Physiological skin oxygen levels: An important criterion for skin cell functionality and therapeutic approaches.

The skin is made up of different layers with various gradients, which maintain a complex microenvironment, particularly in terms of oxygen levels. However, all types of skin cells are cultured in conventional incubators that do not reproduce physiological oxygen levels. Instead, they are cultured at atmospheric oxygen levels, a condition that is far removed from physiology and may lead to the generation of free radicals known to induce skin ageing. This review aims to summarize the current literature on the effect of physiological oxygen levels on skin cells, highlight the shortcomings of current in vitro models, and demonstrate the importance of respecting skin oxygen levels. We begin by clarifying the terminology used about oxygen levels and describe the specific distribution of oxygen in the skin. We review and discuss how skin cells adapt their oxygen consumption and metabolism to oxygen levels environment, as well as the changes that are induced, particularly, their redox state, life cycle and functions. We examine the effects of oxygen on both simple culture models and more complex reconstructed skin models. Finally, we present the implications of oxygen modulation for a more therapeutic approach.

Improvement of Antioxidant Defences in Keratinocytes Grown in Physioxia: Comparison of 2D and 3D Models.

Keratinocytes prevent skin photoaging by ensuring the defence against oxidative stress, an excessive production of reactive oxygen species (ROS). They are localized within the epidermis where the oxygen level (1-3% O2), named physioxia, is low compared to other organs. Oxygen is essential for life but also generates ROS. Most of the in vitro studies on keratinocyte antioxidant capacities are performed under atmospheric oxygen, named normoxia, which is very far from the physiological microenvironment, thus submitting cells to an overoxygenation. The present study is aimed at investigating the antioxidant status of keratinocyte grown under physioxia in both 2D and 3D models. First, we show that the basal antioxidant profiles of keratinocytes display important differences when comparing the HaCaT cell line, primary keratinocytes (NHEK), reconstructed epidermis (RHE), and skin explants. Physioxia was shown to promote a strong proliferation of keratinocytes in monolayers and in RHE, resulting in a thinner epidermis likely due to a slowdown in cell differentiation. Interestingly, cells in physioxia exhibited a lower ROS production upon stress, suggesting a better protection against oxidative stress. To understand this effect, we studied the antioxidant enzymes and reported a lower or equivalent level of mRNA for all enzymes in physioxia conditions compared to normoxia, but a higher activity for catalase and superoxide dismutases, whatever the culture model. The unchanged catalase amount, in NHEK and RHE, suggests an overactivation of the enzyme in physioxia, whereas the higher amount of SOD2 can explain the strong activity. Taken together, our results demonstrate the role of oxygen in the regulation of the antioxidant defences in keratinocytes, topic of particular importance for studying skin aging. Additionally, the present work points out the interest of the choice of both the keratinocyte culture model and the oxygen level to be as close as possible to the in situ skin.

Tumour angiogenesis normalized by myo-inositol trispyrophosphate alleviates hypoxia in the microenvironment and promotes antitumor immune response.

Pathologic angiogenesis directly responds to tumour hypoxia and controls the molecular/cellular composition of the tumour microenvironment, increasing both immune tolerance and stromal cooperation with tumour growth. Myo-inositol-trispyrophosphate (ITPP) provides a means to achieve stable normalization of angiogenesis. ITPP increases intratumour oxygen tension (pO2 ) and stabilizes vessel normalization through activation of endothelial Phosphatase-and-Tensin-homologue (PTEN). Here, we show that the tumour reduction due to the ITPP-induced modification of the tumour microenvironment by elevating pO2 affects the phenotype and properties of the immune infiltrate. Our main observations are as follows: a relative change in the M1 and M2 macrophage-type proportions, increased proportions of NK and CD8+ T cells, and a reduction in Tregs and Th2 cells. We also found, in vivo and in vitro, that the impaired access of PD1+ NK cells to tumour cells is due to their adhesion to PD-L1+ /PD-L2+ endothelial cells in hypoxia. ITPP treatment strongly reduced PD-L1/PD-L2 expression on CD45+/CD31+ cells, and PD1+ cells were more numerous in the tumour mass. CTLA-4+ cell numbers were stable, but level of expression decreased. Similarly, CD47+ cells and expression were reduced. Consequently, angiogenesis normalization induced by ITPP is the mean to revert immunosuppression into an antitumor immune response. This brings a key adjuvant effect to improve the efficacy of chemo/radio/immunotherapeutic strategies for cancer treatment.

