Directions for improvement of substitute heart valves: National Heart, Lung, and Blood Institute's Working Group report on heart valves.
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Equipment Design↗
Biomedical subjects
L V McIntire
Publications and source records attributed to L V McIntire.
Endothelial P-selectin and VCAM-1 each can function as primary adhesive mechanisms for T cells under conditions of flow.
This study demonstrates that endothelial P-selectin and vascular cell adhesion molecule 1 (VCAM-1), but not intercellular adhesion molecule 1 (ICAM-1), are capable of supporting extensive primary adhesion of T cells under flow. To address this issue, we used human umbilical vein endothelial cells (HUVECs) stimulated with histamine, interleukin-4 (IL-4), or interferon-gamma (IFN-gamma) that provide essentially a P-selectin, VCAM-1, or ICAM-1 surface, respectively, in a physiologically relevant cell type. Monoclonal antibody (mAb) blockade studies were carried out to confirm the specificity of these adhesive interactions and rule out a number of other potentially important adhesion molecules. Quantitation of adhesion showed that almost all of the interacting T cells rolled on histamine-stimulated HUVECs or CHO-P cell monolayers. In contrast, approximately 20% of the total interacting T cells with 24-h IL-4-treated HUVECs were firmly adherent. mAb blocking experiments revealed that T cell adhesion to IL-4-treated HUVECs is alpha 4-VCAM-1 dependent. Furthermore, mAb 4B9 directed against domain 1 of VCAM-1 eliminated adhesion, suggesting that alpha 4 integrins may not interact with either the alternatively spliced domain 4 of VCAM-1 or fibronectin in this process. At a wall shear stress of 2 dyn/cm2, the mean T cell rolling velocities were significantly lower on 24-h IL-4-activated HUVECs (10.2 +/- 2.6 microgm/s) compared with either CHO-P cells (15.6 +/- 3.1 microm/s) or histamine-stimulated HUVECs (16.6 +/- 6.1 microm/s). ICAM-1, expressed on the surface of 24-h IFN-gamma-activated HUVECs pretreated with an anti-VCAM-1 mAb to eliminate any VCAM-1-dependent contribution, did not support T cell adhesion under shear conditions. Together these data indicate that T cell primary adhesion can be mediate d by both endothelial P-selectin and VCAM-1 but not ICAM-1. alpha 4 integrins are highly versatile molecules, capable of initiating T cell rolling interactions and mediating firm arrest on activated endothelium.
Animals↗
Differential adhesion of metastatic RAW117 large-cell lymphoma cells under static or hydrodynamic conditions: role of integrin alpha(v) beta3.
RGD-containing substrates were used to study static and hydrodynamic adhesion of murine RAW117 large-cell lymphoma sublines with differential liver-metastatic potentials. Highly liver-metastatic RAW117-H10 cells had higher rates of static adhesion to vitronectin, fibronectin and (GRGDS)4 than poorly metastatic RAW117-P and moderately liver-metastatic RAW117-L17 cells. Under hydrodynamic conditions, adhesion stabilization was more rapid for H10 cells compared to P or L17 cells. Among the RGD peptides, only the polymeric RGD peptide (GRGDS)4 mediated strong static adhesion of H10 cells. Interestingly, all the RGD peptides mediated adhesion stabilization for H10 cells but still not for L17 or P cells under hydrodynamic conditions. Integrin alpha(v) beta3 was involved in stabilizing hydrodynamic adhesion to (GRGDS)4, monomeric RGD peptide R1, but was less important in static adhesion to monomeric RGD peptides. Differential adhesion to liver sinusoidal endothelial cell-derived extracellular matrix (H10 >> L17 > P) was observed under hydrodynamic but not static conditions. Integrin alpha(v) beta3 was also important in hydrodynamic adhesion to liver sinusoidal endothelial cell-derived extracellular matrix. We believe that strong static and hydrodynamic adhesion of H10 cells and their capability of altering adhesive behavior in response to fluid shear may contribute to liver metastasis.
Amino Acid Sequence↗
Intracellular pH changes in human aortic smooth muscle cells in response to fluid shear stress.
