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.

[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.

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.

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.

Phenotypic and functional differences between human liver cancer endothelial cells and liver sinusoidal endothelial cells.

BACKGROUND/AIMS: The phenotypic and functional characteristics of microvascular endothelial cells derived from human liver cancer (HLCEC) were analyzed in vitro and compared with those of human liver sinusoidal endothelial cells (LSEC). METHODS AND RESULTS: Flow-cytometric and real-time PCR analysis indicated that expressions of tumor necrosis factor receptor (TNFR) p75, alphavbeta3 and alphavbeta5 were increased, while those of TNFR p55 and intercellular-adhesion molecule 1 (ICAM-1) were decreased in HLCEC compared with LSEC. The functional analysis indicated that HLCEC exhibited higher angiogenic ability than LSEC, including proliferation capacity, ability to form capillary-like networks and release of matrix metalloproteinases. In response to tumor necrosis factor, LSEC exhibited a significant dose-dependent cytotoxicity, while HLCEC did not. Moreover, the coagulant and fibrinolytic capacity was increased in HLCEC. In addition, tumor cell adherence was significantly higher on HLCEC than on LSEC, while leukocyte adherence was lower on HLCEC than on LSEC. The cytoadherence of HLCEC was inhibited by antibodies against alphavbeta3 and alphavbeta5,and of LSEC by antibodies against ICAM-1. CONCLUSION: These results indicate that tumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue. These differences are valuable for understanding tumor angiogenesis and metastasis.

[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.

Decreased proliferation of mesenchymal stem cells in corticosteroid-induced osteonecrosis of femoral head.

The ability of self-repair in patients with corticosteroid-induced osteonecrosis of the femoral head is limited, and it has been suggested the cause is likely relevant to the poor proliferation activity of mesenchymal stem cells in the femoral head region. This study measured the number and proliferation activity of human mesenchymal stem cells in patients both with and without corticosteroid-induced osteonecrosis of the femoral head. Bone marrow was collected from the proximal femur in patients with steroid-induced osteonecrosis of the femoral head (osteonecrosis group, n=18) and patients with new femoral neck fractures without osteonecrosis (control group, n=11). Mesenchymal stem cells were isolated by density gradient centrifugation, and then selected by the adhesive method. The MTT reduction assay method was used to evaluate the level of proliferation. Cells from osteonecrosis patients showed reduced proliferation ability compared with the control patients. The percentage of cells in the S+G2/M phase was decreased significantly (P<.01) in the osteonecrosis group. The decreased proliferation ability of mesenchymal stem cells may play a role in the low repair capacity of steroid-induced osteonecrosis of femoral head. The altered function of mesenchymal stem cells may be responsible for the pathogenesis and progression of osteonecrosis.

Treatment of gingival defects with gingival mesenchymal stem cells derived from human fetal gingival tissue in a rat model.

BACKGROUND: The study aimed to evaluate the efficacy and safety of gingival mesenchymal stem cells (GMSCs) from human fetal gingival tissue used for treating gingival defects in a rat model. METHODS: GMSCs were isolated from human fetal gingival tissue and identified by flow cytometry for nestin, Oct4, vimentin, NANOG, CD105, and CD90. The immunogenicity of GMSCs was analyzed by mixed lymphocyte reactions; the tumorigenicity of GMSCs was evaluated by xenotransplanting into nude mice. The gingival defect animal model was established by mechanical resection in rats. GMSCs were transplanted into the defective area, and the regeneration of gingival tissue was observed twice weekly. Four weeks after transplantation, the gingival tissue was surgically cut down, and the graft was analyzed by immunohistochemistry staining for human mitochondrial antigens and rat CD3 and CD20. RESULTS: GMSCs from human fetal gingival tissue positively expressed nestin, Oct4, vimentin, NANOG, CD105, and CD90. There was no cell aggregation after mixed lymphocyte reactions, and interleukin-2 did not increase. Inoculation of GMSCs into nude mice for 6 months showed no tumor formation. GMSCs were transplanted into the gingiva defects of rats. One week after transplantation, the defect area was reduced, and after 3 weeks the morphology and color of local gingival tissue was similar to normal gingival tissue, and gingival height was the same as the normal control group. CONCLUSIONS: Using GMSCs from human fetal gingival tissue to treat gingival defects is a safe and effective innovative treatment method.

The role of adhesion molecules, alpha v beta 3, alpha v beta 5 and their ligands in the tumor cell and endothelial cell adhesion.

