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Biomedical subjects

Elazer R Edelman

Publications and source records attributed to Elazer R Edelman.

At least 19 recordsLinked to original sources
PubMed2007

Endosomal receptor kinetics determine the stability of intracellular growth factor signalling complexes.

There is an emerging paradigm that growth factor signalling continues in the endosome and that cell response to a growth factor is defined by the integration of cell surface and endosomal events. As activated receptors in the endosome are exposed to a different set of binding partners, they probably elicit differential signals compared with when they are at the cell surface. As such, complete appreciation of growth factor signalling requires understanding of growth factor-receptor binding and trafficking kinetics both at the cell surface and in endosomes. Growth factor binding to surface receptors is well characterized, and endosomal binding is assumed to follow surface kinetics if one accounts for changes in pH. Yet, specific binding kinetics within the endosome has not been examined in detail. To parse the factors governing the binding state of endosomal receptors we analysed a whole-cell mathematical model of epidermal growth factor receptor trafficking and binding. We discovered that the stability of growth factor-receptor complexes within endosomes is governed by three primary independent factors: the endosomal dissociation constant, total endosomal volume and the number of endosomal receptors. These factors were combined into a single dimensionless parameter that determines the endosomal binding state of the growth factor-receptor complex and can distinguish different growth factors from each other and different cell states. Our findings indicate that growth factor binding within endosomal compartments cannot be appreciated solely on the basis of the pH-dependence of the dissociation constant and that the concentration of receptors in the endosomal compartment must also be considered.

Endosomes↗
PubMed2007

Vascular bed origin dictates flow pattern regulation of endothelial adhesion molecule expression.

Endothelial cell phenotypes markedly differ, depending upon function and vascular bed of origin. Differences might account for specific susceptibility to pathological conditions. As leukocyte adhesion to activated endothelium is the initiating event in a range of diseases, we compared the influence of vascular bed-specific flow patterns on adhesion molecule expression in human saphenous vein (HSVEC) and coronary artery endothelial cells (HCAEC). In vitro, immune cell attachment was increased 1.6-fold when tumor necrosis factor (TNF)-alpha-stimulated HSVEC were exposed to coronary artery flow in place of physiological venous flow and 1.9-fold higher compared with attachment to cytokine-stimulated HCAEC exposed to coronary artery flow. This was associated with increased concentrations of soluble E-selectin, VCAM-1, and ICAM-1 in supernatants of HSVEC exposed to coronary artery flow compared with HCAEC exposed to the same flow pattern. Venous and coronary artery flow both increased TNF-alpha-induced E-selectin and ICAM-1 expression on HSVEC, but only coronary artery flow increased VCAM-1 expression. In marked contrast to HSVEC, venous and coronary artery flow attenuated TNF-alpha-induced E-selectin and VCAM-1 expression on HCAEC, whereas coronary artery flow further induced ICAM-1 on cytokine-stimulated HCAEC. With the exception of cytokine-induced ICAM-1, adhesion molecule expression on HSVEC exposed to coronary artery flow exceeded expression on HCAEC. Thus ICAM-1 expression involves complex flow-dependent and -independent pathways with marked dissimilarities between the two endothelial cell types studied. Interestingly, Kruppel-like factor (KLF) 4 overexpression in HCAEC and HSVEC significantly reduced TNF-alpha-induced E-selectin and VCAM-1 expression in static conditions, while ICAM-1 expression remained constant. Furthermore, both flow patterns induced KLF2 and KLF4 expression in HCAEC and HSVEC. Venous and coronary artery flow differentially influence endothelial adhesion molecule and transcription factor expression, depending on the vascular bed of origin. Differences in adhesion molecule expression and subsequent immune cell adhesion between HSVEC and HCAEC may contribute to different susceptibility to pathological conditions.

Blood Flow Velocity↗
PubMed2006

Cell-matrix contact prevents recognition and damage of endothelial cells in states of heightened immunity.

