Procaspase 8 and Bax are up-regulated by distinct pathways in Streptococcal pyrogenic exotoxin B-induced apoptosis.

We have previously identified integrin alpha(v)beta(3) and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S, a mutant of SPE B that binds to Fas only. In the current study we found that after binding to alpha(v)beta(3), SPE B stimulated the tyrosine phosphorylation of JAK2 and STAT1. STAT1 tyrosine phosphorylation was inhibited by a JAK2 inhibitor, AG490, short interfering RNA (siRNA) silencing of JAK2, and anti-alpha(V)beta(3) antibody. AG490 also decreased the binding of tyrosine-phosphorylated STAT1 to the procaspase 8 promoter, decreasing procaspase 8 expression, suggesting that SPE B up-regulates procaspase 8 expression via the JAK2/STAT1 pathway. Alternatively, both SPE B and G308S increased STAT1 phosphorylation at serine 727, which was inhibited by anti-Fas antibody, a p38 inhibitor, SB203580, and siRNA silencing of p38. In addition, SPE B and G308S increased binding of serine-phosphorylated STAT1 to the Bax promoter and Bax expression, which was decreased by SB203580. SPE B and G308S-stimulated Bax expression was also inhibited by anti-Fas antibody. These findings suggest that Fas mediate SPE B-induced Bax expression through p38. Silencing of JAK2 or p38 by siRNA blocked procaspase 8 expression, whereas only p38 siRNA decreased Bax expression. Furthermore, JAK2 inhibition and p38 inhibition reduced SPE B-induced apoptosis, but only p38 inhibition blocked G308S-induced apoptosis.

Molecular mimicry between streptococcal pyrogenic exotoxin B and endothelial cells.

Molecular mimicry between group A streptococcus and host antigens has important roles in the development of post-streptococcal sequelae, including glomerulonephritis and rheumatic heart disease (RHD). The etiology of RHD involves host cross-reactivity with M proteins and carbohydrate antigens. In this study, we show that anti-streptococcal pyrogenic exotoxin B (SPE B) antibodies exhibited characteristics of autoantibodies, which cross-react with endothelial cells. Immunoglobulin G (IgG) deposition and complement activation were observed in the heart valve of SPE B-immunized mice. In addition, apoptosis in the heart valve was detected in SPE B-immunized mice. An anti-SPE B monoclonal antibody (mAb) 10G showed cross-reactivity with human microvascular endothelial (HMEC-1) cells and mouse valve endothelial cells. Passive immunization with mAb 10G also caused IgG deposition, complement activation, and apoptotic cell death in the mouse heart valve. We conducted peptide array and ELISA using synthetic peptides to identify the SPE B antigenic epitope recognized by mAb 10G. Results showed that the major epitope of mAb 10G is localized to amino-acid residues 296-310 of SPE B (P7-8). The cross-reactivity of mAb 10G with endothelial cells was inhibited using P7-8 peptides for competition. These results suggest that anti-SPE B antibodies cross-react with endothelial cells, and that a dominant epitope is located within the amino-acid residues 296-310 of SPE B. Moreover, we found that mAb 10G can also bind to N-acetyl-ß-D-glucosamine (GlcNAc) conjugated with bovine serum albumin (BSA), but not to BSA or M1 protein. Competition assay showed that the binding activity of mAb 10G with GlcNAc-BSA and P7-8 of SPE B was inhibited by pretreatment with GlcNAc-BSA or P7-8 peptides. Therefore, our results suggest that conformational molecular mimicry may exist between SPE B and GlcNAc.

Oxidative stress and metal ions regulate a ferritin-like gene, dpr, in Streptococcus pyogenes.

