Brain delivery remains a challenge for the clinical translation of therapeutic nanomedicines, particularly in focal diseases with specific delivery needs, such as stroke. In this scenario, clinically relevant endovascular interventions are recently being proposed as strategies to enhance delivery into specific cerebral vascular territories. In this study, we assess the feasibility of endovascular delivery and magnetic retention of biocompatible magnetic nanocapsules (NCs) in cerebral circulation models that better predict human responses. More specifically, polymeric NCs synthesized with magnetic properties (superparamagnetic oxide nanoparticles, SPIONs) and fluorescent (Cy5) moieties were infused into pigs via a femoral microcatheter reaching the brain vasculature and showing greater efficacy in targeting the ipsilateral brain hemisphere with preferential accumulation in microvessels when compared to intravenous administration which resulted in very little accumulation. Transient adverse effects related to hemodynamic instability upon nanocapsule administration were observed in both administration groups related to acute complement activation. Successful endovascular brain NC delivery is further demonstrated in a 3D-vascular model of the human large arterial vessel brain supply, with successful NC accumulation in the target arterial segment (the proximal middle cerebral artery) with sensible enhancement when using local magnetic fields. This study demonstrates the feasibility of endovascular NC delivery for focal brain nanotargeting via clinically relevant and minimally invasive procedures and proves the advantages of using magnetized nanomaterials to improve local vascular NC retention. Further safety and efficacy studies, including drug nanocapsule formulations, are needed to establish the clinical relevance of the proposed approach.

Ischemic stroke is a major global health burden, leading to considerable mortality and long-term disability. Endovascular thrombectomy and mechanical recanalization have revolutionized acute stroke care. Nonetheless, many patients experience poor long-term neurological outcomes, which are often attributed to the no-reflow phenomenon and activation of inflammatory cascades. The perioperative period of endovascular thrombectomy, managed under either general anesthesia or conscious sedation, represents a critical window where anesthetic strategies may influence recovery through hemodynamic control and possibly immune modulation. This consensus review was generated by an international multidisciplinary expert group and synthesizes preclinical and clinical evidence to evaluate the promise of various immunomodulatory strategies for improving functional outcomes in patients with ischemic stroke following endovascular thrombectomy. Our goal is to provide a foundational reference for future research and development of novel perioperative immune therapies for patients with endovascular thrombectomy.

The central nervous system was once viewed as immunologically privileged, but it is now recognized that brain-immune interactions are dynamic and critical. After central nervous system injury or disease, microglia and perivascular macrophages survey the damaged tissue, while diverse immune cells infiltrate through various barriers, driving neurovascular dysfunction and neuroinflammation. Depending on timing and phenotype, systemic immune responses can also promote brain repair and recovery. Recent studies show that brain-peripheral organ communication profoundly affects both injury progression and healing. These insights redefine the neurovascular-immune interface and the concept of the brain's immune system. This review examines immune cell infiltration and highlights emerging brain-peripheral circuits that regulate neuroinflammation and recovery after stroke.

Neuroprotection after ischemic stroke has been focused on targeting one pathway of the ischemic cascade. In this study, we have hypothesized that combination therapy with alpha-1 antitrypsin (A1AT) and a blocker of tumor necrosis factor (TNFα) could be beneficial in the acute phases after ischemia. Following a detailed safety assessment of the co-administration of both drugs, we tested their neuroprotective effect in a transient mouse model of proximal middle cerebral artery occlusion (MCAo) by evaluating infarct extension and functional outcomes. Anti-TNFα (20 mg/kg) and A1AT were administered at different doses (ranging from 60 mg/kg to 700 mg/kg), as a single therapy during occlusion or at different time-points following reperfusion. Results showed that the administration of A1AT (60 mg/kg) in combination with anti-TNFα (20 mg/kg) was safe and effective when given during occlusion by reducing infarct volume at 24 h by 27% compared with the vehicle group (p = 0.0001). In conclusion, the synergy of the anti-apoptotic and anti-inflammatory properties of both drugs can reduce infarct volume in a stroke mouse model when given in the hyperacute phase. This approach shows promise as an early intervention strategy for stroke patients and underscores the potential of drug repurposing to develop new stroke treatments.

