scholarly journals Utilizing a Kidney-Targeting Peptide to Improve Renal Deposition of a Pro-Angiogenic Protein Biopolymer

Pharmaceutics ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 542 ◽  
Author(s):  
Fakhri Mahdi ◽  
Alejandro R. Chade ◽  
Gene L. Bidwell
Keyword(s):  
Endothelial Cells ◽  
Cell Types ◽  
Endothelial Cell Proliferation ◽  
Microvascular Endothelial Cell ◽  
Similar Distribution ◽  
Target Tissue ◽  
Cell Binding ◽  
Targeting Peptide

Elastin-like polypeptides (ELP) are versatile protein biopolymers used in drug delivery due to their modular nature, allowing fusion of therapeutics and targeting agents. We previously developed an ELP fusion with vascular endothelial growth factor (VEGF) and demonstrated its therapeutic efficacy in translational swine models of renovascular disease and chronic kidney disease. The goal of the current work was to refine renal targeting and reduce off-target tissue deposition of ELP–VEGF. The ELP–VEGF fusion protein was modified by adding a kidney-targeting peptide (KTP) to the N-terminus. All control proteins (ELP, KTP–ELP, ELP–VEGF, and KTP–ELP–VEGF) were also produced to thoroughly assess the effects of each domain on in vitro cell binding and activity and in vivo pharmacokinetics and biodistribution. KTP–ELP–VEGF was equipotent to ELP–VEGF and free VEGF in vitro in the stimulation of primary glomerular microvascular endothelial cell proliferation, tube formation, and extracellular matrix invasion. The contribution of each region of the KTP–ELP–VEGF protein to the cell binding specificity was assayed in primary human renal endothelial cells, tubular epithelial cells, and podocytes, demonstrating that the VEGF domain induced binding to endothelial cells and the KTP domain increased binding to all renal cell types. The pharmacokinetics and biodistribution of KTP–ELP–VEGF and all control proteins were determined in SKH-1 Elite hairless mice. The addition of KTP to ELP slowed its in vivo clearance and increased its renal deposition. Furthermore, addition of KTP redirected ELP–VEGF, which was found at high levels in the liver, to the kidney. Intrarenal histology showed similar distribution of all proteins, with high levels in blood vessels and tubules. The VEGF-containing proteins also accumulated in punctate foci in the glomeruli. These studies provide a thorough characterization of the effects of a kidney-targeting peptide and an active cytokine on the biodistribution of these novel biologics. Furthermore, they demonstrate that renal specificity of a proven therapeutic can be improved using a targeting peptide.

scholarly journals Tropism Modification of Adenovirus Vectors by Peptide Ligand Insertion into Various Positions of the Adenovirus Serotype 41 Short-Fiber Knob Domain

2006 ◽  
Vol 81 (6) ◽  
pp. 2688-2699 ◽  
Author(s):  
Andrea Hesse ◽  
Daniela Kosmides ◽  
Roland E. Kontermann ◽  
Dirk M. Nettelbeck
Keyword(s):  
Gene Transfer ◽  
Cell Types ◽  
Short Fiber ◽  
Peptide Ligand ◽  
Cell Binding ◽  
Adenovirus Serotype ◽  
C Terminus ◽  
Genetic Targeting

ABSTRACT Recombinant adenoviruses have emerged as promising agents in therapeutic gene transfer, genetic vaccination, and viral oncolysis. Therapeutic applications of adenoviruses, however, would benefit substantially from targeted virus cell entry, for example, into cancer or immune cells, as opposed to the broad tropism that adenoviruses naturally possess. Such tropism modification of adenoviruses requires the deletion of their natural cell binding properties and the incorporation of cell binding ligands. The short fibers of subgroup F adenoviruses have recently been suggested as a tool for genetic adenovirus detargeting based on the reduced infectivity of corresponding adenovectors with chimeric fibers in vitro and in vivo. The goal of our study was to determine functional insertion sites for peptide ligands in the adenovirus serotype 41 (Ad41) short fiber knob. With a model peptide, CDCRGDCFC, we could demonstrate that ligand incorporation into three of five analyzed loops of the knob, namely, EG, HI, and IJ, is feasible without a loss of fiber trimerization. The resulting adenovectors showed enhanced infectivity for various cell types, which was superior to that of viruses with the same peptide fused to the fiber C terminus. Strategies to further augment gene transfer efficacy by extension of the fiber shaft, insertion of tandem copies of the ligand peptide, or extension of the ligand-flanking linkers failed, indicating that precise ligand positioning is pivotal. Our study establishes that internal ligand incorporation into a short-shafted adenovirus fiber is feasible and suggests the Ad41 short fiber with ligand insertion into the top (IJ loop) or side (EG and HI loops) of the knob domain as a novel platform for genetic targeting of therapeutic adenoviruses.