The smooth muscle cell (SMC) layers of human arteries may be exposed to blood flow after endothelium denudation, for example, following balloon angioplasty treatment. These SMCs are also constantly subjected to pressure driven transmural fluid flow. Flow-induced shear stress can alter SMC growth and metabolism. Signal transduction mechanisms involved in these flow effects on SMCs are still poorly understood. In this work, the hypothesis that shear stress alters the intracellular pH (pHi) of SMC is examined. When exposed to venous and arterial levels of shear stress, human aortic smooth muscle cells (hASMC) undergo alkalinization. The alkalinization plateau persisted even after 20 min of cell exposure to flow. Addition of amiloride (10 micromoles) or its 5-(N-ethyl-N-isopropyl) analog (EIPA, 10 micromoles), both Na+/H+ exchanger inhibitors, attenuated intracellular alkalinization, suggesting the involvement of the Na+/H+ exchanger in this response. The same concentrations of these inhibitors did not show an effect on pHi of hASMCs in static culture. 4-Acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid (SITS, 1 mM), a Cl-/HCO3- exchange inhibitor, affected the pHi of hASMCs both in static and flow conditions. Our results suggest that flow may perturb the Na+/H+ exchanger leading to an alkalinization of hASMCs, a different response from the flow-induced acidification seen with endothelial cells at the same levels of shear stress. Understanding the flow-induced signal transduction pathways in the vascular cells is of great importance in the tissue engineering of vascular grafts. In the case of SMCs, the involvement of pHi changes in nitric oxide production and proliferation regulation highlights further the significance of such studies.
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
Effects of Integrelin on platelet function in flow models of arterial thrombosis.
The effects of intravenous Integrelin, an antagonist of platelet glycoprotein (GP) IIb-IIIa, were studied ex vivo in flow models of platelet adhesion/aggregation in patients undergoing angioplasty. Blood was collected from each patient before, during, and after a 24-h Integrelin infusion (0.75 micrograms/kg/min; plasma Integrelin levels, 312-759 ng/ml). The effects of Integrelin administered in vivo were evaluated by using two different models of platelet thrombus formation: (a) platelet adhesion onto von Willebrand factor (vWF)/collagen, followed by platelet aggregation, in a perfusion system; and (b) direct platelet aggregation induced by elevated levels of shear stress imposed by a cone-and-plate viscometer. Neither aspirin nor heparin, also given to the patients, affected platelet adhesion or aggregation in these flow models. In the perfusion studies, platelet adhesion to vWF/collagen I was not inhibited by in vivo Integrelin. In contrast, in each of the six patients studied by using blood collected after 45 min of Integrelin infusion, there was a decrease in the size of platelet aggregates compared with patient baseline samples. In the viscometer experiments, shear-induced platelet aggregation was reduced by 61-71% in samples collected 45 min into the Integrelin infusion (plasma Integrelin levels: 759 +/- 69 ng/ml) compared with baseline samples. Within 24-48 h after termination of the Integrelin, direct shear-induced platelet aggregation and platelet aggregation subsequent to adhesion returned to or near baseline in each of the patients studied. We conclude that Integrelin administered in vivo inhibits both platelet aggregation subsequent to initial platelet adhesion and direct shear-induced platelet aggregation under pathologic flowing conditions, After discontinuation of the drug, these inhibitory effects do not persist in vivo as long as the inhibitory effect on aggregation produced by c7E3 (the monoclonal antibody against GPIIb-IIIa).
Blood Platelets↗
Effects of fluid dynamic forces on vascular cell adhesion.
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Blood Platelets↗
Adhesion of platelets to surface-bound fibrinogen under flow.