BACKGROUND: Autoimmunity may exacerbate vascular disease, particularly in the form of anti-endothelial cell (EC) antibodies. The increased morbidity of cardiovascular diseases in concert with diabetes mellitus, hypertension, and other systemic illnesses may reflect the increase presence and potency of these antibodies. Matrix-embedded ECs act as powerful regulators of vascular repair accompanied by significant reduction in expected systemic and local inflammation. We compared the immune response against free and matrix-embedded ECs in naïve mice and mice with heightened EC immune reactivity. METHODS AND RESULTS: Mice were presensitized to EC with repeated (days 0, 21, 35) subcutaneous injections of saline-suspended porcine EC (PAE) (5 x 10(5) cells). Controls received saline injections. On day 42, mice received 5 x 10(5) matrix-embedded or free PAEs. Circulating PAE-specific antibodies and effector T-cells were analyzed via flow cytometry, and xenoreactive lymphocytes via ELISPOT, 90 days after implantation. PAE-specific antibody-titers, frequency of CD4+-effector cells, and xenoreactive splenocytes were 2- to 4-fold lower (P<0.0001) when naïve mice were injected with matrix-embedded instead of saline-suspended PAEs. Though basal levels of circulating antibodies were significantly elevated after serial PAE injections (2210+/-341 mean fluorescence intensity, day 42) and almost doubled again 90 days after injection of a fourth set of free PAEs, antibody levels declined by half in recipients of matrix-embedded PAEs at day 42 (P<0.0001). Levels of CD4+-effector cells and xenoreactive splenocytes showed similar results. CONCLUSIONS: Implantation of free PAE elicits a significant immune response in naïve mice and even more pronounced in mice with predeveloped anti-endothelial immunity. Matrix-embedding protects xenogeneic ECs against immune reaction in naïve mice and to a similar extent in mice with heightened immune reactivity. Matrix-embedded EC might offer a promising approach for treatment of advanced cardiovascular disease.

Animals↗
PubMed2005

Local and systemic drug competition in drug-eluting stent tissue deposition properties.

The efficacy of drug-eluting stents (DES) requires delivery of potent compounds directly to the underlying arterial tissue. The commercially available DES drugs rapamycin and paclitaxel bind specifically to their respective therapeutic targets, FKBP12 and polymerized microtubules, while also associating in a more general manner with other tissue elements. As it is binding that provides biological effect the question arises as to whether other locally released or systemically circulating drugs can displace DES drugs from their tissue binding domains. Specific and general binding sites for both drugs are distributed across the media and adventitia with higher specific binding associated with the higher specific binding site densities in the media. The ability of rapamycin and paclitaxel to compete for specific protein binding and general tissue deposition was assessed for both compounds simultaneously and in the presence of other commonly administered cardiac drugs. Drugs classically used to treat standard cardiovascular diseases, such as hypertension and hypercoaguability, displace rapamycin and paclitaxel from general binding sites, possibly decreasing tissue reserve capacity for locally delivered drugs. Paclitaxel and rapamycin do not affect the other's binding to their biologically relevant specific protein targets, but can generally displace each other from tissue at three log order molar excess, decreasing arterials loads by greater than 50%. Local competitive binding therefore should not limit the placement of rapamycin and paclitaxel eluting stents in close proximity.

Animals↗
PubMed2005

Proangiogenic stimulation of bone marrow endothelium engages mTOR and is inhibited by simultaneous blockade of mTOR and NF-kappaB.

Most bone marrow (BM) malignancies develop in association with an angiogenic phenotype and increased numbers of endothelial cells. The molecular mechanisms involved in the modulation and recruitment of BM endothelium are largely unknown and may provide novel therapeutic targets for neoplastic diseases. We observed that angiogenic stimulation of BM endothelial cells activates mTOR and engages its downstream pathways 4E-BP1 and S6K1, which are inhibited by the mTOR-specific blockers rapamycin and CCI-779. Both mTOR blockers significantly inhibit growth factor- and leukemia-induced proliferation of BM endothelium by inducing G0/G1 cell-cycle arrest. This effect is associated with down-regulation of cyclin D1 and cdk2 phosphorylation, and up-regulation of the cdk inhibitors p27(kip1) and p21(cip1). Under conditions that reproduce the biomechanical fluidic environment of the BM, CCI-779 is equally effective in inhibiting BM endothelial-cell proliferation. Finally, simultaneous blockade of mTOR and NF-kappaB pathways synergize to significantly inhibit or abrogate the proliferative responses of BM endothelial cells to mitogenic stimuli. This study identifies mTOR as an important pathway for the proangiogenic stimulation of BM endothelium. Modulation of this pathway may serve as a valid therapeutic intervention in BM malignancies evolving in association with an angiogenic phenotype.

