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Subcutaneous injection of the tumor cell suspension is a simple and commonly used tool for studying tumor development in vivo. However, subcutaneous models poorly resemble tumor complexity due to the fast growth not reflecting the natural course. Here, we describe an application of the new spheroid-plug model to combine the simplicity of subcutaneous injection with improved resemblance to natural tumor progression. Spheroid-plug model relies on in vitro formation of tumor spheroids, followed by injection of single tumor spheroid subcutaneously in Matrigel matrix. In spheroid-plug model, tumors grow slower in comparison to tumors formed by injection of cell suspension as assessed by 3D ultrasonography (USG) and in vivo bioluminescence measurements. The slower tumor growth rate in spheroid-plug model is accompanied by reduced necrosis. The spheroid-plug model ensures increased and more stable vascularization of tumor than classical subcutaneous tumor model as demonstrated by 3D USG Power Doppler examination. Flow cytometry analysis showed that tumors formed from spheroids have enhanced infiltration of endothelial cells as well as hematopoietic and progenitor cells with stem cell phenotype (c-Kit+ and Sca-1+). They also contain more tumor cells expressing cancer stem cell marker CXCR4. Here, we show that spheroid-plug model allows investigating efficiency of anticancer drugs. Treatment of spheroid-plug tumors with known antiangiogenic agent axitinib decreased their size and viability. The antiangiogenic activity of axitinib was higher in spheroid-plug model than in classical model. Our results indicate that spheroid-plug model imitates natural tumor growth and can become a valuable tool for cancer research.
Hypoxia-inducing pathologies as cancer develop pathologic and inefficient angiogenesis which rules tumor facilitating microenvironment, a key target for therapy. As such, the putative ability of endothelial precursor cells (EPCs) to specifically home to hypoxic sites of neovascularization prompted to design optimized, site-specific, cell-mediated, drug-/gene-targeting approach. Thus, EPC lines were established from aorta-gonad-mesonephros (AGM) of murine 10.5 dpc and 11.5 dpc embryo when endothelial repertoire is completed. Lines representing early endothelial differentiation steps were selected : MAgEC10.5 and MagEC11.5. Distinct in maturation, they differently express VEGF receptors, VE-cadherin and chemokine/receptors. MAgEC11.5, more differentiated than MAgEC 10.5, displayed faster angiogenesis in vitro, different response to hypoxia and chemokines. Both MAgEC lines cooperated to tube-like formation with mature endothelial cells and invaded tumor spheroids through a vasculogenesis-like process. In vivo, both MAgEC-formed vessels established blood flow. Intravenously injected, both MAgECs invaded Matrigel(TM)-plugs and targeted tumors. Here we show that EPCs (MAgEC11.5) target tumor angiogenesis and allow local overexpression of hypoxia-driven soluble VEGF-receptor2 enabling drastic tumor growth reduction. We propose that such EPCs, able to target tumor angiogenesis, could act as therapeutic gene vehicles to inhibit tumor growth by vessel normalization resulting from tumor hypoxia alleviation.
The microenvironment that surrounds tumor cells is characterized by hypoxic conditions and extracellular acidity. These hostile conditions induce crucial changes in cell behavior and can promote the secretion of many soluble factors such as growth factors, cytokines and enzymes. The lysosomal aspartyl-endopeptidase cathepsin D (CD) is a marker of poor prognosis in breast cancer and is associated with a metastatic risk. In this study, the transport of CD was investigated in a model of breast cancer cells line (MCF-7) cultivated under hypoxia and acidification of media. CD secretion was assessed using Western blot analysis and protease activity was measured in conditioned culture media. We demonstrate that cultured MCF-7 cells secrete an active 52 kDa pCD precursor and report that under hypoxia there was an increased amount of pCD secreted. More surprisingly, extracellular acidification (pH 6 and 5.6) induced the secretion of the fully-mature and active (34 kDa + 14 kDa) double chain CD. Our findings reflect the fact that chemical anomalies influence the secretion path of CD in a breast cancer cell model, resulting in altered trafficking of the mature form. This important result may provide new arguments in favor of the role of extracellular CD in the degradation of the matrix proteins that constitute the breast tumor microenvironment.
The tumor microenvironment is a complex system, playing an important role in tumor development and progression. Besides cellular stromal components, extracellular matrix fibers, cytokines, and other metabolic mediators are also involved. In this review we outline the potential role of hypoxia, a major feature of most solid tumors, within the tumor microenvironment and how it contributes to immune resistance and immune suppression/tolerance and can be detrimental to antitumor effector cell functions. We also outline how hypoxic stress influences immunosuppressive pathways involving macrophages, myeloid-derived suppressor cells, T regulatory cells, and immune checkpoints and how it may confer tumor resistance. Finally, we discuss how microenvironmental hypoxia poses both obstacles and opportunities for new therapeutic immune interventions.
The cancer stem cell (CSC) model has recently been approached also in renal cell carcinoma (RCC). A few populations of putative renal tumor-initiating cells (TICs) were identified, but they are indifferently understood ; however, the first and most thoroughly investigated are CD105-positive CSCs. The article presents a detailed comparison of all renal CSC-like populations identified by now as well as their presumable origin. Hypoxic activation of hypoxia-inducible factors (HIFs) contributes to tumor aggressiveness by multiple molecular pathways, including the governance of immature stem cell-like phenotype and related epithelial-to-mesenchymal transition (EMT)/de-differentiation, and, as a result, poor prognosis. Due to intrinsic von Hippel-Lindau protein (pVHL) loss of function, clear-cell RCC (ccRCC) develops unique pathological intra-cellular pseudo-hypoxic phenotype with a constant HIF activation, regardless of oxygen level. Despite satisfactory evidence concerning pseudo-hypoxia importance in RCC biology, its influence on putative renal CSC-like largely remains unknown. Thus, the article discusses a current knowledge of HIF-1 alpha/2 alpha signaling pathways in the promotion of undifferentiated tumor phenotype in general, including some experimental findings specific for pseudo-hypoxic ccRCC, mostly dependent from HIF-2 alpha oncogenic functions. Existing gaps in understanding both putative renal CSCs and their potential connection with hypoxia need to be filled in order to propose breakthrough strategies for RCC treatment.
