Ines Martin-Padura

Deletion of the p66Shc longevity gene reduces systemic and tissue oxidative stress, vascular cell apoptosis, and early atherogenesis in mice fed a high-fat diet

Several experimental and clinical studies have shown that oxidized low-density lipoprotein and oxidation-sensitive mechanisms are central in the pathogenesis of vascular dysfunction and atherogenesis. Here, we have used p66Shc−/− and WT mice to investigate the effects of high-fat diet on both systemic and tissue oxidative stress and the development of early vascular lesions. To date, the p66Shc−/− mouse is the unique genetic model of increased resistance to oxidative stress and prolonged life span in mammals. Computer-assisted image analysis revealed that chronic 21% high-fat treatment increased the aortic cumulative early lesion area by ≈21% in WT mice and only by 3% in p66Shc−/− mice. Early lesions from p66Shc−/− mice had less content of macrophage-derived foam cells and apoptotic vascular cells, in comparison to the WT. Furthermore, in p66Shc−/− mice, but not WT mice, we found a significant reduction of systemic and tissue oxidative stress (assessed by isoprostanes, plasma low-density lipoprotein oxidizability, and the formation of arterial oxidation-specific epitopes). These results support the concept that p66Shc−/− may play a pivotal role in controlling systemic oxidative stress and vascular diseases. Therefore, p66Shc might represent a molecular target for therapies against vascular diseases.

Deletion of p66shc Gene Protects Against Age-Related Endothelial Dysfunction

Background—Enhanced production of reactive oxygen species (ROS) has been recognized as the major determinant of age-related endothelial dysfunction. The p66shc protein controls cellular responses to oxidative stress. Mice lacking p66shc (p66shc−/−) have increased resistance to ROS and a 30% prolonged life span. The present study investigates age-dependent changes of endothelial function in this model. Methods and Results—Aortic rings from young and old p66shc−/− or wild-type (WT) mice were suspended for isometric tension recording. Nitric oxide (NO) release was measured by a porphyrinic microsensor. Expression of endothelial NO synthase (eNOS), inducible NOS (iNOS), superoxide dismutase, and nitrotyrosine-containing proteins was assessed by Western blotting. Nitrotyrosine residues were also identified by immunohistochemistry. Superoxide (O2−) production was determined by coelenterazine-enhanced chemiluminescence. Endothelium-dependent relaxation in response to acetylcholine was age-dependently impaired in WT mice but not in p66shc−/− mice. Accordingly, an age-related decline of NO release was found in WT but not in p66shc−/− mice. The expression of eNOS and manganese superoxide dismutase was not affected by aging either in WT or in p66shc−/− mice, whereas iNOS was upregulated only in old WT mice. It is interesting that old WT mice displayed a significant increase of O2− production as well as of nitrotyrosine expression compared with young animals. Such age-dependent changes were not found in p66shc−/− mice. Conclusions—We report that inactivation of the p66shc gene protects against age-dependent, ROS-mediated endothelial dysfunction. These findings suggest that the p66shc is part of a signal transduction pathway also relevant to endothelial integrity and may represent a novel target to prevent vascular aging.

Genetic deletion of p66Shc adaptor protein prevents hyperglycemia-induced endothelial dysfunction and oxidative stress

Increased production of reactive oxygen species (ROS) and loss of endothelial NO bioavailability are key features of vascular disease in diabetes mellitus. The p66Shc adaptor protein controls cellular responses to oxidative stress. Mice lacking p66Shc (p66Shc−/−) have increased resistance to ROS and prolonged life span. The present work was designed to investigate hyperglycemia-associated changes in endothelial function in a model of insulin-dependent diabetes mellitus p66Shc−/− mouse. p66Shc−/− and wild-type (WT) mice were injected with citrate buffer (control) or made diabetic by an i.p. injection of 200 mg of streptozotocin per kg of body weight. Streptozotocin-treated p66Shc−/− and WT mice showed a similar increase in blood glucose. However, significant differences arose with respect to endothelial dysfunction and oxidative stress. WT diabetic mice displayed marked impairment of endothelium-dependent relaxations, increased peroxynitrite (ONOO−) generation, nitrotyrosine expression, and lipid peroxidation as measured in the aortic tissue. In contrast, p66Shc−/− diabetic mice did not develop these high-glucose-mediated abnormalities. Furthermore, protein expression of the antioxidant enzyme heme oxygenase 1 and endothelial NO synthase were up-regulated in p66Shc−/− but not in WT mice. We report that p66Shc−/− mice are resistant to hyperglycemia-induced, ROS-dependent endothelial dysfunction. These data suggest that p66Shc adaptor protein is part of a signal transduction pathway relevant to hyperglycemia vascular damage and, hence, may represent a novel therapeutic target against diabetic vascular complications.