Delayed cerebral ischemia (DCI) is a major cause of morbidity following aneurysmal subarachnoid hemorrhage (aSAH), yet early prediction remains challenging. This study aimed to identify blood-based protein biomarkers within 24 h of intensive care unit admission associated with DCI using high-throughput proteomics. We conducted a prospective longitudinal study including 86 patients with aSAH, of whom 28 developed DCI. For this exploratory analysis, we matched eight patients who developed DCI with eight controls without DCI based on age, sex, and severity scores. Plasma samples were analyzed using the Olink Explore 3072 platform targeting 2,943 proteins. Differential expression analysis was performed using linear Bayesian models with false discovery rate correction. We identified 15 significantly dysregulated proteins (P < 0.01) in patients with DCI. Key downregulated proteins included THSD1, CA3, and PROK1, which are associated with vascular integrity and endothelial function. Upregulated proteins included BGN, IFNG, and CSF2, which are related to innate immunity and neuroinflammation. Novel candidates, such as CLSTN3 and DOCK9, also showed altered expression. Protein–protein interaction and gene ontology enrichment analyses revealed involvement in inflammatory, immune, and metabolic pathways. Our findings suggest a distinct early molecular signature in patients with aSAH who develop DCI, characterized by proinflammatory and neurovascular dysfunction markers. These candidate biomarkers warrant further validation in larger cohorts and may provide preliminary insight into biological processes associated with DCI. Their potential clinical value will require confirmation in independent studies.

OBJECTIVE:This study was undertaken to identify novel blood-based biomarkers associated with the early diagnosis of status epilepticus (SE) by exploring proteomic alterations in plasma extracellular vesicles (EVs). METHODS:We conducted an observational and prospective study in a two-phase design: a discovery proteomic analysis of plasma EVs from 16 patients (eight with SE and eight matched non-SE), followed by a validation phase using basic immunodetection techniques in a separate cohort of 160 patients (80 SE, 80 non-SE) for selected proteins in plasma. Proteomic profiling was performed with liquid chromatography-tandem mass spectrometry, and protein interaction networks were explored using STRING and Cytoscape software platforms. RESULTS:In the discovery phase, 2046 proteins were identified, and 977 met inclusion criteria. CDH1, APOC4, and IGHG2 showed the highest differential expression in SE patients. Network analysis revealed strong interactions involving SLC4A1 and SRC. In the validation cohort, levels of APOC4, CDH1, SLC4A1, and S100B were significantly higher in SE patients (p < .05). In the multivariate logistic regression model, only APOC4, S100B, and SLC4A1 remained independently associated with SE diagnosis. A combined model including these proteins predicted SE with 84.6% accuracy (95% confidence interval = 64.3%-94.9%). Other proteins, including NSE and HMGB1, showed no significant differences. SIGNIFICANCE:This study identifies new plasma biomarkers that may support the early diagnosis of SE, particularly in emergency settings where access to electroencephalography is limited. These findings suggest potential applicability to clinical practice; nevertheless, prospective validation in independent and larger cohorts is required.

Blood biomarkers in stroke patients seek to guide decision-making in clinical practice, but research on those that respond to brain recovery and their relationship with rehabilitation is still limited. Our aim was to explore the value of known neuroplasticity-related molecules, such as endostatin, growth and differentiation factor-10 (GDF-10), urokinase-type plasminogen activator (uPA) and the uPA receptor (uPAR), as blood biomarkers of recovery during poststroke rehabilitation. In an observational, prospective and multicenter study, biomarker levels were assessed after stroke in a cohort of 62 stroke patients with outcome evaluations and blood sampling before starting rehabilitation and during therapy at 1, 3 and 6 months of first visit, and in 43 control subjects. Serum levels were determined by Enzyme-Linked Immunosorbent Assay (ELISA) together with a complete battery of sensorimotor and functional tests/scales: modified Rankin Scale (mRS), Barthel Index (BI), Fugl-Meyer Assessment (FMA) for the upper extremity, Functional Ambulation Categories (FAC), Chedoke Arm and Hand Activity Inventory (CAHAI), 10-m walk test and the Medical Research Council (MRC). Statistical mixed linear models were built to investigate its prognostic value. The results revealed that, compared to controls, only endostatin was significantly increased at baseline after stroke (p < 0.01). Interestingly, the highest baseline GDF-10 or uPAR values were related to unfavorable scores during the complete follow-up (p < 0.05 for walking speed or MRC with GDF-10, and for FMA or MRC with uPAR), whereas decreased endostatin or increased GDF-10 biomarker changes at first month of rehabilitation were related to greater sensorimotor and functional improvements during follow-up (p < 0.05 for FMA or MRC with endostatin, and CAHAI or BI with GDF-10). Our results position endostatin, GDF-10 and uPAR as potential blood biomarkers to monitor recovery during rehabilitation after stroke.

