Cerebral ischaemia and matrix metalloproteinase-9 modulate the angiogenic function of early and late outgrowth endothelial progenitor cells

The enhancement of endogenous angiogenesis after stroke will be critical in neurorepair therapies where endothelial progenitor cells (EPCs) might be key players. Our aim was to determine the influence of cerebral ischaemia and the role of matrix metalloproteinase-9 (MMP-9) on the angiogenic function of EPCs. Permanent focal cerebral ischaemia was induced by middle cerebral artery (MCA) occlusion in MMP-9/knockout (MMP-9/KO) and wild-type (WT) mice. EPCs were obtained for cell counting after ischaemia (6 and 24 hrs) and in control animals. Matrigel™ assays and time-lapse imaging were conducted to monitor angiogenic function of WT and MMP9-deficient EPCs or after treatment with MMP-9 inhibitors. Focal cerebral ischaemia increased the number of early EPCs, while MMP-9 deficiency decreased their number in non-ischaemic mice and delayed their release after ischaemia. Late outgrowth endothelial cells (OECs) from ischaemic mice shaped more vessel structures than controls, while MMP-9 deficiency reduced the angiogenic abilities of OECs to form vascular networks, in vitro. Treatment with the MMP inhibitor GM6001 and the specific MMP-9 inhibitor I also decreased the number of vessel structures shaped by both human and mouse WT OECs, while exogenous MMP-9 could not revert the impaired angiogenic function in MMP-9/KO OECs. Finally, time-lapse imaging showed that the extension of vascular networks was influenced by cerebral ischaemia and MMP-9 deficiency early during the vascular network formation followed by a dynamic vessel remodelling. We conclude that focal cerebral ischaemia triggers the angiogenic responses of EPCs, while MMP-9 plays a key role in the formation of vascular networks by EPCs

Local administration of porcine immunomodulatory, chemotactic and angiogenic extracellular vesicles using engineered cardiac scaffolds for myocardial infarction

The administration of extracellular vesicles (EV) from mesenchymal stromal cells (MSC) is a promising cell-free nanotherapy for tissue repair after myocardial infarction (MI). However, the optimal EV delivery strategy remains undetermined. Here, we designed a novel MSC-EV delivery, using 3D scaffolds engineered from decellularised cardiac tissue as a cell-free product for cardiac repair. EV from porcine cardiac adipose tissue-derived MSC (cATMSC) were purified by size exclusion chromatography (SEC), functionally analysed and loaded to scaffolds. cATMSC-EV markedly reduced polyclonal proliferation and pro-inflammatory cytokines production (IFNγ, TNFα, IL12p40) of allogeneic PBMC. Moreover, cATMSC-EV recruited outgrowth endothelial cells (OEC) and allogeneic MSC, and promoted angiogenesis. Fluorescently labelled cATMSC-EV were mixed with peptide hydrogel, and were successfully retained in decellularised scaffolds. Then, cATMSC-EV-embedded pericardial scaffolds were administered in vivo over the ischemic myocardium in a pig model of MI. Six days from implantation, the engineered scaffold efficiently integrated into the post-infarcted myocardium. cATMSC-EV were detected within the construct and MI core, and promoted an increase in vascular density and reduction in macrophage and T cell infiltration within the damaged myocardium. The confined administration of multifunctional MSC-EV within an engineered pericardial scaffold ensures local EV dosage and release, and generates a vascularised bioactive niche for cell recruitment, engraftment and modulation of short-term post-ischemic inflammation

Importance of Angiogenin and Endothelial Progenitor Cells After Rehabilitation Both in Ischemic Stroke Patients and in a Mouse Model of Cerebral Ischemia

