Human CD44 Research Articles

High-resolution crystal structures of alternate forms of the human CD44 hyaluronan-binding domain reveal a site for protein interaction.

Two new crystal structures of the extracellular hyaluronan-binding domain of human CD44 are described at high resolution. A hexagonal crystal form at 1.60 Å resolution and a monoclinic form at 1.08 Å resolution both have two molecules in the asymmetric unit arranged about a similar noncrystallographic twofold axis of symmetry. These structures are compared with those previously reported at 2.20 Å resolution to show that the fold is quite resistant to structural deformation in different crystal environments. Unexpectedly, a short peptide is found in the monoclinic crystals at a site remote from the known hyaluronan-binding groove. The peptide with a valine at the carboxy-terminus must have co-purified from the bacterial expression host and binds on the opposite side of the domain from the known hyaluronan-binding groove. This opportunistic binding may identify a site of interaction used as CD44 assembles with other proteins to accomplish effective signaling regarding changes to the extracellular environment.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.

Absence of lymphatic vessels in the developing human sclera

The adult sclera is free of lymphatic vessels, but contains a net of blood vessels. Whether and when this selectively lymphangiogenic privilege is achieved during embryologic development is not known yet. Therefore, we investigated the developing human sclera for blood- and lymphatic vessels in 34 abortions/stillborns (12–38 weeks of gestation). The probes were subdivided into three groups (group 1: 12–18 weeks of gestation, n = 10; group 2: 19–23 weeks of gestation, n = 13; group 3: 24–38 weeks of gestation, n = 11), and prepared for paraffin sections followed by immunohistochemistry against CD31 to detect blood vessels, and against lymphatic vessel endothelial hyaluronan receptor-1 (LYVE1)/podoplanin to detect lymphatic vessels. We could show, that in the human episclera distinct CD31 + blood vessels are present as early as week of gestation 13. Their amount increased during pregnancy, whereas stromal CD31 + blood vessels were elevated in early pregnancy and regressed with ongoing pregnancy. In the lamina fusca CD31 + blood vessels were absent at any time point investigated. Single LYVE1 + cells were identified primarily in the episclera; their amount decreased significantly with increasing gestational ages (group 1 compared to group 3: p < 0.01). However, LYVE1+/podoplanin + lymphatic vessels were not detectable in the sclera at any gestational ages analyzed. In contrast to the conjunctiva where LYVE1+/podoplanin + lymphatic vessels were detectable as early as week 17, the amount of LYVE1 + cells in the sclera was highest in early pregnancy (group 1), with a significant decrease during continuing pregnancy (p < 0.001). These findings are the first evidence for a fetal lymphangiogenic privilege of the sclera and show, that the fetal human sclera contains CD31 + blood vessels, but is primarily alymphatic. Our findings suggest a strong expression of selectively antilymphangiogenic factors, making the developing sclera a potential model to discern antilymphangiogenic mechanisms.

Endothelial cells enhance the in vivo bone-forming ability of osteogenic cell sheets

Addressing the problem of vascularization is of vital importance when engineering three-dimensional (3D) tissues. Endothelial cells are increasingly used in tissue-engineered constructs to obtain prevascularization and to enhance in vivo neovascularization. Rat bone marrow stromal cells were cultured in thermoresponsive dishes under osteogenic conditions with human umbilical vein endothelial cells (HUVECs) to obtain homotypic or heterotypic cell sheets (CSs). Cells were retrieved as sheets from the dishes after incubation at 20 °C. Monoculture osteogenic CSs were stacked on top of homotypic or heterotypic CSs, and subcutaneously implanted in the dorsal flap of nude mice for 7 days. The implants showed mineralized tissue formation under both conditions. Transplanted osteogenic cells were found at the new tissue site, demonstrating CS bone-inductive effect. Perfused vessels, positive for human CD31, confirmed the contribution of HUVECs for the neovascularization of coculture CS constructs. Furthermore, calcium quantification and expression of osteocalcin and osterix genes were higher for the CS constructs, with HUVECs demonstrating the more robust osteogenic potential of these constructs. This work demonstrates the potential of using endothelial cells, combined with osteogenic CSs, to increase the in vivo vascularization of CS-based 3D constructs for bone tissue engineering purposes.