Controlled Adipose-derived Stromal Cells Differentiation into Adipose and Endothelial Cells in a 3D Structure Established by Cell-assembly Technique

2009 ◽  
Vol 24 (1_suppl) ◽  
pp. 31-47 ◽  
Author(s):  
Mingen Xu ◽  
Yongnian Van ◽  
Haixia Liu ◽  
Rui Yag ◽  
Xiaohong Wang
Keyword(s):  
Endothelial Cells ◽  
3D Structure ◽  
Spherical Shape ◽  
Cell Types ◽  
Tissue Growth ◽  
Cell Assembly ◽  
Adipose Tissue Engineering ◽  
Assembly Technique

One of the major obstacles in engineering thick and complex tissues while vascularizing tissues in vitro is to maintain cell viability during tissue growth and structural organization. Adipose-derived stromal (ADS) cells were used to establish a multicellular system through a cell-assembly technique. Attempts were made to control ADS cells differentiation into different targeted cell types according to their positions within an orderly 3D structure. Oil red 0 staining confirmed that the ADS cells in the structure differentiated into adipocytes with a spherical shape while immunostaining tests confirmed that the endothelial growth factor induced ADS cells on the walls of channels differentiated into mature endothelial cells and then organized into tubular structures throughout the engineered 3D structure. The endothelin-1 and nitric oxide release rules of the endothelial cells were coincidental with those in vivo. This study provides a new approach to engineer orderly endothelial vessel networks in vitro and has potential applications in adipose-tissue engineering.

Role of inducible nitric oxide synthase in the regulation of leucocyte recruitment

2000 ◽  
Vol 100 (1) ◽  
pp. 1-12 ◽  
Author(s):  
Michael J. HICKEY
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Nitric Oxide Synthase ◽  
Cell Types ◽  
Adhesive Interactions ◽  
Oxide Synthase ◽  
Leucocyte Recruitment

Constitutively produced nitric oxide released by endothelial cells has been shown to act as an endogenous agent which inhibits the rolling and adhesion of leucocytes in the microcirculation. However, during various types of inflammation, expression of the inducible form of nitric oxide synthase (iNOS) can dramatically increase the amount of nitric oxide present in tissues. Furthermore, as iNOS can be expressed by a wide variety of cell types, the distribution of nitric oxide is likely to be altered relative to that in unstimulated tissue. Under these conditions, it is less well understood whether iNOS-derived nitric oxide retains the anti-adhesive capabilities of constitutively produced nitric oxide. This review summarizes work done to examine this issue. Three main approaches have been used. In vitro studies have examined the role of iNOS in adhesive interactions between stimulated endothelial cells and leucocytes, providing evidence of an anti-adhesive effect of iNOS. In addition, the role of iNOS has been examined in vivo in animal models of inflammation using pharmacological iNOS inhibitors. These experiments were extended by the advent of the iNOS-deficient (iNOS-/-) mouse. Intravital microscopy studies of these mice have indicated that, under conditions of low-dose endotoxaemia, iNOS-derived nitric oxide can inhibit leucocyte rolling and adhesion. The potential mechanisms for these effects are discussed. In contrast, several other studies have observed either no effect or an enhancing effect of iNOS on inflammatory leucocyte recruitment. Taken together, these studies suggest that the importance of iNOS in modulating leucocyte recruitment can vary according to the type of inflammatory response.

