GroEL of Porphyromonas gingivalis-induced microRNAs accelerate tumor neovascularization by downregulating thrombomodulin expression in endothelial progenitor cells.

We found that GroEL in Porphyromonas gingivalis accelerated tumor growth and increased mortality in tumor-bearing mice; GroEL promoted proangiogenic function, which may be the reason for promoting tumor growth. To understand the regulatory mechanisms by which GroEL increases the proangiogenic function of endothelial progenitor cells (EPCs), we explored in this study. In EPCs, MTT assay, wound-healing assay, and tube formation assay were performed to analyze its activity. Western blot and immunoprecipitation were used to study the protein expression along with next-generation sequencing for miRNA expression. Finally, a murine tumorigenesis animal model was used to confirm the results of in vitro. The results indicated that thrombomodulin (TM) direct interacts with PI3 K/Akt to inhibit the activation of signaling pathways. When the expression of TM is decreased by GroEL stimulation, molecules in the PI3 K/Akt signaling axis are released and activated, resulting in increased migration and tube formation of EPCs. In addition, GroEL inhibits TM mRNA expression by activating miR-1248, miR-1291, and miR-5701. Losing the functions of miR-1248, miR-1291, and miR-5701 can effectively alleviate the GroEL-induced decrease in TM protein levels and inhibit the proangiogenic abilities of EPCs. These results were also confirmed in animal experiments. In conclusion, the intracellular domain of the TM of EPCs plays a negative regulatory role in the proangiogenic capabilities of EPCs, mainly through direct interaction between TM and PI3 K/Akt to inhibit the activation of signaling pathways. The effects of GroEL on tumor growth can be reduced by inhibiting the proangiogenic properties of EPCs through the inhibition of the expression of specific miRNAs.

Tumor Necrosis Factor Superfamily 14 (LIGHT) Restricts Neovascularization by Decreasing Circulating Endothelial Progenitor Cells and Function.

Tumor necrosis factor superfamily 14 (TNFSF14) is also known as the LT-related inducible ligand (LIGHT). It can bind to the herpesvirus invasion mediator and lymphotoxin-ß receptor to perform its biological activity. LIGHT has multiple physiological functions, including strengthening the synthesis of nitric oxide, reactive oxygen species, and cytokines. LIGHT also stimulates angiogenesis in tumors and induces the synthesis of high endothelial venules; degrades the extracellular matrix in thoracic aortic dissection, and induces the expression of interleukin-8, cyclooxygenase-2, and cell adhesion molecules in endothelial cells. While LIGHT induces tissue inflammation, its effects on angiogenesis after tissue ischemia are unclear. Thus, we analyzed these effects in the current study. In this study, the animal model of hind limb ischemia surgery in C57BL/6 mice was performed. Doppler ultrasound, immunohistochemical staining, and Western blotting were employed to analyze the situation of angiogenesis. In addition, human endothelial progenitor cells (EPCs) were used for in vitro studies to analyze the possible mechanisms. The results in the animal study showed that LIGHT injection inhibited angiogenesis in ischemic limbs. For the in vitro studies, LIGHT inhibited the expression of integrins and E-selectin; decreased migration and tube formation capabilities, mitochondrial respiration, and succinate dehydrogenase activity; and promoted senescence in EPCs. Western blotting revealed that the impairment of EPC function by LIGHT may be due to its effects on the proper functioning of the intracellular Akt signaling pathway, endothelial nitrite oxide synthase (eNOS), and mitochondrial respiration. In conclusion, LIGHT inhibits angiogenesis after tissue ischemia. This may be related to the clamped EPC function.

Far-Infrared Therapy Decreases Orthotopic Allograft Transplantation Vasculopathy.

