Recombinant human GLP-1(rhGLP-1) alleviating renal tubulointestitial injury in diabetic STZ-induced rats.

GLP-1-based treatment improves glycemia through stimulation of insulin secretion and inhibition of glucagon secretion. Recently, more and more findings showed that GLP-1 could also protect kidney from diabetic nephropathy. Most of these studies focused on glomeruli, but the effect of GLP-1 on tubulointerstitial and tubule is not clear yet. In this study, we examined the renoprotective effect of recombinant human GLP-1 (rhGLP-1), and investigated the influence of GLP-1 on inflammation and tubulointerstitial injury using diabetic nephropathy rats model of STZ-induced. The results showed that rhGLP-1 reduced urinary albumin without influencing the body weight and food intake. rhGLP-1 could increased the serum C-peptide slightly but not lower fasting blood glucose significantly. In diabetic nephropathy rats, beside glomerular sclerosis, tubulointerstitial fibrosis was very serious. These lesions could be alleviated by rhGLP-1. rhGLP-1 decreased the expression of profibrotic factors collagen I, α-SMA, fibronectin, and inflammation factors MCP-1 and TNFα in tubular tissue and human proximal tubular cells (HK-2 cells). Furthermore, rhGLP-1 significantly inhibited the phosphorylation of NF-κB, MAPK in both diabetic tubular tissue and HK-2 cells. The inhibition of the expression of TNFα, MCP-1, collagen I and α-SMA in HK-2 cells by GLP-1 could be mimicked by blocking NF-κB or MAPK. These results indicate that rhGLP-1 exhibit renoprotective effect by alleviation of tubulointerstitial injury via inhibiting phosphorylation of MAPK and NF-κB. Therefore, rhGLP-1 may be a potential drug for treatment of diabetic nephropathy.

Transplantation of human fetal pancreatic progenitor cells ameliorates renal injury in streptozotocin-induced diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is a severe complication of diabetes mellitus (DM). Pancreas or islet transplantation has been reported to prevent the development of DN lesions and ameliorate or reverse existing glomerular lesions in animal models. Shortage of pancreas donor is a severe problem. Islets derived from stem cells may offer a potential solution to this problem. OBJECTIVE: To evaluate the effect of stem cell-derived islet transplantation on DN in a rat model of streptozotocin-induced DM. METHODS: Pancreatic progenitor cells were isolated from aborted fetuses of 8 weeks of gestation. And islets were prepared by suspension culture after a differentiation of progenitor cells in medium containing glucagon-like peptide-1 (Glp-1) and nicotinamide. Then islets were transplanted into the liver of diabetic rats via portal vein. Blood glucose, urinary volume, 24 h urinary protein and urinary albumin were measured once biweekly for 16 weeks. Graft survival was evaluated by monitoring human C-peptide level in rat sera and by immunohistochemical staining for human mitochondrial antigen and human C-peptide in liver tissue. The effect of progenitor-derived islets on filtration membrane was examined by electron microscopy and real-time polymerase chain reaction (PCR). Immunohistochemical staining, real-time PCR and western blot were employed for detecting fibronectin, protein kinase C beta (PKCß), protein kinase A (PKA), inducible nitric oxide synthase (iNOS) and superoxide dismutase (SOD). RESULTS: Islet-like clusters derived from 8th gestational-week human fetal pancreatic progenitors survived in rat liver. And elevated serum level of human C-peptide was detected. Blood glucose, 24 h urinary protein and urinary albumin were lower in progenitor cell group than those in DN or insulin treatment group. Glomerular basement membrane thickness and fibronectin accumulation decreased significantly while podocytes improved morphologically in progenitor cell group. Furthermore, receptor of advanced glycation end products and PKCß became down-regulated whereas PKA up-regulated by progenitor cell-derived islets. And iNOS rose while SOD declined. CONCLUSIONS: DN may be reversed by transplantation of human fetal pancreatic progenitor cell-derived islets. And fetal pancreatic progenitor cells offer potential resources for cell replacement therapy.

Colocalization of insulin and glucagon in insulinoma cells and developing pancreatic endocrine cells.

