Identification of tissue-specific cell death using methylation patterns of circulating DNA.

Minimally invasive detection of cell death could prove an invaluable resource in many physiologic and pathologic situations. Cell-free circulating DNA (cfDNA) released from dying cells is emerging as a diagnostic tool for monitoring cancer dynamics and graft failure. However, existing methods rely on differences in DNA sequences in source tissues, so that cell death cannot be identified in tissues with a normal genome. We developed a method of detecting tissue-specific cell death in humans based on tissue-specific methylation patterns in cfDNA. We interrogated tissue-specific methylome databases to identify cell type-specific DNA methylation signatures and developed a method to detect these signatures in mixed DNA samples. We isolated cfDNA from plasma or serum of donors, treated the cfDNA with bisulfite, PCR-amplified the cfDNA, and sequenced it to quantify cfDNA carrying the methylation markers of the cell type of interest. Pancreatic ß-cell DNA was identified in the circulation of patients with recently diagnosed type-1 diabetes and islet-graft recipients; oligodendrocyte DNA was identified in patients with relapsing multiple sclerosis; neuronal/glial DNA was identified in patients after traumatic brain injury or cardiac arrest; and exocrine pancreas DNA was identified in patients with pancreatic cancer or pancreatitis. This proof-of-concept study demonstrates that the tissue origins of cfDNA and thus the rate of death of specific cell types can be determined in humans. The approach can be adapted to identify cfDNA derived from any cell type in the body, offering a minimally invasive window for diagnosing and monitoring a broad spectrum of human pathologies as well as providing a better understanding of normal tissue dynamics.

S1PR3 Signaling Drives Bacterial Killing and Is Required for Survival in Bacterial Sepsis.

RATIONALE: Efficient elimination of pathogenic bacteria is a critical determinant in the outcome of sepsis. Sphingosine-1-phosphate receptor 3 (S1PR3) mediates multiple aspects of the inflammatory response during sepsis, but whether S1PR3 signaling is necessary for eliminating the invading pathogens remains unknown. OBJECTIVES: To investigate the role of S1PR3 in antibacterial immunity during sepsis. METHODS: Loss- and gain-of-function experiments were performed using cell and murine models. S1PR3 levels were determined in patients with sepsis and healthy volunteers. MEASUREMENTS AND MAIN RESULTS: S1PR3 protein levels were up-regulated in macrophages upon bacterial stimulation. S1pr3-/- mice showed increased mortality and increased bacterial burden in multiple models of sepsis. The transfer of wild-type bone marrow-derived macrophages rescued S1pr3-/- mice from lethal sepsis. S1PR3-overexpressing macrophages further ameliorated the mortality rate of sepsis. Loss of S1PR3 led to markedly decreased bacterial killing in macrophages. Enhancing endogenous S1PR3 activity using a peptide agonist potentiated the macrophage bactericidal function and improved survival rates in multiple models of sepsis. Mechanically, the reactive oxygen species levels were decreased and phagosome maturation was delayed in S1pr3-/- macrophages due to impaired recruitment of vacuolar protein-sorting 34 to the phagosomes. In addition, S1RP3 expression levels were elevated in monocytes from patients with sepsis. Higher levels of monocytic S1PR3 were associated with efficient intracellular bactericidal activity, better immune status, and preferable outcomes. CONCLUSIONS: S1PR3 signaling drives bacterial killing and is essential for survival in bacterial sepsis. Interventions targeting S1PR3 signaling could have translational implications for manipulating the innate immune response to combat pathogens.

Distinct designer diamines promote mitophagy, and thereby enhance healthspan in C. elegans and protect human cells against oxidative damage.

Impaired mitophagy is a primary pathogenic event underlying diverse aging-associated diseases such as Alzheimer and Parkinson diseases and sarcopenia. Therefore, augmentation of mitophagy, the process by which defective mitochondria are removed, then replaced by new ones, is an emerging strategy for preventing the evolvement of multiple morbidities in the elderly population. Based on the scaffold of spermidine (Spd), a known mitophagy-promoting agent, we designed and tested a family of structurally related compounds. A prototypic member, 1,8-diaminooctane (VL-004), exceeds Spd in its ability to induce mitophagy and protect against oxidative stress. VL-004 activity is mediated by canonical aging genes and promotes lifespan and healthspan in C. elegans. Moreover, it enhances mitophagy and protects against oxidative injury in rodent and human cells. Initial structural characterization suggests simple rules for the design of compounds with improved bioactivity, opening the way for a new generation of agents with a potential to promote healthy aging.

