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The periosteum is the layer of cells that covers nearly the entire surface of every bone. Upon infection, injury or malignancy the bone surface undergoes new growth-the periosteal reaction-but the mechanism and physiological role of this process remain unknown 1,2 . Here we show that the periosteal reaction protects against cancer invasion into the bone. Histological analyses of human lesions of head and neck squamous cell carcinomas (HNSCCs) show that periosteal thickening occurs in proximity to the tumour. We developed a genetically dissectible mouse model of HNSCC and demonstrate that inducible depletion of periosteal cells accelerates cancerous invasion of the bone. Single-cell RNA sequencing reveals that expression of the gene encoding the protease inhibitor TIMP1 is markedly increased in the periosteum at the pre-invasive stage. This increase is due to upregulation of HIF1α expression in the tumour microenvironment, and increased TIMP1 inactivates matrix-degrading proteases, promoting periosteal thickening to inhibit cancer invasion. Genetic deletion of Timp1 impairs periosteal expansion, exacerbating bone invasion and decreasing survival in tumour-bearing mice. Together, these data show that the periosteal reaction may act as a functional stromal barrier against tumour progression, representing a unique example of tissue immunity mediated by stromal cells., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Finding peaks in chromatograms and determining their start and end points (peak picking) is a core task in chromatography based biotechnology. Construction of peak-picking neural networks by deep learning was, however, hampered from the preparation of exact peak-picked or "labeled" chromatograms since the exact start and end points were often unclear in overlapping peaks in real chromatograms. We present a design of a fake chromatogram generator, along with a method for deep learning of peak-picking neural networks. Fake chromatograms were generated by generation of fake peaks, random sampling of peak positions from feature distributions, and merging with real blank sample chromatograms. Information on the exact start and end points, as labeled on the fake chromatograms, were effective for training and evaluating peak-picking neural networks. The peak-picking neural networks constructed herein outperformed conventional peak-picking software and showed comparable performance with that of experienced operators for processing the widely targeted metabolome data. Results of this study indicate that generation of fake chromatograms would be crucial for developing peak-picking neural networks and a key technology for further improvement of peak picking neural networks., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Receptor activator of NF-κB ligand (RANKL) is a multifunctional cytokine known to affect immune and skeletal systems, as well as oncogenesis and metastasis 1-4 . RANKL is synthesized as a membrane-bound molecule, and cleaved into its soluble form by proteases 5-7 . As the soluble form of RANKL does not contribute greatly to bone remodelling or ovariectomy-induced bone loss 8 , whether soluble RANKL has a role in pathological settings remains unclear. Here we show that soluble RANKL promotes the formation of tumour metastases in bone. Mice that selectively lack soluble RANKL (Tnfsf11 ΔS/ΔS ) 5-7,9 have normal bone homoeostasis and develop a normal immune system but display markedly reduced numbers of bone metastases after intracardiac injection of RANK-expressing melanoma and breast cancer cells. Deletion of soluble RANKL does not affect osteoclast numbers in metastatic lesions or tumour metastasis to non-skeletal tissues. Therefore, soluble RANKL is dispensable for physiological regulation of bone and immune systems, but has a distinct and pivotal role in the promotion of bone metastases.

Pericytes are mesenchymal cells that surround the endothelial cells of small vessels in various organs. These cells express several markers, such as NG2, CD146, and PDGFRβ, and play an important role in the stabilization and maturation of blood vessels. It was also recently revealed that like mesenchymal stem cells (MSCs), pericytes possess multilineage differentiation capacity, especially myogenic, adipogenic, and fibrogenic differentiation capacities. Although some previous studies have reported that pericytes also have osteogenic potential, the osteogenesis of pericytes can still be further elucidated. In the present study, we established novel methods for isolating and culturing primary murine pericytes. An immortalized pericyte line was also established. Multilineage induction of the pericyte line induced osteogenesis, adipogenesis, and chondrogenesis of the cells in vitro. In addition, pericytes that were injected into the fracture site of a bone fracture mouse model contributed to callus formation. Furthermore, in vivo pericyte-lineage-tracing studies demonstrated that endogenous pericytes also differentiate into osteoblasts and osteocytes and contribute to bone fracture healing as a cellular source of osteogenic cells. Pericytes can be a promising therapeutic candidate for treating bone fractures with a delayed union or nonunion as well as bone diseases causing bone defects.

Bone is one of common metastasis sites for many types of cancer. In bone metastatic microenvironment, tumor-bone interactions play a significant role in the regulation of osteolytic or osteoblastic bone metastasis. In order to investigate the direct interaction between tumor cells and bone tissue, it is essential to generate appropriate animal models that mimic the behavior of tumor cells in bone metastatic lesions. Calvarial implantation model (bone invasion model) is a newly-established animal model that accurately recapitulates the behavior of tumor cells in the tumor-bone microenvironment. The surgical technique for tumor cell implantation is simpler than intracardiac, intra-arterial, or intraosseous injection techniques. This model can be useful for the identification of key factors driving tumor-induced osteolytic or osteoblastic changes., Competing Interests: Competing interestsAll authors declare no conflicts of interest., (Copyright © 2019 The Authors; exclusive licensee Bio-protocol LLC.)