Through GWAS studies we identified PATJ associated with functional outcome after ischemic stroke (IS). The aim of this study was to determine PATJ role in brain endothelial cells (ECs) in the context of stroke outcome. PATJ expression analyses in patient’s blood revealed that: (i) the risk allele of rs76221407 induces higher expression of PATJ, (ii) PATJ is downregulated 24 h after IS, and (iii) its expression is significantly lower in those patients with functional independence, measured at 3 months with the modified Rankin scale ((mRS) ≤2), compared to those patients with marked disability (mRS = 4–5). In mice brains, PATJ was also downregulated in the injured hemisphere at 48 h after ischemia. Oxygen-glucose deprivation and hypoxia-dependent of Hypoxia Inducible Factor-1α also caused PATJ depletion in ECs. To study the effects of PATJ downregulation, we generated PATJ-knockdown human microvascular ECs. Their transcriptomic profile evidenced a complex cell reprogramming involving Notch, TGF-ß, PI3K/Akt, and Hippo signaling that translates in morphological and functional changes compatible with endothelial to mesenchymal transition (EndMT). PATJ depletion caused loss of cell-cell adhesion, upregulation of metalloproteases, actin cytoskeleton remodeling, cytoplasmic accumulation of the signal transducer C-terminal transmembrane Mucin 1 (MUC1-C) and downregulation of Notch and Hippo signaling. The EndMT phenotype of PATJ-depleted cells was associated with the nuclear recruitment of MUC1-C, YAP/TAZ, β-catenin, and ZEB1. Our results suggest that PATJ downregulation 24 h after IS promotes EndMT, an initial step prior to secondary activation of a pro-angiogenic program. This effect is associated with functional independence suggesting that activation of EndMT shortly after stroke onset is beneficial for stroke recovery.

We synthesize highly crystalline citrate-coated iron oxide superparamagnetic nanoparticles that are stable and readily dispersible in water by an extremely fast microwave-assisted route and investigate the uptake of magnetic nanoparticles by endothelial cells. Nanoparticles form large aggregates when added to complete endothelial cell medium. The size of the aggregates was controlled by adjusting the ionic strength of the medium. The internalization of nanoparticles into endothelial cells was then investigated by transmission electron microscopy, magnetometry and chemical analysis, together with cell viability assays. Interestingly, a sevenfold more efficient uptake was found for systems with larger nanoparticle aggregates, which also showed significantly higher magnetic resonance imaging effectiveness without compromising cell viability and functionality. We are thus presenting an example of a straightforward microwave synthesis of citrate-coated iron oxide nanoparticles for safe endothelial progenitor cell labeling and good magnetic resonance cell imaging with potential application for magnetic cell guidance and in vivo cell tracking.

