Dll4 Inhibition Promotes Graft Retention in Fat Grafting Enriched with Adipose-Derived Stem Cells.

Autologous fat grafting is among the safest and most effective treatments for soft-tissue restoration and augmentation, and many efforts have been made to improve its efficiency, including adipose-derived stem cell (ASC) supplementation. Here, we investigated the role of Notch ligand Delta-like ligand 4 (Dll4) in angiogenesis within grafted fat and its effect on graft retention, as well as the effect of Dll4 inhibition on ASC supplementation. Using a murine fat graft model, we investigated the expression of Dll4 in fat grafts and assessed the graft volume, vascularity, and perfusion within the graft, and ASC differentiation patterns depending on the blockade of Dll4. The underlying mechanism of Dll4 inhibition on ASC supplemented fat grafts was investigated using transcriptome analysis. Dll4 was highly expressed in vascular endothelial cells (ECs) within grafted fat, where Dll4-blocking antibody treatment-induced angiogenesis, promoting fat graft retention. In addition, its effect on fat graft retention was synergistically improved when ASCs were concomitantly supplemented. The expression of junctional proteins was increased in ECs, and inflammatory processes were downregulated in grafted fat upon ASC supplementation and Dll4 inhibition. Dll4 inhibition induced vascularization within the grafted fat, thereby promoting graft retention and exhibiting synergistic effects with concomitant ASC supplementation. This study serves as a basis for developing new potential therapeutic approaches targeting Dll4 to improve graft retention after cell-assisted transfer.

Biological Role of Vitamin K-With Particular Emphasis on Cardiovascular and Renal Aspects.

Vitamin K (VK) plays many important functions in the body. The most important of them include the contribution in calcium homeostasis and anticoagulation. Vascular calcification (VC) is one of the most important mechanisms of renal pathology. The most potent inhibitor of this process-matrix Gla protein (MGP) is VK-dependent. Chronic kidney disease (CKD) patients, both non-dialysed and hemodialysed, often have VK deficiency. Elevated uncarboxylated matrix Gla protein (ucMGP) levels indirectly reflected VK deficiency and are associated with a higher risk of cardiovascular events in these patients. It has been suggested that VK intake may reduce the VC and related cardiovascular risk. Vitamin K intake has been suggested to reduce VC and the associated cardiovascular risk. The role and possibility of VK supplementation as well as the impact of anticoagulation therapy on VK deficiency in CKD patients is discussed.

Del-1 enhances therapeutic efficacy of bacterial cancer immunotherapy by blocking recruitment of tumor-infiltrating neutrophils.

BACKGROUND: Bacterial-mediated cancer immunotherapy (BCI) elicits a more robust initial immune response than conventional immunotherapy, but does not prevent tumor recurrence and metastasis. BCI is associated with recruitment of tumor-infiltrating neutrophils, which could suppress the therapeutic efficacy of this modality. Development endothelial locus 1 (Del-1), a potent inhibitor of neutrophil recruitment, antagonizes lymphocyte function-associated antigen-1 on the vascular endothelium. Here, we aimed to determine the effect of Del-1-secreting S.t△ppGpp on anti-tumor activity and tumor-infiltrating neutrophil recruitment in a mouse model of colon cancer. METHODS: We investigated the anti-cancer activity of Del-1-secreting engineered Salmonella (△ppGpp S. Typhimurium) in the mice colon cancer models. RESULTS: In the present study, we identified that Del-1-secreting engineered Salmonella had more potent anti-cancer activity compared with normal S.t△ppGpp without Del-1 secretion. We postulated that Del-1 expression increased M1 macrophage recruitment to tumors by decreasing tumor-infiltrating neutrophils. This approach could enhance the anti-cancer effects of S.t△ppGpp. CONCLUSIONS: Collectively, the approach of using engineered bacteria that deliver Del-1 to block tumor-infiltrating neutrophil recruitment is a potential therapeutic approach.

Stromal cell-derived DEL-1 inhibits Tfh cell activation and inflammatory arthritis.

