Post-stroke administration of H2 relaxin reduces functional deficits, neuronal apoptosis and immune cell infiltration into the mouse brain.

Brain inflammation and apoptosis contribute to neuronal damage and loss following ischaemic stroke, leading to cognitive and functional disability. It is well-documented that the human gene-2 (H2)-relaxin hormone exhibits pleiotropic properties via its cognate receptor, Relaxin Family Peptide Receptor 1 (RXFP1), including anti-inflammatory and anti-apoptotic effects, thus making it a potential therapeutic for stroke. Hence, the current study investigated whether post-stroke H2-relaxin administration could improve functional and histological outcomes. 8-12-week-old male C57BL/6 mice were subjected to sham operation or photothrombotic stroke and intravenously-administered with either saline (vehicle) or 0.02, 0.2 or 2 mg/kg doses of recombinant H2-relaxin at 6, 24 and 48 h post-stroke. Motor function was assessed using the hanging wire and cylinder test pre-surgery, and at 24 and 72 h post-stroke. Brains were removed after 72 h and infarct volume was assessed via thionin staining, and RXFP1 expression, leukocyte infiltration and apoptosis were determined by immunofluorescence. RXFP1 was identified on neurons, astrocytes and macrophages, and increased post-stroke. Whilst H2-relaxin did not alter infarct volume, it did cause a dose-dependent improvement in motor function at 24 and 72 h post-stroke. Moreover, 2 mg/kg H2-relaxin significantly decreased the number of apoptotic cells as well as macrophages and neutrophils within the ischaemic hemisphere, but did not alter T or B cells numbers. The anti-inflammatory and anti-apoptotic effects of H2-relaxin when administered at 6 h post-cerebral ischaemia may provide a novel therapeutic option for patients following ischaemic stroke.

Simultaneous late-gadolinium enhancement and T1 mapping of fibrosis and a novel cell-based combination therapy in hypertensive mice.

Fibrosis is a hallmark of chronic hypertension and disrupts the viability of human bone marrow-derived mesenchymal stromal cells (BM-MSCs) post-transplantation. This study thus, determined whether the anti-fibrotic drug, serelaxin (RLX), could enhance the therapeutic effects of BM-MSCs or BM-MSC-derived exosomes (BM-MSC-EXO) in hypertensive mice. Left ventricular (LV) fibrosis in particular was assessed using conventional histological staining and non-invasive cardiac magnetic resonance imaging (CMRI). CMRI was employed using a novel magnetisation prepared 2 rapid acquisition gradient echo (MP2RAGE) sequence to simultaneously perform late gadolinium enhancement imaging and T1 mapping. Adult male C57BL/6 mice were uninephrectomised, received deoxycorticosterone acetate and saline to drink (1 K/DOCA/salt) for 21 days, whilst control mice were given normal drinking water for the same time-period. On day 14 post-injury, subgroups of 1 K/DOCA/salt-hypertensive mice were treated with RLX alone or in combination with BM-MSCs or BM-MSC-EXO; or the mineralocorticoid receptor antagonist, spironolactone. At day 21 post-injury, LV and kidney histopathology was assessed, whilst LV fibrosis and function were additionally analysed by CMRI and echocardiography. 1 K/DOCA/salt-hypertensive mice developed kidney tubular injury, inflammation, fibrosis, and more moderate LV hypertrophy, fibrosis and diastolic dysfunction. RLX and BM-MSCs combined provided optimal protection against these pathologies and significantly reduced picrosirius red-stained organ fibrosis and MP2RAGE analysis of LV fibrosis. A significant correlation between MP2RAGE analysis and histologically-stained interstitial LV fibrosis was detected. It was concluded that the MP2RAGE sequence enhanced the non-invasive CMRI detection of LV fibrosis. Furthermore, combining RLX and BM-MSCs may represent a promising treatment option for hypertensive cardiorenal syndrome.

Comparing the renoprotective effects of BM-MSCs versus BM-MSC-exosomes, when combined with an anti-fibrotic drug, in hypertensive mice.

