Anti-CD31/PECAM-1 (T709) Anticorps (A25599)

CD31, an immunoglobulin-like molecule expressed by leukocytes and endothelial cells, is thought to contribute to the physiological regulation T cell homeostasis due to the presence of two immunotyrosine-based inhibitory motifs in its cytoplasmic tail. Indeed, loss of CD31 expression leads to uncontrolled T cell-mediated inflammation in a variety of experimental models of disease and certain CD31 polymorphisms correlate with increased disease severity in human graft-versus-host disease and atherosclerosis. The molecular mechanisms underlying CD31-mediated regulation of T cell responses have not yet been clarified. We here show that CD31-mediated signals attenuate T cell chemokinesis both in vitro and in vivo. This effect selectively affects activated/memory T lymphocytes, in which CD31 is clustered on the cell membrane where it segregates to the leading edge. We provide evidence that this molecular segregation, which does not occur in naïve T lymphocytes, might lead to cis-CD31 engagement on the same membrane and subsequent interference with the chemokine-induced PI3K/Akt signalling pathway. We propose that CD31-mediated modulation of memory T cell chemokinesis is a key mechanism by which this molecule contributes to the homeostatic regulation of effector T cell immunity.

OBJECTIVE:

Diabetic nephropathy (DN) is a serious complication for patients with diabetes mellitus (DM). Emerging evidence suggests that complement C3a is involved in the progression of DN. The aim of this study was to investigate the effect of C3a Receptor Agonist (C3aRA) on DN and its potential mechanism of action in rats with type 2 diabetes mellitus (T2DM).

METHODS:

T2DM was induced in SD rats by a high fat diet (HFD) plus repeated low dose streptozocin (STZ) injections. T2DM rats were treated with vehicle or C3aRA for 8 weeks. Biochemical analysis, HE and PAS stains were performed to evaluate the renal function and pathological changes. Human renal glomerular endothelial cells (HRGECs) were cultured and treated with normal glucose (NG), high glucose (HG), HG+C3a, HG+C3a+C3aRA and HG+C3a+BAY-11-7082 (p-IKBα Inhibitor) or SIS3 (Smad3 Inhibitor), respectively. Real-time PCR, immunofluorescent staining and western blot were performed to detect the mRNA and protein levels, respectively.

RESULTS:

T2DM rats showed worse renal morphology and impaired renal function compared with control rats, including elevated levels of serum creatinine (CREA), blood urea nitrogen (BUN) and urine albumin excretion (UACR), as well as increased levels of C3a, C3aR, IL-6, p-IKBα, collagen I, TGF-β and p-Smad3 in the kidney of T2DM rats and C3a-treated HRGECs. In contrast, C3aRA treatment improved renal function and morphology, reduced CREA, UACR and the intensity of PAS and collagen I staining in the kidney of T2DM rats, and decreased C3a, p-IKBα, IL-6, TGF-β, p-Smad3 and collagen I expressions in HRGECs and T2DM rats.

CONCLUSION:

C3a mediated pro-inflammatory and pro-fibrotic responses and aggravated renal injury in T2DM rats. C3aRA ameliorated T2DN by inhibiting IKBα phosphorylation and cytokine release, and also TGF-β/Smad3 signaling and ECM deposition. Therefore, complement C3a receptor is a potential therapeutic target for DN.

AIMS:

Transducer and activator of transcription-3 (STAT3) plays an important role in tumor cell invasion and metastasis. The aim of the present study was to investigate the effects of STAT3 knockdown in nude mouse xenografts of pancreatic cancer cells and underlying gene expression.

METHODS:

A STAT3 shRNA lentiviral vector was constructed and infected into SW1990 cells. qRT-PCR and western immunoblot were performed to detect gene expression. Nude mouse xenograft assays were used to assess changes in phenotypes of these stable cells in vivo. HE staining was utilized to evaluate tumor cell invasion and immunohistochemistry was performed to analyze gene expression.