The secreted protein developmental endothelial locus 1 (DEL-1) regulates inflammatory cell recruitment and protects against inflammatory pathologies in animal models. Here, we investigated DEL-1 in inflammatory arthritis using collagen-induced arthritis (CIA) and collagen Ab-induced arthritis (CAIA) models. In both models, mice with endothelium-specific overexpression of DEL-1 were protected from arthritis relative to WT controls, whereas arthritis was exacerbated in DEL-1-deficient mice. Compared with WT controls, mice with collagen VI promoter-driven overexpression of DEL-1 in mesenchymal cells were protected against CIA but not CAIA, suggesting a role for DEL-1 in the induction of the arthritogenic Ab response. Indeed, DEL-1 was expressed in perivascular stromal cells of the lymph nodes and inhibited Tfh and germinal center B cell responses. Mechanistically, DEL-1 inhibited DC-dependent induction of Tfh cells by targeting the LFA-1 integrin on T cells. Overall, DEL-1 restrained arthritis through a dual mechanism, one acting locally in the joints and associated with the anti-recruitment function of endothelial cell-derived DEL-1; the other mechanism acting systemically in the lymph nodes and associated with the ability of stromal cell-derived DEL-1 to restrain Tfh responses. DEL-1 may therefore be a promising therapeutic for the treatment of inflammatory arthritis.

Excitation of Putative Glutamatergic Neurons in the Rat Parabrachial Nucleus Region Reduces Delta Power during Dexmedetomidine but not Ketamine Anesthesia.

BACKGROUND: Parabrachial nucleus excitation reduces cortical delta oscillation (0.5 to 4 Hz) power and recovery time associated with anesthetics that enhance γ-aminobutyric acid type A receptor action. The effects of parabrachial nucleus excitation on anesthetics with other molecular targets, such as dexmedetomidine and ketamine, remain unknown. The hypothesis was that parabrachial nucleus excitation would cause arousal during dexmedetomidine and ketamine anesthesia. METHODS: Designer Receptors Exclusively Activated by Designer Drugs were used to excite calcium/calmodulin-dependent protein kinase 2α-positive neurons in the parabrachial nucleus region of adult male rats without anesthesia (nine rats), with dexmedetomidine (low dose: 0.3 µg · kg-1 · min-1 for 45 min, eight rats; high dose: 4.5 µg · kg-1 · min-1 for 10 min, seven rats), or with ketamine (low dose: 2 mg · kg-1 · min-1 for 30 min, seven rats; high dose: 4 mg · kg-1 · min-1 for 15 min, eight rats). For control experiments (same rats and treatments), the Designer Receptors Exclusively Activated by Designer Drugs were not excited. The electroencephalogram and anesthesia recovery times were recorded and analyzed. RESULTS: Parabrachial nucleus excitation reduced delta power in the prefrontal electroencephalogram with low-dose dexmedetomidine for the 150-min analyzed period, excepting two brief periods (peak median bootstrapped difference [clozapine-N-oxide - saline] during dexmedetomidine infusion = -6.06 [99% CI = -12.36 to -1.48] dB, P = 0.007). However, parabrachial nucleus excitation was less effective at reducing delta power with high-dose dexmedetomidine and low- and high-dose ketamine (peak median bootstrapped differences during high-dose [dexmedetomidine, ketamine] infusions = [-1.93, -0.87] dB, 99% CI = [-4.16 to -0.56, -1.62 to -0.18] dB, P = [0.006, 0.019]; low-dose ketamine had no statistically significant decreases during the infusion). Recovery time differences with parabrachial nucleus excitation were not statistically significant for dexmedetomidine (median difference for [low, high] dose = [1.63, 11.01] min, 95% CI = [-20.06 to 14.14, -20.84 to 23.67] min, P = [0.945, 0.297]) nor low-dose ketamine (median difference = 12.82 [95% CI: -3.20 to 39.58] min, P = 0.109) but were significantly longer for high-dose ketamine (median difference = 11.38 [95% CI: 1.81 to 24.67] min, P = 0.016). CONCLUSIONS: These results suggest that the effectiveness of parabrachial nucleus excitation to change the neurophysiologic and behavioral effects of anesthesia depends on the anesthetic's molecular target.