In recent years, special attention has been paid to finding new pro-angiogenic factors which could be used in gene therapy of vascular diseases such as critical limb ischaemia (CLI). Angiogenesis, the formation of new blood vessels, is a complex process dependent on different cytokines, matrix proteins, growth factors and other pro- or anti-angiogenic stimuli. Numerous lines of evidence suggest that key mediators of angiogenesis, vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) together with fibroblast growth factor2 (FGF2) are involved in regulation of the normal and pathological process of angiogenesis. However, less information is available on the complex interactions between these and other angiogenic factors. The aim of this study was to characterise the effect of fibroblast growth factor2 on biological properties of human endothelial progenitor cells with respect to the expression level of other regulatory cytokines. Ectopic expression of FGF2 in EP cells stimulates their pro-angiogenic behaviour, leading to increased proliferation, migration and tube formation abilities. Moreover, we show that the expression profile of VEGF and other pro-angiogenic cytokines, such as HGF, MCP2, and interleukins, is affected differently by FGF2 in EPC. In conclusion, we provide evidence that FGF2 directly affects not only the biological properties of EP cells but also the expression pattern and secretion of numerous chemocytokines. Our results suggest that FGF2 could be applied in therapeutic approaches for CLI and other ischaemic diseases of the vascular system in vivo.
The ruthenium polypyridyl complexes [Ru(dip)2(bpy/bpy-2-nitroIm)]2+ (dip = 4,7-diphenyl-1,10-phenanthroline, bpy = 2,2[prime or minute]-bipyridine, bpy-2-nitroIm = 4-[3-(2-nitro-1H-imidazol-1-yl)propyl]) were found to be ca. ten times more cytotoxic against breast cancer (4T1) and human lung adenocarcinoma epithelial cells (A549) than a well-known anticancer drug, cisplatin. Even though the Ru complexes were quite cytotoxic towards FVB mouse lung microvascular endothelial cells (MLuMEC FVB) their efflux from these non transformed cells was much more efficient than from cancer ones. Both Ru complexes accumulated in cells. The cellular uptake of both Ru complexes occurs through passive diffusion while the nitroimidazole derivative is also endocytosed. They arrest cell growth in the S-phase and induce apoptosis. Such cell response can result from activation of oxidative stress by Ru complexes. The modulation of the mRNA expression profile for genes which might be involved in metastasis and angiogenesis processes by Ru complexes was analyzed for both cancer (4T1) and endothelial (MLuMEC FVB) cells. Ru complexes appeared to have a distinct impact on cell adhesion and migration as well as they affect endothelial cell vasculature. They are not only cytotoxic but are also potentially invasive and anti-metastatic agents. This work illustrates the putative future development of polypyridyl ruthenium.
The tumour microenvironment, long considered as determining cancer development, still offers research fields to define hallmarks of cancer. An early key-step, the “angiogenic switch”, allows tumour growth. Pathologic angiogenesis is a cancer hallmark as it features results of tumour-specific properties that can be summarised as a response to hypoxia. The hypoxic state occurs when the tumour mass reaches a volume sufficient not to permit oxygen diffusion inside the tumour centre. Thus tumour cells turn on adaptation mechanisms to the low pO(2) level, inducing biochemical responses in terms of cytokines/chemokines/receptors and consequently recruitment of specific cell types, as well as cell-selection inside the tumour. Moreover, these changes are orchestrated by the microRNA balance strongly reflecting the hypoxic milieu and mediating the cross-talk between endothelial and tumour cells. MicroRNAs control of the endothelial precursor-vascular settings shapes the niche for selection of cancer stem cells.
Here, we examine the photophysical properties of five ruthenium(II) complexes comprising two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and functionalized bipyridine (R(1)bpy-R(2), where R(1)= H or CH3, R(2)= H, CH(3), COO(-),4-[3-(2-nitro-1H-imidazol-1-yl)propyl] or 1,3-dicyclohexyl-1-carbonyl-urea) towards development of luminescence probes for cellular imaging. These complexes have been shown to interact with albumin and the formed adducts exhibited up to eightfold increase in the luminescence quantum yield as well as the average lifetime of emission. It was demonstrated that they cannot bind to DNA through the intercalation mode and its luminescence in the presence of DNA is quenching. Cell viability experiments indicated that all complexes possess significant dose-dependent cytotoxicity (with IC(5)(0) 5-19 muM) on 4T1 breast cancer cell line and their anti-proliferative activity correlates very well with their lipophilicity. Cellular uptake was studied by measuring the ruthenium content in cells using ICP-MS technique. As expected, the better uptake is directly related to higher lipophilicity of doubly charged ruthenium complexes while uptake of monocationic one is much lower in spite of the highest lipophilicity. Additionally staining properties were assessed using flow cytometry and fluorescence microscopy. These experiments showed that complex with 1,3-dicyclohexyl-1-carbonyl-urea substituent exhibits the best staining properties in spite of the lowest luminescence quantum yield in buffered solution (pH 7.4). Our results point out that both the imaging and cytotoxic properties of the studied ruthenium complexes are strongly influence by the level of internalization and protein interaction.
Polyphenols are strong antioxidant molecules allowing prevention of skin photo-ageing damages, but their use is limited due to low solubility and toxicity towards skin cells. We postulated that enzymatic glucosylation could improve their solubility, stability and, consequently, their efficacy. The aim of this work was to study changes induced by addition of a glucose moiety on two polyphenols displaying very different chemical structures [caffeic acid (CA), epigallocatechin-3-gallate (EGCG) and there glucosylated form, Glc-CA and Glc-EGCG] by assessing their cytotoxic properties and their antioxidant and anti-inflammatory activities.
Their antioxidant effect was assessed first by the classical DPPH radical-scavenging method. Then, a panel of human skin cells (keratinocytes, melanocytes, fibroblasts and endothelial cells) was used to evaluate their effect on cell toxicity and their antioxidant activities. With this aim, a photo-ageing model based on UV irradiation of skin cells was established. Molecule activity was assessed on reactive oxygen species (ROS) production, on superoxide dismutase (SOD) and catalase activities and, finally, on inflammatory factor production IL-6, IL-8 and IL-1β.
In an acellular model, antioxidant activity assessed by DPPH method was strongly reduced for Glc-CA compared to CA, whereas it remained the same for Glc-EGCG compared to EGCG. Glucosylated derivatives did not display more toxic effect on various skin cells. Moreover, toxicity was even strongly reduced for caffeic acid upon glucosylation. The efficacy of glucosyl-compounds against UV-induced ROS production was preserved, both with pre- and post-UV treatments. Particularly, a better antioxidant efficacy was shown by Glc-EGCG, vs. EGCG, on keratinocytes. In addition, an induction of SOD and catalase activity was clearly observed for Glc-CA. Both glucosyl-polyphenols display the same activity as their parent molecule in decreasing inflammatory factor production.