Failure of anti tumor-derived endothelial cell immunotherapy depends on augmentation of tumor hypoxia

We have previously demonstrated that Tenascin-C (TNC)(+) human neuroblastoma (NB) cells transdifferentiate into tumor-derived endothelial cells (TDEC), which have been detected both in primary tumors and in tumors formed by human NB cell lines in immunodeficient mice. TDEC are genetically unstable and may favor tumor progression, suggesting that their elimination could reduce tumor growth and dissemination. So far, TDEC have never been targeted by antibody-mediated immunotherapy in any of the tumor models investigated. To address this issue, immunodeficient mice carrying orthotopic NB formed by the HTLA-230 human cell line were treated with TDEC-targeting cytotoxic human (h)CD31, that spares host-derived endothelial cells, or isotype-matched mAbs. hCD31 mAb treatment did not affect survival of NB-bearing mice, but increased significantly hypoxia in tumor microenvironment, where apoptotic and proliferating TDEC coexisted, indicating the occurrence of vascular remodeling. Tumor cells from hCD31 mAb treated mice showed i) up-regulation of epithelial-mesenchymal transition (EMT)-related and vascular mimicry (VM)-related gene expression, ii) expression of endothelial (i.e. CD31 and VE-cadherin) and EMT-associated (i.e. Twist-1, N-cadherin and TNC) immunophenotypic markers, and iii) up-regulation of high mobility group box-1 (HMGB-1) expression. In vitro experiments with two NB cell lines showed that hypoxia was the common driver of all the above phenomena and that human recombinant HMGB-1 amplified EMT and TDEC trans-differentiation. In conclusion, TDEC targeting with hCD31 mAb increases tumor hypoxia, setting the stage for the occurrence of EMT and of new waves of TDEC trans-differentiation. These adaptive responses to the changes induced by immunotherapy in the tumor microenvironment allow tumor cells to escape from the effects of hCD31 mAb.

Establishment of hypoxia induction in an in vivo animal replacement model for experimental evaluation of pancreatic cancer.

Transplantation of tumor xenografts to fertilized chicken eggs is a promising animal replacement method, which has successfully been used for xenotransplantation of pancreatic ductal adenocarcinoma (PDA) cells. PDA is characterized by a pronounced tumor hypoxia, which mediates aggressive growth, therapy resistance and cancer stem cell (CSC) features. For in vivo experimental evaluation of hypoxia-targeting therapeutic strategies, the xenografting of tumors to chicken eggs combined with the induction of hypoxia is necessary. However, the chicken embryos do not survive the conventional method of hypoxia induction by a gas mixture of 1% O2, 5% CO2, 94% N2, not even when hypoxia is applied for only 30 min. Therefore, we employed chemical induction of hypoxia by the hypoxia mimetic agent cobalt chloride (CoCl2). Whereas CoCl2 did not further increase tumor growth, it mediated the induction of carbonic anhydrase IX (CAIX) in the tumor xenografts and led to enhanced expression of the human CSC markers CD133, Sox2 and CD44. Side-effects in chicken embryos were not observed as evaluated by H&E staining of embryo-derived liver sections and the determination of the embryo weight. These results suggest the successful induction of hypoxia in chicken eggs and xenografted tumors by CoCl2. For therapeutic intervention and as a control, we treated the eggs with the plant-derived anti-inflammatory agent triptolide, which recently showed promising effects toward hypoxia-induced tumor progression in experimental PDA. Triptolide abolished tumor growth and the CoCl2-induced hypoxic effects, without inducing obvious side-effects. Collectively, our data present a new in vivo animal replacement method for the successful induction of tumor hypoxia in PDA.

Regulatory factor X1 is a new tumor suppressive transcription factor that acts via direct downregulation of CD44 in glioblastoma

