Forced expression of mouse progerin attenuates the osteoblast differentiation interrupting ß-catenin signal pathway in vitro.

Nuclear protein, lamin A, which is a component of inner membrane on nucleoplasm, plays a role in nuclear formation and cell differentiation. The expression of mutated lamin A, termed progerin, causes a rare genetic aging disorder, Hutchinson-Gilford progeria syndrome, which shows abnormal bone formation with the decrease in a number of osteoblasts and osteocytes. However, exact molecular mechanism how progerin exerts depressive effects on osteogenesis has not been fully understood. Here, we created mouse lamin A dC50 cDNA encoding progerin that lacks 50 amino acid residues at C-terminus, transfected it in mouse preosteoblast-like MC3T3-E1 cells, and examined the changes in osteoblast phenotype. When lamin A dC50-expressed cells were cultured with differentiation-inductive medium, alkaline phosphatase (ALP) activity and mRNA levels of major osteoblast markers, type I collagen (Col1), bone sialoprotein (BSP), dentine matrix protein 1 (DMP1), and Runx2 were significantly decreased, and no mineralized nodules were detected as seen in control cells expressing empty vector. In the culture with mineralization-inductive medium, mRNA levels of BSP, osteocalcin, DMP1, Runx2, and osterix were strongly decreased parallel with loss of mineralization in lamin A dC50-expressed cells, while mineralized nodules appear at 21 days in control cells. Furthermore, lamin A dC50 expression was depressed nuclear localization of ß-catenin with the decrease of GSK-3ß phosphorylation level. These results suggest that lamin A dC50 depresses osteoblast differentiation in both early and late stages, and it negatively regulates ß-catenin activity interacting with GSK-3ß in cytoplasm.

MP4CO, a pegylated hemoglobin saturated with carbon monoxide, is a modulator of HO-1, inflammation, and vaso-occlusion in transgenic sickle mice.

Transgenic sickle mice expressing ß(S) hemoglobin have activated vascular endothelium in multiple organs that exhibits enhanced expression of NF-ĸB and adhesion molecules and promotes microvascular stasis in sickle, but not normal, mice in response to hypoxia/reoxygenation (H/R), or heme. Induction of heme oxygenase-1 (HO-1) or administration of its products, carbon monoxide (CO) or biliverdin, inhibits microvascular stasis in sickle mice. Infusion of human hemoglobin conjugated with polyethylene glycol and saturated with CO (MP4CO) markedly induced hepatic HO-1 activity and inhibited NF-ĸB activation and H/R-induced microvascular stasis in sickle mice. These effects were mediated by CO; saline or MP4 saturated with O2 (MP4OX) had little to no effect on H/R-induced stasis, though unmodified oxyhemoglobin exacerbated stasis. The HO-1 inhibitor, tin protoporphyrin, blocked MP4CO protection, consistent with HO-1 involvement in the protection afforded by MP4CO. MP4CO also induced nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), an important transcriptional regulator of HO-1 and other antioxidant genes. In a heterozygous (hemoglobin-AS) sickle mouse model, intravenous hemin induced cardiovascular collapse and mortality within 120 minutes, which was significantly reduced by MP4CO, but not MP4OX. These data demonstrate that MP4CO induces cytoprotective Nrf2 and HO-1 and decreases NF-ĸB activation, microvascular stasis, and mortality in transgenic sickle mouse models.

Impact of acellular hemoglobin-based oxygen carriers on brain apoptosis in rats.

