Cold Storage Preservation and Warm Ischaemic Injury to Isolated Arterial Segments: Endothelial Cell Injury

Injury to endothelial calls is thought to be important to the development of the vascular lesion of chronic rejection. It was the aim of this study to develop a semiquantitative method to assess endothelial injury in arterial grafts and to document the injury produced by cold storage preservation and additional warm ischaemia. Twelve- and 24-h cold preservation of rat aortic segments, together with an additional 1 h of warm ischaemia, were assessed. Electron micrographs of representative endothelial cells were scored for cytoplasmic, nuclear and mitochondrial injury. The overall injury score was obtained by addition of the individual scores. Storage for up to 24 h in University of Wisconsin (UW) and Terasaki did not produce any injury. Twenty-four hours of storage in Euro-Collins resulted in endothelial cell death. Injury occurred after 12 h of storage in Ross, Collins and normal saline, and the injury increased following 24 h of storage. One hour of warm ischaemia did not increase the injury. Injury to endothelial cells varies with the preservation solution used and the time of cold storage, so that both the type of solution and the storage time should be taken into account in clinical studies looking at the influence of cold ischaemia time and graft outcome.

Expression of Heparan Sulphate N-deacetylase/N-sulphotransferase by Vascular Smooth Muscle Cells

Heparan sulphate is an important mediator in determining vascular smooth muscle cell (SMC) phenotype. The sulphation pattern of the heparan sulphate chains is critical to their function. We have examined the initial step in the biosynthesis of the sulphated domains mediated by the enzyme heparan sulphate N-deacetylase/N-sulphotransferase (NDST). Rabbit aortic SMC in primary culture exhibited NDST enzyme activity and expressed NDST-1 in their Golgi apparatus, with maximal expression in SMC 2 days after dispersal in primary culture confirmed by Western blot analysis. Endothelial cells, macrophages and fibroblasts expressed NDST-1 but had generally less intense staining than SMC, although SMC expression decreased with culture. The uninjured rat aorta also showed widespread expression of NDST-1. After balloon de-endothelialisation, NDST-1 could not be detected in SMC of the neointima in the early stages of neointimal formation, but was re-expressed at later time points (after 12 weeks). In human coronary arteries, SMC of the media and the diffuse intimal thickening expressed NDST-1, while SMC in the atherosclerotic plaque were negative for NDST-1. We conclude that SMC may regulate their heparan sulphate sulphation at the level of expression of the enzyme heparan sulphate NDST in a manner related to their phenotypic state.

Mast cells and oral inflammation

Mast cells are mobile granule-containing secretory cells that are distributed preferentially about the microvascular endothelium in oral mucosa and dental pulp. The enzyme profile of mast cells in oral tissues resembles that of skin, with most mast cells expressing the serine proteases tryptase and chymase. Mast cells in oral tissues contain the pro-inflammatory cytokine tumour necrosis factor-alpha in their granules, and release of this promotes leukocyte infiltration during evolving inflammation in several conditions, including lichen planus, gingivitis, pulpitis, and periapical inflammation, through induction of endothelial-leukocyte adhesion molecules. Mast cell synthesis and release of other mediators exerts potent immunoregulatory effects on other cell types, while several T-lymphocyte-derived cytokines influence mast cell migration and mediator release. Mast cell proteases may contribute to alterations in basement membranes in inflammation in the oral cavity, such as the disruptions that allow cytotoxic lymphocytes to enter the epithelium in oral lichen planus. A close relationship exists among mast cells, neural elements, and laminin, and this explains the preferential distribution of mast cells in tissues. Mast cells are responsive to neuropeptides and, through their interaction with neural elements, form a neural immune network with Langerhans cells in mucosal tissues. This facilitates mast cell degranulation in response to a range of immunological and non-immunological stimuli. Because mast cells play a pivotal role in inflammation, therapies that target mast cell functions could have value in the treatment of chronic inflammatory disorders in the oral cavity.

Annexin II regulates multivesicular endosome biogenesis in the degradation pathway of animal cells

Proteins of the annexin family are believed to be involved in membrane-related processes, but their precise functions remain unclear. Here, we have made use of several experimental approaches, including pathological conditions, RNA interference and in vitro transport assays, to study the function of annexin II in the endocytic pathway. We find that annexin II is required for the biogenesis of multivesicular transport intermediates destined for late endosomes, by regulating budding from early endosomes-but not the membrane invagination process. Hence, the protein appears to be a necessary component of the machinery controlling endosomal membrane dynamics and multivesicular endosome biogenesis. We also find that annexin II interacts with cholesterol and that its subcellular distribution is modulated by the subcellular distribution of cholesterol, including in cells from patients with the cholesterol-storage disorder Niemann-Pick C. We conclude that annexin II forms cholesterol-containing platforms on early endosomal membranes, and that these platforms regulate the onset of the degradation pathway in animal cells.