Our results demonstrated that enzymatic glucosylation of CA and EGCG led to an improved or preserved antioxidant activity in a cellular model of UV-induced skin ageing, despite the decrease in instantaneous antioxidant properties observed for Glc-CA. Glc-EGCG is specifically more active on keratinocytes, suggesting a specific targeting. Such glucosylated polyphenols displaying improved physicochemical and biological properties should be better candidates than natural ones for use in food additives and cosmetics.
Proangiogenic enzyme thymidine phosphorylase (TP) is a promising target for anticancer therapy, yet its action in non-small cell lung carcinoma (NSCLC) is not fully understood. To elucidate its role in NSCLC tumor growth, NCI-H292 lung mucoepidermoid carcinoma cells and endothelial cells were engineered to overexpress TP by viral vector transduction. NSCLC cells with altered expression of transcription factor Nrf2 or its target gene heme oxygenase-1 (HO-1) were used to study the regulation of TP and the findings from pre-clinical models were related to gene expression data from clinical NSCLC specimens. Overexpression of Nrf2 or HO-1 resulted in upregulation of TP in NCI-H292 cells, an effect mimicked by treatment with an antioxidant N-acetylcysteine and partially reversed by HO-1 knockdown. Overexpression of TP attenuated cell prolifera\tion and migration in vitro, but simultaneously enhanced angiogenic potential of cancer cells supplemented with thymidine. The latter was also observed for SK-MES-1 squamous cell carcinoma and NCI-H460 large cell carcinoma cells. TP-overexpressing NCI-H292 tumors in vivo exhibited better oxygenation and higher expression of IL-8, IL-1 beta and IL-6. TP overexpression in endothelial cells augmented their angiogenic properties which was associated with enhanced generation of HO-1 and VEGF. Correlation of TP with the expression of HO-1 and inflammatory cytokines was confirmed in clinical samples of NSCLC. Altogether, the increased expression of IL-1 beta and IL-6 together with proangiogenic effects of TP-expressing NSCLC on endothelium can contribute to tumor growth, implying TP as a target for antiangiogenesis in NSCLC.
The lack of oxygen is a major reason for the resistance of tumor cells to treatments such as radiotherapies. A large number of recent publications on non-thermal plasma applications in medicine report cell behavior modifications and modulation of soluble factors. This in vivo study tested whether such modifications can lead to vascular changes in response to plasma application. Two in situ optical-based methods were used simultaneously, in real time, to assess the effect of non-thermal plasma on tissue vasculature. Tissue oxygen partial pressure (pO(2)) was measured using a time-resolved luminescence-based optical probe, and the microvascular erythrocyte flow was determined by laser Doppler flowmetry. When plasma treatment was applied on mouse skin, a rapid pO(2) increase (up to 4 times) was subcutaneously measured and correlated with blood flow improvement. Such short duration, i.e. 5 min, plasma-induced effects were shown to be locally restricted to the treated area and lasted over 120 min. Further investigations should elucidate the molecular mechanisms of these processes. However, improvement of oxygenation and perfusion open new opportunities for tumor treatments in combination with radiotherapy, and for tumor blood vessel normalization based strategies.
VEGFs are found at high levels in hypoxic tumors. As major components directing pathologic neovascularization, they regulate stromal reactions. Consequently, novel strategies targeting and inhibiting VEGF overproduction upon hypoxia offer considerable potential for modern anticancer therapies controlling rather than destroying tumor angiogenesis. Here, we report the design of a vector expressing the soluble form of VEGF receptor-2 (sVEGFR2) driven by a hypoxia-responsive element (HRE)-regulated promoter. To enable in vivo imaging by infrared visualization, mCherry and IFP1.4 coding sequences were built into the vector. Plasmid construction was validated through transfection into embryonic human kidney HEK293 and murine B16F10 melanoma cells. sVEGFR2 was expressed in hypoxic conditions only, confirming that the gene was regulated by the HRE promoter. sVEGFR2 was found to bind efficiently and specifically to murine and human VEGF-A, reducing the growth of tumor and endothelial cells as well as impacting angiogenesis in vitro. The hypoxia-conditioned sVEGFR2 expression was shown to be functional in vivo : Tumor angiogenesis was inhibited and, on stable transfection of B16F10 melanoma cells, tumor growth was reduced. Enhanced expression of sVEGFR2 was accompanied by a modulation in levels of VEGF-A. The resulting balance reflected the effect on tumor growth and on control of angiogenesis. A concomitant increase of intratumor oxygen tension also suggested an influence on vessel normalization. The possibility to express an angiogenesis regulator as sVEGFR2, in a hypoxia-conditioned manner, significantly opens new strategies for tumor vessel–controlled normalization and the design of adjuvants for combined cancer therapies. Mol Cancer Ther ; 13(1) ; 165–78. ©2013 AACR.
The developmentofthePlasmaGun,generatingPulsedAtmospheric-pressurePlasmaStreamsinside long andsmalldiametertubes flushed withlowgas flow rates,isreportedwithaspecialemphasisonthe first demonstrationandperspectivesfor in vivo endoscopic applications.Thesafedeliveryandantitumor action ofplasmahavebeenachievedfor in vivo colorectal andpancreaticcancers.Plasmadeliveryand plasma triggeredinflammation insidemouselungswereperformedthroughanendoscopicprotocol. The studyofplasmasplittingortransferacrossmetallicsectionsordielectricbarriersisdocumentedand open upopportunitiesforselfguidedandlowinvasiveplasmaendoscopy.
AIMS : Heme oxygenase-1 (HO-1, HMOX1) can prevent tumor initiation ; while in various tumors, it has been demonstrated to promote growth, angiogenesis, and metastasis. Here, we investigated whether HMOX1 can modulate microRNAs (miRNAs) and regulate human non-small cell lung carcinoma (NSCLC) development. RESULTS : Stable HMOX1 overexpression in NSCLC NCI-H292 cells up-regulated tumor-suppressive miRNAs, whereas it significantly diminished the expression of oncomirs and angiomirs. The most potently down-regulated was miR-378. HMOX1 also up-regulated p53, down-regulated angiopoietin-1 (Ang-1) and mucin-5AC (MUC5AC), reduced proliferation, migration, and diminished angiogenic potential. Carbon monoxide was a mediator of HMOX1 effects on proliferation, migration, and miR-378 expression. In contrast, stable miR-378 overexpression decreased HMOX1 and p53 ; while enhanced expression of MUC5AC, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and Ang-1, and consequently increased proliferation, migration, and stimulation of endothelial cells. Adenoviral delivery of HMOX1 reversed miR-378 effect on the proliferation and migration of cancer cells. In vivo, HMOX1 overexpressing tumors were smaller, less vascularized and oxygenated, and less metastatic. Overexpression of miR-378 exerted opposite effects. Accordingly, in patients with NSCLC, HMOX1 expression was lower in metastases to lymph nodes than in primary tumors. INNOVATION AND CONCLUSION : In vitro and in vivo data indicate that the interplay between HMOX1 and miR-378 significantly modulates NSCLC progression and angiogenesis, suggesting miR-378 as a new therapeutic target. REBOUND TRACK : This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16, 293-296, 2012) with the following serving as open reviewers : James F. George, Mahin D. Maines, Justin C. Mason, and Yasufumi Sato.
