Extracellular vesicles and their microRNA cargo in ischaemic stroke.

Acute ischaemic stroke (AIS) is a leading cause of death and disability. MicroRNAs (miRNAs) are short non-coding RNAs which hold the potential to act as a novel biomarker in AIS. The majority of circulating miRNAs are actively encapsulated by extracellular vesicles (EVs) produced by many cells and organs endogenously. EVs released by mesenchymal stem cells (MSCs) have been extensively studied for their therapeutic potential. In health and disease, EVs are vital for intercellular communication, as the cargo within EVs can be exchanged between neighbouring cells or transported to distant sites. It is clear here from both current preclinical and clinical studies that AIS is associated with specific EV-derived miRNAs, including those transported via MSC-derived EVs. In addition, current studies provide evidence to show that modulating levels of specific EV-derived miRNAs in AIS provides a novel therapeutic potential of miRNAs in the treatment of stroke. Commonalities exist in altered miRNAs across preclinical and clinical studies. Of those EV-packaged miRNAs, miRNA-124 was described both as an EV-packaged biomarker and as a potential EV-loaded therapeutic in experimental models. Alterations of miRNA-17 family and miRNA-17-92 cluster were identified in preclinical, clinical and MSC-EV-mediated neuroprotection in experimental stroke. Finally, miRNA-30d and -30a were found to mediate therapeutic effect when overexpressed from MSC and implicated as a biomarker clinically. Combined, EV-derived miRNAs will further our understanding of the neuropathological processes triggered by AIS. In addition, this work will help determine the true clinical value of circulating EV-packaged miRNAs as biomarkers of AIS or as novel therapeutics in this setting.

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS.

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

Small vessels, dementia and chronic diseases - molecular mechanisms and pathophysiology.

Cerebral small vessel disease (SVD) is a major contributor to stroke, cognitive impairment and dementia with limited therapeutic interventions. There is a critical need to provide mechanistic insight and improve translation between pre-clinical research and the clinic. A 2-day workshop was held which brought together experts from several disciplines in cerebrovascular disease, dementia and cardiovascular biology, to highlight current advances in these fields, explore synergies and scope for development. These proceedings provide a summary of key talks at the workshop with a particular focus on animal models of cerebral vascular disease and dementia, mechanisms and approaches to improve translation. The outcomes of discussion groups on related themes to identify the gaps in knowledge and requirements to advance knowledge are summarized.

The glymphatic system and multiple sclerosis: An evolving connection.

Multiple sclerosis (MS) is a complex autoimmune disorder that affects the central nervous system, resulting in demyelination and an array of neurological manifestations. Recently, there has been significant scientific interest in the glymphatic system, which operates as a waste-clearance system for the brain. This article reviews the existing literature, and explores potential links between the glymphatic system and MS, shedding light on its evolving significance in the context of MS pathogenesis. The authors consider the pathophysiological implications of glymphatic dysfunction in MS, the impact of disrupted sleep on glymphatic function, and the bidirectional relationship between MS and sleep disturbances. By offering an understanding of the intricate interplay between the glymphatic system and MS, this review provides valuable insights which may lead to improved diagnostic techniques and more effective therapeutic interventions.

Hypertension & dementia: Pathophysiology & potential utility of antihypertensives in reducing disease burden.

