogenesis. These results suggest that DEF-1 is an important component of a signal transduction process that is involved in the differentiation of fibroblasts and possibly of other types of cells.