Direct binding of recombinant plasminogen kringle 1-3 to angiogenin inhibits angiogenin-induced angiogenesis in the chick embryo CAM.

Angiogenin is one of the most potent angiogenesis-inducing proteins. Angiostatin is one of the most potent angiogenesis inhibitors, and it contains the first four kringle domains of plasminogen (K1-4). Recombinant human plasminogen kringle 1-3 (rK1-3) was expressed in Escherichia coli and purified to homogeneity. The binding of t-4-aminomethylcyclohexanecarboxylic acid with the purified kringle 1-3 was determined by changes in intrinsic fluorescence. rK1-3 exhibits comparable ligand-binding properties as native human plasminogen kringle 1-3. The purified rK1-3 inhibits neovascularization in the chick embryo chorioallantoic membrane (CAM) assay. Interaction of angiogenin with rK1-3 was examined by immunological binding assay and surface plasmon resonance kinetic analysis, and the equilibrium dissociation constants for the complex, Kd, are 0.89 and 0.18 microM, respectively. rK1-3 inhibits angiogenin-induced angiogenesis in the chick embryo CAM in a concentration-dependent manner. These results indicate that rK1-3 directly binds to angiogenin and thus rK1-3 inhibits the angiogenic activity of angiogenin.

The effect of sonication on simulated osteoarthritis. Part I: effects of 1 MHz ultrasound on uptake of hyaluronan into the rabbit synovium.

High molecular weight (MW) hyaluronan (HA) preparation is considered to be more biologically active than HAs of lower MWs. However, many of the HA preparations currently used to treat osteoarthritis (OA) have lower MWs by the enhanced penetration of HA molecules into the synovial lining cells. In this study, we determined the effectiveness of sonophoresis on the delivery of high MW HA into synovial membrane using an animal model of OA. A total of 1000 kDa (HA1000) and 3000 kDa (HA3000) HA were labeled with fluorescein and injected into the knees of rabbits. Low-intensity continuous ultrasound at 1 MHz, 400 mW/cm2 was applied to the knees for 10 min treatment bid. Synovial fluid analysis revealed increased absorption and fluorescence microscopy showed deeper penetration of both HA1000 and HA3000, more so with the latter. Histologic examination indicated that ultrasound treatment resulted in no apparent damage to the synovial membrane. These results suggest that simultaneous sonication with HA injection might compensate for the short half-life of HA. Consequently, this dual treatment would render HA a far more effective tool in the management of OA.

The effect of sonication on simulated osteoarthritis. Part II: alleviation of osteoarthritis pathogenesis by 1 MHz ultrasound with simultaneous hyaluronate injection.

In our previous study, we demonstrated the effects of ultrasound (US) on the delivery of hyaluronan (HA) into the synovium, even at molecular sizes as high as 3000 kDA. We hypothesized that a combined therapy with US and HA would have synergistic effects on alleviating the pathogenesis of osteoarthritis (OA). In the present study, we evaluated the effectiveness of sonication on the progress of induced OA in rabbits. We measured the cartilage degradation and inflammation, synovial fluid amount along with protein content and, finally, performed histologic analyses of the integrity of the cartilage and synovium. Low-intensity continuous US at 1 MHz, 400 mW/cm2 was applied to the knees for 10 min bid. Combined treatment with US and HA most strikingly reduced total synovial fluid volume and also significantly alleviated the OA-induced accretion of total protein, proteoglycan and prostaglandin E2 in the synovial fluid. It also attenuated the release of collagen type II and matrix metalloproteinase-3 in the OA-induced joint to normal levels. Histopathology revealed that combined HA and US treatment also reduced the severity of OA-induced structural damages in the cartilage and synovium. The effectiveness of HA with regard to the alleviation of OA pathogenic changes can be greatly enhanced by the simultaneous treatment with HA and US.

Tissue-engineered cartilage using fibrin/hyaluronan composite gel and its in vivo implantation.