BACKGROUND: Extracellular hemoglobin (Hb)-based oxygen carriers (HBOCs) are under extensive consideration as oxygen therapeutics. Their effects on cellular mechanisms related to apoptosis are of particular interest, because the onset of proapoptotic pathways may give rise to tissue damage. STUDY DESIGN AND METHODS: The objective was to assess whether the properties of the Hb that replaces blood during an isovolemic hemodilution would modulate apoptotic-response mechanisms in rat brain and whether such signaling favors cytoprotection or damage. We exposed rats to exchange transfusion (ET; 50% blood volume and isovolemic replacement with Hextend [negative colloid control], MP4OX [PEGylated HBOC with high oxygen affinity], and ααHb [αα-cross-linked HBOC with low oxygen affinity; n=4-6/group]). Sham rats acted as control. Animals were euthanized at 2, 6, and 12 hours after ET; brain tissue was harvested and processed for analysis. RESULTS: In MP4OX animals, the number of neurons that overexpressed the hypoxia-inducible factor (HIF)-1α was higher than in ααHb, particularly at the early time points. In addition, MP4OX was associated with greater phosphorylation of protein kinase B (Akt), a well-known cytoprotective factor. Indeed, the degree of apoptosis, measured as terminal deoxynucleotidyl transferase-positive neurons and caspase-3 cleavage, ranked in order of MP4OX < Hextend < ααHb. CONCLUSION: Even though both HBOCs showed increased levels of HIF-1α compared to shams or Hextend-treated animals, differences in signaling events resulted in very different outcomes for the two HBOCs. ααHb-treated brain tissue showed significant neuronal damage, measured as apoptosis. This was in stark contrast to the protection seen with MP4OX, apparently due to recruitment of Akt and neuronal specific HIF-1α pathways.

Prostate-apoptosis response-4 phosphorylation in vascular smooth muscle.

The protein prostate-apoptosis response (Par)-4 has been implicated in the regulation of smooth muscle contraction, based largely on studies with the A7r5 cell line. A mechanism has been proposed whereby Par-4 binding to MYPT1 (the myosin-targeting subunit of myosin light chain phosphatase, MLCP) blocks access of zipper-interacting protein kinase (ZIPK) to Thr697 and Thr855 of MYPT1, whose phosphorylation is associated with MLCP inhibition. Phosphorylation of Par-4 at Thr155 disrupts its interaction with MYPT1, exposing the sites of phosphorylation in MYPT1 and leading to MLCP inhibition and contraction. We tested this "padlock" hypothesis in a well-characterized vascular smooth muscle system, the rat caudal artery. Par-4 was retained in Triton-skinned tissue, suggesting a tight association with the contractile machinery, and indeed Par-4 co-immunoprecipitated with MYPT1. Treatment of Triton-skinned tissue with the phosphatase inhibitor microcystin (MC) evoked phosphorylation of Par-4 at Thr155, but did not induce its dissociation from the contractile machinery. Furthermore, analysis of the time courses of MC-induced phosphorylation of MYPT1 and Par-4 revealed that MYPT1 phosphorylation at Thr697 or Thr855 preceded Par-4 phosphorylation. Par-4 phosphorylation was inhibited by the non-selective kinase inhibitor staurosporine, but not by inhibitors of ZIPK, Rho-associated kinase or protein kinase C. In addition, Par-4 phosphorylation did not occur upon addition of constitutively-active ZIPK to skinned tissue. We conclude that phosphorylation of Par-4 does not regulate contraction of this vascular smooth muscle tissue by inducing dissociation of Par-4 from MYPT1 to allow phosphorylation of MYPT1 and inhibition of MLCP.

Stimulation of bone formation in the expanding premaxillary suture with a GSK-3ß inhibitor.

OBJECTIVES: Glycogen synthase kinase-3ß (GSK-3ß)/ß-catenin signaling mediates osteogenesis in response to mechanical loading. We tested the hypothesis that local administration of a GSK-3ß inhibitor could stimulate new bone formation in the expanding premaxillary suture. MATERIALS AND METHODS: Thirty-five Sprague-Dawley rats were subjected to premaxillary suture expansion using a helix spring. The experimental rats were given one or two local injections of SB-415286, a small-molecule GSK-3ß inhibitor. Animals were administered calcein and sacrificed on day 7 to quantify new bone formation. To evaluate the proliferation and differentiation of osteoblasts, rats were labeled with bromodeoxyuridine on day 1 and sacrificed on day 2 or 4. ß-catenin expression was evaluated by immunohistochemical staining. RESULTS: Two injections of SB-415286 led to an elevation of ß-catenin expression and an increase in the number of proliferating osteoblasts in expanding sutures on day 2 and day 4. Consequently, new bone formation in the suture increased significantly on day 7. CONCLUSIONS: These results suggest that local delivery of a GSK-3ß inhibitor could stimulate bone formation in the expanding premaxillary suture by eliciting ß-catenin signaling. GSK-3ß could be a pharmaceutical target for improving the effect of orthodontic treatments such as rapid palatal expansion.