Bacteria encounter oxidative stress by exposure to reactive oxygen species (ROS) present in the aerobic environment and during immune responses. In Streptococcus pyogenes, Dpr has been identified as a stress protein conferring resistance to hydrogen peroxide and multiple other stresses. The expression of Dpr is under perR (peroxide stress response regulator) control. However, the exact molecular mechanism of PerR regulation of Dpr is not clear. In this study, a perR deletion mutant was constructed by double cross-over mutagenesis. The profile of Dpr expression, performed by Western blot assay, revealed growth-phase dependency under normal culture conditions. Dpr expression decreased under iron-restricted conditions, whereas iron, zinc, nickel, and hydrogen peroxide induced its expression. The perR mutant does not induce Dpr as well when exposed to environmental signals. PerR binds the promoter region of dpr. Increased iron and hydrogen peroxide concentrations decreased PerR binding to the promoter region of dpr, suggesting that regulation of Dpr by environmental signals is mediated by PerR directly.

Recurrent DICER1 Hotspot Mutations in Malignant Thyroid Gland Teratomas: Molecular Characterization and Proposal for a Separate Classification.

Thyroid gland teratomas are rare tumors that span a wide clinicopathologic spectrum. Although benign and immature teratomas arise in infants and young children and generally have good outcomes, malignant teratomas affect adults and follow an aggressive course. This divergent behavior raises the possibility that benign/immature and malignant teratomas are separate entities rather than different grades of a single tumor. However, the histogenesis and molecular underpinnings of thyroid gland teratomas are poorly understood regardless of grade. In this study, we performed next-generation sequencing on 8 thyroid gland teratomas, including 4 malignant, 3 benign, and 1 immature. We identified DICER1 hotspot mutations in all 4 malignant cases (100%) but not in any benign/immature cases (0%). No clinically significant mutations in other genes were found in either group. We also performed immunohistochemistry to characterize the primitive components of malignant teratomas. Not only did all cases consistently contain immature neural elements (synaptophysin and INSM1 positive), but also spindled cells with rhabdomyoblastic differentiation (desmin and myogenin positive) and bland epithelial proliferations of thyroid follicular origin (TTF-1 and PAX8 positive). Although DICER1 mutations have previously been implicated in multinodular hyperplasia and well-differentiated thyroid carcinomas, these findings demonstrate the first recurrent role for DICER1 in primitive thyroid tumors. The combined neural, rhabdomyoblastic, and homologous epithelial elements highlighted in this series of malignant thyroid gland teratomas parallel the components of DICER1-mutated tumors in other organs. Overall, these molecular findings further expand the differences between benign/immature teratomas and malignant teratomas, supporting the classification of these tumors as separate entities.

emm1/sequence type 28 strains of group A streptococci that express covR at early stationary phase are associated with increased growth and earlier SpeB secretion.

Streptococcus pyogenes (group A streptococcus [GAS]) is a versatile human pathogen, and emm1/sequence type 28 (ST28) is the most frequently isolated type from GAS infections. The emm1/ST28 strain is associated with necrotizing fasciitis and streptococcal toxic shock syndrome. Growth-phase regulation is one of the important regulatory mechanisms in GAS, which controls gene expression at restricted phases of growth. CovRS, a two-component regulatory system, is considered the regulator of streptococcal pyrogenic exotoxin B (SpeB) and is thought to be activated in the exponential phase of growth. In the present study, Northern hybridization analysis showed that 52% of the analyzed GAS strains expressed covR at the exponential phase, but 48% of the strains expressed covR at the early stationary phase of growth. Strains transcribing covR at the early stationary phase showed better growth and earlier SpeB expression than the other group of strains. Multilocus sequence typing and pulsed-field gel electrophoresis analysis showed only emm1/ST28 strains (which comprise a clonal cluster) were expressing covR at the early stationary phase of growth, indicating that emm1/ST28 strains have special characteristics which may be related to their worldwide distribution.

Changing epidemiology of Streptococcus pyogenes emm types and associated invasive and noninvasive infections in Southern Taiwan.