The biological functions of regulatory factor (RF)X1, a transcription factor, are not known. Since the RFX1 gene is often epigenetically silenced and clusters of differentiation (CD)44 proteins that regulate cancer cell biology are increased in human glioblastomas, we designed this study to determine whether RFX1 could regulate CD44 expression in glioblastoma. Regulatory factor X1 was overexpressed in 4 human glioblastoma cell lines. CD44 expression and cell proliferation, apoptosis, and invasion were assayed under in vitro conditions. In vivo growth of human glioblastoma xenografts was determined in mice. The expression of RFX1 and CD44 in human glioblastoma tissues was quantified. A putative RFX1 binding sequence existed in the first exon of the human CD44 gene. The transcription activity of the DNA fragment containing this putative sequence was decreased in cells overexpressing RFX1. Regulatory factor X1 bound to the CD44 gene in glioblastoma cells. It reduced CD44 expression and activated Akt and extracellular signal-regulated kinase, signaling molecules downstream of CD44 to regulate cell proliferation and survival. Overexpression of RFX1 inhibited the survival, proliferation, and transwell invasion of glioblastoma cells and in vivo growth of human glioblastoma xenografts. CD44 overexpression reversed RFX1 effects on cell proliferation. Finally, CD44 protein levels were inversely correlated with RFX1 protein levels in human glioblastoma tissues. These results suggest that RFX1 directly regulates CD44 expression. This mechanism may contribute to RFX1's effects on proliferation, survival, and invasion of glioblastoma cells. Our results provide initial evidence that RFX1 may be an important target/regulator of the malignancy of glioblastoma.

A9.12 Effects of endothelin-1 and its receptor antagonism on the endothelial-to-mesenchymal transition in cultured human dermal microvascular endothelial cells

Background and ObjectivesThe early events involved in the pathogenesis of many connective tissue diseases, such as systemic sclerosis (SSc) include endothelial/microvascular damage and activation of myofibroblasts responsible for excessive deposition...

In Vivo Evaluation of Wound Bed Reaction and Graft Performance After Cold Skin Graft Storage

Surplus harvested skin grafts are routinely stored at 4 to 6°C in saline for several days in plastic surgery. The purpose of this study was to evaluate the influence of storage on human skin graft performance in an in vivo intravital microscopic setting after transplantation. Freshly harvested human full-thickness skin grafts and split-thickness skin grafts (STSGs) after storage of 0, 3, or 7 days in moist saline at 4 to 6°C were transplanted into the modified dorsal skinfold chamber, and intravital microscopy was performed to evaluate vessel morphology and angiogenic change of the wound bed. The chamber tissue was harvested 10 days after transplantation for evaluation of tissue integrity and inflammation (hematoxylin and eosin) as well as for immunohistochemistry (human CD31, murine CD31, Ki67, Tdt-mediated dUTP-biotin nick-end labelling). Intravital microscopy results showed no differences in the host angiogenic response between fresh and preserved grafts. However, STSGs and full-thickness skin grafts exhibited a trend toward different timing and strength in capillary widening and capillary bud formation. Preservation had no influence on graft quality before transplantation, but fresh STSGs showed better quality 10 days after transplantation than 7-day preserved grafts. Proliferation and apoptosis as well as host capillary in-growth and graft capillary degeneration were equal in all groups. These results indicate that cells may activate protective mechanisms under cold conditions, allowing them to maintain function and morphology. However, rewarming may disclose underlying tissue damage. These findings could be translated to a new approach for the design of full-thickness skin substitutes.

Nodal Promotes the Self-Renewal of Human Colon Cancer Stem Cells via an Autocrine Manner through Smad2/3 Signaling Pathway

Colorectal cancer is one of the most common and fatal tumors. However, molecular mechanisms underlying carcinogenesis of colorectal cancer remain largely undefined. Here, we explored the expression and function of Nodal in colon cancer stem cells (CCSCs). Nodal and its receptors were present in numerous human colorectal cancer cell lines. NODAL and ALK-4 were coexpressed in human colon cancerous tissues, and NODAL, CD24, and CD44, markers for CCSCs, were expressed at higher levels in human colon cancerous tissues than adjacent noncancerous colon tissues. Human CCSCs were isolated by magnetic activated cell sorting using anti-CD24 and anti-CD44. Nodal transcript and protein were hardly detectable in CD44- or CD24-negative human colorectal cancer cell lines, whereas Nodal and its receptors were present in CCSCs. Notably, Nodal facilitated spheroid formation of human CCSCs, and phosphorylation of Smad2 and Smad3 was activated by Nodal in cells of spheres derived from human CCSCs. Collectively, these results suggest that Nodal promotes the self-renewal of human CCSCs and mediate carcinogenesis of human colorectal cancer via an autocrine manner through Smad2/3 pathway. This study provides a novel insight into molecular mechanisms controlling fate of human CCSCs and offers new targets for gene therapy of human colorectal cancer.

