Publications by authors named "Megan A Evans"

21 Publications

  • Page 1 of 1

Large-Scale Multivariate Analysis to Interrogate an Animal Model of Stroke: Novel Insights Into Poststroke Pathology.

Stroke 2021 Nov 8;52(11):3661-3669. Epub 2021 Oct 8.

Clinical Trials, Imaging and Informatics Division, Stroke and Ageing Research, Department of Medicine, School of Clinical Sciences at Monash Health (H.M., T.G.P.), Monash University, Clayton, Victoria, Australia.

[Figure: see text].
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http://dx.doi.org/10.1161/STROKEAHA.121.036500DOI Listing
November 2021

Anomalous Aortic Origin of a Coronary Artery Repair Through an Anterior Minithoracotomy.

Innovations (Phila) 2021 Sep-Oct;16(5):480-484. Epub 2021 Aug 1.

3065 Department of Surgery, Duke University Hospitals, Durham, NC, USA.

Objective: The benefits of minimally invasive adult cardiac surgery are well established. Nevertheless, minimally invasive congenital cardiac procedures, even for adult patients, are uncommon. In 2018, we started repairing anomalous aortic origin of a coronary artery (AAOCA) through a 5 cm anterior minithoracotomy when possible to improve cosmesis and avoid sternal precautions. We hypothesized this approach was safe and reliable.

Methods: A 5 cm incision was made in the right second intercostal space. The incision was carried down to the pericardium while preserving the internal mammary artery. With the pericardium in view, the second and third ribs were disarticulated. Central cardiopulmonary bypass was established, and the repair was carried out based on the patient's anatomy. The technique was modified to a left anterior minithoracotomy for 1 patient who required pulmonary artery translocation. At any point, if the dissection or repair was not progressing appropriately, the minimally invasive exposure was converted to a partial or traditional median sternotomy.

Results: Between June 2018 and June 2019, 11 patients underwent minimally invasive anomalous coronary repair. Four patients (3 with body mass index >30) were converted to traditional sternotomy due to poor visualization. Postoperatively, 1 patient required coronary artery bypass after 335 days, due to extensive collaterals and stable angina. Otherwise, at a median follow-up of 437 days (IQR 340 to 480), patients had resumed baseline activity without recurrent symptoms.

Conclusions: Minimally invasive AAOCA repair may be appealing, although surgeons should be cautious given the high conversion rate.
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http://dx.doi.org/10.1177/15569845211031541DOI Listing
November 2021

The Cancer Therapy-Related Clonal Hematopoiesis Driver Gene Promotes Inflammation and Non-Ischemic Heart Failure in Mice.

Circ Res 2021 Sep 28;129(6):684-698. Epub 2021 Jul 28.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA (Y.Y., E.M.-Y., K.-D.M., N.C., A.H.P., H.O., K.H., H.D., M.A.E., M.S., Y.W., S.S., K.W.).

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http://dx.doi.org/10.1161/CIRCRESAHA.121.319314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8409899PMC
September 2021

Murine models of clonal hematopoiesis to assess mechanisms of cardiovascular disease.

Cardiovasc Res 2021 Jun 23. Epub 2021 Jun 23.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Clonal hematopoiesis (CH) is a phenomenon whereby somatic mutations confer a fitness advantage to hematopoietic stem and progenitor cells (HSPC) and thus facilitate their aberrant clonal expansion. These mutations are carried into progeny leukocytes leading to a situation whereby a substantial fraction of an individual's blood cells originate from the HSPC mutant clone. Although this condition rarely progresses to a hematological malignancy, circulating blood cells bearing the mutation have the potential to affect other organ systems as they infiltrate into tissues under both homeostatic and disease conditions. Epidemiological and clinical studies have revealed that CH is highly prevalent in the elderly and is associated with an increased risk of cardiovascular disease and mortality. Recent experimental studies in murine models have assessed the most commonly mutated "driver" genes associated with CH, and have provided evidence for mechanistic connections between CH and cardiovascular disease. A deeper understanding of the mechanisms by which specific CH mutations promote disease pathogenesis is of importance, as it could pave the way for individualized therapeutic strategies targeting the pathogenic CH gene mutations in the future. Here, we review the epidemiology of CH and the mechanistic work from studies using murine disease models, with a particular focus on the strengths and limitations of these experimental systems. We intend for this review to help investigators select the most appropriate models to study CH in the setting of cardiovascular disease.
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http://dx.doi.org/10.1093/cvr/cvab215DOI Listing
June 2021

Bone Marrow Transplantation Procedures in Mice to Study Clonal Hematopoiesis.