Orthotopic allograft transplantation (OAT) is a major strategy for solid heart and kidney failure. However, the recipient's immunity-induced chronic rejection induces OAT vasculopathy that results in donor organ failure. With the exception of immunosuppressive agents, there are currently no specific means to inhibit the occurrence of OAT vasculopathy. On the other hand, far-infrared (FIR) therapy uses low-power electromagnetic waves given by FIR, with a wavelength of 3-25 µm, to improve human physiological functions. Previous studies have shown that FIR therapy can effectively inhibit inflammation. It has also been widely used in adjuvant therapy for various clinical diseases, especially cardiovascular diseases, in recent years. Thus, we used this study to explore the feasibility of FIR in preventing OAT vasculopathy. In this study, the model of transplantation of an aorta graft from PVG/Seac rat to ACI/NKyo rat, and in vitro model of human endothelial progenitor cells (EPCs) was used. In this report, we presented that FIR therapy decreased the serious of vasculopathy in OAT-recipient ACI/NKyo rats via inhibiting proliferation of smooth muscle cells, accumulation of collagen, and infiltration of fibroblast in the vessel wall; humoral and cell-mediated immune responses were decreased in the spleen. The production of inflammatory proteins/cytokines also decreased in the plasma. Additionally, FIR therapy presented higher mobilization and circulating EPC levels associated with vessel repair in OAT-recipient ACI/NKyo rats. In vitro studies demonstrated that the underlying mechanisms of FIR therapy inhibiting OAT vasculopathy may be associated with the inhibition of the Smad2-Slug axis endothelial mesenchymal transition (EndoMT). Thus, FIR therapy may be the strategy to prevent chronic rejection-induced vasculopathy.

Impact of Metal-Organic Frameworks on Protein Expression.

The influence of excessive metal-organic frameworks (MOFs) on the translation process was investigated in this study. Different concentrations and sizes of MOFs were tested through adding them to a cell-free expression system and observing the downstream expression signal. Furthermore, a proteomics analysis has been employed to explore and reveal the influence of MOFs on the protein expression, which can provide significant referential value for developing a delivery system with MOFs.

A Novel Relative High-Density Lipoprotein Index to Predict the Structural Changes in High-Density Lipoprotein and Its Ability to Inhibit Endothelial-Mesenchymal Transition.

Therapeutic elevation of high-density lipoprotein (HDL) is thought to minimize atherogenesis in subjects with dyslipidemia. However, this is not the case in clinical practice. The function of HDL is not determined by its concentration in the plasma but by its specific structural components. We previously identified an index for the prediction of HDL functionality, relative HDL (rHDL) index, and preliminarily explored that dysfunctional HDL (rHDL index value > 2) failed to rescue the damage to endothelial progenitor cells (EPCs). To confirm the effectiveness of the rHDL index for predicting HDL functions, here we evaluated the effects of HDL from patients with different rHDL index values on the endothelial-mesenchymal transition (EndoMT) of EPCs. We also analyzed the lipid species in HDL with different rHDL index values and investigated the structural differences that affect HDL functions. The results indicate that HDL from healthy adults and subjects with an rHDL index value < 2 protected transforming growth factor (TGF)-ß1-stimulated EndoMT by modulating Smad2/3 and Snail activation. HDL from subjects with an rHDL index value > 2 failed to restore the functionality of TGF-ß1-treated EPCs. Lipidomic analysis demonstrated that HDL with different rHDL index values may differ in the composition of triglycerides, phosphatidylcholine, and phosphatidylinositol. In conclusion, we confirmed the applicability of the rHDL index value to predict HDL function and found structural differences that may affect the function of HDL, which warrants further in-depth studies.

Transferring Plasmon Effect on a Biological System: Expression of Biological Polymers in Chronic Rejection and Inflammatory Rat Model.

