Christian Stockmann

A role for VEGF as a negative regulator of pericyte function and vessel maturation

Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively. PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators. Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rβ signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rβ and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rβ/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.Angiogenesis does not only depend on endothelial cell invasion and proliferation: it also requires pericyte coverage of vascular sprouts for vessel stabilization. These processes are coordinated by vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) through their cognate receptors on endothelial cells and vascular smooth muscle cells (VSMCs), respectively. PDGF induces neovascularization by priming VSMCs/pericytes to release pro-angiogenic mediators. Although VEGF directly stimulates endothelial cell proliferation and migration, its role in pericyte biology is less clear. Here we define a role for VEGF as an inhibitor of neovascularization on the basis of its capacity to disrupt VSMC function. Specifically, under conditions of PDGF-mediated angiogenesis, VEGF ablates pericyte coverage of nascent vascular sprouts, leading to vessel destabilization. At the molecular level, VEGF-mediated activation of VEGF-R2 suppresses PDGF-Rbeta signalling in VSMCs through the assembly of a previously undescribed receptor complex consisting of PDGF-Rbeta and VEGF-R2. Inhibition of VEGF-R2 not only prevents assembly of this receptor complex but also restores angiogenesis in tissues exposed to both VEGF and PDGF. Finally, genetic deletion of tumour cell VEGF disrupts PDGF-Rbeta/VEGF-R2 complex formation and increases tumour vessel maturation. These findings underscore the importance of VSMCs/pericytes in neovascularization and reveal a dichotomous role for VEGF and VEGF-R2 signalling as both a promoter of endothelial cell function and a negative regulator of VSMCs and vessel maturation.

Deletion of vascular endothelial growth factor in myeloid cells accelerates tumorigenesis

Angiogenesis and the development of a vascular network are required for tumour progression, and they involve the release of angiogenic factors, including vascular endothelial growth factor (VEGF-A), from both malignant and stromal cell types. Infiltration by cells of the myeloid lineage is a hallmark of many tumours, and in many cases the macrophages in these infiltrates express VEGF-A. Here we show that the deletion of inflammatory-cell-derived VEGF-A attenuates the formation of a typical high-density vessel network, thus blocking the angiogenic switch in solid tumours in mice. Vasculature in tumours lacking myeloid-cell-derived VEGF-A was less tortuous, with increased pericyte coverage and decreased vessel length, indicating vascular normalization. In addition, loss of myeloid-derived VEGF-A decreases the phosphorylation of VEGF receptor 2 (VEGFR2) in tumours, even though overall VEGF-A levels in the tumours are unaffected. However, deletion of myeloid-cell VEGF-A resulted in an accelerated tumour progression in multiple subcutaneous isograft models and an autochthonous transgenic model of mammary tumorigenesis, with less overall tumour cell death and decreased tumour hypoxia. Furthermore, loss of myeloid-cell VEGF-A increased the susceptibility of tumours to chemotherapeutic cytotoxicity. This shows that myeloid-derived VEGF-A is essential for the tumorigenic alteration of vasculature and signalling to VEGFR2, and that these changes act to retard, not promote, tumour progression.

Differential activation and antagonistic function of HIF-α isoforms in macrophages are essential for NO homeostasis

Hypoxic response and inflammation both involve the action of the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha. Previous studies have revealed that both HIF-alpha proteins are in a number of aspects similarly regulated post-translationally. However, the functional interrelationship of these two isoforms remains largely unclear. The polarization of macrophages controls functionally divergent processes; one of these is nitric oxide (NO) production, which in turn is controlled in part by HIF factors. We show here that the HIF-alpha isoforms can be differentially activated: HIF-1alpha is induced by Th1 cytokines in M1 macrophage polarization, whereas HIF-2alpha is induced by Th2 cytokines during an M2 response. This differential response was most evident in polarized macrophages through HIF-alpha isoform-specific regulation of the inducible NO synthase gene by HIF-1alpha, and the arginase1 gene by HIF-2alpha. In silico modeling predicted that regulation of overall NO availability is due to differential regulation of HIF-1alpha versus HIF-2alpha, acting to, respectively, either increase or suppress NO synthesis. An in vivo model of endotoxin challenge confirmed this; thus, these studies reveal that the two homologous transcription factors, HIF-1alpha and HIF-2alpha, can have physiologically antagonistic functions, but that their antiphase regulation allows them to coordinately regulate NO production in a cytokine-induced and transcription-dependent fashion.