Inhibition of GSK3 abolishes bacterial-induced periodontal bone loss in mice.

The tissue destruction that characterizes periodontitis is driven by the host response to bacterial pathogens. Inhibition of glycogen synthase kinase 3ß (GSK3ß) in innate cells leads to suppression of Toll-like receptor (TLR)-initiated proinflammatory cytokines under nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 transcriptional control and promotion of cyclic adenosine monophosphate response element-binding (CREB)-dependent gene activation. Therefore, we hypothesized that the cell permeable GSK3-specific inhibitor, SB216763, would protect against alveolar bone loss induced by the key periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), in a murine model. B6129SF2/J mice either were infected orally with P. gingivalis ATCC 33277; or treated with SB216763 and infected with P. gingivalis; sham infected; or exposed to vehicle only (dimethyl sulfoxide [DMSO]); or to GSK3 inhibitor only (SB216763). Alveolar bone loss and local (neutrophil infiltration and interleukin [IL]-17) and systemic (tumor necrosis factor [TNF], IL-6, Il-1ß and IL-12/IL-23 p40) inflammatory indices also were monitored. SB216763 unequivocally abrogated mean P. gingivalis-induced bone resorption, measured at 14 predetermined points on the molars of defleshed maxillae as the distance from the cementoenamel junction to the alveolar bone crest (p < 0.05). The systemic cytokine response, the local neutrophil infiltration and the IL-17 expression were suppressed (p < 0.001). These data confirm the relevance of prior in vitro phenomena and establish GSK3 as a novel, efficacious therapeutic preventing periodontal disease progression in a susceptible host. These findings also may have relevance to other chronic inflammatory diseases and the systemic sequelae associated with periodontal infections.

Toll-like receptor 3 activation promotes desensitization of histamine response in human gingival fibroblasts: Poly (I:C) induces histamine receptor desensitization in human gingival fibroblasts.

Viruses are associated with the development of periodontal disease, particularly during periods of suppressed cellular immunity. For this reason, we evaluated the hypothesis that viral components regulate the actions of histamine, an important mediator of immune responses. We assessed the effect of Poly (I:C) on histamine-mediated intracellular calcium mobilization in human gingival fibroblasts. Our results show that histamine induces an increase in intracellular calcium concentrations in a dose-dependent manner. This response was blocked when cells were incubated in the presence of Poly (I:C). In addition, phorbol esters, a diacylglycerol analog, mimics the inhibitory actions of Poly (I:C) in response to histamine. The effect of Poly (I:C) was reversed by Stuarosporine (1 µM), GÖ6983 (7 µM), Bisindolylmaleimide (1 µM) [a protein inhibitor (PKC)], and SB 203580 (3 µM) (a p38-MAPK inhibitor). These findings suggest that Poly (I:C) regulates histamine-induced calcium mobilization through activation of PKC and p38.

Exploiting maleimide-functionalized hyaluronan hydrogels to test cellular responses to physical and biochemical stimuli.