Background: Rehabilitation therapy is the only available treatment for stroke survivors presenting neurological deficits; however, the underlying molecules and mechanisms associated with functional/motor improvement during rehabilitation are poorly understood.Objective: Our aim is to study the modulation of angiogenin and endothelial progenitor cells (EPCs) as repair-associated factors in a cohort of stroke patients and mouse models of rehabilitation after cerebral ischemia.Methods: The clinical study included 18 ischemic strokes admitted to an intensive rehabilitation therapy (IRT) unit, 18 non-ischemic controls and brain samples from three deceased patients. Angiogenin and EPCs were measured in blood obtained before and up to 6 months after IRT together with an extensive evaluation of the motor/functional status. In parallel, C57BL/6 mice underwent middle cerebral artery occlusion, and the pasta matrix reaching-task or treadmill exercises were used as rehabilitation models. Angiogenin RNA expression was measured after 2 or 12 days of treatment together with cell counts from EPCs cultures.Results: Brain angiogenin was identified in both human and mouse tissue, whereas serum levels increased after 1 month of IRT in association with motor/functional improvement. EPC populations were increased after stroke and remained elevated during follow-up after IRT. The mouse model of rehabilitation by the task-specific pasta matrix exercise increased the number of EPCs at 2 days and increased angiogenin expression after 12 days of rehabilitation.Conclusions: Angiogenin and EPCs are modulated by rehabilitation after cerebral ischemia, suggesting that both angiogenin and EPCs could serve as biomarkers of improvement during rehabilitation or future therapeutic targets

RANK signaling increases after anti-HER2 therapy contributing to the emergence of resistance in HER2-positive breast cancer

Background: Around 15-20% of primary breast cancers are characterized by HER2 protein overexpression and/or HER2 gene amplification. Despite the successful development of anti-HER2 drugs, intrinsic and acquired resistance represents a major hurdle. This study was performed to analyze the RANK pathway contribution in HER2-positive breast cancer and anti-HER2 therapy resistance. Methods: RANK and RANKL protein expression was assessed in samples from HER2-positive breast cancer patients resistant to anti-HER2 therapy and treatment-naive patients. RANK and RANKL gene expression was analyzed in paired samples from patients treated with neoadjuvant dual HER2-blockade (lapatinib and trastuzumab) from the SOLTI-1114 PAMELA trial. Additionally, HER2-positive breast cancer cell lines were used to modulate RANK expression and analyze in vitro the contribution of RANK signaling to anti-HER2 resistance and downstream signaling. Results: RANK and RANKL proteins are more frequently detected in HER2-positive tumors that have acquired resistance to anti-HER2 therapies than in treatment-naive ones. RANK (but not RANKL) gene expression increased after dual anti-HER2 neoadjuvant therapy in the cohort from the SOLTI-1114 PAMELA trial. Results in HER2-positive breast cancer cell lines recapitulate the clinical observations, with increased RANK expression observed after short-term treatment with the HER2 inhibitor lapatinib or dual anti-HER2 therapy and in lapatinib-resistant cells. After RANKL stimulation, lapatinib-resistant cells show increased NF-κB activation compared to their sensitive counterparts, confirming the enhanced functionality of the RANK pathway in anti-HER2-resistant breast cancer. Overactivation of the RANK signaling pathway enhances ERK and NF-κB signaling and increases lapatinib resistance in different HER2-positive breast cancer cell lines, whereas RANK loss sensitizes lapatinib-resistant cells to the drug. Our results indicate that ErbB signaling is required for RANK/RANKL-driven activation of ERK in several HER2-positive cell lines. In contrast, lapatinib is not able to counteract the NF-κB activation elicited after RANKL treatment in RANK-overexpressing cells. Finally, we show that RANK binds to HER2 in breast cancer cells and that enhanced RANK pathway activation alters HER2 phosphorylation status. Conclusions: Our data support a physical and functional link between RANK and HER2 signaling in breast cancer and demonstrate that increased RANK signaling may contribute to the development of lapatinib resistance through NF-κB activation. Whether HER2-positive breast cancer patients with tumoral RANK expression might benefit from dual HER2 and RANK inhibition therapy remains to be elucidated