The plasmon-activated water (PAW) that reduces hydrogen bonds is made of deionized reverse osmosis water (ROW). However, compared with ROW, PAW has a significantly higher diffusion coefficient and electron transfer rate constant in electrochemical reactions. PAW has a boiling point of97 °C and specific heat of0.94; the energy of PAW is also 1121 J/mol higher than ordinary water. The greater the force of hydrogen bonds between H2O, the larger the volume of the H2O cluster, and the easier it is to lose the original characteristics. The hydrogen bonding force of PAW is weak, so the volume of its cluster is small, and it exists in a state very close to a single H2O. PAW has a high permeability and diffusion rate, which can improve the needs of biological applications and meet the dependence of biological organisms on H2O when performing physiological functions. PAW can successfully remove free radicals, and efficiently reduce lipopolysaccharide (LPS)-induced monocytes to release nitric oxide. PAW can induce expression of the antioxidant gene Nrf2 in human gingival fibroblasts, lower amyloid burden in mice with Alzheimer's disease, and decrease metastasis in mice grafted with Lewis lung carcinoma cells. Because the transferring plasmon effect may improve the abnormality of physiological activity in a biological system, we aimed to evaluate the influence of PAW on orthotopic allograft transplantation (OAT)-induced vasculopathy in this study. Here, we demonstrated that daily intake of PAW lowered the progression of vasculopathy in OAT-recipient ACI/NKyo rats by inhibiting collagen accumulation, proliferation of smooth muscle cells and fibroblasts, and T lymphocyte infiltration in the vessel wall. The results showed reduced T and B lymphocytes, plasma cells, and macrophage activation in the spleen of the OAT-recipient ACI/NKyo rats that were administered PAW. In contrast to the control group, the OAT-recipient ACI/NKyo rats that were administered PAW exhibited higher mobilization and levels of circulating endothelial progenitor cells associated with vessel repair. We use the transferring plasmon effect to adjust and maintain the biochemical properties of water, and to meet the biochemical demand of organisms. Therefore, this study highlights the therapeutic roles of PAW and provides more biomedical applicability for the transferring plasmon effect.

Dipeptidyl Peptidase-4 Inhibitor Decreases Allograft Vasculopathy Via Regulating the Functions of Endothelial Progenitor Cells in Normoglycemic Rats.

PURPOSE: Chronic rejection induces the occurrence of orthotopic allograft transplantation (OAT) vasculopathy, which results in failure of the donor organ. Numerous studies have demonstrated that in addition to regulating blood sugar homeostasis, dipeptidyl peptidase-4 (DPP-4) inhibitors can also provide efficacious therapeutic and protective effects against cardiovascular diseases. However, their effects on OAT-induced vasculopathy remain unknown. Thus, the aim of this study was to investigate the direct effects of sitagliptin on OAT vasculopathy in vivo and in vitro. METHODS: The PVG/Seac rat thoracic aorta graft to ACI/NKyo rat abdominal aorta model was used to explore the effects of sitagliptin on vasculopathy. Human endothelial progenitor cells (EPCs) were used to investigate the possible underlying mechanisms. RESULTS: We demonstrated that sitagliptin decreases vasculopathy in OAT ACI/NKyo rats. Treatment with sitagliptin decreased BNP and HMGB1 levels, increased GLP-1 activity and stromal cell-derived factor 1α (SDF-1α) expression, elevated the number of circulating EPCs, and improved the differentiation possibility of mononuclear cells to EPCs ex vivo. However, in vitro studies showed that recombinant B-type natriuretic peptide (BNP) and high mobility group box 1 (HMGB1) impaired EPC function, whereas these phenomena were reversed by glucagon-like peptide 1 (GLP-1) receptor agonist treatment. CONCLUSIONS: We suggest that the mechanisms underlying sitagliptin-mediated inhibition of OAT vasculopathy probably occur through a direct increase in GLP-1 activity. In addition to the GLP-1-dependent pathway, sitagliptin may regulate SDF-1α levels and EPC function to reduce OAT-induced vascular injury. This study may provide new prevention and treatment strategies for DPP-4 inhibitors in chronic rejection-induced vasculopathy.

Dark channel prior based video dehazing algorithm with sky preservation and its embedded system realization for ADAS applications.