Cancers as wounds that do not heal: differences and similarities between renal regeneration/repair and renal cell carcinoma.

Cancers have been described as wounds that do not heal, suggesting that the two share common features. By comparing microarray data from a model of renal regeneration and repair (RRR) with reported gene expression in renal cell carcinoma (RCC), we asked whether those two processes do, in fact, share molecular features and regulatory mechanisms. The majority (77%) of the genes expressed in RRR and RCC were concordantly regulated, whereas only 23% were discordant (i.e., changed in opposite directions). The orchestrated processes of regeneration, involving cell proliferation and immune response, were reflected in the concordant genes. The discordant gene signature revealed processes (e.g., morphogenesis and glycolysis) and pathways (e.g., hypoxia-inducible factor and insulin-like growth factor-I) that reflect the intrinsic pathologic nature of RCC. This is the first study that compares gene expression patterns in RCC and RRR. It does so, in particular, with relation to the hypothesis that RCC resembles the wound healing processes seen in RRR. However, careful attention to the genes that are regulated in the discordant direction provides new insights into the critical differences between renal carcinogenesis and wound healing. The observations reported here provide a conceptual framework for further efforts to understand the biology and to develop more effective diagnostic biomarkers and therapeutic strategies for renal tumors and renal ischemia.

Self-Assembling Myristoylated Human α-Defensin 5 as a Next-Generation Nanobiotics Potentiates Therapeutic Efficacy in Bacterial Infection.

The increasing prevalence of antibacterial resistance globally underscores the urgent need to the update of antibiotics. Here, we describe a strategy for inducing the self-assembly of a host-defense antimicrobial peptide (AMP) into nanoparticle antibiotics (termed nanobiotics) with significantly improved pharmacological properties. Our strategy involves the myristoylation of human α-defensin 5 (HD5) as a therapeutic target and subsequent self-assembly in aqueous media in the absence of exogenous excipients. Compared with its parent HD5, the C-terminally myristoylated HD5 (HD5-myr)-assembled nanobiotic exhibited significantly enhanced broad-spectrum bactericidal activity in vitro. Mechanistically, it selectively killed Escherichia coli ( E. coli) and methicillin-resistant Staphylococcus aureus (MRSA) through disruption of the cell wall and/or membrane structure. The in vivo results further demonstrated that the HD5-myr nanobiotic protected against skin infection by MRSA and rescued mice from E. coli-induced sepsis by lowering the systemic bacterial burden and alleviating organ damage. The self-assembled HD5-myr nanobiotic also showed negligible hemolytic activity and substantially low toxicity in animals. Our findings validate this design rationale as a simple yet versatile strategy for generating AMP-derived nanobiotics with excellent in vivo tolerability. This advancement will likely have a broad impact on antibiotic discovery and development efforts aimed at combating antibacterial resistance.

Lyn is a target gene for prostate cancer: sequence-based inhibition induces regression of human tumor xenografts.

The Src-related protein kinase Lyn plays an important role in B-cell activation. However, several lines of evidence suggest that it is also involved in the control of cellular proliferation and the inhibition of apoptosis. We have discovered that Lyn is expressed in normal prostate epithelia, in 95% of primary human prostate cancer (PC) specimens examined, and in all of the PC cell lines that we assayed. Moreover, Lyn knockout mice display abnormal prostate gland morphogenesis, which suggests that Lyn plays an important role in prostate epithelium development and implies that Lyn is a candidate target for specific therapy for PC. Using a drug-design strategy to construct sequence-based peptide inhibitors, a Lyn-specific inhibitor, KRX-123, targeting a unique interaction site within Lyn, was synthesized. KRX-123 was found to inhibit cellular proliferation in three hormone-refractory PC cell lines, DU145, PC3, and TSU-Pr1 with IC(50) values of 2-4 micro M. In vivo, tumor volume of DU145 explants in nude mice was significantly reduced after once-a-week injections of KRX-123, at a dose of 10 mg/kg, for a period of 5 weeks. Histological analyses of the treated tumors indicated extensive apoptosis. Thus, we suggest that Lyn inhibition may serve as a prime target for the treatment of hormone-refractory PC.

A synthetic heparin-mimicking polyanionic compound inhibits central nervous system inflammation.