Dementia is a common cause of disability and dependency among the elderly due to its progressive neurodegenerative nature. As there is currently no curative therapy, it is of major importance to identify new ways to reduce its prevalence. Hypertension is recognised as a modifiable risk factor for dementia, particularly for the two most common subtypes; vascular dementia (VaD) and Alzheimer's disease (AD). From the current literature, identified through a comprehensive literature search of PubMed and Cochrane Library, this review aims to establish the stage in adulthood when hypertension becomes a risk for cognitive decline and dementia, and whether antihypertensive treatment is effective as a preventative therapy. Observational studies generally found hypertension in mid-life (age 45-64) to be correlated with an increased risk of cognitive decline and dementia incidence, including both VaD and AD. Hypertension manifesting in late life (age ≥ 65) was demonstrated to be less of a risk, to the extent that incidences of high blood pressure (BP) in the very elderly (age ≥ 75) may even be related to reduced incidence of dementias. Despite the evidence linking hypertension to dementia, there were conflicting findings as to whether the use of antihypertensives was beneficial for its prevention and this conflicting evidence and inconsistent results could be due to the methodological differences between the reviewed observational and randomised controlled trials. Furthermore, dihydropyridine calcium channel blockers and potassium-sparing diuretics were proposed to have neuroprotective properties in addition to BP lowering. Overall, if antihypertensives are confirmed to be beneficial by larger-scale homogenous trials with longer follow-up durations, treatment of hypertension, particularly in mid-life, could be an effective strategy to considerably lower the prevalence of dementia. Furthermore, greater clarification of the neuroprotective properties that some antihypertensives possess will allow for better clinical practice guidance on the choice of antihypertensive class for both BP lowering and dementia prevention.

Unscheduled changes in pre-clinical stroke model housing contributes to variance in physiological and behavioural data outcomes: A post hoc analysis.

Ischaemic stroke presents a significant problem worldwide with no neuroprotective drugs available. Many of the failures in the search for neuroprotectants are attributed to failure to translate from pre-clinical models to humans, which has been combatted with rigorous pre-clinical stroke research guidelines. Here, we present post hoc analysis of a pre-clinical stroke trial, conducted using intraluminal filament transient middle cerebral artery occlusion in the stroke-prone spontaneously hypertensive rat, whereby unscheduled changes were implemented in the animal housing facility. These changes severely impacted body weight post-stroke resulting in a change from the typical body weight of 90.6% of pre-surgery weight post-stroke, to on average 80.5% of pre-surgery weight post-stroke. The changes also appeared to impact post-stroke blood pressure, with an increase from 215.4 to 240.3 mmHg between housing groups, and functional outcome post-stroke, with a 38% increased latency to contact in the sticky label test. These data highlight the importance of tightly controlled housing conditions when using physiological or behavioural measurements as a primary outcome.

Polypathologies and Animal Models of Traumatic Brain Injury.

Traumatic brain injury (TBI) is an important health issue for the worldwide population, as it causes long-term pathological consequences for a diverse group of individuals. We are yet to fully elucidate the significance of TBI polypathologies, such as neuroinflammation and tau hyperphosphorylation, and their contribution to the development of chronic traumatic encephalopathy (CTE) and other neurological conditions. To advance our understanding of TBI, it is necessary to replicate TBI in preclinical models. Commonly used animal models include the weight drop model; these methods model human TBI in various ways and in different animal species. However, animal models have not demonstrated their clinical utility for identifying therapeutic interventions. Many interventions that were successful in improving outcomes for animal models did not translate into clinical benefit for patients. It is important to review current animal models and discuss their strengths and limitations within a TBI context. Modelling human TBI in animals encounters numerous challenges, yet despite these barriers, the TBI research community is working to overcome these difficulties. Developments include advances in biomarkers, standardising, and refining existing models. This progress will improve our ability to model TBI in animals and, therefore, enhance our understanding of TBI and, potentially, how to treat it.

Systematic review: Association between circulating microRNA expression & stroke.

This systematic review aimed to establish the range and quality of clinical and preclinical evidence supporting the association of individual microRNAs, and the use of microRNA expression in the diagnosis and prognosis of ischaemic or haemorrhagic stroke. Electronic databases were searched from 1993 to October 2021, using key words relevant to concepts of stroke and microRNA. Studies that met specific inclusion and exclusion criteria were selected for data extraction. To minimise erroneous associations, findings were restricted to microRNAs reported to change in more than two independent studies. Of the papers assessed, 155 papers reported a change in microRNA expression observed in more than two independent studies. In ischaemic studies, two microRNAs were consistently differentially expressed in clinical samples (miR-29b & miR-146a) and four were altered in preclinical samples (miR-137, miR-146a, miR-181b & miR-223-3p). Across clinical and preclinical haemorrhagic studies, four microRNAs were downregulated consistently (miR-26a, miR-126, miR-146a & miR-155). Across included studies, miR-126 and miR-146a were the only two microRNAs to be differentially expressed in clinical and preclinical cohorts following ischaemic or haemorrhagic stroke. Further studies, employing larger populations with consistent methodologies, are required to validate the true clinical value of circulating microRNAs as biomarkers of ischaemic and haemorrhagic stroke.