Tumor metastasis is a complex process involving the interaction between tumor cells and endothelial cells in which some adhesion molecules play an important role. It was our aim to investigate the role of the adhesion molecules, alpha v beta 3 and alpha v beta 5 and their ligands, developmental endothelial locus-1 (Del-1) and L1, in tumor cell adhesion to endothelial cells in vitro. In this study, the expression and regulation of alpha v beta 3, alpha v beta 5 and intercellular adhesion molecule -1 on liver sinusoidal endothelial cells and liver cancer endothelial cells (T3A) were analyzed by real-time PCR and fluorescent-activated cell sorter. The expression and regulation of the integrin ligands, Del-1 and L1, in six tumor cell lines were analyzed by real-time PCR and western blot. We found the expressions of alpha v beta 3 and alpha v beta 5 were higher on T3A than that on liver sinusoidal endothelial cells, whereas expression of intercellular adhesion molecule-1 was lower on T3A than that on liver sinusoidal endothelial cells. After 24 h hypoxia, the expressions of alpha v beta 3 and alpha v beta 5 were upregulated on T3A and liver sinusoidal endothelial cells; the expression of intercellular adhesion molecule-1 was increased on liver sinusoidal endothelial cells, but remained unchanged on T3A. Del-1 and L1 expression levels were obviously diverse in various tumor cell lines and differentially modulated after 12 h hypoxia. The adhesion of tumor cells with Del-1 and L1 expression was higher in T3A than that in liver sinusoidal endothelial cells, and was significantly increased under hypoxic conditions. Interestingly, the tumor cell adherence could be inhibited by antibodies against alpha v beta 5 and alpha v beta 5, but not by an antibody against intercellular adhesion molecule-1. The adhesion of tumor cells without Del-1 and L1 expression was also higher on T3A than that on liver sinusoidal endothelial cells, but the adhesion could not be inhibited by antibodies against alpha v beta 5, alpha v beta 5 or intercellular adhesion molecule-1, suggesting that other receptors are involved. In conclusion, alpha v beta 5, alpha v beta 5 and their ligands Del-1 and L1 play an important role in the process of tumor cells moving from the original place.

[Phenotypic and functional characteristics of endothelial cells derived from human liver cancer].

OBJECTIVE: To analyze the phenotypic and functional characteristics of endothelial (T3A) cells derived from human hepatocellular cell carcinoma. METHODS: Endothelial cells were isolated from human hepatocellular carcinoma specimens. The identification of T3A cells was performed by checking von Willebrand Factor (vWF), CD31, CD34 and Dil-Ac-LDL uptake. The cell surface fenestrations, a specific morphological feature of tumor derived EC, were investigated by scanning and transmission electron microscopy. The phenotypic characteristics of T3A cells were analyzed by fluorescence-activated cell sorter (FACS) and were further conformed by real-time PCR at transcription level. Furthermore, tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity was evaluated by 3-(4, 5-dimethythiazolyl) -2, -diphenyl-2H-tetrazolium-bromide (MTT) assay; Matrix metalloproteinase secretion was detected by zymography; Angiogenic ability in vitro was analyzed by culturing T3A cells in three-dimensional Matrigel plug. Coagulant and fibrinolytic activities were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: The isolated T3A cells exhibited classic "spindle-shape" morphology and monolayer growth and contact inhibition properties. Immunofluorescent staining showed that T3A cells expressed vWF, CD31, CD34, and uptake of Dil-Ac-LDL at a high level. The cell surface fenestrations were observed on T3A cells by scanning and transmission electron microscopy. By FACS and real-time PCR, T3A cells were found to express alphav3, alphavbeta5 and TNF receptor p75 at high levels, and TNF receptor p55 and ICAM-1 at low levels, as compared with those in human liver sinusoidal endothelial cells (LSEC). In response to TNFalpha, LSEC exhibited a dose-dependent cytotoxicity, while T3A cells were resistant. Gelatin zymography showed that MMP-2 activity was higher in T3A cells than that in LSEC. In a three-dimensional plug of Matrigel, T3A cells exhibited stronger angiogenic ability as compared with LSEC. In addition, T3A cells released more tissue factor (TF), tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor (PAI-1) and urine plasminogen activator (u-PA) than LSEC in response to TNFalpha. CONCLUSION: Tumor-derived endothelial cells are phenotypically and functionally different from those derived from normal liver tissue.

[Expression of severe acute respiratory syndrome coronavirus receptors, ACE2 and CD209L in different organ derived microvascular endothelial cells].