RESULTS:

STAT3 shRNA successfully silenced expression of STAT3 mRNA and protein in SW1990 cells compared to control cells. Growth rate of the STAT3-silenced tumor cells in nude mice was significantly reduced compared to in the control vector tumors and parental cells-generated tumors. Tumor invasion into the vessel and muscle were also suppressed in the STAT3-silenced tumors compared to controls. Collagen IV expression was complete and continuous surrounding the tumors of STAT3-silenced SW1990 cells, whereas collagen IV expression was incomplete and discontinuous surrounding the control tumors. Moreover, microvessel density was significantly lower in STAT3-silenced tumors than parental or control tumors of SW1990 cells. In addition, MMP-7 expression was reduced in STAT3-silenced tumors compared to parental SW1990 xenografts and controls. In contrast, expression of IL-1β and IgT7α was not altered.

CONCLUSION:

These data clearly demonstrate that STAT3 plays an important role in regulation of tumor growth, invasion, and angiogenesis, which could be act by reducing MMP-7 expression in pancreatic cancer cells.

Smoking is one of the most harmful lifestyles in the world. Very few studies have investigated the effects of melatonin in smoke-induced vascular injury. This study was designed to investigate whether melatonin could protect rats and humans from smoke-induced vascular injury. 32 male rats and a double-blind randomized controlled trial (RCT) containing 63 participants formed the subjects of this study. In rats, 10 mg/kg of melatonin was intraperitoneally injected. Blood samples and abdominal artery were harvested two weeks later. Melatonin decreased the expression of platelet endothelial cell adhesion molecule-1 (CD31), intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelin-1 (ET-1) compared with the smoke exposed group (P < 0.05), whereas endothelial nitric oxide synthase (eNOS), nuclear erythroid 2-related factor 2 (Nrf2), NAD(P)H quinone oxidoreductase 1 (NQO-1), catalytic glutamate cysteine ligase (GCLC) and heme oxygenase-1 (HO-1) recovered markedly (P < 0.05). In humans, 3 mg/day of melatonin was taken orally by the participants. Blood samples were drawn at baseline and after two weeks of treatment. Compared with the oral placebo group, melatonin decreased the concentration of fibrinogen (Fbg) (P = 0.04) and free fatty acids (FFA) (P = 0.04) in smokers, along with the decreased expression of ICAM-1, VCAM-1 and ET-1 (P = 0.004, P = 0.001, P < 0.0001, respectively). In contrast, Nrf2 and HO-1 expression were markedly increased (P = 0.0001, P = 0.0049, respectively) after smokers took melatonin orally. In summary, our present data suggest that melatonin could ameliorate smoke-induced vascular injury.

We investigated the expression of human chorionic gonadotropin (HCG) and its effects on vasculogenic mimicry (VM) formation in ovarian cancer cells under normoxic and hypoxic conditions in three-dimensional matrices preconditioned by an endothelial-trophoblast cell co-culture system. The co-culture model was established using human umbilical vein endothelial cells (HUVECs) and HTR-8 trophoblast cells in a three-dimensional culture system. The co-cultured cells were removed with NH4OH, and ovarian cancer cells were implanted into the preconditioned matrix. VM was identified morphologically and by detecting vascular markers expressed by cancer cells. The specificity of the effects of exogenous HCG in the microenvironment was assessed by inhibition with a neutralizing anti-HCG antibody. HCG siRNA was used to knock down endogenous HCG expression in OVCAR-3 ovarian cancer cells. HTR-8 cells 'fingerprinted' HUVECs to form capillary-like tube structures in co-cultures. In the preconditioned HCG-rich microenvironment, the number of vessel-like network structures formed by HCG receptor-positive OVCAR-3 cells and the expression levels of CD31, VEGF and factor VIII were significantly increased. The preconditioned HCG-rich microenvironment significantly increased the expression of hypoxia inducible factor-1α (HIF‑1α) and VM formation in OVCAR-3 cells under hypoxic conditions. Treatment with a neutralizing anti-HCG antibody but not HCG siRNA significantly inhibited the formation of vessel-like network structures. HCG in the microenvironment contributes to OVCAR-3 differentiation into endothelioid cells in three-dimensional matrices preconditioned with an endothelial-trophoblast cell co-culture system. HCG may synergistically enhance hypoxia-induced vascular markers and HIF-1α expression. These findings would provide perspectives on new therapeutic targets for ovarian cancer.

