Cav-1 Protein Levels in Serum and Infarcted Brain Correlate with Hemorrhagic Volume in a Mouse Model of Thromboembolic Stroke, Independently of rt-PA Administration.

Thrombolytic therapy with recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for acute ischemic stroke. However, its administration is still limited due to the associated increased risk of hemorrhagic transformation (HT). rt-PA may exacerbate blood-brain barrier (BBB) injury by several mechanisms that have not been fully elucidated. Caveolin-1 (Cav-1), a major structural protein of caveolae, has been linked to the endothelial barrier function. The effects of rt-PA on Cav-1 expression remain largely unknown. Here, Cav-1 protein expression after ischemic conditions, with or without rt-PA administration, was analyzed in a murine thromboembolic middle cerebral artery occlusion (MCAO) and in brain microvascular endothelial bEnd.3 cells subjected to oxygen/glucose deprivation (OGD). Our results show that Cav-1 is overexpressed in endothelial cells of infarcted area and in bEnd.3 cell line after ischemia but there is disagreement regarding rt-PA effects on Cav-1 expression between both experimental models. Delayed rt-PA administration significantly reduced Cav-1 total levels from 24 to 72 h after reoxygenation and increased pCav-1/Cav-1 at 72 h in the bEnd.3 cells while it did not modify Cav-1 immunoreactivity in the infarcted area at 24 h post-MCAO. Importantly, tissue Cav-1 positively correlated with Cav-1 serum levels at 24 h post-MCAO and negatively correlated with the volume of hemorrhage after infarction, the latter supporting a protective role of Cav-1 in cerebral ischemia. In addition, the negative association between baseline serum Cav-1 levels and hemorrhagic volume points to a potential usefulness of baseline serum Cav-1 levels to predict hemorrhagic volume, independently of rt-PA administration.

Evaluation of long-term rt-PA effects on bEnd.3 endothelial cells under ischemic conditions; changes in ZO-1 expression and glycosylation of the bradykinin B2 receptor.

Recombinant tissue plasminogen activator (rt-PA) has proven effective in the treatment of acute ischemic stroke, despite the increased risk of hemorrhagic transformation (HT), its major associated complication. Although it is known that HT is related to blood brain barrier (BBB) disruption, the underlying mechanisms are not well established. We assessed time-dependent effects of rt-PA on the bEnd.3 murine brain endothelial cell line subjected either to normoxia or to 2.5 h of oxygen and glucose deprivation (OGD), evaluating a longer period than has previously been done, beyond 6 h post-reoxygenation. Parameters of cell viability, metabolic activity, ionic and transcellular permeability, as well as levels of claudin-5, zonula occludens-1 (ZO-1) and bradykinin B2 receptor (B2R) protein expression were analyzed at 24, 48 and 72 h post-reoxygenation with or without the administration of rt-PA. rt-PA treatment increased both the ionic and transcellular permeability until 72 h and did not modify cell viability or metabolic activity or the expression of claudin-5, ZO-1 and B2R under normoxia at any analyzed time. Under OGD conditions, rt-PA exacerbated OGD effects on metabolic activity from 48 to 72 h, increased transcellular permeability from 24 to 72 h, significantly decreased ZO-1 protein levels at the plasma membrane and increased B2R glycosylation at 72 h post-reoxygenation. Our findings suggest that a long-term analysis is necessary to elucidate time-dependent molecular mechanisms associated to BBB breakdown due to rt-PA administration under ischemia. Thus, protective BBB therapies after ischemic stroke and rt-PA treatment should be explored at least until 72 h after OGD and rt-PA administration.

Evaluation of common housekeeping proteins under ischemic conditions and/or rt-PA treatment in bEnd.3 cells.

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is the only pharmacological approved treatment for ischemic stroke, despite its associated increasing risk of hemorrhagic transformation. Since many of rt-PA effects in blood-brain barrier (BBB) are not well characterized, the study of protein changes in BBB cells after rt-PA administration may help to understand its adverse effects. Our aim was to analyze protein levels of four commonly used housekeeping proteins: ß-Actin, α-Tubulin, GAPDH and HPRT in bEnd.3 endothelial cell line subjected to oxygen and glucose deprivation (OGD) conditions and rt-PA treatment to determine their reliability as Western blot loading controls. bEnd.3 monolayers were subjected to 2.5 h of OGD and reperfusion with/without 20 µg/ml of rt-PA. At 3, 6, 24 and 72 h post-OGD, protein levels were analyzed by Western blot using Stain-Free technology. OGD significantly decreased ß-Actin, α-Tubulin, GAPDH and HPRT protein levels at 3, 6, 24 and 72 h post-OGD without significant rt-PA treatment effects except for the GAPDH levels increase in control condition at 3 h post-OGD. The present study clearly demonstrated that ß-Actin, α-Tubulin, GAPDH and HPRT proteins are not suitable as loading controls for Western Blot analysis in bEnd.3 cells after OGD. SIGNIFICANCE: We reported altered levels of ß-Actin, α-Tubulin, GAPDH and HPRT housekeeping proteins in bEnd.3 endothelial cell line after an ischemic insult. Therefore, we demonstrated that these proteins are not suitable as loading controls for Western Blot analysis in our experimental conditions and we recommended the use of Stain-Free gels as an alternative to traditional housekeeping proteins normalization.