J Vis Exp 2021 05 26(171). Epub 2021 May 26.

Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine; Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine;

Clonal hematopoiesis is a prevalent age-associated condition that results from the accumulation of somatic mutations in hematopoietic stem and progenitor cells (HSPCs). Mutations in driver genes, that confer cellular fitness, can lead to the development of expanding HSPC clones that increasingly give rise to progeny leukocytes harboring the somatic mutation. Because clonal hematopoiesis has been associated with heart disease, stroke, and mortality, the development of experimental systems that model these processes is key to understanding the mechanisms that underly this new risk factor. Bone marrow transplantation procedures involving myeloablative conditioning in mice, such as total-body irradiation (TBI), are commonly employed to study the role of immune cells in cardiovascular diseases. However, simultaneous damage to the bone marrow niche and other sites of interest, such as the heart and brain, is unavoidable with these procedures. Thus, our lab has developed two alternative methods to minimize or avoid possible side effects caused by TBI: 1) bone marrow transplantation with irradiation shielding and 2) adoptive BMT to non-conditioned mice. In shielded organs, the local environment is preserved allowing for the analysis of clonal hematopoiesis while the function of resident immune cells is unperturbed. In contrast, the adoptive BMT to non-conditioned mice has the additional advantage that both the local environments of the organs and the hematopoietic niche are preserved. Here, we compare three different hematopoietic cell reconstitution approaches and discuss their strengths and limitations for studies of clonal hematopoiesis in cardiovascular disease.
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http://dx.doi.org/10.3791/61875DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8439117PMC
May 2021

A Single-Cell Analysis of DNMT3A-Mediated Clonal Hematopoiesis in Heart Failure.

Circ Res 2021 01 21;128(2):229-231. Epub 2021 Jan 21.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, VA.

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http://dx.doi.org/10.1161/CIRCRESAHA.120.318575DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7839253PMC
January 2021

Clonal haematopoiesis and cardiovascular disease: how low can you go?

Eur Heart J 2021 01;42(3):266-268

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, 415 Lane Road, Charlottesville, VA 22908, USA.

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http://dx.doi.org/10.1093/eurheartj/ehaa848DOI Listing
January 2021

Tet2-mediated clonal hematopoiesis in nonconditioned mice accelerates age-associated cardiac dysfunction.

JCI Insight 2020 03 26;5(6). Epub 2020 Mar 26.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Clonal hematopoiesis of indeterminate potential is prevalent in elderly individuals and associated with increased risks of all-cause mortality and cardiovascular disease. However, mouse models to study the dynamics of clonal hematopoiesis and its consequences on the cardiovascular system under homeostatic conditions are lacking. We developed a model of clonal hematopoiesis using adoptive transfer of unfractionated ten-eleven translocation 2-mutant (Tet2-mutant) bone marrow cells into nonirradiated mice. Consistent with age-related clonal hematopoiesis observed in humans, these mice displayed a progressive expansion of Tet2-deficient cells in multiple hematopoietic stem and progenitor cell fractions and blood cell lineages. The expansion of the Tet2-mutant fraction was also observed in bone marrow-derived CCR2+ myeloid cell populations within the heart, but there was a negligible impact on the yolk sac-derived CCR2- cardiac-resident macrophage population. Transcriptome profiling revealed an enhanced inflammatory signature in the donor-derived macrophages isolated from the heart. Mice receiving Tet2-deficient bone marrow cells spontaneously developed age-related cardiac dysfunction characterized by greater hypertrophy and fibrosis. Altogether, we show that Tet2-mediated hematopoiesis contributes to cardiac dysfunction in a nonconditioned setting that faithfully models human clonal hematopoiesis in unperturbed bone marrow. Our data support clinical findings that clonal hematopoiesis per se may contribute to diminished health span.
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http://dx.doi.org/10.1172/jci.insight.135204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7213793PMC
March 2020

Genetics of age-related clonal hematopoiesis and atherosclerotic cardiovascular disease.