OBJECTIVE: To investigate the expression of the 2 severe acute respiratory syndrome coronavirus (SARS-CoV) receptors, angiotensin-converting enzyme 2 (ACE2) and CD209L in different human organ/tissue derived microvascular endothelial cells. METHODS: Endothelial cells from the microvessels in human brain, lung, hepatic sennoside, fat adipose tissue, adrenal gland, esophagus, lymph nodes, and bone were culture. RT-PCR, Western blotting and immunocytochemistry were used to detect the expression of ACE2 and CD209L receptors. RESULTS: Both SARS-CoV receptors of ACE2 and CD209L were expressed in the 8 organ/tissue-derived endothelial cells. The expression of ACE2 receptor was the highest in the human lung microvascular endothelial cells, and lowest in the lymphatic endothelial cells. The expression of CD209L was relatively higher in the human lymphatic endothelial cells. CONCLUSION: The organ derived microvascular endothelial cells are the important target of SARS-CoV. The pathological injury of lung and lymph system induced by SARS-CoV may be mediated respectively by different receptors of SARS-CoV.

[Characteristics of endothelial cells derived from human hepatic cavernous hemangioma in vitro].

OBJECTIVE: To investigate the characteristics of endothelial cells derived from human cavernous hemangioma in morphology, phenotypes and functions. METHODS: Endothelial cells were isolated from human hepatic cavernous hemangioma. The morphological, and phenotypical and functional features of these cells were analyzed by transmission electron microscopy, fluorescence-activated cell sorter, RT-PCR, zymography, and confocal microscopy. Human liver sinusoidal endothelial cells (LSEC) were used as control. RESULTS: As compared with the LSEC, abnormally expanded endoplasmic reticulums and similarly arranged cytoplasmic vacuoles were found in the endothelial cells derived from hepatic cavernous hemangioma (HCHEC) by transmission electron microscopy. Flow cytometry showed that expression of alphavbeta3 was significantly increased in the HCHEC. The mRNA of vascular endothelial cell growth factor and angiopoietin 1 were more abundant in HCHEC than that in LSEC. Functional analysis indicated that the HCHEC exhibited strong activated angiogenesis capacity and formed abnormal capillary-like structures. HCHEC produced more pro-matrix metalloproteinase 2 (MMP-2) and the activated MMP-2 form as compared with the LSEC. Confocal microscopy revealed that MMP-2 was concentrated in those cytoplasmic granules of the HCHEC and was consistent with the distribution of the expanded endoplasmic reticulums. CONCLUSION: The endothelial cells derived from human cavernous hemangioma differ from the normal endothelial cells in morphology, phenotypes and functions.

Overexpressed Derlin-1 inhibits ER expansion in the endothelial cells derived from human hepatic cavernous hemangioma.

Proteins that are unfolded or misfolded in the endoplasmic reticulum (ER) must be targeted for refolding or degradation to maintain the homeostasis of the ER. Derlin-1 was reportedly implicated in the retro-translocation of misfolded proteins from the ER to the cytosol for degradation. In this report, we showed that Derlin-1 was down-regulated in the endothelial cells derived from human hepatic cavernous hemangioma (CHEC) compared with other tested cells. Electron microscopy analysis showed that ER was aberrantly enlarged in CHEC cells, but not in other tested cells. When overexpressed, Derlin-1 induced the dilated ER to return normal size. This ER dynamic was associated with the activation of unfolded protein response (UPR). In CHEC cells where Derlin-1 was down-regulated, increased expression of the immunoglobulin heavy chain-binding protein (Bip) and UPR-specific splicing of X-box DNAbinding protein 1 (XBP1) mRNA were detected, as compared with that in other tested cells, indicating that UPR was activated. After Derlin-1 overexpression, the extent of UPR activation diminished, as evidenced by decreased expression of Bip, reduced amount of the spliced form of XBP1 (XBP1s), and elevated expression of the unspliced form of XBP1 (XBP1u). Taken together, these findings provide another example of a single protein being able to affect ER dynamic in mammalian cells, and an insight into the possible molecular mechanism(s).

[Expression of alpha 1 anti-trypsin in proximal tubular epithelial cell line].

OBJECTIVE: To investigate the expression and regulation of alpha 1 anti-trypsin (AAT) in tubular epithelial cells. METHODS: The expression of AAT in tubular epithelial cell line HKC was detected with indirect immunofluorescence assay and confirmed by reverse transcription polymerase chain reaction (PCR) in transcription level respectively, and PCR product was sequenced. In order to observe the response of HCK to LPS, the different concentrations of LPS (0, 0.5, 1.0, 2.0 microg/ml) were used in the research and the regulation of AAT in HKC was semi-quantitatively detected with Western Blot and Real-Time PCR respectively. RESULTS: Indirect immunofluorescence staining showed that AAT was positive in HKC, but negative in renal interstitial fibroblast. By PCR, a significant messenger RNA band of AAT was found in HKC, but not in renal interstitial fibroblast. DNA sequencing indicated that the sequence of PCR product is consistent with AAT mRNA sequence in Gene Bank. Real-time PCR showed that the expression of AAT mRNA was up-regulated (fluorescence intensity ratio is 3.43 +/- 0.88 versus 1.22 +/- 0.20; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 4 h, and Western Blot showed that the synthesis of AAT significantly increased (band density ratio is 0.88 +/- 0.12 versus 0.59 +/- 0.05; P < 0.01) in HKC stimulated with 2.0 microg/ml LPS for 8 h, as compared with unstimulated HKC. 0.5, 1.0 g/ml of LPS has no effect on the expression of AAT mRNA and AAT protein in HKC. CONCLUSIONS: HKC express AAT and the expression can be up-regulated by LPS.

