Growth-associated protein 43 and neural cell adhesion molecule expression following bone marrow-derived mesenchymal stem cell transplantation in a rat model of ischemic brain injury

BACKGROUND： Transplantation of bone marrow-derived mesenchymal stem cells （BMSCs） improves motor functional recovery, but the mechanisms remain unclear. OBJECTIVE： To investigate expression of growth-associated protein 43 （GAP-43） and neural cell adhesion molecule following BMSC transplantation to the lateral ventricle in rats with acute focal cerebral ischemic brain damage. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment using immunohistochemistry was performed at the laboratories of Department of Neurology, Renmin Hospital of Wuhan University and Doctoral Scientific Research Work Station of C-BONS PHARMA, Hubei Province, China, from January 2007 to December 2008. MATERIALS： Monoclonal mouse anti-rat 5-bromo-2-deoxyuridine and neural cell adhesion molecule antibodies were purchased from Sigma, USA; monoclonal mouse anti-rat GAP-43 antibody was purchased from Wuhan Boster, China. METHODS： Rat models of right middle cerebral artery occlusion were established using the thread method. At 1 day after middle cerebral artery occlusion, 20μL culture solution, containing 5×10^5 BMSCs, was transplanted to the left lateral ventricle using micro-injection. MAIN OUTCOME MEASURES： Scores of neurological impairment were measured to assess neural function. Expression of GAP-43 and neural cell adhesion molecule at the lesion areas was examined by immunohistochemistry. RESULTS： GAP-43 and neural cell adhesion molecule expression was low in brain tissues of the sham-operated group, but expression increased at the ischemic boundary （P 〈 0.05）. Transplantation of BMSCs further enhanced expression of GAP-43 and neural cell adhesion molecule （P 〈 0.05） and remarkably improved neurological impairment of ischemic rats （P 〈 0.05）. CONCLUSION： BMSC transplantation promoted neurological recovery in rats by upregulating expression of GAP-43 and neural cell adhesion molecule.

Nerve bundle formation during the promotion of peripheral nerve regeneration:collagenⅥ-neural cell adhesion molecule 1 interaction

The formation of nerve bundles,which is partially regulated by neural cell adhesion molecule 1(NCAM1),is important for neural network organization during peripheral nerve regeneration.However,little is known about how the extracellular matrix(ECM)microenvironment affects this process.Here,we seeded dorsal root ganglion tissue blocks on different ECM substrates of peripheral nerve ECM-derived matrixgel,Matrigel,laminin 521,collagen I,and collagen IV,and observed well-aligned axon bundles growing in the peripheral nerve ECM-derived environment.We confirmed that NCAM1 is necessary but not sufficient to trigger this phenomenon.A protein interaction assay identified collagen VI as an extracellular partner of NCAM1 in the regulation of axonal fasciculation.Collagen VI interacted with NCAM1 by directly binding to the FNIII domain,thereby increasing the stability of NCAM1 at the axolemma.Our in vivo experiments on a rat sciatic nerve defect model also demonstrated orderly nerve bundle regeneration with improved projection accuracy and functional recovery after treatment with 10 mg/m L Matrigel and 20μg/m L collagen VI.These findings suggest that the collagen VI-NCAM1 pathway plays a regulatory role in nerve bundle formation.This study was approved by the Animal Ethics Committee of Guangzhou Medical University(approval No.GY2019048)on April 30,2019.

Alterations in the polysialylated neural cell adhesion molecule and retinal ganglion cell density in mice with diabetic retinopathy