INHIBITION OF CULTURED CELL GROWTH BY VASCULAR ENDOTHELIAL CADHERIN (CADHERIN-5/VE-CADHERIN)

Endothelial cell proliferation is inhibited by the establishment of cell to cell contacts. Adhesive molecules at junctions could therefore play a role in transferring negative growth signals. The transmembrane protein VE-cadherin (vascular endothelial cadherin/cadherin-S) is selectively expressed at intercellular clefts in the endothelium. The intracellular domain interacts with cytoplasmic proteins called catenins that transmit the adhesion signal and contribute to the anchorage of the protein to the actin cytoskeleton. Transfection of VE-cadherin in both Chinese hamster ovary (CHO) and L929 cells confers inhibition of cell growth. Truncation of VE-cadherin cytoplasmic region, responsible for linking catenins, does not affect VE-cadherin adhesive properties but abolishes its effect on cell growth. Seeding human umbilical vein endothelial cells or VE-cadherin transfectants on a recombinant VE-cadherin amino-terminal fragment inhibited their proliferation. These data show that VE-cadherin homotypic engagement at junctions participates in density dependent inhibition of cell growth. This effect requires both the extracellular adhesive domain and the intracellular catenin binding region of the molecule.

Human acute leukemia cells injected in NOD/LtSz-scid/IL-2Rγ null mice generate a faster and more efficient disease compared to other NOD/scid-related strains

Transplantation of human acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) primary cells and cell lines in different strains of immunodeficient mice has led to preclinical models extensively used to investigate acute leukemia stem cells, biology and drug sensitivity. We studied the engraftment kinetics of AML and ALL cell lines and primary cells in 3 strains of NOD.CB17‐Prkdcscid (NOD/scid, NS)‐related mice (NOD.Cg‐PrkdcscidB2mtm1Unc/J, abbreviated NOD/scid/β2 null, NSB; and NOD.Cg‐PrkdcscidIl2rgtm1Wjll/SzJ, abbreviated NOD/scid/IL‐2Rγ null, NSG). The engraftment of human malignant cells was investigated by means of clinicopathological criteria, flow cytometry, PCR and immunohistochemistry. In NSG mice, we observed a significantly faster development of leukemia‐related symptoms and a higher percentage of leukemia cells in the blood, in the marrow and in the spleen. The leukemia‐related angiogenic switch (measured as the number of circulating endothelial cells and progenitors) was faster in NSG compared to NS and NSB mice. These models will be instrumental to studies on leukemia‐initiating stem cells, leukemia biology, preclinical treatment studies, and to obtain patient‐specific preclinical models to design and investigate patient‐tailored therapies.

Increased DC trafficking to lymph nodes and contact hypersensitivity in junctional adhesion molecule-A–deficient mice

Junctional adhesion molecule-A (JAM-A) is a transmembrane adhesive protein expressed at endothelial junctions and in leukocytes. In the present work, we found that DCs also express JAM-A. To evaluate the biological relevance of this observation, Jam-A(-/-) mice were generated and the functional behavior of DCs in vitro and in vivo was studied. In vitro, Jam-A(-/-) DCs showed a selective increase in random motility and in the capacity to transmigrate across lymphatic endothelial cells. In vivo, Jam-A(-/-) mice showed enhanced DC migration to lymph nodes, which was not observed in mice with endothelium-restricted deficiency of the protein. Furthermore, increased DC migration to lymph nodes was associated with enhanced contact hypersensitivity (CHS). Adoptive transfer experiments showed that JAM-A-deficient DCs elicited increased CHS in Jam-A(+/+) mice, further supporting the concept of a DC-specific effect. Thus, we identified here a novel, non-redundant role of JAM-A in controlling DC motility, trafficking to lymph nodes, and activation of specific immunity.