[Characteristics of human adrenal microvascular endothelial cells in morphology, phenotypes, and function].

OBJECTIVE: To investigate the phenotypic and functional characteristics of human adrenal microvascular endothelial cells (AdrEC). METHODS: AdrEC were isolated and purified from a sample of human adrenal tissue by sub-cell clone method. The cells identified by flow cytometry for classical endothelial markers von Willebrand factor (vWF) and CD31, uptake of Dil-labeled acetylated low density lipoprotein (Dil-Ac-LDL), as well as phenotypes. The cell fenestrations were checked by scanning electron microscopy. The expressions of endogenous vascular endothelial growth factor (VEGF) mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. The glucocorticoid-induced cytotoxicities in different organs-derived microvascular endothelial cells were compared. RESULTS: Human AdrEC expressed those classical endothelial markers such as vWF, CD31, and uptake of Dil-Ac-LDL. The phenotypic analysis indicated that alpha-1 proteinase inhibitor, tumor necrosis factor receptor p55, and intercellular adhesion molecule-1 were expressed in human AdrEC. Scanning electron microscopy demonstrated that there were many microvilli and fenestrations on cellular surface. RT-PCR and immunocytochemistry showed that there was expression of endogenous VEGF in AdrEC. In response to glucocorticoid-induced cytotoxicity, microvascular endothelial cells (MVEC) derived from human brain were highly susceptible, MVEC derived from human lung and human liver sinusoidal endothelial cells were sub-sensitive, while AdrEC were highly resistant. CONCLUSION: Human AdrEC are specially differentiated and have characteristics that are different from other organ-derived MVEC in phenotypes and functions.

Endothelial progenitor cells from human fetal aorta cure diabetic foot in a rat model.

OBJECTIVE: Recent evidence has suggested that circulating endothelial progenitor cells (EPCs) can repair the arterial endothelium during vascular injury. However, a reliable source of human EPCs is needed for therapeutic applications. In this study, we isolated human fetal aorta (HFA)-derived EPCs and analyzed the capacity of EPCs to differentiate into endothelial cells. In addition, because microvascular dysfunction is considered to be the major cause of diabetic foot (DF), we investigated whether transplantation of HFA-derived EPCs could treat DF in a rat model. METHODS: EPCs were isolated from clinically aborted fetal aorta. RT-PCR, fluorescence-activated cell sorting, immunofluorescence, and an enzyme-linked immunosorbent assay were used to examine the expressions of CD133, CD34, CD31, Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), von Willebrand Factor (vWF), and Endothelial Leukocyte Adhesion Molecule-1 (ELAM-1). Morphology and Dil-uptake were used to assess function of the EPCs. We then established a DF model by injecting microcarriers into the hind-limb arteries of Goto-Kakizaki rats and then transplanting the cultured EPCs into the ischemic hind limbs. Thermal infrared imaging, oxygen saturation apparatus, and laser Doppler perfusion imaging were used to monitor the progression of the disease. Immunohistochemistry was performed to examine the microvascular tissue formed by HFA-derived EPCs. RESULTS: We found that CD133, CD34, and VEGFR2 were expressed by HFA-derived EPCs. After VEGF induction, CD133 expression was significantly decreased, but expression levels of vWF and ELAM-1 were markedly increased. Furthermore, tube formation and Dil-uptake were improved after VEGF induction. These observations suggest that EPCs could differentiate into endothelial cells. In the DF model, temperature, blood flow, and oxygen saturation were reduced but recovered to a nearly normal level following injection of the EPCs in the hind limb. Ischemic symptoms also improved. Injected EPCs were preferentially and durably engrafted into the blood vessels. In addition, anti-human CD31+-AMA+-vWF+ microvasculars were detected after transplantation of EPCs. CONCLUSION: Early fetal aorta-derived EPCs possess strong self-renewal ability and can differentiate into endothelial cells. We demonstrated for the first time that transplanting HFA-derived EPCs could ameliorate DF prognosis in a rat model. These findings suggest that the transplantation of HFA-derived EPCs could serve as an innovative therapeutic strategy for managing DF.