Accumulating evidence indicates that heat shock protein (HSP) 60 is strongly associated with the pathology of atherosclerosis (AS). However, the precise mechanisms by which HSP60 promotes atherosclerosis remain unclear. In the present study, we found that HSP60 mRNA and protein expression levels in the thoracic aorta are enhanced not only in a mouse model of AS but also in high-fat diet (HFD) mice. HSP60 expression and secretion was activated by platelet-derived growth factor-BB (PDGF-BB) and interleukin (IL)-8 in both human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs). HSP60 was found to induce VSMC migration, and exposure to HSP60 activated ERK MAPK signaling. U0126, an inhibitor of ERK, reduced VSMC migration. The HSP60-stimulated VSMCs were found to express TLR4 mRNA but not TLR2 mRNA. Knockdown of TLR4 by siRNA reduced HSP60-induced VSMC migration and HSP60-induced ERK activation. Finally, HSP60 induced IL-8 secretion in VSMCs. Together these results suggest that HSP60 is involved in the stimulation of VSMC migration, via TLR4 and ERK MAPK activation. Meanwhile, activation of HSP60 is one of the most powerful methods of sending a 'danger signal' to the immune system to generate IL-8, which assists in the management of an infection or disease.

Acute lung injury (ALI) is a critical manifestation of sepsis/septic shock. Heat shock protein A12B (HSPA12B), an endothelial cell-expressed heat shock protein, shows a negative regulation of lipopolysaccharide (LPS)-induced inflammation in myocardium and endothelial cells. However, it is unclear whether HSPA12B exerts protective effects against ALI during sepsis/septic shock. In this study, we treated HSPA12B transgenic mice (Tg) and wild type littermates (WT) with LPS for 6h to induce endotoxemia. LPS treatment significantly caused pulmonary injuries as evidenced by microarchitecture destruction, vascular leakage and neutrophil recruitment in lungs of WT mice. However, the LPS-induced pulmonary injuries were significantly attenuated in Tg mice. Moreover, the LPS-induced activation of extracellular signal-regulated kinases (ERKs) and upregulation of intercellular adhesion molecule-1 (ICAM-1) and Cyclooxygenase-2 (Cox-2) were inhibited in Tg lungs compared with that in WT mice. Additionally, Tg lungs showed a significant lower level of vascular endothelial growth factor (VEGF) compared with WT mice. Our results demonstrate a pulmonary protective effect of HSPA12B against endotoxin challenge, which indicates management of HSPA12B expression could serve as a potential therapeutic target for ALI during sepsis/septic shock.

The chemokine (C-X-C motif) ligand 1 (CXCL1) regulates tumor-stromal interactions and tumor invasion. However, the precise role of CXCL1 on gastric tumor growth and patient survival remains unclear. In the current study, protein expressions of CXCL1, vascular endothelial growth factor (VEGF) and phospho-signal transducer and activator of transcription 3 (p-STAT3) in primary tumor tissues from 98 gastric cancer patients were measured by immunohistochemistry (IHC). CXCL1 overexpressed cell lines were constructed using Lipofectamine 2000 reagent or lentiviral vectors. Effects of CXCL1 on VEGF expression and local tumor growth were evaluated in vitro and in vivo. CXCL1 was positively expressed in 41.4% of patients and correlated with VEGF and p-STAT3 expression. Higher CXCL1 expression was associated with advanced tumor stage and poorer prognosis. In vitro studies in AGS and SGC-7901 cells revealed that CXCL1 increased cell migration but had little effect on cell proliferation. CXCL1 activated VEGF signaling in gastric cancer (GC) cells, which was inhibited by STAT3 or chemokine (C-X-C motif) receptor 2 (CXCR2) blockade. CXCL1 also increased p-STAT3 expression in GC cells. In vivo, CXCL1 increased xenograft local tumor growth, phospho-Janus kinase 2 (p-JAK2), p-STAT3 levels, VEGF expression and microvessel density. These results suggested that CXCL1 increased local tumor growth through activation of VEGF signaling which may have mechanistic implications for the observed inferior GC survival. The CXCL1/CXCR2 pathway might be potent to improve anti-angiogenic therapy for gastric cancer.