Among endothelial cells (ECs), subpopulations are mainly distinguished in terms of maturation, tissue specialization and functions. Heterogeneity is an important characteristic of endothelial cells responsible for organ-specific cell and molecule delivery. Endothelial cell heterogeneity is a key to embryonic development as well as cell recruitment in adult organism during the immune response ; it determines also the pathologic spreading of diseases, such as cancer invasion and infectious disease progression among species. Heterogeneity is also a feature of intra-organ specialization of endothelial cells. ECs are highly reactive to the microenvironment and their condition reflects healthy vs. diseased states. They respond to tissue hypoxia which brings a new parameter to endothelial heterogeneity. Hypoxia changes the phenotype and biology of ECs by turning on angiogenesis to restore physioxia. Highly responsive to hypoxia are the endothelial precursor cells (EPCs) and the selected cancer stem cell (CSC) populations, the most aggressive dedifferentiated tumor cells. They cooperate with one another and contribute to the vascular mimicry process of angiogenesis. This has a most effective impact on tumor cells escape and spreading.
Normal chondrocytes display susceptibility to lysis by natural killer (NK) cells and this phenomenon may play a role in some inflammatory cartilage disorders. The mechanisms of chondrocyte recognition and killing by NK cells remain unclear. Using flow cytometry and immunohistochemical staining we found that normal human articular chondrocytes constitutively express a ligand for NKp44, one of stimulatory NK cell receptors involved in recognition and killing of target cells. Expression of NKp44 ligand by normal articular chondrocytes is not involved in their killing by unstimulated NK cells ; however, it is responsible for anti-chondrocyte cytotoxicity mediated by long-term activated NK cells. Thus, expression of NKp44 ligand may play a role in chondrocyte destruction in course of chronic inflammatory cartilage disorders.
This chapter describes a short historical overview of the progress in endothelium research and point the importance of organ-selective characteristics according to the present knowledge about endothelium biology. Uncovering the advantages that the endothelial cell properties and characteristics provide for the development of future targeted therapies, the review describes why mature endothelial cells due to their organ-specificity can be useful to target diseased organs.
In the same line, endothelium properties will be exploited to make the endothelial cells a disease marker, e.g., in diabetes, stroke, cancer, inflammation, or ischemia and to provide a potential diagnostic indicator for the estimation of metastatic progression. New perspectives are thus opened by endothelial cells that can be considered both as a reporter and a target. These features can be combined with new cell-mediated and cell-targeted therapeutics designed to correct angiogenesis. Examples of such possible applications are detailed in the repair of tumor angiogenesis with help of endothelial cell precursors through their ability to target the pathologic angiogenesis and participate to normalization of the pathologic vasculature. The hypothesis that normalized angiogenesis may provide an efficient treatment, working as adjuvant to classical therapies, is being developed. The objective is to reach a mechanical stabilization that should result in an advantageous change of the tumor microenvironment.
We have created unique near-infrared (NIR)–emitting nanoscale metal-organic frameworks (nano-MOFs) incorporating a high density of Yb3+ lanthanide cations and sensitizers derived from phenylene. We establish here that these nano-MOFs can be incorporated into living cells for NIR imaging. Specifically, we introduce bulk and nano-Yb-phenylenevinylenedicarboxylate-3 (nano-Yb-PVDC-3), a unique MOF based on a PVDC sensitizer-ligand and Yb3+ NIR-emitting lanthanide cations. This material has been structurally characterized, its stability in various media has been assessed, and its luminescent properties have been studied. We demonstrate that it is stable in certain specific biological media, does not photobleach, and has an IC50 of 100 μg/mL, which is sufficient to allow live cell imaging. Confocal microscopy and inductively coupled plasma measurements reveal that nano-Yb-PVDC-3 can be internalized by cells with a cytoplasmic localization. Despite its relatively low quantum yield, nano-Yb-PVDC-3 emits a sufficient number of photons per unit volume to serve as a NIR-emitting reporter for imaging living HeLa and NIH 3T3 cells. NIR microscopy allows for highly efficient discrimination between the nano-MOF emission signal and the cellular autofluorescence arising from biological material. This work represents a demonstration of the possibility of using NIR lanthanide emission for biological imaging applications in living cells with single-photon excitation.
Inefficient immune response is a major glitch during tumor growth and progression. Chaotic and leaky blood vessels created in the process of angiogenesis allow tumor cells to escape and extricate anti-cancer immunity. Proangiogenic characteristics of hypoxic tumor microenvironment maintained by low oxygen tension attract endothelial progenitor cells, drive expansion of cancer stem cells, and deviantly differentiate monocyte descendants. Such cellular milieu further boosts immune tolerance and eventually appoint immunity for cancer advantage. Blood vessel normalization strategies that equilibrate oxygen levels within tumor and fix abnormal vasculature bring exciting promises to future anticancer therapies especially when combined with conventional chemotherapy. Recently, a new group of microRNAs (miRs) engaged in angiogenesis, called angiomiRs and hypoxamiRs, emerged as new therapeutic targets in cancer. Some of those miRs were found to efficiently regulate cancer immunity and their dysregulation efficiently programs aberrant angiogenesis and cancer metastasis. The present review highlights new findings in the field of miRs proficiency to normalize aberrant angiogenesis and to restore anti-tumor immune responses.
Among innovative strategies developed for cancer treatments, gene therapies stand of great interest despite their well-known limitations in targeting, delivery, toxicity or stability. The success of any given genetherapy is highly dependent on the carrier efficiency. New approaches are often revisiting the mythic trojan horse concept to carry therapeutic nucleic acid, i.e. DNAs, RNAs or small interfering RNAs, to pathologic tumor site. Recent investigations are focusing on engineering carrying modalities to overtake the above limitations bringing new promise to cancer patients. This review describes recent advances and perspectives for gene therapies devoted to tumor treatment, taking advantage of available knowledge in biotechnology and medicine.
Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.
The skin is a multifunctional organ and a first line of defense actively protecting from environmental stress caused by injury, microbial treat, UV irradiation and environmental toxins. Diverse cutaneous cell types together with extracellular matrix elements and factors create a dynamic scene for cellular communication crucial in vital processes such as wound healing, inflammation, angiogenesis, immune response. Direct functional success of skin equilibrium depends on its microenvironment settings and particularly the local oxygen tension. Indeed, skin entire milieu is characterized by and highly dependent on its low oxygen tension called physioxia as emphasized in this review. In the context of skin physioxia, we review and propose here new approaches to minimize age-related changes in skin state and function. We particularly emphasize carbohydrate-mediated interactions and new 3D models of engineered skin substitutes. We highlight newly emerged tools and targets including stem cells, miRNAs, matrix metalloproteinases, mitochondria and natural antioxidants that are promising in prevention of skin ageing and disease restraint. In the era of advanced dermatology, new attempts are bringing us closer to ’well being’ perception.
Tumor microenvironment is a complex and highly dynamic milieu that provides very important clues on tumor development and progression mechanisms. Tumor-associated endothelial cells play a key role in stroma organization. They achieve tumor angiogenesis, a formation of tumor-associated (angiogenic) vessels mainly through sprouting from locally preexisting vessels and/or recruitment of bone marrow-derived endothelial progenitor cells. This process participates to supply nutritional support and oxygen to the growing tumor.
Endothelial cells constitute the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby controlling leukocyte recruitment, tumor cell behavior and metastasis formation.
Hypoxia, a critical parameter of the tumor microenvironment, controls endothelial/tumor cell interactions and is the key to tumor angiogenesis development. Under hypoxic stress, tumor cells produce factors that promote angiogenesis, vasculogenesis, tumor cell motility, metastasis and cancer stem cell selection.
Targeting tumor vessels is a therapeutic strategy that has lately been fast evolving from antiangiogenesis to vessel normalization as discussed in this review. We shall focus on the pivotal role of endothelial cells within the tumor microenvironment, the specific features and the part played by circulating endothelial precursors cells. Attention is stressed on their recruitment to the tumor site and their role in tumor angiogenesis where they are submitted to miRNAs-mediated de/regulation. Here the compensation of the tumor deregulated angiogenic miRNAs – angiomiRs - is emphasized as a potential therapeutic approach. The strategy is to over express anti-angiomiRs in the tumor angiogenesis site upon selective delivery by precursor endothelial cells as miRs carriers.
Non-thermal plasma (NTP) is generated by ionizing neutral gas molecules/atoms leading to a highly reactive gas at ambient temperature containing excited molecules, reactive species and generating transient electric fields. Given its potential to interact with tissue or cells without a significant temperature increase, NTP appears as a promising approach for the treatment of various diseases including cancer. The aim of our study was to evaluate the interest of NTP both in vitro and in vivo. To this end, we evaluated the antitumor activity of NTP in vitro on two human cancer cell lines (glioblastoma U87MG and colorectal carcinoma HCT-116). Our data showed that NTP generated a large amount of reactive oxygen species (ROS), leading to the formation of DNA damages. This resulted in a multiphase cell cycle arrest and a subsequent apoptosis induction. In addition, in vivo experiments on U87MG bearing mice showed that NTP induced a reduction of bioluminescence and tumor volume as compared to nontreated mice. An induction of apoptosis was also observed together with an accumulation of cells in S phase of the cell cycle suggesting an arrest of tumor proliferation. In conclusion, we demonstrated here that the potential of NTP to generate ROS renders this strategy particularly promising in the context of tumor treatment.
Endothelial progenitor cells (EPCs) modulate postnatal vascularization and contribute to vessel regeneration in adults. Stem cells and progenitor cells were found in umbilical cord blood, bone marrow, and mobilized peripheral blood cells, from where they were isolated and cultured. However, the yield of progenitor cells is usually not sufficient for clinical application and the quality of progenitor cells varies. The aim of the study was the immortalization of early progenitor cells with high proliferative potential, capable to differentiate to EPCs and, further, toward endothelial cells. Two cell lines, namely HEPC-CB. 1 and HEPC-CB. 2 (human endothelial progenitor cells-cord blood) were isolated. As assessed by specific antibody labeling and flow cytometric analysis, they express a panel of stem cell markers : CD133, CD13, CD271, CD90 and also endothelial cell markers : CD202b, CD309 (VEGFR2), CD146, CD105, and CD143 but they do not present markers of finally differentiated endothelial cells : CD31, vWf, nor CD45 which is a specific hematopoietic cell marker. Using the multiplex Cytometric Bead Assay, the simultaneous production of proangiogenic cytokines IL8, angiogenin, and VEGF was demonstrated in normoxia and was shown to be increased by hypoxia. Both cell lines, similarly as mature endothelial cells, underwent in vitro pre-angiogenic process, formed pseudovessel structures and present an accelerated angiogenesis in hypoxic conditions. To date, these are the first CD133 positive established cell lines from human cord blood cells. (C) 2011 International Society for Advancement of Cytometry.
Chemical and physical stimuli trigger a cutaneous response by first inducing the main epidermal cells, keratinocytes, to produce specific mediators that are responsible for the initiation of skin inflammation. Activation modulates cell communication, namely leucocyte recruitment and blood-to-skin extravasation through the selective barrier of the vascular ECs (endothelial cells). In the present study, we describe an in vitro model which takes into account the various steps of human skin inflammation, from keratinocyte activation to the adhesion of leucocytes to dermal capillary ECs.
5,6-Dimethylxanthenone-4- acetic acid (1) is scheduled for phase III clinical trials as a vascular disrupting agent. However, its biochemical receptor(s) have yet to be identified. In this report, the synthesis of azido analogues of I that could be used for photoaffinity labeling of proteins as an approach toward identifying its molecular targets is described. While 5-azidoxantfienone-4-acetic acid (2) and 5-azido-6-methylxantheone-4-acetic acid (3) were found to have biological activities similar to that of 1, 6-azido-5-methylxanthenone-4-acetic acid (4) was unstable and could not be evaluated.