The immunomodulating capacity of heparin led us to test the effect of the synthetic heparin-mimicking and low anticoagulant compound RG-13577 on the course of experimental autoimmune encephalomyelitis (EAE) and central nervous system (CNS) inflammation. EAE was induced in SJL mice by inoculation with whole mouse spinal cord homogenate. RG-13577, delivered intraperitoneally, inhibited the clinical signs of acute EAE and markedly ameliorated inflammation in the spinal cord, primarily by inhibiting heparanase activity in lymphocytes and astrocytes and thus impairing lymphocyte traffic. RG-13577 treatment was effective when started on day of disease induction or day 7 after induction. The low molecular weight heparin, enoxaparin, tested under the same conditions, exerted only a minor insignificant inhibitory effect. RG-13577 also inhibited the tyrosine phosphorylation of several proteins, particularly Erk1 and Erk2 of the MAP kinase signaling pathways associated with inflammation and cell proliferation. RG-13577 blocked the activity of sPLA(2) and inhibited CNS PGE(2) production both in vivo and in vitro.

Inhibition of anastomotic intimal hyperplasia by a synthetic nonsulphated heparin-mimicking compound.

Despite extensive research in the design of endovascular catheters and advanced surgical techniques, stenosis recurs in a large percentage of patients undergoing angioplasty or anastomosis. Hence, neointimal hyperplasia, caused by migration and proliferation of vascular smooth muscle cells (SMC), remains a significant limitation to the relief of obstructive-occlusive vascular disease. It has been previously demonstrated that heparin displaces active basic fibroblast growth factor (bFGF) from the lumenal surface of blood vessels. Sequestration of the displaced bFGF by injured areas of the vessel wall is inhibited in the presence of a synthetic nonsulphated heparin-mimicking polyanionic compound (RG-13577). This compound also induces a phenotype transformation of coronary SMC into a metabolically active hypertropic status that could promote repair processes after balloon angioplasty while inhibiting cell proliferation. In this paper, the result of a continuous administration of compound RG-13577 both in the rat carotid catheter injury model and in a newly developed rat model of surgical arterial vascular injury (anastomosis) is reported: it causes a profound inhibition of intimal hyperplasia in both models. A combined treatment with heparin/heparan sulphate mimetics and halofuginone, a potent inhibitor of collagen synthesis, extracellular matrix deposition and SMC proliferation, is expected to inhibit restenosis through inhibition of both signals/activities induced by soluble molecules (ie, heparin-binding growth factors) and components of the extracellular matrix (ie, type I collagen).

Paradoxical effects of aurintricarboxylic acid and RG-13577: acute thrombosis and in-stent stenosis in a passive-coated stent.

PURPOSE: To investigate if a platelet inhibitor (aurintricarboxylic acid [ATA]) and a heparin-mimicking antagonist (RG-13577) of basic fibroblast growth factor 2 (bFGF2) could be combined as a stable compound and attached to conventional bare metal stents to hinder thrombus formation and inflammatory reactions of stenting. METHODS: Fifteen domestic pigs were stented with RG-13577/ATA-coated (n=6), ATA-coated (n=12), and bare metal stents (n=12) in the left anterior descending (LAD) and left circumflex (LCX) coronary arteries. All surviving pigs were evaluated with contrast angiography and intravascular ultrasonography (IVUS) after 4 weeks. Histological analysis of the stented arteries was performed after hematoxylin-eosin staining. Tissue factor (TF) staining and scanning electron microscopy (SEM) were performed in animals with acute stent thrombosis. RESULTS: Five of the 6 animals receiving an RG-13577/ATA-coated stent experienced acute stent thrombosis, while no adverse events occurred in the animals of the other 2 groups. Follow-up angiography did not show significant in-stent stenosis in either bare or ATA-coated stents. However, histomorphometry revealed larger neointimal area (3.54+/-0.69 mm2 versus 1.82+/-0.27 mm2, p<0.05) and outward plaque area (1.56+/-0.34 mm2 versus 0.61+/-0.12 mm2, p<0.05) in ATA-coated stents. Three-dimensional IVUS analysis showed analogous results, with significantly larger neointimal volume and outward plaque volume in ATA-coated stents. There was a slight increase in TF staining around the stent struts, while SEM showed increased platelet adhesion and activity in RG-13577/ATA-coated stents versus the ATA-coated and bare metal stents. CONCLUSION: RG-13577/ATA-coated stents lead to acute stent thrombosis. The ATA coating alone did not lead to acute events, but resulted in higher neointimal hyperplasia and expansive remodeling. These results underline the importance of preclinical studies before using new coated stents in human arteries.