The importance of scaffold biomaterials has been emphasized for in vitro culture of tissue-engineered cartilage in a three-dimensional (3D) environment. In this study, we examined the feasibility of fibrin glue, mixed with hyaluronic acid (HA) as a composite scaffold. Fibrin glue has been a useful cell delivery matrix for cartilage tissue engineering and HA is a key component of normal articular cartilage. Our hypothesis is that compared to fibrin itself, a fibrin/HA composite can have significantly enhanced properties, due mainly to the added benefits of HA in the matrix. Pieces of cartilage were isolated from rabbit knees and the chondrocytes were harvested through enzymatic digestion. Both fibrin and fibrin/HA composite were prepared and subsequently implanted in nude mice (n = 9, each group) for 1, 2, and 4 weeks, respectively. The retrieved specimens were then analyzed and the results were compared. Cartilage-like tissue formation was detected earlier with fibrin/HA specimens. They produced significantly higher amounts of the extracellular matrix (ECM) molecules, GAG, and collagen at each time point than those in fibrin. Interestingly, the fibrin/HA composite was also competent in maintaining its initial size. Histology--Safranin O/fast green and Alcian blue--of the retrieved specimens found more intense, uniform staining in the fibrin/HA composites. Analysis of the gene expression of the ECM molecules also confirmed the benefits of the composite with added HA in the maintenance of phenotypic stability. The present study suggests that fibrin/HA composite may serve as a dependable cell delivery vehicle as well as a structural basis for tissue-engineered cartilage.

Antisense inhibition of transglutaminase 2 affects development of mouse embryo submandibular gland in organ culture.

Tissue transglutaminase (TGase 2) has been implicated in numerous cellular functions, i.e., apoptosis, differentiation, extracellular matrix protein cross-linking and organogenesis. Earlier report of the strong transient expression of TGase 2 localized at the anchoring sites of muscle bundles of human embryo and recent findings of a similar transient expression of the TGase 2 in the salivary myoepithelial cells of mouse embryo indicated a definitive role of TGase 2 in the cytodifferentiation of myoepithelial cells. To understand functional role(s) of TGase 2 in the organogenesis of salivary gland, antisense inhibition of TGase 2 expression was performed in the organ culture of mouse embryo submandibular gland. The antisense of TGase 2 transfection tested using oral keratinocyte cell line, KB cells, elicited significant inhibition of cellular transglutaminase expression. The same antisense treatment of submandibular glands in organ culture also resulted in the suppression of cellular TGase 2 expression as indicated by weak immunoreaction against anti-TGase 2 in the myoepithelial cells of submandibular glands in contrast to strong reaction in those of the normal and sense-treated glands. Antisense to TGase 2 treatment induced retarded growth of salivary epithelium in 1 week and severe aberrant growth of salivary ducts and acini in 2 weeks and also expression of apoptotic inducer, Bax specifically localized in the myoepithelial cells, suggesting apoptotic state of myoepithelial cells. These data suggest that the antisense inhibition of TGase 2 expression affects the cytodifferentiation of ductal cells and myoepithelial cells, and resulted in severe retardation of tubuloalveolar structure formation of salivary gland.

Transglutaminase 2 expression in the salivary myoepithelial cells of mouse embryo.

Earlier a strong transient expression of transglutaminase 2 (TGase 2) localized at the anchoring sites of muscle bundles in human embryo was observed. In this study, we report a similar transient expression of the TGase 2 in the salivary myoepithelial cells of mouse embryo by immunohistochemistry, RNA in situ hybridisation, and RT-PCR. From 35 submandibular glands of mouse embryos and postnatal mice, a consistent expression of TGase 2 in the myoepithelial cells via a stage-specific manner was identified by mono-clonal antibody to TGase 2 immunostaining. A similar expression pattern of TGase 2 in the myoepithelial cells was also observed by RNA in situ hybridisation analysis. The expression of TGase 2 in the salivary epithelium and mesenchyme during the prenatal 14.5-15.5 days was found minimally diffusely spread and became intensely focalised in the myoepithelial cells of salivary acini and ducts during the prenatal 16.5-18.5 days but thereafter gradually decreased until postnatal 7 days and remained weak in postnatal 3 weeks. Such transient rise and fall expressions of TGase 2 were also found with the sequential amount of RT-PCR products during the same period. The alpha-smooth muscle actin (alpha-SMA) as a positive control in the myoepithelial cells of mouse submandibular glands was consistently expressed during the prenatal and postnatal period. These results of transient expression of TGase 2 in the myoepithelial cells coincided with the formation of the dendritic basket structure in the periphery of acini and ducts, suggest a possible catalytic role of transglutaminase in a newly formed cellular matrixes during the cytodifferentiating stage of mouse prenatal and neonatal submandibular glands.

