Influència de les cèl·lules endotelials progenitores sobre la modulació espaial i temporal de l’angiogènesi i la vasculogènesi després de l’ictus isquèmic humà

L'ictus o infart cerebral es produeix com a resultat d'una alteració del flux sanguini cerebral que pot ser transitori, si es restableix espontàniament o mitjançant accions terapèutiques, o permanent. Aquesta malaltia suposa una de les primeres causes de mortalitat, juntament amb les malalties cardíaques i el càncer, en el món occidental. Actualment l'únic tractament disponible per a l'ictus isquèmic és l'administració de l'activador recombinant del plasminogen tissular (rt-PA) dintre de les 4,5h primeres hores des de l'inici dels símptomes. Tot i la seva eficàcia, evitant una mort/dependència de cada set pacients tractats menys del 5% del pacients amb ictus isquèmic es poden beneficiar d'aquesta teràpia. Per tant, són importants nous estudis dirigits a identificar noves dianes terapèutiques que ens permetin aplicar nous tractaments més efectius i segurs. La neuroreparació, és una estratègia terapèutica que permet recuperar tant la zona de penombra com formar nous circuits neuronals per restablir les funcions sensorials i motores perdudes. Processos com l'angiogènesi i la vasculogènesi tenen un paper clau en aquesta recuperació. És per aquest motiu, que l'objectiu d'aquesta tesi és estudiar el paper que juguen les cèl·lules endotelials progenitores (EPCs) i els nivells plasmàtics de diferents promotors i inhibidors angiogènics en el pronòstic clínic dels pacients que han patit un ictus isquèmic comparat amb controls sans. Es van fer extraccions sanguinies a pacients que havien patit un ictus isquèmic i havien rebut tractament trombolítico a diferents temps després de l'ictus i es van determinar els nivells en plasma mitjançant ELISA de PDGF-BB, HGF, FGF, VEGF, HB-EFG, PDGF-AA, KGF, TPO , VEGF-R1, VEGF-R2, TSP-1, TSP-2, endostatina, angiostatina, SDF- 1a i GM-CSF. Aquests valors es van correlacionar amb diferents parametres clínics (milloria, risc de TH) i escales neurològiques (NIHSS, rankin). Les EPCs es van aïllar a partir de la fracció mononuclear de pacients (en fase subaguda i fase aguda) i controls i la seva quantificació es va realitzar mitjançant el cultiu cel·lular i la citometria de flux. Es van fenotipar els diferents tipus de EPCs i es va determinar la seva funcionalitat mitjançant assajos de tubulogènesi en Matrigel . També es va analitzar la capacitat de secreció de factors angiogènics d'aquestes cèl·lules, així com la seva expressió gènica. Els resultats d'aquesta tesi mostren els nivells circulants d'EPCs determinats mitjançant citometria de flux es troben elevats en pacients amb ictus isquèmic comparat amb controls. De la mateixa manera, trobem una major capacitat de diferenciació a cèl·lula endotelial en els cultius cel·lulars de pacients amb ictus. Les EPCs obtingudes de pacients en la fase subaguda de l'ictus mostren una major funcionalitat in vitro que les obtingudes durant la fase aguda, i podrien se més eficients en teràpies de neuroreparació. Diferents promotors i inhibidors angiogènics es troben modulats després de l'ictus isquèmic. El nivells en plasma de PDGF-BB, d'endostatina i de TSP-2 i GM-CSF es troben augmentats en la fase hiperaguda de l'ictus comparat amb una població control.Una major angiogènesi endògena determinada pels nivells de VEGF, VEGF/endostatina o PDGF-BB/angiostatina s'associen a un menor dèficit neurològic a curt termini, sense incrementar el risc de TH. En canvi, nivells elevats de l'inhibidor angiogènic endostatina s'associen a un pitjor pronòstic funcional dels pacients al tercer mes.Stroke is the third leading cause of death and the most common cause of permanent disability in adults worldwide. In the acute phase of stroke an energy loss caused by reduction of cerebral blood flow triggers multiple brain-cell death pathways. Currently, the only available treatment for stroke is the use of thrombolytic drugs to restore brain perfusion during the hyperacute phase. The National Institutes of Neurological Disorders Study (NINDS) and the European Cooperative Acute Stroke Study (ECASS) have described the beneficial effects of intravenous tissue plasminogen activator (tPA) administered up to 3–4.5 h after symptom onset of ischemic stroke. However, despite its proven efficacy, tPA is only given to less than 5% of stroke patients due to its narrow therapeutic window and the fact that it carries an increased risk of intracranial hemorrhage. Therefore, there is an urgent need for new stroke therapies applicable beyond the hyperacute phase. One promising therapeutic strategy is to induce neovascularization in the brain. Our aim was study the role of endothelial progenitor cells (EPCs) and the association between plasma levels of different angiogenic promoters and inhibitors and clinical outcome of ischemic stroke patients. Blood samples were drawn from tPA-stroke patients and controls at different time-points after ischemic event and plasma level of PDGF-BB, HGF, FGF, VEGF, HB-EFG, PDGF-AA, KGF, TPO , VEGF-R1, VEGF-R2, TSP-1, TSP-2, endostatina, angiostatina, SDF-1a and GM-CSF was determined. Angiogenic promoters and inhibitors levels were associated with different clinical parameters (TH, NIHSS and mRS scales). EPCs levels in each group were quantified by flow cytometry and cell culture assays. Immunophenotyping was performed using multiple markers (UEA-lectin, CD133, vWF and KDR) and tubulogenic function was assessed with the Matrigel® assay. The produced angiogenic factors were quantified by multiple ELISA and RT-PCR. Fluorescence activated cell sorting (FACS) revealed higher levels of circulating CD133+/CD34+/KDR+/CD45+ cells in the acute strokes as compared to the control subjects (p=0.02). On the other hand, more EPCs grew in cell culture from subacute strokes (p=0.016) than from controls. The endothelial and progenitor lineages of the EPCs were confirmed by immunophenotyping. Interestingly, the appearance of outgrowth EPCs (OECs) correlated positively to stroke severity (p=0.013). Finally, greater capacity to induce vasculogenesis in vitro was found in EPCs from subacute strokes (p=0.03), which we attribute to a higher expression and secretion of angiogenic factors. Baseline PDGF-BB, endostatin and thrombospondin-2 levels were higher in stroke patients than in controls (p<0.05). Most angiogenesis biomarkers showed a decrease during the first 24h alter stroke and a long recovery to baseline levels. A pro-angiogenic balance was associated with lower NIHSS scores and less intracranial hemorrhagic complications. Interestingly, a high baseline endostatin level was associated to long-term functional dependency