BACKGROUND: Beyond neuronal injury, cell death pathways may also contribute to vascular injury after stroke. We examined protein networks linked to major cell death pathways and identified SLC22A17 (solute carrier family 22 member 17) as a novel mediator that regulates endothelial tight junctions after ischemia and inflammatory stress. METHODS: Protein-protein interactions and brain enrichment analyses were performed using STRING, Cytoscape, and a human tissue-specific expression RNA-seq database. In vivo experiments were performed using mouse models of transient focal cerebral ischemia. Human stroke brain tissues were used to detect SLC22A17 by immunostaining. In vitro experiments were performed using human brain endothelial cultures subjected to inflammatory stress. Immunostaining and Western blot were used to assess responses in SLC22A17 and endothelial tight junctional proteins. Water content, dextran permeability, and electrical resistance assays were used to assess edema and blood-brain barrier (BBB) integrity. Gain and loss-of-function studies were performed using lentiviral overexpression of SLC22A17 or short interfering RNA against SLC22A17, respectively. RESULTS: Protein-protein interaction analysis showed that core proteins from apoptosis, necroptosis, ferroptosis, and autophagy cell death pathways were closely linked. Among the 20 proteins identified in the network, the iron-handling solute carrier SLC22A17 emerged as the mediator enriched in the brain. After cerebral ischemia in vivo, endothelial expression of SLC22A17 increases in both human and mouse brains along with BBB leakage. In human brain endothelial cultures, short interfering RNA against SLC22A17 prevents TNF-α (tumor necrosis factor alpha)–induced ferroptosis and downregulation in tight junction proteins and disruption in transcellular permeability. Notably, SLC22A17 could repress the transcription of tight junctional genes. Finally, short interfering RNA against SLC22A17 ameliorates BBB leakage in a mouse model of focal cerebral ischemia. CONCLUSIONS: Using a combination of cell culture, human stroke samples, and mouse models, our data suggest that SLC22A17 may play a role in the control of BBB function after cerebral ischemia. These findings may offer a novel mechanism and target for ameliorating BBB injury and edema after stroke.

The combustion of fossil fuels, mainly by diesel engines, generates Diesel Exhaust Particles (DEP) which are the main source of Particulate Matter (PM), a major air pollutant in urban areas. These particles are a risk factor for stroke with 5.6% of cases attributed to PM exposure. Our aim was to evaluate the effect of DEP exposure on clot formation and lysis in the context of stroke. An ex-vivo clot formation and lysis turbidimetric assay has been conducted in human and mouse plasma samples from ischemic stroke or control subjects exposed to DEP or control conditions. Experimental DEP exposure was achieved by nasal instillation in mice, or by ex-vivo exposure in human plasma. Results show consistent pro-thrombogenic features in plasma after human ischemic stroke and mouse cerebral ischemia (distal MCAo), boosted by the presence of DEP. Otherwise, thrombolysis times were increased after ischemia in chronically exposed mice but not in the DEP exposed group. Finally, subjects living in areas with high PM levels presented accelerated thrombolysis compared to those living in low polluted areas. Overall, our results point at a disbalance of the thrombogenic/lytic system in presence of DEP which could impact on ischemic stroke onset, clot size and thrombolytic treatment.