A significant portion of human and rat insulinomas coexpress multiple hormones. This character termed as multihormonality is also observed in some early pancreatic endocrine cells which coexpress insulin and glucagon, suggesting an incomplete differentiation status of both cells. Here we demonstrate that insulinoma cells INS-1 and INS-1-derived single cell clone INS-1-15 coexpressed insulin and glucagon in a portion of cells. These two hormones highly colocalized in the intracellular vesicles within a cell. Due to the existence of both PC1/3 and PC2 in INS-1-derived cells, proglucagon could be processed into glucagon, GLP-1 and GLP-2. These glucagon-family peptides and insulin were secreted simultaneously corresponding to the elevating glucose concentrations. The coexpression and partial colocalization of insulin and glucagon was also observed in rat fetal pancreatic endocrine cells, but the colocalization rate was generally lower and more diverse, suggesting that in the developing pancreatic endocrine cells, insulin and glucagon may be stored in nonidentical pools of secreting vesicles and might be secreted discordantly upon stimulus.

C-peptide ameliorates renal injury in type 2 diabetic rats through protein kinase A-mediated inhibition of fibronectin synthesis.

Type 1 diabetes mellitus (T1DM) is characterized by the deficiencies of insulin and C-peptide. Mounting evidences have proved the beneficial effects of C-peptide on the renal function in T1DM. However, it is still controversial about the roles of C-peptide in T2DM nephropathy since the level of C-peptide fluctuates greatly at different stages of T2DM. In the present study, we found that the serum C-peptide concentration was much lower in GK rats with diabetic nephropathy than that in normal counterparts. A sustained supplementation of C-peptide at a physiological level could ameliorate urinary albumin, independent of blood glucose control. C-peptide treatment improved glomerulosclerosis and podocyte morphology and reduced the thickness of glomerular basement membrane as compared with saline treatment control. Moreover, it decreased fibronectin synthesis in diabetic glomeruli and in cultured rat mesangial cells accompanied by a down-regulation of RAGE and an up-regulation of PKA. Interestingly, H-89, a PKA inhibitor, could reverse the inhibition effect of C-peptide on fibronectin production in cultured mesangial cells. These findings suggest that C-peptide level is low in T2DM rats with nephropathy and a treatment with a physiological dose of C-peptide can prevent renal injury in diabetic GK rats.

In vitro and in vivo evidence for amphotericin B as a P-glycoprotein substrate on the blood-brain barrier.

Amphotericin B (AMB) has been a mainstay therapy for fungal infections of the central nervous system, but its use has been limited by its poor penetration into the brain, the mechanism of which remains unclear. In this study, we aimed to investigate the role of P-glycoprotein (P-gp) in AMB crossing the blood-brain barrier (BBB). The uptake of AMB by primary brain capillary endothelial cells in vitro was significantly enhanced after inhibition of P-gp by verapamil. The impact of two model P-gp inhibitors, verapamil and itraconazole, on brain/plasma ratios of AMB was examined in both uninfected CD-1 mice and those intracerebrally infected with Cryptococcus neoformans. In uninfected mice, the brain/plasma ratios of AMB were increased 15 min (3.5 versus 2.0; P < 0.05) and 30 min (5.2 versus 2.8; P < 0.05) after administration of verapamil or 45 min (6.0 versus 3.9; P < 0.05) and 60 min (5.4 versus 3.8; P < 0.05) after itraconazole administration. The increases in brain/plasma ratios were also observed in infected mice treated with AMB and P-gp inhibitors. The brain tissue fungal CFU in infected mice were significantly lower in AMB-plus-itraconazole or verapamil groups than in the untreated group (P < 0.005), but none of the treatments protected the mice from succumbing to the infection. In conclusion, we demonstrated that P-gp inhibitors can enhance the uptake of AMB through the BBB, suggesting that AMB is a P-gp substrate.

A bacteria deriving peptide modified dendrigraft poly-l-lysines (DGL) self-assembling nanoplatform for targeted gene delivery.

