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.

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.

Separation of crystal violet dyes and its application to pen ink analysis using CZE and MEKC methods.

A crystal violet (CV) standard was irradiated under a Hg-Cd lamp for different exposure times to obtain various N-demethylation products. CZE effectively separated the photodegradation products based on molecular weight differences. In contrast, micellar EKC (MEKC), using SDS as the surfactant, was ineffective because the binding constants of the demethylation products and SDS were too close for separation. Nevertheless, MEKC analysis of ink has applications in forensic science because MEKC separated neutral components in the inks. Thus, MEKC can be used to obtain an ink "fingerprint" since each ink is unique depending on the location and time it was made. In contrast, CZE is useful for dating inks because CV is the primary ink dye and it photodegrades slowly.

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.

UV light-emitting diode-induced fluorescence detection combined with online sample concentration techniques for capillary electrophoresis.

The application of an ultraviolet (UV) light-emitting diode (LED) to on-line sample concentration/fluorescence detection in capillary electrophoresis (CE) is described. The utility of a UV-LED (peak emission wavelength at 380 nm, approximately 2 mW) for fluorescence detection was demonstrated by examining both a naturally fluorescent (riboflavin) compound and a nonfluorescent compound (tryptophan), respectively. The detection limit for riboflavin was determined to be 0.2 ppm by the normal MEKC mode, which was improved to 3-7 ppb when dynamic pH-junction technique was applied. On the other hand, the detection limit of the tryptophan derivative was determined to be 1.5 ppm using the MEKC mode, which was improved to 3 ppb when the sweeping-MEKC mode was applied. In an analysis of an actual sample, the concentrations of riboflavin in beer, and tryptophan in urine and milk samples were determined, respectively.

Large-volume sample sweeping with a high theoretical plate number using a coupled-capillary in capillary electrophoresis.

A large-volume sample injection (> 5 microL) with an extremely high theoretical plate number (N > 10(7)) was achieved when the sweeping-MEKC mode and a coupled-capillary (100 - 50 microm i.d.) were simultaneously used in a capillary electrophoresis (CE) separation. A low-cost and compact violet-LED ( approximately 2 mW) was used as the fluorescence excitation source. As a result, the theoretical plate numbers of the detected peaks (two model compounds: naphthalene-2,3-dicarboxaldehyde derivatized-dopamine and -norepinephrine) were 1.0 x 10(7) and 7.4 x 10(6), respectively. The limits of detection (at S/N = 3) of these were determined to be 2.8 x 10(-10) M (92 ppt) and 2.3 x 10(-10) M (83 ppt), respectively.

Thrombomodulin regulates monocye differentiation via PKCδ and ERK1/2 pathway in vitro and in atherosclerotic artery.

Thrombomodulin (TM) modulates the activation of protein C and coagulation. Additionally, TM regulates monocyte migration and inflammation. However, its role on monocyte differentiation is still unknown. We investigated the effects of TM on monocyte differentiation. First, we found that TM was increased when THP-1 cells were treated with phorbol-12-myristate-13-acetate (PMA). Overexpression of TM enhanced the macrophage markers, CD14 and CD68 expression in PMA-induced THP-1. TM siRNA depressed the PMA-induced increase of p21Cip1/WAF1 via ERK1/2-NF-kB p65 signaling. TM regulated cytoskeletal reorganization via its interaction with paxillin, cofilin, LIMK1, and PYK2. In addition, PMA-induced p21Cip1/WAF1 expression, CD14-positive cell labeling intensity and ERK1/2 phosphorylation were markedly inhibited when protein kinase C-δ (PKCδ) was knocked down. We identified that TM directly interacts with PKCδ. PKCδ was highly expressed in human atherosclerotic arteries and colocalized with TM in CD68-positive infiltrated macrophages of plaques, indicating that the coordination between TM and PKCδ in macrophages participated in atherogenesis. TM may act as a scaffold for PKCδ docking, which keeps PKCδ in the region close to the monocyte membrane to promote the activation of ERK1/2. Taken together, our findings suggest that TM-PKCδ interaction may contribute to cardiovascular disorders by affecting monocye differentiation, which may develop future therapeutic applications.

The role of calpain-myosin 9-Rab7b pathway in mediating the expression of Toll-like receptor 4 in platelets: a novel mechanism involved in α-granules trafficking.