Curr Opin Cardiol 2020 05;35(3):219-225

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia, USA.

Purpose Of Review: While advanced age is the major risk factor for the development of atherosclerotic cardiovascular disease (ASCVD), we have a poor understanding of how aging promotes the progression of this disease. Recent evidence suggests that the age-dependent accumulation of somatic mutations in hematopoietic cells may represent a new causal risk factor for ASCVD.

Recent Findings: A hallmark of aging is the accumulation of somatic DNA mutations in all tissues of the body. Accordingly, evidence shows that hematopoietic stem/progenitor cells accumulate somatic mutations as a function of age in nonsymptomatic individuals. When these mutations occur in driver genes that provide a selective advantage to the hematopoietic stem/progenitor cells, they undergo a clonal expansion and progressively give rise to blood leukocytes that harbor these mutations. This phenomenon, referred to as clonal hematopoiesis, has been associated with the increased risk of mortality, hematologic malignancy, ASCVD, and related diseases. Notably, many individuals exhibiting clonal hematopoiesis carry single 'driver' mutations in preleukemic genes including DNA methyltransferase 3a, ten-eleven translocation 2, additional sex combs like 1, and Janus kinase 2. Experimental studies show that these mutations in some of these genes can alter the inflammatory properties of the leukocyte and contribute to the pathogenesis of ASCVD.

Summary: We review recent epidemiological and experimental findings on the association between age-related clonal hematopoiesis and ASCVD by focusing on prevalent driver gene mutations.
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http://dx.doi.org/10.1097/HCO.0000000000000726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7138732PMC
May 2020

Cardiovascular Disease, Aging, and Clonal Hematopoiesis.

Annu Rev Pathol 2020 01 5;15:419-438. Epub 2019 Nov 5.

Hematovascular Biology Center and the Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA; email:

Traditional risk factors are incompletely predictive of cardiovascular disease development, a leading cause of death in the elderly. Recent epidemiological studies have shown that human aging is associated with an increased frequency of somatic mutations in the hematopoietic system, which provide a competitive advantage to a mutant cell, thus allowing for its clonal expansion, a phenomenon known as clonal hematopoiesis. Unexpectedly, these mutations have been associated with a higher incidence of cardiovascular disease, suggesting a previously unrecognized connection between somatic mutations in hematopoietic cells and cardiovascular disease. Here, we provide an up-to-date review of clonal hematopoiesis and its association with aging and cardiovascular disease. We also give a detailed report of the experimental studies that have been instrumental in understanding the relationship between clonal hematopoiesis and cardiovascular disease and have shed light on the mechanisms by which hematopoietic somatic mutations contribute to disease pathology.
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http://dx.doi.org/10.1146/annurev-pathmechdis-012419-032544DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7104598PMC
January 2020

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models.

J Vis Exp 2019 10 3(152). Epub 2019 Oct 3.

Hematovascular Biology Center, Robert M. Berne Cardiovascular Research Center, University of Virginia School of Medicine;