Currentin vitrothree-dimensional (3D) models of liver tissue have been limited by the inability to study the effects of specific extracellular matrix (ECM) components on cell phenotypes. This is in part due to limitations in the availability of chemical modifications appropriate for this purpose. For example, hyaluronic acid (HA), which is a natural ECM component within the liver, lacks key ECM motifs (e.g. arginine-glycine-aspartic acid (RGD) peptides) that support cell adhesion. However, the addition of maleimide (Mal) groups to HA could facilitate the conjugation of ECM biomimetic peptides with thiol-containing end groups. In this study, we characterized a new crosslinkable hydrogel (i.e. HA-Mal) that yielded a simplified ECM-mimicking microenvironment supportive of 3D liver cell culture. We then performed a series of experiments to assess the impact of physical and biochemical signaling in the form of RGD peptide incorporation and transforming growth factorß(TGF-ß) supplementation, respectively, on hepatic functionality. Hepatic stellate cells (i.e. LX-2) exhibited increased cell-matrix interactions in the form of cell spreading and elongation within HA-Mal matrices containing RGD peptides, enabling physical adhesions, whereas hepatocyte-like cells (HepG2) had reduced albumin and urea production. We further exposed the encapsulated cells to soluble TGF-ßto elicit a fibrosis-like state. In the presence of TGF-ßbiochemical signals, LX-2 cells became activated and HepG2 functionality significantly decreased in both RGD-containing and RGD-free hydrogels. Altogether, in this study we have developed a hydrogel biomaterial platform that allows for discrete manipulation of specific ECM motifs within the hydrogel to better understand the roles of cell-matrix interactions on cell phenotype and overall liver functionality.

Rho-kinase inhibition attenuates calcium-induced contraction in ß-escin but not Triton X-100 permeabilized rabbit femoral artery.

K+-depolarization (KCl) of smooth muscle has long been known to cause Ca2+-dependent contraction, but only recently has this G protein-coupled receptor (GPCR)-independent stimulus been associated with rhoA kinase (ROCK)-dependent myosin light chain (MLC) phosphatase inhibition and Ca2+ sensitization. This study examined effects of ROCK inhibition on the concentration-response curves (CRCs) generated in femoral artery by incrementally adding increasing concentrations of KCl to intact tissues, and Ca2+ to tissues permeabilized with Triton X-100, ß-escin and α-toxin. For a comparison, tissue responses were assessed also in the presence of protein kinase C (PKC) and MLC kinase inhibition. The ROCK inhibitor H-1152 induced a strong concentration-dependent inhibition of a KCl CRC. A relatively low GF-109203X concentration (1 µM) sufficient to inhibit conventional PKC isotypes also inhibited the KCl CRC but did not affect the maximum tension. ROCK inhibitors had no effect on the Ca2+ CRC induced in Triton X-100 or α-toxin permeabilized tissues, but depressed the maximum contraction induced in ß-escin permeabilized tissue. GF-109203X at 1 µM depressed the maximum Ca2+-dependent contraction induced in α-toxin permeabilized tissue and had no effect on the Ca2+ CRC induced in Triton X-100 permeabilized tissue. The MLC kinase inhibitor wortmannin (1 µM) strongly depression the Ca2+ CRCs in tissues permeabilized with Triton X-100, α-toxin and ß-escin. H-1152 inhibited contractions induced by a single exposure to a submaximum [Ca2+] (pCa 6) in both rabbit and mouse femoral arteries. These data indicate that ß-escin permeabilized muscle preserves GPCR-independent, Ca2+- and ROCK-dependent, Ca2+ sensitization.

DePEGylation studies: PEG-RBC stability in conditions consistent with massive transfusion.

Each year the United States population receives an estimated 12 to 14 million units of packed red blood cells (RBCs) and whole blood. It is estimated that 33% of transfusions associated with trauma are with unmatched type O RBCs (UORBC). UORBCs have been proven effective and relatively safe however, by masking RBC surface antigens the risk of transfusion reaction may be further decreased. It is, therefore, important to evaluate and validate the stability of antigen masked RBCs, which may play a part in avoiding transfusion reactions. These antigen-masked RBCs would be regularly subjected to abnormal in vivo conditions commonly associated with massive transfusion such as lactic acidosis, bacteremia, and in vitro irradiation, which is frequently used to sterilize and decrease T Lymphocyte counts in RBC units before transfusion. This study compared two methods of masking RBC antigens by PEGylation: maleimide-PEGylation and cyanuric chloride-PEGylation. RBC PEGylation effectively masks the Rh(D) antigen and PEG-RBC bond stability was evaluated by comparison of pre and post exposure agglutination with anti-D sera. While the stability of maleimide-PEG-RBCs remained unaffected, the cyanuric chloride-PEG-RBCs remained stable in the bacteremia and irradiation studies, but critical concentrations of lactic acid caused dePEGylation. Further studies are warranted to ensure in vivo stability.

