Pavel Uhrin

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.

Disruption of the protein C inhibitor gene results in impaired spermatogenesis and male infertility

Protein C inhibitor (PCI) is a nonspecific, heparin-binding serpin (serine protease inhibitor) that inactivates many plasmatic and extravascular serine proteases by forming stable 1:1 complexes. Proteases inhibited by PCI include the anticoagulant activated protein C, the plasminogen activator urokinase, and the sperm protease acrosin. In humans PCI circulates as a plasma protein but is also present at high concentrations in organs of the male reproductive tract. The biological role of PCI has not been defined so far. However, the colocalization of high concentrations of PCI together with several of its target proteases in the male reproductive tract suggests a role of PCI in reproduction. We generated mice lacking PCI by homologous recombination. Here we show that PCI(-/-) mice are apparently healthy but that males of this genotype are infertile. Infertility was apparently caused by abnormal spermatogenesis due to destruction of the Sertoli cell barrier, perhaps due to unopposed proteolytic activity. The resulting sperm are malformed and are morphologically similar to abnormal sperm seen in some cases of human male infertility. This animal model might therefore be useful for analyzing the molecular bases of these human conditions.

Cardiac malformations and myocardial abnormalities in podoplanin knockout mouse embryos: Correlation with abnormal epicardial development.

Epicardium and epicardium‐derived cells have been shown to be necessary for myocardial differentiation. To elucidate the function of podoplanin in epicardial development and myocardial differentiation, we analyzed podoplanin knockout mouse embryos between embryonic day (E) 9.5 and E15.5 using immunohistochemical differentiation markers, morphometry, and three‐dimensional reconstructions. Podoplanin null mice have an increased embryonic lethality, possibly of cardiac origin. Our study reveals impairment in the development of the proepicardial organ, epicardial adhesion, and spreading and migration of the epicardium‐derived cells. Mutant embryos show a hypoplastic and perforated compact and septal myocardium, hypoplastic atrioventricular cushions resulting in atrioventricular valve abnormalities, as well as coronary artery abnormalities. The epicardial pathology is correlated with reduced epithelial–mesenchymal transformation caused by up‐regulation of E‐cadherin, normally down‐regulated by podoplanin. Our results demonstrate a role for podoplanin in normal cardiac development based on epicardial–myocardial interaction. Abnormal epicardial differentiation and reduced epithelial–mesenchymal transformation result in deficient epicardium‐derived cells leading to myocardial pathology and cardiac anomalies. Developmental Dynamics 237:847–857, 2008.

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.

Sexing and multiple genotype analysis from a single cell of bovine embryo

We described a procedure for multiple genotype analysis (determination of sex and of three genetic markers) from a single cell derived from bovine preimplantation embryo. It consists of primer extension preamplification-polymerase chain reaction (PEP-PCR) and subsequent single assay or multiplex PCR. A single blastomere that was isolated by microaspiration from bovine embryos at the 16- to 32-cell stage then was lysed and was subjected to the PEP-PCR. When testing 75 embryos, efficiency of genotyping by standard PCR for kappa-casein, growth hormone (GH) and prolactin (PRL) polymorphic alleles was 91, 88 and 89%, respectively. Sexing efficiency in the multiplex PCR was 91%, based on the amplification of Y-specific locus using kappa-casein internal standard. The microaspiration of a single blastomere was shown not to be invasive for the embryos. It did not alter their development potential in vitro (P > 0.05), as was seen by obtaining a similar percentage of embryos developing further into the blastocyst stage in the group subjected to biopsy (44/75, 59%) and in the control group of embryos (30/50, 60%).

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.

Podoplanin deficient mice show a rhoa-related hypoplasia of the sinus venosus myocardium including the sinoatrial node

We investigated the role of podoplanin in development of the sinus venosus myocardium comprising the sinoatrial node, dorsal atrial wall, and primary atrial septum as well as the myocardium of the cardinal and pulmonary veins. We analyzed podoplanin wild‐type and knockout mouse embryos between embryonic day 9.5–15.5 using immunohistochemical marker podoplanin; sinoatrial‐node marker HCN4; myocardial markers MLC‐2a, Nkx2.5, as well as Cx43; coelomic marker WT‐1; and epithelial‐to‐mesenchymal transformation markers E‐cadherin and RhoA. Three‐dimensional reconstructions were made and myocardial morphometry was performed. Podoplanin mutants showed hypoplasia of the sinoatrial node, primary atrial septum, and dorsal atrial wall. Myocardium lining the wall of the cardinal and pulmonary veins was thin and perforated. Impaired myocardial formation is correlated with abnormal epithelial‐to‐mesenchymal transformation of the coelomic epithelium due to up‐regulated E‐cadherin and down‐regulated RhoA, which are controlled by podoplanin. Our results demonstrate an important role for podoplanin in development of sinus venosus myocardium. Developmental Dynamics 238:183–193, 2009.

Annexin VI isoforms are differentially expressed in mammalian tissues

Purified annexin VI migrates as a closely spaced doublet when separated by SDS-PAGE. Immunolocalization of annexin VI in heart demonstrates staining at different defined subcellular compartments. Moss et al. identified two cDNAs, one having an insert of 18 bases encoding VAAEIL at the beginning of repeat domain seven. We have identified the splicing site of the murine annexin VI gene. It contains a single small exon of 18 bases. PCR amplification of reverse transcribed (RT) mRNA demonstrates that, in all tissues tested, the mRNA isoform containing the insert is predominant. Site-directed antibody was produced and affinity purified against peptides reflecting the insert and deletion sequences. The steady-state isoform ratio of the annexin VI protein is consistent with the RT-PCR data. Chromatographic experiments demonstrate that the annexin VI protein isoforms have biochemical differences. These differences may target the individual isoforms to unique cellular compartments or alter functional properties.