Fibrosis, a hallmark of chronic kidney disease (CKD), impairs the viability of human bone marrow derived-mesenchymal stromal cells (BM-MSCs) post-transplantation. To address this, we demonstrated that combining BM-MSCs with the anti-fibrotic drug, serelaxin (RLX), enhanced BM-MSC-induced renoprotection in preclinical CKD models. Given the increased interest and manufacturing advantages to using stem cell-derived exosomes (EXO) as therapeutics, this study determined whether RLX could enhance the therapeutic efficacy of BM-MSC-EXO, and compared the renoprotective effects of RLX and BM-MSC-EXO versus RLX and BM-MSCs in mice with hypertensive CKD. Adult male C57BL/6 mice were uninephrectomised, received deoxycorticosterone acetate and given saline to drink (1K/DOCA/salt) for 21 days. Control mice were uninephrectomised and given normal drinking water for the same time-period. Subgroups of 1K/DOCA/salt-hypertensive mice were then treated with either RLX (0.5 mg/kg/day) or BM-MSC-EXO (25 µg/mouse; equivalent to 1-2 × 106 BM-MSCs/mouse) alone; combinations of RLX and BM-MSC-EXO or BM-MSCs (1 × 106/mouse); or the mineralocorticoid receptor antagonist, spironolactone (20 mg/kg/day), from days 14-21. 1K/DOCA/salt-hypertensive mice developed kidney tubular damage, inflammation and fibrosis, and impaired kidney function 21 days post-injury. Whilst RLX alone attenuated the 1K/DOCA/salt-induced fibrosis, BM-MSC-EXO alone only diminished measures of tissue inflammation post-treatment. Comparatively, the combined effects of RLX and BM-MSC-EXO or BM-MSCs demonstrated similar anti-fibrotic efficacy, but RLX and BM-MSCs offered broader renoprotection over RLX and/or BM-MSC-EXO, and comparable effects to spironolactone. Only RLX and BM-MSCs, but not RLX and/or BM-MSC-EXO, also attenuated the 1K/DOCA/salt-induced hypertension. Hence, although RLX improved the renoprotective effects of BM-MSC-EXO, combining RLX with BM-MSCs provided a better therapeutic option for hypertensive CKD.

Combining mesenchymal stem cells with serelaxin provides enhanced renoprotection against 1K/DOCA/salt-induced hypertension.

BACKGROUND AND PURPOSE: Fibrosis is a hallmark of chronic kidney disease (CKD) that significantly contributes to renal dysfunction, and impairs the efficacy of stem cell-based therapies. This study determined whether combining bone marrow-derived mesenchymal stem cells (BM-MSCs) with the renoprotective effects of recombinant human relaxin (serelaxin) could therapeutically reduce renal fibrosis in mice with one kidney/deoxycorticosterone acetate/salt (1K/DOCA/salt)-induced hypertension, compared with the effects of the ACE inhibitor, perindopril. EXPERIMENTAL APPROACH: Adult male C57BL/6 mice were uni-nephrectomised and received deoxycorticosterone acetate and saline to drink (1K/DOCA/salt) for 21 days. Control mice were uni-nephrectomised but received water over the same time period. Sub-groups of 1K/DOCA/salt-injured mice (n = 5-8 per group) were treated with either serelaxin (0.5 mg·kg-1 ·day-1 ) or BM-MSCs (1 × 106 per mouse) alone; both treatments combined (with 0.5 × 106 or 1 × 106 BM-MSCs per mouse); or perindopril (2 mg·kg-1 ·day-1 ) from days 14-21. KEY RESULTS: 1K/DOCA/salt-injured mice developed elevated BP and hypertension-induced renal damage, inflammation and fibrosis. BM-MSCs alone reduced the injury-induced fibrosis and attenuated BP to a similar extent as perindopril. Serelaxin alone modestly reduced renal fibrosis and effectively reduced tubular injury. Strikingly, the combined effects of BM-MSCs (at both doses) with serelaxin significantly inhibited renal fibrosis and proximal tubular epithelial injury while restoring renal architecture, to a greater extent than either therapy alone, and over the effects of perindopril. CONCLUSION AND IMPLICATIONS: Combining BM-MSCs and serelaxin provided broader renoprotection over either therapy alone or perindopril and might represent a novel treatment for hypertensive CKD.

Aldosterone-induced hypertension is sex-dependent, mediated by T cells and sensitive to GPER activation.