Bone metastatic lesions are classified as osteoblastic or osteolytic lesions. Prostate and breast cancer patients frequently exhibit osteoblastic-type and osteolytic-type bone metastasis, respectively. In metastatic lesions, tumor cells interact with many different cell types, including osteoblasts, osteoclasts, and mesenchymal stem cells, resulting in an osteoblastic or osteolytic phenotype. However, the mechanisms responsible for the modification of bone remodeling have not been fully elucidated. MicroRNAs (miRNAs) are transferred between cells via exosomes and serve as intercellular communication tools, and numerous studies have demonstrated that cancer-secreted miRNAs are capable of modifying the tumor microenvironment. Thus, cancer-secreted miRNAs can induce an osteoblastic or osteolytic phenotype in the bone metastatic microenvironment. In this study, we performed a comprehensive expression analysis of exosomal miRNAs secreted by several human cancer cell lines and identified eight types of human miRNAs that were highly expressed in exosomes from osteoblastic phenotype-inducing prostate cancer cell lines. One of these miRNAs, hsa-miR-940, significantly promoted the osteogenic differentiation of human mesenchymal stem cells in vitro by targeting ARHGAP1 and FAM134A Interestingly, although MDA-MB-231 breast cancer cells are commonly known as an osteolytic phenotype-inducing cancer cell line, the implantation of miR-940-overexpressing MDA-MB-231 cells induced extensive osteoblastic lesions in the resulting tumors by facilitating the osteogenic differentiation of host mesenchymal cells. Our results suggest that the phenotypes of bone metastases can be induced by miRNAs secreted by cancer cells in the bone microenvironment., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Sepsis/endotoxemia is elicited by the circulatory distribution of pathogens/endotoxins into whole bodies, and causes profound effects on human health by causing inflammation in multiple organs. Mitochondrial damage is one of the characteristics of the cellular degeneration observed during sepsis/endotoxemia. Elimination of damaged mitochondria through the autophagy-lysosome system has been reported in the liver, indicating that autophagy should play an important role in liver homeostasis during sepsis/endotoxemia. An increased appearance of mitochondrial DNA and proteins in the plasma is another feature of sepsis/endotoxemia, suggesting that damaged mitochondria are not only eliminated within the cells, but also extruded through currently unknown mechanisms. Here we provide evidence for the secretion of mitochondrial proteins and DNA from lipopolysaccharide (LPS)-stimulated rat hepatocytes as well as mouse embryonic fibroblasts (MEFs). The secretion of mitochondrial contents is accompanied by the secretion of proteins that reside in the lumenal space of autolysosomes (LC3-II and CTSD/cathepsin D), but not by a lysosomal membrane protein (LAMP1). The pharmacological inhibition of autophagy by 3MA blocks the secretion of mitochondrial constituents from LPS-stimulated hepatocytes. LPS also stimulates the secretion of mitochondrial as well as autolysosomal lumenal proteins from wild-type (Atg5(+/+)) MEFs, but not from atg5(-/-) MEFs. Furthermore, we show that direct exposure of purified mitochondria activates polymorphonuclear leukocytes (PMNs), as evident by the induction of IL1B/interlekin-1β, a pro-inflammatory cytokine. Taken together, the data suggest the active extrusion of mitochondrial contents, which provoke an inflammatory response of immune cells, through the exocytosis of autolysosomes by cells stimulated with LPS.

DNA transcription, replication, and repair are regulated by histone acetylation, a process that requires the generation of acetyl-coenzyme A (CoA). Here, we show that all the subunits of the mitochondrial pyruvate dehydrogenase complex (PDC) are also present and functional in the nucleus of mammalian cells. We found that knockdown of nuclear PDC in isolated functional nuclei decreased the de novo synthesis of acetyl-CoA and acetylation of core histones. Nuclear PDC levels increased in a cell-cycle-dependent manner and in response to serum, epidermal growth factor, or mitochondrial stress; this was accompanied by a corresponding decrease in mitochondrial PDC levels, suggesting a translocation from the mitochondria to the nucleus. Inhibition of nuclear PDC decreased acetylation of specific lysine residues on histones important for G1-S phase progression and expression of S phase markers. Dynamic translocation of mitochondrial PDC to the nucleus provides a pathway for nuclear acetyl-CoA synthesis required for histone acetylation and epigenetic regulation., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Rationale: Bronchopulmonary dysplasia (BPD) and emphysema are characterized by arrested alveolar development or loss of alveoli; both are significant global health problems and currently lack effective therapy. Bone marrow-derived mesenchymal stem cells (BMSCs) prevent adult lung injury, but their therapeutic potential in neonatal lung disease is unknown., Objectives: We hypothesized that intratracheal delivery of BMSCs would prevent alveolar destruction in experimental BPD., Methods: In vitro, BMSC differentiation and migration were assessed using co-culture assays and a modified Boyden chamber. In vivo, the therapeutic potential of BMSCs was assessed in a chronic hyperoxia-induced model of BPD in newborn rats., Measurements and Main Results: In vitro, BMSCs developed immunophenotypic and ultrastructural characteristics of type II alveolar epithelial cells (AEC2) (surfactant protein C expression and lamellar bodies) when co-cultured with lung tissue, but not with culture medium alone or liver. Migration assays revealed preferential attraction of BMSCs toward oxygen-damaged lung versus normal lung. In vivo, chronic hyperoxia in newborn rats led to air space enlargement and loss of lung capillaries, and this was associated with a decrease in circulating and resident lung BMSCs. Intratracheal delivery of BMSCs on Postnatal Day 4 improved survival and exercise tolerance while attenuating alveolar and lung vascular injury and pulmonary hypertension. Engrafted BMSCs coexpressed the AEC2-specific marker surfactant protein C. However, engraftment was disproportionately low for cell replacement to account for the therapeutic benefit, suggesting a paracrine-mediated mechanism. In vitro, BMSC-derived conditioned medium prevented O(2)-induced AEC2 apoptosis, accelerated AEC2 wound healing, and enhanced endothelial cord formation., Conclusions: BMSCs prevent arrested alveolar and vascular growth in part through paracrine activity. Stem cell-based therapies may offer new therapeutic avenues for lung diseases that currently lack efficient treatments.