Direct imaging of glymphatic transport using H217O MRI.

The recently proposed glymphatic pathway for solute transport and waste clearance from the brain has been the focus of intense debate. By exploiting an isotopically enriched MRI tracer, H217O, we directly imaged glymphatic water transport in the rat brain in vivo. Our results reveal glymphatic transport that is dramatically faster and more extensive than previously thought and unlikely to be explained by diffusion alone. Moreover, we confirm the critical role of aquaporin-4 channels in glymphatic transport.

Vascular-targeting antioxidant therapy in a model of hypertension and stroke.

Oxidative stress is implicated in the pathogenesis of hypertension and stroke. Superoxide is produced by NAD(P)H oxidase in the vasculature and reduces nitric oxide bioavailability, which leads to increased blood pressure. The objective of this study was to determine whether targeting an antioxidant peptide to the vasculature would increase the antioxidant effect and reduce systolic blood pressure (SBP) in a model of genetic hypertension, the stroke-prone spontaneously hypertensive rat. Vascular-targeting peptides CRPPR and CSGMARTKC were identified by phage display in mice. These peptides retain their selectivity across species and target the aorta (CRPPR) and cardiac vasculature (CSGMARTKC) in the stroke-prone spontaneously hypertensive rat. These vascular-targeting peptides were linked to the antioxidant peptide gp91ds, which selectively inhibits assembly of NAD(P)H oxidase, thereby reducing superoxide production. SBP was determined for 1 week before treatment followed by 3 weeks of study duration before euthanasia. SBP in the control animals increased from 178.1 ± 4.1 mmHg to 201.6 ± 9.0 mmHg. The SBP of the animals treated with gp91ds alone, HIV-tat-gp91ds, and CSGMARTKC-gp91ds increased from 177.8 ± 3.5 mmHg, 179.8 ± 4.7 mmHg, and 177.9 ± 5.2 mmHg, respectively, to 201.6 ± 10.8 mmHg, 200.3 ± 11.7 mmHg and 205.7 ± 10.9 mmHg, respectively. This increase in SBP was significantly attenuated in animals receiving CRPPR-gp91ds (maximum SBP 187.5 mmHg ± 5.2, *P , 0.001 versus other treatment groups and control group). Additionally, animals treated with CRPPR-gp91ds, CSGMARTKC-gp91ds, and gp91ds alone showed significantly improved nitric oxide bioavailability determined by large vessel myography. Therefore, targeting an antioxidant to the aortic vasculature in vivo using peptides can significantly improve nitric oxide bioavailability and attenuate the time-dependent and progressive increase in SBP in the stroke-prone spontaneously hypertensive rat. This study has demonstrated the importance and potential benefit of targeting a biologically active peptide in the context of a preclinical model of endothelial dysfunction and hypertension.

The counter regulatory axis of the renin angiotensin system in the brain and ischaemic stroke: Insight from preclinical stroke studies and therapeutic potential.

Stroke is the 2nd leading cause of death worldwide and the leading cause of physical disability and cognitive issues. Although we have made progress in certain aspects of stroke treatment, the consequences remain substantial and new treatments are needed. Hypertension has long been recognised as a major risk factor for stroke, both haemorrhagic and ischaemic. The renin angiotensin system (RAS) plays a key role in blood pressure regulation and this, plus local expression and signalling of RAS in the brain, both support the potential for targeting this axis therapeutically in the setting of stroke. While historically, focus has been on suppressing classical RAS signalling through the angiotensin type 1 receptor (AT1R), the identification of a counter-regulatory axis of the RAS signalling via the angiotensin type 2 receptor (AT2R) and Mas receptor has renewed interest in targeting the RAS. This review describes RAS signalling in the brain and the potential of targeting the Mas receptor and AT2R in preclinical models of ischaemic stroke. The animal and experimental models, and the route and timing of intervention, are considered from a translational perspective.

