Johannes M. Breuss

Discovery and resupply of pharmacologically active plant-derived natural products: A review.

Medicinal plants have historically proven their value as a source of molecules with therapeutic potential, and nowadays still represent an important pool for the identification of novel drug leads. In the past decades, pharmaceutical industry focused mainly on libraries of synthetic compounds as drug discovery source. They are comparably easy to produce and resupply, and demonstrate good compatibility with established high throughput screening (HTS) platforms. However, at the same time there has been a declining trend in the number of new drugs reaching the market, raising renewed scientific interest in drug discovery from natural sources, despite of its known challenges. In this survey, a brief outline of historical development is provided together with a comprehensive overview of used approaches and recent developments relevant to plant-derived natural product drug discovery. Associated challenges and major strengths of natural product-based drug discovery are critically discussed. A snapshot of the advanced plant-derived natural products that are currently in actively recruiting clinical trials is also presented. Importantly, the transition of a natural compound from a “screening hit” through a “drug lead” to a “marketed drug” is associated with increasingly challenging demands for compound amount, which often cannot be met by re-isolation from the respective plant sources. In this regard, existing alternatives for resupply are also discussed, including different biotechnology approaches and total organic synthesis. While the intrinsic complexity of natural product-based drug discovery necessitates highly integrated interdisciplinary approaches, the reviewed scientific developments, recent technological advances, and research trends clearly indicate that natural products will be among the most important sources of new drugs also in the future.

Oxidized Phospholipids Stimulate Angiogenesis Via Autocrine Mechanisms, Implicating a Novel Role for Lipid Oxidation in the Evolution of Atherosclerotic Lesions

Angiogenesis is a common feature observed in advanced atherosclerotic lesions. We hypothesized that oxidized phospholipids (OxPLs), which accumulate in atherosclerotic vessels can stimulate angiogenesis. We found that oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) stimulated the formation of sprouts from endothelial cell spheroids and promoted growth of capillaries into Matrigel plugs in mice. OxPLs stimulated expression of vascular endothelial growth factor (VEGF) in vivo and in several normal and tumor cell types in vitro. In addition, OxPAPC upregulated cyclooxygenase (COX)-2 and interleukin (IL)-8. COX-2 inhibitors, as well as blocking antibodies to IL-8 suppressed activation of sprouting by OxPAPC. We conclude that OxPAPC stimulates angiogenesis via autocrine mechanisms involving VEGF, IL-8, and COX-2–generated prostanoids. Our data suggest that accumulation of OxPLs may contribute to increased growth of blood capillaries in advanced lesions, thus leading to progression and destabilization of atherosclerotic plaques.

Vascular Endothelial Growth Factor Is Induced by the Inflammatory Cytokines Interleukin-6 and Oncostatin M in Human Adipose Tissue In Vitro and in Murine Adipose Tissue In Vivo

Objectives—It is believed that adipose tissue acts as an endocrine organ by producing inflammatory mediators and thereby contributes to the increased cardiovascular risk seen in obesity. A link between adipose tissue mass and angiogenesis has been suggested. Vascular endothelial growth factor (VEGF) seems to be implicated in this process. Members of the glycoprotein (gp)130 ligand family regulate VEGF expression in other cells. Methods and Results—We used tissue explants as well as primary cultures of preadipocytes and adipocytes from human subcutaneous and visceral adipose tissue to investigate whether the gp130 ligands oncostatin M (OSM), interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and cardiotrophin-1 (CT-1) regulate VEGF expression in human adipose tissue. Human subcutaneous and visceral adipose tissue responded to treatment with IL-6 and OSM with a significant increase in VEGF production. Human preadipocytes were isolated from subcutaneous and visceral adipose tissue. Adipocyte-differentiation was induced by hormone-supplementation. All cell types responded to IL-6 and OSM with a robust increase in VEGF protein production and a similar increase in VEGF-specific mRNA. Furthermore, IL-1&bgr; synergistically enhanced the effect of OSM on VEGF production. AG-490, a JAK/STAT inhibitor, abolished the OSM-dependent VEGF induction almost completely. In mice, IL-6 and OSM increased serum levels of VEGF and VEGF mRNA and vessel density in adipose tissue. Conclusion—We speculate that the inflammatory cytokines IL-6 and OSM might support angiogenesis during adipose tissue growth by upregulating VEGF.