Dark Channel Prior (DCP) is one of the significant dehazing methods based upon the observation of the key features of the haze-free images. But it has disadvantages; high computational complexity, over-enhancement in the sky region, flickering artefacts in video processing, and poor dehazing. Therefore, we propose improved solutions to solve the aforementioned drawbacks. First, we adopt the fast one-dimensional filter, look-up table, and program optimization to reduce the computational complexity. Next, we follow by using a part of the guided filter for sky detection and to preserve the sky region from noise by avoiding over recovery. Then, we propose an airlight update strategy and adjust the radius of a guided filter to reduce the flickering artifacts and also propose an airlight estimation method to produce the better dehazing result as the final step of our algorithm. The improved results from our proposed algorithm are stable and are obtained from the real-time processing suitable for ADAS, surveillance, and monitoring systems. The implementation of the proposed algorithm has yielded a processing speed of 75 fps and 23 fps respectively on an NVIDIA Jetson TX1 embedded platform and Renesas R-Car M2, both on D1 (720x480) resolution videos.

B-type natriuretic peptide enhances fibrotic effects via matrix metalloproteinase-2 expression in the mouse atrium in vivo and in human atrial myofibroblasts in vitro.

B-type natriuretic peptide (BNP) was approved by the US Food and Drug Administration in 2001 for the treatment of heart failure. However, the effects of BNP in clinical applications are controversial and uncertain. Recently, study indicated that high BNP levels are associated with an increased risk of developing atrial fibrillation. In this study, we investigated the direct effects of BNP on TNF-α-induced atrial fibrosis mice, as well as its effects on human atrial myofibroblasts. We found that injecting TNF-α-induced mice with recombinant human BNP enhanced atrial fibrosis via matrix metalloproteinase-2 (MMP-2) expression and collagen accumulation. Furthermore, we found that BNP stimulated MMP-2 expression in human atrial myofibroblasts. Treatment of human atrial myofibroblasts with cycloheximide had no effect on this outcome; however, treatment of cells with MG132 enhanced BNP-induced MMP-2 expression, indicating that protein stability and inhibition of proteasome-mediated protein degradation pathways are potentially involved. Inhibition of SIRT1 significantly decreased BNP-induced MMP-2 expression. Additionally, confocal and coimmunoprecipitation data indicated that BNP-regulated MMP-2 expression are likely to be mediated through direct interaction with SIRT1, which is thought to deacetylate MMP-2 and to increase its protein stability in human atrial myofibroblasts. Finally, we confirmed that SIRT1 is expressed and cytoplasmically redistributed as well as colocalized with MMP-2 in mouse fibrotic atrial tissue. We suggest a possible fibrosis-promoting role of BNP in the atrium, although the antifibrotic properties of BNP in the ventricle have been reported in previous studies, and that the coordination between MMP-2 and SIRT1 in BNP-induced atrial myofibroblasts participates in atrial fibrosis.

Dysfunctional high density lipoprotein failed to rescue the function of oxidized low density lipoprotein-treated endothelial progenitor cells: a novel index for the prediction of HDL functionality.

Lipid metabolic disorders play critical roles in atherogenesis. Traditionally, it has been suggested that reduced high density lipoprotein (HDL) levels might be an important morbidity indicator for cardiovascular diseases. Therefore, it has been argued that therapeutically raising HDL levels may reduce atherogenesis in patients with dyslipidemia. However, recent clinical trials to elevate serum HDL levels by pharmacologic approaches failed to demonstrate clinical efficacy. Thus, to investigate the functionality of HDL and to explore the possible clinical relevance as well as to define an effective indicator that can represent HDL function may provide another key and reference to disclose the clinical treatment of dyslipidemia. We analyzed the association between the data of dichlorofluorescein assay (assay the functionality of HDL), the effect of HDL on oxidized low density lipoprotein (oxLDL)-stimulated endothelial progenitor cells (EPCs) in vitro, levels of circulating EPCs, and ex vitro EPC colony forming units of each case, we defined the indicator (relative HDL index (RHDL index) = dichlorofluorescein assay result of each subject/dichlorofluorescein assay reading of our young healthy controls) that may represent functionality of HDL. HDL from healthy adults protected oxLDL-treated EPCs by modulating p38 mitogen-activated protein kinase and Rho activation and by promoting nitric oxide production. HDL from subject with RHDL index ≧2 also failed to restore the functionality of oxLDL-treated EPCs via cell-signaling pathways in vitro. The RHDL index significantly correlated with patients' circulating EPC number or EPC colony forming units ex vivo. In conclusions, we explored the RHDL index as a score to predict a patient's EPC functions in vivo and ex vitro.