AIM：To investigate the impact of polysialylated neural cell adhesion molecule（PSA-NCAM）on the survival of retinal ganglion cells（RGCs）in the experimentally induced diabetes in mice.METHODS：Diabetes was induced in 2.5 months old Swiss Webster mice by intraperitoneal injection of streptozotocin（STZ,90 mg/kg）once daily for two consecutive days.Examination of the proteins of interest in the retinas from diabetic mice at 2mo after diabetes induction was performed using immunohistochemistry and Western blot analysis.RGCs were counted in the wholemounted retinas,and Brn3a marker was used.RESULTS：Examination of retinas from diabetic mice at 2mo after diabetes induction revealed a considerable reduction in RGC density.Our experiments also demonstrated a redistribution of PSA-NCAM in the retina of diabetic animals.PSA-NCAM immunoreactivity was diminished in the inner part of the retina where RGCs were located.In contrast,an enhanced PSA-NCAM immunoreactivity was detected in the outer layers of the retina.PSA-NCAM signal was co-localized with glial fibrillary acidic protein immunoreactivity in the Müller cell branches.Previous studies have shown that matrix metalloproteinase-9（MMP-9）is responsible for the reduction in PSA-NCAM levels in neuronal cells.The reduced levels of PSA-NCAM in inner layers（nerve fiber layer,ganglion cell layer）were accompanied by the increased expression of MMP-9.In contrast,in the outer retinal layers,the expression of MMP-9 was much less pronounced.CONCLUSION：MMP-9 induces PSA-NCAM shedding in the inner part of the retina and the decreased level of PSA-NCAM in the inner part of the retina might be,at least in part,responsible for the loss of RGCs in diabetic mice.

Expression changes of nerve cell adhesion molecules L1 and semaphorin 3A after peripheral nerve injury

The expression of nerve cell adhesion molecule L1 in the neuronal growth cone of the central nervous system is strongly associated with the direction of growth of the axon, but its role in the regeneration of the peripheral nerve is still unknown. This study explored the problem in a femoral nerve section model in rats. L1 and semaphorin 3A m RNA and protein expressions were measured over the 4-week recovery period. Quantitative polymerase chain reaction showed that nerve cell adhesion molecule L1 expression was higher in the sensory nerves than in motor nerves at 2 weeks after injury, but vice versa for the expression of semaphorin 3A. Western blot assay results demonstrated that nerve cell adhesion molecule L1 expression was higher in motor nerves than in the sensory nerves at the proximal end after injury, but its expression was greater in the sensory nerves at 2 weeks. Semaphorin 3A expression was higher in the motor nerves than in the sensory nerves at 3 days and 1 week after injury. Nerve cell adhesion molecule L1 and semaphorin 3A expressions at the distal end were higher in the motor nerves than in the sensory nerves at 3 days, 1 and 2 weeks. Immunohistochemical staining results showed that nerve cell adhesion molecule L1 expression at the proximal end was greater in the sensory nerves than in the motor nerves; semaphorin 3A expression was higher in the motor nerves than in the sensory nerves at 2 weeks after injury. Taken together, these results indicated that nerve cell adhesion molecules L1 and semaphorin 3A exhibited different expression patterns at the proximal and distal ends of sensory and motor nerves, and play a coordinating role in neural chemotaxis regeneration.

Cognitive disorder and changes in cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury

BACKGROUND： Learning and memory damage is one of the most permanent and the severest symptoms of traumatic brain injury; it can seriously influence the normal life and work of patients. Some research has demonstrated that cognitive disorder is closely related to nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor. OBJECTIVE： To summarize the cognitive disorder and changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury. RETRIEVAL STRATEGY： A computer-based online search was conducted in PUBMED for English language publications containing the key words ＂brain injured, cognitive handicap, acetylcholine, N-methyl-D aspartate receptors, neural cell adhesion molecule, brain-derived neurotrophic factor＂ from January 2000 to December 2007. There were 44 papers in total. Inclusion criteria： ① articles about changes in nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor following brain injury; ② articles in the same researching circle published in authoritative journals or recently published. Exclusion criteria： duplicated articles. LITERATURE EVALUATION： References were mainly derived from research on changes in these four factors following brain injury. The 20 included papers were clinical or basic experimental studies. DATA SYNTHESIS： After craniocerebral injury, changes in these four factors in brain were similar to those during recovery from cognitive disorder, to a certain degree. Some data have indicated that activation of nicotine cholinergic receptors, N-methyl-D aspartate receptors, neural cell adhesion molecule, and brain-derived neurotrophic factor could greatly improve cognitive disorder following brain injury. However, there are still a lot of questions remaining; for example, how do these factors change at different time points after brain injury, and what is the relationship between associated factors and cognitive disorder. CONCLUSION： It is necessary to comprehensively study some associated factors, to analyze their changes and their relationship with cognitive disorder following brain injury, and to investigate their effects at different time points after brain injury.