The GTPase-activating protein-related domain of neurofibromin interacts with MC1R and regulates pigmentation-mediated signaling in human melanocytes.

The melanocortin 1 receptor (MC1R) is a G-protein coupled receptor (GPCR) which plays a major role in controlling melanogenesis. A large body of evidence indicates that GPCRs are part of large protein complexes that are critical for their signal transduction properties. Among proteins which may affect MC1R signaling, neurofibromin (Nf1), a GTPase activating protein (GAP) for Ras, is of special interest as it regulates adenylyl cyclase activity and ERK signaling, two pathways involved in MC1R signaling. Moreover, mutations in this gene encoding Nf1 are responsible for neurofibromatosis type I, a disease inducing hyperpigmented flat skin lesions. Using co-immunoprecipitation and Bioluminescence Resonance Energy Transfer experiments we demonstrated a physical interaction of Nf1 with MC1R. In particular, the GAP domain of Nf1 directly and constitutively interacts with MC1R in melanocytes. Pharmacologic and genetic approaches revealed that the GAP activity of Nf1 is important to regulate intracellular signaling pathways involved in melanogenesis and, consequently, melanogenic enzyme expression and melanin production. These finding shed new light on the understanding and cure of skin pigmentation disorders.

Deleterious Effects of an Air Pollutant (NO2) on a Selection of Commensal Skin Bacterial Strains, Potential Contributor to Dysbiosis?

The skin constitutes with its microbiota the first line of body defense against exogenous stress including air pollution. Especially in urban or sub-urban areas, it is continuously exposed to many environmental pollutants including gaseous nitrogen dioxide (gNO2). Nowadays, it is well established that air pollution has major effects on the human skin, inducing various diseases often associated with microbial dysbiosis. However, very few is known about the impact of pollutants on skin microbiota. In this study, a new approach was adopted, by considering the alteration of the cutaneous microbiota by air pollutants as an indirect action of the harmful molecules on the skin. The effects of gNO2 on this bacterial skin microbiota was investigated using a device developed to mimic the real-life contact of the gNO2 with bacteria on the surface of the skin. Five strains of human skin commensal bacteria were considered, namely Staphylococcus aureus MFP03, Staphylococcus epidermidis MFP04, Staphylococcus capitis MFP08, Pseudomonas fluorescens MFP05, and Corynebacterium tuberculostearicum CIP102622. Bacteria were exposed to high concentration of gNO2 (10 or 80 ppm) over a short period of 2 h inside the gas exposure device. The physiological, morphological, and molecular responses of the bacteria after the gas exposure were assessed and compared between the different strains and the two gNO2 concentrations. A highly significant deleterious effect of gNO2 was highlighted, particularly for S. capitis MFP08 and C. tuberculostearicum CIP102622, while S. aureus MFP03 seems to be the less sensitive strain. It appeared that the impact of this nitrosative stress differs according to the bacterial species and the gNO2 concentration. Thus the exposition to gNO2 as an air pollutant could contribute to dysbiosis, which would affect skin homeostasis. The response of the microbiota to the nitrosative stress could be involved in some pathologies such as atopic dermatitis.

Soluble Guanylate Cyclase Inhibitors Discovered among Natural Compounds.

Soluble guanylate cyclase (sGC) is the human receptor of nitric oxide (NO) in numerous kinds of cells and produces the second messenger 3',5'-cyclic guanosine monophosphate (cGMP) upon NO binding to its heme. sGC is involved in many cell signaling pathways both under healthy conditions and under pathological conditions, such as angiogenesis associated with tumor growth. Addressing the selective inhibition of the NO/cGMP pathway is a strategy worthwhile to be investigated for slowing down tumoral angiogenesis or for curing vasoplegia. However, sGC inhibitors are lacking investigation. We have explored a chemical library of various natural compounds and have discovered inhibitors of sGC. The selected compounds were evaluated for their inhibition of purified sGC in vitro and sGC in endothelial cells. Six natural compounds, from various organisms, have IC50 in the range 0.2-1.5 µM for inhibiting the NO-activated synthesis of cGMP by sGC, and selected compounds exhibit a quantified antiangiogenic activity using an endothelial cell line. These sGC inhibitors can be used directly as tools to investigate angiogenesis and cell signaling or as templates for drug design.