Catecholamine-induced heart injury in mice: differential effects of isoproterenol and phenylephrine

-The involvement of catecholamines in stress-induced heart injury is well documented. However, the contribution of adrenergic receptor types is less understood. Both the profile of plasma marker enzyme activities (lactate dehydrogenase-1 and aspartate transaminase) and the distribution and morphology of the lesions observed in tissue sections of adrenaline-injected mice resembled those of stressed (restraint and cold exposed) mice. Next, we compared the effect of isoproterenol (ß-adrenergic agonist) and phenylephrine (α1-adrenergic agonist) on both heart function and tissue injury. In Langendorff-perfused rat hearts, α1-adrenergic receptors made a minor contribution to the tonic effect of adrenaline, as indicated by the lack of effect on the heart rate and the delayed negative inotropic effect of phenylephrine. However, in whole mice, phenylephrine but not isoproterenol, induced an increase of both lactate dehydrogenase-1 and aspartate transaminase activities. Hearts of phenylephrine-injected mice showed necrotic lesions in subendocardial areas of the left ventricle. In addition a scattered focal leukocyte infiltration around single apoptotic-like myocytes was observed in the ventricle wall. Hearts of isoproterenol-injected mice showed a similar number of apoptotic-like myocytes, but a much lower number of necrotic areas, than phenylephrine-injected animals. Our results suggest that the cardiotonic effect of catecholamines involves mainly the ß-adrenergic receptors. However, the acute catecholamine-induced heart injury involves mainly α1-adrenergic receptor

Influència de les cèl·lules endotelials progenitores sobre la modulació espaial i temporal de l'angiogènesi i la vasculogènesi després de l'ictus isquèmic humà