FoxM1 is a specific transcription factor that has an important function in aggressive human carcinomas, including cervical cancer. However, the specific function and internal molecular mechanism in cervical cancer remain unclear. In this study, RNAi-mediated FoxM1 knockdown inhibited cell growth. This process also decreased the migration and invasion activities of HeLa cells in vitro. Downregulation of FoxM1 inhibited tumor growth and angiogenesis in vivo. In addition, the expressions of uPA, matrix metalloproteinase (MMP)-2, MMP-9 and VEGF were significantly decreased in vitro and in vivo. These results suggested that the inactivation of FoxM1 could be a novel therapeutic target for cervical cancer treatment.

The membrane protein tyrosine phosphatase receptor U (PTPRU) has been shown to function as a negative regulator of adhesion and proliferation in certain cancer cell types, primarily through its dephosphorylation of β-catenin and inhibition of subsequent downstream signaling. In the present study, we set out to characterize the role of PTPRU in glioma and found that, while the expression of full-length PTPRU protein is low in these tumors, a number of non-full-length PTPRU isoforms are highly expressed. Among these isoforms, one in particular is localized to the nucleus, and its expression is increased in glioma tissues in a manner that positively correlates with malignancy grade. Short hairpin RNA knockdown of endogenous PTPRU in human and rat glioma cell lines suppressed proliferation, survival, invasion, migration, adhesion and vasculogenic tube formation in vitro, as well as intracranial tumor progression in vivo. In addition, knocking down PTPRU reduced tyrosine phosphorylation (pY) and transcriptional activity of β-catenin, and we were able to specifically rescue the cell migration defect by expressing a LEF1-β-catenin fusion protein in PTPRU-depleted cells. PTPRU knockdown also led to increased tyrosine pY of the E3 ubiquitin ligase c-Cbl and to the destabilization of several focal adhesion proteins. Taken together, our findings demonstrate that endogenous PTPRU promote glioma progression through their effect on β-catenin and focal adhesion signaling.

Tumor cycling hypoxia is now a well-recognized phenomenon in animal and human solid tumors. However, how tumor cycling hypoxia impacts chemotherapy is unclear. In the present study, we explored the impact and the mechanism of cycling hypoxia on tumor microenvironment-mediated chemoresistance. Hoechst 33342 staining and hypoxia-inducible factor-1 (HIF-1) activation labeling together with immunofluorescence imaging and fluorescence-activated cell sorting were used to isolate hypoxic tumor subpopulations from human glioblastoma xenografts. ABCB1 expression, P-glycoprotein function, and chemosensitivity in tumor cells derived from human glioblastoma xenografts or in vitro cycling hypoxic stress-treated glioblastoma cells were determined using Western blot analysis, drug accumulation and efflux assays, and MTT assay, respectively. ABCB1 expression and P-glycoprotein function were upregulated under cycling hypoxia in glioblastoma cells concomitant with decreased responses to doxorubicin and BCNU. However, ABCB1 knockdown inhibited these effects. Moreover, immunofluorescence imaging and flow cytometric analysis for ABCB1, HIF-1 activation, and Hoechst 3342 in glioblastoma revealed highly localized ABCB1 expression predominantly in potentially cycling hypoxic areas with HIF-1 activation and blood perfusion in the solid tumor microenvironment. The cycling hypoxic tumor cells derived from glioblastoma xenografts exhibited higher ABCB1 expression, P-glycoprotein function, and chemoresistance, compared with chronic hypoxic and normoxic cells. Tumor-bearing mice that received YC-1, an HIF-1α inhibitor, exhibited suppressed tumor microenvironment-induced ABCB1 induction and enhanced survival rate in BCNU chemotherapy. Cycling hypoxia plays a vital role in tumor microenvironment-mediated chemoresistance through the HIF-1-dependent induction of ABCB1. HIF-1 blockade before and concurrent with chemotherapy could suppress cycling hypoxia-induced chemoresistance.

