Samuel Joseph Leibovich

Production of vascular endothelial growth factor by murine macrophages: regulation by hypoxia, lactate, and the inducible nitric oxide synthase pathway.

Murine thioglycolate-induced peritoneal macrophages (MPMs) and the murine RAW264.7 macrophage-like cell line (RAW cells) constitutively produce vascular endothelial growth factor (VEGF). VEGF production is increased under hypoxic conditions or after cell activation with interferon-gamma (IFNgamma) and endotoxin (lipopolysaccharide, LPS). In contrast, tumor necrosis factor-alpha is produced only by IFNgamma/LPS-activated cells. Lactate (25 mmol/L) does not increase VEGF production by these cells. However, hypoxia, lactate, and IFNgamma/LPS-activated MPMs express angiogenic activity, whereas normoxic, nonactivated MPMs do not. Lack of angiogenic activity is not due to an antiangiogenic factor(s) in the medium of these cells. Angiogenic activity produced by hypoxia and lactate-treated MPMs is neutralized by anti-VEGF antibody, which also neutralizes most of the angiogenic activity produced by IFNgamma/LPS-activated MPMs. The inducible nitric oxide synthase inhibitors Ng-nitro-L-arginine-methyl ester (1.5 mmol/L) and aminoguanidine (1 mmol/L) block production of angiogenic activity by MPMs and RAW cells. In RAW cells, Ng-nitro-L-arginine-methyl ester and AG block IFNgamma/LPS-activated, but not constitutive, VEGF production, whereas in MPMs, neither constitutive nor IFNgamma/LPS-activated VEGF synthesis is affected. Synthesis of tumor necrosis factor-alpha is also unaffected. In contrast to normoxic, nonactivated MPMs, inducible nitric oxide synthase-inhibited, IFNgamma/LPS-activated MPMs produce an antiangiogenic factor(s). We conclude that VEGF is a major contributor to macrophage-derived angiogenic activity, and that activation by hypoxia, lactate, or IFNgamma/LPS switches macrophage-derived VEGF from a nonangiogenic to an angiogenic state. This switch may involve a posttranslational modification of VEGF, possibly by the process of ADP-ribosylation. ADP-ribosylation by MPM cytosolic extracts or by cholera toxin switches rVEGF165 from an angiogenic to a nonangiogenic state. In IFNgamma/LPS-activated MPMs, the inducible nitric oxide synthase-dependent pathway also regulates the expression of an antiangiogenic factor(s) that antagonizes the bioactivity of VEGF and provides an additional regulatory pathway controlling the angiogenic phenotype of macrophages.

Leptin upregulates VEGF in breast cancer via canonic and non-canonical signalling pathways and NFκB/HIF-1α activation

High levels of VEGF and leptin are strongly linked to worse prognosis of breast cancer. Leptin signalling upregulates VEGF in human and mouse mammary tumor cells (MT), but the specific molecular mechanisms are largely unknown. Pharmacologic and genetic approaches were used to dissect the mechanism of leptin regulation of VEGF protein and mRNA in MT (4T1, EMT6 and MMT). A series of VEGF-promoter Luc-reporters (full-length and transcription factor-binding deletions) were transfected into MT to analyze leptin regulation of VEGF transcription. Deletion analysis of VEGF promoter and RNA knockdown shows that HIF-1alpha and NFkappaB are essentials for leptin regulation of VEGF. Leptin activation of HIF-1alpha was mainly linked to canonic (MAPK, PI-3K) and non-canonic (PKC, JNK and p38 MAP) signalling pathways. Leptin non-canonic signalling pathways (JNK, p38 MAP and to less extent PKC) were linked to NFkappaB activation. SP1 was involved in leptin regulation of VEGF in 4T1 cells. AP1 was not involved and AP2 repressed leptin-induced increase of VEGF. Overall, these data suggest that leptin signalling regulates VEGF mainly through HIF-1alpha and NFkappaB. These results delineate a comprehensive mechanism for leptin regulation of VEGF in MT. Disruption of leptin signalling could be used as a novel way to treat breast cancer.

Suppression of PLCβ2 by Endotoxin Plays a Role in the Adenosine A2A Receptor-Mediated Switch of Macrophages from an Inflammatory to an Angiogenic Phenotype

Toll-like receptor (TLR) 2, 4, 7, and 9 agonists, together with adenosine A(2A) receptor (A(2A)R) agonists, switch macrophages from an inflammatory (M1) to an angiogenic (M2-like) phenotype. This switch involves induction of A(2A)Rs by TLR agonists, down-regulation of tumor necrosis factor alpha (TNFalpha) and interleukin-12, and up-regulation of vascular endothelial growth factor (VEGF) and interleukin-10 expression. We show here that the TLR4 agonist lipopolysaccharide (LPS) induces rapid and specific post-transcriptional down-regulation of phospholipase C(PLC)beta1 and beta2 expression in macrophages by de-stabilizing their mRNAs. The PLCbeta inhibitor U73122 down-regulates TNFalpha expression by macrophages, and in the presence of A(2A)R agonists, up-regulates VEGF, mimicking the synergistic action of LPS with A(2A)R agonists. Selective down-regulation of PLCbeta2, but not PLCbeta1, using small-interfering RNA resulted in increased VEGF expression in response to A(2A)R agonists, but did not suppress TNFalpha expression. Macrophages from PLCbeta2(-/-) mice also expressed increased VEGF in response to A(2A)R agonists. LPS-mediated suppression of PLCbeta1 and beta2 is MyD88-dependent. In a model of endotoxic shock, LPS (35 microg/mouse, i.p.) suppressed PLCbeta1 and beta2 expression in spleen, liver, and lung of wild-type but not MyD88(-/-) mice. These studies indicate that LPS suppresses PLCbeta1 and beta2 expression in macrophages in vitro and in several tissues in vivo. These results suggest that suppression of PLCbeta2 plays an important role in switching M1 macrophages into an M2-like state.