Achieving effective gene therapy for glioma depends on gene delivery systems. The gene delivery system should be able to cross the blood-brain barrier (BBB) and further target glioma at its early stage. Active brain tumor targeted delivery can be achieved using the "Trojan horse" technology, which involves either endogenous ligands or extraneous substances that can recognize and bind to specific receptors in target sites. This method facilitates receptor-mediated endocytosis to cross the BBB and enter into glioma cells. Dendrigraft poly-l-lysines (DGLs), which are novel nonviral gene vectors, are conjugated to a peptide (sequence: EPRNEEK) derived from Streptococcus pneumonia, a pathogen causing meningitis. This process yields peptide-modified nanoparticles (NPs) after DNA loading. Cellular uptake and in vivo imaging results indicate that EPRNEEK peptide-modified NPs have a better brain tumor targeted effect compared with a pentapeptide derived from endogenous laminin after intravenous injection. The mechanism of this effect is further explored in the present study. Besides, EPRNEEK peptide-modified NPs also exhibited a prolonged median survival time. In conclusion, the EPRNEEK peptide-modified DGL NPs exhibit potential as a nonviral platform for efficient, noninvasive, and safe brain glioma dual-targeted gene delivery.

Cytoadherence of Plasmodium berghei-infected red blood cells to murine brain and lung microvascular endothelial cells in vitro.

Sequestration of infected red blood cells (iRBC) within the cerebral and pulmonary microvasculature is a hallmark of human cerebral malaria (hCM). The interaction between iRBC and the endothelium in hCM has been studied extensively and is linked to the severity of malaria. Experimental CM (eCM) caused by Plasmodium berghei ANKA reproduces most features of hCM, although the sequestration of RBC infected by P. berghei ANKA (PbA-iRBC) has not been completely delineated. The role of PbA-iRBC sequestration in the severity of eCM is not well characterized. Using static and flow cytoadherence assays, we provide the first direct in vitro evidence for the binding of PbA-iRBC to murine brain and lung microvascular endothelial cells (MVEC). We found that basal PbA-iRBC cytoadherence to MVECs was significantly higher than that of normal red blood cells (NRBC) and of RBC infected with P. berghei K173 (PbK173-iRBC), a strain that causes noncerebral malaria (NCM). MVEC prestimulation with tumor necrosis factor (TNF) failed to promote any further significant increase in mixed-stage iRBC adherence. Interestingly, enrichment of the blood for mature parasites significantly increased PbA-iRBC binding to the MVECs prestimulated with TNF, while blockade of VCAM-1 reduced this adhesion. Our study provides evidence for the firm, flow-resistant binding to endothelial cells of iRBC from strain ANKA-infected mice, which develop CM, and for less binding of iRBC from strain K173-infected mice, which develop NCM. An understanding of P. berghei cytoadherence may help elucidate the importance of sequestration in the development of CM and aid the development of antibinding therapies to help reduce the burden of this syndrome.

Angiopep-conjugated nanoparticles for targeted long-term gene therapy of Parkinson's disease.

PURPOSE: To prepare an angiopep-conjugated dendrigraft poly-L-lysine (DGL)-based gene delivery system and evaluate the neuroprotective effects in the rotenone-induced chronic model of Parkinson's disease (PD). METHODS: Angiopep was applied as a ligand specifically binding to low-density lipoprotein receptor-related protein (LRP) which is overexpressed on blood-brain barrier (BBB), and conjugated to biodegradable DGL via hydrophilic polyethyleneglycol (PEG), yielding DGL-PEG-angiopep (DPA). In vitro characterization was carried out. The neuroprotective effects were evaluated in a chronic parkinsonian model induced by rotenone using a regimen of multiple dosing intravenous administrations. RESULTS: The successful synthesis of DPA was demonstrated via (1)H-NMR. After encapsulating the therapeutic gene encoding human glial cell line-derived neurotrophic factor (hGDNF), DPA/hGDNF NPs showed a sphere-like shape with the size of 119 ± 12 nm and zeta potential of 8.2 ± 0.7 mV. Angiopep-conjugated NPs exhibited higher cellular uptake and gene expression in brain cells compared to unmodified counterpart. The pharmacodynamic results showed that rats in the group with five injections of DPA/hGDNF NPs obtained best improved locomotor activity and apparent recovery of dopaminergic neurons compared to those in other groups. CONCLUSION: This work provides a practical non-viral gene vector for long-term gene therapy of chronic neurodegenerative disorders.