After platelet activation, fibrinogen mediates platelet-platelet interactions leading to platelet aggregation. In addition, fibrinogen can also function as a cell adhesion molecule, providing a substratum for adhesion of platelets and endothelial cells. In this report, we studied the adhesion of platelets to surface-immobilized fibrinogen under flow in different shear rates. Heparinized whole blood containing mepacrine-labeled platelets was perfused for two minutes at various wall shear rates from 250 to 2,000 s-1 in a parallel plate flow chamber. The number of adherent fluorescent platelets was quantitated every 15 seconds with an epifluorescent videomicroscope and digital image processing system. When compared with platelet adhesion and aggregation seen on glass surfaces coated with type I bovine collagen, a significant increase in platelet adhesion was observed on immobilized fibrinogen up to wall shear rates of 800 s-1. The adherent platelets formed a single layer on fibrinogen-coated surfaces. Under identical conditions, no significant adhesion was observed on fibronectin- or vitronectin-coated surfaces. Although platelet adhesion to collagen was substantially inhibited by the platelet inhibitors prostaglandin E1 and theophylline, these inhibitors had no effect on platelet adhesion to fibrinogen. Platelets adhered to recombinant homodimeric wild-type (gamma 400-411) fibrinogen, but not to the recombinant homodimeric gamma' variant of fibrinogen. Platelet adhesion to recombinant fibrinogen with RGD to RGE mutations at positions alpha 95-97 and alpha 572-574 was similar to that with plasma-derived fibrinogen. These results show that platelets adhere to fibrinogen-coated surfaces under moderate wall shear rates, that the interaction is mediated by the fibrinogen 400-411 sequence at the carboxy-terminus of the gamma chain, and that the interaction is independent of platelet activation and the RGD sequences in the alpha chain.
Alprostadil↗
Neutrophil adhesion to 24-hour IL-1-stimulated endothelial cells under flow conditions.
In this study, we examine neutrophil adhesion under flow conditions to cultured HUVEC stimulated for 4 or 24 h with IL-1. Interactions are measured using videomicroscopy and a parallel-plate flow system which is capable of distinguishing primary adhesion and rolling from secondary (firm) adhesion. We find that after 24 h, E-selectin does not contribute to primary adhesion, in contrast to a significant contribution after 4 h. Endothelial cell P-selectin does not contribute at either time point. Blocking or removing L-selectin from the neutrophil surface decreases adhesion 50 to 70% at either time point, and neuraminidase treatment of neutrophils decreases adhesion by 85%. These results suggest that after a 24-h stimulation, primary adhesion depends on a distinct selectin-like interaction in which a HUVEC receptor binds carbohydrates on neutrophil glycoproteins, including L-selectin. We also find that secondary adhesion in this system can be inhibited 90% with Ab blockade of CD18/ICAM-1 interactions after a 24-h stimulation, and that a combination of IL-1 and IL-4 selectively down-regulates the pathway for primary adhesion at 24 h. These results demonstrate that neutrophils adhere using different receptor pathways following 4- and 24-h stimulations, and provide experimental data characterizing some properties of the receptors involved. One of the pathways that is evident at 24 h appears to be a novel selectin-like interaction.
Blood Flow Velocity↗
Adhesion of sickle red blood cells and damage to interleukin-1 beta stimulated endothelial cells under flow in vitro.
Two factors that are hypothesized to contribute to vasoocclusive crises in sickle cell anemia are increased sickle red blood cell-endothelial cell interactions and damage to endothelium. Despite considerable study, the mechanisms by which erythrocyte-endothelial interactions occur and the role of endothelial damage have not yet been fully elucidated. In this report, we demonstrate that adhesion and damage may be related in a model of vasoocclusion in sickle cell anemia. Phase contrast microscopy coupled to digital image processing was used to determine the adhesion of sickle red blood cells to 1-, 4-, and 24-hour interleukin-I beta (IL-1 beta) stimulated endothelial calls in a parallel plate flow chamber. Morphological alterations to activated endothelial cells after the perfusion of sickle erythrocytes were also identified. Pretreatment of monolayers with 50 pg/mL of IL-1 beta for 1, 4, and 24 hours caused approximately 16-fold increases in adhesion of sickle cells to activated endothelium at all time points. Results with an Arginine-glycine aspartic acid (RGD) peptide and monoclonal antibodies indicated a role for three different endothelial cell receptors: alpha v beta 3 after 1 hour of IL-1 beta stimulation; E-selectin after 4 hours of IL-1 beta stimulation; and vascular cell adhesion molecule-1 after prolonged exposure to cytokines. Perfusion of sickle, but not normal, erythrocytes resulted in alteration of endothelial morphology. Approximately 6% to 8% damage was observed on 4- and 24-hour IL-1 beta stimulated endothelial cells after the perfusion of sickle cells. Damage to 24-hour activated endothelial cells showed a positive correlation (r = .899) with the number of adherent sickle erythrocytes.