Manipulating genes in hematopoietic stem cells using conventional transgenesis approaches can be time-consuming, expensive, and challenging. Benefiting from advances in genome editing technology and lentivirus-mediated transgene delivery systems, an efficient and economical method is described here that establishes mice in which genes are manipulated specifically in hematopoietic stem cells. Lentiviruses are used to transduce Cas9-expressing lineage-negative bone marrow cells with a guide RNA (gRNA) targeting specific genes and a red fluorescence reporter gene (RFP), then these cells are transplanted into lethally-irradiated C57BL/6 mice. Mice transplanted with lentivirus expressing non-targeting gRNA are used as controls. Engraftment of transduced hematopoietic stem cells are evaluated by flow cytometric analysis of RFP-positive leukocytes of peripheral blood. Using this method, ~90% transduction of myeloid cells and ~70% of lymphoid cells at 4 weeks after transplantation can be achieved. Genomic DNA is isolated from RFP-positive blood cells, and portions of the targeted site DNA are amplified by PCR to validate the genome editing. This protocol provides a high-throughput evaluation of hematopoiesis-regulatory genes and can be extended to a variety of mouse disease models with hematopoietic cell involvement.
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http://dx.doi.org/10.3791/59977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7249700PMC
October 2019

microRNA-367-3p regulation of GPRC5A is suppressed in ischemic stroke.

J Cereb Blood Flow Metab 2020 06 11;40(6):1300-1315. Epub 2019 Jul 11.

Department of Medicine, Vanderbilt University Medical Center, Nashville, TN, USA.

Ischemic stroke is a major cause of mortality and long-term disability with limited treatment options, and a greater understanding of the gene regulatory mechanisms underlying ischemic stroke-associated neuroinflammation is required for new therapies. To study ischemic stroke , mice were subjected to sustained ischemia by intraluminal filament-induced middle cerebral artery occlusion (MCAo) for 24 h without reperfusion or transient ischemia for 30 min followed by 23.5 h reperfusion, and brain miRNA and mRNA expression changes were quantified by TaqMan OpenArrays and gene (mRNA) expression arrays, respectively. Sustained ischemia resulted in 18 significantly altered miRNAs and 392 altered mRNAs in mouse brains compared to Sham controls; however, the transient ischemic condition was found to impact only 6 miRNAs and 126 mRNAs. miR-367-3p was found to be significantly decreased in brain homogenates with sustained ischemia. G protein-coupled receptor, family C, group 5, member A (), a miR-367-3p target gene, was found to be significantly increased with sustained ischemia. In primary neurons, inhibition of endogenous miR-367-3p resulted in a significant increase in expression. Moreover, miR-367-3p was found to be co-expressed with in human neurons. Results suggest that loss of miR-367-3p suppression of GPRC5A may contribute to neuroinflammation associated with ischemic stroke.
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http://dx.doi.org/10.1177/0271678X19858637DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238381PMC
June 2020

IL-33 modulates inflammatory brain injury but exacerbates systemic immunosuppression following ischemic stroke.

JCI Insight 2018 09 20;3(18). Epub 2018 Sep 20.

Cardiovascular Disease Program, Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Clayton, Victoria, Australia.

Stroke triggers a complex inflammatory process in which the balance between pro- and antiinflammatory mediators is critical for the development of the brain infarct. However, systemic changes may also occur in parallel with brain inflammation. Here we demonstrate that administration of recombinant IL-33, a recently described member of the IL-1 superfamily of cytokines, promotes Th2-type effects following focal ischemic stroke, resulting in increased plasma levels of Th2-type cytokines and fewer proinflammatory (3-nitrotyrosine+F4/80+) microglia/macrophages in the brain. These effects of IL-33 were associated with reduced infarct size, fewer activated microglia and infiltrating cytotoxic (natural killer-like) T cells, and more IL-10-expressing regulatory T cells. Despite these neuroprotective effects, mice treated with IL-33 displayed exacerbated post-stroke lung bacterial infection in association with greater functional deficits and mortality at 24 hours. Supplementary antibiotics (gentamicin and ampicillin) mitigated these systemic effects of IL-33 after stroke. Our findings highlight the complex nature of the inflammatory mechanisms differentially activated in the brain and periphery during the acute phase after ischemic stroke. The data indicate that a Th2-promoting agent can provide neuroprotection without adverse systemic effects when given in combination with antibiotics.
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http://dx.doi.org/10.1172/jci.insight.121560DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237219PMC
September 2018

Amnion epithelial cells - a novel therapy for ischemic stroke?

Neural Regen Res 2018 Aug;13(8):1346-1349

Department of Physiology, Anatomy & Microbiology, La Trobe University, Melbourne, Australia.