A total of 242 isolates were recovered from 76 patients with invasive diseases, 89 with scarlet fever, and 77 with pharyngitis. The most frequent emm types were types 12 (43.4%), 4 (18.2%), and 1 (16.9%). emm12 reemerged in 2005 and peaked in 2007. emm11 was recovered only from patients with invasive disease.

An iron-binding protein, Dpr, decreases hydrogen peroxide stress and protects Streptococcus pyogenes against multiple stresses.

Streptococcus pyogenes does not produce catalase, but it can grow in aerobic environments and survive in the presence of peroxide. One of the stress proteins of this organism, peroxide resistance protein (Dpr), has been studied to examine its role in resistance to hydrogen peroxide, but the protective mechanism of Dpr is not clear. The aim of this study was to characterize the dpr gene and its role in dealing with different stresses. A dpr deletion mutant was constructed by double-crossover mutagenesis. The dpr mutant was more sensitive to H(2)O(2), and complementation could partially restore the defect in the mutant. Pretreatment with the iron chelator deferoxamine mesylate rescued the survival activity of the mutant under oxidative stress conditions. The dpr mutant also showed a low survival rate in the long-term stationary phase, when it was treated with extreme acids, and under alkaline pH conditions compared to the wild-type strain. The growth of the dpr mutant was slower than that of the wild-type strain in iron-limiting conditions. The dpr mutant showed high sensitivity to iron and zinc but not to manganese, copper, nickel, and calcium. Recombinant Dpr protein was purified and showed iron-binding activity, whereas no DNA-binding activity was found. These data indicate that an iron-binding protein, Dpr, provides protection from hydrogen peroxide stress by preventing the Fenton reaction, and Dpr was identified as a novel stress protein that protects against several stresses in group A streptococci.

Streptococcal pyrogenic exotoxin B-induced apoptosis in A549 cells is mediated through alpha(v)beta(3) integrin and Fas.

Our previous work suggested that streptococcal pyrogenic exotoxin (SPE) B-induced apoptosis is mediated through a receptor-like mechanism. In this study, we have identified alpha(v)beta(3) and Fas as the SPE B receptors for this function. The SPE B fragment without the RGD motif and G308S, a SPE B mutant with the RSD motif, induced less apoptosis than did native SPE B, suggesting that the RGD motif is critical for SPE B-induced apoptosis. Fluorescein isothiocyanate-SPE B binding assays and immunoprecipitation analysis showed that SPE B specifically interacted with alpha(v)beta(3). Anti-alpha(v)beta(3) antibody partially inhibited SPE B-induced apoptosis but had no effect on G308S-induced apoptosis. In addition, Fas binding to SPE B was verified in an affinity column and an immunoprecipitation analysis. Anti-Fas antibody inhibited SPE B- and G308S-induced apoptosis in a dose-dependent manner, suggesting that Fas-mediated SPE B-induced apoptosis also occurs RGD independently. Both anti-alpha(v)beta(3) and anti-Fas antibodies synergistically inhibited SPE B-induced apoptosis. The apoptotic cascades were activated by SPE B and G308S, with a little delay by the latter. After SPE B binding, the cell surface level of alpha(v)beta(3), but not of Fas, was decreased. The decreased alpha(v)beta(3) level was restored by treatment with the proteasome inhibitor MG132, suggesting a SPE B-mediated endocytosis of integrin alpha(v)beta(3) via the ubiquitin-proteasome system. Taken together, our results demonstrate that SPE B-induced apoptosis is mediated through alpha(v)beta(3) integrin and Fas in a synergistic manner.

The transcriptional terminator sequences downstream of the covR gene terminate covR/S operon transcription to generate covR monocistronic transcripts in Streptococcus pyogenes.

CovR/S is an important two component regulatory system, which regulates about 15% of the gene expression in Streptococcus pyogenes. The covR/S locus was identified as an operon generating an RNA transcript around 2.5-kb in size. In this study, we found the covR/S operon produced three RNA transcripts (around 2.5-, 1.0-, and 0.8-kb in size). Using RNA transcriptional terminator sequence prediction and transcriptional terminator analysis, we identified two atypical rho-independent terminator sequences downstream of the covR gene and showed these terminator sequences terminate RNA transcription efficiently. These results indicate that covR/S operon generates covR/S transcript and monocistronic covR transcripts.