HomeStrokeVol. 54, No. 4Retinoic Acid–Loaded Nanoparticles Promote Neurovascular Protection in Stroke Free AccessResearch ArticlePDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissionsDownload Articles + Supplements ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toSupplemental MaterialFree AccessResearch ArticlePDF/EPUBRetinoic Acid–Loaded Nanoparticles Promote Neurovascular Protection in Stroke Marta Machado-Pereira, Alba Grayston, Miguel Garcia-Gabilondo, Vitor Francisco, Ana Cristóvão, João Marto, Helena Vieira, Miguel Viana-Baptista, Lino Ferreira, Liliana Bernardino, Anna Rosell and Raquel Ferreira Marta Machado-PereiraMarta Machado-Pereira https://orcid.org/0000-0002-8986-8679 Health Sciences Research Centre (M.M.-P., A.C., L.B., R.F.), University of Beira Interior, Portugal. , Alba GraystonAlba Grayston https://orcid.org/0000-0002-1466-0099 Health Sciences Research Centre (M.M.-P., A.C., L.B., R.F.), University of Beira Interior, Portugal. Neurovascular Research Laboratory, Vall d'Hebron Institut de Recerca, Universitat Autónoma de Barcelona, Spain (A.G., M.G.-G., A.R.). , Miguel Garcia-GabilondoMiguel Garcia-Gabilondo https://orcid.org/0000-0002-2015-2621 Neurovascular Research Laboratory, Vall d'Hebron Institut de Recerca, Universitat Autónoma de Barcelona, Spain (A.G., M.G.-G., A.R.). , Vitor FranciscoVitor Francisco https://orcid.org/0000-0002-2704-9950 Center for Neuroscience and Cell Biology (V.F., L.F.), University of Coimbra, Portugal. Faculty of Medicine (V.F., L.F.), University of Coimbra, Portugal. , Ana CristóvãoAna Cristóvão https://orcid.org/0000-0001-5806-4478 NeuroSoV, UBImedical (A.C.), University of Beira Interior, Portugal. , João MartoJoão Marto https://orcid.org/0000-0003-2277-5950 Department of Neurology, Hospital Egas Moniz, Centro Hospitalar de Lisboa Ocidental, Portugal (J.M., M.V.-B.). NMS Research, Nova Medical School Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal (J.M., H.V., M.V.-B., R.F.). , Helena VieiraHelena Vieira https://orcid.org/0000-0001-9415-3742 NMS Research, Nova Medical School Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal (J.M., H.V., M.V.-B., R.F.). Applied Molecular Biosciences Unit (H.V.), NOVA School of Science and Technology, Universidade NOVA de Lisboa, Portugal. Associate Laboratory i4HB – Institute for Health and Bioeconomy, Department of Chemistry (H.V.), NOVA School of Science and Technology, Universidade NOVA de Lisboa, Portugal. , Miguel Viana-BaptistaMiguel Viana-Baptista https://orcid.org/0000-0001-6166-2073 Department of Neurology, Hospital Egas Moniz, Centro Hospitalar de Lisboa Ocidental, Portugal (J.M., M.V.-B.). NMS Research, Nova Medical School Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal (J.M., H.V., M.V.-B., R.F.). , Lino FerreiraLino Ferreira https://orcid.org/0000-0001-8985-9302 Center for Neuroscience and Cell Biology (V.F., L.F.), University of Coimbra, Portugal. Faculty of Medicine (V.F., L.F.), University of Coimbra, Portugal. , Liliana BernardinoLiliana Bernardino Liliana Bernardino, PhD, Health Sciences Research Centre, University of Beira Interior, Portugal, Email E-mail Address: [email protected] https://orcid.org/0000-0003-3395-5973 Health Sciences Research Centre (M.M.-P., A.C., L.B., R.F.), University of Beira Interior, Portugal. , Anna RosellAnna Rosell Neurovascular Research Laboratory, Vall d'Hebron Institut de Recerca, Universitat Autónoma de Barcelona, Spain (A.G., M.G.-G., A.R.). and Raquel FerreiraRaquel Ferreira Correspondence to: Raquel Ferreira, PhD, NOVA Medical School Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Campo dos Mártires da Pátria 130, 1169-056 Portugal, Email E-mail Address: [email protected] https://orcid.org/0000-0003-1014-0499 Health Sciences Research Centre (M.M.-P., A.C., L.B., R.F.), University of Beira Interior, Portugal. NMS Research, Nova Medical School Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Portugal (J.M., H.V., M.V.-B., R.F.). Now with Technophage, Lisbon, Portugal (R.F.). Originally published13 Mar 2023https://doi.org/10.1161/STROKEAHA.122.041839Stroke. 2023;54:e149–e151Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: March 13, 2023: Ahead of Print The role of Ago2 (argonaute-2) in stroke is insufficiently described. Loss of this increases endothelial cell death and impairs tubule formation.1 Conversely, the abnormal human brain vasculature overexpresses Ago2, and we have highlighted its clinical potential as a systemic microRNA carrier; signaling pathways remain unexplored in stroke.2 The actions of retinoic acid (RA) are complex to manage (poor solubility, photosensitivity, narrow therapeutic window), but its encapsulation in trackable polymeric nanoparticles (RA polymeric nanoparticles [RA-NP]) has enhanced its efficacy.3 In vitro, 3 µg/mL RA-NP (not the free molecule [0.12 µM]), recuperated Ago2 after oxygen and glucose deprivation, followed by a recovery period intended to mimic reperfusion (OGD/R; Figure [A]). Normalizing intracellular Ago2 in an inflammatory context recuperates the neurovascular unit, via the NO pathway, which also intersects with RA signaling.3,4 After OGD/R, RA-NP normalized the phosphorylation of Akt and overexpression of endothelial nitric oxide synthase (eNOS; Figure [B]) and consequent NO overproduction (Figure [C]). By silencing Ago2 or by inhibiting its activity, NO production was also enhanced, producing the first evidence that RA-NP can operate via Ago2 and the regulation of the Akt (protein kinase B)/eNOS/NO pathway. In vivo,5 a single intravenous injection of RA-NP (20 µg/g), equivalent to 0.246 µg/g free RA, reached the brain without systemic toxicity and reduced infarct volume, three days poststroke (Figure [D]). In this period, RA-NP normalized microglial anti-ionized calcium binding adaptor molecule-1 and astrocytic GFAP (anti-glial fibrillary acidic protein) levels (Figure [E]), when inflammation is more exacerbated. Seven days poststroke, treated mice recovered grip strength (Figure [D]), and in parallel, RA-NP increased vessel density (Figure [F]), suggesting a promising proangiogenic effect in the peri-infarct region. Therefore, RA-NP could be envisioned as a potential therapeutic systemic agent aimed at treating neurovascular and/or neuroinflammatory conditions.Download figureDownload PowerPointFigure. Retinoic acid polymeric nanoparticles (RA-NP) modulate markers relevant for the recovery of the ischemic brain. RA-NP recuperated Ago2 (argonaute-2; A), pAkt (phosphorylated- Akt), and eNOS (B) in primary brain endothelial cells after oxygen and glucose deprivation followed by a recovery period (OGD/R), resulting in normalized NO production (C). Using a model of transient middle cerebral artery occlusion, RA-NP reduced infarct volume and increased grip strength (D). RA-NP normalized microglial and astrocytic markers (anti-ionized calcium binding adaptor molecule-1 [Iba-1] and GFAP [anti-glial fibrillary acidic protein]) 3 days postinjection (E), and increased vessel density compromised by stroke 7 days postinjection (F). CT indicates contralateral; IP, ipsilateral; and siAgo2, small interfering ribonucleic acid for argonaute-2.MethodsExpanded Methods are detailed in the in Supplemental Material.4,5 All experiments were performed in accordance with the National Institutes of Health and European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (European Union directive number 2010/63/EU) for the care and use of laboratory animals, the Ethics Committee for Animal Experimentation of the Vall d'Hebron Research Institute, according to Spanish legislation.The data that support the findings of this study are available from the corresponding author upon reasonable request, according to the American Heart Association Journals' implementation of the Transparency and Openness Promotion Guidelines.Article InformationSources of FundingSupported by Portuguese Platform of BioImaging, POCI-01-0145-FEDER-022122; FCT, UID/Multi/00709/2013, SFRH/BD/137440/2018, ISCIII, FI17/00073; PTDC/BTM- SAL/5174/2020, EXPL/BTM-ORG/1348/2021; INTER-REG Atlantic Area (EAPA_791/2018_ NEUROATLANTIC Project), INTER-REG V A España Portugal (POCTEP; 0624_2IQBIONEURO_6_E), European Regional Development Fund; SLT017/20/000197 from AGAUR; RICORS-Stroke Network from ISCIII (RD21/0006/0007); IF/00178/2015/CP1300/CT0001; PERIS-SLT017/20/000197 (Generalitat Catalunya).Supplemental MaterialSupplemental MethodsDisclosures None.FootnotesFor Sources of Funding and Disclosures, see page e150.Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/STROKEAHA.122.041839.Correspondence to: Raquel Ferreira, PhD, NOVA Medical School Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Campo dos Mártires da Pátria 130, 1169-056 Portugal, Email raquel.ferreira@nms.unl.ptLiliana Bernardino, PhD, Health Sciences Research Centre, University of Beira Interior, Portugal, Email libernardino@fcsaude.ubi.ptReferences1. Asai T, Suzuki Y, Matsushita S, Yonezawa S, Yokota J, Katanasaka Y, Ishida T, Dewa T, Kiwada H, Nango M, et al. Disappearance of the angiogenic potential of endothelial cells caused by Argonaute2 knockdown.Biochem Biophys Res Commun. 2008; 368:243–248. doi: 10.1016/j.bbrc.2008.01.074CrossrefGoogle Scholar2. Ferreira R, Santos T, Amar A, Gong A, Chen TC, Tahara SM, Giannotta SL, Hofman FM. Argonaute-2 promotes miR-18a entry in human brain endothelial cells.J Am Heart Assoc. 2014; 3:e000968. doi: 10.1161/JAHA.114.000968LinkGoogle Scholar3. Ferreira R, Napoli J, Enver T, Bernardino L, Ferreira L. Advances and challenges in retinoid delivery systems in regenerative and therapeutic medicine.Nat Commun. 2020; 11:1–14. doi: 10.1038/s41467-020-18042-2CrossrefGoogle Scholar4. Machado-Pereira M, Saraiva C, Bernardino L, Cristóvão AC, Ferreira R. Argonaute- 2 protects the neurovascular unit from damage caused by systemic inflammation.J Neuroinflammation. 2022; 19:1–17. doi: 10.1186/s12974-021-02324-7CrossrefGoogle Scholar5. Morancho A, García-Bonilla L, Barceló V, Giralt D, Campos-Martorell M, Garcia S, Montaner J, Rosell A. A new method for focal transient cerebral ischaemia by distal compression of the middle cerebral artery.Neuropathol Appl Neurobiol. 2012; 38:617–627. doi: 10.1111/j.1365-2990.2012.01252.xCrossrefMedlineGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetails April 2023Vol 54, Issue 4 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/STROKEAHA.122.041839PMID: 36912140 Originally publishedMarch 13, 2023 Keywordsnanoparticlesribonucleoproteininflammationendothelial cellstrokePDF download Advertisement SubjectsCell Signaling/Signal TransductionCerebrovascular Disease/StrokeIschemic Stroke

