Platelet Endothelial Cell Adhesion Research Articles

Role of aneuploid circulating tumor cells and CD31+ circulating tumor endothelial cells in predicting and monitoring anti-angiogenic therapy efficacy in advanced NSCLC.

Prognosticating the efficacy of anti‐angiogenic therapy through longitudinal monitoring and early detection of treatment resistance in cancer patients remain highly challenging. In this study, co‐detection and comprehensive phenotypic and karyotypic molecular characterization of aneuploid circulating tumor cells (CTCs) and circulating tumor endothelial cells (CTECs) were conducted on non‐small cell lung cancer (NSCLC) patients receiving bevacizumab plus chemotherapy. Prognostic values of the cell‐based significant univariate risk factors identified by Cox regression analyses were progressively investigated. Subjects showing an increase in total post‐therapeutic platelet endothelial cell adhesion molecule‐1 (CD31)– CTCs and CD31+ CTECs exhibited a significantly reduced median progression‐free survival (mPFS) and overall survival. Further stratification analyses indicated that pretherapeutic patients bearing vimentin (Vim)+ CTECs (mesenchymal M‐type) at baseline revealed a significantly shortened mPFS compared with patients with Vim– CTECs. Post‐therapeutic patients harboring epithelial cell adhesion molecule (EpCAM)+ CTCs and CTECs (epithelial E‐type), regardless of Vim expression or not, showed a significantly reduced mPFS. Post‐therapeutic patients possessing de novo EpCAM+/Vim+ (hybrid E/M‐type) CTECs displayed the shortest mPFS. Patients harboring either pre‐ or post‐therapeutic EpCAM–/Vim– null CTECs (N‐type) exhibited a better response to therapy compared to patients harboring EpCAM+ and/or Vim+ CTECs. The presented results support the notion that baseline Vim+ CTECs and post‐therapeutic EpCAM+ CTCs and CTECs are predictive biomarkers for longitudinal monitoring of response to anti‐angiogenesis combination regimens in NSCLC patients.

Hyaluronic acid-functionalized poly-lactic acid (PLA) microfibers regulate vascular endothelial cell proliferation and phenotypic shape expression

This work was designed to evaluate the efficacy of hyaluronic acid (HA) functionalized tubular poly-lactic acid (PLA) microfibers in directing the luminal pre-endothelialization of vascular endothelial cells (ECs). Tubular HA/PLA microfibers with hierarchical architecture were prepared by electrospinning and chemical cross-linking process. A layer of HA microfibrous film coating was fixed on the inner wall surface of the tubular HA/PLA microfibers, resulting in higher anisotropy wettability and relatively lower surface energy and roughness. We confirmed that HA coating on PLA microfibers surface have reduced hemolytic activity and coagulation degree. Mouse vascular ECs exhibited surface-dependent differences in cell elongation and proliferation (HA/PLA > PLA). Compared with PLA microfibers, the gene expression levels of platelet EC adhesion molecule-1 (PECAM-1/CD31) and vascular endothelial growth factor (VEGF) in ECs of HA/PLA microfibers surface were up-regulated. Immunostaining analysis revealed that the surface of HA/PLA nanofibers supported the expression of mature vascular EC phenotype CD31 protein. In vitro co-culture analysis showed that the luminal pre-endothelialization induced vascular smooth muscle cells (SMCs) to maintain their phenotypic shape and establish natural behavior patterns in the hierarchical tubular scaffold. These studies indicate that the biophysical cues of scaffolds are potent regulators of vascular EC endothelialization.

A Three-Dimensional Microfluidic Device for Monitoring Cancer and Chemotherapy-Associated Platelet Activation.

Platelet activation and the risk of thrombosis are increased in cancer patients, especially after chemotherapy. Our previous studies indicated that chemotherapy-induced platelet activation is largely due to endothelial cell damage. Thus, simple in vitro tests, such as aggregometry, are not desirable tests to predict platelet responsiveness to different chemotherapeutic agents because other contributory factors, such as tumor cells, endothelial cells, and the flow rate of platelets, also contribute to the formation of cancer-associated thrombosis. Therefore, developing a platelet detection system, which includes all possible risk parameters, is necessary. In the present study, we described a microengineered microfluidic system that contained a drug concentration generator, cancer cell culture chip, and three-dimensional (3D) circular microvascular model covered with a confluent endothelial layer and perfused with human platelets at a stable flow rate. Doxorubicin was injected through two injection sites. Endothelial cell injury was evaluated by counting, cell cytoskeleton observation, and the level of IACM1 and ET-1 in endothelial cells or a culture medium. Prestained platelets were perfused into the artificial blood vessel, and platelet-endothelial cell adhesion was measured. We found that (i) MCF7 cell-released factors had a cytotoxicity effect on both endothelial cells and platelets. (ii) We confirmed that doxorubicin-induced platelet activation was endothelial cell-dependent. (iii) A lower dosage of doxorubicin (0–2.0 μM) induced platelet activation, while a higher dosage of doxorubicin (2.0–4.0 μM) led to platelet death. Our findings indicated that platelet-endothelial cell adhesion could be used as a diagnostic marker of platelet activation, providing a simple and rapid detective way to predict platelet responsiveness before or during chemotherapy.