[Effects of platelets on the adhesion between leukocytes and liver sinusoidal endothelium cells as well as on the transendothelial migration of leukocytes undergoing hypoxia-reoxygenation].

OBJECTIVE: To investigate the effects of platelet on intercellular adhesion between leukocyte and liver sinusoidal endothelial cell(LSEC) and the transendothelial migration under the hypoxia-reoxygenation condition, as well as the role of relevant adhesion molecules. METHOD: LSEC was cultured for 24 hours under hypoxia condition and then reoxygenated for 2 hours (hypoxia-reoxygenation, HR). This hypoxia-reoxygenation model was used to simulate the clinical liver ischemia-reperfusion injury process (IRI). Platelets and leukocytes were labeled with fluorescence dye, and then the adhesion was detected by fluorescence microscope, fluorescence plate reader and laser scanning confocal microscope. Antibody blockage experiment was used to analyze the relevant adhesion molecules. RESULTS: The adhesion between platelets and LSEC was increased significantly after HR. The fluorescence intensity of adherent platelets increased from 142.10 ± 7.53 to 289.17 ± 20.00(P < 0.01). After H-R treatment and the addition of platelets, the number of adherent leukocytes increased markedly, and a significant statistical difference (360.71 ± 23.47 and 186.39 ± 17.96, P < 0.01) was found in comparing with the platelet deficient group. These adhesion processes could be blocked respectively by anti-GPIb, anti-GPIIb, anti-GPIIIa, anti-P-selectin, anti-CD31, anti-ICAM-1, anti-VCAM-1 and anti-ELAM-1. Confocal microscopy showed that platelets located between leukocytes and LSEC, and mediated their adhesion process. However, the adhesion of platelets to LSEC decreased the transendothelial migration of leukocytes (227.79 ± 16.51 and 167.27 ± 10.92, P < 0.05). CONCLUSION: During ischemia-reperfusion condition platelets adhere to the surface of LSEC, and then further mediate more adhesion processes between leukocytes and endothelial cells, as well as inhibit the transendothelial migration of leukocytes. The consequence is that large numbers of leukocytes were sequestrated within hepatic sinus, and deteriorate liver ischemia-reperfusion injury.

Differential activations of PKC/PKA related to microvasculopathy in diabetic GK rats.

Microvasculopathy is the most serious and predictable threat to the health of diabetic patients, which often results in end-stage renal disease, blindness, and limb amputations. Up to the present, the underlying mechanisms have remained elusive. Here, it was found that the differential activations of PKC/PKA were involved in diabetic microvasculopathy in diabetic GK rats. By real-time PCR, Western blot, immunohistochemistry, and enzyme activity assay, upregulation of PKC was prominent in kidney but was not significant in liver and brain. The expression and activity of PKA were lowered in kidney but comparable in brain and liver during diabetic nephropathy. Furthermore, the generation of reactive oxygen species, production of nitric oxide, and expression of inducible nitric oxide synthase induced by advanced glycation end products were inhibited by PKCß inhibitor LY-333531 or a PKA agonist in rat glomerular microvascular endothelial cells. Finally, albuminuria was significantly lowered by a PKA agonist and boosted by a PKA antagonist. It suggested that the differential activations of PKC/PKA related to microvasculopathy in diabetes and that activation of PKA may protect the diabetic microvasculature.

Antibody library-based tumor endothelial cells surface proteomic functional screen reveals migration-stimulating factor as an anti-angiogenic target.

Angiogenesis is critical for cancer development and metastasis. Here we have employed a functional antibody library-based proteomic screen to identify proteins that participate in and might be used as therapeutic targets for tumor-related angiogenesis. Mice were immunized with human esophageal cancer endothelial cells (HECEC). The antibody library was established with the mouse spleen cells the serum of which had most anti-angiogenic effect. Monoclonal antibodies were subjected to an immunoreactive and functional screen and monoclonal antibodies that reacted strongly with cell surface antigens of HECECs and influenced their behavior were selected. Antigens that recognized by the antibodies were obtained by immunoprecipitation and then identified by mass spectrometry analysis. Migration-stimulating factor (MSF), the antigen of 1D2 antibody was identified using this approach. Further studies demonstrated that the 1D2 antibody suppressed MSF-effected migration and adhesion of HECECs on fibronectin matrix. Biodistribution assay showed that MSF targeting antibody 1D2 could specifically home to the xenograft with humanized blood vessel. Targeting treatment with 1D2 antibody significantly suppressed tumor growth through inhibition of human tumor-related angiogenesis. These results indicate that the functional antibody library-based proteomic screen can successfully identify proteins that involved in tumor-related angiogenesis and MSF may be a target for the anti-angiogenic treatment of the esophageal cancer.

