Biomedical subjects

Dmitry A Soloviev

Publications and source records attributed to Dmitry A Soloviev.

4 recordsLinked to original sources

PubMed2006

Cell adhesion and migration assays.

Adhesion and migration are basic responses of living cells to environmental stimuli. Such responses are central to a broad range of physiological processes, such as the immune response, repair of injured tissues, and prevention of excessive bleeding. Cell adhesion and migration also contributes to pathologies, including vascular and inflammatory diseases, as well as tumor growth and metastasis. These cellular responses depend on engagement of adhesion receptors by components of the extracellular matrix or molecules present on the surface of other cells. Hence, cell adhesion and migration assays are crucial methods in cell biology. In this chapter, several detailed protocols describing cell adhesion and migration assays are presented, and advantages and disadvantages of each method are discussed.

Cardiovascular Diseases↗

PubMed2004

Integrin alphaMbeta2 orchestrates and accelerates plasminogen activation and fibrinolysis by neutrophils.

Plasmin, the pivotal thrombolytic enzyme, is generated on the surface of many cell types, where urokinase receptor (uPAR)-bound urokinase (uPA) activates cell-bound plasminogen (Plg). It has been reported that neutrophils mediate endogenous thrombolysis involving a uPA-dependent mechanism, and we previously demonstrated that both uPAR and integrin alpha(M)beta(2) recognize uPA to control cell migration and adhesion. In the present study, we report that the alpha(M)beta(2) regulates neutrophil-dependent fibrinolysis. Phorbol 12-myristate 13-acetate (PMA)-stimulated but not resting neutrophils dissolved fibrin clots, and this activity was not only uPA- and Plg-dependent but also alpha(M)beta(2)-dependent. Purified alpha(M)beta(2) directly bound uPA (K(d) = 40 nm) and Plg (K(d) = 1 microm) in a dose-dependent and saturable manner. In Plg activation assays, addition of purified alpha(M)beta(2), but not a control protein, to a single chain uPA (sc-uPA)/Plg mixture, decreased the K(m) from 2 to 0.1 microm, thereby augmenting the overall reaction efficiency by 50-fold. The binding of sc-uPA to alpha(M)beta(2) was critical for the alpha(M)beta(2)-mediated enhancement of plasmin (Plm) generation, because this effect was lost when WT-sc-uPA was replaced with a kringle-less mutant (DeltaK-sc-uPA), which does not bind to alpha(M)beta(2). Plm inactivation by alpha(2)-antiplasmin was significantly delayed when Plm was preincubated with purified, soluble alpha(M)beta(2). When Plg was added to PMA-stimulated neutrophils, both uPA and Plg were co-immunoprecipitated with alpha(M)beta(2.) Thus, assembly of Plg and uPA on integrin alpha(M)beta(2) regulates Plm activity and, thereby, plays a crucial role in neutrophil-mediated thrombolysis.

Cell Adhesion↗

PubMed2004

Leukocyte engagement of fibrin(ogen) via the integrin receptor alphaMbeta2/Mac-1 is critical for host inflammatory response in vivo.

The leukocyte integrin alpha(M)beta(2)/Mac-1 appears to support the inflammatory response through multiple ligands, but local engagement of fibrin(ogen) may be particularly important for leukocyte function. To define the biological significance of fibrin(ogen)-alpha(M)beta(2) interaction in vivo, gene-targeted mice were generated in which the alpha(M)beta(2)-binding motif within the fibrinogen gamma chain (N(390)RLSIGE(396)) was converted to a series of alanine residues. Mice carrying the Fibgamma(390-396A) allele maintained normal levels of fibrinogen, retained normal clotting function, supported platelet aggregation, and never developed spontaneous hemorrhagic events. However, the mutant fibrinogen failed to support alpha(M)beta(2)-mediated adhesion of primary neutrophils, macrophages, and alpha(M)beta(2)-expressing cell lines. The elimination of the alpha(M)beta(2)-binding motif on fibrin(ogen) severely compromised the inflammatory response in vivo as evidenced by a dramatic impediment in leukocyte clearance of Staphylococcus aureus inoculated into the peritoneal cavity. This defect in bacterial clearance was due not to diminished leukocyte trafficking but rather to a failure to fully implement antimicrobial functions. These studies definitively demonstrate that fibrin(ogen) is a physiologically relevant ligand for alpha(M)beta(2), integrin engagement of fibrin(ogen) is critical to leukocyte function and innate immunity in vivo, and the biological importance of fibrinogen in regulating the inflammatory response can be appreciated outside of any alteration in clotting function.

Animals↗

PubMed2002

Convergence of the adhesive and fibrinolytic systems: recognition of urokinase by integrin alpha Mbeta 2 as well as by the urokinase receptor regulates cell adhesion and migration.

Previous studies demonstrated that integrin alpha(M)beta(2) (CD11b/18, Mac-1) forms a physical complex with the urokinase-type plasminogen activator receptor (uPAR/CD87) on leukocytes. In this study, we used human peripheral blood neutrophils and transfected cells expressing alpha(M)beta(2), uPAR, or both receptors to show that the integrin can directly interact with urokinase (uPA). We demonstrate that alpha(M)beta(2) supported adhesion and migration of these cells to uPA, and, in each case, blockade of alpha(M)beta(2) suppressed the response. Within uPA, both the kringle and proteolytic domains are recognized by alpha(M)beta(2), which are distinct from the growth factor domain that binds to uPAR. Within the alpha(M) subunit of the integrin, the I domain interacts with uPA, which is distinct from the region that interacts with uPAR. On cells expressing uPAR and alpha(M)beta(2), both receptors mediated adhesion and migration. This cooperation was particularly apparent in the responses of neutrophils to uPA, where blockade of alpha(M)beta(2) reduced uPAR-mediated responses and engagement of uPAR enhanced recognition of uPA by alpha(M)beta(2). Thus, recognition of uPA by alpha(M)beta(2) allows for formation of a multicontact trimolecular complex, in which a single uPA ligand may bind simultaneously to both uPAR and alpha(M)beta(2). This complex may play an important role in the control of inflammatory cell migration and vascular homeostasis.

Cell Adhesion↗