Ischemic stroke is a major cause of death and disability worldwide. Translation into the clinical setting of neuroprotective agents showing promising results in pre-clinical studies has systematically failed. One possible explanation is that the animal models used to test neuroprotectants do not properly represent the population affected by stroke, as most of the pre-clinical studies are performed in healthy young male mice. Therefore, we aimed to determine if the response to cerebral ischemia differed depending on age, sex and the presence of comorbidities. Thus, we explored proteomic and transcriptomic changes triggered during the hyperacute phase of cerebral ischemia (by transient intraluminal middle cerebral artery occlusion) in the brain of: (1) young male mice, (2) young female mice, (3) aged male mice and (4) diabetic young male mice. Moreover, we compared each group's proteomic and transcriptomic changes using an integrative enrichment pathways analysis to disclose key common and exclusive altered proteins, genes and pathways in the first stages of the disease. We found 61 differentially expressed genes (DEG) in male mice, 77 in females, 699 in diabetics and 24 in aged mice. Of these, only 14 were commonly dysregulated in all groups. The enrichment pathways analysis revealed that the inflammatory response was the biological process with more DEG in all groups, followed by hemopoiesis. Our findings indicate that the response to cerebral ischemia regarding proteomic and transcriptomic changes differs depending on sex, age and comorbidities, highlighting the importance of incorporating animals with different phenotypes in future stroke research studies.