CD44 Monoclonal Antibody-Enhanced Clearance Of Chronic Myeloid Leukemia Stem Cells From The Malignant Niche

IntroductionLeukemia stem cells (LSC) in chronic myeloid leukemia (CML) are important in disease progression and relapse. Conventional therapy with tyrosine kinase inhibitors (TKIs), although very effective at reducing bulk CML cells, frequently fail to eliminate LSC residing in the protective bone marrow niche. Thus, there is an unmet need for combination therapy that simultaneously targets bulk disease and eliminates LSC in order to prevent disease progression and relapse. Stem cell niches are often rich in hyaluronic acid. CD44, the main receptor for hyaluronic acid, and some of its splice variants are frequently overexpressed on cancer stem cells, including LSC. In this study we aimed to evaluate the in vivo anti-LSC activity of CD44 monoclonal antibody (mAb) RG7356. Methods and ResultsFor anti-CD44 LSC inhibition studies, immunodeficient RAG2-/-γc-/- neonatal mice were intrahepatically transplanted with human BC LSC from imatinib responsive and resistant patients. Once engraftment was verified, mice were treated with control IgG1 (30mg/kg/week), dasatinib (25mg/kg/day), CD44 mAb (RG7356) (30mg/kg/week) or a combination of CD44 mAb (RG7356) and dasatinib. The mice were sacrificed after two weeks of therapy and bone marrow, spleen, peripheral blood and myeloid sarcomas were analyzed by FACS to assess CML LSC burden. In the imatinib responsive patient, CD44 mAb single agent therapy showed an 85% reduction of LSC in bone marrow, 98% reduction in spleen and 91% reduction in peripheral blood. CD44 mAb therapy thus showed an efficacy equal to that of dasatinib, which showed an 87%, 98% and 98% reduction in bone marrow, spleen and peripheral blood respectively. Combination therapy (CD44 mAb + dasatinib) completely eradicated any trace of CML LSC in all tissues analyzed. In the imatinib resistant patient, dasatinib very effectively reduced the number of myeloid sarcomas and blasts but did not inhibit LSC survival in bone marrow. In contrast, CD44 mAb single agent therapy effectively reduced the number of CML LSC to 51% in bone marrow, 96% in spleen and 93% in peripheral blood. ConclusionsThis study demonstrates that a human CD44 specific mAb, RG7356, significantly reduces CML LSC survival. Notably, TKI resistance of CML LSC, can be overcome by treatment with a human CD44 specific mAb, RG7356, as it sensitizes CML LSC residing in malignant niches to dasatinib. From these results, RG7356 appears to be an excellent antibody for future combination clinical studies aimed at eradicating CML. Disclosures:Runza:employee of Roche Diagnostics GmbH: Employment. Weigand:Roche Diagnostics GmbH: Employment. Jamieson:J&J: Research Funding; Sanofi: Consultancy; Roche: Research Funding.

Abstract B237: CD105: A therapeutic target for sarcomas.

Abstract Purpose: Endoglin/CD105 is a homodimeric transmembrane glycoprotein that is expressed on the proliferating endothelium of solid tumors. TRC105, the chimeric IgG1 antibody with high affinity binding for human CD105, has recently undergone human testing. This exploratory study sought to validate CD105 as a potential therapeutic target in sarcomas. Methods: We performed immunohistochemical staining for CD105 expression in 146 retrospectively identified paraffin embedded sarcoma patient samples in our tumor registry, utilizing an anti-CD105 antibody (clone 4G11, Novocastra). The diagnoses were verified and samples were examined for CD105 expression in the malignant vasculature as well as tumor cells. Results: The vast majority of specimen expressed CD105 in the tumor vasculature, 134 of 137 evaluable cases. CD105 expression in the cells of the varied sarcoma histologies themselves were as follows: 19/20 angiosarcomas, 6/7 Ewing sarcomas, 8/20 undifferentiated pleomorphic sarcomas, 6/20 uterine leiomyosarcomas (LMS), 4/20 osteosarcomas, 3/20 non-uterine LMS, 3/20 synovial sarcomas. None of the 19 evaluated chondrosarcoma cells expressed CD105. Thirteen of 19 angiosarcomas showed CD105 expression in &amp;gt;50% of tumor cells, 3 of 19 showed CD105 expression in 25-49% of the tumor cells and 3 of 19 showed CD105 expression in 5-24% of tumor cells. Conclusions: CD105 targeting appears to be a rational antiangiogenic therapeutic approach in sarcomas, with particular emphasis on angiosarcomas. A clinical trial utilizing TRC105 in soft tissue sarcomas is currently under development. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B237. Citation Format: Karen Fritchie, Steven Attia, Scott Okuno, Carola Arndt, Steven Robinson. CD105: A therapeutic target for sarcomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B237.