The in vitro and in vivo antifungal activities of T-2307, a novel arylamidine, were evaluated and compared with those of fluconazole, voriconazole, micafungin, and amphotericin B. T-2307 exhibited broad-spectrum activity against clinically significant pathogens, including Candida species (MIC range, 0.00025 to 0.0078 microg/ml), Cryptococcus neoformans (MIC range, 0.0039 to 0.0625 microg/ml), and Aspergillus species (MIC range, 0.0156 to 4 microg/ml). Furthermore, T-2307 exhibited potent activity against fluconazole-resistant and fluconazole-susceptible-dose-dependent Candida albicans strains as well as against azole-susceptible strains. T-2307 exhibited fungicidal activity against some Candida and Aspergillus species and against Cryptococcus neoformans. In mouse models of disseminated candidiasis, cryptococcosis, and aspergillosis, the 50% effective doses of T-2307 were 0.00755, 0.117, and 0.391 mg.kg(-1).dose(-1), respectively. This agent was considerably more active than micafungin and amphotericin B against candidiasis and than amphotericin B against cryptococcosis, and its activity was comparable to the activities of micafungin and amphotericin B against aspergillosis. The results of preclinical in vitro and in vivo evaluations performed thus far indicate that T-2307 could represent a potent injectable agent for the treatment of candidiasis, cryptococcosis, and aspergillosis.

Patent ductus arteriosus (PDA) complicates the hospital course of premature infants. Impaired oxygen (O2)-induced vasoconstriction in preterm ductus arteriosus (DA) contributes to PDA and results, in part, from decreased function/expression of O2-sensitive, voltage-gated potassium channels (Kv) in DA smooth muscle cells (DASMCs). This paradigm suggests that activation of the voltage-sensitive L-type calcium channels (CaL), which increases cytosolic calcium ([Ca2+]i), is a passive consequence of membrane depolarization. However, effective Kv gene transfer only partially matures O2 responsiveness in preterm DA. Thus, we hypothesized that CaL are directly O2 sensitive and that immaturity of CaL function in preterm DA contributes to impaired O2 constriction. We show that preterm rabbit DA rings have reduced O2- and 4-aminopyridine (Kv blocker)-induced constriction. Preterm rabbit DASMCs have reduced O2-induced whole-cell calcium current (ICa) and [Ca2+]i. BAY K8644, a CaL activator, increased O2 constriction, ICa, and [Ca]i in preterm DASMCs to levels seen at term but had no effect on human and rabbit term DA. Preterm rabbit DAs have decreased gamma and increased alpha subunit protein expression. We conclude that the CaL in term rabbit and human DASMCs is directly O2 sensitive. Functional immaturity of CaL O2 sensitivity contributes to impaired O2 constriction in premature DA and can be reversed by BAY K8644.

In pulmonary arterial hypertension (PAH), antiapoptotic, proliferative, and inflammatory diatheses converge to create an obstructive vasculopathy. A selective down-regulation of the Kv channel Kv1.5 has been described in human and animal PAH. The resultant increase in intracellular free Ca(2+) ([Ca(2+)](i)) and K(+) ([K(+)](i)) concentrations explains the pulmonary artery smooth muscle cell (PASMC) contraction, proliferation and resistance to apoptosis. The recently described PASMC hyperpolarized mitochondria and increased bcl-2 levels also contribute to apoptosis resistance in PAH. The cause of the Kv1.5, mitochondrial, and inflammatory abnormalities remains unknown. We hypothesized that these abnormalities can be explained in part by an activation of NFAT (nuclear factor of activated T cells), a Ca(2+)/calcineurin-sensitive transcription factor. We studied PASMC and lungs from six patients with and four without PAH and blood from 23 PAH patients and 10 healthy volunteers. Compared with normal, PAH PASMC had decreased Kv current and Kv1.5 expression and increased [Ca(2+)](i), [K(+)](i), mitochondrial potential (Delta Psi m), and bcl-2 levels. PAH but not normal PASMC and lungs showed activation of NFATc2. Inhibition of NFATc2 by VIVIT or cyclosporine restored Kv1.5 expression and current, decreased [Ca(2+)](i), [K(+)](i), bcl-2, and Delta Psi m, leading to decreased proliferation and increased apoptosis in vitro. In vivo, cyclosporine decreased established rat monocrotaline-PAH. NFATc2 levels were increased in circulating leukocytes in PAH versus healthy volunteers. CD3-positive lymphocytes with activated NFATc2 were seen in the arterial wall in PAH but not normal lungs. The generalized activation of NFAT in human and experimental PAH might regulate the ionic, mitochondrial, and inflammatory remodeling and be a therapeutic target and biomarker.

Background: Constriction of the ductus arteriosus (DA) is initiated at birth by inhibition of O2-sensitive K+ channels in DA smooth muscle cells. Subsequent membrane depolarization and calcium influx through L-type calcium channels initiates functional closure. We hypothesize that Rho-kinase activation is an additional mechanism that sustains DA constriction., Methods and Results: The effect of increased PO2 on the activity and expression of Rho-kinase was assessed in DAs from neonates with hypoplastic left-heart syndrome (n=15) and rabbits (339 term and 99 preterm rabbits). Rho-kinase inhibitors (Y-27632 and fasudil) prevent and reverse O2 constriction. Heterogeneity exists in the sensitivity of constrictors (PO2=endothelin=phenylephrine>KCl) and of fetal vessels (DA=pulmonary artery>aorta) to Rho-kinase inhibition. Inhibition of L-type calcium channels (nifedipine) or removal of extracellular calcium inhibits approximately two thirds of O2 constriction. Residual DA constriction reflects calcium sensitization, which persists after removal of extracellular calcium and blocking of sarcoplasmic reticulum Ca2+-ATPase. In term DA, an increase in PO2 activates Rho-kinase and thereby increases RhoB and ROCK-1 expression. Activation of Rho-kinase in DA smooth muscle cells is initiated by a PO2-dependent, rotenone-sensitive increase in mitochondrion-derived reactive O2 species. O2 effects on Rho-kinase are mimicked by exogenous H2O2. In preterm DAs, immaturity of mitochondrial reactive oxygen species generation is associated with reduced and delayed O2 constriction and lack of PO2-dependent upregulation of Rho-kinase expression., Conclusions: O2 activates Rho-kinase and increases Rho-kinase expression in term DA smooth muscle cells by a redox-regulated, positive-feedback mechanism that promotes sustained vasoconstriction. Conversely, Rho-kinase inhibitors may be useful in maintaining DA patency, as a bridge to congenital heart surgery.

