Biomedical subjects

Michael Sobel

Publications and source records attributed to Michael Sobel.

11 recordsLinked to original sources

PubMed2006

Heparin-II domain of fibronectin is a vascular endothelial growth factor-binding domain: enhancement of VEGF biological activity by a singular growth factor/matrix protein synergism.

We describe extracellular interactions between fibronectin (Fn) and vascular endothelial growth factor (VEGF) that influence integrin-growth factor receptor crosstalk and cellular responses. In previous work, we found that VEGF bound specifically to fibronectin (Fn) but not vitronectin or collagens. Herein we report that VEGF binds to the heparin-II domain of Fn and that the cell-binding and VEGF-binding domains of Fn, when physically linked, are necessary and sufficient to promote VEGF-induced endothelial cell proliferation, migration, and Erk activation. Using recombinant Fn domains, the C-terminal heparin-II domain of Fn (type III repeats 13 to 14) was identified as a key VEGF-binding site. Mutation of the heparin-binding residues on FnIII(13-14) abolished VEGF binding, and peptides corresponding to the heparin-binding sequences in FnIII(13-14) inhibited VEGF binding to Fn. Fn fragments containing both the alpha5beta1 integrin-binding domain (III 9 to 10) and the VEGF-binding domain (III 13 to 14) significantly enhanced VEGF-induced EC migration and proliferation and induced strong phosphorylation of the VEGF receptor and Erk. Neither the cell-binding or VEGF-binding fragment of Fn alone had comparable VEGF-promoting effects. These results suggest that the mechanism of VEGF/Fn synergism is mediated extracellularly by the formation of a novel VEGF/Fn complex requiring both the cell-binding and VEGF-binding domains linked in a single molecular unit. These data also highlight a new function for the Fn C-terminal heparin-binding domain that may have important implications for angiogenesis and tumor growth.

Binding Sites↗

PubMed2006

The "linker" region (amino acids 38-47) of the disintegrin elegantin is a novel inhibitory domain of integrin alpha5beta1-dependent cell adhesion on fibronectin: evidence for the negative regulation of fibronectin synergy site biological activity.

Disintegrins are a family of potent inhibitors of cell-cell and cell-matrix adhesion. In this study we have identified a region of the disintegrin elegantin, termed the "linker domain" (amino acids 38-47), with inhibitory activity toward alpha(5)beta(1)-mediated cell adhesion on fibronectin (Fn). Using a chimeric structure-function approach in which sequences of the functionally distinct disintegrin kistrin were introduced into the elegantin template at targeted sites, a loss of inhibitory function toward alpha(5)beta(1)-mediated adhesion on Fn was observed when the elegantin linker domain was substituted. Subsequent analysis comparing the inhibitory efficacies of the panel of elegantin-kistrin chimeras toward CHO alpha(5) cell adhesion on recombinant Fn III(6-10) fragments showed that the loss of inhibitory activity associated with the disruption of the elegantin linker domain was dependent upon the presence of a functional Fn III(9) synergy site within the Fn III(6-10) substrate. This suggested that the elegantin linker domain inhibits primarily the activity of the Fn synergy domain in promoting alpha(5)beta(1) integrin-mediated cell adhesion. Construction of a cyclic peptide corresponding to the entire region of the elegantin linker domain showed that this domain has intrinsic alpha(5)beta(1) inhibitory activity comparable with the activity of the RGDS peptide. These data demonstrate a novel biological function for a disintegrin domain that antagonizes integrin-mediated cell adhesion.

ADAM Proteins↗

PubMed2005

Novel hepatocyte growth factor (HGF) binding domains on fibronectin and vitronectin coordinate a distinct and amplified Met-integrin induced signalling pathway in endothelial cells.

BACKGROUND: The growth of new blood vessels in adult life requires the initiation of endothelial cell migration and proliferation from pre-existing vessels in addition to the recruitment and differentiation of circulating endothelial progenitor cells. Signals emanating from growth factors and the extracellular matrix are important in regulating these processes. RESULTS: Here we report that fibronectin (FN) and vitronectin (VN) modulate the responses of endothelial cells to HGF (Scatter Factor), an important pro-angiogenic mediator. Novel binding sites for HGF were identified on both FN and VN that generate molecular complexes with enhanced biological activity and these were identified in the supernatants of degranulated platelet suspensions implicating their release and formation in vivo. In the absence of co-stimulation with an ECM glycoprotein, HGF could not promote endothelial cell migration but retained the capacity to induce a proliferative response utilising the Map kinase pathway. Through promoting Met-Integrin association, HGF-FN and HGF-VN complexes coordinated and enhanced endothelial cell migration through activation of the PI-3 kinase pathway involving a Ras-dependent mechanism whereas a Ras-independent and attenuated migratory response was promoted by co-stimulation of cells with HGF and a non-binding partner ECM glycoprotein such as collagen-1. CONCLUSIONS: These studies identify a novel mechanism and pathway of HGF signalling in endothelial cells involving cooperation between Met and integrins in a Ras dependent manner. These findings have implications for the regulation of neovascularization in both health and disease.