Characterization of CBL-CIPK signaling complexes and their involvement in cold response in tea plant.

Calcineurin B-like (CBL) proteins, a class of Ca2+-binding proteins, play vital roles in calcium signal transduction by interacting specifically with CBL-interacting protein kinases (CIPKs), and these two gene families and their interacting complexes are involved in regulating plant responses to various environmental stimuli. In the present study, eight CBL and 25 CIPK genes were identified in tea plant and divided into four and five subfamilies, respectively. Analysis of the expression of these genes in response to abiotic stresses (mature leaves treated with cold, salinity, and PEG and young shoots treated with cold) revealed that CsCBL1/3/5 and CsCIPK1/4/5/6a/7/8/10b/10c/12/14a/19/23a/24 could be induced by at least two stresses. Under cold stress, CsCBL9 and CsCIPK4/6a/6b/7/11/14b/19/20 were upregulated in both mature leaves and young shoots, CsCBL1/3/5 and CsCIPK1/8/10a/10b/10c/12/14a/23a/24 were induced only in mature leaves, and CsCIPK5/25 were induced only in young shoots. Yeast two-hybrid analysis showed that CsCBL1 could interact with CsCIPK1/10b/12 but not with CsCIPK6a/7/11/14b/20. CsCBL9 was found to interact with CsCIPK1/10b/12/14b but not with CsCIPK6a/7/11/20. These results suggest divergent responses to cold stress regulated by CBL-CIPK complexes between tea plant and Arabidopsis, as well as between mature leaves and young shoots in tea plant. A model of Ca2+-CsCBL-CsCIPK module-mediated abiotic stress signaling in tea plant is proposed.

Dehydrocostus lactone inhibits NLRP3 inflammasome activation by blocking ASC oligomerization and prevents LPS-mediated inflammation in vivo.

Uncontrolled activation of NLRP3 inflammasome initiates a series of human inflammatory diseases. Targeting NLRP3 inflammasome has attracted considerable attention in developing potential therapeutic interventions. Here, we reported that dehydrocostus lactone (DCL), a main component of Saussurea lappa from the traditional Chinese medicine, inhibited NLRP3 inflammasome-mediated caspase-1 activation and subsequent interleukin (IL)-1ß production in primary mouse macrophages and human peripheral blood mononuclear cells and exerted an inhibitory effect on NLRP3-driven inflammation. Mechanistically, DCL significantly blocked the ASC oligomerization, which is essential for the assembly of activated inflammasome. Importantly, in vivo experiments showed that DCL reduced IL-1ß secretion and peritoneal neutrophils recruitment in LPS-mediated inflammation mouse model, which is demonstrated to be NLRP3 dependent. These results suggest that DCL is a potent pharmacological inhibitor of NLRP3 inflammasome and may be developed as a therapeutic drug for treating NLRP3-associated diseases.

Genetic analyses uncover pleiotropic compensatory roles for Drosophila Nucleobindin-1 in inositol trisphosphate-mediated intracellular calcium homeostasis.

Nucleobindin-1 is an EF-hand calcium-binding protein with a distinctive profile, predominantly localized to the Golgi in insect and wide-ranging vertebrate cell types, alike. Its putative involvements in intracellular calcium (Ca2+) homeostasis have never been phenotypically characterized in any model organism. We have analyzed an adult-viable mutant that completely disrupts the G protein α-subunit binding and activating (GBA) motif of Drosophila Nucleobindin-1 (dmNUCB1). Such disruption does not manifest any obvious fitness-related, morphological/developmental, or behavioral abnormalities. A single copy of this mutation or the knockdown of dmnucb1 in restricted sets of cells variously rescues pleiotropic mutant phenotypes arising from impaired inositol 1,4,5-trisphosphate receptor (IP3R) activity (in turn depleting cytoplasmic Ca2+ levels across diverse tissue types). Additionally, altered dmNUCB1 expression or function considerably reverses lifespan and mobility improvements effected by IP3R mutants, in a Drosophila model of amyotrophic lateral sclerosis. Homology modeling-based analyses further predict a high degree of conformational conservation in Drosophila, of biochemically validated structural determinants in the GBA motif that specify in vertebrates, the unconventional Ca2+-regulated interaction of NUCB1 with Gαi subunits. The broad implications of our findings are hypothetically discussed, regarding potential roles for NUCB1 in GBA-mediated, Golgi-associated Ca2+ signaling, in health and disease.