Cycling hypoxia is a well-recognized phenomenon within animal and human solid tumors. It mediates tumor progression and radiotherapy resistance through mechanisms that involve reactive oxygen species (ROS) production. However, details of the mechanism underlying cycling hypoxia-mediated radioresistance remain obscure. We have previously shown that in glioblastoma, NADPH oxidase subunit 4 (Nox4) is a critical mediator involved in cycling hypoxia-mediated ROS production and tumor progression. Here, we examined the impact of an in vivo tumor microenvironment on Nox4 expression pattern and its impact on radiosensitivity in GBM8401 and U251, two glioblastoma cell lines stably transfected with a dual hypoxia-inducible factor-1 (HIF-1) signaling reporter construct. Furthermore, in order to isolate hypoxic tumor cell subpopulations from human glioblastoma xenografts based on the physiological and molecular characteristics of tumor hypoxia, several techniques were utilized. In this study, the perfusion marker Hoechst 33342 staining and HIF-1 activation labeling were used together with immunofluorescence imaging and fluorescence-activated cell sorting (FACS). Our results revealed that Nox4 was predominantly highly expressed in the endogenous cycling hypoxic areas with HIF-1 activation and blood perfusion within the solid tumor microenvironment. Moreover, when compared to the normoxic or chronic hypoxic cells, the cycling hypoxic tumor cells derived from glioblastoma xenografts have much higher Nox4 expression, ROS levels, and radioresistance. Nox4 suppression in intracerebral glioblastoma-bearing mice suppressed tumor microenvironment-mediated radioresistance and enhanced the efficiency of radiotherapy. In summary, our findings indicated that cycling hypoxia-induced Nox4 plays an important role in tumor microenvironment-promoted radioresistance in glioblastoma; hence, targeting Nox4 may be an attractive therapeutic strategy for blocking cycling hypoxia-mediated radioresistance.

Many of the ligands involved in developmental processes require HS (heparan sulfate) to modulate signal transduction. hHS6ST2 (human heparan sulfate D-glucosaminyl 6-O-sulfotransferase-2) is a Golgi-resident enzyme that usually acts on GlcA/IdoA(2S)-GlcNAc/NS disaccharide-6-sulfate modifications within the HS sequence. Emerging evidence indicates the importance of 6-O-sulfation in a number of developmental processes. However, any correlation with cancer-related events remains largely unexplored. In the present study, we found that hHS6ST2, but not other variants, was activated in human PC (pancreatic cancer). shRNA (short hairpin RNA)-mediated silencing of endogenous hHS6ST2 expression in the PC cell line PANC-1 inhibited cell invasion and migration. hHS6ST2 knockdown also resulted in markedly reduced tumorigenesis in immunocompromised mice. To specifically explore the molecular alterations resulting from depletion of hHS6ST2-generated 6-O-sulfation, we employed two-dimensional gel electrophoresis technology followed by nano-HPLC-ESI (electrospray ionization)-tandem MS to separate and identify total proteins from PC cells. Our data suggest that hHS6ST2 potentiates Notch signalling in PC cells. We also identified a role for hHS6ST2 in the growth and tumorigenicity of these cells which, at least in part, acts through Notch-mediated EMT (epithelial-mesenchymal transition) and angiogenesis. The results of the present study suggest that hHS6ST2 could be an attractive target for PC therapy.