Tumour growth inhibition and anti-angiogenic effects using curcumin correspond to combined PDE2 and PDE4 inhibition

2015 ◽  
Vol 113 (02) ◽  
pp. 319-328 ◽  
Author(s):  
Abdurazzag Abusnina ◽  
Thérèse Keravis ◽  
Qingwei Zhou ◽  
Hélène Justiniano ◽  
Annelise Lobstein ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Tumour Growth ◽  
Umbilical Vein ◽  
Camp Level ◽  
Pde4 Inhibitor ◽  
Endothelial Cell Proliferation ◽  
And Migration ◽  
Proliferation And Migration

SummaryVascular endothelial growth factor (VEGF) plays a major role in angiogenesis by stimulating endothelial cells. Increase in cyclic AMP (cAMP) level inhibits VEGF-induced endothelial cell proliferation and migration. Cyclic nucleotide phosphodiesterases (PDEs), which specifically hydrolyse cyclic nucleotides, are critical in the regulation of this signal transduction. We have previously reported that PDE2 and PDE4 up-regulations in human umbilical vein endothelial cells (HUVECs) are implicated in VEGF-induced angiogenesis and that inhibition of PDE2 and PDE4 activities prevents the development of the in vitro angiogenesis by increasing cAMP level, as well as the in vivo chicken embryo angiogenesis. We have also shown that polyphenols are able to inhibit PDEs. The curcumin having anti-cancer properties, the present study investigated whether PDE2 and PDE4 inhibitors and curcumin could have similar in vivo anti-tumour properties and whether the anti-angiogenic effects of curcumin are mediated by PDEs. Both PDE2/PDE4 inhibitor association and curcumin significantly inhibited in vivo tumour growth in C57BL/6N mice. In vitro, curcumin inhibited basal and VEGF-stimulated HUVEC proliferation and migration and delayed cell cycle progression at G0/G1, similarly to the combination of selective PDE2 and PDE4 inhibitors. cAMP levels in HUVECs were significantly increased by curcumin, similarly to rolipram (PDE4 inhibitor) and BAY-60–550 (PDE2 inhibitor) association, indicating cAMP-PDE inhibitions. Moreover, curcumin was able to inhibit VEGF-induced cAMP-PDE activity without acting on cGMP-PDE activity and to modulate PDE2 and PDE4 expressions in HUVECs. The present results suggest that curcumin exerts its in vitro anti-angiogenic and in vivo antitumour properties through combined PDE2 and PDE4 inhibition.

Origin and diversity of embryonic endothelium/endocardium

2018 ◽  
Author(s):  
LeShana SaintJean ◽  
H.S. Baldwin
Keyword(s):  
Endothelial Cells ◽  
Heart Development ◽  
Vascular Endothelial Cells ◽  
Coronary Vessels ◽  
Cell Types ◽  
Genetic Profile ◽  
Vascular Endothelial ◽  
Tremendous Progress

The endocardium represents a distinct population of endothelial cells that arises during the initiation of heart development. Endocardial cells can easily be distinguished from most of the other cardiac cell types. However, endocardial and vascular endothelial cells contain a similar genetic profile that limits the ability to study each group independently. Despite these limitations, tremendous progress has been made in identifying the different roles of endocardial cells throughout heart development. Initial studies focused on the origin of endocardial cells and their role in valvulogenesis, trabeculation, and formation of the ventricular and atrial septum. With the advancement of microscopy and the availability of endocardial specific reporter models (in vitro and in vivo) we have obtained more insight into the molecular, structural, and functional complexity of the endocardium. Additional studies have demonstrated how the endocardium is also involved in the development of coronary vessels within the compact myocardium and in heart regeneration.

scholarly journals Bovine Organospecific Microvascular Endothelial Cell Lines as New and Relevant In Vitro Models to Study Viral Infections

2020 ◽  
Vol 21 (15) ◽  
pp. 5249 ◽  
Author(s):  
Anne-Claire Lagrée ◽  
Fabienne Fasani ◽  
Clotilde Rouxel ◽  
Marine Pivet ◽  
Marie Pourcelot ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Lines ◽  
T Antigen ◽  
In Vitro Studies ◽  
Microvascular Endothelial Cell ◽  
Target Cells ◽  
Microvascular Endothelial