Eotaxin-2 induces monocytic apoptosis in patients who have undergone coronary artery bypass surgery and in THP-1 cells in vitro regulated by thrombomodulin.

Cardiopulmonary bypass (CPB) induces cytokine production and causes postoperative monocytic inflammatory responses, which are associated with patient outcomes. In fact, monocytes regulate immunity through dynamic networks of survival and cellular apoptosis as well as thrombomodulin (TM)-associated differenciiation. Whether CPB affects the plasma level of eotaxin-2, a potent chemoattractant, or stimulates monocyte apoptosis among patients undergoing elective coronary artery bypass graft (CABG) surgery is also unknown. Thus, we aimed to investigate this subject and explored the feasible roles of TM in the phenomena. Firstly, clinical data showed that after CABG surgery, patients with lower plasma eotaxin-2 levels and higher TM expression levels exhibited reduced monocytic apoptosis, compared with that in patients with lower TM expression levels. Subsequently, to explore the hypothesis that eotaxin-2 induces monocytic apoptosis mediation by TM expression, we used in vitro monocytic THP-1 cells. The results indicated that treatment of THP-1 cells with eotaxin-2 markedly increased apoptosis. Knockdown of TM significantly increased, and overexpression of TM significantly reversed eotaxin-2-induced monocyte apoptosis, which was compared with that of only eotaxin-2-treated THP-1 cells. TM may regulate mitochondria-mediated apoptosis by its PI3K/Akt axis signaling pathway, which acts as an extinguisher for p53 and BAX activation, as well as limit further downstream release of cytochrome c and cleavage of caspases 8 and 3; we suggest that TM interacts with the cofilin cytoskeleton, which further supports a role for TM in eotaxin-induced THP-1 cell apoptosis. Based on clinical observation and in vitro study, we conclude that TM expression on monocytes is associated with their apoptosis. The above mechanisms may be relevant to clinical phenomena in which patients exhibiting more monocytic apoptosis are complicated by higher plasma levels of eotaxin-2 and lower TM expression on monocytes after CABG surgery.

Self-assembled hemispherical nanowell arrays for superhydrophobic antireflection coatings.

The present study reports an inexpensive and simple bottom-up technology for fabricating superhydrophobic antireflection coatings with highly ordered hemispherical nanowell structures, which are assembled by a scalable Langmuir-Blodgett technology. The subwavelength gratings lead to a gradual change in the effective refractive index, substantially suppressing specular reflection over the entire visible wavelength range. After surface modification with fluorinated silane, the resulting nanowell arrays exhibit superhydrophobic surface with high static water contact angle (154°) and low water contact angle hysteresis (7°). The experimental results on the wettability can be qualitatively interpreted by adopting the Cassie's dewetting model. Moreover, the effect of the nanowell shape on the antireflective and superhydrophobic properties has also been investigated in the study.

Fabrication of biodegradable polyurethane microspheres by a facile and green process.

