Annalisa Pucci

Cutting edge: IL-1beta mediates the proangiogenic activity of osteopontin-activated human monocytes.

Inflammation plays an important role in the onset of angiogenesis. In the present study, we show that osteopontin (OPN), a proinflammatory mediator involved in tissue repair, induces IL-1β up-regulation in human monocytes. This was accompanied by the enhanced production of TNF-α, IL-8, and IL-6, a decreased release of IL-10, and increased p38 phosphorylation. The supernatants of OPN-treated monocytes were highly angiogenic when delivered on the chick embryo chorioallantoic membrane. The angiogenic response was completely abrogated by a neutralizing anti-IL-1 Ab, thus indicating that this cytokine represents the major proangiogenic factor expressed by OPN-activated monocytes. Accordingly, rIL-1β mimicked the proangiogenic activity of OPN-treated monocyte supernatants, and IL-1R (type I) was found to be expressed in the chorioallantoic membrane. In conclusion, OPN-activated monocytes may contribute to the onset of angiogenesis through a mechanism mediated by IL-1β.

Thrombin regulates the expression of proangiogenic cytokines via proteolytic activation of protease-activated receptor-1

In addition to its central role in blood coagulation and hemostasis, human alpha-thrombin is a growth factor for a variety of cell types, including monocytes and endothelial cells, involved in the control of angiogenesis. Different cytokines produced by mononuclear cells have been implicated in angiogenic processes associated with tissue repair and certain human malignancies. We have previously shown that thrombin enhances proliferative responses in T lymphocytes. More recently, we reported that interferon-gamma-differentiated monocytes have increased expression of protease-activated receptor-1 (PAR-1) and increased thrombin binding. Since cytokines may be involved directly and indirectly in angiogenesis, we initiated studies to determine thrombin effects on the induction of cytokines, such as interleukin (IL)-1 and IL-6, in human mononuclear cells. IL-1 and IL-6 protein expression was significantly enhanced by thrombin (P<.05), as determined by enzyme-linked immunosorbent assay (ELISA). Treating mononuclear cells with the PAR-1 peptide, SFLLRN, has effects similar to those of thrombin. Thus, it appears that these thrombin effects are mediated through activation of PAR-1. These results confirm that thrombin is a strong activator of monocytes and could be involved in angiogenesis by inducing cytokines that could enhance the angiogenic process in tissue repair.

Thrombin‐mediated IL‐10 up‐regulation involves protease‐activated receptor (PAR)‐1 expression in human mononuclear leukocytes

Thrombin, the key enzyme of the coagulation cascade, exerts cellular effects through activation of the protease‐activated receptors (PARs). Interleukin (IL)‐10, besides its anti‐inflammatory properties, is considered a major denominator of the immunosuppressive effect during human endotoxemia. We have recently shown that thrombin inhibits IL‐12 production in human mononuclear cells and that such inhibition is accompanied by IL‐10 up‐regulation. To our knowledge, there are no data available to show that thrombin mediates IL‐10 production by its interactions with PAR‐1. We here report that human α‐thrombin enhances IL‐10 expression in human peripheral blood mononuclear cells and in established monocytic cell lines and that this up‐regulation requies PAR‐1 expression. The use of proteolytically inactive thrombin reveals that such enhancement requires thrombin proteolytic activity. Addition of PAR‐1 agonist peptides, such as SFLLRN, results in a significant increase of IL‐10 production. PAR‐1 expression is required for thrombin‐induced IL‐10 production, as shown by experiments performed with antisense or sense PAR‐1 oligonucleotides. Treatment with thrombin or SFLLRN of monocytic cell lines, such as U937 and Mono Mac‐6, results in an increased IL‐10 production. This suggests that the observed IL‐10 up‐regulation may be the result of a direct interaction with monocytes. The observation that thrombin‐mediated up‐regulation of IL‐10 may require the expression of the PAR‐1 receptor identifies a new, functional link between inflammation and coagulation. Our results may also contribute to better design therapeutic strategies to treat several disorders, characterized by the presence of inflammatory as well as coagulant responses.