Microvascular endothelial cells constitute potential targets for exogenous microorganisms, in particular for vector-borne pathogens. Their phenotypic and functional variations according to the organs they are coming from provide an explanation of the organ selectivity expressed in vivo by pathogens. In order to make available relevant tools for in vitro studies of infection mechanisms, our aim was to immortalize bovine organospecific endothelial cells but also to assess their permissivity to viral infection. Using transfection with SV40 large T antigen, six bovine microvascular endothelial cell lines from various organs and one macrovascular cell line from an umbilical cord were established. They display their own panel of endothelial progenitor/mature markers, as assessed by flow cytometry and RT-qPCR, as well as the typical angiogenesis capacity. Using both Bluetongue and foot-and-mouth disease viruses, we demonstrate that some cell lines are preferentially infected. In addition, they can be transfected and are able to express viral proteins such as BTV8-NS3. Such microvascular endothelial cell lines bring innovative tools for in vitro studies of infection by viruses or bacteria, allowing for the study of host-pathogen interaction mechanisms with the actual in vivo target cells. They are also suitable for applications linked to microvascularization, such as anti-angiogenic and anti-tumor research, growing fields in veterinary medicine.

scholarly journals Preparation and Characterization of a Novel Chimeric Protein VEGI-CTT inEscherichia coli

2008 ◽  
Vol 2008 ◽  
pp. 1-9 ◽  
Author(s):  
Jiping Cai ◽  
Ruili Wei ◽  
Jinwei Cheng
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Recombinant Expression ◽  
Tumor Therapy ◽  
Vascular Endothelial Cell ◽  
Chimeric Protein ◽  
Growth Inhibitor ◽  
Endothelial Cell Proliferation

Vascular endothelial cell growth inhibitor (VEGI) is a recently identified antiangiogenic cytokine that belongs to the TNF superfamily, and could effectively inhibit endothelial cell proliferation and angiogenesis. Synthetic peptide CTT (CTTHWGFTLC) has been found to suppress invasion and migration of both tumor and endothelial cells by potent and selective inhibition of MMP-2 and MMP-9. To prepare chimeric protein VEGI-CTT for more potent antitumor therapy, the recombinant expression vector pET-VEGI-CTT was constructed. This fusion protein was expressed in inclusion bodies inE. coliBL21 (DE3), and was refolded and purified by immobilized metal affinity chromatography using His-tag. Purified VEGI-CTT protein was characterized by proliferation assays of the endothelial cells and casein degradation assay in vitro. The results demonstrated that chimeric protein VEGI-CTT had a potent activity of antiangiogenesis through inhibiting the proliferation of endothelial cells, and could effectively reduce the activity of MMP-2 and MMP-9. The preliminarily in vivo study demonstrated that chimeric protein VEGI-CTT had more potent antitumor activity than VEGI and/or CTT peptide against CA46 human lymphoma xenografts in nude mice. Thus, these facts that are derived from the present study suggest that the chimeric protein VEGI-CTT may be used for tumor therapy in the future.

Abstract 376: Downregulation of Endogenous Apelinergic Axis Mediates Endothelial and Organismal Senescence

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Rahul Rai ◽  
Asish K Ghosh ◽  
Layton H Smith ◽  
Douglas E Vaughan
Keyword(s):  
Endothelial Cells ◽  
Replicative Senescence ◽  
Cell Types ◽  
Ang Ii ◽  
Systematic Assessment ◽  
Multiple Cell ◽  
Deficient Mice