Differential regulation of HIF‐1α isoforms in murine macrophages by TLR4 and adenosine A2A receptor agonists

Adenosine A2AR and TLR agonists synergize to induce an “angiogenic switch” in macrophages, down‐regulating TNF‐α and up‐regulating VEGF expression. This switch involves transcriptional regulation of VEGF by HIF‐1, transcriptional induction of HIF‐1α by LPS (TLR4 agonist), and A2AR‐dependent post‐transcriptional regulation of HIF‐1α stability. Murine HIF‐1α is expressed as two mRNA isoforms: HIF‐1αI.1 and ‐I.2, which contain alternative first exons and promoters. HIF‐1αI.2 is expressed ubiquitously, and HIF‐1αI.1 is tissue‐specific. We investigated the regulation of these isoforms in macrophages by TLR4 and A2AR agonists. HIF‐1αI.1 is induced strongly compared with HIF‐1αI.2 upon costimulation with LPS and A2AR agonists (NECA or CGS21680). In unstimulated cells, the I.1 isoform constituted ∼4% of HIF‐1α transcripts; in LPS and NECA‐ or CGS21680‐treated macrophages, this level was ∼15%, indicating a substantial contribution of HIF‐1αI.1 to total HIF‐1α expression. The promoters of both isoforms were induced by LPS but not enhanced further by NECA, suggesting A2AR‐mediated post‐transcriptional regulation. LPS/NECA‐induced expression of HIF‐1αI.1 was down‐regulated by Bay 11‐7085 (NF‐κB inhibitor) and ZM241385 (A2AR antagonist). Although VEGF and IL‐10 expression by HIF‐1αI.1−/− macrophages was equivalent to that of wild‐type macrophages, TNF‐α, MIP‐1α, IL‐6, IL‐12p40, and IL‐1β expression was significantly greater, suggesting a role for HIF‐1αI.1 in modulating expression of these cytokines. A2AR expression in unstimulated macrophages was low but was induced rapidly by LPS in a NF‐κB‐dependent manner. LPS‐induced expression of A2ARs and HIF‐1α and A2AR‐dependent HIF‐1α mRNA and protein stabilization provide mechanisms for the synergistic effects of LPS and A2AR agonists on macrophage VEGF expression.

Developmental ablation of Id1 and Id3 genes in the vasculature leads to postnatal cardiac phenotypes

The Id1 and Id3 genes play major roles during cardiac development, despite their expression being confined to non-myocardial layers (endocardium-endothelium-epicardium). We previously described that Id1Id3 double knockout (dKO) mouse embryos die at mid-gestation from multiple cardiac defects, but early lethality precluded the studies of the roles of Id in the postnatal heart. To elucidate postnatal roles of Id genes, we ablated the Id3 gene and conditionally ablated the Id1 gene in the endothelium to generate conditional KO (cKO) embryos. We observed cardiac phenotypes at birth and at 6 months of age. Half of the Id cKO mice died at birth. Postnatal demise was associated with cardiac enlargement and defects in the ventricular septum, trabeculation and vasculature. Surviving Id cKO mice exhibited fibrotic vasculature, cardiac enlargement and decreased cardiac function. An abnormal vascular response was also observed in the healing of excisional skin wounds of Id cKO mice. Expression patterns of vascular, fibrotic and hypertrophic markers were altered in the Id cKO hearts, but addition of Insulin-Like Growth Factor binding protein-3 (IGFbp3) reversed gene expression profiles of vascular and fibrotic, but not hypertrophic markers. Thus, ablation of Id genes in the vasculature leads to distinct postnatal cardiac phenotypes. These findings provide important insights into the role/s of the endocardial network of the endothelial lineage in the development of cardiac disease, and highlight IGFbp3 as a potential link between Id and its vascular effectors.

The Role of Macrophages in the Placenta

Placenta formation occurs through a complex and coordinated effort between the fetus’s extraembryonic tissues and the gravid endometrial tissues. Many macrophages are present in the placenta throughout pregnancy and have been detected as early as day 10 of pregnancy (Chang et al., 1993). Placental macrophages include Hofbauer cells of the fetal chorionic villi and decidual macrophages of the maternal decidua basalis (Figure 1) (Bulmer & Johnson, 1984). Functions of placental macrophages include the production of substances that regulate local immune reactions (such as factors that regulate maternal immunological tolerance and fetal protection) and that promote angiogenesis in the placenta during its development (Mues et al., 1989; Sevala et al., 2007). Although they represent a significant presence on both the maternal and fetal sides of the placenta, placental macrophage functions have not been completely elucidated and still remain a significantly studied area of investigation.