Two different compositions of water-based biodegradable polyurethane (PU) in the form of homogeneous nanoparticles (NPs) were synthesized using biodegradable polyesters as the soft segment. The first PU (PU01) was based on poly(ε-caprolactone) (PCL) diol and the second PU (PU02) was based on 40% PCL diol and 60% polyethylene butylene adipate diol. The PU NP dispersions with different solid contents were sprayed into liquid nitrogen and resuspended in water to generate elastic microspheres (50-60 µm) with different nanoporosities. In vitro degradation analysis revealed that microspheres of PU02 (i.e., PU02 MS) degraded faster than those of PU01 (PU01 MS). Methylene blue was encapsulated during microsphere formation and the release was investigated. Microspheres made from a lower content (10%) of PU02 dispersion (i.e., PU02 MS_10) showed a greater burst release of methylene blue in 6 h, whereas those made from a higher content (30%) of PU01 dispersion (i.e., PU01 MS_30) revealed a prolonged release with a significantly lower burst release. Biocompatibility evaluation using L929 fibroblasts demonstrated that cells were attached and proliferated on microspheres after 24 h. On the other hand, microspheres may further self-assemble into films and scaffolds. Surface modification of microspheres by chitosan may modify the self-assembly behavior of microspheres. Microspheres could be stacked to form scaffolds with different macroporosities. Fibroblasts were successfully seeded and grown in the microsphere-stacked scaffolds. We concluded that the biodegradable and elastic microspheres may be facilely produced from a green and sustainable process with potential applications in drug release and three-dimensional cell culture.

Improving specific activity and thermostability of Escherichia coli phytase by structure-based rational design.

Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications.

Improving the specific activity of ß-mannanase from Aspergillus niger BK01 by structure-based rational design.

ß-Mannanase has found various biotechnological applications because it is capable of degrading mannans into smaller sugar components. A highly potent example is the thermophilic ß-mannanase from Aspergillus niger BK01 (ManBK), which can be efficiently expressed in industrial yeast strains and is thus an attractive candidate for commercial utilizations. In order to understand the molecular mechanism, which helps in strategies to improve the enzyme's performance that would meet industrial demands, 3D-structural information is a great asset. Here, we present the 1.57Å crystal structure of ManBK. The protein adopts a typical (ß/α)8 fold that resembles the other GH5 family members. Polysaccharides were subsequently modeled into the substrate binding groove to identify the residues and structural features that may be involved in the catalytic reaction. Based on the structure, rational design was conducted to engineer ManBK in an attempt to enhance its enzymatic activity. Among the 23 mutants that we constructed, the most promising Y216W showed an 18±2.7% increase in specific activity by comparison with the wild type enzyme. The optimal temperature and heat tolerance profiles of Y216W were similar to those of the wild type, manifesting a preserved thermostability. Kinetic studies showed that Y216W has higher kcat values than the wild type enzyme, suggesting a faster turnover rate of catalysis. In this study we applied rational design to ManBK by using its crystal structure as a basis and identified the Y216W mutant that shows great potentials in industrial applications.

MK-0626, a dipeptidyl peptidase-4 inhibitor, improves neovascularization by increasing both the number of circulating endothelial progenitor cells and endothelial nitric oxide synthetase expression.

Current treatment modalities for critical limb ischemia (CLI) are of limited benefit; therefore, advances in therapeutic vasculogenesis may open an important new avenue for the treatment of CLI. This study examines the therapeutic potential of the DPP-4 inhibitor MK-0626 as a regulator of vasculogenesis in vivo. MK-0626 was administered daily to C57CL/B6 mice and eGFP-labeled bone marrow-transplanted ICR mice that had undergone hind limb ischemia surgery. Laser Doppler imaging and flow cytometry were used to evaluate the degree of neo-vasculogenesis and the number of circulating endothelial progenitor cells (EPCs), respectively. Cell surface markers of EPCs and the level of endothelial nitric oxide synthase (eNOS) were studied in the vessels. Mice that received MK-0626 had an elevated level of glucagon- like peptide-1 (GLP-1) and a decreased level of dipeptidyl peptidase-4 (DPP-4) in their plasma, in addition to an ischemia-induced increase in the level of stromal cell-derived factor-1 (SDF-1). In C57CL/B6 mice, blood flow in the ischemic limb was significantly improved by treatment with MK-0626. The number of circulating EPCs and both the synthesis and phosphorylation of eNOS were also increased in ischemic thigh muscle after MK-0626 treatment. In contrast, similar effects of MK-0626 were not observed in B6.129P2-Nos3(tm1Unc)/J mice (an eNOS knockout mouse). Additionally, MK-0626 treatment promoted the mobilization and homing of EPCs to ischemic tissue in eGFP transgenic mouse bone marrow-transplanted ICR mice. We conclude that both the number of circulating EPCs and neo-vasculogenesis are increased in response to DPP-4 inhibitor treatment and that this occurs via an eNOS-dependent mechanism. The results highlight the therapeutic vasculogenesis potential of the DPP-4 inhibitor MK-0626 using a hind limb ischemia mouse model.