The emerging potential of cold atmospheric plasma in skin biology.

The maintenance of skin integrity is crucial to ensure the physiological barrier against exogenous compounds, microorganisms and dehydration but also to fulfill social and aesthetic purposes. Besides the development of new actives intended to enter a formulation, innovative technologies based on physical principles have been proposed in the last years. Among them, Cold Atmospheric Plasma (CAP) technology, which already showed interesting results in dermatology, is currently being studied for its potential in skin treatments and cares. CAP bio-medical studies gather several different expertise ranging from physics to biology through chemistry and biochemistry, making this topic hard to pin. In this review we provide a broad survey of the interactions between CAP and skin. In the first section, we tried to give some fundamentals on skin structure and physiology, related to its essential functions, together with the main bases on cold plasma and its physicochemical properties. In the following parts we dissected and analyzed each CAP parameter to highlight the already known and the possible effects they can play on skin. This overview aims to get an idea of the potential of cold atmospheric plasma technology in skin biology for the future developments of dermo-cosmetic treatments, for example in aging prevention.

LINGO family receptors are differentially expressed in the mouse brain and form native multimeric complexes.

Leucine-rich repeat and immunoglobin-domain containing (LRRIG) proteins that are commonly involved in protein-protein interactions play important roles in nervous system development and maintenance. LINGO-1, one of this family members, is characterized as a negative regulator of neuronal survival, axonal regeneration, and oligodendrocyte precursor cell (OPC) differentiation into mature myelinating oligodendrocytes. Three LINGO-1 homologs named LINGO-2, LINGO-3, and LINGO-4 have been described. However, their relative expression and functions remain unexplored. Here, we show by in situ hybridization and quantitative polymerase chain reaction that the transcripts of LINGO homologs are differentially expressed in the central nervous system. The immunostaining of brain slices confirmed this observation and showed the co-expression of LINGO-1 with its homologs. Using BRET (bioluminescence resonance energy transfer) analysis, we demonstrate that LINGO proteins can physically interact with each of the other ones with comparable affinities and thus form the oligomeric states. Furthermore, co-immunoprecipitation experiments indicate that LINGO proteins form heterocomplexes in both heterologous systems and cortical neurons. Since LINGO-1 is a promising target for the treatment of demyelinating diseases, its ability to form heteromeric complexes reveals a new level of complexity in its functioning and opens the way for new strategies to achieve diverse and nuanced LINGO-1 regulation.

Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections.

Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

Activity of the human immortalized endothelial progenitor cell line HEPC-CB.1 supporting in vitro angiogenesis.

The human HEPC-CB.1 cell line with many characteristics of endothelial progenitor cells (EPC) was tested for its proangiogenic properties as a potentially therapeutic compound. HEPC-CB.1 cells' potential to differentiate into endothelial cells was revealed after treating the cells with a mixture of ATRA, cAMP and VEGF, as shown by the reduced expression levels of CD133, CD271 and CD90 antigens, augmentation of CD146 and CD31, and a decrease in cell clonogenicity. The cooperation of HEPC-CB.1 with the endothelial cell line HSkMEC.2 resulted in the formation of a common network. Tube formation was significantly more effective when resulting from HEPC-CB.1 and HSkMEC.2 cell co-culture as compared to a monoculture of each cell line. The exocrine mechanism of HEPC-CB.1 and HSkMEC.2 cross talk by secreted factors was evidenced using the HEPC-CB.1 supernatant to increase the efficacy of HSkMEC.2 tube formation. The proangiogenic factors produced by HEPC-CB.1 were identified using cytokine antibody array. Out of 120 examined factors, the HEPC-CB.1 cell line produced 63, some with known angiogenic activity. As in vivo the angiogenic process occurs at low oxygen tension, it was observed that in hypoxia, the production of defined factors was augmented. The presented results demonstrate that HEPC-CB.1 cells are able to both cooperate and integrate in a newly formed network and produce factors that help the network formation. The results suggest that HEPC-CB.1 cells are indeed endothelial progenitors and may prove to be an effective tool in regenerative medicine.