Descripció del recurs: 27 octubre 2011L'ictus o infart cerebral es produeix com a resultat d'una alteració del flux sanguini cerebral que pot ser transitori, si es restableix espontàniament o mitjançant accions terapèutiques, o permanent. Aquesta malaltia suposa una de les primeres causes de mortalitat, juntament amb les malalties cardíaques i el càncer, en el món occidental. Actualment l'únic tractament disponible per a l'ictus isquèmic és l'administració de l'activador recombinant del plasminogen tissular (rt-PA) dintre de les 4,5h primeres hores des de l'inici dels símptomes. Tot i la seva eficàcia, evitant una mort/dependència de cada set pacients tractats menys del 5% del pacients amb ictus isquèmic es poden beneficiar d'aquesta teràpia. Per tant, són importants nous estudis dirigits a identificar noves dianes terapèutiques que ens permetin aplicar nous tractaments més efectius i segurs. La neuroreparació, és una estratègia terapèutica que permet recuperar tant la zona de penombra com formar nous circuits neuronals per restablir les funcions sensorials i motores perdudes. Processos com l'angiogènesi i la vasculogènesi tenen un paper clau en aquesta recuperació. És per aquest motiu, que l'objectiu d'aquesta tesi és estudiar el paper que juguen les cèl·lules endotelials progenitores (EPCs) i els nivells plasmàtics de diferents promotors i inhibidors angiogènics en el pronòstic clínic dels pacients que han patit un ictus isquèmic comparat amb controls sans. Es van fer extraccions sanguinies a pacients que havien patit un ictus isquèmic i havien rebut tractament trombolítico a diferents temps després de l'ictus i es van determinar els nivells en plasma mitjançant ELISA de PDGF-BB, HGF, FGF, VEGF, HB-EFG, PDGF-AA, KGF, TPO , VEGF-R1, VEGF-R2, TSP-1, TSP-2, endostatina, angiostatina, SDF- 1a i GM-CSF. Aquests valors es van correlacionar amb diferents parametres clínics (milloria, risc de TH) i escales neurològiques (NIHSS, rankin). Les EPCs es van aïllar a partir de la fracció mononuclear de pacients (en fase subaguda i fase aguda) i controls i la seva quantificació es va realitzar mitjançant el cultiu cel·lular i la citometria de flux. Es van fenotipar els diferents tipus de EPCs i es va determinar la seva funcionalitat mitjançant assajos de tubulogènesi en Matrigel . També es va analitzar la capacitat de secreció de factors angiogènics d'aquestes cèl·lules, així com la seva expressió gènica. Els resultats d'aquesta tesi mostren els nivells circulants d'EPCs determinats mitjançant citometria de flux es troben elevats en pacients amb ictus isquèmic comparat amb controls. De la mateixa manera, trobem una major capacitat de diferenciació a cèl·lula endotelial en els cultius cel·lulars de pacients amb ictus. Les EPCs obtingudes de pacients en la fase subaguda de l'ictus mostren una major funcionalitat in vitro que les obtingudes durant la fase aguda, i podrien se més eficients en teràpies de neuroreparació. Diferents promotors i inhibidors angiogènics es troben modulats després de l'ictus isquèmic. El nivells en plasma de PDGF-BB, d'endostatina i de TSP-2 i GM-CSF es troben augmentats en la fase hiperaguda de l'ictus comparat amb una població control.Una major angiogènesi endògena determinada pels nivells de VEGF, VEGF/endostatina o PDGF-BB/angiostatina s'associen a un menor dèficit neurològic a curt termini, sense incrementar el risc de TH. En canvi, nivells elevats de l'inhibidor angiogènic endostatina s'associen a un pitjor pronòstic funcional dels pacients al tercer mes.Stroke is the third leading cause of death and the most common cause of permanent disability in adults worldwide. In the acute phase of stroke an energy loss caused by reduction of cerebral blood flow triggers multiple brain-cell death pathways. Currently, the only available treatment for stroke is the use of thrombolytic drugs to restore brain perfusion during the hyperacute phase. The National Institutes of Neurological Disorders Study (NINDS) and the European Cooperative Acute Stroke Study (ECASS) have described the beneficial effects of intravenous tissue plasminogen activator (tPA) administered up to 3-4.5 h after symptom onset of ischemic stroke. However, despite its proven efficacy, tPA is only given to less than 5% of stroke patients due to its narrow therapeutic window and the fact that it carries an increased risk of intracranial hemorrhage. Therefore, there is an urgent need for new stroke therapies applicable beyond the hyperacute phase. One promising therapeutic strategy is to induce neovascularization in the brain. Our aim was study the role of endothelial progenitor cells (EPCs) and the association between plasma levels of different angiogenic promoters and inhibitors and clinical outcome of ischemic stroke patients. Blood samples were drawn from tPA-stroke patients and controls at different time-points after ischemic event and plasma level of PDGF-BB, HGF, FGF, VEGF, HB-EFG, PDGF-AA, KGF, TPO , VEGF-R1, VEGF-R2, TSP-1, TSP-2, endostatina, angiostatina, SDF-1a and GM-CSF was determined. Angiogenic promoters and inhibitors levels were associated with different clinical parameters (TH, NIHSS and mRS scales). EPCs levels in each group were quantified by flow cytometry and cell culture assays. Immunophenotyping was performed using multiple markers (UEA-lectin, CD133, vWF and KDR) and tubulogenic function was assessed with the Matrigel® assay. The produced angiogenic factors were quantified by multiple ELISA and RT-PCR. Fluorescence activated cell sorting (FACS) revealed higher levels of circulating CD133+/CD34+/KDR+/CD45+ cells in the acute strokes as compared to the control subjects (p=0.02). On the other hand, more EPCs grew in cell culture from subacute strokes (p=0.016) than from controls. The endothelial and progenitor lineages of the EPCs were confirmed by immunophenotyping. Interestingly, the appearance of outgrowth EPCs (OECs) correlated positively to stroke severity (p=0.013). Finally, greater capacity to induce vasculogenesis in vitro was found in EPCs from subacute strokes (p=0.03), which we attribute to a higher expression and secretion of angiogenic factors. Baseline PDGF-BB, endostatin and thrombospondin-2 levels were higher in stroke patients than in controls (p 0.05). Most angiogenesis biomarkers showed a decrease during the first 24h alter stroke and a long recovery to baseline levels. A pro-angiogenic balance was associated with lower NIHSS scores and less intracranial hemorrhagic complications. Interestingly, a high baseline endostatin level was associated to long-term functional dependency