A promiscuous conformational switch in the secondary multidrug transporter MdfA.

Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter. Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active transport is triggered promiscuously by merely occupying the Mdr-binding site.

Blood component ridable and CD44 receptor targetable nanoparticles based on a maleimide-functionalized chondroitin sulfate derivative.

Chondroitin sulfate A-deoxycholic acid-polyethylene glycol-maleimide (CSA-DOCA-PEG-MAL; CDPM) nanostructures were designed for the transient binding of MAL with thiol in blood components and cell membranes, in addition to the CD44 receptor targeting, for the therapy of breast cancer. The spontaneous binding of free thiol groups in plasma proteins and blood cells with the MAL group of CDPM was significantly higher than that of CSA-DOCA-PEG (CDP). Enhanced cellular uptake and the in vitro antiproliferation efficacy of docetaxel (D)-loaded CDPM (CDPM/D) nanoparticles (NPs) in MCF-7 cells indicated dual-targeting effects based on MAL-thiol reactions and CSA-CD44 receptor interactions. Following intravenous injection in rats, reduced clearance and an elevated half-life of the drug was observed in the CDPM/D NPs compared to the CDP/D NPs. Taken together, MAL modification of CDP NPs could be a promising approach not only to enhance tumor targeting and penetration but also to extend the blood circulation time of anticancer drugs.

Liposomal formulation of alpha-tocopheryl maleamide: in vitro and in vivo toxicological profile and anticancer effect against spontaneous breast carcinomas in mice.

The vitamin E analogue alpha-tocopheryl succinate (alpha-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of alpha-tocopheryl maleamide (alpha-TAM), an esterase-resistant analogue of alpha-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of alpha-TAM towards cancer cells (MCF-7, B16F10) compared to alpha-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that alpha-TAM was cytotoxic to non-malignant cells (e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both alpha-TOS and alpha-TAM to solve the problem with cytotoxicity of free alpha-TAM (neurotoxicity and anaphylaxis), as well as the low solubility of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal alpha-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of alpha-TAM and alpha-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of alpha-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of alpha-TOS. Thus, the liposomal formulation of alpha-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.

Probing actin polymerization by intermolecular cross-linking.

We have used N,N'-1,4-phenylenebismaleimide, a bifunctional sulfhydryl cross-linking reagent, to probe the oligomeric state of actin during the early stages of its polymerization into filaments. We document that one of the first steps in the polymerization of globular monomeric actin (G-actin) under a wide variety of ionic conditions is the dimerization of a significant fraction of the G-actin monomer pool. As polymerization proceeds, the yield of this initial dimer ("lower" dimer with an apparent molecular mass of 86 kD by SDS-PAGE [LD]) is attenuated, while an actin filament dimer ("upper" dimer with an apparent molecular mass of 115 kD by SDS-PAGE [UD] as characterized [Elzinga, M., and J. J. Phelan. 1984. Proc. Natl. Acad. Sci. USA. 81:6599-6602]) is formed. This shift from LD to UD occurs concomitant with formation of filaments as assayed by N-(1-pyrenyl)iodoacetamide fluorescence enhancement and electron microscopy. Isolated cross-linked LD does not form filaments, while isolated cross-linked UD will assemble into filaments indistinguishable from those polymerized from unmodified G-actin under typical filament-forming conditions. The presence of cross-linked LD does not effect the kinetics of polymerization of actin monomer, whereas cross-linked UD shortens the "lag phase" of the polymerization reaction in a concentration-dependent fashion. Several converging lines of evidence suggest that, although accounting for a significant oligomeric species formed during early polymerization, the LD is incompatible with the helical symmetry defining the mature actin filament; however, it could represent the interfilament dimer found in paracrystalline arrays or filament bundles. Furthermore, the LD is compatible with the unit cell structure and symmetry common to various types of crystalline actin arrays (Aebi, U., W. E. Fowler, G. Isenberg, T. D. Pollard, and P. R. Smith. 1981. J. Cell Biol. 91:340-351) and might represent the major structural state in which a mutant beta-actin (Leavitt, J., G. Bushar, T. Kakunaga, H. Hamada, T. Hirakawa, D. Goldman, and C. Merril. 1982. Cell. 28:259-268) is arrested under polymerizing conditions.

