Catherine Grillon

Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia

•  Introduction ‐  Imaging of hypoxic areas ‐  Hypoxia markers ‐  Positron emission tomography (PET) ‐  Near‐infrared spectroscopy (NIRS) ‐  Magnetic resonance spectroscopy (MRS) ‐  Electron paramagnetic resonance spectroscopy (EPR) •  Oxygen partial pressure measurement ‐  Polarographic sensor ‐  Optical sensor ‐  Mass spectrometry •  What does tumour hypoxia mean? •  Small molecules and hypoxia ‐  Chemotherapeutic drugs ‐  Radiation sensitizers: the nitroimidazoles ‐  Hypoxia prodrugs: tirapazimine and anthraquinone ‐  pO2 modulator: myo‐inositol trispyrophosphate (ITPP) ‐  Hypoxia mimetics: dimethyloxallyl glycine, desferrioxamine and metal ions •  What does physioxia mean? ‐  From air to blood ‐  In brain ‐  In lungs ‐  In skin ‐  In intestinal tissue ‐  In liver ‐  In kidney ‐  In muscle ‐  In bone marrow ‐  In umbilical cord blood ‐  To summarize physioxia •  Cellular and molecular consequences of physioxia versus normoxia and hypoxia ‐  Hypoxia inducible factors actions ‐  Role of microRNAs in hypoxia‐dependent regulations ‐  Cell adhesion molecules: their regulation by oxygen partial pressure ‐  Soluble molecules: example of angiogenin •  Conclusion

Nitric oxide modulates the expression of endothelial cell adhesion molecules involved in angiogenesis and leukocyte recruitment.

Tumor angiogenesis and immune response have in common to be cell recognition mechanisms, which are based on specific adhesion molecules and dependent on nitric oxide (NO(•)). The aim of the present study is to deepen the mechanisms of angiogenesis and inflammation regulation by NO(•) to find out the molecular regulation processes that govern endothelial cell permeability and leukocyte transmigration. Effects of NO(•), either exogenous or produced in hypoxic conditions, were studied on microvascular endothelial cells from skin and lymph node because of their strong involvement in melanoma progression. We found that NO(•) down-regulation of pseudo-vessel formation was linked to a decrease in endothelial cell ability to adhere to each other which can be explain, in part, by the inhibition of PECAM-1/CD31 expression. On the other hand, NO(•) was shown to be able to decrease leukocyte adhesion on an endothelial monolayer, performed either in static or in rolling conditions, and to modulate differentially CD34, ICAM-1/CD54, ICAM-2/CD102 and VCAM-1/CD106 expression. In conclusion, during angiogenesis and leukocyte recruitment, NO(•) regulates cell interactions by controlling adhesion molecule expression and subsequently cell adhesion. Moreover, each endothelial cell type presents its own organospecific response to NO(•), reflecting the functions of the tissue they originate from.

Stable tumor vessel normalization with pO2 increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment

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.

Human keratinocyte membrane lectins: characterization and modulation of their expression by cytokines

Summary— 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 α‐l‐fucosyl or α‐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 α‐l‐rhamnosyl‐BSA. Keratinocytes were adapted to grow on collagen; harvesting conditions allowing the analysis of keratinocytes by flow cytometry are described. This technique allows the quantification of the binding at 4°C, and the estimation of the endocytosis of F‐, neoglycoproteins: F‐, α‐l‐Rha‐BSA and F‐, α‐l‐Fuc‐BSA were efficiently internalized. Thereafter, α‐l‐rhamnose‐substituted liposomes containing 5‐(6)carboxyfluorescein were prepared in order to follow the delivery of the fluorescent dye into cells. This was measured both by flow cytometry and by spectrofluorimetry. The expression of surface lectins was checked upon action of cytokines (IL1α, IL1β, IL2 and TNF) which are known as biological response modifiers of keratinocytes.

CD133 positive progenitor endothelial cell lines from human cord blood

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.

Hypoxia control to normalize pathologic angiogenesis: Potential role for endothelial precursor cells and miRNAs regulation

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.

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. Mol Cancer Ther; 13(1); 165–78. ©2013 AACR.

Trojan horse at cellular level for tumor gene therapies.

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 gene-therapy 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.

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.

In vitro effect of acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) analogs resistant to angiotensin I-converting enzyme on hematopoietic stem cell and progenitor cell proliferation.

The tetrapeptide Acetyl‐N‐Ser‐Asp‐Lys‐Pro (AcSDKP), an inhibitor of hematopoietic stem cell proliferation, is known to reduce in vivo the damage resulting from treatment with chemotherapeutic agents or ionizing radiation on the stem cell compartment. Recently, AcSDKP has been shown to be a physiological substrate of the N‐active site of angiotensin I‐converting enzyme (ACE). Four analogs of the tetrapeptide expressing a high stability towards ACE degradation in vitro have been synthesized in order to provide new molecules likely to improve the myeloprotection displayed by AcSDKP. These analogs are three pseudopeptides with a modified peptidic bond, Ac‐SerΨ(CH2‐NH)Asp‐Lys‐Pro, Ac‐Ser‐AspΨ(CH2‐NH)Lys‐Pro, Ac‐Ser‐Asp‐LysΨ(CH2‐N)Pro, and one C‐terminus modified peptide (AcSDKP‐NH2). We report here that these analogs reduce in vitro the proportion of murine colony‐forming units‐granulocyte/macrophage in S‐phase and inhibit the entry into cycle of high proliferative potential colony‐forming cells. The efficacy of AcSDKP analogs in preventing in vitro primitive hematopoietic stem cells from entering into cycle suggests that these molecules could be new candidates for the powerful inhibition of hematopoietic stem and progenitor cell proliferation in vivo.