Tumor secreted extracellular vesicles (EVs) are potent intercellular signaling platforms. They are responsible for the accommodation of the premetastatic niche (PMN) to support cancer cell engraftment and metastatic growth. However, complex cancer cell composition within the tumor increases also the heterogeneity among cancer secreted EVs subsets, a functional diversity that has been poorly explored. This phenomenon is particularly relevant in highly plastic and heterogenous triple‐negative breast cancer (TNBC), in which a significant representation of malignant cancer stem cells (CSCs) is displayed. Herein, we selectively isolated and characterized EVs from CSC or differentiated cancer cells (DCC; EVsCSC and EVsDCC, respectively) from the MDA‐MB‐231 TNBC cell line. Our results showed that EVsCSC and EVsDCC contain distinct bioactive cargos and therefore elicit a differential effect on stromal cells in the TME. Specifically, EVsDCC activated secretory cancer associated fibroblasts (CAFs), triggering IL‐6/IL‐8 signaling and sustaining CSC phenotype maintenance. Complementarily, EVsCSC promoted the activation of α‐SMA+ myofibroblastic CAFs subpopulations and increased the endothelial remodeling, enhancing the invasive potential of TNBC cells in vitro and in vivo. In addition, solely the EVsCSC mediated signaling prompted the transformation of healthy lungs into receptive niches able to support metastatic growth of breast cancer cells.

