scholarly journals Culture of isolated bovine megakaryocytes on reconstituted basement membrane matrix leads to proplatelet process formation

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 912-924 ◽  
Author(s):  
KS Topp ◽  
F Tablin ◽  
J Levin
Keyword(s):  
Basement Membrane ◽  
Bone Marrow Cells ◽  
Human Platelets ◽  
Velocity Sedimentation ◽  
Thin Filaments ◽  
Membrane Matrix ◽  
Process Formation ◽  
Vitro Model

Abstract We have enriched for bovine megakaryocytes and identified a culture system that may provide an in vitro model for platelet formation. Mature megakaryocytes with an unusually high ploidy distribution were obtained after differential centrifugation and velocity sedimentation of bone marrow cells through gradients of bovine serum albumin (BSA). The cell membranes of isolated megakaryocytes and megakaryocytes in vivo stained with antisera to human platelets and human platelet membrane GPIIIa. The microenvironment of bovine megakaryocytes in vivo was investigated using antibodies to types I and IV collagen and laminin. In an attempt to duplicate the microenvironment in vitro, bovine megakaryocytes were cultured on a reconstituted basement membrane matrix (Matrigel). The cells adhered to the gel, extended radial lamellipodia, and occasionally formed lengthy pseudopodia. Ultrastructural examination of these cells showed widening and coalescence of the megakaryocyte demarcation membranes (DMS), and inclusion of platelet granules, thin filaments, and microtubules in the processes. Very few DMS vesicles were present distally in the processes. The culture of megakaryocytes on a reconstituted basement membrane may closely model the in vivo megakaryocyte microenvironment and allow the study of thrombocytopoiesis in vitro.

scholarly journals Culture of isolated bovine megakaryocytes on reconstituted basement membrane matrix leads to proplatelet process formation

Blood ◽  
1990 ◽  
Vol 76 (5) ◽  
pp. 912-924 ◽  
Author(s):  
KS Topp ◽  
F Tablin ◽  
J Levin
Keyword(s):  
Basement Membrane ◽  
Bone Marrow Cells ◽  
Human Platelets ◽  
Velocity Sedimentation ◽  
Thin Filaments ◽  
Membrane Matrix ◽  
Process Formation ◽  
Vitro Model

We have enriched for bovine megakaryocytes and identified a culture system that may provide an in vitro model for platelet formation. Mature megakaryocytes with an unusually high ploidy distribution were obtained after differential centrifugation and velocity sedimentation of bone marrow cells through gradients of bovine serum albumin (BSA). The cell membranes of isolated megakaryocytes and megakaryocytes in vivo stained with antisera to human platelets and human platelet membrane GPIIIa. The microenvironment of bovine megakaryocytes in vivo was investigated using antibodies to types I and IV collagen and laminin. In an attempt to duplicate the microenvironment in vitro, bovine megakaryocytes were cultured on a reconstituted basement membrane matrix (Matrigel). The cells adhered to the gel, extended radial lamellipodia, and occasionally formed lengthy pseudopodia. Ultrastructural examination of these cells showed widening and coalescence of the megakaryocyte demarcation membranes (DMS), and inclusion of platelet granules, thin filaments, and microtubules in the processes. Very few DMS vesicles were present distally in the processes. The culture of megakaryocytes on a reconstituted basement membrane may closely model the in vivo megakaryocyte microenvironment and allow the study of thrombocytopoiesis in vitro.

scholarly journals Endothelial Cell-Pericyte Interactions Stimulate Basement Membrane Matrix Assembly: Influence on Vascular Tube Remodeling, Maturation, and Stabilization

2011 ◽  
Vol 18 (1) ◽  
pp. 68-80 ◽  
Author(s):  
Amber N. Stratman ◽  
George E. Davis
Keyword(s):  
Growth Factor ◽  
Basement Membrane ◽  
Basement Membranes ◽  
Heparin Binding ◽  
Matrix Assembly ◽  
Matrix Deposition ◽  
Vascular Basement Membrane ◽  
Membrane Matrix

AbstractExtracellular matrix synthesis and deposition surrounding the developing vasculature are critical for vessel remodeling and maturation events. Although the basement membrane is an integral structure underlying endothelial cells (ECs), few studies, until recently, have been performed to understand its formation in this context. In this review article, we highlight new data demonstrating a corequirement for ECs and pericytes to properly deposit and assemble vascular basement membranes during morphogenic events. In EC only cultures or under conditions whereby pericyte recruitment is blocked, there is a lack of basement membrane assembly, decreased vessel stability (with increased susceptibility to pro-regressive stimuli), and increased EC tube widths (a marker of dysfunctional EC-pericyte interactions). ECs and pericytes both contribute basement membrane components and, furthermore, both cells induce the expression of particular components as well as integrins that recognize them. The EC-derived factors—platelet derived growth factor-BB and heparin binding-epidermal growth factor—are both critical for pericyte recruitment to EC tubes and concomitant vascular basement membrane formationin vitroandin vivo. Thus, heterotypic EC-pericyte interactions play a fundamental role in vascular basement membrane matrix deposition, a critical tube maturation event that is altered in key disease states such as diabetes and cancer.