Pulmonary arterial hypertension (PAH) is associated with mutations of bone morphogenetic protein receptor 2 (BMPR2), and BMPR2 expression decreases with the development of experimental PAH. Decreased BMPR2 expression and impaired intracellular BMP signaling in pulmonary artery (PA) smooth muscle cells (PASMC) suppresses apoptosis and promotes proliferation, thereby contributing to the pathogenesis of PAH. We hypothesized that overexpression of BMPR2 in resistance PAs would ameliorate established monocrotaline PAH. Human BMPR2 was inserted into a serotype 5 adenovirus with a green fluorescent protein (GFP) reporter. Dose-dependent transgene expression was confirmed in PASMC using fluorescence microscopy, quantitative RT-PCR, and immunoblots. PAH was induced by injecting Sprague-Dawley rats with monocrotaline (60 mg/kg ip) or saline. On day 14, post-monocrotaline (MCT) rats received 5 x 10(9) plaque-forming units of either Ad-human BMPR2 (Ad-hBMPR2) or Ad-GFP. Transgene expression was confirmed by fluorescence microscopy, quantitative RT-PCR of whole lung samples, and laser-capture microdissected resistance PAs. Invasive hemodynamic and echocardiographic end points of pulmonary hypertension were assessed on day 24. Endogenous BMPR2 mRNA levels were greatest in resistance PAs, and expression declined with MCT PAH. Despite robust hBMPR2 expression in all lung lobes and within resistance PAs of treated rats, hBMPR2 did not lower mean PA pressure, pulmonary vascular resistance index, right ventricular hypertrophy, or remodeling of resistance PAs. Nebulized intratracheal adenoviral gene therapy with hBMPR2 reliably distributed hBMPR2 to resistance PAs but did not ameliorate PAH. Depressed BMPR2 expression may be a marker of PAH but is not central to the pathogenesis of this model of PAH.

The unique metabolic profile of cancer (aerobic glycolysis) might confer apoptosis resistance and be therapeutically targeted. Compared to normal cells, several human cancers have high mitochondrial membrane potential (DeltaPsim) and low expression of the K+ channel Kv1.5, both contributing to apoptosis resistance. Dichloroacetate (DCA) inhibits mitochondrial pyruvate dehydrogenase kinase (PDK), shifts metabolism from glycolysis to glucose oxidation, decreases DeltaPsim, increases mitochondrial H2O2, and activates Kv channels in all cancer, but not normal, cells; DCA upregulates Kv1.5 by an NFAT1-dependent mechanism. DCA induces apoptosis, decreases proliferation, and inhibits tumor growth, without apparent toxicity. Molecular inhibition of PDK2 by siRNA mimics DCA. The mitochondria-NFAT-Kv axis and PDK are important therapeutic targets in cancer; the orally available DCA is a promising selective anticancer agent.

Background: Bronchopulmonary dysplasia (BPD) and pulmonary emphysema, both significant global health problems, are characterized by a loss of alveoli. Vascular endothelial growth factor (VEGF) is a trophic factor required for endothelial cell survival and is abundantly expressed in the lung., Methods and Results: We report that VEGF blockade decreases lung VEGF and VEGF receptor 2 (VEGFR-2) expression in newborn rats and impairs alveolar development, leading to alveolar simplification and loss of lung capillaries, mimicking BPD. In hyperoxia-induced BPD in newborn rats, air space enlargement and loss of lung capillaries are associated with decreased lung VEGF and VEGFR-2 expression. Postnatal intratracheal adenovirus-mediated VEGF gene therapy improves survival, promotes lung capillary formation, and preserves alveolar development in this model of irreversible lung injury. Combined VEGF and angiopoietin-1 gene transfer matures the new vasculature, reducing the vascular leakage seen in VEGF-induced capillaries., Conclusions: These findings underscore the importance of the vasculature in what is traditionally thought of as an airway disease and open new therapeutic avenues for lung diseases characterized by irreversible loss of alveoli through the modulation of angiogenic growth factors.

Rationale: Bronchopulmonary dysplasia (BPD), the chronic lung disease of preterm infants, and pulmonary emphysema, both significant global health problems, are characterized by an arrest in alveolar growth/loss of alveoli structures. Mechanisms that inhibit distal lung growth are poorly understood, but recent studies suggest that impaired vascular endothelial growth factor signaling and reduced nitric oxide (NO) production decreases alveolar and vessel growth in the developing lung, features observed in experimental oxygen-induced BPD. NO exerts its biological activity by stimulating guanosine 3',5'-cyclic monophosphate (cGMP) production., Objectives: Because cGMP is inactivated by phosphodiesterase (PDE) enzymes, we hypothesized that the cGMP-specific PDE5 inhibitor sildenafil would promote angiogenesis and attenuate oxygen-induced lung injury in newborn rats. METHODS, MEASUREMENTS, AND MAIN RESULTS: In vitro, sildenafil (10(-4) M) increased endothelial capillary network formation of human pulmonary endothelial cells exposed to hyperoxia. In vivo, rat pups were randomly exposed from birth to normoxia, hyperoxia (95% O(2), BPD model), and hyperoxia+sildenafil (100 mg/kg/day subcutaneously). Rat pups exposed to hyperoxia showed fewer and enlarged air spaces as well as decreased capillary density, mimicking pathologic features seen in human BPD. These structural anomalies were associated with echographic (decreased pulmonary acceleration time) and structural (right ventricular hypertrophy and increased medial wall thickness) signs of pulmonary hypertension. Sildenafil preserved alveolar growth and lung angiogenesis, and decreased pulmonary vascular resistance, right ventricular hypertrophy and medial wall thickness., Conclusions: Our findings suggest a role for the NO/cGMP pathway during alveolar development. Sildenafil may have therapeutic potential in diseases associated with impaired alveolar structures.