Innovative cell-based therapies, including hepatic tissue engineering following hepatocyte transplantation, are considered as theoretical alternatives to liver transplant or for partial replacement of liver function in patients. However, recent progress in hepatic tissue engineering has been hampered by low initial hepatocyte engraftment and insufficient blood supply in vivo. We developed an intact 3D scaffold of an extracellular matrix (ECM) derived from a decellularized liver lobe, with layer-by-layer (LbL) heparin deposition to avoid thrombosis, which we repopulated with hepatocytes and successfully implanted as a tissue-engineered liver (TEL) into the portal system. The TEL provided sufficient volume for transplantation of cell numbers representing up to 10% of whole-liver equivalents and was perfused by portal vein blood. Treatment of extended hepatectomized rats with a TEL improved liver function and prolonged survival; mean lifespan was extended from 16 to 72 h. At 72 h postoperation, the TEL sustained functional and viable hepatocytes. In conclusion, we propose the TEL as a state-of-the-art substitute for whole-liver transplantation and as a proof of concept for the technology that will eventually allow for the transplantation of a reconstituted liver.

Endostatin has potent anti-endothelial and anti-angiogenic functions. Endostatin was reported to reduce lymphangiogenesis by down-regulating the level of VEGF-C in tumour tissues. However, there is little evidence for the direct function of endostatin on lymphangiogenic endothelial cells and lymphangiogenic vessels. Here, we report that cell surface nucleolin, which was reported as an endostatin receptor mediating its anti-angiogenic and anti-tumour functions, is also selectively expressed on the cell surface of lymphangiogenic endothelial cells both in vitro and in vivo. Treatment of primary mouse lymphatic endothelial cells (mLECs) by endostatin inhibits mLEC migration, tubule formation, and activation of the Erk pathway in mLECs, while neutralization of cell surface nucleolin or nucleolin knockdown results in loss of the anti-lymphatic endothelial activities of endostatin. Also, anti-nucleolin antibody or lentivirus delivered nucleolin siRNA abolishes the anti-lymphangiogenic function of endostatin in the Matrigel plug assay. Endostatin remarkably inhibits tumour-associated lymphangiogenesis, leading to reduced lymphatic metastasis. Systemic blockade of nucleolin notably abolishes the anti-lymphangiogenic and anti-lymphatic metastatic functions of endostatin. Importantly, endostatin does not affect quiescent lymphatics in normal organs, which is consistent with the lack of expression of cell surface nucleolin in quiescent lymphatics. Taken together, our results demonstrate that endostatin directly acts on lymphangiogenic endothelial cells via cell surface nucleolin, which provides a novel mechanism for the inhibition of tumour lymphangiogenesis and lymphatic metastasis by endostatin.

Heat shock protein 90alpha (Hsp90alpha) is a ubiquitously expressed molecular chaperone, which is essential for the maintenance of eukaryote homeostasis. Hsp90alpha can also be secreted extracellularly and is associated with several physiological and pathological processes including wound healing, cancer, infectious diseases and diabetes. Angiogenesis, defined as the sprouting of new blood vessels from pre-existing capillaries via endothelial cell proliferation and migration, commonly occurs in and contributes to the above mentioned processes. However, the secretion of Hsp90alpha from endothelial cells and also its function in angiogenesis are still unclear. Here we investigated the role of extracellular Hsp90alpha in angiogenesis using dermal endothelial cells in vitro and a wound healing model in vivo. We find that the secretion of Hsp90alpha but not Hsp90beta is increased in activated endothelial cells with the induction of angiogenic factors and matrix proteins. Secreted Hsp90alpha localizes on the leading edge of endothelial cells and promotes their angiogenic activities, whereas Hsp90alpha neutralizing antibodies reverse the effect. Furthermore, using a mouse skin wound healing model in vivo, we demonstrate that extracellular Hsp90alpha localizes on blood vessels in granulation tissues of wounded skin and promotes angiogenesis during wound healing. Taken together, our study reveals that Hsp90alpha can be secreted by activated endothelial cells and is a positive regulator of angiogenesis, suggesting the potential application of Hsp90alpha as a stimulator for wound repair.