The severity of Streptococcus pyogenes infections in children is significantly associated with plasma levels of inflammatory cytokines.

Cytokines are intimately involved with the innate and adaptive immune response to bacterial infections. This study was designed to determine the expression of inflammatory cytokines in children by the severity of Streptococcus pyogenes (group A Streptococcus [GAS]) infections. The study population consisted of 16 invasive, 20 noninvasive, and 24 pharyngeal colonization, and 21 healthy controls. All children underwent the laboratory tests and cytokine measurement. GAS isolates were analyzed for emm gene typing. Patients with invasive GAS diseases had significantly higher interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6, IL-8, IL-10, and IL-18 than those with noninvasive diseases, colonization, and healthy controls. There was no difference in tumor necrosis factor (TNF)-alpha, IL-12, and IL-2 levels among the groups. Elevated white blood cell counts and levels of C-reactive protein and C3 were detected only in patients with invasive diseases. emm1 and emm12 predominated in invasive disease and colonization. Children with invasive GAS infections exhibited significant up-regulation of plasma levels of IFN-gamma, IL-1beta, IL-6, IL-8, IL-10, and IL-18, and suppression of TNF-alpha and IL-12 during the acute phase of their illness. An exuberant cytokine response was associated with the severity of illness.

Pathogenetic roles of angiogenic factors in pyogenic granulomas in pregnancy are modulated by female sex hormones.

BACKGROUND: An abundance of microvessels is the major phenotype of pyogenic granuloma, which has been considered a hormone-related lesion based on clinical observations. Although angiogenic factors and inflammatory cytokines have been implied to play roles in the pathogenesis of pyogenic granuloma, their links to female steroid hormones still remain to be elucidated. Since apoptosis is important in limiting inflammation, we also investigated whether steroid hormones could protect granuloma cells from apoptosis and, therefore, lead to overreactive inflammatory response. METHODS: We employed immunoassays in a series of experiments, including human pyogenic granuloma in pregnancy, mouse air pouch granuloma and U937 (monoblastoid) cells in culture to clarify the relationship among vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and female steroid hormones in granuloma formation. The apoptotic rates were analyzed in vivo and in vitro by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling (TUNEL) and flow cytometry, respectively. RESULTS: Both in human and animal studies, the immunoassays (enzyme-linked immunoabsorbent assay [ELISA] and immunohistochemistry) detected significantly more VEGF and bFGF and less TNF-alpha in hormones than the control group, while TUNEL assay revealed less apoptotic cells in groups with pregnancy levels of hormones. In vitro, progesterone enhanced the expression of VEGF in LPS-treated U937 cells. Both estrogen and progesterone inhibited the apoptosis of U937 cells triggered by exogenous TNF-a CONCLUSIONS: Female steroid hormones may have dual effects on the pathogenesis of pyogenic granuloma in pregnancy. The hormones not only enhance the expression of angiogenic factors in inflamed tissue, but also decrease apoptosis of granuloma cells to extend angiogenic effect.

The IL-8 production by Streptococcal pyrogenic exotoxin B.