[Nutrient Sensing and Anorexia via Neuropeptides].

Various neuropeptides play an essential role in the nutrient sensing mechanism and related homeostasis. Nesfatin-1 is a newly identified neuropeptide having anorectic activity, and nesfatin-1-containing neurons are widely distributed in the brain, including the hypothalamus and brain stem. Our previous study showed that dehydration-induced anorectic effects are mediated via the central nesfatin-1 pathway in rats. Our recent studies have also shown that peripheral anorectic peptides (cholecystokinin-8, glucagon-like peptide-1, and leptin) and an antineoplastic agent (cisplatin) caused inhibition of feeding via the central nesfatin-1 pathway in rats. Nesfatin-1-containing neurons in the central nervous system, in particular the hypothalamus and the brain stem, may mediate peripheral nutrient signals and regulate feeding behavior.

SMOC2 inhibits calcification of osteoprogenitor and endothelial cells.

Tissue calcification is an important physiological process required for the normal structure and function of bone. However, ectopic or excessive calcification contributes to diseases such as chondrocalcinosis, to calcium deposits in the skin or to vascular calcification. SMOC2 is a member of the BM-40/osteonectin family of calcium-binding secreted matricellular proteins. Using osteoprogenitor MC3T3-E1 cells stably overexpressing SMOC2, we show that SMOC2 inhibits osteogenic differentiation and extracellular matrix mineralization. Stable Smoc2 knockdown in these cells had no effect on mineralization suggesting that endogenous SMOC2 is not essential for the mineralization process. Mineralization in MC3T3-E1 cells overexpressing mutant SMOC2 lacking the extracellular calcium-binding domain was significantly increased compared to cells overexpressing full length SMOC2. When SMOC2 overexpressing cells were cultured in the presence of extracellular calcium supplementation, SMOC2's inhibitory effect on calcification was rescued. Our observations were translationally validated in primary human periosteal-derived cells. Furthermore, SMOC2 was able to impair mineralization in transdifferentiated human umbilical vein endothelial cells. Taken together, our data indicate that SMOC2 can act as an inhibitor of mineralization. We propose a possible role for SMOC2 to prevent calcification disorders.

Role of Nesfatin-1 in the Reproductive Axis of Male Rat.

Nesfatin-1 is an important molecule in the regulation of reproduction. However, its role in the reproductive axis in male animals remains to be understood. Here, we found that nesfatin-1 was mainly distributed in the arcuate nucleus (ARC), paraventricular nucleus (PVN), periventricular nucleus (PeN), and lateral hypothalamic area (LHA) of the hypothalamus; adenohypophysis and Leydig cells in male rats. Moreover, the concentrations of serum nesfatin-1 and its mRNA in hypothalamo-pituitary-gonadal axis (HPGA) vary with the age of the male rat. After intracerebroventricular injection of nesfatin-1, the hypothalamic genes for gonadotrophin releasing hormone (GnRH), kisspeptin (Kiss-1), pituitary genes for follicle-stimulate hormone ß(FSHß), luteinizing hormone ß(LHß), and genes for testicular steroidogenic acute regulatory (StAR) expression levels were decreased significantly. Nesfatin-1 significantly increased the expression of genes for 3ß-hydroxysteroid dehydrogenase (3ß-HSD), 17ß-hydroxysteroid dehydrogenase (17ß-HSD), and cytochrome P450 cleavage (P450scc) in the testis of pubertal rats, but their levels decreased in adult rats (P < 0.05), along with the serum FSH, LH, and testosterone (T) concentrations. After nesfatin-1 addition in vitro, T concentrations of the supernatant were significantly higher than that in the control group. These results were suggestive of the role of nesfatin-1 in the regulation of the reproductive axis in male rats.