UK consensus on pre-clinical vascular cognitive impairment functional outcomes assessment: Questionnaire and workshop proceedings.

Assessment of outcome in preclinical studies of vascular cognitive impairment (VCI) is heterogenous. Through an ARUK Scottish Network supported questionnaire and workshop (mostly UK-based researchers), we aimed to determine underlying variability and what could be implemented to overcome identified challenges. Twelve UK VCI research centres were identified and invited to complete a questionnaire and attend a one-day workshop. Questionnaire responses demonstrated agreement that outcome assessments in VCI preclinical research vary by group and even those common across groups, may be performed differently. From the workshop, six themes were discussed: issues with preclinical models, reasons for choosing functional assessments, issues in interpretation of functional assessments, describing and reporting functional outcome assessments, sharing resources and expertise, and standardization of outcomes. Eight consensus points emerged demonstrating broadly that the chosen assessment should reflect the deficit being measured, and therefore that one assessment does not suit all models; guidance/standardisation on recording VCI outcome reporting is needed and that uniformity would be aided by a platform to share expertise, material, protocols and procedures thus reducing heterogeneity and so increasing potential for collaboration, comparison and replication. As a result of the workshop, UK wide consensus statements were agreed and future priorities for preclinical research identified.

Preclinical Validation of the Therapeutic Potential of Glasgow Oxygen Level Dependent (GOLD) Technology: a Theranostic for Acute Stroke.

In acute stroke patients, penumbral tissue is non-functioning but potentially salvageable within a time window of variable duration and represents target tissue for rescue. Reperfusion by thrombolysis and/or thrombectomy can rescue penumbra and improve stroke outcomes, but these treatments are currently available to a minority of patients. In addition to the utility of Glasgow Oxygen Level Dependent (GOLD) as an MRI contrast capable of detecting penumbra, its constituent perfluorocarbon (PFC) oxygen carrier, combined with normobaric hyperoxia, also represents a potential acute stroke treatment through improved oxygen delivery to penumbra. Preclinical studies were designed to test the efficacy of an intravenous oxygen carrier, the perfluorocarbon emulsion Oxycyte® (O-PFC), combined with normobaric hyperoxia (50% O2) in both in vitro (neuronal cell culture) and in vivo rat models of ischaemic stroke. Outcome was assessed through the quantification of lipid peroxidation and oxidative stress levels, mortality, infarct volume, neurological scoring and sensorimotor tests of functional outcome in two in vivo models of stroke. Additionally, we investigated evidence for any positive or negative interactions with the thrombolytic recombinant tissue plasminogen activator (rt-PA) following embolus-induced stroke in rats. Treatment with intravenous O-PFC + normobaric hyperoxia (50% O2) provided evidence of reduced infarct size and improved functional recovery. It did not exacerbate oxidative stress and showed no adverse interactions with rt-PA. The positive results and lack of adverse effects support human trials of O-PFC + 50% O2 normobaric hyperoxia as a potential therapeutic approach. Combined with the diagnostic data presented in the preceding paper, O-PFC and normobaric hyperoxia is a potential theranostic for acute ischaemic stroke.

Altered Extracellular Vesicle MicroRNA Expression in Ischemic Stroke and Small Vessel Disease.