Transcriptional characterisation of human lung cells identifies novel mesenchymal lineage markers.

The lung mesenchyme gives rise to multiple distinct lineages of cells in the mature respiratory system, including smooth muscle cells of the airway and vasculature. However, a thorough understanding of the specification and mesenchymal cell diversity in the human lung is lacking. We completed single-cell RNA sequencing analysis of fetal human lung tissues. Canonical correlation analysis, clustering, cluster marker gene identification and t-distributed stochastic neighbour embedding representation was performed in Seurat. Cell populations were annotated using ToppFun. Immunohistochemistry and in situ hybridisation were used to validate spatiotemporal gene expression patterns for key marker genes. We identified molecularly distinct populations representing "committed" fetal human lung endothelial cells, pericytes and smooth muscle cells. Early endothelial lineages expressed "classic" endothelial cell markers (platelet endothelial cell adhesion molecule/CD31 and claudin 5), while pericytes expressed platelet-derived growth factor receptor-β, Thy-1 membrane glycoprotein and basement membrane molecules (collagen IV, laminin and proteoglycans). We observed a large population of "nonspecific" human lung mesenchymal progenitor cells characterised by expression of collagen I and multiple elastin fibre genes (ELN, MFAP2 and FBN1). We closely characterised the diversity of mesenchymal lineages defined by α2-smooth muscle actin (ACTA2) expression. Two cell populations, with the highest levels of ACTA2 transcriptional activity, expressed unique sets of markers associated with airway or vascular smooth muscle cells. Spatiotemporal analysis of these marker genes confirmed early and persistent spatial specification of airway (HHIP, MYLK and IGF1) and vascular (NTRK3 and MEF2C) smooth muscle cells in the developing human lung. Our data suggest that specification of distinct airway and vascular smooth muscle cell phenotypes is established early in development and can be identified using the markers we provide.

Loss of endothelial surface molecules in the central retinal artery and retina with methamphetamine

ObjectiveThe purpose of this study is to investigate the retinal effects of methamphetamine.IntroductionMETH, a highly addictive stimulant of neurotransmitters is associated with cardiovascular and neurological diseases. METH‐induced ophthalmic complications are also present but have been insufficiently investigated.Materials and methodsC57BL/6J mice were administrated progressively increasing doses of METH (0 to 6 mg/kg) by repetitive intraperitoneal injections for 5 days (4 times per day). Norepinephrine levels in plasma were measured by ELISA. Cross‐sections of the central retinal artery (CRA) and retina were stained with hematoxylin and eosin. Expression of various proteins was determined by immunostain and immunoblot in the CRA and retina.ResultsElevated norepinephrine levels in plasma were induced by METH (6.49±1.72 ng/mL, n=5), compared with saline treatment (1.38±0.48, P<0.001, n=5). After treatment of METH, the ocular pupil was dilated and retinal thicknesses of the outer nuclear layer, outer plexiform layer, inner nuclear layer, and inner plexiform layer were decreased. Platelet endothelial cell adhesion molecule‐1 (PECAM‐1) protein expression level was decreased in the CRA and retina of METH‐treated mice, along with the endothelial proteoglycans glypican‐1 and syndecan‐1. Moreover, a regulator of the extracellular matrix, membrane type 1 matrix metalloproteinase (MMP‐14), but not MMP‐2 and MMP‐9, was increased in the retina by METH treatment.ConclusionThese results suggest that METH administration is involved in retinal degeneration with a vascular loss of PECAM‐1 and the glycocalyx, and an increase of MMP‐14 in the CRA and retina.Support or Funding InformationThis work was supported by funding from the National Institute of Health (NIH) EY025632.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Assembly of a mechanosensitive complex is required to initiate sprouting angiogenesis