Sex-Specific Protection of Osteoarthritis by Deleting Cartilage Acid Protein 1.

Cartilage acidic protein 1 (CRTAC1) was recently identified as an elevated protein in the synovial fluid of patients with osteoarthritis (OA) by a proteomic analysis. This gene is also upregulated in both human and mouse OA by transcriptomic analysis. The objective of this study was to characterize the expression and function of CRTAC1 in OA. Here, we first confirm the increase of CRTAC1 in cartilage biopsies from OA patients undergoing joint replacement by real-time PCR and immunohistochemistry. Furthermore, we report that proinflammatory cytokines interleukin-1beta and tumor necrosis factor alpha upregulate CRTAC1 expression in primary human articular chondrocytes and synovial fibroblasts. Genetic deletion of Crtac1 in mice significantly inhibited cartilage degradation, osteophyte formation and gait abnormalities of post-traumatic OA in female, but not male, animals undergoing the destabilization of medial meniscus (DMM) surgery. Taken together, CRTAC1 is upregulated in the osteoarthritic joint and directly induced in chondrocytes and synovial fibroblasts by pro-inflammatory cytokines. This molecule is necessary for the progression of OA in female mice after DMM surgery and thus represents a potential therapy for this prevalent disease, especially for women who demonstrate higher rates and more severe OA.

Peripheral injection of bombesin induces c-Fos in NUCB2/nesfatin-1 neurons.

As anorexigenic hormones bombesin and nucleobindin2 (NUCB2)/nesfatin-1 decrease food intake in rodents. Both hormones have been described in brain nuclei that play a role in the modulation of hunger and satiety, like the paraventricular nucleus of the hypothalamus (PVN) and the nucleus of the solitary tract (NTS). However, the direct interaction of the two hormones is unknown so far. The aim of study was to elucidate whether bombesin directly interacts with NUCB2/nesfatin-1 neurons in the PVN and NTS. Therefore, we injected bombesin intraperitoneally (ip) at two doses (26 and 32nmol/kg body weight) and assessed c-Fos activation in the PVN, arcuate nucleus (ARC) and NTS compared to vehicle treated rats (0.15M NaCl). We also performed co-localization studies with oxytocin or tyrosine hydroxylase. Bombesin at both doses increased the number of c-Fos positive neurons in the PVN (p<0.05) and NTS (p<0.05) compared to vehicle, while in the ARC no modulation was observed (p>0.05). In the PVN and NTS the number of c-Fos positive neurons colocalized with NUCB2/nesfatin-1 increased after bombesin injection compared to vehicle treatment (p<0.05). Moreover, an increase of activated NUCB2/nesfatin-1 immunoreactive neurons that co-expressed oxytocin in the PVN (p<0.05) or tyrosine hydroxylase in the NTS (p<0.05) was observed compared to vehicle. Our results show that peripherally injected bombesin activates NUCB2/nesfatin-1 neurons in the PVN and NTS giving rise to a possible interaction between bombesin and NUCB2/nesfatin-1 in the modulation of food intake.

The interaction between Staphylococcus aureus SdrD and desmoglein 1 is important for adhesion to host cells.

Staphylococcus aureus is known as a frequent colonizer of the skin and mucosa. Among bacterial factors involved in colonization are adhesins such as the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs). Serine aspartate repeat containing protein D (SdrD) is involved in adhesion to human squamous cells isolated from the nose. Here, we identify Desmoglein 1 (Dsg1) as a novel interaction partner for SdrD. Genetic deletion of sdrD in S. aureus NCTC8325-4 through allelic replacement resulted in decreased bacterial adherence to Dsg1- expressing HaCaT cells in vitro. Complementary gain-of-function was demonstrated by heterologous expression of SdrD in Lactococcus lactis, which increased adherence to HaCaT cells. Also ectopic expression of Dsg1 in HEK293 cells resulted in increased adherence of S. aureus NCTC8325-4 in vitro. Increased adherence of NCTC8325-4, compared to NCTC8325-4ΔsdrD, to the recombinant immobilized Dsg1 demonstrated direct interaction between SdrD and Dsg1. Specificity of SdrD interaction with Dsg1 was further verified using flow cytometry and confirmed binding of recombinant SdrD to HaCaT cells expressing Dsg1 on their surface. These data demonstrate that Dsg1 is a host ligand for SdrD.