Hypoxia Inhibits Proliferation of Human Dermal Lymphatic Endothelial Cells via Downregulation of Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 Expression

Objective:Lymphatic endothelial cell(LEC)proliferation is essential for lymphangiogenesis.Hypoxia induces lymphangiogenesis,but it directly inhibits LEC proliferation and the underlying mechanisms have not been fully understood.The aim of this study was to investigate the role of carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1)in hypoxia-repressed LEC proliferation.Methods:Human dermal lymphatic endothelial cells(HDLECs)were cultured under normoxic or hypoxic conditions,and cell proliferation was determined using MTT or CCK-8 assays.CEACAM1 expression was silenced by siRNA transfection.Activation of mitogen-activated protein kinases(MAPKs)was examined by Western blotting and blocked by specific inhibitors.Results:Under hypoxia,HDLECs proliferation was suppressed and CEACAM1 expression was downregulated.Silence of CEACAM1 in normoxia inhibited HDLECs proliferation and did not further decrease proliferation in HDLECs in response to hypoxia,suggesting that CEACAM1 may mediate hypoxia-induced inhibition of HDLECs proliferation.In addition,silence of CEACAM1 increased phosphorylation of MAPK molecules:extracellular signal-regulated kinase(ERK),p38 MAPK and Jun N-terminal kinase(JNK)in HDLECs.However,only inhibition of the JNK pathway rescued the reduction of HDLEC proliferation induced by CEACAM1 silence.Conclusion:Our results suggested that hypoxia downregulates CEACAM1 expression by activation of the JNK pathway,leading to inhibition of HDLEC proliferation.These findings may help to understand the mechanisms of LEC-specific response to hypoxia and develop novel therapies for pathological lymphangiogenesis.

Re-expression of Cell Adhesion Molecule Inhibits Growth and Induces Apoptosis of Human Pancreatic Cancer Cell Line PANC-1

This study examined the expression of cell adhesion molecule 1 （CADM1） in pancreatic cancer and the possible mechanism. The expression of CADM 1 was detected by immunohistochemistry in tissues of pancreatic cancer, pancreatitis, and normal pancreas. The plasmid pcDNA3.1-Hy- gro（＋）/CADM1 was transfected into PANC-1 cells （a pancreatic cancer cell line）. The expression of CADM1 in the transfected cells was determined by RT-PCR and Western blotting. Cell growth was measured by the MTT method and cell apoptosis by flow cytometry. The results showed that CADM1 was weakly expressed in tissues of pancreatic cancer in contrast to its high expression in normal pancreatic and pancreatitis tissues. The expression level of CADM in pancreatic caner was intensely correlated with the differentiation degree, lymph node metastasis and TNM stages. The growth of CADMl-transfected PANC-1 cells was significantly suppressed in vitro by a G1 cell cycle arrest and apoptosis occurrence. It was concluded that re-expression of CADM1 inhibits the growth of pancreatic cancer cells and induces their apoptosis in vitro. As a tumor suppressor gene, CADM1 plays an important role in the occurrence, progression and metastasis of pancreatic cancer.

Local translation of cell adhesion molecules in axons

During development and regeneration,axonal growth depends on a rapid response to extracellular growth and guidance molecules.One mechanism underlying this rapid response is local protein synthesis（Jung et al.,2012）.Local protein synthesis is a highly tuned,

Loss of membranous carcinoembryonic antigen-related cell adhesion molecule 1 expression is related to decreased relapse-free survival of hepatocellular carcinoma following liver transplantation

Background Loss of carcinoembryonic antigen-related cell adhesion molecule 1 （CEACAM1） expression is an adverse prognostic factor in hepatocellular carcinoma （HCC）. The purpose of this study was to investigate the expression of CEACAM1 and its effect on relapse-free survival （RFS） following liver transplantation （LT） for HCC. Methods Expression of CEACAM1 was immunohistochemically detected in HCC specimens from 48 patients. The relationship between CEACAM1 expression and clinicopathologic variables, as well as tumor recurrence, was further analyzed. Results Of the 48 HCC specimens, membranous CEACAM1 expression was detected in 25 specimens and cytoplasmic CEACAM1 expression was detected in 19 specimens. Four specimens had loss of CEACAM1 expression. Loss of membranous CEACAM1 expression was significantly associated with tumor size, tumor number, and serum （z-fetoprotein levels （all P 〈0.05）. Patients with loss of membranous CEACAM1 had significantly poorer RFS than patients with membranous expression, determined via Kaplan-Meier analysis （P=0.027）. Multivariate analysis revealed that loss of membranous CEACAM1 expression might be an independent prognostic factor of RFS for HCC patients after liver transplantation （P=0.037）. Conclusion Loss of membranous CEACAM1 expression in HCC was closely associated with aggressive tumor biology and might be a relapsing biomarker of HCC treated with LT.

