Processing of cell-bound insulin by capillary and macrovascular endothelial cells in culture

1985 ◽  
Vol 248 (2) ◽  
pp. E244-E251 ◽  
Author(s):  
K. D. Dernovsek ◽  
R. S. Bar
Keyword(s):  
Endothelial Cells ◽  
High Performance ◽  
Potential Role ◽  
Umbilical Vein ◽  
Gel Filtration ◽  
Storage Site ◽  
Surface Receptors ◽  
Chromatography Analysis ◽  
Degradation Rates ◽  
Cellular Processing

The processing of cell-bound insulin was determined in endothelial cells cultured from three large blood vessels (human umbilical vein, bovine pulmonary artery, and bovine aorta) and one microvascular source (bovine fat capillaries). Cells were exposed to monoiodinated TyrA14-insulin, the rates of dissociation of cell-bound TyrA14-insulin determined, and cell alteration of insulin assessed by gel filtration and high-performance liquid chromatography analysis. We found that 1) overall degradation rates of insulin are low for all cultured endothelial cells, 2) cell-bound insulin is rapidly processed to a nonreceptor compartment and then rapidly dissociated from all cells, primarily as biologically intact insulin, and 3) degradation of cell-bound insulin, although relatively low, does occur in endothelial cells with the least degradation by capillary cells. The presence of specific surface receptors for insulin on endothelial cells coupled with rapid cellular processing of intact insulin is consistent with a potential role for endothelial cells in either the transport of intact insulin out of the bloodstream or as a regional storage site for intact hormone.

scholarly journals Collagen scaffolds functionalized with triple-helical peptides support 3D HUVEC culture

2020 ◽  
Vol 7 (5) ◽  
pp. 471-482
Author(s):  
Jean-Daniel Malcor ◽  
Emma J Hunter ◽  
Natalia Davidenko ◽  
Daniel V Bax ◽  
Ruth Cameron ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Three Dimensional ◽  
Cell Types ◽  
Cross Linking ◽  
Collagen Scaffolds ◽  
Mechanical Stiffness ◽  
Helical Peptides ◽  
Surface Receptors ◽  
Engineered Tissues

Abstract Porous biomaterials which provide a structural and biological support for cells have immense potential in tissue engineering and cell-based therapies for tissue repair. Collagen biomaterials that can host endothelial cells represent promising tools for the vascularization of engineered tissues. Three-dimensional collagen scaffolds possessing controlled architecture and mechanical stiffness are obtained through freeze–drying of collagen suspensions, followed by chemical cross-linking which maintains their stability. However, cross-linking scaffolds renders their biological activity suboptimal for many cell types, including human umbilical vein endothelial cells (HUVECs), by inhibiting cell–collagen interactions. Here, we have improved crucial HUVEC interactions with such cross-linked collagen biomaterials by covalently coupling combinations of triple-helical peptides (THPs). These are ligands for collagen-binding cell-surface receptors (integrins or discoidin domain receptors) or secreted proteins (SPARC and von Willebrand factor). THPs enhanced HUVEC adhesion, spreading and proliferation on 2D collagen films. THPs grafted to 3D-cross-linked collagen scaffolds promoted cell survival over seven days. This study demonstrates that THP-functionalized collagen scaffolds are promising candidates for hosting endothelial cells with potential for the production of vascularized engineered tissues in regenerative medicine applications.

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism

2003 ◽  
Vol 284 (6) ◽  
pp. H1978-H1984 ◽  
Author(s):  
M. Ursula Norman ◽  
Shane B. Reeve ◽  
Vincent Dive ◽  
A. Ian Smith ◽  
Rebecca A. Lew
Keyword(s):  
Endothelial Cells ◽  
High Performance ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Hybrid Cell ◽  
Fluorescent Substrate ◽  
Intact Cells ◽  
Aortic Endothelial Cells

The closely related metalloendopeptidases EC 3.4.24.15 (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.

