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.This work was supported by grants from Instituto de Salud Carlos III and co-fnanced by the European Development Regional Fund “A Way to Achieve Europe” Health Strategic Action Program PI13/02258 and PI17/02123 (MC), PI20/00535 (IL), and Spanish Stroke Research Network RETICS RD12/0014/0010 (MC), and RD16/0019/0003 (JS), RD16/0019/0004 (MC), and RD16/0019/0009 (IL); from Regional Madrid Government B2017/BMD- 3688 (IL); from Spanish Ministry of Science and Innovation PID2019-106581RBI00 (MAM); from Leducq Foundation for Cardiovascular Research TNE-19CVD01 (MAM); and from Fundación La Caixa HR17_00527 (MAM). P. Comajoan was a recipient of a predoctoral fellowship from the University of Girona (IF-UdG 2015).S

Efecte de l'administració de l'rt-PA en condicions isquèmiques in vitro i in vivo: Cav-1 com a potencial biomarcador de volum d'infart

Recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for the treatment of acute ischaemic stroke. However, the application of this therapy is limited to <5-7% of patients due to the associated increased risk of haemorrhagic transformation (HT). Although it is known that HT is related to rt-PA-induced blood brain barrier (BBB) disruption, the underlying mechanisms are not well established. The obtained results show that long-term studies are needed to elucidate time-dependent molecular mechanisms associated to BBB breakdown, and to explore protective BBB therapies after ischaemic stroke and rt-PA treatment. On the other hand, it has been demonstrated that OGD induces significant alterations to loading control proteins for Western Blot analysis proposing Stain-Free technology as an alternative normalization method to traditional housekeeping proteins. Finally, serum Cav-1 levels could represent a potential biomarker predicting the ischaemic outcome before rt-PA administrationL'rt-PA és l’únic fàrmac aprovat per tractar l’ictus isquèmic agut. No obstant, l’estreta finestra terapèutica, deguda al risc associat de transformació hemorràgica (TH) provoca que només s’apliqui a <5-7% dels pacients. La TH està relacionada amb la disrupció de la barrera hematoencefàlica (BHE) deguda a l’rt-PA però els mecanismes subjacents encara no estan del tot establerts. Els resultats obtinguts mostren que es requereixen estudis a llarg termini per tal de dilucidar els mecanismes dependents del temps associats a la disrupció de la BHE, i explorar noves teràpies protectores per al tractament de l’ictus isquèmic. S’ha demostrat que la POG provoca canvis significatius en els nivells proteics de controls de càrrega per “Western blot” i es presenta la tecnologia “Stain-Free” com a una alternativa a la normalització tradicional. Finalment, els nivells sèrics de Cav-1 podrien representar un potencial biomarcador predictor del pronòstic després d’una isquèmia en absència d’rt-PAS'ha extret el capítol de resultats del pdf de la tesi fins a la seva publicació en forma d'article. Results chapter removed from pdf file until publicatio

Efecte de l'administració de l'rt-PA en condicions isquèmiques in vitro i in vivo: Cav-1 com a potencial biomarcador de volum d'infart

Recombinant tissue plasminogen activator (rt-PA) is currently the only FDA-approved drug for the treatment of acute ischaemic stroke. However, the application of this therapy is limited to <5-7% of patients due to the associated increased risk of haemorrhagic transformation (HT). Although it is known that HT is related to rt-PA-induced blood brain barrier (BBB) disruption, the underlying mechanisms are not well established. The obtained results show that long-term studies are needed to elucidate time-dependent molecular mechanisms associated to BBB breakdown, and to explore protective BBB therapies after ischaemic stroke and rt-PA treatment. On the other hand, it has been demonstrated that OGD induces significant alterations to loading control proteins for Western Blot analysis proposing Stain-Free technology as an alternative normalization method to traditional housekeeping proteins. Finally, serum Cav-1 levels could represent a potential biomarker predicting the ischaemic outcome before rt-PA administrationL'rt-PA és l’únic fàrmac aprovat per tractar l’ictus isquèmic agut. No obstant, l’estreta finestra terapèutica, deguda al risc associat de transformació hemorràgica (TH) provoca que només s’apliqui a <5-7% dels pacients. La TH està relacionada amb la disrupció de la barrera hematoencefàlica (BHE) deguda a l’rt-PA però els mecanismes subjacents encara no estan del tot establerts. Els resultats obtinguts mostren que es requereixen estudis a llarg termini per tal de dilucidar els mecanismes dependents del temps associats a la disrupció de la BHE, i explorar noves teràpies protectores per al tractament de l’ictus isquèmic. S’ha demostrat que la POG provoca canvis significatius en els nivells proteics de controls de càrrega per “Western blot” i es presenta la tecnologia “Stain-Free” com a una alternativa a la normalització tradicional. Finalment, els nivells sèrics de Cav-1 podrien representar un potencial biomarcador predictor del pronòstic després d’una isquèmia en absència d’rt-PAS'ha extret el capítol de resultats del pdf de la tesi fins a la seva publicació en forma d'article. Results chapter removed from pdf file until publicatio