Adult↗
PubMed2005

Matrix embedding alters the immune response against endothelial cells in vitro and in vivo.

BACKGROUND: Endothelial cell (EC) dysfunction represents the first manifestation of atherosclerotic disease. Restoration of endothelium via seeding or transfection is hampered by local alterations in flow, inflammation, and metabolic activation. Perivascular EC matrix implants are shielded from these forces and still control vascular repair. The host immune response to such implants, however, remains largely unknown. We investigated the effect of embedding of ECs within 3-dimensional matrices on host immune responses in vitro and in vivo. METHODS AND RESULTS: We compared expression of major histocompatibility complex (MHC), costimulatory, and adhesion molecules by free aortic ECs or ECs embedded in Gelfoam matrices by flow-cytometry. T-cell proliferation was assessed by [3H] thymidine incorporation. Humoral immune response (ELISA and FACS analysis) and cellular (histopathology) infiltration were investigated after subcutaneous injection of free porcine aortic ECs (PAEs) or of a Gelfoam/EC block, or after concomitant injection of PAEs adjacent to Gelfoam in rats. Aortic ECs embedded in Gelfoam expressed lower levels of MHC class II, costimulatory, and adhesion molecules compared with free ECs (P<0.001), and induced 3-fold less proliferation of human CD4+ T-cells (P<0.0005). Implantation of a Gelfoam/EC block in rats nearly abrogated the immune response with 1.75- to 9.0-fold downregulation in tumor necrosis factor-alpha, interleukin-6, monocyte chemotactic protein-1, and PAE-specific immunoglobulin G (P<0.005) and 3.3- to 4.5-fold reduction in leukocytic tissue infiltration. Injecting PAEs adjacent to Gelfoam induced a significant response comparable to that of free implanted PAEs. CONCLUSIONS: Embedding ECs within 3-dimensional matrices alters the host immune response by inhibiting expression of MHC class II, costimulatory, and adhesion molecules, offering the rationale to develop novel therapies for vascular diseases.

Animals↗
PubMed2005

Vascular neointimal formation and signaling pathway activation in response to stent injury in insulin-resistant and diabetic animals.

Diabetes and insulin resistance are associated with increased disease risk and poor outcomes from cardiovascular interventions. Even drug-eluting stents exhibit reduced efficacy in patients with diabetes. We now report the first study of vascular response to stent injury in insulin-resistant and diabetic animal models. Endovascular stents were expanded in the aortae of obese insulin-resistant and type 2 diabetic Zucker rats, in streptozotocin-induced type 1 diabetic Sprague-Dawley rats, and in matched controls. Insulin-resistant rats developed thicker neointima (0.46+/-0.08 versus 0.37+/-0.06 mm2, P=0.05), with decreased lumen area (2.95+/-0.26 versus 3.29+/-0.15 mm2, P=0.03) 14 days after stenting compared with controls, but without increased vascular inflammation (ED1+ tissue macrophages). Insulin-resistant and diabetic rat vessels did exhibit markedly altered signaling pathway activation 1 and 2 weeks after stenting, with up to a 98% increase in p-ERK (anti-phospho ERK) and a 54% reduction in p-Akt (anti-phospho Akt) stained cells. Western blotting confirmed a profound effect of insulin resistance and diabetes on Akt and ERK signaling in stented segments. p-ERK/p-Akt ratio in stented segments uniquely correlated with neointimal response (R2=0.888, P=0.04) in insulin-resistant and type 1 and 2 diabetic rats, but not in lean controls. Transfemoral aortic stenting in rats provides insight into vascular responses in insulin resistance and diabetes. Shifts in ERK and Akt signaling related to insulin resistance may reflect altered tissue repair in diabetes accompanied by a shift in metabolic:proliferative balance. These findings may help explain the increased vascular morbidity in diabetes and suggest specific therapies for patients with insulin resistance and diabetes.