We have previously identified alpha(v)beta(3) and Fas as receptors for the streptococcal pyrogenic exotoxin B (SPE B), and G308S, a mutant of SPE B with RSD motif, which interacts with Fas only. This study aims to evaluate how SPE B interacts with cells to induce the production of IL-8. Our results showed that following exposure to SPE B or G308S, the levels of IL-8 protein and mRNA were increased and the increase was inhibited by the addition of anti-Fas antibody, suggesting that the increased production of IL-8 by SPE B is mediated through Fas receptor. In the presence of G308S, the association of FADD and procaspase 8, and activation of NF-kappaB were also detected. The application of siRNA of FADD and of procaspase 8 could inhibit the NF-kappaB activity. The proteolytic activity of caspase 8 was required for the NF-kappaB activity. Further studies showed that G308S could increase the phosphorylation of ERK and the translocation of NF-kappaB into the nucleus, and the inhibition of ERK phosphorylation decreased the IL-8 production, mRNA expression and activation of NF-kappaB. In addition, siRNA of procaspase 8 could inhibit the G308S-induced cleavage of MEKK1, binding of MEKK1 to caspase 8, activation of ERK and the NF-kappaB activity. Taken together, the production of IL-8 by SPE B in A549 cells is mediated by Fas, and followed by the activation of FADD, caspase 8, MEKK1, ERK and NF-kappaB.

Solution structure and backbone dynamics of streptopain: insight into diverse substrate specificity.

Streptococcal pyrogenic exotoxin B (SPE B) is a cysteine protease expressed by Streptococcus pyogenes. The D9N, G163S, G163S/A172S, and G239D mutant proteins were expressed to study the effect of the allelic variants on their protease activity. In contrast to other mutants, the G239D mutant was approximately 12-fold less active. The Gly-239 residue is located within the C-terminal S230-G239 region, which cannot be observed in the x-ray structure. The three-dimensional structure and backbone dynamics of the 28-kDa mature SPE B (mSPE B) were determined. Unlike the x-ray structure of the 40-kDa zymogen SPE B (proSPE B), we observed the interactions between the C-terminal loop and the active site residues in mSPE B. The structural differences between mSPE B and proSPE B were the conformation of the C-terminal loop and the orientation of the catalytic His-195 residue, suggesting that activation and inactivation of SPE B is involved in the His-195 side-chain rotation. Dynamics analysis of mSPE B and the mSPE B/inhibitor complexes showed that the catalytic and C-terminal loops were the most flexible regions with low order parameter values of 0.5 to 0.8 and exhibited the motion on the ps/ns timescale. These findings suggest that the flexible C-terminal loop of SPE B may play an important role in controlling the substrate binding, resulting in its broad substrate specificity.

Streptococcal pyrogenic exotoxin B cleaves human S-adenosylhomocysteine hydrolase and induces hypermethioninemia.

Group A Streptococcus is a common pathogen that causes pharyngitis, impetigo, myositis, and lethal streptococcal toxic shock syndrome. Streptococcal pyrogenic exotoxin B (SPE B) is strongly associated with the severity of disease. SPE B is a cysteine protease and matures itself by autocatalysis. We found that SPE B was directly associated with human S-adenosylhomocysteine hydrolase (AdoHcyase), an essential factor for a delayed-type immune response. AdoHcyase protein levels and enzymatic activities were significantly higher in human cells infected with the Streptococcus pyogenes SW510 speB mutant strain than in cells infected with the NZ131 wild-type strain. SPE B also inactivated AdoHcyase, shown by a decrease in homocysteine, the main product of AdoHcyase. We found that in vivo and in vitro, SPE B induced hypermethioninemia, which is caused by an AdoHcyase defect. We also found that AdoHcyase is a substrate of SPE B cysteine protease. SPE B, therefore, potentially causes immunosuppression by cleaving AdoHcyase.

The fate of SPE B after internalization and its implication in SPEB-induced apoptosis.

After streptococcal pyrogenic exotoxin B (SPE B) induces apoptosis, its fate is unknown. Using confocal time-course microscopy at 37 degrees C, we detected green fluorescence 20 min after adding FITC-SPE B. Orange fluorescence, an indication of co-localization of SPE B with lysosomes which were labeled with a red fluorescent probe, was maximal at 40 min and absent by 60 min. SPE B was co-precipitated with clathrin, which is consistent with endocytotic involvement. Western blotting assay also indicated that uptake of SPE B was maximal at 40 min and disappeared after 60 min. However, in the presence of chloroquine, a lysosome inhibitor, the uptake of SPE B was not detectable. The disappearance of TCA-precipitated FITC-SPE B was parallel to the appearance of TCA soluble FITC-SPE B; in the presence of chloroquine, however, no SPE B degradation occurred. Chloroquine increased the level of SPE B-induced apoptosis by inhibiting the degradation of SPE B. These results suggest that the internalization and degradation of SPE B in cells may be a host defense system that removes toxic substances by sacrificing the exposed cells.

