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

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Induced Pluripotent Stem Cells as Vasculature Forming Entities

Journal of Clinical Medicine ◽

10.3390/jcm8111782 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1782 ◽

Cited By ~ 4

Author(s):

Antonio Palladino ◽

Isabella Mavaro ◽

Carmela Pizzoleo ◽

Elena De Felice ◽

Carla Lucini ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Regenerative Medicine ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Tissue Growth ◽

Safety Issues ◽

Induced Pluripotent

Tissue engineering (TE) pursues the ambitious goal to heal damaged tissues. One of the most successful TE approaches relies on the use of scaffolds specifically designed and fabricated to promote tissue growth. During regeneration the guidance of biological events may be essential to sustain vasculature neoformation inside the engineered scaffold. In this context, one of the most effective strategies includes the incorporation of vasculature forming cells, namely endothelial cells (EC), into engineered constructs. However, the most common EC sources currently available, intended as primary cells, are affected by several limitations that make them inappropriate to personalized medicine. Human induced Pluripotent Stem Cells (hiPSC), since the time of their discovery, represent an unprecedented opportunity for regenerative medicine applications. Unfortunately, human induced Pluripotent Stem Cells-Endothelial Cells (hiPSC-ECs) still display significant safety issues. In this work, we reviewed the most effective protocols to induce pluripotency, to generate cells displaying the endothelial phenotype and to perform an efficient and safe cell selection. We also provide noteworthy examples of both in vitro and in vivo applications of hiPSC-ECs in order to highlight their ability to form functional blood vessels. In conclusion, we propose hiPSC-ECs as the preferred source of endothelial cells currently available in the field of personalized regenerative medicine.

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

Clinical Science ◽

10.1042/cs1000001 ◽

2000 ◽

Vol 100 (1) ◽

pp. 1-12 ◽

Cited By ~ 34

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.

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Origin and diversity of embryonic endothelium/endocardium

10.1093/med/9780198757269.003.0005 ◽

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.

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

Pharmaceutics ◽

10.3390/pharmaceutics11100542 ◽

2019 ◽

Vol 11 (10) ◽

pp. 542 ◽

Cited By ~ 1

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.

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

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.376 ◽

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.

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The galactose-specific receptor system in rat liver during development

Development ◽

10.1242/dev.100.1.13 ◽

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.

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Endocytosis of ricin by rat liver cells in vivo and in vitro is mainly mediated by mannose receptors on sinusoidal endothelial cells

Biochemical Journal ◽

10.1042/bj2910749 ◽

1993 ◽

Vol 291 (3) ◽

pp. 749-755 ◽

Cited By ~ 54

Author(s):

S Magnússon ◽

T Berg

Keyword(s):

Endothelial Cells ◽

Mannose Receptor ◽

Cell Types ◽

Plant Toxin ◽

Sinusoidal Endothelial Cells ◽

Liver Uptake ◽

Parenchymal Cells ◽

Mannose Receptors

Upon intravenous injection into rats, the plant toxin ricin was rapidly cleared from the circulation by the liver. Among the different liver cell populations, most of the injected ricin associated with the sinusoidal endothelial cells (EC), whereas the liver parenchymal cells (PC) and Kupffer cells (KC) yielded minor contributions to the total liver uptake in vivo. Co-injection of mannan strongly inhibited ricin uptake by the EC, showing that it was mediated by mannose receptors. On the other hand, co-injection of lactose, which inhibits the galactose-specific association of ricin with cells, enhanced ricin uptake by the EC. The carbohydrate-dependency of the EC contribution to the uptake of ricin in vivo was reflected in the carbohydrate-dependency of the uptake in vivo by whole liver. In vitro, the EC also endocytosed ricin more efficiently than did the PC or KC. Whereas uptake in vitro in the EC was mainly mannose-specific, uptake in the two other cell types was mainly galactose-specific. Western blotting showed that the mannose receptors of liver non-parenchymal cells are identical with the mannose receptor previously isolated from alveolar macrophages. The mannose receptors are expressed at a higher level in EC than in KC. Ligand blotting showed that, in the presence of lactose, the mannose receptor is the only protein in the EC that binds ricin, and the binding is mannose-specific and Ca(2+)-dependent.