PURIFICATION AND CHARACTERIZATION OF PAI-1 FROM HUMAN ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
N A Booth ◽  
I R MacGregor ◽  
N R Hunter ◽  
B Bennett
Keyword(s):  
Endothelial Cells ◽  
Specific Activity ◽  
Umbilical Vein ◽  
Gel Filtration ◽  
Rabbit Antiserum ◽  
Protein Band ◽  
Single Chain ◽  
Step Procedure ◽  
Umbilical Vein Endothelial Cells ◽  
Pai 1

Platelet α-granules and endothelial cells contain an inhibitor of plasminogen activator, which inhibits both t-PA and u-PA. The inhibitor (PAI-1) is detectable after SDS-PAGE and zymography on fibrin/plasmin-ogen/u-PA detector gels. We have purified endothelial PAI-1 by a simple two-step procedure. Serum-free conditioned medium from human umbilical vein endothelial cells, grown in microcarrier culture, was fractionated on Sephadex CM-50, a cation exchanger, followed by gel filtration on Sephacryl S-200. Aprotinin was included throughout the procedure to maintain the activity of the inhibitor. The PAI-1 was purified 2000-fold with a recovery of about 7%. The purified protein had a specific activity of 8500 U/mg protein and the activity could be stimulated 14-fold by 4M guanidine. The purified PAI-1, of M 48000, was a single-chain glycoprotein.The ptoduct wasapparently homogeneous on a silver-stained SDS-polyacrylamide gel, the protein band co-migrating with PAI activity. Further, a rabbit antiserum raised against the purified PAI-1 revealed only a single band on immunoblots of material from each stage of the purification. The immunoglobulin fraction ofthe antiserum, incorporated into the detector gel for zymographic analysis, neutralised the inhibitor from plasma, platelets and endothelial cells, confirming their identity. Preincubation of PAI-1 from these sources with the immunoglobulin prevented formation of a complex with t-PA or u-PA. This purification procedure, in which no denaturants are employed, provides a homogeneous preparation of PAI-1 that is useful for studies on the stimulatory effects of denaturants. The antiserum raised has allowed the development of a sensitive ELISA, specific for PAI-1.

Ecto-enzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells

1997 ◽  
Vol 273 (3) ◽  
pp. C918-C927 ◽  
Author(s):  
J. Mateo ◽  
M. T. Miras-Portugal ◽  
P. Rotllan
Keyword(s):  
Endothelial Cells ◽  
Kinetic Parameters ◽  
High Performance ◽  
Vascular Endothelial Cells ◽  
Vascular Endothelial ◽  
Diadenosine Polyphosphates ◽  
Chromatography Analysis ◽  
Extracellular Signaling ◽  
Effects Of Ph ◽  
Membrane Bound

We investigated the extracellular degradation of diadenosine polyphosphates (ApnA) by cultured adrenomedullary endothelial cells using fluorogenic analogs of ApnA, the di(1,N6-ethenoadenosine) 5',5"'-P1,Pn-polyphosphates [epsilon-(ApnA)]. Kinetic parameters of epsilon-(ApnA) cleavage and effects of pH, ions, and inhibitors were determined by continuous fluorometric assays, using suspensions of endothelial cells grown on Cytodex-1 microspheres. Ecto-enzyme kinetic parameters for epsilon-(Ap3A), epsilon-(Ap4A), and epsilon-(Ap5A) hydrolysis are as follows: Michaelis-Menten constants of 0.39 +/- 0.07, 0.42 +/- 0.09, and 0.37 +/- 0.05 microM respectively, and maximal velocities of 26.1 +/- 6.8, 74.2 +/- 16.4, and 24.4 +/- 3.4 pmol.min-1.10(6) cells-1, respectively. ApnA and guanosine 5',5"'-P1,P4-tetraphosphate behave as competitor substrates of epsilon-(Ap4A) hydrolysis. The ectoenzyme is activated by Mg2+ and Mn2+ and inhibited by Ca2+, F-, adenosine 5'-tetraphosphate, adenosine 5'-O-(3-thiotriphosphate), and suramin. Optimum pH is around 9.0. High-performance liquid chromatography analysis reveals that the ecto-enzyme hydrolyzes epsilon-(ApnA) to give epsilon-adenosine-5'(n-1)-phosphate and epsilon-AMP, which are then further catabolized up to epsilon-adenosine via the membrane-bound nucleotidase system ecto-ATPase, ecto-ADPase (or apyrase), and ecto-5'-nucleotidase. The endothelial ecto-diadenosine polyphosphate hydrolase studied here exhibits different kinetic parameters and sensitivity to ions with respect to the enzyme from the tissue-related neurochromaffin cells. These different properties may be important in the extracellular signaling by ApnA.

scholarly journals One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

2020 ◽  
Author(s):  
Yongling Qin ◽  
Qiqian Li ◽  
Fengfeng Luo ◽  
Yue Fu ◽  
Haiyan He
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Chromatography Analysis ◽  
Sds Page ◽  
One Step ◽  
Optimal Reaction Temperature ◽  
Fusarium Chlamydosporum ◽  
Layer Chromatography ◽  
Optimal Reaction

Abstract A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4-10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

scholarly journals Localization of endothelin-1-like immunoreactivity in human placenta.