Active transport of microRNAs (miRNA) in extracellular vesicles (EV) occurs in disease. Circulating EV-packaged miRNAs in the serum of stroke patients were compared to stroke mimics with matched cardio- and cerebrovascular risk factors, with corroboration of results in a pre-clinical model. An unbiased miRNA microarray was performed in stroke vs. stroke mimic patients (n = 39). Results were validated (n = 173 patients) by real-time quantitative polymerase chain reaction. miRNA expression was quantified in total serum/EV (n = 5-7) of naïve adult spontaneously hypertensive stroke-prone rats (SHRSP), their normotensive reference strain (Wistar Kyoto, WKY) and in circulating EV (n = 3), peri-infarct brain (n = 6), or EV derived from this region (n = 3) in SHRSP following transient middle cerebral artery occlusion (tMCAO). Circulating EV concentration did not differ between stroke and stroke mimic patients. The microarray identified many altered EV-packaged miRNAs: levels of miRNA-17-5p, -20b-5p and -93-5p (miRNA-17 family members) and miRNA-27b-3p were significantly (p ≤ 0.05) increased in stroke vs. stroke mimic patients. Patients with small vessel disease (SVD) consistently had the highest miRNA levels. Circulating EV concentration was unaltered between naïve SHRSP and WKY but levels of miRNA-17-5p and -93-5p were significantly increased in SHRSP. tMCAO in SHRSP did not further alter circulating EV miRNA-17 family member expression and nor did it change total miRNA-17 family levels in peri-infarct brain tissue or in EV isolated from this region at 24 h post-tMCAO. Changes in EV packaged miRNA expression was validated in patients with stroke, particularly those with SVD and corroborated pre-clinically. Together, altered circulating EV levels of miRNA-17 family members may reflect the chronic sequelae underlying cerebrovascular SVD rather than the acute ischemic stroke itself.

Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats.

BACKGROUND: Vectors based on adeno-associated virus-8 (AAV8) have shown efficiency and efficacy for liver-directed gene therapy protocols following intravascular injection, particularly in relation to haemophilia gene therapy. AAV8 has also been proposed for gene therapy targeted at skeletal and cardiac muscle, again via intravascular injection. It is important to assess vector targeting at the level of virion accumulation and transgene expression in multiple species to ascertain potential issues relating to species variation in infectivity profiles. METHODS: We used AAV8 vectors expressing human factor IX (FIX) from the liver-specific LP-1 promoter and administered this virus via the intravascular route of injection into 12 week old Wistar Kyoto rats. We assessed FIX levels in serum by ELISA and transgene expression at sacrifice by immunohistochemistry using anti-FIX antibodies. Vector DNA levels in organs we determined by real time PCR. RESULTS: Administration of 1 x 10(11) or 5 x 10(11) scAAV8-LP1-hFIX vector particles/rat resulted in efficient production of physiological hFIX levels, respectively in blood assessed 4 weeks post-injection. This was maintained for the 4 month duration of the study. At 4 months we observed liver persistence of vector with minimal non-hepatic distribution. CONCLUSION: Our results demonstrate that AAV8 is a robust vector for delivering therapeutic genes into rat liver following intravascular injection.

Variability of functional outcome measures used in animal models of stroke and vascular cognitive impairment - a review of contemporary studies.

Despite promising preclinical data, few novel stroke therapies have shown efficacy in man. Efforts to improve standards in conduct and reporting of preclinical research are ongoing. In clinical trials, inconsistency in outcome measures led to regulatory agencies and funders mandating use of a core set of functional outcomes. Our aim was to describe functional outcome measures in preclinical stroke and vascular cognitive impairment (VCI) studies. From 14 high impact journals (January 2005-December 2015 inclusive), 91,956 papers were screened with 1302 full texts analyzed for stroke (ischemic and hemorrhagic) and 56 for VCI studies. In total, 636 (49%) stroke and 37 (66%) VCI papers reported functional outcome measures. There were 74 different functional assessments reported in stroke and 20 in VCI studies. Neurological deficit scores (74%) and Morris water maze (60%) were most commonly used in stroke and VCI, respectively. However, inconsistencies in methods used to assess and score recovery were noted. Neurological and behavioural functional outcome measures are increasingly used in preclinical stroke or VCI studies; however, there is substantial variation in methods. A strict standardized outcome set may not be suitable for translational work, but greater consistency in choice, application and reporting of outcomes may improve the science.

A Unifying Mechanism for Mitochondrial Superoxide Production during Ischemia-Reperfusion Injury.

Ischemia-reperfusion (IR) injury occurs when blood supply to an organ is disrupted--ischemia--and then restored--reperfusion--leading to a burst of reactive oxygen species (ROS) from mitochondria. It has been tacitly assumed that ROS production during IR is a non-specific consequence of oxygen interacting with dysfunctional mitochondria upon reperfusion. Recently, this view has changed, suggesting that ROS production during IR occurs by a defined mechanism. Here we survey the metabolic factors underlying IR injury and propose a unifying mechanism for its causes that makes sense of the huge amount of disparate data in this area and provides testable hypotheses and new directions for therapies.