Treatment of nontraumatic osteonecrosis of the femoral head with the implantation of core decompression and concentrated autologous bone marrow containing mononuclear cells.

BACKGROUND: Since self-limited repair ability of the necrotic lesion may be a cause for failure of the technique, the possibility has been raised that bone marrow mononuclear cells (BMMCs) containing BMSCs implanted into a necrotic lesion of the femoral head with core decompression (CD) may be of benefit in the treatment of this condition. For this reason, we studied the implantation of the concentrated autologous bone marrow containing mononuclear cells in necrotic lesion of the femoral head to determine the effect of the method. METHODS: The study included 45 patients (59 hips, 9 females, 36 males; mean age 37.5 years, range 16-56 years) with stages I-IIIA nontraumatic avascular necrosis of the femoral head according to the system of the Association Research Circulation Osseous. Concentrated bone marrow (30-50 ml) containing mononuclear cells has been gained from autologous bone marrow (100-180 ml) obtained from the iliac crest of patient with the cell processor system. Concentrated bone marrow was injected through a CD channel into the femoral head. The outcome was determined by the changes in the Harris hip score, by progression in radiographic stages, and by the need for hip replacement. The mean follow-up was 27.6 months (range 12-40 months). RESULTS: Pre- and post-operative evaluations showed that the mean Harris hip score increased from 71 to 83. Clinically, the overall success is 79.7%, and hip replacement was done in 7 of the 59 hips (11.9%). Radiologically, 14 of the 59 hips exhibited femoral head collapse or narrowing of the coxofemoral joint space, and the overall failure rate is 23.7%. The number of BMMCs increased from 12.2 +/- 3.2 x 10(6)/ml to 35.2 +/- 12 x 10(6)/ml between pre-concentration and post-concentration. CONCLUSION: The concentrated autologous bone marrow containing mononuclear cells implantation relieves hip pain, prevents the progression of osteonecrosis. Therefore, it may be the treatment of choice particularly in stages I-II nontraumatic osteonecrosis of the femoral head.

[Morphologic and functional characteristics of the immortalized human liver sinusoidal endothelial cell line].

OBJECTIVE: To investigate the morphologic and functional characteristics of the immortalized human liver sinusoidal endothelial cell line (LSEC line). METHODS: Immunofluorescence staining and fluorescence microscopy were used to detect the classic endothelial cell markers in LSEC line, and flow cytometry was used to analyze the purity of the human LSEC line. The morphology (including W-P bodies and surface fenestrations) and phagocytotic capacity of the human LSEC line were observed by transmission and scanning electron microscope. The proliferation curve of the human LSEC line was analyzed by MTT assay. The functional differences between the human LSEC line and human primary LSEC in expression of ELAM-1 and ICAM-1, activities of fibrinolysis (PAI-1, t-PA, u-PA), releasing of IL-6 and IL-8 were compared respectively by enzyme linked immunosorbent assay. Comparison of the susceptibility to hypoxia-reoxygenation induced apoptosis between the human LSEC line and human primary LSEC were investigated by TUNEL. RESULTS: The established human LSEC line maintained a high proliferative ability and has been passaged for more than 80 times in the absence of any growth factors. Immunofluorescence staining showed that the human LSEC line could express classic endothelial cell marks including von Willebrand Factor (vWF), and could take up acetylated low-density lipoproteins (Ac-LDL). The purity of the human LSEC line was confirmed over 95% by flow cytometric analysis. The W-P bodies and the phagocytosis of Dynabeads was demonstrated by transmission electron microscope. And fenestrations could be found cellular surface with scanning electron microscopy. When compared with human primary LSEC, the human LSEC line has an equivalent responsiveness to tumor necrosis factor in up-regulation of ELAM-1 and ICAM-1. The human LSEC line can also release PAI-1, t-PA, u-PA but can not release IL-6 and IL-8 to TNF-alpha. In contrast, human primary LSEC could release IL-6. The human LSEC line showed higher susceptibility to hypoxia-reoxygenation-induced apoptosis, and the percentage of apoptotic cells was as high as (38.4 +/- 6.7)%, while (28.6 +/- 4.5)% and (7.8 +/- 1.2)% respectively in primary LSEC and in human umbilical vein endothelial cells. CONCLUSIONS: The established human LSEC line maintains the special phenotypes and the major functional characteristics, and especially maintains the high susceptibility to hypoxia-reoxygenation-induced apoptosis. Therefore it is feasible to use this cell line for the study of liver ischemia-reperfusion injury.