Binding Sites↗

PubMed2004

Survival after lower-extremity amputation.

BACKGROUND: Lower extremity amputation has long been considered an end-of-life event and it is unclear if survival after amputation has improved over time. STUDY DESIGN: A retrospective cohort comprised from a statewide, hospital discharge database was used to determine if survival after amputation improved with time. The cohort included all patients (older than 18 years) with nontraumatic, lower extremity amputations (1987 to 2000). Survival analysis was used to determine the adjusted hazard ratio of survival as it related to the era of amputation. RESULTS: A total of 13,807 patients (mean age +/- SD, 67 +/- 15, 58.5% men) underwent amputation. The gender and age standardized frequency of amputation remained essentially stable, with a 0.01% increase per year (95% CI, 0.006-0.01%). During followup, 49.2% (6,795/13,807) of patients died, with significantly (p < 0.001) worse outcomes for more proximal levels of amputation. After controlling for potential confounders, including age, gender, level of amputation, comorbid illness, emergency status of procedure, hospital type, and payer of the procedure, patients undergoing amputation in more recent years (1995 to 2000) had a 28% lower hazard of dying (hazard ratio 0.72 [95% CI, 0.67-0.77%) during the study period than those undergoing operation before 1995. Thirty-day survival did not improve by era (p = 0.2), although 1- and 5-year survival after amputation was significantly greater for all levels of amputation (p < 0.001). CONCLUSIONS: Although 30-day survival associated with amputation has remained stable in the state of Washington over the past 14 years, longterm survival after amputation has improved considerably with time. The reasons underlying this improvement should be explored so that further gains may be achieved.

Aged↗

PubMed2004

Fibronectin promotes VEGF-induced CD34 cell differentiation into endothelial cells.

BACKGROUND: Adult endothelial progenitor cells (EPC) may be a useful source for engineering the endothelialization of vascular grafts. However, the optimal factors that promote differentiation of EPCs into endothelium remain to be elucidated. The goal of this current report was to determine which extracellular matrix (ECM) protein might modulate or enhance the effects of EPCs on differentiation into mature endothelium. METHODS: Human EPCs (CD34(+) cells) were cultured in ECM-coated six-well plates in MCDB-131 medium containing vascular endothelial growth factor (VEGF), insulin-like growth factor-1, and basic fibroblast growth factor. After 21 days, differentiated endothelial colonies were confirmed by immunofluorescence for von Willebrand factor (vWF) and vascular-endothelial (VE)-cadherin and mRNA expression of the endothelial markers Flk-1, vWF, and VE-cadherin. Cell migration toward the VEGF-matrix protein combinations was also measured. RESULTS: As judged by positive staining for endothelial markers vWF and VE-cadherin, the combination of VEGF with fibronectin (FN) produced significantly more endothelial colonies (P <.05) than did collagens I or IV or vitronectin. Defined fragments of FN did not enhance VEGF-mediated effects. Fibrinogen produced intermediate stimulation of differentiation. FN also enhanced VEGF-mediated CD34(+) cell migration. Blockade of alpha5beta1, but not alphavbeta3 or alphavbeta5, inhibited both VEGF-mediated CD34(+) cell differentiation and migration. CONCLUSIONS: VEGF and FN together significantly promote the migration and differentiation of CD34(+) cells. This synergism is specific to FN and the alpha5beta1 integrin. Combinations of VEGF and FN may be useful in promoting differentiation of circulating endothelial progenitors into endothelial cells for tissue engineering. Clinical relevance Treatment of injured or diseased tissues with adult stem cells is a promising approach. In particular, bone marrow derived circulating endothelial progenitors (CEP's) have been shown to differentiate into endothelial cells in vitro and promote tissue revascularization of ischemic limbs and myocardium in vivo. Because of the relative ease of obtaining CEP's and as well as its high proliferative rate, CEP's may have clinical potential for endothelialization of prosthetic vascular grafts and revascularization of injured myocardium. However, there is a need to better understand the molecular pathways involved in the proliferation and differentiation of CEP's to take full advantage of its clinical potential.

Adult↗

PubMed2004

Antithrombotic therapy in peripheral arterial occlusive disease: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy.