BACKGROUND:

Endothelial-myofibroblast transition (EndMT) has been implicated in the pathogenesis of diabetic renal fibrosis. In this study, the effect of the complement fragments C3a/C5a and their receptor antagonists C3aRA and C5aRA on EndMT in diabetic kidney disease (DKD) and the possible mechanisms were investigated.

METHODS:

The coexpression of CD31 with α-smooth muscle (α-SMA), C3a receptor (C3aR) and C5a receptor (C5aR) was detected in human renal biopsy tissue obtained from patients with early and advanced DKD and in normal renal tissues from patients with renal-cell carcinoma. The effects of C3aRA and C5aRA on EndMT and the expression of C3a/C3aR, C5a/C5aR, α-SMA, CD31, TGFβ, FN and β-catenin were examined in a streptozotocin (STZ)-induced rat model of DKD and in human renal glomerular endothelial cells (HRGECs) cultured in high glucose and with C3a/C5a, and DKK1 (a Wnt/β-catenin inhibitor).

RESULTS:

Double-labeling of α-SMA, C3aR, C5aR and CD31 was detected in the glomerulus of renal tissues obtained from biopsies of patients with DKD. Upregulated expression of α-SMA, TGF-β, FN and β-catenin and downregulated expression of CD31 were detected in the GECs of diabetic rats. The expression of these proteins was inhibited by treatment with C3aRA/C5aRA. In vitro, C3aRA/C5aRA and DKK1 ameliorated the high glucose-induced EndMT and the subsequent expression of α-SMA, TGFβ, FN and β-catenin in HRGECs.

CONCLUSIONS:

The blockade of C3aR/C5aR and the downstream Wnt/β-catenin pathway may prevent EndMT and alleviate fibrosis in the glomeruli of individuals with DKD.

Copyright © 2015 Elsevier Inc. All rights reserved.

BACKGROUND:

High-intensity focused ultrasound (HIFU) is a widely applied to treatment for unresectable hepatocellular carcinoma. However, insufficient HIFU can result in rapid progression of the residual tumor. The mechanism of such rapid growth of the residual tumor after HIFU ablation is poorly understood.

OBJECTIVE:

The aim of this study was to investigate the dynamic angiogenesis of residual tumor, and the temporal effect and mechanism of the HIF-1, 2α in the residual tumor angiogenesis.

METHODS:

Xenograft tumors of HepG2 cells were created by subcutaneously inoculating nude mice (athymic BALB/c nu/nu mice) with hepatoma cells. About thirty days after inoculation, all mice (except control group) were treated by HIFU and assigned randomly to 7 groups according to various time intervals (1st, 3rd, 5th day (d) and 1st, 2nd, 3rd, 4th week (w)). The residual tumor tissues were obtained from the experimental groups at various time points. Protein levels of HIF-1α, HIF-2α, VEGF-A, and EphA2 were quantified by immunohistochemistry analysis and Western Blot assays, and mRNA levels measured by Q-PCR. Microvascular density was calculated with counting of CD31 positive vascular endothelial cells by immunohistochemical staining.

RESULTS:

Compared with the control group, protein and mRNA levels of HIF-1α reached their highest levels on the 3rd day (P<0.01), then decreased (P<0.05). HIF-2α expression reached its highest level on the 2nd week compared with control group (P<0.01), then decreased (2 w-4 w) (P<0.05). The protein and mRNA levels of VEGF-A and EphA2 in the residual tumor tissues group that received HIFU were significantly decreased until 1 week compared with the control group (P<0.01). However, the levels increased compared to controls in 2-4 weeks (P<0.05). Similar results were obtained for MVD expression (P<0.05).