Endothelial cells (ECs) must respond rapidly to a variety of extracellular cues to initiate sprouting events. We undertook the current study to better understand how mechanical forces (shear stress) combine with biochemical cues to promote sprout outgrowth in a defined 3D model of EC invasion, because while the effects of shear stress have been widely studied in atherosclerosis, the molecular events directed by mechanical forces in angiogenic sprouting are incompletely understood. We initially focused on the known mechanosensitive protein Filamin A, which has been implicated in barrier regulation and vascular development. We found that Filamin A is required for EC sprouting in 3D and localizes to junctions with EC activation. Filamin A formed a complex with vascular endothelial (VE)‐cadherin and platelet endothelial cell adhesion molecule‐1 (PECAM‐1), two known members of mechanosensitive complex, following activation with shear stress and pro‐angiogenic factors. Silencing of Filamin A also disrupted the VE‐cadherin and PECAM‐1 complex, which correlates with decreased sprouting. Filamin A co‐localized with SHP2, a phosphatase previously implicated in barrier formation, following EC activation. SHP2 complexed with junctional molecules Filamin A, VE‐cadherin, and PECAM‐1, and complex formation between SHP2, VE‐cadherin, and PECAM‐1 was lost with Filamin A knockdown. In addition, in the absence of Filamin A expression, SHP2 failed to localize to endothelial junctions. Further, SHP2 silencing significantly reduced sprouting, and defects in sprouting were rescued by delivery of wild‐type SHP2. Altogether these data suggest that Filamin A recruits SHP2 to reinforce a transmembrane complex containing VE‐cadherin and PECAM‐1 that is needed to initiate EC sprouting. These data show for the first time that the formation of this mechanosensitive complex containing VE‐cadherin, PECAM‐1, Filamin A, and SHP2 is needed for successful sprout initiation.Support or Funding InformationWork supported by NIH RO1 HL095786, the Department of Molecular & Cellular Medicine, Texas A&M Health Science Center, and AHA Grant‐in‐Aid, Southwest Affiliate.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

PECAM-1 modulates liver damage induced by synergistic effects of TNF-α and irradiation.

The mechanisms of radiation‐induced liver damage are poorly understood. We investigated if tumour necrosis factor (TNF)‐α acts synergistically with irradiation, and how its activity is influenced by platelet endothelial cell adhesion molecule‐1 (PECAM‐1). We studied murine models of selective single‐dose (25 Gy) liver irradiation with and without TNF‐α application (2 μg/mouse; i.p.). In serum of wild‐type (wt)‐mice, irradiation induced a mild increase in hepatic damage marker aspartate aminotransferase (AST) in comparison to sham‐irradiated controls. AST levels further increased in mice treated with both irradiation and TNF‐α. Accordingly, elevated numbers of leucocytes and increased expression of the macrophage marker CD68 were observed in the liver of these mice. In parallel to hepatic damage, a consecutive decrease in expression of hepatic PECAM‐1 was found in mice that received radiation or TNF‐α treatment alone. The combination of radiation and TNF‐α induced an additional significant decline of PECAM‐1. Furthermore, increased expression of hepatic lipocalin‐2 (LCN‐2), a hepatoprotective protein, was detected at mRNA and protein levels after irradiation or TNF‐α treatment alone and the combination of both. Signal transducer and activator of transcription‐3 (STAT‐3) seems to be involved in the signalling cascade. To study the involvement of PECAM‐1 in hepatic damage more deeply, the liver of both wt‐ and PECAM‐1‐knock‐out‐mice were selectively irradiated (25 Gy). Thereby, ko‐mice showed higher liver damage as revealed by elevated AST levels, but also increased hepatoprotective LCN‐2 expression. Our studies show that TNF‐α has a pivotal role in radiation‐induced hepatic damage. It acts in concert with irradiation and its activity is modulated by PECAM‐1, which mediates pro‐ and anti‐inflammatory signalling.

Effects of UPF1 expression on EMT process by targeting E‑cadherin, N‑cadherin, Vimentin and Twist in a hepatocellular carcinoma cell line.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. It has been reported that HCC has a poor prognosis. In the majority of cases, once metastatic, HCC is incurable. To identify an effective treatment for HCC, it is important to understand the underlying molecular mechanisms of HCC-associated occurrence, proliferation, metastasis and carcinogenesis. In the present study, the role of Up-frameshift 1 (UPF1), a potential tumor suppressor, was investigated in the HCC cell lines. The expression levels of UPF1 in an HCC cell line were examined by reverse transcription-quantitative polymerase chain reaction. The expression levels of 19 key proteins in numerous signaling pathways were detected via protein array analysis in the presence of UPF1 overexpression. The present study further investigated the effects of UPF1 expression levels on the epithelial-mesenchymal transition (EMT) process by targeting E-cadherin, N-cadherin, Vimentin and Twist-related protein 1 (Twist). The results of the present study revealed that UPF1 was significantly downregulated in an HCC cell line. The majority of the proteins exhibited upregulated expression levels in the presence of UPF1 overexpression in the HCC cell line, Huh-7. Key proteins, including cluster of differentiation (CD)31 (platelet endothelial cell adhesion molecule-1), Vimentin, CD44, PCNA, Ki-67, N-Cadherin, Survivin, P53, Met and retinoblastoma exhibited a significant association with UPF1. Furthermore, western blotting indicated that the expression levels of N-cadherin, Vimentin and Twist were notably upregulated while UPF1 was overexpressed; however, E-cadherin was downregulated and opposing observations were reported with protein array analysis. In summary, E-cadherin expression levels were regulated by the manifold, and UPF1, a potential tumor suppressor, may promote the EMT process in Huh-7 HCC cells. The findings of the present study suggested that UPF1 expression levels affected the EMT process by targeting E-cadherin, N-cadherin, Vimentin and Twist.