Streptococcal pyrogenic exotoxin B causes mitochondria damage to polymorphonuclear cells preventing phagocytosis of group A streptococcus.

The streptococcal pyrogenic exotoxin B (SpeB) is known to be involved in group A streptococcus (GAS) survival in blood, but the detailed mechanism is not clear. For clarification of this issue, speB isogenic mutants of strains M6 and M49 were constructed by using an integrational plasmid and confirmed by Southern blot analysis. The resistance to phagocytosis of wild-type strains and their speB isogenic mutants was analyzed. The results demonstrated a five-fold increase in phagocytosis of speB mutants compared to that of wild-type strains in whole blood, but no significant difference in plasma. To further clarify whether this effect is due to a functional SpeB protein, recombinant SpeB (r-SpeB) and a SpeB mutant protein lacking proteinase activity (r-C192S) were purified and incubated with a speB mutant in whole blood. The results showed a two- to threefold increase in resistance to phagocytosis when the M6 speB mutant was incubated with r-SpeB, but not with r-C192S. Incubation with the wild-type strain, speB mutant, or the r-SpeB protein did not affect the total cell number of polymorphonuclear (PMN) cells in whole blood under laboratory conditions. However, the PMN cells' mitochondria showed decreasing dehydrogenase activity and loss of membrane potential after r-SpeB treatment. These data indicate that SpeB could cause the mitochondria damage to the PMN cells, preventing immune clearance at an early infectious stage.

Effects of oligopeptide permease in group a streptococcal infection.

The oligopeptide permease (Opp) of group A streptococci (GAS) is a membrane-associated protein and belongs to the ATP-binding cassette transporter family. It is encoded by a polycistronic operon containing oppA, oppB, oppC, oppD, and oppF. The biological function of these genes in GAS is poorly understood. In order to understand more about the effects of Opp on GAS virulence factors, an oppA isogenic mutant was constructed by using an integrative plasmid to disrupt the opp operon and confirmed by Southern blot hybridization. No transcript was detected in the oppA isogenic mutant by Northern blot analysis and reverse transcriptase PCR. The growth curve for the oppA isogenic mutant was similar to that for wild-type strain A-20. The oppA isogenic mutant not only decreased the transcription of speB, speX, and rofA but also increased the transcription of speF, sagA (streptolysin S-associated gene A), slo (streptolysin O), pel (pleotrophic effect locus), and dppA (dipeptide permease). No effects on the transcription of emm, sda, speJ, speG, rgg, and csrR were found. The phenotypes of the oppA mutant were restored by the oppA revertant and by the complementation strain. The oppA mutant caused less mortality and tissue damage than the wild-type strain when inoculated into BALB/c mice via an air pouch. Based on these data, we suggest that the opp operon plays an important role in the pathogenesis of GAS infection.

Pathogenetic Roles of Angiogenic Factors in Pyogenic Granulornas in Pregnancy Are Modulated by Female Sex Hormones.

