Monica Morini

Neutrophil restraint by green tea: Inhibition of inflammation, associated angiogenesis, and pulmonary fibrosis

Neutrophils play an essential role in host defense and inflammation, but the latter may trigger and sustain the pathogenesis of a range of acute and chronic diseases. Green tea has been claimed to exert anti-inflammatory properties through unknown molecular mechanisms. We have previously shown that the most abundant catechin of green tea, (−)epigallocatechin-3-gallate (EGCG), strongly inhibits neutrophil elastase. Here we show that 1) micromolar EGCG represses reactive oxygen species activity and inhibits apoptosis of activated neutrophils, and 2) dramatically inhibits chemokine-induced neutrophil chemotaxis in vitro; 3) both oral EGCG and green tea extract block neutrophil-mediated angiogenesis in vivo in an inflammatory angiogenesis model, and 4) oral administration of green tea extract enhances resolution in a pulmonary inflammation model, significantly reducing consequent fibrosis. These results provide molecular and cellular insights into the claimed beneficial properties of green tea and indicate that EGCG is a potent anti-inflammatory compound with therapeutic potential.

The HtrA1 serine protease is down-regulated during human melanoma progression and represses growth of metastatic melanoma cells

Differential gene expression of cell lines derived from a malignant melanoma or its autologous lymph node metastasis using cDNA arrays indicated down-regulation of PRSS11, a gene encoding the serine protease HtrA1, a homolog of the Escherichia coli protease HtrA, in the metastatic line. Stable PRSS11 overexpression in the metastatic cell line strongly inhibited proliferation, chemoinvasion and Nm23-H1 protein expression in vitro, as well as cell growth in vivo in nu/nu mice. A polyclonal anti-HtrA1 serum demonstrated a significantly higher expression in primary melanomas when compared to unrelated metastatic lesions in a human melanoma tissue array, and down-modulation of HtrA1 expression in autologous lymph node melanoma metastases in seven out of 11 cases examined. These results suggest that down-regulation of PRSS11 and HtrA1 expression may represent an indicator of melanoma progression.

CXCL1/Macrophage Inflammatory Protein-2-Induced Angiogenesis In Vivo Is Mediated by Neutrophil-Derived Vascular Endothelial Growth Factor-A

The angiogenic activity of CXC-ELR+ chemokines, including CXCL8/IL-8, CXCL1/macrophage inflammatory protein-2 (MIP-2), and CXCL1/growth-related oncogene-α in the Matrigel sponge angiogenesis assay in vivo, is strictly neutrophil dependent, as neutrophil depletion of the animals completely abrogates the angiogenic response. In this study, we demonstrate that mice deficient in the src family kinases, Hck and Fgr (hck−/−fgr−/−), are unable to develop an angiogenic response to CXCL1/MIP-2, although they respond normally to vascular endothelial growth factor-A (VEGF-A). Histological examination of the CXCL1/MIP-2-containing Matrigel implants isolated from wild-type or hck−/−fgr−/− mice showed the presence of an extensive neutrophil infiltrate, excluding a defective neutrophil recruitment into the Matrigel sponges. Accordingly, neutrophils from hck−/−fgr−/− mice normally migrated and released gelatinase B in response to CXCL1/MIP-2 in vitro, similarly to wild-type neutrophils. However, unlike wild-type neutrophils, those from hck−/−fgr−/− mice were completely unable to release VEGF-A upon stimulation with CXCL1/MIP-2. Furthermore, neutralizing anti-VEGF-A Abs abrogated the angiogenic response to CXCL1/MIP-2 in wild-type mice and CXCL1/MIP-2 induced angiogenesis in the chick embryo chorioallantoic membrane assay, indicating that neutrophil-derived VEGF-A is a major mediator of CXCL1/MIP-2-induced angiogenesis. Finally, in vitro kinase assays confirmed that CXCL1/MIP-2 activates Hck and Fgr in murine neutrophils. Taken together, these data demonstrate that CXCL1/MIP-2 leads to recruitment of neutrophils that, in turn, release biologically active VEGF-A, resulting in angiogenesis in vivo. Our observations delineate a novel mechanism by which CXCL1/MIP-2 induces neutrophil-dependent angiogenesis in vivo.