The Immunomodulatory Effect of IrSPI, a Tick Salivary Gland Serine Protease Inhibitor Involved in Ixodes ricinus Tick Feeding.

Ticks are the most important vectors of pathogens affecting both domestic and wild animals worldwide. Hard tick feeding is a slow process-taking up to several days-and necessitates extended control over the host response. The success of the feeding process depends upon injection of tick saliva, which not only controls host hemostasis and wound healing, but also subverts the host immune response to avoid tick rejection that creates a favorable niche for the survival and propagation of diverse tick-borne pathogens. Here, we report on the molecular and biochemical features and functions of an Ixodes ricinus serine protease inhibitor (IrSPI). We characterize IrSPI as a Kunitz elastase inhibitor that is overexpressed in several tick organs-especially salivary glands-during blood-feeding. We also demonstrated that when IrSPI is injected into the host through saliva, it had no impact on tissue factor pathway-induced coagulation, fibrinolysis, endothelial cell angiogenesis or apoptosis, but the protein exhibits immunomodulatory activity. In particular, IrSPI represses proliferation of CD4+ T lymphocytes and proinflammatory cytokine secretion from both splenocytes and macrophages. Our study contributes valuable knowledge to tick-host interactions and provides insights that could be further exploited to design anti-tick vaccines targeting this immunomodulator implicated in I. ricinus feeding.

Developing a circularly permuted variant of Renilla luciferase as a bioluminescent sensor for measuring Caspase-9 activity in the cell-free and cell-based systems.

Biosensors and whole cell biosensors consisting of biological molecules and living cells can sense a special stimulus on a living system and convert it to a measurable signal. A major group of them are the bioluminescent sensors derived from luciferases. This type of biosensors has a broad application in molecular biology and imaging systems. In this project, a luciferase-based biosensor for detecting and measuring caspase-9 activity is designed and constructed using the circular permutation strategy. The spectroscopic method results reveal changes in the biosensor structure. Additionally, its activity is examined in a cell-free coupled assay system. Afterward, the biosensor is utilized for measuring the cellular caspase-9 activity upon apoptosis induction in a cancer cell line. In following the gene of biosensor is sub-cloned into a eukaryotic vector and transfected to HEK293T cell line and then its activity is measured upon apoptosis induction in the presence and absence of a caspase-9 inhibitor. The obtained results show that the designed biosensor detects the caspase-9 activity in the cell-free and cell-based systems.

Psoriasis Treatment Changes the Expression Profile of Selected Caspases and their Regulatory MicroRNAs.

BACKGROUND/AIMS: Psoriasis, an autoimmune diseases of the skin, characterized by patches of abnormal/inflammed skin, although not usually life-threatening, it causes severe discomfort, esthetic impairments, and may lead to impaired social functions and social withdrawal. Besides UV-phototherapy, various anti-inflammatory treatments are applied, depending on the severity of symptoms. In 2008, adalimumab (fully humanized human anti-TNF antibody) was launched for the treatment of psoriasis. In the quest to better understand the pathomechanism of adalimumab's therapeutic effects, and the acquired resistance to the drug, we have investigated how its administration affect the regulation of the expression of selected caspases, including those activated by inflammosome. METHODS: The research was initially carried out on normal human dermal fibroblasts (NHDF) treated with adalimumab for 2, 8 and 24 hours in vitro. Then, expression profile of genes encoding caspases and their regulatory micro-RNAs was determined with the use of oligonucleotide microarray. The validation of the microarray results was carried out by qRT-PCR. The in vitro study was followed by ex-vivo investigation of adalimumab's effects on the expression of caspase-6 in blood of the psoriatic patients. The samples were collected before, and 2 hours after adalimumab's administration and the analysis was determined by qRT-PCR. RESULTS: The result of the analysis indicated that introduction of adalimumab to the NHDF culture resulted in the change of the transcription activity of genes encoding caspases and genes encoding miRNAs. The analysis revealed 5 different miRNA molecules regulating the expression of: CASP2, CASP3 and CASP6. There were no statistically significant differences in the expression of gene encoding caspase-6 in the patients' blood before and 2 hours after the anti-TNF drug administration. CONCLUSION: We have found that adalimumab administration affects caspases expression, thus they may be used as molecular markers for monitoring the therapy with the use of an anti-TNF drugs, including adalimumab. It is likely that the mechanisms responsible for changed expression profiles of genes encoding caspase-2,-3, and -6, may be caused by the upregulation of the respective microRNA molecules. Increased expression of genes encoding specific caspases may induce inflammatory processes, as well as trigger apoptosis. Furthermore, the proapoptotic activity of caspases may be enhanced by miRNA molecules, which exhibit proapoptotic function. The overexpression of such miRNAs was observed in our study.

Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent.

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a MatrigelTM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

Tumor hypoxia modulates podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization.

Podoplanin (PDPN), an O-glycosylated, transmembrane, mucin-type glycoprotein, is expressed by cancer associated fibroblasts (CAFs). In malignant transformation, PDPN is subjected to changes and its role is yet to be established. Here we show that it is involved in modulating the activity of the CCL21/CCR7 chemokine/receptor axis in a hypoxia-dependent manner. In the present model, breast cancer MDA-MB-231 cells and NKL3 cells express the surface CCR7 receptor for CCL21 chemokine which is a potent chemoattractant able to bind to PDPN. The impact of the CCL21/CCR7 axis in the molecular mechanism of the adhesion of NKL3 cells and of MDA-MB-231 breast cancer cells was reduced in a hypoxic tumor environment. In addition to its known effect on migration, CCL21/CCR7 interaction was shown to allow NK cell adhesion to endothelial cells (ECs) and its reduction by hypoxia. A PDPN expressing model of CAFs made it possible to demonstrate the same CCL21/CCR7 axis involvement in the tumor cells to CAFs recognition mechanism through PDPN binding of CCL21. PDPN was induced by hypoxia and its overexpression undergoes a reduction of adhesion, making it an anti-adhesion molecule in the absence of CCL21, in the tumor. CCL21/CCR7 modulated NK cells/ECs and MDA-MB-231 cells/CAF PDPN-dependent interactions were further shown to be linked to hypoxia-dependent microRNAs as miRs: miR-210 and specifically miR-21, miR-29b which influence PDPN expression.

A 3D model of tumour angiogenic microenvironment to monitor hypoxia effects on cell interactions and cancer stem cell selection.

Tumour microenvironment determines the fate of treatments. Reconstitution of tumour conditions is mandatory for alternative in vitro methods devoted to cancer development and the selection of therapeutic strategies. This work describes a 3D model of melanoma growth in its environment. Introducing means to mimic tumour angiogenesis, which turns on tumour progression, the model shows that melanoma tumour spheroids allow reconstitution of solid tumours with stromal cells. Angiogenesis evidenced the differential recruitment of endothelial cells (EC) from early progenitors (EEPCs) to mature ECs. Hypoxia was the key parameter that selected and stabilized melanoma cancer stem like cells (CSCs) phenotype based on aldehyde dehydrogenase expression as the best criterion. The 3D-tumour-model demonstrated the distinct reactivity of ECs toward tumour cells in terms of cellular cross-talk and humoral response. Intra-spheroid cell-to-cell membrane dye exchanges, mediated by intercellular interactions, uncovered the melanoma-to-EEPC cooperation. The resulting changes in tumour milieu were evidenced by the chemokinic composition and hypoxia-related variations in microRNA expression assessed in each cellular component of the spheroids. This method brings new tools to decipher the molecular mechanism of tumour-mediated cell recruitment and for in vitro assessment of therapeutic approaches.

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation.

Active cellular transporters of harmful agents-multidrug resistance (mdr) proteins-are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins-MDR1, BCRP, MRP1, MRP4 and MRP5-in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.

Endothelial precursor cell-based therapy to target the pathologic angiogenesis and compensate tumor hypoxia.

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.

Hypoxia-regulated overexpression of soluble VEGFR2 controls angiogenesis and inhibits tumor growth.

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.

MicroRNAs and tumor vasculature normalization: impact on anti-tumor immune response.

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.