Platelets and a platelet-released factor enhance endothelial barrier

1992 ◽  
Vol 263 (6) ◽  
pp. L670-L678 ◽  
Author(s):  
F. R. Haselton ◽  
J. S. Alexander
Keyword(s):  
Apparent Molecular Weight ◽  
Human Platelets ◽  
Endothelial Barrier ◽  
Sodium Fluorescein ◽  
Similar Response ◽  
Beta Agonists ◽  
Heat Stable ◽  
Vitro Model

The role of platelets in the maintenance of endothelial barrier is examined in an in vitro model of the microvasculature. Human platelets (6,000/microliters) perfused through a cell column of endothelial-covered microcarriers decrease paracellular permeability of sodium fluorescein (mol wt 342) to 63% of baseline values. This effect is reversible and a second application and removal of platelets produces a similar response. This effect occurs within 5 min and reverses within 10 min after platelet removal. The reduction in permeability is not due to mechanical obstruction of endothelial junctions, since the number of recirculating platelets is not reduced and releasate from unstimulated 2-h platelet incubations also decreases permeability. Releasate from platelets stimulated with 0.1 U/ml of thrombin for 15 min have the same permeability reducing effect. In this system, the platelet factors serotonin (10(-3) M) and ADP (10(-4) M) have no effect on permeability. However, the platelet factors adenosine (10(-4) M), ATP (10(-5) M), and beta-agonists decrease permeability. None of these appear to account for platelet permeability activity, since activity is not blocked by agents directed against these mediators (adenosine deaminase, apyrase, 8-phenyltheophylline, or propranolol). The active factor(s) is stable at -20 degrees C, heat stable, sensitive to trypsin, and has an apparent molecular weight > 100. We conclude that unstimulated platelets release a factor(s) that enhances endothelial barrier in vitro and may be important in maintenance of the normal in vivo barrier.

Synthesis of heparan sulfate proteoglycans by the isolated glomerulus

1988 ◽  
Vol 66 (10) ◽  
pp. 1078-1085 ◽  
Author(s):  
Douglas M. Templeton ◽  
Marina Khatchatourian
Keyword(s):  
Basement Membrane ◽  
Heparan Sulfate ◽  
Glomerular Basement Membrane ◽  
Time Course ◽  
In Vitro Model ◽  
Proteoglycan Synthesis ◽  
Proteoglycan Aggregates ◽  
Vitro Model

Incorporation of [35S]sulfate into newly synthesized macromolecules was studied in the isolated rat glomerulus and found to be linear between 6 and 24 h. When whole glomeruli were treated under conditions that dissociate proteoglycan aggregates, >90% of incorporated label was extracted. Of this, 80–90% was found to be the heparan sulfate proteoglycan. Similarly, a linear incorporation of [35S]sulfate into a glomerular basement membrane-enriched fraction was due almost entirely to proteoheparan sulfate. This predominance of heparan sulfate among the newly sulfated glycosaminoglycans has previously been observed in vivo and in the perfused kidney, but different patterns have hitherto been described in vitro. The present results suggest that under certain conditions, the isolated glomerulus is a suitable in vitro model for the study of proteoglycan synthesis. The pattern of incorporation of proteoglycans into the glomerular basement membrane reflects the time course and distribution of their synthesis by the whole glomerulus.

scholarly journals Endothelial-derived PDGF-BB and HB-EGF coordinately regulate pericyte recruitment during vasculogenic tube assembly and stabilization

Blood ◽  
2010 ◽  
Vol 116 (22) ◽  
pp. 4720-4730 ◽  
Author(s):  
Amber N. Stratman ◽  
Amy E. Schwindt ◽  
Kristine M. Malotte ◽  
George E. Davis
Keyword(s):  
Growth Factor ◽  
Basement Membrane ◽  
Growth Factor Receptor ◽  
Platelet Derived Growth Factor ◽  
Heparin Binding ◽  
Matrix Assembly ◽  
Matrix Deposition ◽  
Membrane Matrix

Recently, we reported a novel system whereby human pericytes are recruited to endothelial cell (EC)–lined tubes in 3-dimensional (3D) extracellular matrices to stimulate vascular maturation including basement membrane matrix assembly. Through the use of this serum-free, defined system, we demonstrate that pericyte motility within 3D collagen matrices is dependent on the copresence of ECs. Using either soluble receptor traps consisting of the extracellular ligand-binding domains of platelet-derived growth factor receptor β, epidermal growth factor receptor (EGFR), and ErbB4 receptors or blocking antibodies directed to platelet-derived growth factor (PDGF)–BB, or heparin-binding EGF-like growth factor (HB-EGF), we show that both of these EC-derived ligands are required to control pericyte motility, proliferation, and recruitment along the EC tube ablumenal surface. Blockade of pericyte recruitment causes a lack of basement membrane matrix deposition and, concomitantly, increased vessel widths. Combined inhibition of PDGF-BB and HB-EGF–induced signaling in quail embryos leads to reduced pericyte recruitment to EC tubes, decreased basement membrane matrix deposition, increased vessel widths, and vascular hemorrhage phenotypes in vivo, in support of our findings in vitro. In conclusion, we report a dual role for EC-derived PDGF-BB and HB-EGF in controlling pericyte recruitment to EC-lined tubes during developmental vascularization events.