Mechanisms of Inhibition of Tumor Angiogenesis and Vascular Tumor Growth by Epigallocatechin-3-Gallate

Purpose: Green tea consumption has been linked to a reduced occurrence of some tumor types. Current data indicate that the principal mediator of this chemopreventive effect is epigallocatechin-3-gallate (EGCG), the most abundant polyphenol found in dried tea leaves. Here, we examined the effects of this compound on the two key cell populations typically involved in tumor growth: tumor cells and endothelial cells. Experimental Design: The effects of green tea and EGCG were tested in a highly vascular Kaposi’s sarcoma (KS) tumor model and on endothelial cells in a panel of in vivo and in vitro assays. Results: EGCG inhibited KS-IMM cell growth and endothelial cell growth, chemotaxis, and invasion over a range of doses; high concentrations also induced tumor cell apoptosis. EGCG inhibited the metalloprotease-mediated gelatinolytic activity produced by endothelial cell supernatants and the formation of new capillary-like structures in vitro. Green tea or purified EGCG when administered to mice in the drinking water inhibited angiogenesis in vivo in the Matrigel sponge model and restrained KS tumor growth. Histological analysis of the tumors were consistent with an anti-angiogenic activity of EGCG and green tea. Conclusions: These data suggest that the green tea gallate or its derivatives may find use in the prevention and treatment of vascular tumors in a chemoprevention or adjuvant setting.

Generation of Biologically Active Angiostatin Kringle 1–3 by Activated Human Neutrophils

The contribution of polymorphonuclear neutrophils (PMN) to host defense and natural immunity extends well beyond their traditional role as professional phagocytes. In this study, we demonstrate that upon stimulation with proinflammatory stimuli, human PMN release enzymatic activities that, in vitro, generate bioactive angiostatin fragments from purified plasminogen. We also provide evidence that these angiostatin-like fragments, comprising kringle domain 1 to kringle domain 3 (kringle 1–3) of plasminogen, are generated as a byproduct of the selective proteolytic activity of neutrophil-secreted elastase. Remarkably, affinity-purified angiostatin kringle 1–3 fragments generated by neutrophils inhibited basic fibroblast growth factor plus vascular endothelial growth factor-induced endothelial cell proliferation in vitro, and both vascular endothelial growth factor-induced angiogenesis in the matrigel plug assay and fibroblast growth factor-induced angiogenesis in the chick embryo chorioallantoic membrane assay, in vivo. These results represent the first demonstration that biologically active angiostatin-like fragments can be generated by inflammatory human neutrophils. Because angiostatin is a potent inhibitor of angiogenesis, tumor growth, and metastasis, the data suggest that activated PMN not only act as potent effectors of inflammation, but might also play a critical role in the inhibition of angiogenesis in inflammatory diseases and tumors, by generation of a potent anti-angiogenic molecule.

AAV-mediated gene transfer of tissue inhibitor of metalloproteinases-1 inhibits vascular tumor growth and angiogenesis in vivo

The activity of matrix metalloproteinases (MMPs) is a universal feature of cellular invasion, tumor angiogenesis and metastasis, which is counterbalanced and regulated by the natural tissue inhibitors of MMPs (Timps). Here we show that Timp1 gene transfer delivered by an adeno-associated virus (AAV) vector inhibits tumor growth in a murine xenotransplant model. A human Kaposis sarcoma cell line, forming highly vascularized tumors in vivo and having a high natural permissivity to AAV gene transfer, was transduced to express the Timp1 cDNA. AAV-Timp1-transduced cells secreted high levels of Timp1 that inhibited MMP2 and MMP9 gelatinolytic activity. Following subcutaneous inoculation in nude mice, the AAV-Timp1-transduced cells showed significantly reduced tumor growth when compared to control AAV-LacZ-transduced cells. In addition, direct intratumoral injection of AAV-Timp1 into pre-existing tumors significantly impaired the further expansion of the tumor mass. Histological analyses showed that the AAV-Timp1-transduced tumors had limited development of vascular structures and extensive areas of cell death, suggesting that Timp1 overexpression had an antiangiogenic effect. To further support this conclusion, we demonstrated that AAV-Timp1 transduction significantly reduced endothelial cell migration and the invasion of a Matrigel barrier and strongly inhibited angiogenesis in the chick chorioallantoic membrane assay. These results indicate that transfer and overexpression of the Timp1 gene is a promising therapeutic strategy to target tumor-associated angiogenesis in cancer gene therapy.