Differential Internalization of Amphotericin B - Conjugated Nanoparticles in Human Cells and the Expression of Heat Shock Protein 70

Although a variety of nanoparticles (NPs) functionalized with amphotericin B, an antifungal agent widely used in the clinic, have been studied in the last years their cytotoxicity profile remains elusive. Here we show that human endothelial cells take up high amounts of silica nanoparticles (SNPs) conjugated with amphotericin B (AmB) (SNP-AmB) (65.4 12.4 pg of Si per cell) through macropinocytosis while human fibroblasts internalize relatively low amounts (2.3 0.4 pg of Si per cell) because of their low capacity for macropinocytosis. We further show that concentrations of SNP-AmB and SNP up to 400 mg/mL do not substantially affect fibroblasts. In contrast, endothelial cells are sensitive to low concentrations of NPs (above 10 mg/mL), in particular to SNP-AmB. This is because of their capacity to internalize high concentration of NPs and high sensitivity of their membrane to the effects of AmB. Low-moderate concentrations of SNP-AmB (up to 100 mg/mL) induce the production of reactive oxygen species (ROS), LDH release, high expression of pro-inflammatory cytokines and chemokines (IL-8, IL-6, G-CSF, CCL4, IL-1b and CSF2) and high expression of heat shock proteins (HSPs) at gene and protein levels. High concentrations of SNP-AmB (above 100 ug/mL) disturb membrane integrity and kill rapidly human cells(60% after 5 h). This effect is higher in SNP-AmB than in SNP.

Development of Janus hydrogel microcapsules as novel biomaterials-stem cells construct for 3D co-culture applications

Co-cultures of two or more cell types and biodegradable biomaterials of natural origin have been successfully combined to recreate tissue microenvironments. Segregated co-cultures are preferred over conventional mixed ones in order to better control the degree of homotypic and heterotypic interactions. Hydrogel-based systems in particular, have gained much attention to mimic tissue-specific microenvironments and they can be microengineered by innovative bottom-up approaches such as microfluidics. In this study, we developed bi-compartmentalized (Janus) hydrogel microcapsules of methacrylated hyaluronic acid (MeHA)/methacrylated-chitosan (MeCht) blended with marine-origin collagen by droplet-based microfluidics co-flow. Human adipose stem cells (hASCs) and microvascular endothelial cells (hMVECs) were co-encapsulated to create platforms of study relevant for vascularized bone tissue engineering. A specially designed Janus-droplet generator chip was used to fabricate the microcapsules (<250â μm units) and Janus-gradient co-cultures of hASCs: hMVECs were generated in various ratios (90:10; 75:25; 50:50; 25:75; 10:90), through an automated microfluidic flow controller (Elveflow microfluidics system). Such monodisperse 3D co-culture systems were optimized regarding cell number and culture media specific for concomitant maintenance of both phenotypes to establish effective cell-cell (homotypic and heterotypic) and cell-materials interactions. Cellular parameters such as viability, matrix deposition, mineralization and hMVECs re-organization in tube-like structures, were enhanced by blending MeHA/MeCht with marine-origin collagen and increasing hASCs: hMVECs co-culture gradient had significant impact on it. Such Janus hybrid hydrogel microcapsules can be used as a platform to investigate biomaterials interactions with distinct combined cell populations.

T Cells Bearing a Chimeric Antigen Receptor against Prostate-Specific Membrane Antigen Mediate Vascular Disruption and Result in Tumor Regression.

Aberrant blood vessels enable tumor growth, provide a barrier to immune infiltration, and serve as a source of protumorigenic signals. Targeting tumor blood vessels for destruction, or tumor vascular disruption therapy, can therefore provide significant therapeutic benefit. Here, we describe the ability of chimeric antigen receptor (CAR)-bearing T cells to recognize human prostate-specific membrane antigen (hPSMA) on endothelial targets in vitro as well as in vivo. CAR T cells were generated using the anti-PSMA scFv, J591, and the intracellular signaling domains: CD3ζ, CD28, and/or CD137/4-1BB. We found that all anti-hPSMA CAR T cells recognized and eliminated PSMA(+) endothelial targets in vitro, regardless of the signaling domain. T cells bearing the third-generation anti-hPSMA CAR, P28BBζ, were able to recognize and kill primary human endothelial cells isolated from gynecologic cancers. In addition, the P28BBζ CAR T cells mediated regression of hPSMA-expressing vascular neoplasms in mice. Finally, in murine models of ovarian cancers populated by murine vessels expressing hPSMA, the P28BBζ CAR T cells were able to ablate PSMA(+) vessels, cause secondary depletion of tumor cells, and reduce tumor burden. Taken together, these results provide a strong rationale for the use of CAR T cells as agents of tumor vascular disruption, specifically those targeting PSMA. Cancer Immunol Res; 3(1); 68-84. ©2014 AACR.

Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity.

AIMS: Aldosterone plays a crucial role in cardiovascular disease. 'Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the 'endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. METHODS AND RESULTS: C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high 'endogenous' aldosterone) and in 'exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. CONCLUSION: Obesity-induced endothelial dysfunction depends on the 'endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.

A cell-based model of extracellular-matrix-guided endothelial cell migration during angiogenesis.

Angiogenesis, the formation of new blood vessels sprouting from existing ones, occurs in several situations like wound healing, tissue remodeling, and near growing tumors. Under hypoxic conditions, tumor cells secrete growth factors, including VEGF. VEGF activates endothelial cells (ECs) in nearby vessels, leading to the migration of ECs out of the vessel and the formation of growing sprouts. A key process in angiogenesis is cellular self-organization, and previous modeling studies have identified mechanisms for producing networks and sprouts. Most theoretical studies of cellular self-organization during angiogenesis have ignored the interactions of ECs with the extra-cellular matrix (ECM), the jelly or hard materials that cells live in. Apart from providing structural support to cells, the ECM may play a key role in the coordination of cellular motility during angiogenesis. For example, by modifying the ECM, ECs can affect the motility of other ECs, long after they have left. Here, we present an explorative study of the cellular self-organization resulting from such ECM-coordinated cell migration. We show that a set of biologically-motivated, cell behavioral rules, including chemotaxis, haptotaxis, haptokinesis, and ECM-guided proliferation suffice for forming sprouts and branching vascular trees.

Endothelial Cell-Derived Nitric Oxide Enhances Aerobic Glycolysis in Astrocytes via HIF-1α-Mediated Target Gene Activation.

Astrocytes exhibit a prominent glycolytic activity, but whether such a metabolic profile is influenced by intercellular communication is unknown. Treatment of primary cultures of mouse cortical astrocytes with the nitric oxide (NO) donor DetaNONOate induced a time-dependent enhancement in the expression of genes encoding various glycolytic enzymes as well as transporters for glucose and lactate. Such an effect was shown to be dependent on the hypoxia-inducible factor HIF-1α, which is stabilized and translocated to the nucleus to exert its transcriptional regulation. NO action was dependent on both the PI3K/Akt/mTOR and MEK signaling pathways and required the activation of COX, but was independent of the soluble guanylate cyclase pathway. Furthermore, as a consequence of NO treatment, an enhanced lactate production and release by astrocytes was evidenced, which was prevented by downregulating HIF-1α. Several brain cell types represent possible sources of NO. It was found that endothelial cells, which express the endothelial NO synthase (eNOS) isoform, constitutively produced the largest amount of NO in culture. When astrocytes were cocultured with primary cultures of brain vascular endothelial cells, stabilization of HIF-1α and an enhancement in glucose transporter-1, hexokinase-2, and monocarboxylate transporter-4 expression as well as increased lactate production was found in astrocytes. This effect was inhibited by the NOS inhibitor l-NAME and was not seen when astrocytes were cocultured with primary cultures of cortical neurons. Our findings suggest that endothelial cell-derived NO participates to the maintenance of a high glycolytic activity in astrocytes mediated by astrocytic HIF-1α activation.

Nitrosative stress and corneal transplant endothelial cell death during acute graft rejection.