Bacterial lipopolysaccharide induces HIF-1 activation in human monocytes via p44/42 MAPK and NF-κB

Inflammatory mediators activate the transcriptional complex HIF-1 (hypoxia-inducible factor-1), the key regulator of hypoxia-induced gene expression. Here we report that bacterial LPS (lipopolysaccharide) induces HIF-1alpha mRNA expression and HIF-1alpha protein accumulation in human monocytes as well as in non-differentiated and differentiated cells of the human monocytic cell line THP-1 under normoxic conditions. LPS and hypoxia synergistically activated HIF-1. Whereas LPS increased HIF-1alpha mRNA expression through activation of a NF-kappaB (nuclear factor kappaB) site in the promoter of the HIF-1alpha gene, hypoxia post-translationally stabilized HIF-1alpha protein. HIF-1alpha activation was followed by increased expression of the HIF-1 target gene encoding ADM (adrenomedullin). Knocking down HIF-1alpha by RNA interference significantly decreased ADM expression, which underlines the importance of HIF-1 for the LPS-induced ADM expression in normoxia. Simultaneously with HIF-1 activation, an increase in p44/42 MAPK (mitogen-activated protein kinase) phosphorylation was observed after incubation with LPS. In cells pretreated with the p44/42 MAPK inhibitor PD 98059 or with RNAi (interfering RNA) directed against p44/42 MAPK, LPS-induced HIF-1alpha accumulation and ADM expression were significantly decreased. From these results we conclude that LPS critically involves the p44/42 MAPK and NF-kappaB pathway in the activation of HIF-1, which is an important transcription factor for LPS-induced ADM expression.

Hypoxia-induced erythropoietin production: a paradigm for oxygen-regulated gene expression.

1 The mechanisms controlling the expression of the gene encoding for the hormone erythropoietin (EPO) are exemplary for oxygen‐regulated gene expression. In humans and other mammals, hypoxia modulates EPO levels by increasing expression of the EPO gene. An association between polycythaemia and people living at high altitudes was first reported more than 100 years ago. 2 Since the identification of EPO as the humoral regulator of red blood cell production and the cloning of the EPO gene, considerable progress has been made in understanding the regulation of EPO gene expression. This has finally led to the identification of a widespread cellular oxygen‐sensing mechanism. Central to this mechanism is the transcription factor complex hypoxia‐inducible factor (HIF)‐1. 3 The abundance and activity of HIF‐1, a heterodimer of an α‐ and β‐subunit, is predominantly regulated by oxygen‐dependent post‐translational hydroxylation of the α‐subunit. Non‐heme ferrous iron containing hydroxylases use dioxygen and 2‐oxoglutarate to specifically target proline and an asparagine residue in HIF‐1α. As such, the three prolyl hydroxylases (prolyl hydroxylase domain‐containing protein (PHD) 1, PHD2 and PHD3) and the asparagyl hydroxylase (factor inhibiting HIF (FIH)‐1) act as cellular oxygen sensors. In addition to erythropoiesis, HIF‐1 regulates a broad range of physiologically relevant genes involved in angiogenesis, apoptosis, vasomotor control and energy metabolism. Therefore, the HIF system is implicated in the pathophysiology of many human diseases. 4 In addition to the tight regulation by oxygen tension, temporal and tissue‐specific signals limit expression of the EPO gene primarily to the fetal liver and the adult kidney.

Neuropilin 1 deficiency on CD4+Foxp3+ regulatory T cells impairs mouse melanoma growth

Neuropilin 1 mediates anti-tumor control by promoting regulatory T cell infiltration.

The glial cell response is an essential component of hypoxia-induced erythropoiesis in mice

A key adaptation to environmental hypoxia is an increase in erythropoiesis, driven by the hormone erythropoietin (EPO) through what is traditionally thought to be primarily a renal response. However, both neurons and astrocytes (the largest subpopulation of glial cells in the CNS) also express EPO following ischemic injury, and this response is known to ameliorate damage to the brain. To investigate the role of glial cells as a component of the systemic response to hypoxia, we created astrocyte-specific deletions of the murine genes encoding the hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha and their negative regulator von Hippel-Lindau (VHL) as well as astrocyte-specific deletion of the HIF target gene Vegf. We found that loss of the hypoxic response in astrocytes does not cause anemia in mice but is necessary for approximately 50% of the acute erythropoietic response to hypoxic stress. In accord with this, erythroid progenitor cells and reticulocytes were substantially reduced in number in mice lacking HIF function in astrocytes following hypoxic stress. Thus, we have demonstrated that the glial component of the CNS is an essential component of hypoxia-induced erythropoiesis.