Prostate carcinoma and green tea: (−)epigallocatechin-3-gallate inhibits inflammation-triggered MMP-2 activation and invasion in murine TRAMP model

Green tea infusion has been shown to inhibit metastatic spreading of the transgenic adenocarcinoma of mouse prostate (TRAMP). Investigation on the molecular mechanisms triggered by the main green tea flavonoid, (−)epigallocatechin‐3‐gallate (EGCG), shows that EGCG restrains TRAMP‐C1 cell proliferation in a dose‐dependent manner, at concentrations (IC50 < 0.2 μM) equivalent to those measured in the plasma of moderate green‐tea drinkers. Up to 10 μM, EGCG does not modify the cell‐surface immuno‐localization of MMP‐2, one of the invasion‐instrumental proteinases; but while in default culture conditions these cells secrete mainly pro‐MMP‐2, in the presence of reconstituted basement membrane (Matrigel) they release almost exclusively pro‐MMP‐9. In contrast, when stimulated to traverse Matrigel toward a chemo‐attractant, in addition to pro‐MMP‐9, they secrete pro‐MMP‐2. In the presence of 0.2 μM EGCG, only the level of the latter is markedly lowered in the conditioned medium, in parallel with the invasive behavior (>50%). In vivo, s.c. injection of TRAMP‐C1 cells dispersed in Matrigel gives origin to a tumor mass, whose growth is not inhibited by green‐tea regimen. This growth is contained greater than two‐thirds by LPS‐triggered polymorpho‐nuclear phagocyte (PMN) recruitment but this effect is abolished by green tea. Nevertheless, while tumor‐released pro‐MMP‐2 is activated by co‐incubation of TRAMP‐C1 cells with PMNs, in the presence of 10 μM EGCG the activation is almost abolished. These results suggest that inflammatory involvement of prostate carcinoma could be efficaciously prevented by green tea with a concomitant lowering of the invasive potential.

Angiostatin anti-angiogenesis requires IL-12: the innate immune system as a key target.

BackgroundAngiostatin, an endogenous angiogenesis inhibitor, is a fragment of plasminogen. Its anti-angiogenic activity was discovered with functional assays in vivo, however, its direct action on endothelial cells is moderate and identification of definitive mechanisms of action has been elusive to date. We had previously demonstrated that innate immune cells are key targets of angiostatin, however the pathway involved in this immune-related angiogenesis inhibition was not known. Here we present evidence that IL-12, a principal TH1 cytokine with potent anti-angiogenic activity, is the mediator of angiostatins activity.MethodsFunction blocking antibodies and gene-targeted animals were employed or in vivo studies using the subcutaneous matrigel model of angiogenesis. Quantitative real-time PCR were used to assess modulation of cytokine production in vitro.ResultsAngiostatin inhibts angiogenesis induced by VEGF-TNFα or supernatants of Kaposis Sarcoma cells (a highly angiogenic and inflammation-associated tumor). We found that function-blocking antibodies to IL-12 reverted angiostatin induced angiogenesis inhibition. The use of KO animal models revealed that angiostatin is unable to exert angiogenesis inhibition in mice with gene-targeted deletions of either the IL-12 specific receptor subunit IL-12Rβ2 or the IL-12 p40 subunit. Angiostatin induces IL-12 mRNA synthesis by human macrophages in vitro, suggesting that these innate immunity cells produce IL-12 upon angiostatin stimulation and could be a major cellular mediator.ConclusionOur data demonstrate that an endogenous angiogenesis inhibitor such as angiostatin act on innate immune cells as key targets in inflammatory angiogenesis. Angiostatin proves to be anti-angiogenic as an immune modulator rather than a direct anti-vascular agent. This article is dedicated to the memory of Prof Judah Folkman for his leadership and for encouragement of these studies.