GroEL1, a heat shock protein 60 of Chlamydia pneumoniae, impairs neovascularization by decreasing endothelial progenitor cell function.

The number and function of endothelial progenitor cells (EPCs) are sensitive to hyperglycemia, hypertension, and smoking in humans, which are also associated with the development of atherosclerosis. GroEL1 from Chlamydia pneumoniae has been found in atherosclerotic lesions and is related to atherosclerotic pathogenesis. However, the actual effects of GroEL1 on EPC function are unclear. In this study, we investigate the EPC function in GroEL1-administered hind limb-ischemic C57BL/B6 and C57BL/10ScNJ (a toll-like receptor 4 (TLR4) mutation) mice and human EPCs. In mice, laser Doppler imaging, flow cytometry, and immunohistochemistry were used to evaluate the degree of neo-vasculogenesis, circulating level of EPCs, and expression of CD34, vWF, and endothelial nitric oxide synthase (eNOS) in vessels. Blood flow in the ischemic limb was significantly impaired in C57BL/B6 but not C57BL/10ScNJ mice treated with GroEL1. Circulating EPCs were also decreased after GroEL1 administration in C57BL/B6 mice. Additionally, GroEL1 inhibited the expression of CD34 and eNOS in C57BL/B6 ischemic muscle. In vitro, GroEL1 impaired the capacity of differentiation, mobilization, tube formation, and migration of EPCs. GroEL1 increased senescence, which was mediated by caspases, p38 MAPK, and ERK1/2 signaling in EPCs. Furthermore, GroEL1 decreased integrin and E-selectin expression and induced inflammatory responses in EPCs. In conclusion, these findings suggest that TLR4 and impaired NO-related mechanisms could contribute to the reduced number and functional activity of EPCs in the presence of GroEL1 from C. pneumoniae.

Evaluation of the antibacterial activity and biocompatibility for silver nanoparticles immobilized on nano silicate platelets.

Silver nanoparticles (AgNPs) are known for their bactericidal abilities. The antibacterial potency is dependent on the particle size and dispersion status. In this study, we synthesized AgNP/NSP nanohybrids in two different weight ratios (1/99 and 8/92) using the fully exfoliated clay, i.e., nanosilicate platelets (NSP), as a dispersing agent and carrier for AgNPs. Due to the size of NSP, the immobilized AgNPs do not enter cells readily, which may lower the risk associated with the cellular uptake of AgNPs. The biocompatibility, immunological response, and antimicrobial activities of AgNP/NSP hybrids were evaluated. The results revealed that AgNP/NSP hybrids elicited merely mild inflammatory response and retained the outstanding antibacterial activity. The hybrids were further embedded in poly(ether)urethane (PEU) to increase the biocompatibility. At the same silver content (20 ppm), the PEU-AgNP/NSP nanocomposites were nontoxic to mouse skin fibroblasts, while simultaneously exhibiting nearly complete bacterial growth reduction (99.9%). PEU containing the same amount of free AgNPs did not display such an effect. Our results verify the better biosafety of the AgNPs/NSP hybrids and their polymer nanocomposites for further clinical use.