Interleukin-10 Protects Schwann Cells against Advanced Glycation End Products-Induced Apoptosis via NF-κB Suppression.

Demyelination resulting from Schwann cell injury is a main pathological feature of diabetic neuropathy, and a key contributor to this process may be inflammation due to advanced glycation end products (AGEs). Therefore, protection by anti-inflammation agents is anticipated. In this study, we showed that interleukin-10 (IL-10), an anti-inflammatory cytokine, inhibits apoptosis of Schwann cells induced by AGEs in vitro. We isolated and cultured Schwann cells from rat sciatic nerves. As detected by flow cytometry, apoptosis of Schwann cells markedly increased following incubation with AGEs for 48 h. However, pretreatment with IL-10 inhibited AGE-induced apoptosis. The effect of IL-10 on NF-κB, which is a very important regulator of inflammation, was also evaluated, and results showed high levels of phospho-NF-κB and nuclear localization of NF-κB in cells incubated with AGEs but low levels of phospho-NF-κB and cytoplasmic localization in the cells incubated with IL-10, indicating the activation of NF-κB by AGEs and inhibition of NF-κB by IL-10. Moreover, incubating Schwann cells with an NF-κB inhibitor (caffeic acid phenethyl ester) for 30 min before adding AGEs mimicked IL-10, lowering the amount of reactive oxygen species and activity of caspase-3 and also decreasing apoptosis in Schwann cells. These results indicate that IL-10 may protect Schwann cells against AGE-induced apoptosis by attenuating oxidative stress via the inhibition of activation of NF-κB.

Mitochondrial ND3 as the novel causative gene for Leber hereditary optic neuropathy and dystonia.

Leber hereditary optic neuropathy and dystonia (LDYT) is a mitochondrial disorder associated with variable combinations of vision loss and progressive generalized dystonia. LDYT is a unique oxidative phosphorylation disorder caused by mutations in mitochondrial ND6 or ND4 gene. In this paper, we describe a Chinese family with 18 LDYT patients. The comprehensive nucleotide sequence analysis of the entire mitochondrial genome using resequencing microarray revealed a mutation (mtND3*10197A (m.10197G>A)) substituting a threonine for a highly conserved alanine at codon 47 of MTND3 on the background of haplogroup D4b. Quantitative analysis of the heteroplasmy of the mutation revealed a homoplasmy in the leukocytes of all the affected individuals on the maternal side. This is the first description of the ND3 mutation causing LDYT. The mtND3*10197A (m.10197G>A) mutation has recently been described in French and Korean patients with Leigh syndrome. These findings suggest that the clinical presentations associated with the mtND3*10197A (m.10197G>A) mutation (ND3) are much wider, encompassing those of LDYT and Leigh syndrome.

Abnormal blood vessels formed by human liver cavernous hemangioma endothelial cells in nude mice are suitable for drug evaluation.