Animals↗
PubMed2005

Enhanced T-helper-1 lymphocyte activation patterns in acute coronary syndromes.

OBJECTIVES: We sought to determine whether different stages of coronary artery disease (CAD) are associated with distinct differentiation patterns of activated T cells. BACKGROUND: Atherosclerosis is an inflammatory disease. However, little is known about specific inflammatory cell activation in atherosclerosis, for example, the T-helper (Th)1/Th2-balance. METHODS: We studied 18 patients with stable angina (SA), 28 patients with acute coronary syndrome (ACS) (16 with unstable angina pectoris and 12 with ST-segment elevation myocardial infarction), 19 patients with unheralded myocardial infarction (UH), and 16 control patients. Cytokine patterns and transcription factor signaling pathways of circulating T cells were characterized using reverse transcription polymerase chain reaction and flow cytometry. RESULTS: Although interferon (IFN)-gamma(+)/CD3(+) T cells were approximately 2-fold greater in patients with SA or UH than in control subjects, there was a massive expansion of Th1 cells in patients with ACS (p < 0.001). This increase was paralleled by significantly increased mRNA transcript levels for signal-transducer-and-activator-4 (ACS 1.17 +/- 0.14 relative units [RU]; control patients 0.44 +/- 0.09 RU; SA 0.67 +/- 0.12 RU; UH 0.61 +/- 0.17 RU), interleukin-2 (ACS 1.55 +/- 0.51 RU; control patients 0.21 +/- 0.09 RU; SA 0.54 +/- 0.18 RU; UH 0.45 +/- 0.16 RU), and IFN-gamma in ACS (1.27 +/- 0.39 RU; control patients 0.35 +/- 0.09 RU; SA 0.58 +/- 0.11 RU; UH 0.53 +/- 0.24 RU; p < 0.002). Th2 and Th0 cells were not different across patient subsets. The burden of CAD was identical between SA (1.4 +/- 0.2 diseased vessels, 68 +/- 13% diameter stenosis) and ACS (1.4 +/- 0.2 diseased vessels, 64 +/- 17% diameter stenosis) but significantly greater in patients with UH (2.5 +/- 0.5 diseased vessels, 95 +/- 7% diameter stenosis; p < 0.05). CONCLUSIONS: Patients with UH have a greater burden of obstructive CAD than SA but no greater T-cell activation. Patients with ACS have the same extent of CAD than SA but significantly greater activation of Th1 cells that may contribute to the increasing instability. Differences in circulating Th1 cells might indicate different pathogenic components, leading to ACS and UH.

Aged↗
PubMed2005

Strut position, blood flow, and drug deposition: implications for single and overlapping drug-eluting stents.

BACKGROUND: The intricacies of stent design, local pharmacology, tissue biology, and rheology preclude an intuitive understanding of drug distribution and deposition from drug-eluting stents (DES). METHODS AND RESULTS: A coupled computational fluid dynamics and mass transfer model was applied to predict drug deposition for single and overlapping DES. Drug deposition appeared not only beneath regions of arterial contact with the strut but surprisingly also beneath standing drug pools created by strut disruption of flow. These regions correlated with areas of drug-induced fibrin deposition surrounding DES struts in porcine coronary arteries. Fibrin deposition immediately distal to individual isolated drug-eluting struts was twice as great as in the proximal area and for the stent as a whole was greater in distal segments than proximal segments. Adjacent and overlapping stent struts increased computed arterial drug deposition by far less than the sum of their combined drug load. In addition, drug eluted from the abluminal stent strut surface accounted for only 11% of total deposition, whereas, remarkably, drug eluted from the adluminal surface accounted for 43% of total deposition. Thus, local blood flow alterations and location of drug elution on the strut were far more important in determining arterial wall drug deposition and distribution than were drug load or arterial wall contact with coated strut surfaces. CONCLUSIONS: Simulations that coupled strut configurations with flow dynamics correlated with in vivo effects and revealed that drug deposition occurs less via contact between drug coating and the arterial wall than via flow-mediated deposition of blood-solubilized drug.