GroEL1, from Chlamydia pneumoniae, induces vascular adhesion molecule 1 expression by p37(AUF1) in endothelial cells and hypercholesterolemic rabbit.

The expression of vascular adhesion molecule-1 (VCAM-1) by endothelial cells may play a major role in atherogenesis. The actual mechanisms of chlamydia pneumoniae (C. pneumoniae) relate to atherogenesis are unclear. We investigate the influence of VCAM-1 expression in the GroEL1 from C. pneumoniae-administered human coronary artery endothelial cells (HCAECs) and hypercholesterolemic rabbits. In this study, we constructed the recombinant GroEL1 from C. pneumoniae. The HCAECs/THP-1 adhesion assay, tube formation assay, western blotting, enzyme-linked immunosorbent assay, actinomycin D chase experiment, luciferase reporter assay, and immunohistochemical stainings were performed. The results show that GroEL1 increased both VCAM-1 expression and THP-1 cell adhesives, and impaired tube-formation capacity in the HCAECs. GroEL1 significantly increased the VCAM-1 mRNA stability and cytosolic AU-binding factor 1 (AUF1) level. Overexpression of the p37(AUF1) significantly increased VCAM-1 gene expression in GroEL1-induced bovine aortic endothelial cells (BAECs). GroEL1 prolonged the stability of VCAM-1 mRNA by increasing both p37(AUF1) and the regulation of the 5' untranslated region (UTR) of the VCAM-1 mRNA in BAECs. In hypercholesterolemic rabbits, GroEL1 administration enhanced fatty-streak and macrophage infiltration in atherosclerotic lesions, which may be mediated by elevated VCAM-1 expression. In conclusion, GroEL1 induces VCAM-1 expression by p37(AUF1) in endothelial cells and enhances atherogenesis in hypercholesterolemic rabbits.

Dipeptidyl peptidase-4 inhibitor improves neovascularization by increasing circulating endothelial progenitor cells.

BACKGROUND AND PURPOSE: Current methods used to treat critical limb ischaemia (CLI) are hampered by a lack of effective strategies, therefore, therapeutic vasculogenesis may open up a new field for the treatment of CLI. In this study we investigated the ability of the DPP-4 inhibitor, sitagliptin, originally used as a hypoglycaemic agent, to induce vasculogenesis in vivo. EXPERIMENTAL APPROACH: Sitagliptin were administered daily to C57CL/B6 mice and eGFP transgenic mouse bone marrow-transplanted ICR mice that had undergone hindlimb ischaemic surgery. Laser Doppler imaging and flow cytometry were used to evaluate the degree of neovasculogenesis and circulating levels of endothelial progenitor cells (EPCs) respectively. Cell surface markers of EPCs and endothelial NOS (eNOS) in vessels were studied. KEY RESULTS: Sitagliptin elevated plasma glucagon-like peptide-1 (GLP-1) levels in mice subjected to ischaemia, decreased plasma dipeptidyl peptidase-4 (DPP-4) concentration, and augmented ischaemia-induced increases in stromal cell-derived factor-1 (SDF-1) in a dose-dependent manner. Blood flow in the ischaemic limb was significantly improved in mice treated with sitagliptin. Circulating levels of EPCs were also increased after sitagliptin treatment. Sitagliptin also enhanced the expression of CD 34 and eNOS in ischaemic muscle. In addition, sitagliptin promoted EPC mobilization and homing to ischaemic tissue in eGFP transgenic mouse bone marrow-transplanted ICR mice. CONCLUSION AND IMPLICATIONS: Circulating EPC levels and neovasculogenesis were augmented by the DPP-4 inhibitor, sitagliptin and this effect was dependent on an eNOS-related pathway in a mouse model of hindlimb ischaemia. The results indicate that oral administration of sitagliptin has therapeutic potential as an inducer of vasculogenesis.