Regulation of Angiogenesis by Th1- and Th2-Type Cytokines

Angiogenesis is a complex process, where several cell types and mediators interact to establish a specific microenvironment suitable for the formation of new capillaries from pre-existing vessels. Such biological processes occur in several physiological conditions, such as embryo development and wound healing, as well as in pathological conditions, including tumours and diabetic retinopathy. T lymphocytes, neutrophils and monocytes fully participate in the angiogenic process by secreting cytokines that may control endothelial cell (EC) proliferation, their survival and apoptosis, as well as their migration and activation. Angiogenesis is the result of a net balance between the activities exerted by positive and negative regulators. This balance is conceptually very similar to that of the Th1/Th2 cells that modulate an appropriate and specific immune response. Th1 or Th2 cytokines may control angiogenesis directly, by acting on cell growth and differentiation, indirectly by inducing the release of other cytokines in the microenvironment, and by modulating the expression of specific receptors, involved in the control of angiogenic processes, such as EC proliferation and migration. In this review we will mainly discuss the role of Th1- and Th2-type cytokines in the angiogenic process, emphasizing the complexity of the cytokine and leukocyte/EC network, and highlighting the care that needs to be taken when designing new therapeutic interventions involving Th1 and Th2 cytokines.

p66Shc is involved in promoting HIF‐1α accumulation and cell death in hypoxic T cells

Hypoxia results in adaptationally appropriate alterations of gene expression through the activation of hypoxia‐inducible factor (HIF)‐1 to overcome any shortage of oxygen. Peripheral blood mononuclear cells may be exposed to low oxygen tensions for different times as they migrate between blood and various tissues. We and others have previously shown that T‐cell adaptation to hypoxia is characterized by a modulation of cytokine expression and an inhibition of T‐cell activation. We have recently demonstrated that the adaptor protein p66Shc negatively regulates T‐cell activation and survival. We here show that hypoxia enhances HIF‐1α accumulation and vascular endothelial growth factor production in T cells. Hypoxic T cells expressed high levels of p21WAF1/CIP1, of the pro‐apoptotic molecules BNIP3, a classic HIF target gene, and BAX, as well as low levels of the anti‐apoptotic molecule BCLxl, associated with an induction of cell death. We found out that hypoxic T cells expressed p66Shc. Furthermore, using T‐cell transfectants expressing p66Shc, as well as T cells derived from mice p66Shc−/−, we defined a role of p66Shc in T‐cell responses to hypoxia. Of interest, hypoxic p66Shc‐positive transfectants expressed higher level of HIF‐1α than negative controls. Thus, p66Shc may play an important role in downstream hypoxic signaling, involving HIF‐1α protein accumulation and cell death in T lymphocytes. J. Cell. Physiol. 211: 439–447, 2007.

Hypoxia affects dendritic cell survival: Role of the hypoxia-inducible factor-1α and lipopolysaccharide

Dendritic cells (DC) are the most potent antigen‐presenting cells and during their life cycle they are exposed to different oxygen tensions. Similarly to inflamed and tumor tissues, lymphoid organs are characterized by a hypoxic microenvironment; thus, the modality by which hypoxia may affect DC is important for regulating both the quality and the intensity of the immune response. Here, we show that human monocyte‐derived DC, exposed to hypoxia, expressed high levels of the hypoxia‐inducible factor (HIF)‐1α, associated with upregulation of BNIP3 and BAX expression. This was paralleled with downregulation of the anti‐apoptotic molecule Bcl‐2, enhanced caspase‐3 activity and poly (ADP‐ribose) polymerase cleavage, along with cell death. Transfection of HIF‐1α siRNA protected DC from the effects of hypoxia. Of interest, when hypoxic DC were maturated with lipopolysaccharide (LPS), we did not observe an increased cell death, while HIF‐1α accumulation and BNIP3 expression were still significantly upregulated. In contrast with immature DC, mature DC expressed higher levels of Bcl‐2, and, more importantly, of phosphorylated Akt. Transfection of HIF‐1α siRNA to mature DC resulted in a significant upregulation of Akt phosphorylation as well. Moreover, inhibition of PI3K/Akt pathway resulted in an increased cell death of hypoxic mature DC. We may conclude that a prolonged exposure to hypoxia induces a cell death program which could be prevented by HIF‐1α inhibition and/or LPS maturation. Our results may contribute to further understand the physiology of DC and the molecular mechanisms involved in the survival of DC, with important implications in the regulation of the immune response. J. Cell. Physiol. 227: 587–595, 2012.

Inhibition of Bcr-Abl phosphorylation and induction of apoptosis by pyrazolo[3,4-d]pyrimidines in human leukemia cells.