Chlorinated Lipids Elicit Inflammatory Responses in vitro and in vivo.

Increased endothelial cell adhesion molecule (ECAM) expression, leukocyte-endothelial cell adhesive interactions (LECA), platelet-endothelial cell adhesion (PECA), mast cell activation, production of reactive oxygen species (ROS), and microvascular permeability are hallmarks of the inflammatory response. The infiltration of inflammatory phagocytes is associated with myeloperoxidase (MPO)-dependent production of hypochlorous acid, a reactive chlorinating species that targets membrane lipids to produce halogenated lipids such as 2-chlorohexadecanal (2-ClHDA) and 2-chloropalmitic acid (2-ClPA). Whether these chlorinated lipids contribute to microcirculatory dysfunction is largely unknown. Thus, the objectives of this study were to determine if chlorinated lipids exposure induces such inflammatory responses in an in vitro model employing cultured human intestinal mesenteric vascular endothelial cells (HIMVEC), and in an in vivo model examining responses in small intestinal and mesenteric postcapillary venules of naive rats. Following the addition of either 2-ClPA or 2-ClHDA to the culture medium, HIMVEC displayed increased platelet and neutrophil adherence that was associated with elevated expression of ECAMs and increased permeability. In vivo, chlorinated lipid exposure significantly increased LECA, PECA, ROS production, and albumin leakage, inflammatory events that were associated with mast cell activation and increased tissue MPO activity and expression. Our data provide proof-of-principle that 2-ClPA and 2-ClHDA induce powerful proinflammatory responses both in vitro and in vivo, suggesting the possibility that these chlorinated lipid products of the MPO/ hydrogen peroxide /chloride system may contribute to inflammation noted in neutrophil-dependent, myeloperoxidase-mediated pathologic states such as ischemia/reperfusion, hemorrhagic shock, and sepsis.

Involvement of TIMP-1 in PECAM-1-mediated tumor dissemination.

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is expressed on the vascular endothelium and has been implicated in the late progression of metastatic tumors. The activity of PECAM-1 appears to be mediated by modulation of the tumor microenvironment (TME) and promotion of tumor cell proliferation, rather than through the stimulation of tumor angiogenesis. The present study aimed to extend those initial findings by indicating that the presence of functional PECAM-1 on the endothelium promotes a proliferative tumor cell phenotype in vivo, as well as in tumor cell (B16-F10 melanoma and 4T1 breast cancer cell lines) co-culture assays with mouse endothelial cells (ECs) or a surrogate EC line (REN-MP). The pro-proliferative effects were mediated by soluble endothelial-derived factors that were dependent on PECAM-1 homophilic ligand interactions, but which were independent of PECAM-1-dependent signaling. Further analysis of the conditioned media obtained from tumor/EC and tumor/REN-MP co-cultures identified TIMP metallopeptidase inhibitor-1 (TIMP-1) as a PECAM-1-regulated factor, the targeting of which in the tumor cell/REN-MP system inhibited tumor cell proliferation. In addition, TIMP-1 expression was decreased in metastatic tumors from the lungs of PECAM-1-null mice, thus providing evidence of the in vivo significance of co-culture studies. Taken together, these studies indicated that endothelial PECAM-1, through PECAM-1-dependent homophilic binding interactions, may induce release of TIMP-1 from the endothelium into the TME, thus leading to increased tumor cell proliferation.

Mechanotransduction properties of the cytoplasmic tail of PECAM-1.