Amino Acid Sequence↗
Quantitative effects of peripheral monocytes and nerve growth factor on CNS neural morphometric outgrowth parameters in vitro.
Would healing of the central nervous system (CNS) is a complex process involving interactions between cells from both the vascular and the neural environments, extracellular matrix proteins, and a cocktail of agonistic and antagonistic bioactive molecules. Vascular cells, particularly peripheral monocytes and macrophages, are believed to play an important role in organizing and mediating CNS tissue reactions subsequent to penetrating injuries that compromise the blood-brain barrier. Although many investigators have studied the effect of macrophages and microglia (resident brain macrophages) on neural outgrowth, little is known regarding monocyte effects. We have combined tissue culture, video microscopy, and digital image processing and analysis to quantify morphometric parameters of neurons exposed to monocyte secretory products in vitro. The experimental system developed is simple in design but provides a quantitative understanding of cellular function and molecular mechanisms and has the ability to both study processes of graded complexity and relate cellular function to overall systems behavior. We evaluate the efficacy of the experimental model developed by measuring morphometric parameters of human neural cells (hNT cell line) in the presence of nerve growth factor (NGF). Results suggest that monocyte-conditioned media (MCM) increases neuron outgrowth parameters, such as neuritic output, mean arbor output, neurite branching, and effective cell diameter. Moreover, we show that the bioactive factor present in MCM is not IL-1 and the activity of the factor with respect to neural outgrowth is between that of 10 and 100 ng/ml NGF.
Cell Line↗
Leucocyte adhesion under flow conditions: principles important in tissue engineering.
An understanding of inflammatory responses is important in a wide variety of tissue engineering applications. This review describes the current understanding of a central aspect of inflammatory responses, the adhesion of leucocytes to blood vessel walls prior to their emigration into tissues. These highly specific adhesive interactions are mediated by three main families of receptors: the selectins, integrins, and members of the immunoglobulin superfamily. Under flow conditions, the various receptors make distinct contributions to a multistep process of adhesion in which leucocytes roll, adhere firmly, and eventually transmigrate. Two examples in which these principles are important in tissue engineering research, lymphocyte adherence in transplant rejection and monocyte adherence in atherosclerosis, are discussed in the last part of the paper.
Animals↗
Regulation of melanoma cell adhesion stabilization to fibronectin.
Tumour cell arrest and the formation of stable adhesive interactions between tumour cells and endothelial cells or underlying matrix in the microvasculature are crucial steps in the metastatic process. We have developed a sensitive hydrodynamic adhesion assay to investigate the regulation of melanoma cell adhesion stabilization to the extracellular matrix protein fibronectin. Modulators of human MeWo melanoma Ca2+ concentration and stores, including ionomycin, thapsigargin, dantrolene and caffeine, inhibited cell adhesion stabilization to fibronectin; however, removal of Ca2+ from the extracellular medium did not affect stabilization. The calmodulin inhibitor W-7 and the protein kinase C inhibitor chelerythrine also blocked MeWo adhesion stabilization to fibronectin, as did the tyrosine kinase inhibitor genistein and the cytoskeletal inhibitor cytochalasin D. Manipulation of MeWo cell intracellular CAMP levels had no effect of adhesion stabilization to fibronectin, nor did treatment of cells with phorbol ester, pertussis toxin or cholera toxin. Drug treatments that inhibited adhesion stabilization also had significant effects on the actin cytoskeleton organization of the melanoma cells. This study suggests a role for calcium, calmodulin, protein kinase C and tyrosine kinases in the intracellular regulation of MeWo adhesive stabilization.
Actin Cytoskeleton↗
Cellular interactions among marrow stromal and normal/neoplastic pre-B- and B-lymphoblastic cells.