The pulmonary arteries (PA) in pulmonary arterial hypertension (PAH) are constricted and remodeled;. They have suppressed apoptosis, partly attributable to suppression of the bone morphogenetic protein axis and selective downregulation of PA smooth muscle cell (PASMC) voltage-gated K+ channels, including Kv1.5. The Kv downregulation-induced increase in [K+]i, tonically inhibits caspases, further suppressing apoptosis. Mitochondria control apoptosis and produce activated oxygen species like H2O2, which regulate vascular tone by activating K+ channels, but their role in PAH is unknown. We show that dichloroacetate (DCA), a metabolic modulator that increases mitochondrial oxidative phosphorylation, prevents and reverses established monocrotaline-induced PAH (MCT-PAH), significantly improving mortality. Compared with MCT-PAH, DCA-treated rats (80 mg/kg per day in drinking water on day 14 after MCT, studied on day 21) have decreased pulmonary, but not systemic, vascular resistance (63% decrease, P<0.002), PA medial thickness (28% decrease, P<0.0001), and right ventricular hypertrophy (34% decrease, P<0.001). DCA is similarly effective when given at day 1 or day 21 after MCT (studied day 28) but has no effect on normal rats. DCA depolarizes MCT-PAH PASMC mitochondria and causes release of H2O2 and cytochrome c, inducing a 10-fold increase in apoptosis within the PA media (TUNEL and caspase 3 activity) and decreasing proliferation (proliferating-cell nuclear antigen and BrdU assays). Immunoblots, immunohistochemistry, laser-captured microdissection-quantitative reverse-transcription polymerase chain reaction and patch-clamping show that DCA reverses the Kv1.5 downregulation in resistance PAs. In summary, DCA reverses PA remodeling by increasing the mitochondria-dependent apoptosis/proliferation ratio and upregulating Kv1.5 in the media. We identify mitochondria-dependent apoptosis as a potential target for therapy and DCA as an effective and selective treatment for PAH.

Background: Oxygen (O2)-sensitive K+ channels mediate acute O2 sensing in many tissues. At birth, initial functional closure of the ductus arteriosus (DA) results from O2-induced vasoconstriction. This mechanism often fails in premature infants, resulting in persistent DA, a common form of congenital heart disease. We hypothesized that the basis for impaired O2 constriction in preterm DA is reduced expression and function of O2-sensitive, voltage-gated (Kv) channels., Methods and Results: Preterm rabbit DA rings have reduced O2 constriction (even after inhibition of prostaglandin and nitric oxide synthases), and preterm DA smooth muscle cells (DASMCs) display reduced O2-sensitive K+ current. This is associated with decreased mRNA and protein expression of certain O2-sensitive Kv channels (Kv1.5 and Kv2.1) but equivalent expression of the L-type calcium channel. Transmural Kv1.5 or Kv2.1 gene transfer "rescues" the developmental deficiency, conferring O2 responsiveness to preterm rabbit DAs. Targeted SMC Kv1.5 gene transfer also enhances O2 constriction in human DAs., Conclusions: These data demonstrate a central role for developmentally regulated DASMC O2-sensitive Kv channels in the functional closure of the DA. Modulation of Kv channels may have therapeutic potential in diseases associated with impaired O2 responsiveness, including persistent DA.

Female sexual function is under-studied, and mechanisms of clitoral engorgement-relaxation are incompletely understood. Penile erection results from nitric oxide (NO) -induced cyclic guanosine monophosphate (cGMP) accumulation. cGMP-dependent protein kinase (PKG) activates large-conductance, calcium-activated potassium channels (BK(Ca)), thereby hyperpolarizing and relaxing vascular and trabecular smooth muscle cells, allowing engorgement. We hypothesize rat clitorises relax by a similar mechanism. Rat clitorises express components of the proposed pathway: neuronal and endothelial NO synthases, soluble guanylyl cyclase (sGC), type 5 phosphodiesterase (PDE-5), and BK(Ca) channels. The NO donor diethylamine NONOate (DEANO), the PKG activator 8-pCPT-cGMP, and the PDE-5 inhibitor sildenafil, cause dose-dependent clitoral relaxation that is inhibited by antagonists of PKG (Rp-8-Br-cGMPS) or BK(Ca) channels (iberiotoxin). Electrical field stimulation induces tetrodotoxin-sensitive NO release and relaxation that is inhibited by the Na+ channel blocker tetrodotoxin or sGC inhibitor 1H-(1,2,4)oxadiozolo(4,3-a)quinoxalin-1-one. Human BK(Ca) channels, transferred to Chinese hamster ovary cells via an adenoviral vector, and endogenous rat clitoral smooth muscle K+ current are activated by this PKG-dependent mechanism. Laser confocal microscopy reveals protein expression of BK(Ca) channels on clitoral smooth muscle cells; these cells exhibit BK(Ca) channel activity that is activated by both DEANO and sildenafil. We conclude that neurovascular derived NO causes clitoral relaxation via a PKG-dependent activation of BK(Ca) channels. The BK(Ca) channel is an appealing target for drug therapy of female erectile dysfunction.