After stroke and other brain injuries, there is a high incidence of respiratory complications such as pneumonia or acute lung injury. The molecular mechanisms that drive the brain-lung interaction post-stroke have not yet been elucidated. We performed transient middle cerebral artery occlusion (MCAO) and sham surgery on C57BL/6J mice and collected bronchoalveolar lavage fluid (BALF), serum, brain, and lung homogenate samples 24 h after surgery. A 92 proteins-panel developed by Olink Proteomics® was used to analyze the content in BALF and lung homogenates. MCAO animals had higher protein concentration levels in BALF than sham-controls, but these levels did not correlate with the infarct volume. No alteration in alveolar-capillary barrier permeability was observed. A total of 12 and 14 proteins were differentially expressed between the groups (FDR < 0.1) in BALF and lung tissue homogenates, respectively. Of those, HGF, TGF-α, and CCL2 were identified as the most relevant to this study. Their protein expression patterns were verified by ELISA. This study confirmed that post-stroke lung damage was not associated with increased lung permeability or cerebral ischemia severity. Furthermore, the dysregulation of HGF, TGF-α, and CCL2 in BALF and lung tissue after ischemia could play an important role in the molecular mechanisms underlying stroke-induced lung damage.

Multifunctional magnetic nanocomposites based on mesoporous silica have a wide range of potential applications in catalysis, biomedicine, or sensing. Such particles combine responsiveness to external magnetic fields with other functionalities endowed by the agents loaded inside the pores or conjugated to the particle surface. Different applications might benefit from specific particle morphologies. In the case of biomedical applications, mesoporous silica nanospheres have been extensively studied while nanorods, with a more challenging preparation, have attracted much less attention despite the positive impact on the therapeutic performance shown by seminal studies. Here, we report on a sol-gel synthesis of mesoporous rodlike silica particles of two distinct lengths (1.4 and 0.9 μm) and aspect ratios (4.7 and 2.2) using Pluronic P123 as a structure-directing template and rendering ∼1 g of rods per batch. Iron oxide nanoparticles have been synthesized within the pores yielding maghemite (γ-Fe2O3) nanocrystals of elongated shape (∼7 nm × 5 nm) with a [110] preferential orientation along the rod axis and a superparamagnetic character. The performance of the rods as T2-weighted MRI contrast agents has also been confirmed. In a subsequent step, the mesoporous silica rods were loaded with a cerium compound and their surface was functionalized with fluorophores (fluorescamine and Cyanine5) emitting at λ = 525 and 730 nm, respectively, thus highlighting the possibility of multiple imaging modalities. The biocompatibility of the rods was evaluated in vitro in a zebrafish (Danio rerio) liver cell line (ZFL), with results showing that neither long nor short rods with magnetic particles caused cytotoxicity in ZFL cells for concentrations up to 50 μg/ml. We advocate that such nanocomposites can find applications in medical imaging and therapy, where the influence of shape on performance can be also assessed.

Ischemic stroke is a primary cause of morbidity and mortality worldwide. Beyond the approved thrombolytic therapies, there is no effective treatment to mitigate its progression. Drug repositioning combinational therapies are becoming promising approaches to identify new uses of existing drugs to synergically target multiple disease-response mechanisms underlying complex pathologies. Here, we used a systems biology–based approach based on artificial intelligence and pattern recognition tools to generate in silico mathematical models mimicking the ischemic stroke pathology. Combinational treatments were acquired by screening these models with more than 5 million two-by-two combinations of drugs. A drug combination (CA) formed by ceruletide and alpha-1 antitrypsin showing a predicted value of neuroprotection of 92% was evaluated for their synergic neuroprotective effects in a mouse pre-clinical stroke model. The administration of both drugs in combination was safe and effective in reducing by 39.42% the infarct volume 24 h after cerebral ischemia. This neuroprotection was not observed when drugs were given individually. Importantly, potential incompatibilities of the drug combination with tPA thrombolysis were discarded in vitro and in vivo by using a mouse thromboembolic stroke model with t-PA-induced reperfusion, revealing an improvement in the forepaw strength 72 h after stroke in CA-treated mice. Finally, we identified the predicted mechanisms of action of ceruletide and alpha-1 antitrypsin and we demonstrated that CA modulates EGFR and ANGPT-1 levels in circulation within the acute phase after stroke. In conclusion, we have identified a promising combinational treatment with neuroprotective effects for the treatment of ischemic stroke.