Background: Apelinergic signaling is a recently discovered GPCR mediated pathway. Endothelial cells are the main source of endogenous apelin (apln) while apelin receptor (aplnr) is present on multiple cell types. Since the role of endogenous apelinergic pathway within the context of senescence is largely unknown, we ask if levels of apln- aplnr vary with aging. We also investigate the effects of downregulated apln- aplnr on cellular and organismal aging. Approach and Results: To assess variations in endogenous apln- aplnr with aging, we compared their levels in 1 month (young) and 1 year old (old) WT mice. We noticed significant downregulation of apln- aplnr with chronological senescence in multiple tissues. Expression of apelin was also reduced with replicative senescence of endothelial cells. L-NAME administration, a model of stress induced senescence, also repressed aortic and cardiac apln. To address the mechanism involved in downregulation of apln- aplnr, we administered young wild type mice with Ang II. After a week of Ang II, there was significant downregulation of aortic apln and aplnr. Ang II and TGF-β also repressed apln and aplnr in vitro . Next we investigated the effects of downregulated apln on endothelial cells. In response to shRNA mediated apelin knockdown, cells exhibited slower proliferation and upregulated senescence associated markers. We observed similar results when endothelial aplnr was blocked with an antagonist, ML221. In addition, apln and aplnr deficient mice also exhibited features of cardiovascular aging, including ventricular hypertrophy and lower EF. Importantly, aplnr deficient mice at eight months of age were also hypertensive. Conclusion: We provide a systematic assessment of senescence associated variation in levels of apln- aplnr. We demonstrate the role of Ang II- TGF-β axis in downregulating apln- aplnr during chronological and stress induced senescence in vivo and in vitro . We propose a novel model of Ang II- TGF-β induced senescence. Where in, with aging Ang II and TGF-β repress endogenous apln- aplnr. Downregulation of endogenous apln- aplnr axis decreases beneficial “youthful” effects of apelin, resulting in endothelial dysfunction and accelerated organismal aging.

The galactose-specific receptor system in rat liver during development

Development ◽  
1987 ◽  
Vol 100 (1) ◽  
pp. 13-22
Author(s):  
L. Dini ◽  
L. Conti-Devirgiliis ◽  
S. Russo-Caia
Keyword(s):  
Endothelial Cells ◽  
Ligand Binding ◽  
Rat Liver ◽  
Binding Capacity ◽  
Specific Binding ◽  
Cell Types ◽  
Receptor System ◽  
In Situ Experiments

The number and distribution of galactose-specific binding sites were investigated in rat liver cells during perinatal development. Ligand binding to hepatocytes, macrophages and endothelial cells was followed with in vitro and in situ experiments by electron microscopy, using lactosylated bovine serum albumin adsorbed onto 5 nm colloidal gold particles as ligand. Binding capacity, starting at a late stage of fetal development, is very low both on the hepatocyte and on the macrophage surface, which show single particles statistically distributed. By contrast, bound particles are absent from fetal endothelial cells, which also lack the typical coated regions. In vivo, experiments at 37 degrees C show that endocytosis occurs to some extent in prenatal life. These results indicate that the expression of galactose-specific receptors' activity on the different liver cell types follows different developmental patterns, which are independently modulated.

Keratinocyte growth factor induces angiogenesis and protects endothelial barrier function

1999 ◽  
Vol 112 (12) ◽  
pp. 2049-2057
Author(s):  
P. Gillis ◽  
U. Savla ◽  
O.V. Volpert ◽  
B. Jimenez ◽  
C.M. Waters ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Barrier Function ◽  
Keratinocyte Growth Factor ◽  
Mitogen Activated Protein Kinase ◽  
Microvascular Endothelial Cell ◽  
Vascular Permeability Factor ◽  
Small Vessels

Keratinocyte growth factor (KGF), also called fibroblast growth factor-7, is widely known as a paracrine growth and differentiation factor that is produced by mesenchymal cells and has been thought to act specifically on epithelial cells. Here it is shown to affect a new cell type, the microvascular endothelial cell. At subnanomolar concentrations KGF induced in vivo neovascularization in the rat cornea. In vitro it was not effective against endothelial cells cultured from large vessels, but did act directly on those cultured from small vessels, inducing chemotaxis with an ED50 of 0.02-0.05 ng/ml, stimulating proliferation and activating mitogen activated protein kinase (MAPK). KGF also helped to maintain the barrier function of monolayers of capillary but not aortic endothelial cells, protecting against hydrogen peroxide and vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) induced increases in permeability with an ED50 of 0.2-0.5 ng/ml. These newfound abilities of KGF to induce angiogenesis and to stabilize endothelial barriers suggest that it functions in microvascular tissue as it does in epithelial tissues to protect them against mild insults and to speed their repair after major damage.

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