Paradoxical effects of elastase inhibitor guamerin on the tissue repair of two different wound models: sealed cutaneous and exposed tongue wounds.

Innate elastase inhibitors are known to be putatively involved in the regulation of tissue inflammation by inhibiting polymorphonuclear leukocyte (PMN) derived proteinases. The aim of this study was to evaluate affects of leukocyte elastase suppression and PMN infiltration on wound healing in mouse by administering the recombinant elastase inhibitor guamerin (rEIG) in two different wound models; 1) impaired pin-punctured dorsal mucosa of anterior tongue wound, 60 mice, treated with saline containing rEIG that were fed ad libitum and 2) stable linear excisional cutaneous wound, 40 mice, covered with fibrin sealant containing rEIG. The progress of healing was analyzed by histological methods. The tongue wounds treated with rEIG became edematous around the pin-punctured tongue wound, and influx of inflammatory cells and PMN into the underlying stromal tissue were seen rapidly after wounding and peaked between 2-4 days. Whereas the control mice showed almost no wheal formation in the pin-punctured wound, a far lesser levels of PMN infiltration, and almost complete wound closure in 4 days. In the other model, the liner excisional cutaneous wound treated with fibrin sealant containing rEIG showed early wound constriction, lesser degree of inflammatory cells influx, and complete reepithelialization in 4-5 days, whereas the wound of control mice with the fibrin sealant alone showed contrary delayed reepithelialization, greater degree of inflammatory cell infiltration, and consequencial formation of greater granulation tissue at wound site. Taken together, these data suggest paradoxical effects of rEIG on the wound healing where in the wound exposed to infiltrating milieu of microorganisms in the oral cavity, the rEIG aggravates the wound healing by interfering with other innate defensive factors and extended greater flux of PMNs to inflamed wound site, while in the wound enclosed by fibrin, the rEIG accelerated wound healing by inhibiting the inflammation-generated proteases and the acute inflammatory reaction.

Characterization and biological activities of recombinant human plasminogen kringle 1-3 produced in Escherichia coli.

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is involved in many pathological conditions, for example, tumorigenesis, diabetic retinopathy, and rheumatoid arthritis. Angiostatin, which contains the kringle 1-4 domains of plasminogen, is known to be a potent inhibitor of angiogenesis and a strong suppressor of various solid tumors. In this study, we expressed recombinant protein containing the kringle 1-3 domains of human plasminogen in Escherichia coli and investigated its biological activities. The protein was successfully refolded from inclusion bodies and purified at a 30% overall yield, as a single peak by HPLC. The purified recombinant protein had biochemical properties that were similar to those of the native form, which included molecular size, lysine-binding capacity, and immunoreactivity with a specific antibody. The recombinant protein was also found to strongly inhibit the proliferation of bovine capillary endothelial cells in vitro, and the formation of new capillaries on chick embryos. In addition, it suppressed the growth of primary Lewis lung carcinoma and B16 melanoma in an in vivo mouse model. Our findings suggest that the recombinant kringle 1-3 domains in a prokaryote expression system have anti-angiogenic activities, which may be useful in clinical and basic research in the field of angiogenesis.

Human apolipoprotein(a) kringle V inhibits angiogenesis in vitro and in vivo by interfering with the activation of focal adhesion kinases.