Stroke is a leading cause of death and disability and new therapies are desperately needed. Given the complex nature of ischemic brain injury, it has been postulated that cell-based therapies may be useful. However, cell resources, invasive extraction procedures, immunological rejection, tumorigenesis and ethical challenges make it unlikely that many stem cell types could serve as a practical source for therapy. By contrast, these issues do not pertain to human amnion epithelial cells (hAECs), which are placenta-derived stem cells. We recently assessed the effects of systemically delivered hAECs on stroke outcome using four animal models of stroke. We demonstrated that when injected intravenously after ischemia onset, hAECs migrate preferentially to the spleen and injured brain to limit apoptosis and inflammation, and attenuate early brain infiltration of immune cells, progression of infarction and systemic immunosuppression and to ultimately ameliorate functional deficits. When administration of hAECs is delayed by 1-3 days post-stroke, long-term functional recovery can still be enhanced in young and aged mice of either sex. Moreover, our proof-of-principle findings suggest that hAECs are effective at limiting post-stroke infarct development in non-human primates. Overall, the results suggest that hAECs could be a viable clinical stroke therapy.
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http://dx.doi.org/10.4103/1673-5374.235223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6108203PMC
August 2018

Vitamin D Supplementation Reduces Subsequent Brain Injury and Inflammation Associated with Ischemic Stroke.

Neuromolecular Med 2018 03 23;20(1):147-159. Epub 2018 Feb 23.

Vascular Biology Immunopharmacology Group, Department of Physiology, Anatomy and Microbiology, La Trobe University, Bundoora, VIC, 3083, Australia.

Acute inflammation can exacerbate brain injury after ischemic stroke. Beyond its well-characterized role in calcium metabolism, it is becoming increasingly appreciated that the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-VitD), has potent immunomodulatory properties. Here, we aimed to determine whether 1,25-VitD supplementation could reduce subsequent brain injury and associated inflammation after ischemic stroke. Male C57Bl6 mice were randomly assigned to be administered either 1,25-VitD (100 ng/kg/day) or vehicle i.p. for 5 day prior to stroke. Stroke was induced via middle cerebral artery occlusion for 1 h followed by 23 h reperfusion. At 24 h post-stroke, we assessed infarct volume, functional deficit, expression of inflammatory mediators and numbers of infiltrating immune cells. Supplementation with 1,25-VitD reduced infarct volume by 50% compared to vehicle. Expression of pro-inflammatory mediators IL-6, IL-1β, IL-23a, TGF-β and NADPH oxidase-2 was reduced in brains of mice that received 1,25-VitD versus vehicle. Brain expression of the T regulatory cell marker, Foxp3, was higher in mice supplemented with 1,25-VitD versus vehicle, while expression of the transcription factor, ROR-γ, was decreased, suggestive of a reduced Th17/γδ T cell response. Immunohistochemistry indicated that similar numbers of neutrophils and T cells were present in the ischemic hemispheres of 1,25-VitD- and vehicle-supplemented mice. At this early time point, there were also no differences in the impairment of motor function. These data indicate that prior administration of exogenous vitamin D, even to vitamin D-replete mice, can attenuate infarct development and exert acute anti-inflammatory actions in the ischemic and reperfused brain.
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http://dx.doi.org/10.1007/s12017-018-8484-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834596PMC
March 2018

Acute or Delayed Systemic Administration of Human Amnion Epithelial Cells Improves Outcomes in Experimental Stroke.

Stroke 2018 03 30;49(3):700-709. Epub 2018 Jan 30.