This chapter about antithrombotic therapy for peripheral arterial occlusive disease is part of the seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy: Evidence Based Guidelines. Grade 1 recommendations are strong and indicate that the benefits do, or do not, outweigh risks, burden, and costs, and Grade 2 suggests that individual patients' values may lead to different choices (for a full understanding of the grading see Guyatt et al, CHEST 2004;126:179S-187S). Among the key recommendations in this chapter are the following: For patients with chronic limb ischemia, we recommend lifelong aspirin therapy in comparison to no antiplatelet therapy in patients with clinically manifest coronary or cerebrovascular disease (Grade 1A) and in those without clinically manifest coronary or cerebrovascular disease (Grade 1C+). We recommend clopidogrel over no antiplatelet therapy (Grade 1C+) but suggest that aspirin be used instead of clopidogrel (Grade 2A). For patients with disabling intermittent claudication who do not respond to conservative measures and who are not candidates for surgical or catheter-based intervention, we suggest cilostazol (Grade 2A). We suggest that clinicians not use cilostazol in patients with less-disabling claudication (Grade 2A). In these patients, we recommend against the use of pentoxifylline (Grade 1B). We suggest clinicians not use prostaglandins (Grade 2B). In patients with intermittent claudication, we recommend against the use of anticoagulants (Grade 1A). In patients with acute arterial emboli or thrombosis, we recommend treatment with immediate systemic anticoagulation with unfractionated heparin (UFH) [Grade 1C]. We also recommend systemic anticoagulation with UFH followed by long-term vitamin K antagonist (VKA) in patients with embolism [Grade 1C]). For patients undergoing major vascular reconstructive procedures, we recommend UFH at the time of application of vascular cross-clamps (Grade 1A). In patients undergoing prosthetic infrainguinal bypass, we recommend aspirin (Grade 1A). In patients undergoing infrainguinal femoropopliteal or distal vein bypass, we suggest that clinicians do not routinely use a VKA (Grade 2A). For routine patients undergoing infrainguinal bypass without special risk factors for occlusion, we recommend against VKA plus aspirin (Grade 1A). For those at high risk of bypass occlusion and limb loss, we suggest VKA plus aspirin (Grade 2B). In patients undergoing carotid endarterectomy, we recommend aspirin preoperatively and continued indefinitely (Grade 1A). In nonoperative patients with asymptomatic or recurrent carotid stenosis, we recommend lifelong aspirin (Grade 1C+). For all patients undergoing extremity balloon angioplasty, we recommend long-term aspirin (Grade 1C+).

Arterial Occlusive Diseases↗

PubMed2003

Popliteal vascular injury during total knee arthroplasty.

BACKGROUND: Popliteal vascular injury during total knee arthroplasty (TKA) is rare. Although the etiology, management, and outcome of this injury have not been systematically studied, several anecdotal reviews and small case series have suggested a high morbidity, with tourniquet injury as a predominant etiology. METHODS: We surveyed 190 members of a major regional vascular society concerning their experiences with popliteal vascular injuries sustained during TKA, specifically regarding the presentation, mechanisms of injury, diagnostic studies, surgical exposure and methods of repair, and outcomes. RESULTS: Thirteen surgeons provided data (6.8% response rate) on 19 patients with TKA-related popliteal artery injuries. In contrast to the historical literature, sharp, direct trauma was the predominant etiology of vascular injury. Vascular reconstructive techniques varied, with arterial bypass being the most common. There was no mortality in this series. Eighty-four percent (16/19) of the patients had full recoveries. Limb loss occurred in 2 of the 19 patients (10.5%). CONCLUSION: Popliteal vascular injuries during TKA are primarily the result of direct trauma to the vessel. Prompt diagnosis, extensile vascular exposure through a medial knee surgical incision, and standard reconstructive techniques offer a good prognosis for this limb-threatening complication.

Arthroplasty, Replacement, Knee↗

PubMed2002

Novel vascular endothelial growth factor binding domains of fibronectin enhance vascular endothelial growth factor biological activity.

Interactions between integrins and growth factor receptors play a critical role in the development and healing of the vasculature. This study mapped two binding domains on fibronectin (FN) that modulate the activity of the angiogenic factor, vascular endothelial growth factor (VEGF). Using solid-phase assays and surface plasmon resonance analysis, we identified two novel VEGF binding domains within the N- and C-terminus of the FN molecule. Native FN bound to VEGF enhanced endothelial cell migration and mitogen-activated protein (MAP) kinase activity, but FN that is devoid of the VEGF binding domains failed to do so. Coprecipitation studies confirmed a direct physical association between VEGF receptor-2 (Flk-1) and the FN integrin, alpha5beta1, which required intact FN because FN fragments lacking the VEGF binding domains failed to support receptor association. Thrombin-activated platelets released intact VEGF/FN complexes, which stimulated endothelial cell migration and could be inhibited by soluble high affinity VEGF receptor 1 and antibodies to alpha5beta1 integrin. This study demonstrates that FN is potentially a physiological cofactor for VEGF and provides insights into mechanisms by which growth factor receptors and integrins cooperate to influence cellular behavior.