Vascular endothelial cells (ECs) are a well-known cell system used in the study of mechanobiology. Using cultured ECs, we found that platelet EC adhesion molecule 1 (PECAM-1, CD31), a cell adhesion protein localised to regions of EC-EC contact, was rapidly tyrosine phosphorylated in ECs exposed to shear or cyclic stretch. Src-homology 2 domain-containing protein tyrosine phosphatase 2 (SHP2) binds phosphorylated PECAM-1 and activates the extracellular signal-regulated kinase1/2 (ERK1/2) signalling cascade, a known flow-activated signalling pathway. Although PECAM-1 tyrosine phosphorylation is characterised in ECs exposed to fluid shear stress, it is less well demonstrated in the cells stretched cyclically. Thus, we first show that PECAM-1 is tyrosine-phosphorylated in ECs cyclically stretched. We hypothesise that when an external force is applied to a monolayer of ECs, the force is directly transmitted to PECAM-1 which is then stretched and phosphorylation sites in its cytoplasmic domain are exposed and phosphorylated. This hypothesis requires the presence of any stretchable structure within the PECAM-1 cytoplasmic domain. Force spectroscopy measurements were performed with a construct containing cytoplasmic PECAM-1 domains inserted between I27 motifs, a recombinant string of the structural elements from titin. This strategy allowed us to identify the events in which a single molecule is being pulled and to detect the unravelling of the cytoplasmic domain of PECAM-1 by force. The response by PECAM-1 to mechanical loading was heterogeneous but with magnitudes as high as or higher than the naturally force bearing I27 domains. The PECAM-1 cytoplasmic domain has a structure that can be unfolded by externally applied force and this unfolding of PECAM-1 may be necessary for its phosphorylation, the first step of PECAM-1 mechanosignalling. When EC monolayers are mechanically stimulated, the PECAM-1 found at EC contacts is phosphorylated. We have proposed that under these conditions, the cytoplasmic domain of PECAM-1 is unfolded, which then exposes a phosphorylation site, allowing it to be accessed. The stretch induced unfolding is essential to this model of PECAM-1 mechanosignalling. In this study, we investigate whether the cytoplasmic domain of PECAM-1 has a stretchable structure, and the results are in line with our hypothesis.

The Role of Matrix Metalloproteinases (MMPs) in Platelet Endothelial Cell Adhesion Molecule‐1 (PECAM‐1) Loss in Diabetic Retinopathy: In Vivo and in Vitro Studies

OBJECTIVEThe aim of this study is to understand the mechanisms involved in the diabetes‐induced loss of PECAM‐1 in the retina, and whether it can be attributed to PECAM‐1 cleavage by matrix metalloproteinases (MMPs).INTRODUCTIONPlatelet endothelial cell adhesion molecule‐1 (PECAM‐1) is expressed on the surface of endothelial cells of the vasculature, and has major roles in inflammation, permeability, and angiogenesis. In addition, PECAM‐1 functions in cell signaling, cell‐cell adhesion, and cell survival. However, its physiological role in the pathophysiology of diabetic retinopathy (DR), the leading cause of blindness among working age adults worldwide, has not yet been investigated. We have previously found a significant decrease (50–90%) in retinal PECAM‐1 levels in a diabetic model of type I diabetes in rats.DESIGN AND METHODSFor our animal studies, we injected age‐matched male Wistar rats with either vehicle (control) or streptozotocin (STZ, diabetic), with the latter at 30 mg/kg/day for three consecutive days to elicit a model of type I diabetes. For our in vitro studies, primary rat retinal microvascular endothelial cells (RRMECs) were grown in either normal glucose (NG, 5mM) or high glucose (HG, 25 mM) media, and treated for 6 days with 0 or 10% plasma collected from control or diabetic rats 8 weeks post‐injection. PECAM‐1, and MMP levels or activity, were assessed using immunofluorescence staining, western blotting, or zymography.RESULTSFor RRMECs grown in NG conditions, treatment with 10% diabetic plasma resulted in significantly lower levels of PECAM‐1 than treatment with 10% control plasma (27%, p<0.05). PECAM‐1 loss was further exaggerated when cells were grown under HG conditions and treated with 10% diabetic plasma (74%, p<0.01). In contrast, control plasma added to RRMECs in NG conditions increased the expression of PECAM‐1 in a dose‐dependent manner. These findings indicate that normal plasma contributes to the expression of PECAM‐1, but some factor(s) in diabetic plasma has the reverse effect. MMPs, which are calcium‐dependent zinc‐containing endopeptidases, are known to cleave PECAM‐1. Plasma samples from diabetic rats revealed a significant increase in MMP‐2 levels (2‐fold, p<0.05), and a significant increase in MMP‐7 activity (p<0.01). Moreover, RRMECs showed a significant increase in MMP2 levels (12‐fold, p<0.01) when grown in HG media.CONCLUSIONThese results indicate a possible role for MMPs in the diabetes‐induced loss of PECAM‐1. Further studies will be conducted to investigate the potential benefits of protecting the retinal expression of PECAM‐1 by targeting the actions of MMPs.Support or Funding InformationNIH RO1 EY017599

Allogeneic Neural Stem/Progenitor Cells Derived From Embryonic Stem Cells Promote Functional Recovery After Transplantation Into Injured Spinal Cord of Nonhuman Primates.