Apolipoprotein(a) [apo(a)] contains the largest numbers of kringle domains identified to date. Of these, apo(a) kringle V shows significant sequence homology with plasminogen kringle 5, which is reported to be a potent angiogenesis inhibitor. To determine the effects of apo(a) kringle V on angiogenesis, it was expressed as a soluble protein (termed rhLK8) in Pichia pastoris and its in vitro and in vivo anti-angiogenic properties were examined. rhLK8 inhibited the migration of human umbilical vein endothelial cells in vitro in a dose-dependent manner. This function was associated with the down-regulation of the activation of focal adhesion kinase and the inhibition of the consequent formation of actin stress fibers/focal adhesions. rhLK8 also inhibited new capillary formation in vivo, as assessed by the chick chorioallantoic membrane assay and the Matrigel plug assay. These results indicate that rhLK8 may be an effective angiogenesis inhibitor both in vitro and in vivo.

Inhibition of angiogenesis and angiogenesis-dependent tumor growth by the cryptic kringle fragments of human apolipoprotein(a).

Apolipoprotein(a) (apo(a)) contains tandemly repeated kringle domains that are closely related to plasminogen kringle 4, followed by a single kringle 5-like domain and an inactive protease-like domain. Recently, the anti-angiogenic activities of apo(a) have been demonstrated both in vitro and in vivo. However, its effects on tumor angiogenesis and the underlying mechanisms involved have not been fully elucidated. To evaluate the anti-angiogenic and anti-tumor activities of the apo(a) kringle domains and to elucidate their mechanism of action, we expressed the last three kringle domains of apo(a), KIV-9, KIV-10, and KV, in Escherichia coli. The resultant recombinant protein, termed rhLK68, exhibited a dose-dependent inhibition of basic fibroblast growth factor-stimulated human umbilical vein endothelial cell proliferation and migration in vitro and inhibited the neovascularization in chick chorioallantoic membranes in vivo. The ability of rhLK68 to abrogate the activation of extracellular signal-regulated kinases appears to be responsible for rhLK68-mediated anti-angiogenesis. Furthermore, systemic administration of rhLK68 suppressed human lung (A549) and colon (HCT-15) tumor growth in nude mice. Immunohistochemical examination and in situ hybridization analysis of the tumors showed a significant decrease in the number of blood vessels and the reduced expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin, indicating that suppression of angiogenesis may have played a significant role in the inhibition of tumor growth. Collectively, these results suggest that a truncated apo(a), rhLK68, is a potent anti-angiogenic and anti-tumor molecule.

Anti-angiogenic activity of the recombinant kringle domain of urokinase and its specific entry into endothelial cells.

Urokinase plasminogen activator (uPA) belongs to a family of proteins that contains kringle domain and plays an important role in inflammation, tissue remodeling, angiogenesis, and tumor metastasis by pericellular plasminogen activation. Kringle domains of plasminogen have been shown to demonstrate anti-angiogenic and anti-tumor activities. Here, we report our investigation of the kringle domain of uPA for anti-angiogenic activity and a possible cellular mechanism of action. The recombinant kringle domain of uPA (Asp(45)-Lys(135)) (UK1) inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor (VEGF), or epidermal growth factor. It also inhibited migration of endothelial cells induced by VEGF or uPA, and in vivo angiogenesis on the chick chorioallantoic membrane. It did not block plasminogen activation by activated uPA in clot lysis and chromogenic substrate assays. Neither binding of UK1 to immobilized uPA receptor nor competitive inhibition of uPA binding were confirmed by real-time interaction analysis. However, internalization of UK1 followed by translocation from cytosol to nucleus was determined to be specific to endothelial cells. It also elicited a transient increase of Ca(2+) flux of more than 2-fold within 2 min of exposure in an endothelial cell-specific manner. These results suggest that the kringle domain of uPA exhibits anti-angiogenic activity and that its anti-angiogenic activity may occur through a different mechanism from inhibition of uPA-uPA receptor interaction or uPA proteolytic activity and may be associated with endothelial-cell specific internalization not mediated by the uPA receptor.

Protein kinase CK2 phosphorylates and interacts with deoxyhypusine synthase in HeLa cells.

