47 research outputs found
MicroRNA-148b Targets the TGF-β Pathway to Regulate Angiogenesis and Endothelial-to-Mesenchymal Transition during Skin Wound Healing
Transforming growth factor beta (TGF-β) is crucial for regulation of the endothelial cell (EC) homeostasis. Perturbation of TGF-β signaling leads to pathological conditions in the vasculature, causing cardiovascular disease and fibrotic disorders. The TGF-β pathway is critical in endothelial-to-mesenchymal transition (EndMT), but a gap remains in our understanding of the regulation of TGF-β and related signaling in the endothelium. This study applied a gain- and loss-of function approach and an in vivo model of skin wound healing to demonstrate that miR-148b regulates TGF-β signaling and has a key role in EndMT, targeting TGFB2 and SMAD2. Overexpression of miR-148b increased EC migration, proliferation, and angiogenesis, whereas its inhibition promoted EndMT. Cytokine challenge decreased miR-148b levels in ECs while promoting EndMT through the regulation of SMAD2. Finally, in a mouse model of skin wound healing, delivery of miR-148b mimics promoted wound vascularization and accelerated closure. In contrast, inhibition of miR-148b enhanced EndMT in wounds, resulting in impaired wound closure that was reversed by SMAD2 silencing. Together, these results demonstrate for the first time that miR-148b is a key factor controlling EndMT and vascularization. This opens new avenues for therapeutic application of miR-148b in vascular and tissue repair
The retinoid agonist Tazarotene promotes angiogenesis and wound healing
Therapeutic angiogenesis is a major goal ofregenerative medicine, but no clinically approved small molecule exists that enhancesnew blood vessel formation. Here we show, using a phenotype-driven high-content imaging screen of an annotated chemical library of 1280 bioactive small molecules, that the retinoid agonist Tazarotene, enhances in vitroangiogenesis, promoting branching morphogenesis, and tubule remodeling. The pro-angiogenic phenotype is mediated by Retinoic Acid Receptor (RAR) but not Retinoic X Receptor(RXR) activation, and is characterized by secretion of the pro-angiogenic factors Hepatocyte Growth Factor (HGF), Vascular Endothelial Growth Factor (VEGFA), Plasminogen Activator, Urokinase (PLAU) and Placental Growth Factor (PGF), and reduced secretion of the antiangiogenic factor Pentraxin-3 (PTX3) from adjacent fibroblasts. In vivo, Tazarotene enhanced the growth of mature and functional microvessels in Matrigel implants and wound healing models, and increased blood flow. Notably, in ear punch wound healing model, Tazarotene promoted tissue repair characterized by rapid ear punch closure with normal-appearing skin containing new hair follicles, and maturing collagen fibers. Our study suggests that Tazarotene, an FDA-approved small molecule, could be potentially exploited for therapeutic applications in neovascularization and wound healing
Comprehensive characterization of the Published Kinase Inhibitor Set
Despite the success of protein kinase inhibitors as approved therapeutics, drug discovery has focused on a small subset of kinase targets. Here we provide a thorough characterization of the Published Kinase Inhibitor Set (PKIS), a set of 367 small-molecule ATP-competitive kinase inhibitors that was recently made freely available with the aim of expanding research in this field and as an experiment in open-source target validation. We screen the set in activity assays with 224 recombinant kinases and 24 G protein-coupled receptors and in cellular assays of cancer cell proliferation and angiogenesis. We identify chemical starting points for designing new chemical probes of orphan kinases and illustrate the utility of these leads by developing a selective inhibitor for the previously untargeted kinases LOK and SLK. Our cellular screens reveal compounds that modulate cancer cell growth and angiogenesis in vitro. These reagents and associated data illustrate an efficient way forward to increasing understanding of the historically untargeted kinome
Downregulation of the Long Non-Coding RNA Meg3 Promotes Angiogenesis After Ischemic Brain Injury by Activating Notch Signaling
Delta-like 4 inhibits choroidal neovascularization despite opposing effects on vascular endothelium and macrophages
A Novel High Content Angiogenesis Assay Reveals That Lacidipine, L-Type Calcium Channel Blocker, Induces In Vitro Vascular Lumen Expansion
Angiogenesis is a critical cellular process toward establishing a functional circulatory system capable of delivering oxygen and nutrients to the tissue in demand. In vitro angiogenesis assays represent an important tool for elucidating the biology of blood vessel formation and for drug discovery applications. Herein, we developed a novel, high content 2D angiogenesis assay that captures endothelial morphogenesis’s cellular processes, including lumen formation. In this assay, endothelial cells form luminized vascular-like structures in 48 h. The assay was validated for its specificity and performance. Using the optimized assay, we conducted a phenotypic screen of a library containing 150 FDA-approved cardiovascular drugs to identify modulators of lumen formation. The screening resulted in several L-type calcium channel blockers being able to expand the lumen space compared to controls. Among these blockers, Lacidipine was selected for follow-up studies. We found that the endothelial cells treated with Lacidipine showed enhanced activity of caspase-3 in the luminal space. Pharmacological inhibition of caspase activity abolished the Lacidipine-enhancing effect on lumen formation, suggesting the involvement of apoptosis. Using a Ca2+ biosensor, we found that Lacipidine reduces the intracellular Ca2+ oscillations amplitude in the endothelial cells at the early stage, whereas Lacidipine blocks these Ca2+ oscillations completely at the late stage. The inhibition of MLCK exhibits a phenotype of lumen expansion similar to that of Lacidipine. In conclusion, this study describes a novel high-throughput phenotypic assay to study angiogenesis. Our findings suggest that calcium signalling plays an essential role during lumen morphogenesis. L-type Ca2+ channel blockers could be used for more efficient angiogenesis-mediated therapies