Hypoxic pulmonary vasoconstriction (HPV) is initiated by inhibition of O2-sensitive, voltage-gated (Kv) channels in pulmonary arterial smooth muscle cells (PASMCs). Kv inhibition depolarizes membrane potential (E(M)), thereby activating Ca2+ influx via voltage-gated Ca2+ channels. HPV is weak in extrapulmonary, conduit pulmonary arteries (PA) and strong in precapillary resistance arteries. We hypothesized that regional heterogeneity in HPV reflects a longitudinal gradient in the function/expression of PASMC O2-sensitive Kv channels. In adult male Sprague Dawley rats, constrictions to hypoxia, the Kv blocker 4-aminopyridine (4-AP), and correolide, a Kv1.x channel inhibitor, were endothelium-independent and greater in resistance versus conduit PAs. Moreover, HPV was dependent on Kv-inhibition, being completely inhibited by pretreatment with 4-AP. Kv1.2, 1.5, Kv2.1, Kv3.1b, Kv4.3, and Kv9.3. mRNA increased as arterial caliber decreased; however, only Kv1.5 protein expression was greater in resistance PAs. Resistance PASMCs had greater K+ current (I(K)) and a more hyperpolarized E(M) and were uniquely O2- and correolide-sensitive. The O2-sensitive current (active at -65 mV) was resistant to iberiotoxin, with minimal tityustoxin sensitivity. In resistance PASMCs, 4-AP and hypoxia inhibited I(K) 57% and 49%, respectively, versus 34% for correolide. Intracellular administration of anti-Kv1.5 antibodies inhibited correolide's effects. The hypoxia-sensitive, correolide-insensitive I(K) (15%) was conducted by Kv2.1. Anti-Kv1.5 and anti-Kv2.1 caused additive depolarization in resistance PASMCs (Kv1.5>Kv2.1) and inhibited hypoxic depolarization. Heterologously expressed human PASMC Kv1.5 generated an O2- and correolide-sensitive I(K) like that in resistance PASMCs. In conclusion, Kv1.5 and Kv2.1 account for virtually all the O2-sensitive current. HPV occurs in a Kv-enriched resistance zone because resistance PASMCs preferentially express O2-sensitive Kv-channels.

2'-Beta-methyl- and 2'-beta-hydroxymethyl-2'-deoxy-4'-thionucleosides have been synthesized through PhSeCl-mediated electrophilic glycosidation using 4-thiofuranoid glycals having carbon substituents at the C2-position as a glycosyl donor. Preparation of these glycals were carried out by means of the C2 lithiation of 1-chloro-4-thiofuranoid glycal with LTMP followed by the Birch reduction of the chlorine atom. [reaction: see text]

The ductus arteriosus (DA) is a fetal artery that allows blood ejected from the right ventricle to bypass the pulmonary circulation in utero. At birth, functional closure of the DA is initiated by an O2-induced, vasoconstrictor mechanism which, though modulated by endothelial-derived endothelin and prostaglandins, is intrinsic to the smooth muscle cell (DASMC) [Michelakis et al., Circ. Res. 91 (2002); pp. 478-486]. As pO2 increases, a mitochondrial O2-sensor (electron transport chain complexes I or III) is activated, which generates a diffusible redox mediator (H2O2). H2O2 inhibits voltage-gated K+ channels (Kv) in DASMC. The resulting membrane depolarization activates L-type Ca2+ channels, thereby promoting vasoconstriction. Conversely, inhibiting mitochondrial ETC complexes I or III mimics hypoxia, depolarizing mitochondria, and decreasing H2O2 levels. The resulting increase in K+ current hyperpolarizes the DASMC and relaxes the DA. We have developed two models for study of the DA's O2-sensor pathway, both characterized by decreased O2-constriction and Kv expression: (i) preterm rabbit DA, (ii) ionically-remodeled, human term DA. The O2-sensitive channels Kv1.5 and Kv2.1 are important to DA O2-constriction and overexpression of either channel enhances DA constriction in these models. Understanding this O2-sensing pathway offers therapeutic targets to modulate the tone and patency of human DA in vivo, thereby addressing a common form of congenital heart disease in preterm infants.

Background: Alveolar hypoxia acutely elicits pulmonary vasoconstriction (HPV). Chronic hypoxia (CH), despite attenuating HPV, causes pulmonary hypertension (CH-PHT). HPV results, in part, from inhibition of O2-sensitive, voltage-gated potassium channels (Kv) in pulmonary artery smooth muscle cells (PASMCs). CH decreases Kv channel current/expression and depolarizes and causes Ca2+ overload in PASMCs. We hypothesize that Kv gene transfer would normalize the pulmonary circulation (restore HPV and reduce CH-PHT), despite ongoing hypoxia., Methods and Results: Adult male Sprague-Dawley rats were exposed to normoxia or CH for 3 to 4 weeks and then nebulized orotracheally with saline or adenovirus (Ad5) carrying genes for the reporter, green fluorescent protein reporter+/-human Kv1.5 (cloned from normal PA). HPV was assessed in isolated lungs. Hemodynamics, including Fick and thermodilution cardiac output, were measured in vivo 3 and 14 days after gene therapy by use of micromanometer-tipped catheters. Transgene expression, measured by quantitative RT-PCR, was confined to the lung, persisted for 2 to 3 weeks, and did not alter endogenous Kv1.5 levels. Ad5-Kv1.5 caused no mortality or morbidity, except for sporadic, mild elevation of liver transaminases. Ad5-Kv1.5 restored the O2-sensitive K+ current of PASMCs, normalized HPV, and reduced pulmonary vascular resistance. Pulmonary vascular resistance decreased at day 2 because of increased cardiac output, and remained reduced at day 14, at which time there was concomitant regression of right ventricular hypertrophy and PA medial hypertrophy., Conclusions: Kv1.5 is an important O2-sensitive channel and potential therapeutic target in PHT. Kv1.5 gene therapy restores HPV and improves PHT. This is, to the best of our knowledge, the first example of K+ channel gene therapy for a vascular disease.

Stereoselective C-C bond formation at the anomeric position of uracil and adenine nucleoside has been accomplished through reaction of the anomeric radical, generated by 1,2-acyloxy migration, with a radical acceptor. The present method consists of the following steps: (1) electrophilic addition (bromo-pivaloyloxylation) to 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-protected 1',2'-unsaturated nucleoside, (2) tin radical-mediated reaction of the resulting adduct with a radical acceptor. The use of allyl(tributyl)tin gave the 1'-C-allylated uracil nucleoside 14 in 66% yield together with the unrearranged 2'-C-allylated product 15 (6%). Radical acceptors such as styryl(tributyl)tin and 3-bromo-2-methylacrylonitrile can also be used in the reaction of 5, giving 16 (70%) and 17 (76%) without the formation of unrearranged product. The radical-mediated C-C bond formation of the adenine counterpart 12 was also investigated.