The melanoma cell adhesion molecule (MCAM)/CD146 is expressed as two isoforms differing by their cytoplasmic domain (MCAM long (MCAM-1) and MCAM short (MCAM-s)). MCAM being expressed by endothelial cells and activated T cells, we analyzed its involvement in lymphocyte trafficking. The NK cell line NKL1 was transfected by MCAM isoforms and submitted to adhesion on both the endothelial cell monolayer and recombinant molecules under shear stress. MCAM-1 transfection reduced rolling velocity and increased NKL1 adhesion on the endothelial cell monolayer and VCAM-1.
This study evaluated for the first time the binding of pDNA/polymer complexes (polyplexes) on a human lung microvascular endothelial cell (HLMEC) monolayer under flow conditions. A slide of a HLMEC monolayer was mounted on a parallel flow chamber connected to an open flow system from a reservoir containing fluorescent polyplexes to a syringe.
Heme oxygenase-1 (HO-1), a cytoprotective enzyme, can be induced in tumors in response to anti-cancer therapies. We investigated the role of HO-1 in B16(F10), S91, and Sk-mel188 melanoma cells. Overexpression of HO-1 after transduction with adenoviral vectors increased cell proliferation, resistance to oxidative stress generated by H2O2, and angiogenic potential as determined by induction of endothelial cell divisions. Likewise, cells stably transfected with HO-1 cDNA (B16-HO-1) showed higher proliferation, stress resistance, and angiogenic activity than the wild-type line (B16-WT).
Background Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells. Methods After optimizing growth conditions to obtain a tight HLMEC monolayer, we characterized uptake of polyplexes by flow cytometry and evaluated their transfection efficiency. Polyplexes were formulated as small particles.
Background : Following systemic administration, polyplexes must cross the endothelium barrier to deliver genes to the target cells underneath. To design an efficient gene delivery system into lung epithelium, we evaluated capture and transfection efficiencies of DNA complexed with either Jet-PEI (TM) (PEI-polyplexes) or histidylated polylysine (His-polyplexes) in human lung microvascular endothelial cells (HLMEC) and tracheal epithelial cells.
Interleukin (lL)-7 is a pleiotropic, non-redundant cytokine necessary for the development of B and T lymphocytes, in particular ? d T cell receptor-positive cell differentiation. The cytokine can function as a cofactor during myelopoiesis and the generation of cytotoxic T cells and natural killer cells, can activate monocytes/macrophages, and support the survival of mature T cells.
Human organ-specific microvascular endothelial cells (ECs) were established and used in the present study to investigate their susceptibility to natural killer cell line (NKL)-induced lysis. Our data indicate that although IL-2-stimulated NKL (NKL2) cells adhered to the human peripheral (HPLNEC.B3), mesenteric lymph node (HNILNEQ, brain (HBrMEC), and lung (HLMEC) and skin (HSkMEC.2) ECs, they significantly killed these cells quite differently. A more pronounced lysis of OSECs was also observed when IL-2-stimulated, purified peripheral blood NK cells were used as effector cells. In line with the correlation observed between adhesion pattern and the susceptibility to NKL2-mediated killing, we demonstrated using different chelators that the necessary adhesion step was governed by an Mg2+-dependent, but Ca2+-independent, mechanism as opposed to the subsequent Ca2+- dependent killing.
Numerous adhesion molecules have been described, and the molecular mechanisms of lymphocyte trafficking across the endothelium is starting to be elucidated. Identification of the molecules involved in the organoselectivity of this process would help in the targeting of drug therapy to specific tissues. Adhesion-regulating molecule-1 (ARM-1) is an adhesion-regulating molecule previously identified on T cells. It does not belong to any known families of adhesion molecules. In this study, we show the presence of ARM-1 in endothelial cells, the adhesion partners of lymphocytes.
DMXAA (5,6-dimethylxanthenone-4-acetic acid), the most potent of a series of xanthenone (XAA) analogues developed in this laboratory, is currently undergoing combination clinical trials as an antivascular agent for cancer treatment. XAAs have a complex mode of action, and in vitro assays that are predictive of in vivo antitumor activity have been difficult to develop. In this study, we have utilized a series including XAA, DMXAA, and mono-substituted XAA derivatives to determine firstly whether in vitro NF-kappa B activation of mouse cell lines predicts for the in vivo antitumor potential of this class of agents, and secondly whether the relative activity of these analogues is similar in murine and human cell lines.
Lymphocytes extravasation is determined by their adhesion to the endothelial cells. The process goes along with high degree selectivity and is limited to lymph nodes and lymphatic tissue. It comes to uncontrolled extravasation to the tissues of involved organs in diseases with allergic origin as well in inflammation. Most information concerning adhesive interactions comes from researches dealing with normal lymphocytes. However, this phenomenon plays also crucial role in inflammation, metastasis and other pathologies. The aim of the work was to search for the possible distinct efficacy of adhesive interactions between peripheral blood lymphocytes from children with asthma and endothelial cells isolated from lung, skin and intestine. Isolated peripheral blood leukocytes were overlayered into endothelial cell monolayer. After washing the unbound cells, adhering lymphocytes were collected with endothelial cells. For quantifying the percentage of the particular cell populations cytofluorometric method was applied. The results are presented as a number of adhering lymphocytes per one endothelial cell. It has been shown that lymphocytes from asthmatic children have significantly greater adhesive potential towards endothelial cell lines from lungs and skin as compared with adhesion to endothelium of intestine origin. However, their B lymphocytes subpopulation demonstrated significantly higher percentage of cells adhering to all endothelial cell lines tested, as compared with B lymphocytes from normal controls. There were not statistically different adhesion efficiencies of T lymphocytes and NK cells. These findings indicate that local, tissue specific adhesive leukocyte-endothelial interactions may be of some importance in pathogenesis of allergic diseases.
Interleukin-7 (IL-7) is a pleiotropic, non-redundant cytokine crucial for development of B and T lymphocytes. The cellular response to IL-7 is triggered by binding of the cytokine to its receptor, IL-7R. Until now the expression of the receptor was evidenced only in lymphoid and myeloid cell lineages. The receptor consists of two chains : IL-7 specific a chain (CD127) and the common 7. chain (CD132) which is a component of several other cytokine receptors : IL-2, IL-4, IL-9 and IL-15. The former observation that exogenous IL-7 is biologically active towards murine endothelial cell lines from secondary lymphoid organs was the starting point of our studies. This observation has prompted us to search for the presence of IL-7 receptor in human microvascular endothelial cells. We used in our studies a set of human endothelial cell lines established from various organs.