In vivo biopanning: A methodological approach to identifying novel targeting ligands for delivery of biological agents to the vasculature.

In order for gene delivery to be clinically acceptable, a number of crucial developments need to be made to existing vectors. Significant advances have been made in the identification of novel platform vectors that possess modified tropism to the native vector, directing infectivity away from nontarget tissues such as the liver. In order to fully optimize these detargeted platform vectors, they need to be retargeted toward a chosen, defined site, which will be defined according to the disease studied. The successful transition of targeting peptides identified using in vitro screening protocols to an in vivo disease model may be compromized by the complexity of delivery into an intact biological system. To this end, peptides identified using in vivo biopanning may prove to be of greater clinical significance given that they were identified in the disease model of choice and so should translate more successfully to the intact preclinical model. Exploitation of the heterogeneity of the vascular endothelium using such an approach will go a long way toward improving the efficiency and achieving site-specific gene expression, with important clinical implications for the systemic application of gene delivery.

Differential expression of microRNA-206 and its target genes in preeclampsia.

OBJECTIVES: Preeclampsia is a multisystem disease that significantly contributes to maternal and foetal morbidity and mortality. In this study, we used a nonbiased microarray approach to identify novel circulating miRNAs in maternal plasma that may be associated with preeclampsia. METHODS: Plasma samples were obtained at 16 and 28 weeks of gestation from 18 women who later developed preeclampsia (cases) and 18 matched women with normotensive pregnancies (controls). We studied miRNA expression profiles in plasma and subsequently confirmed miRNA and target gene expression in placenta samples. Placental samples were obtained from an independent cohort of 19 women with preeclampsia matched with 19 women with normotensive pregnancies. RESULTS: From the microarray, we identified one miRNA that was significantly differentially expressed between cases and controls at 16 weeks of gestation and six miRNAs that were significantly differentially expressed at 28 weeks. Following qPCR validation, only one miR-206 was found to be significantly increased in 28-week samples in women who later developed preeclampsia (1.4-fold change ±â€Š0.2). The trend for increase in miR-206 expression was mirrored within placental tissue from women with preeclampsia. In parallel, IGF-1, a target gene of miR-206, was also found to be downregulated (0.41 ±â€Š0.04) in placental tissue from women with preeclampsia. miR-206 expression was also detectable in myometrium tissue and trophoblast cell lines. CONCLUSION: Our pilot study has identified miRNA-206 as a novel factor upregulated in preeclampsia within the maternal circulation and in placental tissue.

Adenoviral serotype 5 vectors pseudotyped with fibers from subgroup D show modified tropism in vitro and in vivo.

Adenovirus (Ad5) serotype 5 vectors are commonly used for gene transfer. Preclinical studies have shown that their application to systemic gene delivery, however, is limited by their highly efficient uptake in the liver, principally mediated by receptor-binding sites on the fiber shaft and knob domain. Using Ad to target other sites in vivo requires vectors that lack hepatic tropism. We therefore sought to exploit Ad family diversity to isolate vectors that possessed poor hepatic tropism. We pseudotyped the fibers from Ad16 (subgroup B; Ad5/16), Ad19p (subgroup D; Ad5/19p), and Ad37 (subgroup D; Ad5/37) onto Ad5 capsids and assessed infectivity profiles in vitro in multiple cell types and in vivo in rats. In rat, mouse, and human hepatocytes, Ad5/19p and Ad5/37 both possessed a striking lack of hepatic cell infectivity compared with Ad5. Both vectors were, however, able to transduce human vascular endothelial and smooth muscle cells with efficiencies equal to or greater than that of nonmodified Ad5. We evaluated liver uptake in 12-week-old male rats after intravenous injection. In contrast to a vector with the wild-type Ad5 fiber, Ad5, both Ad5/19p and Ad5/37 produced significantly less virion accumulation (measured at 1 hr and 5 days) and transgene expression in the liver. Thus, Ad5/19p and Ad5/37 may be useful platforms for the development of targeted Ad vectors.