Cavernous hemangioma is vascular malformation with developmental aberrations. It was assumed that the abnormality of endothelial cells contributed greatly to the occurrence of cavernous hemangioma. In our previous study, we have found distinct characteristics of endothelial cells derived from human liver cavernous hemangioma (HCHEC). Here, we reported the abnormal vascular vessels formed by primary HCHEC in nude mice and that the drug podophyllotoxin can destroy HCHEC in vitro and in vivo. HCHEC was isolated from a human liver cavernous hemangioma specimen, and the HCHEC generated a red hemangioma-like mass 7 days after subcutaneously co-inoculating HCHEC and human liver cancer cells (Bel-7402) in nude mice. Lentiviral expression of GFP and immunohistochemistry for human CD31 was used to confirm that the HCHEC formed the blood vessels in nude mice. And the pathological features of vascular vessels formed by HCHEC were very similar to clinical cavernous hemangioma. In addition, by MTT assay, the drug podophyllotoxin was found inhibiting HCHEC viability, and by TUNEL and DNA ladder assays, podophyllotoxin was found inducing apoptosis of HCHEC. Moreover, podophyllotoxin was also effective for destroying the abnormal vascular vessels in the hemangioma-like mass in nude mice. In summary, the HCHEC can form abnormal blood vessels in nude mice, and we can evaluate drugs for cavernous hemangioma by using HCHEC in vitro and in vivo.

[Isolation, purification, and functional evaluation of human pancreatic islet].

OBJECTIVE: To isolate and purify human islet according to the method established by Ricordi and to evaluate the function and safety of these isolated human islets. METHODS: Six pancreases were obtained from human corpses. The islets were isolated by liberase digestion and purificated by Ficoll density gradient centrifugation. The numbers, purity and vitality of the islets were analyzed. The various endocrine cell composition and distribution of the islets were checked by immunofluorescence staining. The glucose-induced insulin secretion was detected by chemiluminescence method. The isolated islets were transplanted under the left renal capsules of 10 streptozocin-induced diabetic nude mice. Twenty days later the left kidneys with transplanted islets of 2 mice with normal blood sugar were resected, and then blood sugar level was observed. An isolated human islet was suspended in RPMI-1066 culture medium for 72 h, then culture of pathogenic micro-organisms, endotoxin and procoagulant activity were detected so as to evaluate the security of the islet products. RESULTS: The mean number of the isolated islets was (229 000 +/- 31 000) islet equivalents (IEQs)/pancreas or (4970 +/- 1620) IEQs/g pancreatic tissue, the mean purity was (59.0 +/- 8.9)%, and the mean vitality was (89 +/- 3)% for the purified islets. Immunofluorescence staining showed that there were 4 types of endocrine cells normally distributed in the islets. The mean insulin stimulation index was 8.1 +/- 4.0 (3.8 - 10.2). The glycemia found in the diabetic nude mice decreased to normal levels from the third day after islet transplantation and maintained normal for over 30 days. The parameters of security in these islet products were under the standard scope. CONCLUSION: Human islets obtained according to Ricordi's method reach the standard for clinical islet transplantation in number, purity, vitality, function, and security.

Protein kinase C and protein kinase A are involved in the protection of recombinant human glucagon-like peptide-1 on glomeruli and tubules in diabetic rats.

AIMS/INTRODUCTION: Blockade or reversal the progression of diabetic nephropathy is a clinical challenge. The aim of the present study was to examine whether recombinant human glucagon-like peptide-1 (rhGLP-1) has an effect on alleviating urinary protein and urinary albumin levels in diabetic rats. MATERIALS AND METHODS: Streptozotocin-induced diabetes rats were treated with rhGLP-1 insulin and saline. Using immunostaining, hematoxylin-eosin, electron microscopy and periodic acid-Schiff staining to study the pathology of diabetic nephropathy, and we carried out quantitative reverse transcription polymerase chain reaction, western blot and immunohistochemistry to identify the differentially expressed proteins. The mechanism was studied through advanced glycation end-products-induced tubular epithelial cells. RESULTS: rhGLP-1 inhibits protein kinase C (PKC)-ß, but increases protein kinase A (PKA), which reduces oxidative stress in glomeruli and in cultured glomerular microvascular endothelial cells. In tubules, rhGLP-1 increased the expression of two key proteins related to re-absorption - megalin and cubilin - which was accompanied by downregulation of PKC-ß and upregulation of PKA. On human proximal tubular epithelial cells, rhGLP-1 enhanced the absorption of albumin, and this was blocked by a PKC activator or PKA inhibitor. CONCLUSIONS: These findings suggest that rhGLP-1 can reverse diabetic nephropathy by protecting both glomeruli and tubules by inhibiting PKC and activating PKA.