Normal and transformed cells home into tissues from the circulation in a very selective way thanks to highly complex molecular mechanisms that govern cell-to-cell interactions and drive the homing of circulating cells so that it is achieved properly. Because this is characterized by a resulting high selectivity, it constitutes a template for targeted drug-, gene- or cell-therapy strategies. Designing a mimetic-based therapy requires the identification of the responsible selective molecules, but also their mechanisms of action and interactions with their ligands together with their biological modulation and regulation. This homing/invasion event is decisive at the level of the endothelium that lines the vessel walls.
Vascular endothelial cells recognize blood-borne circulating cells and allow them to extravasate in a tissue-specific manner. Because this property determines the selectivity of lymphocyte homing, it is fundamental in physiological as well as pathological processes (inflammation, autoimmune diseases, metastasis). As a tool to assess the molecular basis of endothelium selectivity, microvascular endothelial cell lines of distinct tissue origin were established. Endothelial cells, isolated from lymphoid tissues (lymph nodes and appendix) and from nonlymphoid immune sites-intestine, lung, and skin-were immortalized in vitro. Their general endothelial characteristics, such as the presence of von Willebrand factor (wWf), angiotensin-converting enzyme (ACE), VE-cadherin, and the intracellular E-selectin, were preserved.
5,6-Dimethylxanthenone-4-acetic acid, synthesised in this laboratory, reduces tumour blood flow, both in mice and in patients on Phase I trial. We used TUNEL (TdT-mediated dUTP nick end labelling) assays to investigate whether apoptosis induction was involved in its antivascular effect. 5,6-Dimethylxanthenone-4-acetic acid induced dose-dependent apoptosis in vitro in HECPP murine endothelial cells in the absence of up-regulation of mRNA for tumour necrosis factor. Selective apoptosis of endothelial cells was detected in vivo in sections of Colon 38 tumours in mice within 30 min of administration of 5,6-Dimethylxanthenone-4-acetic acid (25 mg kg(-1)). TUNEL staining intensified with time and after 3 h, necrosis of adjacent tumour tissue was observed.
The KDR/flk-1 gene promoter is considered to be endothelial cell-specific. We show its activity in two cancer cell lines of non-endothelial origin : in murine L1 sarcoma and OVP-10 human ovarian carcinoma cell lines. KDR promoter-driven cytosine deaminase gene can be efficiently expressed in these cells leading to sensitization to 5-fluorocytosine, as demonstrated both in vitro and in vivo. Our results indicated that KDR promoter activity is not endothelial cell-exclusive and that this promoter can also be used to obtain specific expression of therapeutic genes in certain cancer cells.
An analytical procedure has been developed for the analysis of intracellular didanosine triphosphate (ddATP). An electrospray ionization tandem mass spectrometer (ESI-MS) was interfaced to liquid chromatography (LC) using a mobile phase CH3OH/H2O (25/75) containing 1% formic acid for the analysis of the 5’-triphosphate metabolite of the antiviral didanosine. In this procedure. ddATP was extracted from CEM-T4 cells, isolated using an exchange anion solid phase extraction procedure, enzymatically dephosphorylated and then analyzed by LC-MS/MS within a I min run time.
A method employing capillary electrophoresis (CE) with tandem mass spectrometry (MS) has been developed for the simultaneous determination, on one hand, of zidovudine (AZT) with stavudine (d4T), and on the other hand, of lamivudine (3TC) with a didanosine metabolite (ddA), four potent human immunodeficiency virus reverse transcriptase (RT-HIV) inhibitors. The influence of several parameters (pH and ionic strength of volatile formic acid-ammonia buffer) as well as the influence of magnesium cation upon electroosmotic flow, electrophoretic mobility and peak efficiency has been studied. The limit of detection (LOD) by this method is 2.5 ppb for AZT and 20 ppb for d4T, 2 ppb for ddA and 5 ppb for 3TC, respectively. This paper illustrates the current importance in CE-ESI/MS/MS technique as a complementary or substituted method to measure levels (at ng/mL) of anti-HIV drugs alone or in combination.
Cyclophilin B (CyPB) is a cyclosporin A (CsA)binding protein mainly located in intracellular vesicles and secreted in biological fluids. In previous works, we demonstrated that CyPB interacts with T lymphocytes and enhances in vitro cellular incorporation and activity of CsA, In addition to its immunosuppressive activity, CsA is able to promote regeneration of damaged peripheral nerves. However, the crossing of the drug from plasma to neural tissue is restricted by the relative impermeability of the blood-brain barrier, To know whether CyPB might also participate in the delivery of CsA into the brain, we have analyzed the interactions of CyPB with brain capillary endothelial cells.
Endogenous lectins are cell receptors, expressed in normal and transformed cells both circulating as well as in organized tissues. Their biological significance was shown in developmentally regulated processes of cell migration, embryonic maturation, differentiation and during various other normal and pathological processes. This work will focus on the role of endogenous lectins and their glycoconjugate ligands in homing of circulating normal and cancer cells. During the normal immune process of lymphocyte recirculation and their journey among the whole body through the secondary lymphoid organs in the search for antigen, lectins are decisive molecules that allow the very first interaction of arrest onto the endothelial cell layer. It has been demonstrated that dual lectin-glycoconjugate interactions were taking part in the initiation of the whole adhesion cascade between adhering and endothelial cell.
Human keratinocytic cells from squamous carcinoma (SCL-1) present, under resting conditions, relatively low amounts of endogenous lectins (sugar-binding proteins). Upon uv irradiation, they express on their cell surface large amounts of endogenous lectin molecules able to bind neoglycoproteins bearing either a-L-rhamnosyl or a-D-glucosyl residues, A similar binding specificity was found with normal human keratinocytes under the same culture conditions. At sunlike doses, uv.A (365 nm) was more efficient than uv.B (312 nm) in the expression of such receptors on the surface of SCL-1 cells, The increased presentation of lectins by SCL-1 cells was transient and reached a maximum 4 h after irradiation.
Vascular endothelial cell addressins play an important role in lymphocyte homing in secondary lymphoid organs and in chronic inflammatory areas, A SV40 large T antigen-immortalized cell line from peripheral lymph nodes, HECa10 [Bizouarne et al., 1993a], was used to characterize the location of addressins with regard to environmental factors and cytokines. For this purpose, two monoclonal antibodies, MECA 79 and MECA 367, specific for peripheral lymph node vascular addressin and for mucosal addressin (Peyer’s patches), respectively, were bound to unstimulated HECa10 cells. Both mucosal and peripheral addressins were detected inside the cells and in cellular extracts of the resting cells. On the cell surface, both addressins could be evidenced on the same cells at a moderate level of expression.