Deoxyhypusine is a modified lysine and formed posttranslationally to be the eukaryotic initiation factor eIF5A by deoxyhypusine synthase, employing spermidine as butylamine donor. Subsequent hydroxylation of this deoxyhypusine-containing intermediate completes the maturation of eIF5A. The previous report showed that deoxyhypusine synthase was phosphorylated by PKC in vivo and the association of deoxyhypusine synthase with PKC in CHO cells was PMA-, and Ca(2+)/phospholipid-dependent. We have extended study on the phosphorylation of deoxyhypusine synthase by protein kinase CK2 in order to define its role on the regulation of eIF5A in the cell. The results showed that deoxyhypusine synthase was phosphorylated by CK2 in vivo as well as in vitro. Endogenous CK2 in HeLa cells and the cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced or decreased by the addition of CK2 effectors such as polylysine, heparin, and poly(Glu, Tyr) 4:1. Phosphoamino acid analysis of this enzyme revealed that deoxyhypusine synthase is mainly phosphorylated on threonine residue and less intensely on serine. These results suggest that phosphorylation of deoxyhypusine synthase is CK2-dependent cellular event as well as PKC-mediated effect. However, there were no observable changes in enzyme activity between the phosphorylated and unphosphorylated forms of deoxyhypusine synthase. Taken together, besides its established function in hypusine modification involving eIF5A substrate, deoxyhypusine synthase and its phosphorylation modification may have other independent cellular functions because of versatile roles of deoxyhypusine synthase.

Differential inhibition of endothelial cell proliferation and migration by urokinase subdomains: amino-terminal fragment and kringle domain.

The serine protease urokinase-type plasminogen activator (uPA) is implicated in pericellular proteolysis in a variety of physiological and pathological processes including angiogenesis and tumor metastasis. The kringle domain of uPA (UK1) has proven to be an anti-angiogenic molecule with unknown mechanism and amino terminal fragment of uPA (u-ATF) with additional growth factor-like domain can be used for blocking interaction of uPA and uPA receptor. Here, we compared anti-angiogenic activities of these two molecules in vitro and in vivo. The recombinant u-ATF from E. coli and refolded in vitro was found to bind to uPAR with high affinity, whereas E. coli-derived UK1 showed no binding by Biacore analysis. In contrast to UK1 having potent inhibitory effect, u-ATF exhibited low inhibitory effect on bovine capillary endothelial cell growth (ED(50)>320 nM). Furthermore, u-ATF inhibition of VEGF-induced migration of human umbilical vein endothelial cell was far less sensitive (IC(50) = 600 nM) than those observed with UK1, and angiogenesis inhibition was marginal in chorioallantoic membrane. These results suggest that kringle domain alone is sufficient for potent anti- angiogenic activity and additional growth factor-like domain diverts this molecule in undergoing different mechanism such as inhibition of uPA/uPAR interaction rather than undergoing distinct anti- angiogenic mechanism driven by kringle domain.

Elafin expression in human fetal and adult submandibular glands.

Elafin, a bifunctional protein, has the NH(2)-terminal domain functions as a transglutaminase substrate for crosslinking to lysine-containing proteins and the COOH-terminal whey acidic protein domain as a potent anti-elastase. Human fetal submandibular glands (n=100) and adult submandibular glands (n=10) were used to elucidate the expression pattern of elafin in the developmental processes of human submandibular gland by immunohistochemistry, in situ hybridization, and western blot analysis. Elafin mRNA was expressed both in the gland epithelium and intralobular mesenchymal tissue of fetal submandibular gland in an early developmental stage (10-18 weeks) and an early intermediate developmental stage (EIDS; 19-24 weeks). The elafin antigen was also found in the intralobular mesenchyme of submandibular gland in the same stages. Thereafter, elafin was transitionally expressed in the ducts and acini of submandibular glands. In the late intermediate developmental stage (LIDS; 25-32 weeks) and the late developmental stage (LDS; 33-40 weeks), elafin became markedly positive in the excretory and striated ducts but weakly positive in the intralobular mesenchymal tissue. The elafin was heavily present in the excretory and striated ducts of adult submandibular gland, while it was sparse in the intralobular mesenchymal tissues. Western blot analysis showed the protein extracts from submandibular glands of EIDS, LIDS, and LDS, adult submandibular gland, fetal tissues (8 weeks), and adult parotid saliva migrated into multiple bands, about 25, 50, 65, and 140 kDa, which were higher than the putative size of elafin protein, 12 kDa. These data suggest that elafin, anti-elastase, is an essential component highly utilized during the morphogenetic processes of fetal salivary gland development and continuously plays a role for the protection of the tubuloalveolar structures of adult salivary gland.