: Previous studies have demonstrated that neural stem/progenitor cells (NS/PCs) promote functional recovery in rodent animal models of spinal cord injury (SCI). Because distinct differences exist in the neuroanatomy and immunological responses between rodents and primates, it is critical to determine the effectiveness and safety of allografted embryonic stem cell (ESC)-derived NS/PCs (ESC-NS/PCs) in a nonhuman primate SCI model. In the present study, common marmoset ESC-NS/PCs were grafted into the lesion epicenter 14 days after contusive SCI in adult marmosets (transplantation group). In the control group, phosphate-buffered saline was injected instead of cells. In the presence of a low-dose of tacrolimus, several grafted cells survived without tumorigenicity and differentiated into neurons, astrocytes, or oligodendrocytes. Significant differences were found in the transverse areas of luxol fast blue-positive myelin sheaths, neurofilament-positive axons, corticospinal tract fibers, and platelet endothelial cell adhesion molecule-1-positive vessels at the lesion epicenter between the transplantation and control groups. Immunoelectron microscopic examination demonstrated that the grafted ESC-NS/PC-derived oligodendrocytes contributed to the remyelination of demyelinated axons. In addition, some grafted neurons formed synaptic connections with host cells, and some transplanted neurons were myelinated by host cells. Eventually, motor functional recovery significantly improved in the transplantation group compared with the control group. In addition, a mixed lymphocyte reaction assay indicated that ESC-NS/PCs modulated the allogeneic immune rejection. Taken together, our results indicate that allogeneic transplantation of ESC-NS/PCs from a nonhuman primate promoted functional recovery after SCI without tumorigenicity. This study demonstrates that allogeneic embryonic stem cell (ESC)-derived neural stem/progenitor cells (NS/PCs) promoted functional recovery after transplantation into the injured spinal cord in nonhuman primates. ESC-NS/PCs were chosen because ESC-NS/PCs are one of the controls for induced pluripotent stem cell-derived NS/PCs and because ESC derivatives are possible candidates for clinical use. This translational research using an allograft model of a nonhuman primate is critical for clinical application of grafting NS/PCs derived from various allogeneic pluripotent stem cells, especially induced pluripotent stem cells, into injured spinal cord at the subacute phase.

Blood–brain barrier: A novel therapeutic target in multiple sclerosis

AbstractMultiple sclerosis is an autoimmune inflammatory disorder of the central nervous system, and the entrance of pathogenic lymphocytes from blood circulation into the brain across the blood–brain barrier (BBB) is accepted as the initial step of pathogenesis. The migration of lymphocytes across the BBB is mediated by their sequential interactions with the brain microvascular endothelial cells, the central component of the BBB, including rolling (loose attachment), adhesion (firm attachment), crawling and transmigration. There are a variety of cell adhesion molecules that are expressed on brain microvascular endothelial cells and are involved in this process. The selectins are assumed to be associated with the rolling of lymphocytes. Vascular cell adhesion molecule‐1 contributes to the adhesion of lymphocytes by binding to very late antigen‐4 on their surface after the stimulation by cytokines. Intercellular adhesion molecule‐1 acts for the adhesion as well and the subsequent crawling of lymphocytes by binding to leukocyte function‐associated antigen‐1 on their surface. Activated leukocyte cell adhesion molecule, melanoma cell adhesion molecule, platelet endothelial cell adhesion molecule‐1, nerve injury‐induced protein‐1 and vascular adhesion protein‐1 are also considered to influence the migratory process of lymphocytes across the BBB, although the detailed mechanisms are not elucidated. Here we review the pathophysiology of multiple sclerosis at the BBB to present a possible molecular targeted therapy in the future, especially focused on the molecules expressing at the brain microvascular endothelial cells.

Regulatory role of glycans in the control of hypoxia-driven angiogenesis and sensitivity to anti-angiogenic treatment.

Abnormal glycosylation is a typical hallmark of the transition from healthy to neoplastic tissues. Although the importance of glycans and glycan-binding proteins in cancer-related processes such as tumor cell adhesion, migration, metastasis and immune escape has been largely appreciated, our awareness of the impact of lectin-glycan recognition in tumor vascularization is relatively new. Regulated glycosylation can influence vascular biology by controlling trafficking, endocytosis and signaling of endothelial cell (EC) receptors including vascular endothelial growth factor receptors, platelet EC adhesion molecule, Notch and integrins. In addition, glycans may control angiogenesis by regulating migration of endothelial tip cells and influencing EC survival and vascular permeability. Recent evidence indicated that changes in the EC surface glycome may also serve "on-and-off" switches that control galectin binding to signaling receptors by displaying or masking-specific glycan epitopes. These glycosylation-dependent lectin-receptor interactions can link tumor hypoxia to EC signaling and control tumor sensitivity to anti-angiogenic treatment.