From the Departments of Pharmacology (M.A.E., H.A.K., H.X.C., C.V.G.-M., K.W.E.T., C.T.C., V.H.B., S.L., Q.N.D., A.V., G.R.D., C.G.S., B.R.S.B.), Obstetrics and Gynaecology (R.L., E.M.W.), Surgery (A.V., G.R.D., C.G.S.), and Medicine (T.G.P., V.K.S.), Australian Regenerative Medicine Institute (W.K., L.T., J.A.B.), and Monash Institute of Pharmaceutical Sciences (C.P.J.H., C.W.P., J.M.H.), Monash University, Victoria, Australia; Department of Physiology, Anatomy and Microbiology, La Trobe University, Victoria, Australia (M.A.E., H.A.K., Q.N.D., A.V., G.R.D., C.G.S.); The Ritchie Centre, Hudson Institute of Medical Research, Victoria, Australia (R.L., E.M.W.); Stroke Unit (T.G.P., V.K.S., H.M.) and Monash Women's Services (E.M.W.), Monash Health, Victoria, Australia; Menzies Research Institute, Tasmania, Australia (V.K.S.); Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore (T.V.A., D.Y.F., L.P.); School of Pharmacy, Sungkyunkwan University, Seoul, South Korea (T.V.A.); School of Health and Biomedical Sciences, RMIT University, Victoria, Australia (S.S.); Department of Anatomy, Brain Health Research Centre and Brain Research New Zealand (S.N., E.K.G., A.N.C.) and Department of Pathology (S.N.;S.Y.;A.N.C.), University of Otago, Dunedin, New Zealand; and Faculty of Pharmacy, University of Sydney, NSW, Australia (A.N.C.).

Background And Purpose: Human amnion epithelial cells (hAECs) are nonimmunogenic, nontumorigenic, anti-inflammatory cells normally discarded with placental tissue. We reasoned that their profile of biological features, wide availability, and the lack of ethical barriers to their use could make these cells useful as a therapy in ischemic stroke.

Methods: We tested the efficacy of acute (1.5 hours) or delayed (1-3 days) poststroke intravenous injection of hAECs in 4 established animal models of cerebral ischemia. Animals included young (7-14 weeks) and aged mice (20-22 months) of both sexes, as well as adult marmosets of either sex.

Results: We found that hAECs administered 1.5 hours after stroke in mice migrated to the ischemic brain via a CXC chemokine receptor type 4-dependent mechanism and reduced brain inflammation, infarct development, and functional deficits. Furthermore, if hAECs administration was delayed until 1 or 3 days poststroke, long-term functional recovery was still augmented in young and aged mice of both sexes. We also showed proof-of-principle evidence in marmosets that acute intravenous injection of hAECs prevented infarct development from day 1 to day 10 after stroke.

Conclusions: Systemic poststroke administration of hAECs elicits marked neuroprotection and facilitates mechanisms of repair and recovery.
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http://dx.doi.org/10.1161/STROKEAHA.117.019136DOI Listing
March 2018

Diet-induced vitamin D deficiency has no effect on acute post-stroke outcomes in young male mice.

J Cereb Blood Flow Metab 2018 11 23;38(11):1968-1978. Epub 2017 Aug 23.

1 Cardiovascular Disease Program and Department of Pharmacology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria, Australia.

Recent observational studies have reported that patients with low circulating levels of vitamin D experience larger infarct volumes and worse functional outcomes after ischemic stroke compared to those with sufficient levels. However, it is unknown whether a causal relationship exists between low vitamin D levels and poor stroke outcome. This study aimed to assess the effect of vitamin D deficiency on acute outcomes post-stroke. Male C57Bl6 mice (six week old) were assigned to either a control or vitamin D deficient diet for four weeks prior to stroke. Stroke was induced by 1 h middle cerebral artery occlusion (MCAO) with reperfusion. At 24 h, we assessed functional outcomes, infarct volume, quantified immune cells in the brain by immunofluorescence and examined susceptibility to lung infection. ELISAs showed that the plasma level of hydroxyvitamin D was 85% lower in mice fed the vitamin D-deficient diet compared with the control group. Despite this, vitamin D deficiency had no impact on functional outcomes or infarct volume after stroke. Further, there were no differences in the numbers of infiltrating immune cells or bacterial load within the lungs. These data suggest that diet-induced vitamin D deficiency has no effect on acute post-stroke outcomes.
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http://dx.doi.org/10.1177/0271678X17719208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6259312PMC
November 2018

Aldosterone-induced oxidative stress and inflammation in the brain are mediated by the endothelial cell mineralocorticoid receptor.