The direct interaction between B-cells, both normal and neoplastic, and marrow stromal cells (MSC) or MSC-secreted extracellular matrix proteins is believed to play a decisive role in lymphopoiesis as well as in the sustained proliferation and homing of lymphoid malignancies. Although information has been accruing on possible obligatory cell-cell and cell-extracellular matrix adhesion molecules, our current state of knowledge regarding B-cell interaction with the marrow microenvironment is rather limited. We discuss in this review what is currently known regarding adhesion molecules involved in the apposition between B-cells and MSC. In addition, we describe how the cellular interactions are modulated by cytokines present in the marrow microenvironment. Further, we present possible signal transduction pathways activated by B-cell/MSC apposition and attempt to integrate in vitro data with in vivo observations. We close this review by providing implications for novel clinical and therapeutic applications based on the current knowledge of B-cell/MSC adhesion mechanisms and signaling pathways.
B-Lymphocytes↗
Platelet adhesion and aggregation on human type VI collagen surfaces under physiological flow conditions.
Type VI collagen is a subendothelial constituent that binds von Willebrand factor (vWF) and platelets. The interaction of platelets with type VI collagen and the roles of platelet glycoprotein (GP) receptors and vWF were studied under flow conditions using epi-fluorescent videomicroscopy coupled with digital image processing. We found that surface coverage was less than 6% on collagen VI at a relatively high-wall shear rate (1,000 s-1) and was approximately 60% at a low-wall shear rate (100 s-1). The molecular mechanisms involved in low-shear platelet binding were studied using monoclonal antibodies to platelet GPIb and GPIIb-IIIa, and polymeric aurin tricarboxylic acid. Anti-GPIIb-IIIa was the most effective in eliminating adhesion (surface coverage, 0.8%), followed by anti-GPIb (4.3%), and ATA (12.6%). Experiments with von Willebrand disease blood indicate that vWF is involved in platelet adhesion to collagen VI at 100 s-1. In the absence of vWF, there may be direct binding of platelet GPIIb-IIIa complexes to collagen VI. Adhesion and aggregation on collagen VI are different in shear rate dependence from collagen I. Our results suggest a possible role for collagen VI and vWF in platelet adhesion and aggregation in vascular regions with low shear rates.
Animals↗
Comparative real-time effects on platelet adhesion and aggregation under flowing conditions of in vivo aspirin, heparin, and monoclonal antibody fragment against glycoprotein IIb-IIIa.
BACKGROUND: A real-time in vitro system of human platelet thrombosis under arterylike flowing conditions similar to those produced in vivo by angioplasty would be useful for the evaluation of potential antiarterial thrombotic agents in association with in vivo trials. Aspirin, heparin, and the chimeric monoclonal antibody antigen-binding fragment 7E3 (c7E3 Fab) directed against platelet glycoprotein (GP) IIb-IIIa have been used in attempts to delay or prevent thrombotic reocclusion of coronary arteries after angioplasty. We compared the effects of these agents administered in vivo on GPIb-mediated platelet adhesion to von Willebrand factor (vWF)/collagen type I (as in atherosclerotic subendothelium) and on subsequent GPIIb-IIIa-fibrinogen/vWF-mediated platelet aggregation under flowing conditions analogous to those in constricted coronary arteries. METHODS AND RESULTS: Citrated whole blood containing mepacrine-labeled platelets from patients and healthy donors was perfused for 1 minute at an abnormally elevated shear rate of 1500 seconds-1 (arterial wall shear stress of 50 to 60 dynes/cm2) at 37 degrees C over collagen I/vWF. The number of adherent fluorescent platelets was quantified every 15 seconds with a low-light-level video camera and epifluorescent microscopy. After 5 healthy donors had ingested 975 mg aspirin, platelet adhesion was unaffected in the aspirin-treated blood compared with the control blood in all experiments (10 of 10), and subsequent aggregation was unchanged in most runs (8 of 10). The blood of 3 aspirin-treated patients undergoing angioplasty was analyzed before and after a 12,000-U heparin injection and 2 minutes, 2 hours, and 24 hours after infusion of 0.25 mg/kg of c7E3 Fab. In these patients, the bolus of heparin did not inhibit either platelet adhesion to collagen I/vWF or subsequent aggregation. In contrast, there was > 50% inhibition of platelet aggregation 2 minutes after the infusion of c7E3 Fab in all 3 patients, and inhibition persisted in 2 of the 3 patients at 2 hours and 24 hours after c7E3 Fab. CONCLUSIONS: In contrast to aspirin or heparin, the in vivo injection of c7E3 Fab considerably reduces platelet aggregate formation mediated by the binding of fibrinogen, vWF, or some other ligand to platelet GPIIb-IIIa under conditions of abnormally increased shear stress analogous to those in narrowed coronary arteries. Platelet adherence to collagen I/vWF is not affected. This study describes an in vitro model of arterial injury (similar to angioplasty) that uses human blood to compare directly, in real time, the precise relative effects of aspirin, heparin, and c7E3 Fab on platelet adhesion and subsequent aggregation.