The angiogenic gene profile of circulating endothelial progenitor cells from ischemic stroke patients.

Our study shows that OECs from stroke patients present higher levels of pro-angiogenic factors at early stages, decreasing in mature OECs when they become more similar to mature microvascular endothelial cells

The angiogenic gene profile of circulating endothelial progenitor cells from ischemic stroke patients

Altres ajuts: AR is supported by the Miguel Servet programme (CP09/00265) from the Spanish Ministry of Health (Instituto de Salud Carlos III). This work has been funded by Instituto de Salud Carlos III, grant PI10/00694 and the Spanish stroke research network RENEVAS (RD06/0026/0010) co-financed by the European Regional Development Fund (ERDF). This work has been supported also by the European Union's Seventh Framework Programme (FP7/2007-2013) under grant agreements n° 201024 and n° 202213 (European Stroke Network).The identification of circulating endothelial progenitor cells (EPCs) has introduced new possibilities for cell-based treatments for stroke. We tested the angiogenic gene expression of outgrowth endothelial cells (OECs), an EPC subtype capable to shape vessel structures. OECs (at colony or mature stages) from ischemic stroke patients (n=8) were characterized using the RT 2 Profiler TM human angiogenesis PCR Array, and human microvascular endothelial cells (hCMEC/D3) were used as an expression reference of endothelial cells. Colony-OECs showed higher expression of CCL2, ID3, IGF-1, MMP9, TGFBR1, TNFAIP2, TNF and TGFB1. However, BAI-1, NRP2, THBS1, MMP2 and VEGFC expression was increased in mature-OECs (p<0.05). ID3 (p=0.008) and TGFBR1 (p=0.03) genes remained significantly overexpressed in colony-OECs compared to mature-OECs or hCMEC/D3. MMP9 levels were significantly increased in colony-OECs (p=0.025) compared to mature-OECs. Moreover, MMP-2, VEGF-C, THBS1 and NRP-2 gene expression was also significantly increased in mature-OECs compared to hCMEC/D3 (p<0.05). Some of these genes were positively validated by RT-PCR. Our study shows that OECs from stroke patients present higher levels of pro-angiogenic factors at early stages, decreasing in mature OECs when they become more similar to mature microvascular endothelial cells