Vascular Endothelial Growth Factor Receptor-2–Induced Initial Endothelial Cell Migration Depends on the Presence of the Urokinase Receptor

The angiogenic response of endothelial cells initiated by different growth factors is accompanied by assembly of cell surface–bound proteolytic machinery as a prerequisite for focal invasion. We have shown previously how the vascular endothelial growth factor (VEGF) initiates proteolysis by activation of pro-urokinase (pro-PA) via the VEGF receptor-2 (VEGFR-2). We now show that the cell surface receptor of the uPA-system, the urokinase receptor (uPAR), is redistributed to focal adhesions at the leading edge of endothelial cells in response to VEGF. VEGF165 and VEGF-E, both interacting with VEGFR-2, but not PlGF exclusively stimulating VEGFR-1, induce within minutes internalization of uPAR via an LDL receptor–like molecule, dependent on generation of active uPA and the presence of plasminogen activator inhibitor-1 (PAI-1). uPAR seems to play a pivotal role in VEGFR-2–induced endothelial cell migration because cleavage of surface uPAR impaired the migratory response of endothelial cells toward VEGF-E, but not toward PlGF.

Sustained Expression of Early Growth Response Protein-1 Blocks Angiogenesis and Tumor Growth

Transient induction of the transcription factor early growth response protein-1 (EGR-1) plays a pivotal role in the transcriptional response of endothelial cells to the angiogenic growth factors vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which are produced by most tumors and are involved in the angiogenic switch. We report here that sustained expression of EGR-1 by recombinant adenoviruses in endothelial cells, however, leads to the specific induction of potent feedback inhibitory mechanisms, including strong up-regulation of transcriptional repressors, negative cell cycle check point effectors, proteins with established antiangiogenic activity, and several proapoptotic genes. Sustained EGR-1 expression consistently leads to an antiangiogenic state characterized by an altered responsiveness to VEGF and bFGF and a striking inhibition of sprouting and tubule formation in vitro. Furthermore, EGR-1-expressing viruses potently inhibit cell invasion and vessel formation in the murine Matrigel model and repress tumor growth in a murine fibrosarcoma model. We propose that gene therapy involving sustained EGR-1 expression may constitute a novel therapeutic principle in the treatment of cancer due to the simultaneous induction of multiple pathways of antiangiogenesis, growth arrest, and apoptosis induction in proliferating cells leading to preferential inhibition of angiogenesis and tumor growth.

The N Terminus of Mannose 6-Phosphate/Insulin-like Growth Factor 2 Receptor in Regulation of Fibrinolysis and Cell Migration

Leukocyte migration to sites of inflammation is a multistep process involving transient adhesion to the endothelium followed by cell surface-controlled proteolysis for transmigration through the vessel wall and chemotactic movement within tissues. One of the key players in this machinery appears to be the urokinase-type plasminogen activator (uPA)/uPA receptor system. The role of uPA and its receptor (CD87) in plasminogen (Plg) activation, cell adhesion, and chemotaxis is well established; however, less is known of how these activities are regulated. Here we provide evidence that the mannose 6-phosphate/insulin-like growth factor 2 receptor (CD222) controls CD87-mediated functions. Expression of human CD222 in CD222−/− mouse fibroblasts down-regulated Plg activation, cell adhesion, and chemotaxis induced by the uPA/CD87 system. In addition, we demonstrate that the N-terminal region of CD222, which is similar to the Plg-binding site of streptokinase, plays a crucial role in binding of CD87 and Plg. A peptide derived from this region in CD222 is able to disrupt the physical interaction of CD222 with CD87 and, furthermore, mimics the inhibitory effects of CD222 on CD87 functions. Taken together, our results indicate a novel role for CD222 in regulation of fibrinolysis, cell adhesion, and migration.