BACKGROUND: Nitrosative stress takes place in endothelial cells (EC) during corneal acute graft rejection. The purpose of this study was to evaluate the potential role of peroxynitrite on corneal EC death. METHODS: The effect of peroxynitrite was evaluated in vivo. Fifty, 250, and 500 microM in 1.5 microL of the natural or denatured peroxynitrite in 50 microM NaOH, 50 microM NaOH alone, or balanced salt solution were injected into the anterior chamber of rat eyes (n=3/group). Corneal toxic signs after injection were assessed by slit-lamp, in vivo confocal imaging, pachymetry, and EC count. The effect of peroxynitrite was also evaluated on nitrotyrosine and leucocyte elastase inhibitor/LDNase II immunohistochemistry. Human corneas were incubated with peroxynitrite and the effect on EC viability was evaluated. A specific inducible nitric oxide synthase inhibitor (iNOS) was administered systemically in rats undergoing allogeneic corneal graft rejection and the effect on EC was evaluated by EC count. RESULTS: Rat eyes receiving as little as 50 microM peroxynitrite showed a specific dose-dependent toxicity on EC. We observed an intense nitrotyrosine staining of human and rat EC exposed to peroxynitrite associated with leucocyte elastase inhibitor nuclear translocation, a noncaspase dependent apoptosis reaction. Specific inhibition of iNOS generation prevented EC death and enhanced EC survival of the grafted corneas. However, inhibition of iNOS did not have a significant influence on the incidence of graft rejection. CONCLUSION: Nitrosative stress during acute corneal graft rejection in rat eyes induces a noncaspase dependent apoptotic death in EC. Inhibition of nitric oxide production during the corneal graft rejection has protective effects on the corneal EC survival.

Endothelial cell integrins and COX-2: mediators and therapeutic targets of tumor angiogenesis.

Vascular integrins are essential regulators and mediators of physiological and pathological angiogenesis, including tumor angiogenesis. Integrins provide the physical interaction with the extracellular matrix (ECM) necessary for cell adhesion, migration and positioning, and induce signaling events essential for cell survival, proliferation and differentiation. Integrins preferentially expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, are considered as relevant targets for anti-angiogenic therapies. Anti-integrin antibodies and small molecular integrin inhibitors suppress angiogenesis and tumor progression in many animal models, and are currently tested in clinical trials as anti-angiogenic agents. Cyclooxygense-2 (COX-2), a key enzyme in the synthesis of prostaglandins and thromboxans, is highly up-regulated in tumor cells, stromal cells and angiogenic endothelial cells during tumor progression. Recent experiments have demonstrated that COX-2 promotes tumor angiogenesis. Chronic intake of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors significantly reduces the risk of cancer development, and this effect may be due, at least in part, to the inhibition of tumor angiogenesis. Endothelial cell COX-2 promotes integrin alphaVbeta3-mediated endothelial cell adhesion, spreading, migration and angiogenesis through the prostaglandin-cAMP-PKA-dependent activation of the small GTPase Rac. In this article, we review the role of integrins and COX-2 in angiogenesis, their cross talk, and discuss implications relevant to their targeting to suppress tumor angiogenesis.

The complement inhibitor low molecular weight dextran sulfate prevents TLR4-induced phenotypic and functional maturation of human dendritic cells.

Low molecular weight dextran sulfate (DXS) has been reported to inhibit the classical, alternative pathway as well as the mannan-binding lectin pathway of the complement system. Furthermore, it acts as an endothelial cell protectant inhibiting complement-mediated endothelial cell damage. Endothelial cells are covered with a layer of heparan sulfate (HS), which is rapidly released under conditions of inflammation and tissue injury. Soluble HS induces maturation of dendritic cells (DC) via TLR4. In this study, we show the inhibitory effect of DXS on human DC maturation. DXS significantly prevents phenotypic maturation of monocyte-derived DC and peripheral myeloid DC by inhibiting the up-regulation of CD40, CD80, CD83, CD86, ICAM-1, and HLA-DR and down-regulates DC-SIGN in response to HS or exogenous TLR ligands. DXS also inhibits the functional maturation of DC as demonstrated by reduced T cell proliferation, and strongly impairs secretion of the proinflammatory mediators IL-1beta, IL-6, IL-12p70, and TNF-alpha. Exposure to DXS leads to a reduced production of the complement component C1q and a decreased phagocytic activity, whereas C3 secretion is increased. Moreover, DXS was found to inhibit phosphorylation of IkappaB-alpha and activation of NF-kappaB. These findings suggest that DXS prevents TLR-induced maturation of human DC and may therefore be a useful reagent to impede the link between innate and adaptive immunity.