Positive regulation of steroidogenic acute regulatory protein gene expression through the interaction between Dlx and GATA-4 for testicular steroidogenesis

Split hand/foot malformation (SHFM) is syndromic ectrodactyly often associated with mental retardation and/or craniofacial defects. Several clinical reports previously described urogenital dysplasia such as micropenis, hypospadias, and small testis in SHFM patients. Genetic lesions in the Dlx5 and Dlx6 (Dlx5/6) locus are associated with the human genetic disorder SHFM type 1. Although Dlx5/6 are expressed in the testis, their possible function of Dlx5/6 during testis differentiation has not been described. In this study, we show that Dlx5/6 are expressed in the fetal Leydig cells during testis development. We examined the effect of Dlx5 expression on the promoter activation of the steroidogenic acute regulatory protein (StAR) gene, which is essential for gonadal and adrenal steroidogenesis, in a Leydig cell line. Dlx5 efficiently activates the StAR promoter when GATA-4, another transcription factor essential for testicular steroidogenesis, was coexpressed. The transcriptional activation required the GATA-4-recognition element in the StAR promoter region and Dlx5 can physically interact with GATA-4. Furthermore, we herein show that the double inactivation of Dlx5 and Dlx6 in the mouse leads to decreased testosterone level and abnormal masculinization phenotype. These results suggest that Dlx5 and Dlx6 participate in the control of steroidogenesis during testis development. The findings of this study may open the way to analyze human congenital birth defects.

Mutually exclusive expression of DLX2 and DLX5/6 is associated with the metastatic potential of the human breast cancer cell line MDA-MB-231

BackgroundThe DLX gene family encodes for homeobox transcription factors involved in the control of morphogenesis and tissue homeostasis. Their expression can be regulated by Endothelin1 (ET1), a peptide associated with breast cancer invasive phenotype. Deregulation of DLX gene expression was found in human solid tumors and hematologic malignancies. In particular, DLX4 overexpression represents a possible prognostic marker in ovarian cancer. We have investigated the role of DLX genes in human breast cancer progression.MethodsMDA-MB-231 human breast carcinoma cells were grown in vitro or injected in nude mice, either subcutaneously, to mimic primary tumor growth, or intravenously, to mimic metastatic spreading. Expression of DLX2, DLX5 and DLX6 was assessed in cultured cells, either treated or not with ET1, tumors and metastases by RT-PCR. In situ hybridization was used to confirm DLX gene expression in primary tumors and in lung and bone metastases. The expression of DLX2 and DLX5 was evaluated in 408 primary human breast cancers examining the GSE1456 and GSE3494 microarray datasets. Kaplan-Meier estimates for disease-free survival were calculated for the patients grouped on the basis of DLX2/DLX5 expression.ResultsBefore injection, or after subcutaneous growth, MDA-MB-231 cells expressed DLX2 but neither DLX5 nor DLX6. Instead, in bone and lung metastases resulting from intravenous injection we detected expression of DLX5/6 but not of DLX2, suggesting that DLX5/6 are activated during metastasis formation, and that their expression is alternative to that of DLX2. The in vitro treatment of MDA-MB-231 cells with ET1, resulted in switch from DLX2 to DLX5 expression. By data mining in microarray datasets we found that expression of DLX2 occurred in 21.6% of patients, and was significantly correlated with prolonged disease-free survival and reduced incidence of relapse. Instead, DLX5 was expressed in a small subset of cases, 2.2% of total, displaying reduced disease-free survival and high incidence of relapse which was, however, non-significantly different from the other groups due to the small size of the DLX+ cohort. In all cases, we found mutually exclusive expression of DLX2 and DLX5.ConclusionsOur studies indicate that DLX genes are involved in human breast cancer progression, and that DLX2 and DLX5 genes might serve as prognostic markers.