CONCLUSION:

Insufficient HIFU ablation promotes the angiogenesis in residual carcinoma tissue over time. The data indicate that the HIF-1, 2α/VEGFA/EphA2 pathway is involved.

BACKGROUND & AIMS:

Unusual hypervascularity is a hallmark of human hepatocellular carcinoma (HCC). Although microRNA-214 (miR-214) is upregulated in other human cancers, it is downregulated in HCC. We elucidated the biological and clinical significance of miR-214 downregulation in HCC.

METHODS:

MicroRNAs deregulated in HCC were identified using array-based microRNA profiling. A luciferase reporter assay confirmed target association between miR-214 and the hepatoma-derived growth factor (HDGF). Tube formation and in vivo angiogenesis assays validated the roles of miR-214/HDGF in angiogenesis.

RESULTS:

miR-214 downregulation was associated with higher tumor recurrence and worse clinical outcomes. Ectopic expression of miR-214 suppressed xenograft tumor growth and microvascularity of the tumors and their surrounding tissues. The genes downregulated by ectopic expression of miR-214 were involved in the regulation of apoptosis, cell cycle, and angiogenesis. Integrated analysis disclosed HDGF as a downstream target of miR-214. Conditioned medium of HCC cells contained bioactivity to stimulate tube formation of human umbilical vein endothelial cells, which was abolished by pretreatment of the conditioned media with HDGF antibodies, suppression of HDGF expression or ectopic expression of miR-214 in the donor HCC cells. The angiogenic activity of the conditioned media, lost by ectopic expression of miR-214 in the donor cells, was restored by supplementation with recombinant HDGF. In vivo tumor angiogenesis assays showed significant suppression of tumor vascularity by ectopic expression of miR-214.

CONCLUSIONS:

A novel role of microRNA in tumorigenesis is identified. Downregulation of miR-214 contributes to the unusual hypervascularity of HCC via activation of the HDGF paracrine pathway for tumor angiogenesis.

Copyright © 2012 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

PURPOSE:

High-grade gliomas are closely related to the mesenchymal phenotype which might be explained by unorthodox differentiation of glioma cancer stem cells (gCSCs). We reasoned that other non-neural stem cells, especially mesenchymal stem cells (MSCs), might play a role in expressing mesenchymal phenotype of high-grade gliomas. Thus we hypothesized that cells resembling MSCs exist in glioma specimens.

METHODS:

We created a mouse (m) orthotopic glioma model using human gCSCs. Single-cell suspensions were isolated from glioma specimens and cultured according to the methods for mMSCs or gliomaspheres. These cells were analyzed by fluorescence-activated cell sorting (FACS) for surface markers associated with mMSCs or gCSCs. Glioma stroma (GS)-MSCs were exposed to mesenchymal differentiation conditions. To decide the location of GS-MSCs, sections of orthotopic glioma models were analyzed by immunofluorescent labeling.

RESULTS:

GS-MSCs were isolated which were morphologically similar to mMSCs. FACS analysis showed that the GS-MSCs had similar surface markers to mMSCs (stem cell antigen-1 [Sca-1](+), CD9(+), CD45(-), CD11b(-), CD31(-), and nerve/glial antigen 2 [NG2](-)). GS-MSCs were capable of mesenchymal differentiation. Immunofluorescent labeling indicated that GS-MSCs are located around blood vessels, are distinct from endothelial cells, and have features that partially overlap with vascular pericytes.

CONCLUSIONS:

Our results indicate that cells similar to mMSCs exist in glioma specimens. The GS-MSCs might be located around vessels, which suggests that GS-MSCs may provide the mesenchymal elements of the vascular niche. GS-MSCs may represent non-neural stem cells that act as an important source of mesenchymal elements, particularly during the growth of gliomas.