Selectin ligands on leukemic cells

RESUME L'infiltration tissulaire par les cellules leucémiques, responsable de leucostase, est une complication grave de la leucémie aiguë hyperleucocytaire. Elle peut entraîner une détresse respiratoire et des troubles neurologiques de mauvais pronostic. Pendant longtemps, la prolifération intravasculaire des cellules leucémiques et l'augmentation de la viscosité étaient considérées comme en étant responsables, et le traitement reposait sur une cytoréduction rapide par leucaphérèse. Actuellement, l'interaction entre les cellules leucémiques et l'endothélium vasculaire est plutôt considérée comme la cause de ce phénomène. En effet, les cellules leucémiques peuvent induire l'expression des sélectives endothéliales. Les sélectives initient le roulement des leucocytes avant leur adhésion ferme et leur migration dans les tissus. Elles reconnaissent des ligands spécifiques exprimés à la surface des leucocytes, comme PSGL-1 qui est un ligand commun des sélectives. Cependant, plusieurs études suggèrent que d'autres ligands de la E-sélective soient exprimés par les leucocytes. L'interaction des cellules leucémiques avec la E- et la P- sélective est corrélée avec l'expression de la molécule CLA, reconnue par l'anticorps HECA-452. L'immunopurification des ligands de la E-sélective avec cet anticorps a permis d'isoler, des cellules THP1 et U937, une protéine de 170 kDa, ainsi qu'une autre protéine de 250 kDa des cellules U937, en plus de PSGL-1. Ces protéines ont également été purifiées avec la protéine de fusion Esélective/IgM. CD43 et CD44 semblent être des ligands de la E-sélective sur certaines lignées, mais leur interaction avec la E-sélective n'est pas toujours retrouvée. De plus, cette étude a permis de montrer que ces ligands de la E-sélectiné sont exprimés dans les rafts lipidiques, comme PSGL-1 et la L-sélective des neutrophiles. Ces deux nouveaux ligands sont en cours d'identification. Ils pourraient représenter une nouvelle cible dans le traitement de la leucostase, mais aussi lors d'inflammation chronique ou de métastases. ABSTRACT Leukostasis is alife-threatening complication of acute leukemia, that results from tissue infiltration of leukemic blasts that migrate out of blood flow and interfere with normal tissue functions. The process leading to these complications has been attributed to the overcrowding of leukemic cells in the microcirculation. However, leukostasis more likely results from the adhesive interactions between leukemic blasts and the endothelium. Activated endothelium express adhesion molecules like P- and E-selectin, and leukemic cells themselves can induce the expression of E-selectin on endothelial cells. Selectins are essential in initiating the rolling of intravascular cells on endothelium before firm adhesion and transmigration outside of blood vessels. They interact with specific ligands on leukocyte cell surface. P-selectin glycoprotein ligand-1 (PSGL-1) is common ligand for E-, P- and L-selectin. Recently, CD44, ESL-1 and CD44 were shown to cooperate. ìn supporting mouse neutrophil adhesion to E-selectin. Other E-selectin ligands remain to be identified in humans. Leukemic cells were screened in order to characterize human E-selectin ligands. The interactions of E- and P-selectin correlate with the expression of CLA epitope. Therefore, HECA-452 mAb that recognizes CLA was used for immunopurification. Aglycoprotein of 170 kDa was purified from THP1 and U937 cells, and a protein of 250 kDa from U937 cells. These proteins were also purified by affinity binding to E-selectin/IgM chimera. PSGL-1 bound to E-selectin as expected, but CD43 and CD44 were not always adsorbed on E-selectin chimera, depending on cell types. E-selectin ligands were also shown to be in lipid rafts in leukemic cells, like PSGL-1 and L-selectin in human neutrophils. The 170 kDa protein has been sequenced, and three interesting ligands were among the candidates: ESL-1, CD44 and podocalyxin. These ligands are under investigation, and may represent a new therapeutic target in leukostasis, inflammation or cancer metastasis.

Cytotoxic effects of combination of oxidosqualene cyclase inhibitors with atorvastatin in human cancer cells.

Ten oxidosqualene cyclase inhibitors with high efficacy as cholesterol-lowering agents and of different chemical structure classes were evaluated as potential anticancer agents against human cancer cells from various tissue origins and nontumoral human-brain-derived endothelial cells. Inhibition of cancer cell growth was demonstrated at micromolar concentrations, comparable to the concentrations of statins necessary for antitumor effect. Human glioblastoma cells were among the most sensitive cells. These compounds were also able to decrease the proliferation of angiogenic brain-derived endothelial cells, as a model of tumor-induced neovasculation. Additive effects in human glioblastoma cells were also demonstrated for oxidosqualene cyclase inhibitors in combination with atorvastatin while maintaining selectivity against endothelial cells. Thus, not only statins targeting the 3-hydroxy-3-methylglutaryl coenzyme A reductase but also inhibitors of oxidosqualene cyclase decrease tumor growth, suggesting new therapeutic opportunities of combined anti-cholesterol agents for dual treatment of glioblastoma.