Background: Left internal mammary arteries (LIMAs) synthesize endothelium-derived hyperpolarizing factor (EDHF), a short-lived K(+) channel activator that persists after inhibition of nitric oxide (NO) and prostaglandin synthesis. EDHF hyperpolarizes and relaxes smooth muscle cells (SMCs). The identity of EDHF in humans is unknown. We hypothesized that EDHF (1) is 11,12-epoxyeicosatrienoic acid (11,12-EET); (2) is generated by cytochrome P450-2C, CYP450-2C; and (3) causes relaxation by opening SMC large-conductance Ca(2+)-activated K(+) channels (BK(Ca))., Methods and Results: The identity of EDHF and its mechanism of action were assessed in 120 distal human LIMAs and 20 saphenous veins (SVs) obtained during CABG. The predominant EET synthesized by LIMAs is 11,12-EET. Relaxations to exogenous 11,12-EET and endogenous EDHF are of similar magnitudes. Inhibition of EET synthesis by chemically distinct CYP450 inhibitors (17-octadecynoic acid, N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide), or a selective EET antagonist (4,15-epoxyeicosa-5(Z)-enoic acid) impairs EDHF relaxation. 11,12-EET activates a BK(Ca) current and hyperpolarizes LIMA SMCs. Inhibitors of BK(Ca) but not inward-rectifier or small-conductance K(Ca) channels abolish relaxation to endogenous EDHF and exogenous 11,12-EET. BK(Ca) and CYP450-2C mRNA and proteins are more abundant in LIMAs than in SVs, perhaps explaining the lack of EDHF activity of the SV. Laser capture microdissection and quantitative RT-PCR demonstrate that BK(Ca) channels are primarily in vascular SMCs, whereas the CYP450-2C enzyme is present in both the endothelium and SMCs., Conclusions: In human LIMAs, EDHF is 11,12-EET produced by an EDHF synthase CYP450-2C and accounting for approximately 40% of net endothelial relaxation. 11,12-EET causes relaxation by activating SMC BK(Ca) channels.

The 4-thiofuranoid glycal 6 was converted into 1-chlorinated derivative 7 through LDA lithiation. When 7 was treated with LTMP, successful C-2 lithiation occurred, and subsequent treatment of the lithiated species with MeI gave 8. Dechlorination of 8 with Na/liq.NH3 yielded the 2-methyl glycal 9. Glycosidation of 9 with silylated thymine was mediated with PhSeCl as an electrophile, and gave the beta-isomer 10 stereoselectively. Removal of the 2'-phenylselenenyl group of 10 was effected with tributyltin radical, and subsequent deprotection furnished the target 2'-beta-methyl 4'-thiothymidine (11). In a similar manner, the corresponding cytosine analogue 12 could also be synthesized.

Fetal to maternal blood flow matching in the placenta, necessary for optimal fetal blood oxygenation, may occur via hypoxic fetoplacental vasoconstriction (HFPV). We hypothesized that HFPV is mediated by K(+) channel inhibition in fetoplacental vascular smooth muscle, as occurs in several other O(2)-sensitive tissues. With the use of an isolated human placental cotyledon perfused at a constant flow rate, we found that hypoxia reversibly increased perfusion pressure by >20%. HFPV was unaffected by cyclooxygenase or nitric oxide synthase inhibition. HFPV and 4-aminopyridine, an inhibitor of voltage-dependent K(+) (K(v)) channels, increased pressure in a nonadditive manner, suggesting they act via a common mechanism. Iberiotoxin, a large conductance Ca(2+)-sensitive K(+) (BK(Ca)) channel inhibitor, had little effect on normoxic pressure. Immunoblotting and RT-PCR showed expression of several putative O(2)-sensitive K(+) channels in peripheral fetoplacental vessels. In patch-clamp experiments with smooth muscle cells isolated from peripheral fetoplacental arteries, hypoxia reversibly inhibited K(v) but not BK(Ca) or ATP-dependent currents. We conclude that human fetoplacental vessels constrict in response to hypoxia. This response is largely mediated by hypoxic inhibition of K(v) channels in the smooth muscle of small fetoplacental arteries.

Functional closure of the human ductus arteriosus (DA) is initiated within minutes of birth by O2 constriction. It occurs by an incompletely understood mechanism that is intrinsic to the DA smooth muscle cell (DASMC). We hypothesized that O2 alters the function of an O2 sensor (the mitochondrial electron transport chain, ETC) thereby increasing production of a diffusible redox-mediator (H2O2), thus triggering an effector mechanism (inhibition of DASMC voltage-gated K+ channels, Kv). O2 constriction was evaluated in 26 human DAs (12 female, aged 9+/-2 days) studied in their normal hypoxic state or after normoxic tissue culture. In fresh, hypoxic DAs, 4-aminopyridine (4-AP), a Kv inhibitor, and O2 cause similar constriction and K+ current inhibition (I(K)). Tissue culture for 72 hours, particularly in normoxia, causes ionic remodeling, characterized by decreased O2 and 4-AP constriction in DA rings and reduced O2- and 4-AP-sensitive I(K) in DASMCs. Remodeled DAMSCs are depolarized and express less O2-sensitive channels (including Kv2.1, Kv1.5, Kv9.3, Kv4.3, and BK(Ca)). Kv2.1 adenoviral gene-transfer significantly reverses ionic remodeling, partially restoring both the electrophysiological and tone responses to 4-AP and O2. In fresh DASMCs, ETC inhibitors (rotenone and antimycin) mimic hypoxia, increasing I(K) and reversing constriction to O2, but not phenylephrine. O2 increases, whereas hypoxia and ETC inhibitors decrease H2O2 production by altering mitochondrial membrane potential (DeltaPsim). H2O2, like O2, inhibits I(K) and depolarizes DASMCs. We conclude that O2 controls human DA tone by modulating the function of the mitochondrial ETC thereby varying DeltaPsim and the production of H2O2, which regulates DASMC Kv channel activity and DA tone.