The IMPROVE guidelines (Ischaemia Models: Procedural Refinements Of in Vivo Experiments)

Most in vivo models of ischaemic stroke target the middle cerebral artery and a spectrum of stroke severities, from mild to substantial, can be achieved. This review describes opportunities to improve the in vivo modelling of ischaemic stroke and animal welfare. It provides a number of recommendations to minimise the level of severity in the most common rodent models of middle cerebral artery occlusion, while sustaining or improving the scientific outcomes. The recommendations cover basic requirements pre-surgery, selecting the most appropriate anaesthetic and analgesic regimen, as well as intraoperative and post-operative care. The aim is to provide support for researchers and animal care staff to refine their procedures and practices, and implement small incremental changes to improve the welfare of the animals used and to answer the scientific question under investigation. All recommendations are recapitulated in a summary poster (see supplementary information)

New adipokines vaspin and omentin. Circulating levels and gene expression in adipose tissue from morbidly obese women

<p>Abstract</p> <p>Background</p> <p>Vaspin and omentin are recently described molecules that belong to the adipokine family and seem to be related to metabolic risk factors. The objectives of this study were twofold: to evaluate vaspin and omentin circulating levels and mRNA expression in subcutaneous and visceral adipose tissues in non-diabetic morbidly obese women; and to assess the relationship of vaspin and omentin with anthropometric and metabolic parameters, and other adipo/cytokines.</p> <p>Design</p> <p>We analysed vaspin and omentin circulating levels in 71 women of European descent (40 morbidly obese [BMI ≥ 40 kg/m²] and 31 lean [BMI ≤ 25]). We assessed vaspin and omentin gene expression in paired samples of visceral and subcutaneous abdominal adipose tissue from 46 women: 40 morbidly obese and 6 lean. We determined serum vaspin and plasma omentin levels with an Enzyme-Linked Immunosorbent Assay and adipose tissue mRNA expression by real time RT-PCR.</p> <p>Results</p> <p>Serum vaspin levels in the morbidly obese were not significantly different from those in controls. They correlated inversely with levels of lipocalin 2 and interleukin 6. Vaspin mRNA expression was significantly higher in the morbidly obese, in both subcutaneous and visceral adipose tissue.</p> <p>Plasma omentin levels were significantly lower in the morbidly obese and they correlated inversely with glucidic metabolism parameters. Omentin circulating levels, then, correlated inversely with the metabolic syndrome (MS). Omentin expression in visceral adipose tissue was significantly lower in morbidly obese women than in controls.</p> <p>Conclusions</p> <p>The present study indicates that vaspin may have a compensatory role in the underlying inflammation of obesity. Decreased omentin circulating levels have a close association with MS in morbidly obese women.</p

Neutrophil depletion reduces edema formation and tissue loss following traumatic brain injury in mice

Background: Brain edema as a result of secondary injury following traumatic brain injury (TBI) is a major clinical concern. Neutrophils are known to cause increased vascular permeability leading to edema formation in peripheral tissue, but their role in the pathology following TBI remains unclear. Methods: In this study we used controlled cortical impact (CCI) as a model for TBI and investigated the role of neutrophils in the response to injury. The outcome of mice that were depleted of neutrophils using an anti-Gr-1 antibody was compared to that in mice with intact neutrophil count. The effect of neutrophil depletion on blood-brain barrier function was assessed by Evan's blue dye extravasation, and analysis of brain water content was used as a measurement of brain edema formation (24 and 48 hours after CCI). Lesion volume was measured 7 and 14 days after CCI. Immunohistochemistry was used to assess cell death, using a marker for cleaved caspase-3 at 24 hours after injury, and microglial/macrophage activation 7 days after CCI. Data were analyzed using Mann-Whitney test for non-parametric data. Results: Neutrophil depletion did not significantly affect Evan's blue extravasation at any time-point after CCI. However, neutrophil-depleted mice exhibited a decreased water content both at 24 and 48 hours after CCI indicating reduced edema formation. Furthermore, brain tissue loss was attenuated in neutropenic mice at 7 and 14 days after injury. Additionally, these mice had a significantly reduced number of activated microglia/macrophages 7 days after CCI, and of cleaved caspase-3 positive cells 24 h after injury. Conclusion: Our results suggest that neutrophils are involved in the edema formation, but not the extravasation of large proteins, as well as contributing to cell death and tissue loss following TBI in mice