Protein kinase C activation promotes microtubule advance in neuronal growth cones by increasing average microtubule growth lifetimes.

We describe a novel mechanism for protein kinase C regulation of axonal microtubule invasion of growth cones. Activation of PKC by phorbol esters resulted in a rapid, robust advance of distal microtubules (MTs) into the F-actin rich peripheral domain of growth cones, where they are normally excluded. In contrast, inhibition of PKC activity by bisindolylmaleimide and related compounds had no perceptible effect on growth cone motility, but completely blocked phorbol ester effects. Significantly, MT advance occurred despite continued retrograde F-actin flow-a process that normally inhibits MT advance. Polymer assembly was necessary for PKC-mediated MT advance since it was highly sensitive to a range of antagonists at concentrations that specifically interfere with microtubule dynamics. Biochemical evidence is presented that PKC activation promotes formation of a highly dynamic MT pool. Direct assessment of microtubule dynamics and translocation using the fluorescent speckle microscopy microtubule marking technique indicates PKC activation results in a nearly twofold increase in the typical lifetime of a MT growth episode, accompanied by a 1.7-fold increase and twofold decrease in rescue and catastrophe frequencies, respectively. No significant effects on instantaneous microtubule growth, shortening, or sliding rates (in either anterograde or retrograde directions) were observed. MTs also spent a greater percentage of time undergoing retrograde transport after PKC activation, despite overall MT advance. These results suggest that regulation of MT assembly by PKC may be an important factor in determining neurite outgrowth and regrowth rates and may play a role in other cellular processes dependent on directed MT advance.

Hemospan: design principles for a new class of oxygen therapeutic.

Hemoglobin-based oxygen carriers have been under development for decades, but safety concerns have prevented commercial approval. Early designs for modified hemoglobins by polymerization or intramolecular cross-linking reactions increased molecular size and decreased oxygen affinity, but all exhibited side effects of vasoconstriction and reduced blood flow. A new strategy has been established by applying principles of oxygen transport to cell-free hemoglobin. Sangart has developed a new oxygen therapeutic, Hemospan, using site-specific, poly(ethylene) glycol conjugation chemistry designed on two principles: (i) increased macromolecular size to prolong intravascular retention time, and (ii) increased oxygen affinity to prevent premature oxygen offloading in arterioles. In contrast to early-generation products, Hemospan infusion maintains normal arteriolar vascular tone and capillary flow. Phase I and Phase II clinical trials have been completed, showing that Hemospan is well-tolerated in humans, with evidence of efficacy to impart hemodynamic stability in surgical patients under anesthesia. Phase III trials in orthopedic surgery have recently completed enrollment in Europe.

Effects of maleimide-polyethylene glycol-modified human hemoglobin (MP4) on tissue necrosis in SKH1-hr hairless mice.

OBJECTIVE: Tissue hypoxia after blood loss, replantation and flap reperfusion remains a challenging task in surgery. Normovolemic hemodilution improves hemorheologic properties without increasing oxygen carrying capacity. Red blood cell transfusion is the current standard of treatment with its attendant risks. The aim of this study was to investigate the potential of the chemically modified hemoglobin, MP4, to reduce skin flap necrosis and its effect on selected blood markers and kidneys. MATERIALS AND METHODS: Tissue ischemia was induced in the ear of hairless mice (n=26). Hemodilution was performed by replacing one third of blood volume with the similar amount of MP4, dextran, or blood. The extent of non-perfused tissue was assessed by intravital fluorescent microscopy. RESULTS: Of all groups, MP4 showed the smallest area of no perfusion (in percentage of the ear +/- SEM: 16.3% +/- 2.4), the control group the largest (22.4% +/- 3.5). Leukocytes showed a significant increase in the MP4 and dextran group (from 8.7 to 13.6 respectively 15.4*109/l). On histology no changes of the kidneys could be observed. CONCLUSION: MP4 causes an increase of leukocytes, improves the oxygen supply of the tissue and shows no evidence of renal impairment.