In Vitro Selection of Modified RNA Aptamers Against CD44 Cancer Stem Cell Marker

Cancer stem cells (CSCs) are a subset of tumor cells that has the ability to self-renew and to generate the diverse cells that comprise the tumor mass. The cell-surface glycoprotein CD44 is one of the most common surface markers used to identify CSCs. Aptamers are synthetic oligonucleotides selected from pools of random sequences that can bind to a wide range of targets with high affinity and specificity. In this study, the systematic evolution of ligands by exponential enrichment (SELEX) technology was used to isolate RNA aptamers using human recombinant full-length CD44 protein and 2'-F-pyrimidine modified RNA library with a complexity of around 10(14) different molecules. Following 11 iterative rounds of SELEX, the selected aptamers were cloned and sequenced. Three different sequences were identified. The binding specificities for one of these RNA aptamers was assessed using representative breast cancer cell lines expressing CD44; namely, MDA-MB-231, MCF7, and T47D. The selected RNA aptamer (Apt1) was found to interact specifically with such cancer cells when analyzed by flow cytometry and fluorescent microscopy, with different intensities of fluorescence reflecting the level of CD44 expression on the surface of these cells. It can be concluded that the selected aptamers can be used to target CD44 positive cells, including cancer stem cells, for detection, sorting, and enrichment and for drug delivery purposes.

Hox genes are involved in vascular wall-resident multipotent stem cell differentiation into smooth muscle cells

Human vascular wall-resident CD44+ multipotent stem cells (VW-MPSCs) within the vascular adventitia are capable to differentiate into pericytes and smooth muscle cells (SMC). This study demonstrates HOX-dependent differentiation of CD44(+) VW-MPSCs into SMC that involves epigenetic modification of transgelin as a down-stream regulated gene. First, HOXB7, HOXC6 and HOXC8 were identified to be differentially expressed in VW-MPSCs as compared to terminal differentiated human aortic SMC, endothelial cells and undifferentiated pluripotent embryonic stem cells. Silencing these HOX genes in VW-MPSCs significantly reduced their sprouting capacity and increased expression of the SMC markers transgelin and calponin and the histone gene histone H1. Furthermore, the methylation pattern of the TAGLN promoter was altered. In summary, our findings suggest a role for certain HOX genes in regulating differentiation of human VW-MPSC into SMCs that involves epigenetic mechanisms. This is critical for understanding VW-MPSC–dependent vascular disease processes such as neointima formation and tumor vascularization.

Endothelial cells do not arise from tumor-initiating cells in human hepatocellular carcinoma

BackgroundConventional models of carcinogenesis suggest that tumors recruit blood vessel formation from normal host tissues. This concept has recently been challenged by prominent studies of glioblastoma, which suggest that intratumoral endothelial cells (ECs) may arise from cancer stem cells/tumor-initiating cells (TICs). Hepatocellular carcinoma (HCC) is a common, highly vascularized tumor with few effective therapies, against which anti-angiogenic strategies are being actively explored. TICs are felt to play a role in HCC pathobiology, but their contributions to tumor vasculature have not been studied.MethodsWe examined human HCCs in settings that selected for tumor formation from functionally defined TICs, and in which the origin of intratumoral ECs from TICs as opposed to host tissues could be clearly distinguished. We generated HCC nodules in the livers of immunodeficient mice by intrasplenic injection of HCC cells from cell lines and patient specimens and studied the tumor ECs by immunohistochemistry for mouse and human markers. We then used immunohistochemistry for EC markers in combination with fluorescence in situ hybridization (FISH) for X and Y chromosomes to study the endothelium of recurrent HCC specimens resected from sex-mismatched liver allografts of patients who had undergone liver transplantation for HCC.ResultsWe observed that all ECs in intrahepatic human HCC xenografts expressed mouse rather than human CD31. FISH analysis of recurrent HCCs resected from patients with sex-mismatched liver allografts revealed that all CD31+ and CD34+ intratumoral ECs originated from the donor allograft rather than the tumor.ConclusionsThese observations suggest that the vasculature of human HCC arises from normal host tissues rather than from TICs, supporting ongoing efforts to target angiogenesis in HCC as it is currently understood, and suggesting that the contribution of TICs to the vasculature of other cancers is disease-specific.