scholarly journals Blood platelet formation in vitro. The role of the cytoskeleton in megakaryocyte fragmentation

1990 ◽  
Vol 97 (1) ◽  
pp. 59-70 ◽  
Author(s):  
F. Tablin ◽  
M. Castro ◽  
R.M. Leven
Keyword(s):  
Membrane Vesicles ◽  
Peripheral Zone ◽  
Membrane System ◽  
Blood Platelet ◽  
Collagen Gels ◽  
Process Formation ◽  
Vitro Model ◽  
Platelet Formation

We have developed a unique in vitro model that promotes differentiation of megakaryocytes into platelets. When megakaryocytes isolated from guinea pig bone marrow were cultured on hydrated rat tail collagen gels, cells spontaneously formed elongated, beaded processes that fragmented to yield cytoplasmic pieces with the same size and internal composition as individual platelets. Addition of nocodazole at the initiation of cultures blocked process formation, while addition of nocodazole to cells with previously established processes resulted in their retraction. The addition of taxol to cultures resulted in abnormally thick processes that were tightly adherent to the underlying substratum, and did not bead or fragment. Cytochalasin D accelerated process formation and fragmentation of megakaryocytes cultured on collagen gels by twofold. On the basis of these results, we propose a model for platelet formation in culture that involves the following steps: adherence of megakaryocytes to the underlying extracellular matrix; dilation of the demarcation membrane system and breakdown of the actin-rich peripheral zone; microtubule-based extension of pseudopodia, which are no longer adherent to the substratum; and fragmentation into platelets by the coalescence and fusion of demarcation membrane vesicles with the plasma membrane. We feel that this distinctive culture system closely approximates thrombocytopoiesis in vivo, thus allowing detailed elucidation of this important process.

The Effects of Brinolase (Fibrinolytic Enzyme from Aspergillus Oryzae) on Platelet Aggregation of Dog and Man

1972 ◽  
Vol 28 (01) ◽  
pp. 031-048 ◽  
Author(s):  
W. H. E Roschlau ◽  
R Gage
Keyword(s):  
Platelet Aggregation ◽  
Aspergillus Oryzae ◽  
Degradation Products ◽  
Blood Platelet ◽  
Human Platelets ◽  
Fibrinolytic Enzyme ◽  
Inhibitory Effects ◽  
Blood Platelet Aggregation

SummaryInhibition of blood platelet aggregation by brinolase (fibrinolytic enzyme from Aspergillus oryzae) has been demonstrated with human platelets in vitro and with dog platelets in vivo and in vitro, using both ADP and collagen as aggregating stimuli. It is suggested that the optimal inhibitory effects of brinolase occur indirectly through the generation of plasma fibrinogen degradation products, without compromising platelet viability, rather than by direct proteolysis of platelet structures.

scholarly journals Functional and Transcriptional Adaptations of Blood Monocytes Recruited to the Cystic Fibrosis Airway Microenvironment In Vitro

2021 ◽  
Vol 22 (5) ◽  
pp. 2530
Author(s):  
Bijean D. Ford ◽  
Diego Moncada Giraldo ◽  
Camilla Margaroli ◽  
Vincent D. Giacalone ◽  
Milton R. Brown ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Bactericidal Activity ◽  
Scavenger Receptor ◽  
Receptor Expression ◽  
Blood Monocytes ◽  
Bacterial Killing ◽  
Blood Neutrophils ◽  
Vitro Model

Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.

scholarly journals Radiofrequency Irradiation Modulates TRPV1-Related Burning Sensation in Rosacea

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1424
Author(s):  
Seyeon Oh ◽  
Myeongjoo Son ◽  
Joonhong Park ◽  
Donghwan Kang ◽  
Kyunghee Byun
Keyword(s):  
Burning Sensation ◽  
In Vivo Model ◽  
Molecular Evidence ◽  
Exposed Skin ◽  
Heat Treated ◽  
Vitro Model ◽  
Effective Use ◽  
Inflammatory Molecules

Rosacea is a skin inflammatory condition that is accompanied by not only redness and flushing but also unseen symptoms, such as burning, stinging, and itching. TRPV1 expression in UVB-exposed skin can lead to a painful burning sensation. Upregulated TRPV1 expression helps release neuropeptides, including calcitonin gene-related peptide, pituitary adenylate cyclase-activating polypeptide, and vasoactive intestinal peptide, which can activate macrophage and inflammatory molecules. In this study, we found that radiofrequency (RF) irradiation reduced TRPV1 activation and neuropeptide expression in a UVB-exposed in vivo model and UVB- or heat-treated in an in vitro model. RF irradiation attenuated neuropeptide-induced macrophage activation and inflammatory molecule expression. Interestingly, the burning sensation in the skin of UVB-exposed mice and patients with rosacea was significantly decreased by RF irradiation. These results can provide experimental and molecular evidence on the effective use of RF irradiation for the burning sensation in patients with rosacea.

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