p66ShcA Modulates Tissue Response to Hindlimb Ischemia

Background—Oxidative stress plays a pivotal role in ischemia and ischemia/reperfusion injury. Because p66ShcA-null (p66ShcA−/−) mice exhibit both lower levels of intracellular reactive oxygen species and increased resistance to cell death induced by oxidative stress, we investigated whether tissue damage that follows acute ischemia or ischemia/reperfusion was altered in p66ShcA−/− mice. Methods and Results—Unilateral hindlimb ischemia was induced by femoral artery dissection, and ischemia/reperfusion was induced with an elastic tourniquet. Both procedures caused similar changes in blood perfusion in p66ShcA wild-type (p66ShcAwt) and p66ShcA−/− mice. However, significant differences in tissue damage were found: p66ShcAwt mice displayed marked capillary density decrease and muscle fiber necrosis. In contrast, in p66ShcA−/− mice, minimal capillary density decrease and myofiber death were present. When apoptosis after ischemia was assayed, significantly lower levels of apoptotic endothelial cells and myofibers were found in p66ShcA−/− mice. In agreement with these data, both satellite muscle cells and endothelial cells isolated from p66ShcA−/− mice were resistant to apoptosis induced by simulated ischemia in vitro. Lower apoptosis levels after ischemia in p66ShcA−/− cells correlated with decreased levels of oxidative stress both in vivo and in vitro. Conclusions—p66ShcA plays a crucial role in the cell death pathways activated by acute ischemia and ischemia/reperfusion, indicating p66ShcA as a potential therapeutic target for prevention and treatment of ischemic tissue damage.

Residual dormant cancer stem-cell foci are responsible for tumor relapse after antiangiogenic metronomic therapy in hepatocellular carcinoma xenografts

Hepatocellular carcinoma (HCC) is the fifth most common solid tumor and the third leading cause of cancer-related deaths. Currently available chemotherapeutic options are not curative due in part to tumor resistance to conventional therapies. We generated orthotopic HCC mouse models in immunodeficient NOD/SCID/IL2rγ null mice by injection of human alpha-feto protein (hAFP)- and/or luciferase-expressing HCC cell lines and primary cells from patients, where tumor growth and spread can be accurately monitored in a non-invasive way. In this model, low-dose metronomic administration of cyclophosphamide (LDM-CTX) caused complete regression of the tumor mass. A significant increase in survival (P<0.0001), reduced aberrant angiogenesis and hyperproliferation, and decrease in the number of circulating tumor cells were found in LDM-CTX-treated animals, in comparison with untreated mice. Co-administration of LDM-CTX with anti-VEGF therapy further improved the therapeutic efficacy. However, the presence of residual circulating hAFP levels suggested that some tumor cells were still present in livers of treated mice. Immunohistochemistry revealed that those cells had a hAFP+/CD13+/PCNA− phenotype, suggesting that they were dormant cancer stem cells (CSC). Indeed, discontinuation of therapy resulted in tumor regrowth. Moreover, in-vitro LDM-CTX treatment reduced hepatosphere formation in both number and size, and the resulting spheres were enriched in CD13+ cells indicating that these cells were particularly resistant to therapy. Co-treatment of the CD13-targeting drug, bestatin, with LDM-CTX leads to slower tumor growth and a decreased tumor volume. Therefore, combining a CD13 inhibitor, which targets the CSC-like population, with LDM-CTX chemotherapy may be used to eradicate minimal residual disease and improve the treatment of liver cancer.

A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination

RAB5A and RAB4 promote breast tumor cell dissemination by controlling the trafficking of proteins necessary for localized invadosome formation.

Complementary Populations of Human Adipose CD34+ Progenitor Cells Promote Growth, Angiogenesis, and Metastasis of Breast Cancer

Obesity is associated with an increased frequency, morbidity, and mortality of several types of neoplastic diseases, including postmenopausal breast cancer. We found that human adipose tissue contains two populations of progenitors with cooperative roles in breast cancer. CD45(-)CD34(+)CD31(+)CD13(-)CCRL2(+) endothelial cells can generate mature endothelial cells and capillaries. Their cancer-promoting effect in the breast was limited in the absence of CD45(-)CD34(+)CD31(-)CD13(+)CD140b(+) mesenchymal progenitors/adipose stromal cells (ASC), which generated pericytes and were more efficient than endothelial cells in promoting local tumor growth. Both endothelial cells and ASCs induced epithelial-to-mesenchymal transition (EMT) gene expression in luminal breast cancer cells. Endothelial cells (but not ASCs) migrated to lymph nodes and to contralateral nascent breast cancer lesions where they generated new vessels. In vitro and in vivo, endothelial cells were more efficient than ASCs in promoting tumor migration and in inducing metastases. Granulocyte colony-stimulating factor (G-CSF) effectively mobilized endothelial cells (but not ASCs), and the addition of chemotherapy and/or of CXCR4 inhibitors did not increase endothelial cell or ASC blood mobilization. Our findings suggest that adipose tissue progenitor cells cooperate in driving progression and metastatic spread of breast cancer.