Brain Res 2016 Apr 26;1637:146-153. Epub 2016 Feb 26.

Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Building 13E, Monash University, Wellington Rd, Clayton, Victoria 3800, Australia. Electronic address:

Elevated aldosterone levels, which promote cerebral vascular oxidative stress, inflammation, and endothelial dysfunction, may increase stroke risk, independent of blood pressure and other risk factors. The main target receptor of aldosterone, the mineralocorticoid receptor (MR), is expressed in many cell types, including endothelial cells. Endothelial cell dysfunction is thought to be an initiating step contributing to cardiovascular disease and stroke; however the importance of MR expressed on endothelial cells in the brain is unknown. Here we have examined whether endothelial cell MR mediates cerebral vascular oxidative stress and brain inflammation during aldosterone excess. In male mice, aldosterone (0.72 mg/kg/day, 14 days) caused a small increase (~14 mmHg) in blood pressure. The MR blocker spironolactone (25 mg/kg/d, ip) abolished this increase, whereas endothelial cell MR-deficiency had no effect. Aldosterone increased superoxide production capacity in cerebral arteries, and also mRNA expression of the pro-inflammatory cytokines chemokine (C-C motif) ligand 7 (CCL7), CCL8 and interleukin (IL)-1β in the brain. These increases were prevented by both spironolactone treatment and endothelial cell MR-deficiency; whereas IL-1β expression was blocked by spironolactone only. Endothelial cell MR mediates aldosterone-induced increases in cerebrovascular superoxide levels and chemokine expression in the brain, but not blood pressure or brain IL-1β. Endothelial cell-targeted MR antagonism may represent a novel approach to treat cerebrovascular disease and stroke, particularly during conditions of aldosterone excess.
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http://dx.doi.org/10.1016/j.brainres.2016.02.034DOI Listing
April 2016

Effect of a Selective Mas Receptor Agonist in Cerebral Ischemia In Vitro and In Vivo.

PLoS One 2015 5;10(11):e0142087. Epub 2015 Nov 5.

Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria, 3800, Australia.

Functional modulation of the non-AT1R arm of the renin-angiotensin system, such as via AT2R activation, is known to improve stroke outcome. However, the relevance of the Mas receptor, which along with the AT2R forms the protective arm of the renin-angiotensin system, as a target in stroke is unclear. Here we tested the efficacy of a selective MasR agonist, AVE0991, in in vitro and in vivo models of ischemic stroke. Primary cortical neurons were cultured from E15-17 mouse embryos for 7-9 d, subjected to glucose deprivation for 24 h alone or with test drugs, and percentage cell death was determined using trypan blue exclusion assay. Additionally, adult male mice were subjected to 1 h middle cerebral artery occlusion and were administered either vehicle or AVE0991 (20 mg/kg i.p.) at the commencement of 23 h reperfusion. Some animals were also treated with the MasR antagonist, A779 (80 mg/kg i.p.) 1 h prior to surgery. Twenty-four h after MCAo, neurological deficits, locomotor activity and motor coordination were assessed in vivo, and infarct and edema volumes estimated from brain sections. Following glucose deprivation, application of AVE0991 (10-8 M to 10-6 M) reduced neuronal cell death by ~60% (P<0.05), an effect prevented by the MasR antagonist. By contrast, AVE0991 administration in vivo had no effect on functional or histological outcomes at 24 h following stroke. These findings indicate that the classical MasR agonist, AVE0991, can directly protect neurons from injury following glucose-deprivation. However, this effect does not translate into an improved outcome in vivo when administered systemically following stroke.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0142087PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634944PMC
June 2016

Sex-dependent effects of G protein-coupled estrogen receptor activity on outcome after ischemic stroke.

Stroke 2014 Mar 23;45(3):835-41. Epub 2014 Jan 23.

From the Vascular Biology and Immunopharmacology Group, Department of Pharmacology, Monash University, Clayton, Victoria, Australia (B.R.S.B., V.H.B., H.A.K., S.L., H.X.C., C.V.G.-M., E.G., M.A.E., A.A.M., G.R.D., C.G.S.); and School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia (T.V.A.).