Binding Sites↗

PubMed2002

Anticoagulants: to bleed or not to bleed, that is the question.

Thromboembolic vascular diseases remain the main cause of death in Western industrialized societies. Anticoagulants retard the formation, growth, and embolization of thrombi and are effective agents in the prevention and treatment of thromboembolic disease. Anticoagulants in venous thromboembolism have been investigated extensively with rigorous randomized, controlled trials, while the roles for anticoagulants in arterial thromboembolism generally have evolved through natural history studies and empirical practice. Thus, many current guidelines for anticoagulant use in arterial disease are based on successful established routines and rational therapy. To effectively balance the efficacy and risks of anticoagulation, the vascular surgeon needs a thorough understanding of anticoagulant drugs, their mechanisms of action, and their proven and unproven indications. Since the first use of heparin in arterial surgery, a variety of new and different anticoagulants have become available, including low-molecular-weight heparins, heparin-like drugs, hirudins, and thrombin inhibitors. Despite their diverse actions, they all inhibit some portion of the plasma coagulation cascade, thus distinguishing them from platelet inhibitors or fibrinolytics. Every interference with the coagulation cascade carries a risk of minor, major, or fatal hemorrhage. To date, no drug or therapeutic strategy has succeeded fully in dissociating its antithrombotic effects from its risks of bleeding.

Anticoagulants↗

PubMedDate not supplied

Immobilization and clustering of structurally defined oligosaccharides for sugar chips: an improved method for surface plasmon resonance analysis of protein-carbohydrate interactions.

Oligosaccharides are increasingly being recognized as important partners in receptor-ligand binding and cellular signaling. Surface plasmon resonance (SPR) is a very powerful tool for the real-time study of the specific interactions between biological molecules. We report here an advanced method for the immobilization of oligosaccharides in clustered structures for SPR and their application to the analysis of heparin-protein interactions. Reductive amination reactions and linker molecules were designed and optimized. Using mono-, tri-, or tetravalent linker compounds, we incorporated synthetic structurally defined disaccharide units of heparin and immobilized them as ligands for SPR. Their binding to an important hemostatic protein, von Willebrand factor (vWf), and its known heparin-binding domain was quantitatively analyzed. These multivalent ligand conjugates exhibited reproducible binding behavior, with consistency of the surface conditions of the SPR chip. This novel technique for oligosaccharide immobilization in SPR studies is accurate, specific, and easily applicable to both synthetic and naturally derived oligosaccharides.

Animals↗

PubMedDate not supplied

Heparin modulates integrin-mediated cellular adhesion: specificity of interactions with alpha and beta integrin subunits.

Heparin is known to influence the growth, proliferation, and migration of vascular cells, but the precise mechanisms are unknown. We previously demonstrated that unfractionated heparin (UH) binds to the platelet integrin alpha(IIb)beta(3), and enhances ligand binding. To help define the specificity and site(s) of heparin-integrin interactions, we employed the erythroleukemic K562 cell line, transfected to express specific integrins (alpha(v)beta(3), alpha(v)beta(5), and alpha(IIb)beta(3)). By comparing K562 cells expressing a common alpha subunit (Kalpha(v)beta(3), Kalpha(v)beta(5)) with cells expressing a common beta subunit (Kalpha(v)beta(3), Kalpha(IIb)beta(3)), we observed that heparin differentially modulated integrin-mediated adhesion to vitronectin. UH at 0.5-7.5 microg/ml consistently enhanced the adhesion of beta(3) expressing cells (Kalpha(v)beta(3),Kalpha(IIb)beta(3)). In contrast, UH at 0.5-7.5 microg/ml inhibited Kalpha(v)beta(5) adhesion. Experiments using integrin-blocking antibodies, appropriate control ligands, and nontransfected native K562 cells revealed that heparin's actions were mediated by the specific integrins under study. Preincubation of heparin with Kalpha(v)beta(3) cells enhanced adhesion, while preincubation of heparin with the adhesive substrate (vitronectin) had minimal effect. There was a structural specificity to heparin's effect, in that a low molecular weight heparin and chondroitin sulfate showed significantly less enhancement of adhesion. These findings suggest that heparin's modulation of integrin-ligand interactions occurs through its action on the integrin. The inhibitory or stimulatory effects of heparin depend on the beta subunit type, and the potency is dictated by structural characteristics of the glycosaminoglycan.

Antibodies↗