1993 ◽  
Vol 41 (4) ◽  
pp. 535-541 ◽  
Author(s):  
B M Wilkes ◽  
M Susin ◽  
P F Mento
Keyword(s):  
Endothelial Cells ◽  
Human Placenta ◽  
High Performance ◽  
Culture Medium ◽  
Umbilical Vein ◽  
Calf Serum ◽  
Reversed Phase ◽  
Immunoperoxidase Staining ◽  
Immunoperoxidase Technique ◽  
Endothelin 1

We examined the distribution of endothelin-1-like immunoreactivity in human placenta, using the immunoperoxidase technique. A specific polyclonal antibody to endothelin-1 was raised in rabbits, which recognized endothelin-1 and its precursor molecule, big endothelin. Immunoperoxidase staining revealed that endothelin-1-like immunoreactivity was widely distributed in the placenta. Endothelin-1-like immunoreactivity was present in endothelial cells of capillaries of the microvilli and in small- and medium-sized arteries and veins. The distribution of endothelin-1-like immunoreactivity was similar to the distribution of Factor VIII-related antigen, which stains endothelial cells. The nature of endothelin in the human placenta was further examined with cultured umbilical vein endothelial cells. Endothelial cells released endothelin-like material into the culture medium. The amount of endothelin-like material varied directly with time of incubation and the amount of fetal calf serum in the culture medium. Fractionation of the endothelin-1-like material by reversed-phase high-performance liquid chromatography (HPLC) and quantitation by radioimmunoassay (RIA) revealed that endothelin-like immunoreactivity co-eluted with endothelin-1 but not with big endothelin-1. We conclude that endothelin-1-like immunoreactivity is widely distributed in vascular endothelium of the human placenta. These data are compatible with a role for endothelin as an autocrine or paracrine modulator of vascular tone in the human placenta.

Activation of human endothelial cells from specific vascular beds induces the release of a FVIII storage pool

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4902-4909 ◽  
Author(s):  
Tina Shahani ◽  
Renaud Lavend'homme ◽  
Aernout Luttun ◽  
Jean-Marie Saint-Remy ◽  
Kathelijne Peerlinck ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Umbilical Artery ◽  
Umbilical Vein ◽  
Storage Site ◽  
Human Endothelial Cells ◽  
Myristate Acetate ◽  
Main Site ◽  
Vascular Beds ◽  
Lung Endothelial Cells ◽  
And Storage

AbstractAlthough the liver is known to be the main site of factor VIII (FVIII) production, other organs are probably also important for the regulation of FVIII secretion. However, the study of the regulation of extrahepatic FVIII production has been hampered by the lack of definitive identification of human tissues able to secrete FVIII. Recent studies have shown that lung endothelial cells can synthesize FVIII. We therefore studied the production of FVIII by endothelial cells purified from other vascular beds. Because physiologic stress results in a rapid elevation of FVIII, we also investigated whether endothelial cells can store FVIII and secrete it after treatment with agonists. Microvascular endothelial cells from lung, heart, intestine, and skin as well as endothelial cells from pulmonary artery constitutively secreted FVIII and released it after treatment with phorbol-myristate acetate and epinephrine. By contrast, endothelial cells from the aorta, umbilical artery and umbilical vein did not constitutively secrete FVIII or release it after treatment with agonists, probably because of a lack of FVIII synthesis. Extrahepatic endothelial cells from certain vascular beds therefore appear to be an important FVIII production and storage site with the potential to regulate FVIII secretion in chronic and acute conditions.