Induction of pro-angiogenic signaling by a synthetic peptide derived from the second intracellular loop of S1P3 (EDG3).

The G-protein-coupled receptors of the endothelial differentiation gene (EDG) family mediate pro-angiogenic activities, such as endothelial cell proliferation, chemotaxis, and vessel morphogenesis. We synthesized and tested the effects of a 9-amino acid peptide (KRX-725), derived from the second intracellular loop of S1P3 (EDG3). KRX-725 mimics the effects of sphingosine 1-phosphate (S1P), the natural ligand of S1P3, by triggering a Gi-dependent MEK-ERK (mitogen-activated protein kinase kinase and extracellular signal-regulated kinase) signal transduction pathway. Using aortic rings as an ex vivo model of angiogenesis, vascular sprouting was assessed in the presence of KRX-725 or S1P. KRX-725 induced extensive and dense vascular sprouts, which contain an elaborated organization of endothelial and smooth muscle layers, including lumen formation. When KRX-725 or S1P was combined with proangiogenic factors, such as basic fibroblast growth factor (bFGF), stem cell factor, or vascular endothelial growth factor, the effect was synergistic, leading to further enhancement of vascular sprouting. KRX-725 also initiated neovascularization in a mouse corneal pocket assay in vivo and showed synergism with bFGF. The specificity of KRX-725 was demonstrated via peptide-induced receptor internalization of S1P3 but not S1P1. The ability of a short peptide to stimulate extensive angiogenesis and to synergize with pro-angiogenic factors suggests that KRX-725 may serve as a useful agent in treating pathologic conditions such as peripheral vascular disease, cardiac ischemia, or tissue grafts.

Structure-activity relationships of heparin-mimicking compounds in induction of bFGF release from extracellular matrix and inhibition of smooth muscle cell proliferation and heparanase activity.

A series of nine synthetic polyaromatic compounds were synthesized by polymerization of aromatic ring monomers with formaldehyde, which yield substantially ordered backbones with different functional anionic groups (hydroxyl and carboxyl) on the phenol ring. These compounds were tested for their heparin-mimicking activity: (1) inhibition of heparanase activity; (2) inhibition of SMC proliferation; and (3) release of bFGF from the ECM. We demonstrate that compounds that have two hydroxyl groups para and ortho to the carboxylic group and a carboxylic group at a distance of two carbons from the phenol ring inhibit heparanase activity and SMC proliferation, as well as induced an almost complete release of bFGF from ECM. Addition of a methyl group next to the carboxylic group led to a preferential inhibition of heparanase activity. Similar results were obtained with a compound that contains one hydroxyl group para to the carboxylic group and an ether group near the carboxylic group on the phenol ring. Preferential inhibition of SMC proliferation was best achieved when the position of the hydroxyl group is para and ortho to the carboxylic group and the carboxylic group is at a distance of one carbon from the phenol ring. On the other hand, for maximal release of bFGF from ECM, the position of the carboxylic group should be three carbons away from the phenol ring. These new heparin-mimicking compounds may have a potential use in inhibition of tumor metastasis, arteriosclerosis, and inflammation.

Sequence-based design of kinase inhibitors applicable for therapeutics and target identification.

A platform for specifically modulating kinase-dependent signaling using peptides derived from the catalytic domain of the kinase is presented. This technology, termed KinAce, utilizes the canonical structure of protein kinases. The targeted regions (subdomain V and subdomains IX and X) are analyzed and their sequence, three-dimensional structure, and involvement in protein-protein interaction are highlighted. Short myristoylated peptides were derived from the target regions of the tyrosine kinases c-Kit and Lyn and the serine/threonine kinases 3-phosphoinositide-dependent kinase-1 (PDK1) and Akt/protein kinase B (PKB). For each kinase an active designer peptide is shown to selectively inhibit the signaling of the kinase from which it is derived, and to inhibit cancer cell proliferation in the micromolar range. This technology emerges as an applicable tool for deriving sequence-based selective inhibitors for a broad range of protein kinases as hits that may be further developed into drugs. Moreover, it enables identification of novel kinase targets for selected therapeutic indications as demonstrated in the KinScreen application.