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Evidence of Amniotic Epithelial Cell Differentiation toward Hepatic Sinusoidal Endothelial Cells

Cell Transplantation ◽

10.1177/0963689717727541 ◽

2018 ◽

Vol 27 (1) ◽

pp. 23-30 ◽

Cited By ~ 4

Author(s):

Monica Serra ◽

Michela Marongiu ◽

Antonella Contini ◽

Toshio Miki ◽

Erika Cadoni ◽

...

Keyword(s):

Endothelial Cells ◽

Regenerative Medicine ◽

Cell Cycle Progression ◽

Cell Types ◽

Tube Formation ◽

Sinusoidal Endothelial Cells ◽

Liver Repopulation ◽

Hepatic Sinusoidal Endothelial Cells

Amniotic epithelial cells (AECs) represent a useful and noncontroversial source for liver-based regenerative medicine, as they can differentiate into hepatocytes upon transplantation into the liver. However, the possibility that AECs can differentiate into other liver cell types, such as hepatic sinusoidal endothelial cells (HSECs), has never been assessed. In order to test this hypothesis, rat- and human-derived AECs (rAECs and hAECs, respectively) were subjected to endothelial cell tube formation assay in vitro. Moreover, to evaluate differentiation in vivo, the retrorsine (RS) model of liver repopulation was used. Pyrrolizidine alkaloids (including RS) are known to target both hepatocytes and endothelial cells, inducing cell enlargement and inhibition of cell cycle progression. rAECs and hAECs were able to form capillary-like structures when cultured under proangiogenic conditions. For in vivo experiments, rAECs were obtained from dipeptidyl peptidase type IV (DPP-IV, CD26) donors and were transplanted into the liver of recipient CD26 negative animals pretreated with RS. rAEC-derived cells were engrafted in between hepatocytes and resembled HSECs as assessed by morphological analysis and the pattern of expression of CD26. Donor-derived CD26+ cells coexpressed HSEC markers RECA-1 and SE-1, while they lacked expression of typical hepatocyte markers (i.e., cytochrome P450, hepatocyte nuclear factor 4α). As such, these results provide the first evidence that AECs can respond to proangiogenic signals in vitro and differentiate into HSECs in vivo. Furthermore, they support the conclusion that AECs possesses great plasticity and represents a promising tool in the field of regenerative medicine both in the liver and in other organs.

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A 3D-Printed, Modular, and Parallelized Microfluidic System with Customizable ECM Integration to Investigate the Roles of Basement Membrane Topography on Endothelial Cells

10.26434/chemrxiv.12995846.v1 ◽

2020 ◽

Author(s):

Curtis G. Jones ◽

Tianjiao Huang ◽

Jay H. Chung ◽

Chengpeng Chen

Keyword(s):

Endothelial Cells ◽

Basement Membrane ◽

3D Structure ◽

Microfluidic System ◽

Fully Integrated ◽

Membrane Topography ◽

3D Printed ◽

Easy Integration

Because dysfunctions of endothelial cells are involved in many pathologies, in vitro endothelial cell models for pathophysiological and pharmaceutical studies have been a valuable research tool. Although numerous microfluidic-based endothelial models have been reported, they had the cells cultured on a flat surface without considering the possible 3D structure of the native ECM. Endothelial cells rest on the basement membrane in vivo, which contains an aligned microfibrous topography. To better understand and model the cells, it is necessary to know if and how the fibrous topography can affect endothelial functions. With conventional fully integrated microfluidic apparatus, it is difficult to include additional topographies in a microchannel. Therefore, we developed a modular microfluidic system by 3D-printing and electrospinning, which enabled easy integration and switching of desired ECM topographies. Also, with standardized designs, the system allowed for high flow rates up to 4000 µL/min, which covered the full shear stress range for endothelial studies. We found that the aligned fibrous topography on the ECM altered arginine metabolism in endothelial cells, and thus increased nitric oxide production. To the best of our knowledge, this is the most versatile endothelial model that has been reported, and the new knowledge generated thereby lays a groundwork for future endothelial research and modeling.

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