Les cèl·lules progenitores endotelials com a tractament neuroreparador angiovasculogènic en la isquèmia cerebral modulat per la metal·loproteïnasa de matriu 9 (mmp-9)

Avui dia la patologia isquèmica cerebral, amb una fisiopatologia molt complexa, segueix sent una de les causes més importants de mort i incapacitat al món. Actualment l'únic tractament possible són les teràpies trombolítiques amb l’activador del plasminogen de teixit (rt-PA) , que només s'administren en la fase hiperaguda dels ictus isquèmics (<4,5 hores). És per tant necessari investigar noves teràpies aplicables després de la fase hiperaguda per poder augmentar el nombre de pacients tractats. La neuroreparació és una estratègia terapèutica que permet restablir la circulació cerebral i promoure la neuroregeneració. Per a assolir aquests objectius cal potenciar l'angiogènesi i la neurogènesi en el cervell isquèmic. L'objectiu d'aquesta tesi és estudiar el potencial neuroreparador de les cèl·lules progenitores endotelials (EPCs) i el rol de la MMP-9 en la seva modulació. Utilitzant un model d’isquèmia cerebral permanent en ratolí es va estudiar el potencial terapèutic de les EPCs així com dels factors secretats per aquestes. Els resultats mostren la potenciació de les respostes angiogèniques i neurogèniques endògenes en els animals que reben els citats tractaments demostrant per primer cop en un model d’isquèmia cerebral el potencial terapèutic dels factors de creixement que secreten les EPCs, oferint una eina per a teràpies pro-angiogèniques autòlogues basades en factors de creixement i lliures de cèl·lules. Atès que sabem que les metal·loproteïnases de matriu (MMPs) són necessàries per la remodelació extracel·lular que es dóna durant els processos d’angiogènesi i neurogènesi, aquesta tesi aprofundeix en el rol de la MMP-9 com a molècula clau reguladora en les teràpies pro-angiogèniques després de la isquèmia cerebral. Alhora també s’ha estudiat el rol de la isquèmia en l’activació i mobilització de les EPCs. Els resultats d’aquesta tesi mostren que la isquèmia cerebral estimula l’alliberament de les EPCs a la circulació. Per altra banda també hem demostrat com la deficiència de MMP-9 produeix una disminució de les EPCs circulants en animals control, que es reverteix després de l’estímul isquèmic. També s’han realitzat estudis de tubulogènesi in vitro, que demostren un augment de les capacitats angiogèniques en les cèl·lules obtingudes d’animals isquèmics comparat amb no isquèmics així com una disminució d’aquestes en cèl·lules d’animals deficients en MMP-9. La inhibició de les MMPs, i específicament la MMP-9, en EPCs humanes mostren els mateixos resultats, confirmant el rol d’aquesta metal·loproteïnasa en les capacitats angio-vasculogèniques in vitro de les EPCs. Finalment, s’ha posat a punt un model d’isquèmia cerebral transitòria en ratolí, per oclusió distal de l’artèria cerebral mitja mitjançant la seva compressió. Aquest model, amb un infart cortical ben delimitat i amb l’existència de la reperfusió del teixit, presenta una major homologia amb la patologia humana i ens permetrà continuar amb els estudis de neuroreparació en situacions de reperfusió.Nowadays acute ischemic brain disease, which has a complex pathophysiology, remains one of the most important causes of death and disability worldwide. Currently the only possible treatment is the thrombolytic therapy with tissue plasminogen activator (rt-PA), that can be administered only in the hyperacute phase of ischemic stroke (<4.5 hours). Therefore, it is necessary to investigate new therapies that could be applicable after the hyperacute phase to increase the number of treated patients. Neurorepair therapies are therapeutic approaches that allow restoring cerebral circulation and promoting neuroregeneration. To achieve these objectives it is necessary to enhance angiogenesis and neurogenesis in the ischemic brain. The aim of this thesis is to study the neuropair potential of endothelial progenitor cells (EPCS) and the role of MMP-9 in its modulation. Using a model of permanent cerebral ischemia in mice, we studied the therapeutic potential of EPCs and their secreted factors. The results show an increase of angiogenesis in animals receiving the aforementioned treatments, demonstrating for the first time the therapeutic potential of growth factors secreted by EPCs in a model of cerebral ischemia and providing a tool for autologous pro-angiogenic cell-free therapies. Since it is known that matrix metalloproteinases (MMPs) are required for the extracellular remodeling that occurs during angiogenesis and neurogenesis, this thesis explores the role of MMP-9 as a key molecule in the regulation of pro-angiogenic therapies after cerebral ischemia. In addition, the role of ischemia in the activation and mobilization of the EPCs has also been studied. The results of this thesis show that cerebral ischemia stimulates the release of EPCs to circulation. Furthermore we have also demonstrated that MMP-9 deficiency causes a decrease in the number of circulating EPCs in control animals, which is reversed after the ischemic insult. In vitro tubulogenesis studies showed an increased angiogenic capacity in cells obtained from ischemic animals compared with non-ischemic as well as a decrease of these abilities in MMP-9 deficient animals. The pharmacological inhibition of MMPs, specifically MMP-9 in human EPCs showed the same results, confirming the role of this metalloproteinase in the in vitro angio-vasculogenic abilities of EPCs. Finally, we have developed a model of transient cerebral ischemia in mice by occlusion of the distal middle cerebral artery by compression. This model, with a well-defined cortical infarct and the presence of tissue reperfusion, has a greater homology with human disease and allows us to continue the neurorepair studies in the presence of reperfusion