Vitamin K: from coagulation to calcification.

Vitamin K is not only essential for the synthesis of coagulation factors in the liver, but it also strengthens the bones and prevents calcification of the arteries. These effects are mediated through the same mechanism, i.e. carboxylation of Gla target proteins. The discovery of novel Gla proteins that are not associated with blood coagulation or calcium metabolism indicates that vitamin K has additional effects in the pancreas and the central nervous system, for example. As dietary supplements, vitamin K1 of plant origin and vitamins K2 of bacterial origin may exert different effects.

Nimodipine-mediated re-myelination after facial nerve crush injury in rats.

This study aimed to investigate the mechanism of nimodipine-mediated neural repair after facial nerve crush injury in rats. Adult Sprague-Dawley rats were divided into three groups: healthy controls, surgery alone, and surgery plus nimodipine. A facial nerve crush injury model was constructed. Immediately after surgery, the rats in the surgery plus nimodipine group were administered nimodipine, 6 mg/kg/day, for a variable numbers of days. The animals underwent electromyography (EMG) before surgery and at 3, 10, or 20 days after surgery. After sacrifice, nerve samples were stained with hematoxylin and eosin (H&E) and luxol fast blue. The EMG at 20 days revealed an apparent recovery of eletroconductivity, with the surgery plus nimodipine group having a higher amplitude and shorter latency time than the surgery only group. H&E staining showed that at 20 days, the rats treated with nimodipine had an obvious recovery of myelination and reduction in the number of infiltrating cells, suggesting less inflammation, compared with the rats in the surgery only group. Luxol fast blue staining was relatively even in the surgery plus nimodipine group, indicating a protective effect against injury-induced demyelination. Staining for S100 calcium-binding protein B (S-100ß) was not evident in the surgery alone group, but was evident in the surgery plus nimodipine group, indicating that nimodipine reversed the damage of the crush injury. After a facial nerve crush injury, treatment with nimodipine for 20 days reduced the nerve injury by mediating remyelination by Schwann cells. The protective effect of nimodipine may include a reduction of inflammation and an increase in calcium-binding S-100ß protein.

[Cardiovascular calcification inhibitors]. / Inhibiteurs des calcifications cardiovasculaires.

Vascular calcification is a marker of cardiovascular risk increase. Age and specific disease such as diabetes or chronic kidney disease are important factors for calcification genesis. Vascular calcification process is a complex phenomenon, involving several activators and inhibitors factors. Indeed, recent works related to in vitro and in vivo experimental studies have led to a better understanding of calcification process and identification of molecules able to modulate this system. This revue will summarize some of these molecules with a particular interest of those with therapeutic relevance. We will present: i) calcium sensing receptor and its modulation by cinacalcet; ii) pyrophosphate supplementation; iii) fetuin A and overall propensity serum test for calcification and finally; iv) matrix-Gla-protein and the use of vitamin K to prevent vascular calcification progression.

Arabidopsis thaliana†CML25 mediates the Ca(2+) regulation of K(+) transmembrane trafficking during pollen germination and tube elongation.