Comparison of the properties of human CD146+ and CD146− periodontal ligament cells in response to stimulation with tumour necrosis factor α

ObjectivesPeriodontal ligament stem cells (PDLSCs) can be used in periodontal regeneration. Tumour necrosis factor-alpha (TNF-α) participates in the regulation of cell proliferation, apoptosis, differentiation, and migration. However, whether TNF-α can affect the biological features of PDLSCs is still unclear. The objective of this study was to illustrate the biological effects (proliferation, apoptosis, osteogenesis and migration) of TNF-α on human CD146 positive periodontal ligament cells (CD146+PLDCs) and CD146 negative periodontal ligament cells (CD146−PDLCs). MethodsCD146±PDLCs were isolated from human PDLCs and analyzed using a fluorescence-activated cell sorter. The biological effects of TNF-α on CD146±PDLCs were evaluated by CCK-8 assay (proliferation), DAPI staining (apoptosis), alizarin red staining and alkaline phosphatase activities assay (osteogenesis), and wounding assay and transwell assay (migration). ResultsCD146+PDLCs, which expressed MSC surface markers CD105, CD90, CD73, CD44, and Stro-1, showed higher proliferative and osteogenic potential than CD146−PDLCs. TNF-α at a dose of 2.5ng/ml was found to enhance both proliferation and osteogenesis in CD146+PDLCs. At 5ng/ml, TNF-α promoted proliferation, osteogenesis, and apoptosis in CD146+PDLCs and enhanced osteogenesis in CD146−PDLCs. At 10ng/ml, TNF-α only aggravated apoptosis in CD146+PDLCs. The migratory ability of both CD146+PDLCs and CD146−PDLCs was not altered by TNF-α. ConclusionsCD146+PDLCs were subpopulation of MSC. It showed greater proliferative and osteogenic potential than CD146−PDLCs. At low concentration, TNF-α was beneficial to CD146+PDLCs on proliferation and osteogenesis, and at high concentration it was detrimental. CD146−PDLCs were found to be less sensitive to TNF-α.

Enzymatic preparation of κ-carrageenan oligosaccharides and their anti-angiogenic activity

The mixture of κ-neocarrabiose-sulfate, κ-neocarrahexaose-sulfate and κ-neocarraoctaose-sulfate were prepared from κ-carrageenan with enzyme. The anti-tumor and anti-angiogenic activity of obtained κ-carrageenan oligosaccharides (KOS), were explored. The results showed that KOS could inhibit the proliferation, migration and tube formation of ECV304 cells, and could inhibit the growth of new vessels in CAM model. KOS displayed strong anti-tumor activity in both S180 and MCF-7 xenograft models. Only human CD105 was detected in MCF-7 xenograft tumor, moreover KOS could decrease the growing of new blood vessels derived from tumor cell. Real-time PCR results showed that KOS could suppress the mRNA expression of human VEGF, bFGF, bFGFR and CD105 in MCF-7 xenograft tumor. All these results indicated that KOS has anti-tumor and anti-angiogenic activity in vivo and in vitro. Especially K has the potency to inhibit the differentiation of tumor cell to blood vessel endothelial cell.

THU0067 Endothelial-to-Mesenchymal Transition in Cultured Human Dermal Microvascular Endothelial Cells: Effects of Endothelin-1

BackgroundEndothelial/microvascular damage and myofibroblast activation are considered early events in the development of connective tissue diseases, such as systemic sclerosis (SSc) (1). Recent studies showed that vascular endothelial cells may...