BACKGROUND: An abundance of microvessels is the major phenotype of pyogenic granuloma, which has been considered a hormone-related lesion based on clinical observations. Although angiogenic factors and inflammatory cytokines have been implied to play roles in the pathogenesis of pyogenic granuloma, their links to female steroid hormones still remain to be elucidated. Since apoptosis is important in limiting inflammation, we also investigated whether steroid hormones could protect granuloma cells from apoptosis and, therefore, lead to over-reactive inflammatory response. METHODS: We employed immunoassays in a series of experiments, including human pyogenic granuloma in pregnancy, mouse air pouch granuloma and U937 (monoblastoid) cells in culture to clarify the relationship among vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor-alpha (TNF-α), interleukin (IL)- 1 beta and female steroid hormones in granuloma formation. The apoptotic rates were analyzed in vivo and in vitro by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphosphate nick end labeling. (TUNEL) and flow cytometry, respectively. RESULTS: Both in human and animal studies, the immunoassays (enzyme-linked immunoabsorbent assay [ELISA] and irnmunohistochemistry) detected significantly more VEGF and bFGF and less TNF-α in hormones than the control group, while TUNEL assay revealed less apoptotic cells in groups with pregnancy levels of hormones. In vitro, progesterone enhanced the expression of VEGF in LPS-treated U937 cells. Both estrogen and progesterone inhibited the apoptosis of U937 cells triggered by exogenous TNF-α Conclusions: Female steroid hormones may have dual effects on the pathogenesis of pyogenic granuloma in pregnancy. The hormones not only enhance the expression of angiogenic factors in inflamed tissue, but also decrease apoptosis of granuloma cells to extend angiogenic effect. J Periodontol 2002;73:701-708.

Enamel matrix derivative exhibits angiogenic effect in vitro and in a murine model.

OBJECTIVES: Angiogenesis is one of the most critical events in the wound healing process. Any increase in angiogenesis could result in more rapid and complete healing. A recent study found that enamel matrix derivative (EMD) could accelerate early periodontal wound healing. We wanted to clarify whether EMD caused an angiogenic effect and, thus, possibly enhanced wound healing. METHODS: We performed in vitro proliferation and chemotaxis assays on human umbilical vein endothelial cell (HUVEC) cultures, and a tissue culture assay using blood vessel fragments in fibrin gel. Collagen membranes soaked with EMD were implanted subcutaneously in mice to test the in vivo angiogenic effect. RESULTS: While there were no significant differences between the negative control and EMD groups in the proliferation assay, EMD treatment did exhibit a significantly greater dose-dependent chemotactic effect on HUVEC than control group treatments. The tissue culture in fibrin gel showed new blood vessel outgrowths in the EMD groups, but none in the negative control group. In the animal studies, significantly more endothelial cells were detected in the EMD group of mice. CONCLUSIONS: Our findings show that EMD does exhibit some angiogenic effects. However, the underlying molecules and mechanisms are still unidentified. We discuss several possibilities.

Syndecan-1 up-regulated by ephrinB2/EphB4 plays dual roles in inflammatory angiogenesis.

EphrinB2 and EphB4, its cognate receptor, are important in the vascular development of the mouse embryo. Their roles in human inflammatory angiogenesis, however, are not well understood. By examining hyperinflammatory lesions, we saw that ephrinB2 was predominantly expressed in macrophage-like cells and EphB4 in small venules. Because macrophages usually transmigrate through postcapillary venules during inflammation, we wanted to explore the downstream effects of EphB4 after binding to ephrinB2. By using cDNA microarray technique and following reverse transcriptase-polymerase chain reaction (RT-PCR), we found that syntenin and syndecan-1 were up-regulated in EphB4-positive endothelial cells dose dependently and time dependently after stimulation with preclustered ephrinB2. In vitro, ephrinB2 suppressed the angiogenic effects of basic fibroblast growth factor (bFGF) on EphB4-positive endothelial cells, partially due to syndecan-1's competition with fibroblast growth factor receptor (FGFR) for bFGF. However, ephrinB2 exhibited angiogenic effects in vivo, possibly due to an inflammation-associated enzyme-heparanase. The enzymes could convert the inhibitory effect of ephrinB2 on EphB4-positive endothelial cells to an activating effect by removing poorly sulfated side chains of up-regulated syndecan-1 ectodomain. Depending on the presence of heparanases, the roles of syndecan-1 may be opposite in different physiological settings.