Mechanisms Of Viral-Induced Endothelial Injury

1981 ◽  
Author(s):  
H M Friedman
Keyword(s):  
Endothelial Cells ◽  
Herpes Simplex ◽  
Persistent Infection ◽  
Influenza A ◽  
Umbilical Vein ◽  
Complement Components ◽  
Human Umbilical Vein ◽  
Surface Receptors ◽  
The Third

We examined the role of common human viruses as inducors of injury to human umbilical vein or bovine-thoracic aorta endothelial cells grown In vitro. Indicators of infection included endothelial cytopathology, viral replication and viral antigen detection by immunofluorescence. Herpes simplex, adeno, measles and parainfluenza viruses infected both human venous and bovine aorta cells. Mumps, polio, and echo 9 viruses grew only in human venous cells, while coxsackie B4 replicated only in bovine aorta endothelium. Several viruses (CMV, influenza A, RSV) infected neither type of endothelial cell.We examined one virus, herpes simplex type 1 (HSV) in some detail and showed that HSV can establish a persistent infection of bovine aorta endothelial cells in vitro. When HSV was inoculated at a multiplicity of infection (MOI) of 0.1, the amount of infections virus produced by the culture fluctuated over a 3 month period. By the third week after inoculation the endothelial monolayer had recovered and no infectious virus was detected for an additional 2 months. However, by the third month, typical herpetic changes reappeared, 103 plaque forming units of intracellular and supernatant virus was detected, and by infectious cell center assays, approximately 1% of cells produced virus.In separate experiments, human umbilical vein endothelial cells were inoculated with HSV (MOI of 1.0) and examined for induction of surface receptors for the Fc portion of IgG and for C3b. Using 51Cr-labeled sheep erythrocytes coated with IgG or with IgM and purified complement components (C1, C4, C2, C3) an Fc and C3b receptor were detected on the infected cells. These receptors were distinct from one another as demonstrated by blocking experiments using purified Fc fragments, and consisted, at least in part, of viral antigens since antibody to HSV blocked function of both receptors.These studies demonstrate that viruses can produce acute or persistent infection of endothelial cells and can induce receptors for IgG and complement on the cell’s surface.

scholarly journals One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

2020 ◽  
Author(s):  
Yongling Qin ◽  
Qiqian Li ◽  
Fengfeng Luo ◽  
Yue Fu ◽  
Haiyan He
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Chromatography Analysis ◽  
Sds Page ◽  
One Step ◽  
Optimal Reaction Temperature ◽  
Fusarium Chlamydosporum ◽  
Layer Chromatography ◽  
Optimal Reaction

Abstract A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4-10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

scholarly journals One-step purification of two novel thermotolerant β-1,4-glucosidases from a newly isolated strain of Fusarium chlamydosporum HML278 and their characterization

AMB Express ◽  
2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yongling Qin ◽  
Qiqian Li ◽  
Fengfeng Luo ◽  
Yue Fu ◽  
Haiyan He
Keyword(s):  
High Performance ◽  
Gel Filtration ◽  
Hydrolytic Activity ◽  
Chromatography Analysis ◽  
Sds Page ◽  
One Step ◽  
Optimal Reaction Temperature ◽  
Fusarium Chlamydosporum ◽  
Layer Chromatography ◽  
Optimal Reaction

Abstract A newly identified cellulase-producing Fusarium chlamydosporum HML278 was cultivated under solid-state fermentation of sugarcane bagasse, and two new β-glucosides enzymes (BG FH1, BG FH2) were recovered from fermentation solution by modified non-denaturing active gel electrophoresis and gel filtration chromatography. SDS-PAGE analysis showed that the molecular weight of BG FH1 and BG FH2 was 93 kDa and 52 kDa, respectively, and the enzyme activity was 5.6 U/mg and 11.5 U/mg, respectively. The optimal reaction temperature of the enzymes was 60 ℃, and the enzymes were stable with a temperature lower than 70 ℃. The optimal pH of the purified enzymes was 6.0, and the enzymes were stable between pH 4–10. Km and Vmax values ​​were 2.76 mg/mL and 20.6 U/mg for pNPG, respectively. Thin-layer chromatography and high-performance liquid chromatography analysis showed that BG FH1and BG FH2 had hydrolysis activity toward cellobiose and could hydrolyze cellobiose into glucose. In addition, both enzymes exhibited transglycoside activity, which could use glucose to synthesize cellobiose and cellotriose, and preferentially synthesize alcohol. In conclusion, our study demonstrated that F. chlamydosporum HML278 produces heat-resistant β-glucosidases with both hydrolytic activity and transglycosidic activity, and these β-glucosidases have potential application in bioethanol and papermaking industries.

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