Su1725 MiRNA Expression Regulating the Characteristics of Stem-Like Cell in CD44-Positive Gastric Cancer Cells

of xCT, glutamate-cystine transporter. This function enhanced capacity for glutathione (GSH) synthesis and defense against reactive oxygen species (ROS). This mechanism contributes to resistance against cisplatin which is one of ROS-inducible key drug of chemotherapy for gastric cancer (Cancer Cell 19: 387-400, 2011). 5-fluorouracil (5-FU) is another key component of the chemotherapeutic regimen for gastric cancer. Despite significant advances of chemotherapeutic agents and regimens, there are many cases with chemoresistance and mechanisms for the development of chemoresistance still remain elusive. Aim. We designed this study to clarify the relation between the expression of CD44v9 and the sensitivity to 5-FU. Method. Moderately differentiated gastric cancer cells, MKN28, were transfected with pRc/CMV expression plasmid encoding human CD44 standard (CD44s) or CD44v9. Cells stably expressing CD44s (MKN28-CD44s) and CD44v9 (MKN28-CD44v9) were isolated by flowcytometry with antibodies to human CD44s and CD44v9. Intracellular levels of GSH were determined with the luminescent based assay. Cell viabilities of MKN28-CD44s and MKN28-CD44v9 under 5-FU treatment were evaluated with MTS assay, and apoptotic rates of them were examined with cellular DNA fragmentation ELISA. Intracellular ROS accumulation was assessed by flowcytometry with general oxidative stress indicator, CMH2DCFDA. In addition, decrease in sensitivity to 5-FU under cysteine donor, N-acetylcysteine (NAC) treatment was examined by MTS assay. Results. The cell viability of MKN28-CD44v9 under 5-FU treatment was significantly higher than those of MKN28 and MKN28-CD44s (p , 0.05). In addition, the 5-FU-induced apoptotic rate in MKN28-CD44v9 was lower than those of MKN28 and MKN28-CD44s (vs. MKN28: p , 0.05, vs. MKN28-CD44s: p , 0.01). NAC decreased 5-FU-induced cell death in MKN28 and MKN28-CD44s more than MKN28-CD44v9. Although 5-FU treatment increased intracellular ROS level by nearly 2.0-fold in MKN28, MKN28-CD44v9 did not show the increase of ROS level after 5-FU treatment (0.95-fold). Conclusion. 5-FU-induced cell death is dependent on a decrease of intracellular ROS accumulation. Expression of CD44v9 possibly contributes to 5-FU resistance of gastric cancer through its anti-oxidative capacity.

Transplantation of human umbilical cord-derived endothelial progenitor cells promotes re-endothelialization of the injured carotid artery after balloon injury in New Zealand white rabbits

Background Cell transplantation has great potential for promoting endothelial repair and reducing the complications of percutaneous coronary intervention (PCI). The aim of this study was to investigate the effect of transplantation of human umbilical cord blood endothelial progenitor cells (EPCs) on injured arteries. Methods Umbilical cord blood mononuclear cells were obtained from post-partum lying-in women, and EPCs were isolated, cultured, expanded and identified by immunofluorescence. The carotid arterial endothelium of New Zealand white rabbits was injured by dilatation with a 3F balloon, and the EPCs were injected into the lumen of the injured artery in the transplanted group (n=16), while an equal volume of phosphated buffered saline (PBS) was injected into the control group after balloon injury (n=16). The animals were sacrificed after either 2 or 4 weeks, and the grafted cells were identified by double immunofluorescence staining with human nuclear antigen (HNA) and CD31 antibodies. Arterial cross sections were analyzed by pathology, immunohistochemisty and morphometry to evaluate the reparative effects of EPCs. Proliferating cell nuclear antigen (PCNA) and transforming growth factor (TGF)-β1 mRNA expression were detected by reverse transcription-polymerase chain reaction (RT-PCR). Results Fluorescence-labeled EPCs were found in the neointima. The neointimal area and the neointimal/medial area ratio were significantly lower in the transplanted group than in the control group (P &lt;0.05). von Willebrand factor (vWF) immunohistostaining showed more VWF-positive cells in the transplanted animals than in the controls (8.75±2.92 vs. 4.50±1.77, P &lt;0.05). Compared with the control group, the transplanted group had lower expression of PCNA mRNA (0.67±0.11 vs. 1.25±0.40, P &lt;0.01) and higher expression of TGF-β1 mRNA (1.10±0.21 vs. 0.82±0.07, P &lt;0.05). Conclusions EPCs derived from human umbilical cord blood were successfully transplanted into injured vessels. The transplanted EPCs inhibited neointimal hyperplasia and promoted vascular re-endothelialization.