Background And Purpose: Experimental studies indicate that estrogen typically, but not universally, has a neuroprotective effect in stroke. Ischemic stroke increases membrane-bound G protein-coupled estrogen receptor (GPER) distribution and expression in the brain of male but not female mice. We hypothesized that GPER activation may have a greater neuroprotective effect in males than in females after stroke.

Methods: Vehicle (dimethyl sulfoxide), a GPER agonist (G-1, 30 μg/kg), or a GPER antagonist (G-15, 300 μg/kg) were administered alone or in combination to young or aged male mice, or young intact or ovariectomized female mice, 1 hour before or 3 hours after cerebral ischemia-reperfusion. Some mice were treated with a combination of G-1 and the pan-caspase inhibitor, quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh), 1 hour before stroke. We evaluated functional and histological end points of stroke outcome up to 72 hours after ischemia-reperfusion. In addition, apoptosis was examined using cleaved caspase-3 immunohistochemistry.

Results: Surprisingly, G-1 worsened functional outcomes and increased infarct volume in males poststroke, in association with an increased expression of cleaved caspase-3 in peri-infarct neurons. These effects were blocked by G-15 or Q-VD-OPh. Conversely, G-15 improved functional outcomes and reduced infarct volume after stroke in males, whether given before or after stroke. In contrast to findings in males, G-1 reduced neurological deficit, apoptosis, and infarct volume in ovariectomized females, but had no significant effect in intact females.

Conclusions: Future therapies for acute stroke could exploit the modulation of GPER activity in a sex-specific manner.
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http://dx.doi.org/10.1161/STROKEAHA.113.001499DOI Listing
March 2014

Neuroprotective effect of an angiotensin receptor type 2 agonist following cerebral ischemia in vitro and in vivo.

Exp Transl Stroke Med 2012 Aug 24;4(1):16. Epub 2012 Aug 24.

Department of Pharmacology, Monash University, Clayton, VIC, 3800, Australia.

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Background: Intracerebral administration of the angiotensin II type 2 receptor (AT2R) agonist, CGP42112, is neuroprotective in a rat model of ischemic stroke. To explore further its possible cellular target(s) and therapeutic utility, we firstly examined whether CGP42112 may exert direct protective effects on primary neurons following glucose deprivation in vitro. Secondly, we tested whether CGP42112 is effective when administered systemically in a mouse model of cerebral ischemia.

Methods: Primary cortical neurons were cultured from E17 C57Bl6 mouse embryos for 9 d, exposed to glucose deprivation for 24 h alone or with drug treatments, and percent cell survival assessed using trypan blue exclusion. Ischemic stroke was induced in adult male C57Bl6 mice by middle cerebral artery occlusion for 30 min, followed by reperfusion for 23.5 h. Neurological assessment was performed and then mice were euthanized and infarct and edema volume were analysed.

Results: During glucose deprivation, CGP42112 (1x10-8 M and 1x10-7 M) reduced cell death by ~30%, an effect that was prevented by the AT2R antagonist, PD123319 (1x10-6 M). Neuroprotection by CGP42112 was lost at a higher concentration (1x10-6 M) but was unmasked by co-application with the AT1R antagonist, candesartan (1x10-7 M). By contrast, Compound 21 (1x10-8 M to 1x10-6 M), a second AT2R agonist, had no effect on neuronal survival. Mice treated with CGP42112 (1 mg/kg i.p.) after cerebral ischemia had improved functional outcomes over vehicle-treated mice as well as reduced total and cortical infarct volumes.

Conclusions: These results indicate that CGP42112 can directly protect neurons from ischemia-like injury in vitro via activation of AT2Rs, an effect opposed by AT1R activation at high concentrations. Furthermore, systemic administration of CGP42112 can reduce functional deficits and infarct volume following cerebral ischemia in vivo.
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http://dx.doi.org/10.1186/2040-7378-4-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3492080PMC
August 2012
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