Deoxyhypusine synthase is phosphorylated by protein kinase C in vivo as well as in vitro.

Deoxyhypusine synthase catalyzes the first step in the posttranslational synthesis of an unusual amino acid, hypusine, in the eukaryotic translation initiation factor 5A (eIF-5A) precursor protein. We earlier observed that yeast recombinant deoxyhypusine synthase was phosphorylated by protein kinase C (PKC) in vitro (Kang and Chung, 1999) and the phosphorylation rate was synergistically increased to a 3.5-fold following treatment with phosphatidylserine (P.Ser)/diacylglycerol (DAG)/ Ca(2+), suggesting a possible involvement of PKC. We have extended study on the phosphorylation of deoxyhypusine synthase in vivo in different cell lines in order to define its role on the regulation of eIF5A in the cell. Deoxyhypusine synthase was found to be phosphorylated by endogenous kinases in CHO, NIH3T3, and chicken embryonic cells. The highest degree of phosphorylation was found in CHO cells. Moreover, phosphorylation of deoxyhypusine synthase in intact CHO cells was revealed and the expression of phosphorylated deoxyhypusine synthase was significantly diminished by diacyl ethylene glycol (DAEG), a PKC inhibitor, and enhanced by phorbol 12-myristate 13-acetate (PMA) or Ca(2+)/DAG. Endogenous PKC in CHO cell and cell lysate was able to phosphorylate deoxyhypusine synthase and this modification is enhanced by PMA or Ca(2+) plus DAG. Close association of PKC with deoxyhypusine synthase in the CHO cells was evident in the immune coprecipitation and was PMA-, and Ca(2+)/phospholipid dependent. These results suggest that phosphorylation of deoxyhypusine synthase was PKC-dependent cellular event and open a path for possible regulation in the interaction with eIF5A precursor for hypusine synthesis.

Effects of Val34Leu and Val35Leu polymorphism on the enzyme activity of the coagulation factor XIII-A.

Change in fibrin stabilizing activity of factor XIII A subunit (FXIII-A) caused by a specific mutation, Val34Leu, is recently implicated to incidences of pathophysiology of thrombosis. In an effort to understand the effect of Val34Leu on enhanced catalytic role of FXIII-A, wild type human factor XIII A (HFXIII-A) and mutant HFXIII-A: HFXIII-A (V34L), HFXIII-A (V35L) and HFXIII-A (V34L/V35L) cDNA were expressed in E.coli system where the purified recombinant FXIII-A (gammaFXIII-A) showed a similar specific transglutaminase activity comparable to the human native FXIII-A from platelet. Using these gammaFXIII-A mutants, the activation kinetics by thrombin and the enzymatic properties of the activated gammaFXIII-A were characterized. gammaFXIII-A (V34L) and gammaFXIII-A (V34L/V35L) mutants were activated by thrombin much faster than those of wild type gammagFXIII-A and V35L variant. However, the activated gammaFXIII-A and mutants showed the identical catalytic efficiency as measured by in vitro assay. These results suggest that ready activation caused by a specific mutation of neighboring thrombin cleavage site(s) in the activation peptide of FXIII-A like V34L resulted in the real-time amount of the activated factor XIII-A that could influence the outcome of fibrin stabilization in vivo such as alpha2-plasmin inhibitor crosslinking to fibrin, a reaction known to be dependent on the initial concentration of active factor-XIII-A.