L-Tetrahydropalamatine Inhibits Tumor Necrosis Factor-α-Induced Monocyte-Endothelial Cell Adhesion through Downregulation of Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1 Involving Suppression of Nuclear Factor-κ B Signaling Pat

Objective： TO investigate whether I-tetrahydropalmatine （I-THP）, an alkaloid mainly present in Corydalis family, could ameliorate early vascular inflammatory responses in atheroscleroUc processes. Methods： Fluorescently labeled monocytes were co-incubated with human umbilical vein endothelial cells （HUVECs）, which were pretreated with I-THP and then simulated with tumor necrosis factor （TNF）-α in absence of I-THP to determine if I-THP could reduce thecytokine-induced adhesion of monocytes to HUVECs. Then I-THP were further studied the underlying mechanisms through observing the transcriptional and translational level of intercellular adhesion molecule-1 （ICAM-1） and vascular cell adhesion molecule-1 （VCAM-1） and the nuclear translocation of nuclear factor （NF）- K B in HUVECs. Results： L-THP could block TNF-α-induced adhesion of monocytes to HUVECs and could significantly inhibited the expression of ICAM-1 and VCAM-1 on cell surface by 31% and 36% at 30 μmol/L. L-THP pretreatment could also markedly reduce transcriptional and translational level of VCAM-1 as well as mildly reduce the total proteJn and mRNA expressJon levels of ICAM-1. Furthermore, I-THP attenuated TNF- α -stimulated NF- κ B nuclear translocation. Conclusion： These results provide evidences supporting that I-THP could be a promising compound in the prevention and treatment of the early vascular inflammatory reaction in atherosclerosis by inhibiting monocyte adhesion to vascular endothelial cell through down- regulating ICAM-1 and VCAM-1 in vascular endothelial cell based on suppressing NF- κ B.

Cytokine-Induced Cell Surface Expression of Adhesion Molecules in Vascular Endothelial Cells In vitro

Regulation of the adhesion molecules expression by cytokine in vascular endothelial cells was investigated. Human umbilical vein endothelial cells (HUVEC) were stimulated with cytokines, TNF α (1-250 U/ml) or IL 1β (0.1-50 U/ml) for 24 h. HUVEC were also cultured with cytokines, TNF α (100 U/ml) or IL 1β (10 U/ml), for 4-72 h, cell surface expression of adhesion molecules (ICAM 1 and VCAM 1) were detected and quantitated by immunocytochemical methods and computerized imaging analysis technique. Adhesion molecules expression were up regulated by TNF α, IL 1β in a concentration and time dependent manner. Some significant differences were observed between the effects of cytokines on the ICAM 1 and on VCAM 1 expression. Cytokines might directly induce the expression of ICAM 1 and VCAM 1 in vascular endothelial cells. Our observations indicate differential functions of the two adhesion molecules during the evolution of inflammatory responses in stroke.

Changes in serum cellular adhesion molecule and matrix metalloproteinase-9 levels in patients with cerebral infarction following hyperbaric oxygen therapy A case and intergroup control study

BACKGROUND： Animal studies have confirmed that hyperbaric oxygen （HBO） therapy can reduce matrix metalloproteinase activity and blood brain barrier permeability, thereby exhibiting neuroprotective effects. However, at present, consensus does not exist in terms of its clinical efficacy. OBJECTIVE： To validate the significance of changes in serum cellular adhesion molecule and MMP-9 levels in patients with cerebral infarction following HBO therapy. DESIGN, TIME AND SETTING： This randomized, controlled, neurobiochemical study was performed at the Department of Neurology, Affiliated Hospital of Qingdao University Medical College between December 2002 and March 2006. PARTICIPANTS： A total of 112 patients with acute cerebral infarction of internal carotid artery, comprising 64 males and 48 females, averaging （67 ±11） years, were recruited and randomized to a HBO group （n = 50） and a routine treatment group （n = 62）. An additional 30 gender- and age-matched normal subjects, consisting of 17 males and 13 females, averaging （63 ± 9） years, were enrolled as control subjects. METHODS： The routine treatment group received routine drug treatment and rehabilitation exercise. HBO treatment was additionally performed in the HBO group, once a day, for a total of 10 days. MAIN OUTCOME MEASURES： Serum levels of soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, soluble E-selectin, and matrix metalloproteinase-9 were detected by enzyme linked immunosorbent assay. RESULTS： Upon admission, serum levels of soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, soluble E-selectin, and matrix metalloproteinase-9 were significantly increased in patients with cerebral infarction, compared with control subjects （P 〈 0.01）. Following HBO and routine treatments, serum levels of the above-mentioned indices were significantly reduced in the HBO and routine treatment groups （P 〈 0.01）. Moreover, greater efficacy was observed in the HBO group, compared with the routine treatment group （P 〈 0.05 or P 〈 0.01）. CONCLUSION： Intergroup comparison and case-control results indicated that HBO noticeably reduced serum levels of soluble intercellular adhesion molecule, soluble vascular cell adhesion molecule, soluble E-selectin, and matrix metalloproteinase-9.