1,25-(OH)2D3 protects Schwann cells against advanced glycation end products-induced apoptosis through PKA-NF-κB pathway.

AIMS: To explore the effect and mechanism of 1, 25-(OH)2D3 on Schwann cell apoptosis induced by advanced glycation end products. MAIN METHODS: Schwann cells, isolated from rodent sciatic nerve were incubated with AGE-modified bovine serum albumin(AGE) to mimic diabetic conditions and 1,25-(OH)2D3 was used as protector. Cell apoptosis was detected by PI/Annexin-V staining, caspase 3 activity assay and western blotting for caspase 3 and PARP. The activation of protein kinase A (PKA) and nuclear factor kappa-B (NF-κB) was evaluated by western blot. Immunofluorescent staining was used for intercellular location of NF-κB. Cytokine secretion was evaluated by enzyme-linked immunosorbent assay. KEY FINDINGS: Schwann cell apoptosis accelerated after incubating with AGE. However, if combining 1,25-(OH)2D3 with AGE, apoptosis decreased significantly. 1,25-(OH)2D3 enhanced PKA activity, but inhibited AGE-induced nuclear translocation of NF-κB. Furthermore, PKA activator (8-bromoadenoside cyclic adenoside monophosphate, 8-Br-cAMP) or NF-κB inhibitor (caffeic acid phenethyl ester, CAPE) could reduce the apoptosis, decreased cleaved caspase 3 and cleaved PARP, suggesting the involvement of PKA and NF-κB pathways in the protection of 1,25-(OH)2D3 on Schwann cells. Moreover, 8-Br-cAMP and CAPE could inhibit AGE-induced secretion of interleukin(IL)-1ß, prostaglandin E2(PEG2) and cyclooxygenase 2(COX2). Interestingly, 8-Br-cAMP decreased phospho-NF-κB and inhibited nucleus translocation of NF-κB. It hinted at the regulation of PKA to NF-κB. Finally, a pre-treatment of H-89 (an inhibitor of PKA) could block the protection of 1,25-(OH)2D3 on cell apoptosis. In conclusion, 1,25-(OH)2D3 could protect Schwann cell against AGE-induced apoptosis through PKA/NF-κB pathway. SIGNIFICANCE: These findings provide experimental rationales for using vitamin D for diabetic neuropathy.

[Tumor cell-tumor endothelial cell adhesion mediated by alphavbeta3 and alphavbeta5 molecules].

OBJECTIVE: To investigate the role of adhesion molecules alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 in the tumor-endothelial cell adhesion in vitro. METHODS: The expression of alphavbeta3, alphavbeta5 and ICAM-1 in liver sinusoidal endothelial cells (LSEC) and liver cancer endothelial cells (T3A) cultured under normoxia or hypoxia were analyzed by RT-PCR and fluorescent activated cell sorter (FACS). The expression of Del-1 and L1 in six tumor cell lines under normoxia or hypoxia were analyzed by RT-PCR and Western blot, respectively. The adhesion of dye-labeled tumor cells and endothelial LSEC and T3A cells was measured by a fluorescence plate reader after their culture. RESULTS: The expression of alphavbeta3 and alphavbeta5 were higher in T3A cells than that in LSEC cells, and were upregulated under hypoxia, while the expression of ICAM-1 was lower in T3A cells than that in LSEC cells, and was upregulated under hypoxia only in LSEC. The expression of Del-1 and L1 molecules were obviously different in various tumor cell lines and were differentially regulated under hypoxia. The adhesion of tumor cells with Del-1 or L1 expression was higher in T3A cells than that in LSEC cells, and was significantly increased under hypoxia condition. Furthermore, the adhesion of tumor cells to T3A could be inhibited by antibodies against alphavbeta3 and alphavbeta5, or SiRNAs for beta3 and beta5. CONCLUSION: alphavbeta3 and alphavbeta5 and their ligands Del-1 and L1 may play an important role in tumor cell migration.