The Impact of the Immune System on Tumor: Angiogenesis and Vascular Remodeling

Angiogenesis, the formation of new blood vessels, as well as inflammation with massive infiltration of leukocytes are hallmarks of various tumor entities. Various epidemiological, clinical, and experimental studies have not only demonstrated a link between chronic inflammation and cancer onset but also shown that immune cells from the bone marrow such as tumor-infiltrating macrophages significantly influence tumor progression. Tumor angiogenesis is critical for tumor development as tumors have to establish a blood supply in order to progress. Although tumor cells were first believed to fuel tumor angiogenesis, numerous studies have shown that the tumor microenvironment and infiltrating immune cell subsets are important for regulating the process of tumor angiogenesis. These infiltrates involve the adaptive immune system including several types of lymphocytes as well as cells of the innate immunity such as macrophages, neutrophils, eosinophils, mast cells, dendritic cells, and natural killer cells. Besides their known immune function, these cells are now recognized for their crucial role in regulating the formation and the remodeling of blood vessels in the tumor. In this review, we will discuss for each cell type the mechanisms that regulate the vascular phenotype and its impact on tumor growth and metastasis.

Loss of myeloid cell-derived vascular endothelial growth factor accelerates fibrosis

Tissue injury initiates a complex series of events that act to restore structure and physiological homeostasis. Infiltration of inflammatory cells and vascular remodeling are both keystones of this process. However, the role of inflammation and angiogenesis in general and, more specifically, the significance of inflammatory cell-derived VEGF in this context are unclear. To determine the role of inflammatory cell-derived VEGF in a clinically relevant and chronically inflamed injury, pulmonary fibrosis, we deleted the VEGF-A gene in myeloid cells. In a model of pulmonary fibrosis in mice, deletion of VEGF in myeloid cells resulted in significantly reduced formation of blood vessels; however, it causes aggravated fibrotic tissue damage. This was accompanied by a pronounced decrease in epithelial cell survival and a striking increase in myofibroblast invasion. The drastic increase in fibrosis following loss of myeloid VEGF in the damaged lungs was also marked by increased levels of hypoxia-inducible factor (HIF) expression and Wnt/β-catenin signaling. This demonstrates that the process of angiogenesis, driven by myeloid cell-derived VEGF, is essential for the prevention of fibrotic damage.

Resolution of liver fibrosis requires myeloid cell–driven sinusoidal angiogenesis

Angiogenesis is a key feature of liver fibrosis. Although sinusoidal remodeling is believed to contribute to fibrogenesis, the impact of sinusoidal angiogenesis on the resolution of liver fibrosis remains undefined. Myeloid cells, particularly macrophages, constantly infiltrate the fibrotic liver and can profoundly contribute to remodeling of liver sinusoids. We observe that the development of fibrosis is associated with decreased hepatic vascular endothelial growth factor (VEGF) expression as well as sinusoidal rarefication of the fibrotic scar. In contrast, the resolution of fibrosis is characterized by a rise in hepatic VEGF levels and revascularization of the fibrotic tissue. Genetic ablation of VEGF in myeloid cells or pharmacological inhibition of VEGF receptor 2 signaling prevents this angiogenic response and the resolution of liver fibrosis. We observe increased expression of matrix metalloproteases as well as decreased expression of tissue inhibitor of metalloproteases confined to sinusoidal endothelial cells in response to myeloid cell VEGF. Remarkably, reintroduction of myeloid cell–derived VEGF upon recovery restores collagenolytic acitivity and the resolution of fibrosis. Conclusion: We identify myeloid cell–derived VEGF as a critical regulator of extracellular matrix degradation by liver endothelial cells, thereby unmasking an unanticipated link between angiogenesis and the resolution of fibrosis. (Hepatology 2015;61:2042–2055)