Toll-like receptors (TLRs) plays a critical role in innate immunity. In 2004, Aslam R. and Shiraki R. first determined that murine and human platelets express functional TLRs. Additionally, Andonegui G. demonstrated that platelets express TLR4, which contributes to thrombocytopenia. However, the underlying mechanisms of TLR4 expression by platelets have been rarely explored until now. The aim of this study was to identify the mechanism of TLR4 expression underlying thrombin treatment. The human washed platelets were used in this study. According to flowcytometry and western blot analysis, the surface levels of TLR4 were significantly enhanced in thrombin-activated human platelets and decreased by TMB-8, calpeptin, and U73122, but not Y27632 (a Rho-associated protein kinase ROCK inhibitor) indicating that thrombin-mediated TLR4 expression was modulated by PAR/PLC pathway, calcium and calpain. Co-immunoprecipitation (co-IP) assay demonstrated that the interaction between TLR4 and myosin-9 (a substrate of calpain) was regulated by calpain; cleavage of myosin-9 enhanced TLR4 expression in thrombin treated platelets. Transmission electron microscope data indicated that human platelets used α-granules to control TLR4 expression; the co-IP experiment suggested that myosin-9 did not coordinate with Rab7b to negatively regulate TLR4 trafficking in thrombin treated platelets. In summary, phospholipase Cγ-calpain-myosin 9-Rab7b axis was responsible for the mechanism underlying the regulation of TLR4 containing α-granules trafficking in thrombin-stimulated platelets, which was involved in coagulation.

Low-temperature bath/coupled-capillary/sweeping-micellar electrokinetic capillary chromatography for the separation of naphthalene-2,3-dicarboxaldehyde-derivatized dopamine and norepinephrine.

The use of a low-temperature (0 degrees C) bath-assisted coupled capillary for the separation of naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized dopamine and norepinephrine using the sweeping-micellar electrokinetic capillary chromatography (MEKC) mode is described. In this technique, a capillary consisting of two portions with different inside diameters is used. Therefore, the field strength inside the capillary is different. Hence, the electrophoretic migration velocities of the analytes and the electro-osmotic flow (EOF) also are different. Furthermore, when a portion of the capillary (wide portion, used for sweeping) is immersed in a low-temperature bath, the viscosity of the buffer and the retention factor of the analytes inside are increased. Thus, not only are the interactions between the SDS micelles and the analytes increased, but the SDS-analytes also move more slowly. As a result, a more complete separation can be achieved, even when the sample injection volume is large, up to approximately 2 microL. In general, when the volume of an injected sample is larger, the effects of sweeping and separation would become insufficient, especially when the retention values (k) of the analytes are quite different. However, this limitation can be improved when the low-temperature bath/coupled capillary/sweeping-MEKC mode is used.

Full-capillary sample stacking/sweeping-MEKC for the separation of naphthalene-2,3-dicarboxaldehyde-derivatized tryptophan and isoleucine.

In an attempt to improve the sensitivity of detection in capillary electrophoresis (CE), a novel online sample-concentration method, full-capillary sample stacking (FCSS)/sweeping-micellar electrokinetic chromatography (sweeping-MEKC) mode, is proposed. Naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized tryptophan and isoleucine were selected as model compounds. In the initial step, the weakly acidic compounds, dissolved in a low-conductivity buffer (35.1 microS/cm; apparent ph (pH*) in a mixed solution of acetonitrile/methanol/water, 4.6), fill the entire capillary, two vials of a high-conductivity buffer (2.06 mS/cm; pH* 2.0) are placed on each end, and a negative polarity is then applied. Under these conditions, the direction of the electroosmotic flow (EOF) is toward the inlet. Meanwhile, the anionic analytes move in the reverse direction and are neutralized and stacked at the boundary of a dynamic pH-junction (between the sample matrix and the nonmicellar background solution (BGS)). When the sample concentration is completed, the BGS is quickly changed to solutions containing SDS-BGS for the subsequent separation. Since the mobility of SDS-analytes is then greater than the EOF, the following steps occur by the sweeping (for focusing) and MEKC (for separation) mode. Using these steps, a full-capillary sample injection/separation can be achieved.

Comparison of the use of single capillaries and coupled capillaries based on micellar electrokinetic chromatography (MEKC) and sweeping-MEKC modes.

The use of single capillaries (25 and 50 microm inner diameter (ID)) and coupled capillaries of different diameters (100-50 and 75-25 microm ID) based on micellar electrokinetic chromatography (MEKC) and sweeping-MEKC modes is compared and reported. Naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized dopamine was selected as the model compound by examining the fluorescence intensity when a violet (410 +/- 7 nm, 2 mW) light-emitting-diode (LED) was used as the light source. When a single capillary (50 microm ID) was used, the detection limit for NDA-derivatized dopamine was determined to be 2.0 x 10(-7) M (Signal-to-nose ratio S/N = 3) based on the MEKC mode. This was improved to 4.0 x 10(-9) M when the sweeping-MEKC mode was applied. In addition, this can be further improved to 1.0 x 10(-9) M and 5.6 x 10(-10) M when 100-50 and 75-25 microm ID coupled capillaries are used. The use of the coupled capillary is also helpful for improving the separation efficiency. Based on the sweeping-MEKC mode, the number of theoretical plates (N) for the detected peaks were determined to be 6.3 +/- 2.7 x 10(5) by means of a single capillary (50 microm ID). This can be improved to 9.4 +/- 3.6 x 10(5) and 9.4 +/- 0.9 x 10(6) when the 100-50 and 75-25 microm ID coupled capillaries were applied.