Altered Carcinoembryonic Antigen-Related Cell Adhesion Molecule 1/T-Cell Immunoglobulin Mucin-3 Signaling Causes the Dysregulation of Decidual CD8+T Cells in the Third Trimester during Preeclamptic Pregnancies

Objective:To investigate the frequency and function of Tim-3^(+)CD8^(+)T cells in the third trimester of normal pregnancies(NPs)and preeclamptic(PE)pregnancies.Methods:T-cell immunoglobulin mucin-3(Tim-3)expression levels of CD8^(+)T cells in the decidua,peripheral blood,and umbilical cord blood obtained from women showing NPs and PE pregnancies were analyzed using flow cytometry.Decidual CD8^(+)T cells were cultured in the presence of recombinant human carcinoembryonic antigen-related cell adhesion molecule 1(CEACAM1)protein and/or Tim-3-specific neutralizing antibodies for analyzing CD107a and intracellular cytokine expression.The placental CEACAM1 protein expression was analyzed using immunohistochemistry.Results:Tim-3^(+)CD8^(+)T cells were more abundant in the decidua than in the peripheral blood.Tim-3 expression in the decidual CD8^(+)T cells was significantly lower in PE patients.Decidual Tim-3^(+)CD8^(+)T cells from PE patients expressed higher levels of CD107a and the Th1-type cytokine IFN-γ,but lower levels of the Th2-type cytokine IL-4.CEACAM1 altered the CD107a,IFN-γ,and IL-4 levels;this was reversed by anti-Tim-3 antibodies.The CEACAM1 protein levels were lower in the placental tissues of women with PE pregnancies than in those of women with NPs.Conclusions:Abnormal CEACAM1/Tim-3 regulation may participate in the development of PE,accompanied by disturbed Th2 cell predominance and higher cytotoxicity of decidual CD8^(+)T cells.

Bioinformatics research of CD44 and epithelial cell adhesion molecule related genes and pathways in colorectal cancer

Objective To investigate genes and involved biological processes closely associated with stem cell markers of colorectal cancer-epithelial cell adhesion molecule(EpCAM)+and CD44+.Methods By the bioinformatics method,with microarray data of colorectal cancer from gene expression omnibus(GEO)database and R2 platform,the genes significantly related with CD44 and Ep-

Molecular and cellular changes in the post-traumatic spinal cord remodeling after autoinfusion of a genetically-enriched leucoconcentrate in a mini-pig model

Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue.In our previous studies for delivering the therapeutic genes at the site of spinal cord injury,we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses(Ad5/35)carrying recombinant cDNA.In the present study,the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor(VEGF),glial cell line-derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury.Experimental animals were randomly divided into two groups of 4 pigs each:the therapeutic(infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35‐VEGF165,Ad5/35‐GDNF,and Ad5/35‐NCAM1)and control groups(infused with intact leucoconcentrate).The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed:(1)higher sparing of the grey matter and increased survivability of the spinal cord cells(lower number of Caspase-3-positive cells and decreased expression of Hsp27);(2)recovery of synaptophysin expression;(3)prevention of astrogliosis(lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells);(4)higher growth rates of regeneratingβIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region.These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF,GDNF,and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury.Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.