Col·lecció fotogràfica Vicente Reyes: Una finestra al món de l’espectacle de varietats a la Barcelona de la primera meitat del segle XX

Treball de fi de grau d'Humanitats. Curs 2014-2015Directora: Teresa VinardellLa investigació que ha permès dur a terme aquest treball té la intenció de posar en valor una col·lecció fotogràfica que fins ara era invisible. Al dotar-la de significat es mostrarà, alhora, una realitat social i l'existència d'una gran indústria lligada a l’espectacle de varietés o varietats, que va marcar la Barcelona de finals del segle XIX i fins pràcticament els anys 50 del segle passat. Un món que, malauradament, sembla haver quedat pràcticament oblidat avui en dia. Deixant enrere la tendència dominant dins la historiografia que pretén utilitzar la fotografia només a mode d’il·lustració, aquest treball la tracta com una font documental més a partir de la qual es pot destapar la Història

Col·lecció fotogràfica Vicente Reyes: Una finestra al món de l’espectacle de varietats a la Barcelona de la primera meitat del segle XX

Treball de fi de grau d'Humanitats. Curs 2014-2015Directora: Teresa VinardellLa investigació que ha permès dur a terme aquest treball té la intenció de posar en valor una col·lecció fotogràfica que fins ara era invisible. Al dotar-la de significat es mostrarà, alhora, una realitat social i l'existència d'una gran indústria lligada a l’espectacle de varietés o varietats, que va marcar la Barcelona de finals del segle XIX i fins pràcticament els anys 50 del segle passat. Un món que, malauradament, sembla haver quedat pràcticament oblidat avui en dia. Deixant enrere la tendència dominant dins la historiografia que pretén utilitzar la fotografia només a mode d’il·lustració, aquest treball la tracta com una font documental més a partir de la qual es pot destapar la Història