Human recombinant factor VIIa may improve heat intolerance in mice by attenuating hypothalamic neuronal apoptosis and damage.

Intolerance to heat exposure is believed to be associated with hypothalamo-pituitary-adrenocortical (HPA) axis impairment [reflected by decreases in blood concentrations of both adrenocorticotrophic-hormone (ACTH) and corticosterone]. The purpose of this study was to determine the effect of human recombinant factor VIIa (rfVIIa) on heat intolerance, HPA axis impairment, and hypothalamic inflammation, ischemic and oxidative damage, and apoptosis in mice under heat stress. Immediately after heat stress (41.2 °C for 1 h), mice were treated with vehicle (1 mL/kg of body weight) or rfVIIa (65-270 µg/kg of body weight) and then returned to room temperature (26 °C). Mice still alive on day 4 of heat exposure were considered survivors. Cellular ischemia markers (e.g., glutamate, lactate-to-pyruvate ratio), oxidative damage markers (e.g., nitric oxide metabolite, hydroxyl radials), and pro-inflammatory cytokines (e.g., interleukin-6, interleukin-1β, tumor necrosis factor-α) in hypothalamus were determined. In addition, blood concentrations of both ACTH and corticosterone were measured. Hypothalamic cell damage was assessed by determing the neuronal damage scores, whereas the hypothalamic cell apoptosis was determined by assessing the numbers of cells stained with terminal deoxynucleotidyl transferase-mediated αUTP nick-end labeling, caspase-3-positive cells, and platelet endothelial cell adhesion molecula-1-positive cells in hypothalamus. Compared with vehicle-treated heated mice, rfVIIa-treated heated mice had significantly higher fractional survival (8/10 vs 1/10), lesser thermoregulatory deficit (34.1 vs 24.8 °C), lesser extents of ischemic, oxidative, and inflammatory markers in hypothalamus, lesser neuronal damage scores and apoptosis in hypothalamus, and lesser HPA axis impairment. Human recombinant factor VIIa appears to exert a protective effect against heatstroke by attenuating hypothalamic cell apoptosis (due to ischemic, inflammatory, and oxidative damage) in mice.

Doxorubicin-Induced Vascular Toxicity – Targeting Potential Pathways May Reduce Procoagulant Activity

IntroductionPrevious study in mice using real-time intravital imaging revealed an acute deleterious effect of doxorubicin (DXR) on the gonadal vasculature, as a prototype of an end-organ, manifested by a reduction in blood flow and disintegration of the vessel wall. We hypothesized that this pattern may represent the formation of microthrombi. We aimed to further characterize the effect of DXR on platelets’ activity and interaction with endothelial cells (EC) and to examine potential protectants to reduce DXR acute effect on the blood flow.MethodsThe effect of DXR on platelet adhesion and aggregation were studied in vitro. For in vivo studies, mice were injected with either low molecular weight heparin (LMWH; Enoxaparin) or with eptifibatide (Integrilin©) prior to DXR treatment. Testicular arterial blood flow was examined in real-time by pulse wave Doppler ultrasound.ResultsPlatelet treatment with DXR did not affect platelet adhesion to a thrombogenic surface but significantly decreased ADP-induced platelet aggregation by up to 40% (p<0.001). However, there was a significant increase in GPIIbIIIa-mediated platelet adhesion to DXR-exposed endothelial cells (EC; 5.7-fold; p<0.001) reflecting the toxic effect of DXR on EC. The testicular arterial blood flow was preserved in mice pre-treated with LMWH or eptifibatide prior to DXR (P<0.01).ConclusionsDXR-induced acute vascular toxicity may involve increased platelet–EC adhesion leading to EC-bound microthrombi formation resulting in compromised blood flow. Anti-platelet/anti-coagulant agents are effective in reducing the detrimental effect of DXR on the vasculature and thus may serve as potential protectants to lessen this critical toxicity.

Cancer biomarker profiling in patients with chronic hepatitis C virus, liver cirrhosis and hepatocellular carcinoma