Oxygen constriction causes functional closure of the ductus arteriosus (DA) at birth. Although DA closure is crucial for postnatal adaptation, patency of the DA is critical for survival of newborns with duct-dependent cardiac malformations. In these cases, DA patency is achieved by i.v. infusion of prostaglandin E1, which, though effective, is often associated with complications. We hypothesized that sildenafil, a specific phosphodiesterase type 5 inhibitor, is an effective DA vasodilator. In isolated DA rings from term (d 30) fetal rabbits, sildenafil (10(-6)-10(-4) M) and diethylamine NONOate (10(-7)-10(-5) M) induced dose-dependent relaxation of oxygen-constricted DA (-52 +/- 4% and -51 +/- 6%, respectively) that was inhibited by the soluble guanylyl-cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (5 x 10(-5) M). Sildenafil increased cyclic GMP levels. Iberiotoxin (200 nM), an inhibitor of calcium-sensitive potassium channels, decreased the vasodilatory effect of sildenafil and diethylamine NONOate (-30 +/- 2% and -27 +/- 4%, respectively). Oxygen inhibition of whole-cell K+ current and membrane depolarization were partially restored by sildenafil, and this was inhibited by iberiotoxin. Immunohistochemistry and immunoblotting confirmed the presence of phosphodiesterase type 5 and calcium-sensitive potassium channels in the DA smooth muscle cells. This is the first study to demonstrate that sildenafil dilates the DA by increasing soluble guanylyl-cyclase-derived cGMP levels and thereby activating calcium-sensitive potassium channels, causing membrane hyperpolarization. Sildenafil, already approved for human usage, might be an alternative or a useful adjunct to prostaglandin E1 as a bridge to cardiac surgery.

Knots in polypeptide chains have been found in very few proteins. Only two proteins are considered to have a shallow 'trefoil' knot, which tucks a few residues at one end of the chain through a loop exposed on the protein surface. Recently, another protein was found by a mathematical algorithm to have a deep 'figure-of-eight' knot which had not been visually identified. In the present study, the crystal structure of a hypothetical RNA 2'-O-ribose methyltransferase from Thermus thermophilus (RrmA) was determined at 2.4 A resolution and a deep trefoil knot was found for the first time. The present knot is formed by the threading of a 44-residue polypeptide chain through a 41-residue loop and is better defined than the previously reported knots. Two of the three catalytic residues conserved in the 2'-O-ribose methyltransferase family are located in the knotting loop and in the knotted carboxy-terminal chain, which is the first observation that the enzyme active site is constructed right on the knot. On the other hand, the amino-terminal domain exhibits a geometrical similarity to the ribosomal proteins which recognize an internal loop of RNA.

Background: The prognosis of patients with severe pulmonary hypertension (PHT) is poor. To determine prognosis and guide therapy, an acute hemodynamic trial of selective pulmonary vasodilators, usually inhaled nitric oxide (iNO), was performed. We hypothesized that oral sildenafil, a phosphodiesterase-5 inhibitor, is a safe and effective alternative to iNO., Methods and Results: We studied 13 consecutive patients (mean+/-SEM, 44+/-2 years of age; 9 women) referred for consideration of heart-lung transplantation or as a guide to medical therapy. All but one were functional class III or IV. Patients had primary PHT (n=9), pulmonary arterial hypertension (n=2), or secondary PHT (n=2). Hemodynamics and serum cyclic guanosine-monophosphate levels (cGMP) were measured at baseline and at peak effects of iNO (80 ppm), sildenafil (75 mg), and their combination. The decrease in pulmonary vascular resistance was similar with iNO (-19+/-5%) and sildenafil (-27+/-3%), whereas sildenafil+iNO was more effective than iNO alone (-32+/-5%, P<0.003). Sildenafil and sildenafil+iNO increased cardiac index (17+/-5% and 17+/-4%, respectively), whereas iNO did not (-0.2+/-2.0%, P<0.003). iNO increased, whereas sildenafil tended to decrease, pulmonary capillary wedge pressure (+15+/-6 versus -9+/-7%, P<0.0007). Systemic arterial pressure was similar among groups and did not decrease with treatment. cGMP levels increased similarly with iNO and sildenafil, and their combination synergistically elevated cGMP (P<0.0001)., Conclusions: A single oral dose of sildenafil is as effective and selective a pulmonary vasodilator as iNO. Sildenafil may be superior to iNO in that it increases cardiac output and does not increase wedge pressure. Future studies are indicated to establish whether sildenafil could be effective over a longer duration.

We have established a system for hydrogen (H2) production from algal starch via lactic acid using a mixed culture of a lactic acid bacterium, Lactobacillus amylovorus, and a photosynthetic bacterium, Rhodobium marinum A-501. We found that the H2 production from lactate was stimulated in the presence of algal extract, which was obtained from algal biomass homogenate used as a substrate in the system by removing settleable solids including starch. To analyze the stimulating effect of algal extract on H2 production, we developed a kinetic model for H2 production by R. marinum A-501. The model revealed that approximately 20% of lactate was consumed for cell mass production, and the remaining portion was a source of reducing power to drive hydrogen production or other cellular processes. In the presence of algal extract, the model indicated that the conversion efficiency from lactate to the reducing power increased from 0.56 to 0.80 and nitrogenase activity increased up to twofold, resulting in the increase in yield of hydrogen from lactate from 29% to 48%. These results suggest that algal extract can attenuate the limitation process in lactate catabolism by which the supplementation of reducing power to drive H2 production was suppressed.