A series of pyrazolo[3,4‐d]pyrimidines, previously found to be Src inhibitors, was tested for their ability to inhibit proliferation of three Bcr‐Abl‐positive human leukemia cell lines (K‐562, KU‐812, and MEG‐01), on the basis of the experimental evidence that various Src inhibitors are also active against Bcr‐Abl kinase (the so called dual Src/Abl inhibitors). They reduce Bcr‐Abl tyrosine phosphorylation and promote apoptosis of the Bcr‐Abl‐expressing cells. A cell‐free enzymatic assay on isolated c‐Abl confirmed that such compounds directly inhibit Abl activity. Finally, molecular modeling simulations were also performed to hypothesize the binding mode of the compounds into the Abl binding site.

Inhibition of interleukin‐12 expression by α‐thrombin in human peripheral blood mononuclear cells: a potential mechanism for modulating Th1/Th2 responses

In addition to its central role in blood coagulation and hemostasis, human α‐thrombin is a powerful regulator of inflammatory responses and is known to affect cell‐mediated immunity. Interleukin (IL)‐12 is a strong promoter of the development of Th1‐type lymphocytes and its downregulation implies a positive feedback mechanism for development of Th2 responses. We have previously shown that thrombin enhances the release of IL‐6, a Th2‐related cytokine, in human peripheral blood mononuclear cells (PBMC). Here we show that thrombin downregulates IL‐12 production at both protein and mRNA levels in human PBMC. The inhibition of IL‐12 production was accompanied by an enhanced release of IL‐10, which inhibits Th1‐related processes and promotes Th2‐type responses. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, reveals that this downregulation is thrombin‐specific and requires thrombin proteolytic activity. In addition, activation of coagulation inhibits IL‐12 production in whole blood cultures, confirming the tight relationship between the coagulation pathway, where thrombin is a key enzyme, and inflammation. Decreased IL‐12 production appears to be related also to IL‐10 production, since the addition of an anti‐IL‐10 monoclonal antibody to thrombin‐treated PBMC resulted in a partial restoration of IL‐12 production. In conclusion, the observation that thrombin significantly affects the production of IL‐12, as well as of IL‐10, implies a concerted role orchestrated by thrombin in PBMC that could be crucial to effective immunity and inflammation.

Human α‐thrombin stimulates proliferation of interferon‐γ differentiated, growth‐arrested U937 cells, overcoming differentiation‐related changes in expression of p21CIP1/WAF1 and cyclin D1

In addition to its central role in blood coagulation and hemostasis, human α‐thrombin is a growth factor for a variety of cell types. We recently demonstrated that interferon‐γ (IFNγ)‐differentiated U937 cells show increased expression of the proteolytically activated receptor for thrombin (PAR‐1) relative to undifferentiated U937. In the present study we show that cell proliferation is inhibited in IFNγ‐differentiated cells relative to undifferentiated U937. Addition of thrombin to the differentiated cells, however, overcomes the inhibition and restores the cells to a highly proliferative state. Ribonuclease protection assays indicate that the IFNγ‐induced growth arrest is associated with an increased expression of the cyclin‐dependent kinase inhibitor p21CIP1/WAF1 and downregulation of cyclin D1. Treatment of cells with thrombin downregulates p21CIP1/WAF1 expression in these cells and upregulates cyclin D1 mRNA expression, thus overcoming the differentiation‐related effects in a coordinated manner. Treating differentiated cells with the PAR‐1 activation peptide, SFLLRN, stimulates proliferation and has effects similar to those of thrombin on expression of p21CIP1/WAF1. Thus, it appears that these thrombin stimulated proliferative effects are mediated through activation of PAR‐1. These results may help explain how thrombin can overcome growth arrest in normal tissue to initiate tissue repair and why thrombin and thrombin‐like enzymes may contribute to unrestricted proliferation observed in certain malignancies.

The adaptor protein p66shc is a positive regulator in the angiogenic response induced by hypoxic T cells

Immune cells play an important role in the onset of angiogenesis. Here, we report that VEGF represents the major proangiogenic factor expressed by T cells exposed to hypoxia, a common feature of inflammation and tumor microenvironment. The supernatants of hypoxic T cells were highly angiogenic when delivered on the chick embryo CAM. The angiogenic response was abrogated by a neutralizing anti‐VEGF antibody and mimicked by rVEGF. Interestingly, VEGF induction by hypoxia was up‐regulated in Jurkat T cells overexpressing the adaptor protein p66Shc but not the inactive S36 p66Shc mutant, and it was abolished in p66Shc−/− mouse splenocytes. Accordingly, the angiogenic response induced by the supernatants from hypoxic p66Shc−/− splenocytes was reduced dramatically when compared with the wild‐type controls. In conclusion, hypoxic T cells may contribute to the onset of angiogenesis through a novel VEGF‐mediated mechanism, where p66Shc acts as a positive regulator.