VEGF-induced endothelial cell migration requires urokinase receptor (uPAR)-dependent integrin redistribution

AIMS Vascular endothelial growth factor (VEGF)-initiated angiogenesis requires coordinated proteolytic degradation of extracellular matrix provided by the urokinase plasminogen activator/urokinase receptor (uPA/uPAR) system and regulation of cell migration provided by integrin-matrix interaction. In this study, we investigated the mechanisms underlying the uPAR-dependent modulation of VEGF-induced endothelial migration. METHODS AND RESULTS We used flow cytometry to quantify integrins at the cell surface. Stimulation of human and murine endothelial cells with VEGF resulted in internalization of α5β1-integrins. Micropatterning and immunocytochemistry revealed co-clustering of uPAR and α5β1-integrins and retrieval via clathrin-coated vesicles. It was also contingent on receptors of the low-density lipoprotein receptor (LDL-R) family. VEGF-induced integrin redistribution was inhibited by elimination of uPAR from the endothelial cell surface or by inhibitory peptides that block the uPAR-integrin interaction. Under these conditions, the migratory response of endothelial cells upon VEGF stimulation was impaired both in vitro and in vivo. CONCLUSIONS The observations indicate that uPAR is an essential component of the network through which VEGF controls endothelial cell migration. uPAR is a bottleneck through which the VEGF-induced signal must be funnelled for both focused proteolytic activity at the leading edge and for redistribution of integrins.

Indirubin-3′-Monoxime Blocks Vascular Smooth Muscle Cell Proliferation by Inhibition of Signal Transducer and Activator of Transcription 3 Signaling and Reduces Neointima Formation In Vivo

Objective—Our goal was to examine the influence of indirubin-3′-monoxime (I3MO), a natural product–derived cyclin-dependent kinase inhibitor, on vascular smooth muscle cell (VSMC) proliferation in vitro, experimentally induced neointima formation in vivo, and related cell signaling pathways. Methods and Results—I3MO dose-dependently inhibited platelet-derived growth factor (PDGF)–BB-induced VSMC proliferation by arresting cells in the G0/G1 phase of the cell cycle as assessed by 5-bromo-2′-deoxyuridine incorporation and flow cytometry. PDGF-induced activation of the kinases Akt, Erk1/2, and p38MAPK was not affected. In contrast, I3MO specifically blocked PDGF-, interferon-&ggr;-, and thrombin-induced phosphorylation of signal transducer and activator of transcription 3 (STAT3). Human endothelial cells (EA.hy926) responded to I3MO with increased endothelial nitric oxide synthase activity as assessed via [14C]l-arginine/[14C]l-citrulline conversion. The specific STAT3 inhibitor Stattic led to decreased VSMC proliferation, and transient expression of a constitutively active form of STAT3 overcame the I3MO-induced cell cycle arrest in mouse embryonic fibroblasts. In a murine femoral artery cuff model, I3MO prevented neointima formation while reducing STAT3 phosphorylation and the amount of proliferating Ki67-positive cells. Conclusion—I3MO represses PDGF- and thrombin-induced VSMC proliferation and, in vivo, neointima formation, likely because it specifically blocks STAT3 signaling. This profile and its positive effect on endothelial NO production turns I3MO into a promising lead compound to prevent restenosis.

VEGF-initiated angiogenesis and the uPA/uPAR system

Angiogenesis involves a series of tightly regulated cellular processes initiated primarily by the vascular endothelial growth factor (VEGF). The urokinase-type plasminogen activator system, consisting of the urokinase-type plasminogen activator (uPA), its cellular receptor uPAR and its inhibitor PAI-1, participates in the realization of these VEGF-induced processes by activating pericellular proteolysis, increasing vascular permeability and by supporting endothelial cell proliferation and migration.

Identification of plumericin as a potent new inhibitor of the NF-κB pathway with anti-inflammatory activity in vitro and in vivo.

The transcription factor NF‐κB orchestrates many pro‐inflammatory signals and its inhibition is considered a promising strategy to combat inflammation. Here we report the characterization of the natural product plumericin as a highly potent inhibitor of the NF‐κB pathway with a novel chemical scaffold, which was isolated via a bioactivity‐guided approach, from extracts of Himatanthus sucuuba, an Amazonian plant traditionally used to treat inflammation‐related disorders.