Endothelial cells from mouse peripheral lymph nodes were immortalized by cationic liposome-mediated transfection using a plasmid construct containing both the gene coding for the large T antigen of simian virus 40 and a geneticin resistance gene suitable for selection. A cell line (HECa10) was isolated on the basis of its capacity to specifically bind fucoside carrying glycoconjugates ; these cells present the main characteristics of endothelial cells : production of angiotensin converting enzyme and of factor VIII-related antigen.
N-Acetylcysteine and captopril, respectively mucolytic and antihypertensive drugs, contain free sulfhydryl groups. Since in general thiols have well-established radioprotective abilities, we sought putative radioprotective effects of these drugs against therapeutic fast neutrons. We show that pBR322 plasmid DNA is indeed protected against radiolytic strand breakage by both drugs. The oxygen independent protection is consistent with a hydroxyl radical scavenging mechanism. A clonogenicity assay reveals an increase of the survival of SCL-1 cultured keratinocytes irradiated in the presence of the drugs compared with cells irradiated without drugs. Our results suggest possible interferences between treatment with drugs bearing-SH groups and radiotherapy.
The culture of specialized high endothelial cells (HEC) from lymphoid organs (peripheral lymph nodes (PLN) and Peyer’s patches (PP)) was undertaken in order to study and characterize the cell surface molecules which are involved in lymphocyte recognition and allow homing. Cells were stimulated in vivo by a graft versus host (GVH) type of reaction before isolation and culture. The resulting adherent and growing cells were characterized as endothelial cells because of their typical aspect and their ability to produce angiotensin-converting enzyme and factor VIII-related antigen. They possess tissue-specific endothelial addressins. MECA 79 antigen is present on cells isolated from PLN while MECA 367 antigen is detected on cells from PP.
IgD receptor (IgD-R) bearing CD4+ T cells with immunoaugmenting properties in vivo are induced in mice within 24 h after a single injection of dimeric or aggregated IgD. In the present study, we sought to identify the region(s) of IgD responsible for upregulation of IgD-R and for the immunoaugmenting effect of IgD. IgD-R can be upregulated on CD4+ T cells in vitro and in vivo by glutaraldehyde-aggregated mutant IgD or by fragments of enzymatically digested IgD molecules possessing either the C(d)1 domain (Fd(d)) or the C(d)3 domain (Fc(d)). Neoglycoproteins (D-galactose - BSA and N-acetyl-D-glucosamine - BSA), can competitively block upregulation of IgD-R by IgD in vitro. Furthermore, when injected 1 day before antigen, the aggregated IgD derived molecules, KWD1 (which lacks C(d)1), KWD6 (which lacks C(d)1 plus C(d)-hinge), and Fab(d) can all cause augmentation of antigen-specific primary and secondary antibody responses comparable to that achieved with intact aggregated IgD.
Suppression of the immune response, which involves suppressor factors released from specialized T cells, is inhibited by a-L-rhamnose. In this paper, we show the presence of rhamnose-specific receptors on a human CD8+ T cell-rich population and describe a novel method to isolate cells which express a given sugar-binding protein on their surface. We describe the isolation of a-L-rhamnose-specific molecules (rhamnose-binding fractions : RBF) from a water-soluble extract from lymphocytes, their purification by affinity chromatography on immobilized neoglycoproteins containing rhamnose residues. RBF kept their ability to bind rhamnose, as shown by the binding of fluorescein-labeled RBF to rhamnosylated BSA-substituted beads. RBF efficiently suppresses DNA synthesis of mitogen-stimulated human lymphocytes as well as B cell immunoglobulin production. Therefore, these rhamnose-binding molecules appear to be antigen-independent suppressor factors.
In an attempt to identify cell surface molecules involved in recognition phenomena between cells such as keratinocytes and melanocytes and putatively target biological responses modifiers to keratinocytes, we undertook the detection of cell surface sugar specific receptors : membrane lectins. Keratinocyte membrane lectins were found to bind synthetic glycoproteins (neoglycoproteins) carrying either a-L-fucosyl or a-L-rhamnosyl residues. Fluorescence microscopy observations indicate that cultured keratinocytes are able to bind these two neoglycoproteins while frozen sections of human skin labelled with neoglycoprotein-coated covaspheres show that the selectivity of the binding to keratinocytes is restricted to a-L-rhamnosyl-BSA.
Surface lectins, specific for given sugar structures, are expressed on human T cells, as shown by flow cytofluorometry using F-neoglycoproteins bearing either ß- and a-D-galactosyl, ß-D-galactosyl 6-phosphate, or a-L-rhamnosyl groups, but not by F-neoglycoproteins bearing other sugar groups (such as a-D-mannosyl groups). After stimulation with Phaseolus vulgaris mitogen, the number of cells that bind ß-D-galactosyl 6-phosphate groups (6-P-ß-D-Galp lec+ cells) increased fourfold during the first five days ; these cells are helper (CD4+) T cells. Conversely, cells that bind a-L-Rha groups belong to the T suppressor (CD8+) family and their number moderately increased. Upon stimulation by concanavalin A, the number of cells expressing the lectin recognizing a-L-Rha groups increased during the first two days and then decreased within the next two days. These results are discussed with regard to the implication of lymphocyte membrane lectins in the suppressor mechanism and in the homing process.
This paper presents the characterization of a sugar-specific receptor on the surface of human circulating polymorphonuclear cells. With the help of fluorescent neoglycoproteins and flow cytometry, a receptor was identified as being specific for .a.-L-rhamnosyl residues. The number of receptors was 55 000/cell and their affinity reached 2 .times. 108 1 mol-1. This number changes as a function of the biological state of the cells. Indeed, receptor expression was modulated by the presence of other cells. T cells and B cells increased the number of receptors on the granulocyte surface. Expression of the .a.-L-rhamnose-specific lectin was dependent on lymphocyte derived soluble factor(s), which induce(s) growth and differentiation of polymorphonuclear phagocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) specifically produced a significant increase in the number of receptors for .a.-L-rhamnose (2-10-fold/cell). This modulation was independent of protein kinase C activators such as phorbol ester, which produced no effect on .a.-L-rhamnose receptor expression. These findings demonstrate that GM-CSF may stimulate post differentiation functions and properties of mature granulocytes.
Responsable d’équipe , Directeur de recherche , Microenvironnement cellulaire et cibles pharmacologiques