Abciximab↗
Heterotypic adherence between human B-lymphoblastic and pre-B-lymphoblastic cells and marrow stromal cells is a biphasic event: integrin very late antigen-4 alpha mediates only the early phase of the heterotypic adhesion.
Heterotypic adherence between marrow stromal cells (MSC) and lymphoblastic cells is essential for normal lymphopoiesis and malignant lymphoblastic development. However, the detailed molecular mechanisms by which this heterotypic adherence occurs are poorly understood. The cell-cell interactions between a B-lymphoblastic cell line (UTMB-460) and a pre-B-cell line (NALM-6) with MSC were chosen as models to investigate potential mechanisms and adhesion molecules involved in the apposition between normal and malignant lymphoblastic cells and MSC. A parallel-flow detachment assay (PFDA) and a 51Cr detachment assay, coupled with monoclonal antibody (MoAb) blocking experiments, were used to quantify the attachment of lymphoblastic cells to confluent monolayers of MSC. The apposition between MSC and B-lymphoblastic cells (UTMB-460 cells) was investigated for variable time periods, ranging from 1 minute to 4 hours. Results from the temporal study suggest that the heterotypic adherence of the B-lymphoblastic cells to MSC is a biphasic event and the interactions occur rapidly (< or = 1 minute) after the two cells come into contact. More specifically, the early phase of adherence (< or = 15 minutes) solely involves very late antigen-4 alpha (VLA-4 alpha)/vascular cell adhesion molecule 1 (VCAM-1) interactions, as evidenced by the nearly complete inhibition (93%) of UTMB-460 cell adherence in the presence of anti-VLA-4 alpha. The late phase (> or = 30 minutes) proceeds despite the continuous presence of anti-VLA-4 alpha. In addition, the late-phase adherence is not affected by MoAbs to LFA-1, CD44, VCAM-1, E-selectin, or L-selectin, which suggests the possible involvement of other adhesion molecules. Adherence of pre-B-lymphoblastic cells (NALM-6) to MSC is also biphasic. Integrin VLA-4 is again a major player in the early phase of pre-B-lymphoblastic cell/MSC interactions. The early phase of adherence may be important in homing of the malignant lymphoblastic cells to the MSC and the late phase in retention of malignant lymphoblastic cells in the bone marrow.
Antibodies, Monoclonal↗
A cellular automaton model for the proliferation of migrating contact-inhibited cells.
A cellular automaton is used to develop a model describing the proliferation dynamics of populations of migrating, contact-inhibited cells. Simulations are carried out on two-dimensional networks of computational sites that are finite-state automata. The discrete model incorporates all the essential features of the cell locomotion and division processes, including the complicated dynamic phenomena occurring when cells collide. In addition, model parameters can be evaluated by using data from long-term tracking and analysis of cell locomotion. Simulation results are analyzed to determine how the competing processes of contact inhibition and cell migration affect the proliferation rates. The relation between cell density and contact inhibition is probed by following the temporal evolution of the population-average speed of locomotion. Our results show that the seeding cell density, the population-average speed of locomotion, and the spatial distribution of the seed cells are crucial parameters in determining the temporal evolution of cell proliferation rates. The model successfully predicts the effect of cell motility on the growth of isolated megacolonies of keratinocytes, and simulation results agree very well with experimental data. Model predictions also agree well with experimentally measured proliferation rates of bovine pulmonary artery endothelial cells (BPAE) cultured in the presence of a growth factor (bFGF) that up-regulates cell motility.
Algorithms↗