Animals↗
PubMed2005

On the validity of the quasi-steady state approximation of bimolecular reactions in solution.

Two-step binding kinetics are extensively used to study the relative importance of diffusion in biochemical reactions. Classical analysis of this problem assumes ad hoc that the encounter complex is at quasi-steady state (QSS). Using scaling arguments we derive a criterion for the validity of this assumption in the limit of irreversible product formation. We find that the QSS approximation (QSSA) of two-step binding is only valid if the total ligand and receptor concentrations are much smaller than (k2+k-1)/k1, where k1 and k-1 are, respectively, the forward and reverse diffusion encounter rate constants and k2 is the chemical association rate constant. This criterion can be shown to imply that the average time between encounters is much longer than the half-life of the encounter complex and also guarantees that the concentration of the encounter complex is negligible compared to the reactant and product concentrations. Numerical examples of irreversible and reversible cases corroborate our analysis and illustrate that the QSS may be invalid even if k-2<<k2. Our analysis of the irreversible case is shown to carry through to the more rigorous framework of the Smoluchowski theory of diffusion-controlled reactions. This work underscores the need for exercising greater caution in invoking the QSSA.

Animals↗
PubMed2005

Thrombosis modulates arterial drug distribution for drug-eluting stents.

BACKGROUND: Drug-eluting stents deliver potent compounds directly to arterial segments but can become clot laden when deployed. The question arises as to whether thrombi affect drug elution and arterial uptake. METHODS AND RESULTS: Paclitaxel transport and retention were assessed in clots of different blood components. Diffusivity, affected by clot organization, is fastest in fibrin (approximately 347 microm2/s), slower in fibrin-red blood cell clots (34.98 microm2/s), and slowest in whole-blood clots (3.55 microm2/s). Blood cells bind and retain paclitaxel such that levels in clot increase linearly with red cell fraction. At physiological hematocrit, clot retains 3 times the amount of paclitaxel in surrounding solutions. Computational models predict that the potential of thrombus to absorb, retain, and release drug or to act as a barrier to drug delivery depends on clot geometry and strut position in clot relative to the vessel wall. Clot between artery and stent can reduce uptake 10-fold, whereas clot overlying the stent can shield drug from washout, increasing uptake. Model assumptions were confirmed and predictions were validated in a novel rat model that introduces thrombosis within stented aortas where nonocclusive thrombus acts as capacitive space for drug and shifts drug levels to decrease tissue uptake 2-fold. CONCLUSIONS: Thrombus apposed on stents creates large variations in drug uptake and can act to either increase or decrease wall deposition according to the clot and stent geometry. Arterial deposition of drug from stents deployed in clots will be highly variable and unpredictable unless the clot can be adequately controlled or removed.

Animals↗
PubMed2005

Cells in fluidic environments are sensitive to flow frequency.

Virtually all cells accommodate to their mechanical environment. In particular, cells subject to flow respond to rapid changes in fluid shear stress (SS), cyclic stretch (CS), and pressure. Recent studies have focused on the effect of pulsatility on cellular behavior. Since cells of many different tissue beds are constantly exposed to fluid flows over a narrow range of frequencies, we hypothesized that an intrinsic flow frequency that is optimal for determining cell phenotype exists. We report here that cells from various tissue beds (bovine aortic endothelial cells (BAEC), rat small intestine epithelial cells (RSIEC), and rat lung epithelial cells (RLEC)) proliferate maximally when cultured in a perfusion bioreactor under pulsatile conditions at a specific frequency, independent of the applied SS. Vascular endothelial and pulmonary epithelial cell proliferation peaked under 1 Hz pulsatile flow. In contrast, proliferation of gastrointestinal cells, which in their physiological context are subject to no flow or higher wavelength signal, was maximum at 0.125 Hz or under no flow. Moreover, exposure of BAEC to pulsatile flow of varying frequency influenced their nitric oxide synthase activity and prostacyclin production, which reached maximum values at 1 Hz. Notably, the "optimal" frequencies for the cell types examined correspond to the physiologic operating range of the organs from where they were initially derived. These findings suggest that frequency, independent of shear, is an essential determinant of cell response in pulsatile environments.