Phenotype-based high-content chemical library screening identifies small molecules that enhance endothelial cell branching and tubulogenesis
The goal of therapeutic angiogenesis is enhancing new vessel growth that can effectively provide blood supply to ischemic tissues. The endothelial cellular process such as establishment of new connections, branching and tubulogenesis are essential aspects in the growing of a functional vascular network. With the aim of identifying novel small molecule enhancers of these aspects during angiogenesis, we used endothelial tube formation assay to develop an automated assay that enables high-throughput/content screening of small molecule libraries. Methods: The assay is automated and miniaturized into a 384-well format enabling to profile a library of 1280 Pharmacologically Active Compounds (LOPAC1280) for their angiogenic activity. The screen was conducted in duplicate at a final compound concentration of 10 μM using primary Human Umbilical Vein Endothelial Cells. Following cell fixation and staining, the plates were imaged using a high content microscopy system (Operetta). The images were processed and analyzed using fully-automated Metamorph image analysis software. Results: The screen resulted in an initial active compound list of 74 inhibitors and 14 enhancers. Hit characterization revealed 8 enhancer small molecules that reproducibly increased significantly both branching points and total tubule length in a dose dependent manner with low micromolar range (EC50<10μM). Among enhancer hits, there were: GBR-12909 (dopamine reuptake inhibitor), SB 202190 (p38 MAP kinase inhibitor), and Y-27632 (ROCK inhibitor). Moreover, the effect of enhancer hits was examined on multiple cellular aspects of angiogenesis: cell adhesion, proliferation, motility and 3D endothelial sprouting. Conclusions: Our findings suggest that high-throughput/content approach provides a powerful tool to identify new potential targets or to prioritize therapeutic agent candidates for in vivo and clinical testing of pro-angiogenic therapies for ischemic diseases
Phenotype-based high-content chemical library screening identifies small molecules that enhance endothelial cell branching and tubulogenesis
The goal of therapeutic angiogenesis is enhancing new vessel growth that can effectively provide blood supply to ischemic tissues. The endothelial cellular process such as establishment of new connections, branching and tubulogenesis are essential aspects in the growing of a functional vascular network. With the aim of identifying novel small molecule enhancers of these aspects during angiogenesis, we used endothelial tube formation assay to develop an automated assay that enables high-throughput/content screening of small molecule libraries.
Methods: The assay is automated and miniaturized into a 384-well format enabling to profile a library of 1280 Pharmacologically Active Compounds (LOPAC1280) for their angiogenic activity. The screen was conducted in duplicate at a final compound concentration of 10 μM using primary Human Umbilical Vein Endothelial Cells. Following cell fixation and staining, the plates were imaged using a high content microscopy system (Operetta). The images were processed and analyzed using fully-automated Metamorph image analysis software.
Results: The screen resulted in an initial active compound list of 74 inhibitors and 14 enhancers. Hit characterization revealed 8 enhancer small molecules that reproducibly increased significantly both branching points and total tubule length in a dose dependent manner with low micromolar range (EC50<10μM). Among enhancer hits, there were: GBR-12909 (dopamine reuptake inhibitor), SB 202190 (p38 MAP kinase inhibitor), and Y-27632 (ROCK inhibitor). Moreover, the effect of enhancer hits was examined on multiple cellular aspects of angiogenesis: cell adhesion, proliferation, motility and 3D endothelial sprouting.
Conclusions: Our findings suggest that high-throughput/content approach provides a powerful tool to identify new potential targets or to prioritize therapeutic agent candidates for in vivo and clinical testing of pro-angiogenic therapies for ischemic diseases
Phenotypic miRNA screen identifies miRNA-26b to promote the growth and survival of endothelial cells
Endothelial cell proliferation is a crucial event in physiological and pathological angiogenesis. MicroRNAs (miRNAs) have emerged as important modulators of the angiogenic switch. Here, we conducted high content screen of a human miRNA mimic library to identify novel regulators of endothelial cell growth systematically. Several miRNAs were nominated that enhanced or inhibited endothelial cell growth. Out of these, we focused on miR-26b, which is a conserved candidate and expressed in multiple human endothelial cell types. miR-26b overexpression enhances endothelial cell proliferation, migration, and tube formation while inhibition of miR-26b suppressed the proliferative and angiogenic capacity of endothelial cells. A combinatory functional siRNA screening of 48 predicted gene targets revealed that miR-26b enhanced endothelial cell growth and survival through inhibiting PTEN expression. Local administration of miR-26b mimics promoted the growth of new microvessels in the Matrigel plug model. In the mouse model of hindlimb ischemia, miR-26b was found to be downregulated in endothelium in the first week following ischemia, and local overexpression of miR-26b improved the survival of capillaries and muscle fibers in ischemic muscles. Our findings suggest that miR-26b enhances endothelial cell proliferation, survival, and angiogenesis. miR-26b is a potential target for developing novel pro-angiogenic therapeutics in ischemic disease