The detection and diagnosis of hepatocellular carcinoma (HCC) at an early stage may significantly affect the prognosis of HCC patients. Thus, it is necessary to always identify novel putative markers for improving diagnosis. Hepatocarcinogenesis correlates with pathological hepatic angiogenesis. However, each tumor-induced angio-genetic process is influenced by the microenvironment through several pro- and anti-angiogenic factors released from tumor cells, tumor-associated inflammatory cells and/or from the extracellular matrix, and modulated by various signal pathways. In this study, we evaluated the profiling of angiogenic factors using Bio-Plex Pro™ Human Cancer Biomarker Panel1, a 16-plex magnetic bead-based assay, in sera of patients with chronic hepatitisC (CHC) virus, liver cirrhosis (LC) and HCC. Our results demonstrated: i)high levels of hepatocyte growth factor (HGF) and prolactin only in LC and HCC patients, ii)high levels of soluble human epidermal growth factor receptor‑2 (sHER-2/neu; ErbB-2), sIL-6Ra, leptin (LEP) and platelet endothelial cell adhesion molecule‑1 (PECAM-1) in CHC, LC and HCC patients and iii)that sIL-6R correlated with the fibrosis stage in CHC patients, with Child‑Pugh score in those patients with LC and with tumor size in those patients with HCC, confirming that this protein may be used as a predictor of liver damage and of inflammatory process leading to fibrosis, cirrhosis, and subsequently to cancer. Moreover, an interactomic study conducted using the Ingenuity Pathway Analysis (IPA) software proved the existence of a correlation between 5significant proteins [ErbB-2, sIL-6Ra, prolactin (PRL), HGF and LEP] which are involved in the same metabolic pathways.

Serum periostin levels are correlated with progressive skin sclerosis in patients with systemic sclerosis

Periostin, a matricellular protein, serves as a regulator of wound healing and fibrosis. The role of periostin in the pathogenesis of systemic sclerosis (SSc) is unknown. To determine periostin levels in association with severity of skin fibrosis in patients with SSc. Expression of periostin was immunohistochemically examined in skin obtained from patients with SSc and healthy controls. Enzyme-linked immunosorbent assay was performed to evaluate serum periostin levels in association with clinical characteristics in 56 patients with SSc [diffuse cutaneous SSc (dSSc), n=16; and limited cutaneous SSc (lSSc), n=40] and 66 healthy controls. Periostin was strongly expressed in the affected dermis from patients with SSc. Periostin was colocalized in α-smooth muscle actin-positive myofibroblasts and platelet endothelial cell adhesion molecule-1-positive endothelial cells in SSc dermis. Serum levels of periostin in patients with dSSc were markedly elevated compared with those in patients with lSSc and control subjects. Patients with lSSc had increased periostin levels compared with healthy controls. In addition, significantly higher levels of periostin were observed in patients with dSSc with disease duration ≤5 years compared with those with disease duration >5 years. Furthermore, the modified Rodnan total skin thickness score (MRSS) was positively correlated with periostin levels in patients with SSc. Serial analysis revealed a correlation between periostin and MRSS; namely, MRSS decreased in line with decreased periostin levels in some patients with dSSc as the disease progressed. An elevated periostin level in patients with SSc is associated with severity of skin sclerosis. Periostin may be a potential biomarker for progressive skin fibrosis in SSc.

Abstract 9753: Highly Electronegative LDL is Elevated in Patients With Acute Myocardial Infarction and Triggers Platelet Activation and Aggregation

Background: Human LDL can be chromatographically resolved into 5 subfractions. L5, the 5th and most negatively charged subfraction, is capable of inducing atherogenic responses in vitro and in vivo. Platelet activation and aggregation are major events underlying acute thrombosis in myocardial infarction (MI). We examined the relationship between L5 and acute MI and the role of L5 in platelet activation and aggregation. Methods and Results: Plasma L5 concentration ([L5]) was measured and compared between subjects with (1) no risk factors (healthy controls), (2) STEMI, (3) stable coronary artery disease (CAD), and (4) hyperlipidemia (HLP) without CAD. In STEMI subjects, LDL cholesterol (LDL-C) levels were similar to those in CAD and control subjects and lower than those in HLP subjects, but L5/LDL% and [L5] (L5/LDL% × LDL-C) were significantly elevated (Table). L5 from STEMI subjects but not L1 (least electronegative LDL subfraction) induced tissue factor and P-selectin expression in human coronary arterial endothelial cells (ECs) in a concentration-dependent manner; in platelets, L5 increased glycoprotein (GP)IIb/IIIa and P-selectin expression and decreased cAMP production (for all, n=8, P &lt;.01 vs PBS). L5 also increased ADP-stimulated platelet aggregation twofold higher than L1 and induced massive platelet–EC adhesion. Experiments with pharmacologic inhibitors indicated that L5 signals through platelet-activating factor receptor and lectin-like oxidized LDL receptor-1 to reduce cAMP production via Akt and trigger granule release and GPIIb/IIIa activation via PKC-α. Injection of human L5 (5 mg/kg) in C57BL/6 mice twice a week for 6 weeks shortened tail-bleeding time by 43% when compared to L1 (n=3; P &lt;.01) and increased expression of P-selectin and GPIIb/IIIa in mouse platelets. Conclusions: L5 triggers platelet activation and aggregation, as well as adhesion to ECs. The significant elevation of L5 in STEMI strongly suggests L5’s involvement in acute MI.