Animals↗
PubMed2004

Environmental influences on endovascular stent platelet reactivity: an in vitro comparison of stainless steel and gold surfaces.

Thrombosis is an initiating reaction to vascular injury that follows the placement of vascular devices. Platelets play a crucial role in this response. Their interaction with endovascular devices is not solely a function of device properties, but a multifaceted response dependent on several biological factors that interact in the context of a hemodynamic environment. We sought to investigate the role of local environmental variations on determining indices of biocompatibility. Using a recently described in vitro flow apparatus, we separately studied the platelet and coagulative component responses to stainless steel endovascular stents with and without gold coating. When allowed to interact, these biological mediators of thrombosis enabled varied biocompatibility outcomes in a manner that was dependent on flow. Using platelet reactivity as an index, the stainless steel faired better under some conditions, while under other conditions, gold was superior. Considering such impacts of local environment on biocompatibility is important, both in the interpretation of experimental findings, as well as the continued use and optimized development of vascular devices.

Biocompatible Materials↗
PubMed2004

Analysis of compartmental models of ligand-induced endocytosis.

Kinetic models have played a pivotal role in the study of ligand-induced endocytosis. However, an analysis that suggests a systematic way to validate such models is lacking. The current work analyses the base model of ligand-induced endocytosis for three widely used experimental protocols. In protocol I cells initially devoid of ligand are incubated in ligand solution, whereas protocols II and III are desorption experiments in which an initial pool of surface or internalized ligand-receptor complexes, respectively, are released into an elution medium that is initially devoid of ligand. A short-time analysis of protocol I using successive substitutions yielded a corrected pre-factor for the In/Sur plot introduced by Wiley and Cunningham (Cell 25 (1981) 433). In contrast, neglecting the variation in receptor numbers yielded an approximation of protocol I that is valid for long times (e.g. tens of minutes). Similarly, the low cell-concentration limits of protocols II and III are derived by neglecting the concentration of free ligand. The simplicity of these approximations provides a simple and reliable method for estimating the parameters governing ligand kinetics, while their definitive nature implies that they can be used to verify the validity of the base model. This analysis also provides insight on the fast endocytosis and recycling limit of protocol III.

Animals↗
PubMed2004

Specific binding to intracellular proteins determines arterial transport properties for rapamycin and paclitaxel.

Endovascular drug-eluting stents have changed the practice of medicine, and yet it is unclear how they so dramatically reduce restenosis and how to distinguish between the different formulations available. Biological drug potency is not the sole determinant of biological effect. Physicochemical drug properties also play important roles. Historically, two classes of therapeutic compounds emerged: hydrophobic drugs, which are retained within tissue and have dramatic effects, and hydrophilic drugs, which are rapidly cleared and ineffective. Researchers are now questioning whether individual properties of different drugs beyond lipid avidity can further distinguish arterial transport and distribution. In bovine internal carotid segments, tissue-loading profiles for hydrophobic paclitaxel and rapamycin are indistinguishable, reaching load steady state after 2 days. Hydrophilic dextran reaches equilibrium in several hours at levels no higher than surrounding solution concentrations. Both paclitaxel and rapamycin bind to the artery at 30-40 times bulk concentration. Competitive binding assays confirm binding to specific tissue elements. Most importantly, transmural drug distribution profiles are markedly different for the two compounds, reflecting, perhaps, different modes of binding. Rapamycin, which binds specifically to FKBP12 binding protein, distributes evenly through the artery, whereas paclitaxel, which binds specifically to microtubules, remains primarily in the subintimal space. The data demonstrate that binding of rapamycin and paclitaxel to specific intracellular proteins plays an essential role in determining arterial transport and distribution and in distinguishing one compound from another. These results offer further insight into the mechanism of local drug delivery and the specific use of existing drug-eluting stent formulations.

Animals↗