Berberine coated mannosylated liposomes curtail RANKL stimulated osteoclastogenesis through the modulation of GSK3ß pathway via upregulating miR-23a.

Drug-induced microRNAs manifest significant therapeutic approaches; however, such progress in the treatment of osteopathic disorders including osteoporosis and rheumatoid arthritis still remains obscure. Contrarily, non-specific drug delivery, at high doses, increases the risk of side effects and reduces drug therapeutic efficacy. Accordingly, the present study was designed to examine the therapeutic effect of berberine coated mannosylated liposomes (ML-BBR) on RANKL (100 ng/ml) stimulated bone marrow-derived monocytes/macrophages (BMMs) via altering miR-23a expression. Initial studies using confocal microscopy showed successful internalization of ML-BBR in RANKL stimulated BMMs. Treatment with ML-BBR abrogated the increased osteoclast formation in BMM cells via inhibiting phosphorylated glutathione synthase kinase beta (p-GSK3ß) mediated NFATc1 activation. Consequently, ML-BBR also attenuated the expression of bone-degrading enzymes (TRAP, cathepsin K and MMP-9) thereby inhibiting the bone resorptive activity of osteoclasts. Moreover, ML-BBR induced the expression levels of miR-23a at the gene level, which in turn attenuated GSK3ß/p-GSK3ß expression as confirmed via blotting analysis. Further miR-23a inhibition of the GSK3ß phosphorylation was confirmed using luciferase reporter assay. Comparatively, LY2090314 (GSK3ß inhibitor) treatment inhibited the protein level expression of GSK3ß/p-GSK3ß. However, LY2090314 treatment induced a basal level expression of miR-23a owing to the suggestion that ML-BBR has an influential role in upregulating miR-23a level to inhibit GSK-3ß phosphorylation. Cumulatively, our findings endorsed that preferential internalization of ML-BBR by BMMs effectively modulated the RANKL/p-GSK3ß pathway and curtailed the osteoclast-mediated bone erosion possibly through post-transcriptional gene silencing via miR-23a.

CO-MP4, a polyethylene glycol-conjugated haemoglobin derivative and carbon monoxide carrier that reduces myocardial infarct size in rats.

BACKGROUND AND PURPOSE: MP4 (Hemospan) is a Hb-based oxygen therapeutic agent, based on polyethylene-glycol (PEG) conjugation to Hb, undergoing clinical trials as an oxygen carrier. This study describes the functional interaction between MP4 and carbon monoxide (CO), as a CO delivery agent, and the effects of CO-MP4 on myocardial infarct size following ischaemia and reperfusion in rats. EXPERIMENTAL APPROACH: Kinetic measurements of CO-MP4 binding were used to evaluate the effects of PEG modification on Hb subunit structure/function and to calculate CO-MP4 equilibrium constants. CO transport by CO-MP4 was shown by ligand (O2/CO) partitioning between MP4 and red blood cell (RBC)-Hb. Pharmacological effects of CO-MP4 were studied on myocardial infarction in rats. KEY RESULTS: CO binding kinetics show primary structural/functional effects on beta chains in MP4, with alpha chains maintaining the ability to undergo tertiary conformational transition. CO confers long-term, room-temperature stability and is able to rapidly re-equilibrate between MP4 and RBCs. In a rat model of myocardial infarct, in contrast to oxy-MP4, CO-MP4 reduced infarct size when administered prior to the induction of ischaemia. CONCLUSIONS AND IMPLICATIONS: MP4 PEGylation chemistry modifies the individual function of Hb subunits, but results in an overall CO equilibrium constant similar to that for unmodified Hb. CO-MP4 is able to deliver CO to the circulation and reduces ischaemia/reperfusion injury in rats, providing the first evidence for this drug as a CO therapeutic agent.