The concentration alteration of cytosolic-free calcium ([Ca(2+) ]cyt ) is a well-known secondary messenger in plants and plays important roles during pollen grain germination and tube elongation. Here we demonstrate that CML25, a member of calmodulin-like proteins, has Ca(2+) -binding activity and plays a role in pollen grain germination, tube elongation and seed setting. CML25 transcript was abundant in mature pollen grains and pollen tubes, and its product CML25 protein was primarily directed to the cytoplasm. Two independent CML25 loss-of-function T-DNA insertion mutants suffered a major reduction in both the rate of pollen germination and the elongation of the pollen tube. Also, pollen grains of cml25 mutants were less sensitive to the external K(+) and Ca(2+) concentration than wild-type pollen. The disruption of CML25 increased the [Ca(2+) ]cyt in both the pollen grain and the pollen tube, which in turn impaired the Ca(2+) -dependent inhibition of whole-cell inward K(+) currents in protoplasts prepared from these materials (pollen grain and pollen tube). Complementation of cml25-1 mutant resulted in the recovery of wild-type phenotype. Our findings indicate that CML25 is an important transducer in the Ca(2+) -mediated regulation of K(+) influx during pollen germination and tube elongation.

A novel tescalcin-sodium/hydrogen exchange axis underlying sorafenib resistance in FLT3-ITD+ AML.

Internal tandem duplication (ITD) of fms-like tyrosine kinase 3 (FLT3) in acute myeloid leukemia (AML) is associated with inferior clinical prognosis. Sorafenib is effective in clearing leukemic blasts in chemorefractory FLT3-ITD(+) AML, but leukemia progression invariably occurs. Mechanisms of drug resistance are not completely understood. We hypothesized that a gene encoding tescalcin (TESC), known to be upregulated at leukemia progression during continuous sorafenib treatment and activate an Na(+)/H(+) exchanger type-1 (NHE1), may underlie tyrosine kinase inhibitor resistance. TESC was highly expressed in FLT3-ITD(+) AML lines MOLM-13 and MV4-11, and its knockdown by small-interfering RNA lowered intracellular pH (pHi) and induced apoptosis. The results were recapitulated by treatment with an NHE1 inhibitor, 5-(N,N-hexamethylene) amiloride (HMA). Induction of sorafenib resistance in the MOLM-13 cell line (M13-RE) significantly increased its sensitivity to HMA. The later also enhanced suppression of FLT3 signaling by sorafenib in otherwise resistant cell lines. HMA treatment of MOLM-13 and MV4-11 as well as primary FLT3-ITD(+) AML cells significantly reduced leukemia initiation in anti-CD122-primed NOD/SCID mouse xenotransplantation. These observations provided novel information about the pathogenetic role of a TESC-NHE1-pHi axis in mediating sorafenib resistance in AML.

Vitamin K1 to slow vascular calcification in haemodialysis patients (VitaVasK trial): a rationale and study protocol.

BACKGROUND: Patients on haemodialysis (HD) exhibit increased cardiovascular mortality associated with accelerated vascular calcification (VC). VC is influenced by inhibitors such as matrix Gla protein (MGP), a protein activated in the presence of vitamin K. HD patients exhibit marked vitamin K deficiency, and supplementation with vitamin K reduces inactive MGP levels in these patients. The VitaVasK trial analyses whether vitamin K1 supplementation affects the progression of coronary and aortic calcification in HD patients. METHODS: VitaVasK is a prospective, randomized, parallel group, multicentre trial (EudraCT No.: 2010-021264-14) that will include 348 HD patients in an open-label, two-arm design. After baseline multi-slice computed tomography (MSCT) of the heart and thoracic aorta, patients with a coronary calcification volume score of at least 100 will be randomized to continue on standard care or to receive additional supplementation with 5 mg vitamin K1 orally thrice weekly. Treatment duration will be 18 months, and MSCT scans will be repeated after 12 and 18 months. Primary end points are the progression of thoracic aortic and coronary artery calcification (calculated as absolute changes in the volume scores at the 18-month MSCT versus the baseline MSCT). Secondary end points comprise changes in Agatston score, mitral and aortic valve calcification as well as major adverse cardiovascular events (MACE) and all-cause mortality. VitaVask also aims to record MACE and all-cause mortality in the follow-up period at 3 and 5 years after treatment initiation. This trial may lead to the identification of an inexpensive and safe treatment or prophylaxis of VC in HD patients.