RESUMO
Tissue-type plasminogen activator (t-PA) can modulate permeability of the neurovascular unit and exacerbate injury in ischemic stroke. We examined the effects of t-PA using in vitro models of the blood-brain barrier. t-PA caused a concentration-dependent increase in permeability. This effect was dependent on plasmin formation and potentiated in the presence of plasminogen. An inactive t-PA variant inhibited the t-PA-mediated increase in permeability, whereas blockade of low-density lipoprotein receptors or exposed lysine residues resulted in similar inhibition, implying a role for both a t-PA receptor, most likely a low-density lipoprotein receptor, and a plasminogen receptor. This effect was selective to t-PA and its close derivative tenecteplase. The truncated t-PA variant reteplase had a minor effect on permeability, whereas urokinase and desmoteplase were ineffective. t-PA also induced marked shape changes in both brain endothelial cells and astrocytes. Changes in astrocyte morphology coincided with increased F-actin staining intensity, larger focal adhesion size, and elevated levels of phosphorylated myosin. Inhibition of Rho kinase blocked these changes and reduced t-PA/plasminogen-mediated increase in permeability. Hence plasmin, generated on the cell surface selectively by t-PA, modulates the astrocytic cytoskeleton, leading to an increase in blood-brain barrier permeability. Blockade of the Rho/Rho kinase pathway may have beneficial consequences during thrombolytic therapy.
Assuntos
Astrócitos/efeitos dos fármacos , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Fibrinolisina/farmacologia , Ativador de Plasminogênio Tecidual/farmacologia , Quinases Associadas a rho/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/fisiologia , Barreira Hematoencefálica/patologia , Barreira Hematoencefálica/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Fibrinolisina/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Teóricos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Fine movement in the body is controlled by the motor cortex, which signals in a topographically specific manner to neurons in the spinal cord by means of the corticospinal tract (CST). How the correct topography of the CST is established is unknown. To investigate the possibility that the Eph tyrosine kinase receptor EphA4 is involved in this process, we have traced CST axons in mice in which the EphA4 gene has been deleted. The forelimb subpopulation of CST axons is unaffected in the EphA4-/- mice, but the hindlimb subpopulation branches too early within the cord, both temporally and spatially. EphA4 shows a dynamic expression pattern in the environment of the developing CST in the spinal cord: high at the time of forelimb branching and down-regulated before hindlimb branching. To examine whether the fore- and hindlimb subpopulations of CST axons respond differently to EphA4 in their environment, neurons from fore- and hindlimb motor cortex were cultured on a substrate containing EphA4. Neurons from the hindlimb cortex showed reduced branching on the EphA4 substrate compared with their forelimb counterparts. Neurons from the hindlimb cortex express ephrinA5, a high-affinity ligand for EphA4, at higher levels compared with forelimb cortex neurons, and this expression is down-regulated before hindlimb branching. Together, these findings suggest that EphA4 regulates topographic mapping of the CST by controlling the branching of CST axons in the spinal cord.