Integrin binding peptides facilitate growth and interconnected vascular-like network formation of rat primary cortical vascular endothelial cells in vitro

Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults. Integrins are cell surface adhesion receptors mediating important function of cells such as survival, growth and development during tissue organization, differentiation and organogenesis. In this study, we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion, growth, development, and vascular-like network formation of rat primary brain microvascular endothelial cells. Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7. Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates. After 7 days of culture, the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule. 5-Bromo-2′-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation. Among 16 integrins tested, we found that α5β1, αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture. To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis, the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional(3 D) culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor. Following a 7-day 3 D culture, the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy. In the 3 D culture in hydrogels conjugated with the integrin-binding peptide, brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens, which is reminiscent of brain microvascular network in vivo. With the novel integrin-binding array system, we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3 D hydrogel culture system using the binding peptides that specifically bind to the identified integrins, leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3 D culture. This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.

Communication between bone marrow mesenchymal stem cells and multiple myeloma cells:Impact on disease progression

Multiple myeloma(MM)is a hematological malignancy characterized by the accumulation of immunoglobulin-secreting clonal plasma cells at the bone marrow(BM).The interaction between MM cells and the BM microenvironment,and specifically BM mesenchymal stem cells(BM-MSCs),has a key role in the pathophysiology of this disease.Multiple data support the idea that BM-MSCs not only enhance the proliferation and survival of MM cells but are also involved in the resistance of MM cells to certain drugs,aiding the progression of this hematological tumor.The relation of MM cells with the resident BM-MSCs is a two-way interaction.MM modulate the behavior of BM-MSCs altering their expression profile,proliferation rate,osteogenic potential,and expression of senescence markers.In turn,modified BM-MSCs can produce a set of cytokines that would modulate the BM microenvironment to favor disease progression.The interaction between MM cells and BM-MSCs can be mediated by the secretion of a variety of soluble factors and extracellular vesicles carrying microRNAs,long non-coding RNAs or other molecules.However,the communication between these two types of cells could also involve a direct physical interaction through adhesion molecules or tunneling nanotubes.Thus,understanding the way this communication works and developing strategies to interfere in the process,would preclude the expansion of the MM cells and might offer alternative treatments for this incurable disease.

Circulating tumor cells isolation:the "post-EpCAM era"

Circulating tumor cells （CTCs） represent a submicroscopic fraction detached from a primary tumor and in transit to a secondary site. The prognostic significance of CTCs in metastatic cancer patients was demonstrated for the first time more than ten years ago. To date, it seems clear enough that CTCs are highly heterogeneous and dynamically change their shape. Thus, the inadequacy of epithelial cell adhesion molecule （EpCAM） as universal marker for CTCs detection seems unquestionable and alternative methods able to recognize a broader spectrum of phenotypes are definitely needed. In this review the pleiotropic functions of EpCAM are discussed in detail and the role of the molecule in the biology of CTCs is critically dissected.

Impact of chronic exposure to bevacizumab on EpCAM-based detection of circulating tumor cells

Background： Circulating tumor cells （CTCs） are often undetected through the immunomagnetic epithelial cell adhesion molecule （EpCAM）-based CellSearch~ System in breast and colorectal cancer （CRC） patients treated with bevacizumab （BEV）, where low CTC numbers have been reported even in patients with evidence of progression of disease. To date, the reasons for this discrepancy have not been clarified. This study was carried out to investigate the molecular and phenotypic changes in CRC cells after chronic exposure to BEV in vitro. Methods： The human CRC cell line WiDr was exposed to a clinically relevant dose of BEV for 3 months in vitro. The expression of epithelial and mesenchymal markers and EpCAM isoforms was determined by western blotting and immunofluorescence. To evaluate the impact of EpCAM variant isoforms expression on CTC enumeration by CellSearch, untreated and treated colon cancer cells were spiked into 7.5 mL of blood from a healthy donor and enumerated by CellSearch. Results： Chronic exposure of CRC cell line to BEV induced decreased expression of EpCAM 40 kDa isoform and increased expression EpCAM 42 kDa isoform, together with a decreased expression of cytokeratins （CK）, while no evidence of epithelial to mesenchymal transition （EMT） in treated cells was observed. The recovery rate of cells through CellSearch was gradually reduced in course of treatment with BEV, being 84% , 70% and 40% at l, 2 and 3 months, respectively. Conclusions： We hypothesize that BEV may prevent CellSearch from capturing CTCs through altering EpCAM isoforms.