AIMS: The G protein-coupled estrogen receptor 1 (GPER) may modulate some effects of aldosterone. In addition, G-1 (a GPER agonist) can lower blood pressure (BP) and promote T cell-mediated anti-inflammatory responses. This study aimed to test the effects of G-1 and G-15 (a GPER antagonist) on aldosterone-induced hypertension in mice and to examine the cellular mechanisms involved. METHODS AND RESULTS: C57Bl/6 (wild-type, WT), RAG1-deficient and GPER-deficient mice were infused with vehicle, aldosterone (0.72 mg/kg/day S.C. plus 0.9% NaCl for drinking) ± G-1 (0.03 mg/kg/day S.C.) ± G-15 (0.3 mg/kg/day S.C.) for 14 days. G-1 attenuated aldosterone-induced hypertension in male WT but not male GPER-deficient mice. G-15 alone did not alter hypertension but it prevented the anti-hypertensive effect of G-1. In intact female WT mice, aldosterone-induced hypertension was markedly delayed and suppressed compared with responses in males, with BP remaining unchanged until after Day 7. In contrast, co-administration of aldosterone and G-15 fully increased BP within 7 days in WT females. Similarly, aldosterone robustly increased BP by Day 7 in ovariectomized WT females, and in both sexes of GPER-deficient mice. Whereas aldosterone had virtually no effect on BP in RAG1-deficient mice, adoptive transfer of T cells from male WT or male GPER-deficient mice into male RAG1-deficient mice restored the pressor response to aldosterone. This pressor effect could be attenuated by G-1 in RAG1-deficient mice that were reconstituted with either WT or GPER-deficient T cells, suggesting that G-1 does not act via T cells to lower BP. CONCLUSION: Our findings indicate that although aldosterone-induced hypertension is largely mediated by T cells, it can be attenuated by activation of GPER on non-T cells, which accounts for the sex difference in sensitivity to the pressor effect.

Amnion epithelial cells - a novel therapy for ischemic stroke?

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.

Vitamin D3 Supplementation Reduces Subsequent Brain Injury and Inflammation Associated with Ischemic Stroke.

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 D3 (1,25-VitD3), has potent immunomodulatory properties. Here, we aimed to determine whether 1,25-VitD3 supplementation could reduce subsequent brain injury and associated inflammation after ischemic stroke. Male C57Bl6 mice were randomly assigned to be administered either 1,25-VitD3 (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-VitD3 reduced infarct volume by 50% compared to vehicle. Expression of pro-inflammatory mediators IL-6, IL-1ß, IL-23a, TGF-ß and NADPH oxidase-2 was reduced in brains of mice that received 1,25-VitD3 versus vehicle. Brain expression of the T regulatory cell marker, Foxp3, was higher in mice supplemented with 1,25-VitD3 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-VitD3- 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.

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

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.

Endosomal NOX2 oxidase exacerbates virus pathogenicity and is a target for antiviral therapy.

The imminent threat of viral epidemics and pandemics dictates a need for therapeutic approaches that target viral pathology irrespective of the infecting strain. Reactive oxygen species are ancient processes that protect plants, fungi and animals against invading pathogens including bacteria. However, in mammals reactive oxygen species production paradoxically promotes virus pathogenicity by mechanisms not yet defined. Here we identify that the primary enzymatic source of reactive oxygen species, NOX2 oxidase, is activated by single stranded RNA and DNA viruses in endocytic compartments resulting in endosomal hydrogen peroxide generation, which suppresses antiviral and humoral signaling networks via modification of a unique, highly conserved cysteine residue (Cys98) on Toll-like receptor-7. Accordingly, targeted inhibition of endosomal reactive oxygen species production abrogates influenza A virus pathogenicity. We conclude that endosomal reactive oxygen species promote fundamental molecular mechanisms of viral pathogenicity, and the specific targeting of this pathogenic process with endosomal-targeted reactive oxygen species inhibitors has implications for the treatment of viral disease.Production of reactive oxygen species is an ancient antimicrobial mechanism, but its role in antiviral defense in mammals is unclear. Here, To et al. show that virus infection activates endosomal NOX2 oxidase and restricts TLR7 signaling, and that an endosomal NOX2 inhibitor decreases viral pathogenicity.

Intranasal administration of mesenchymoangioblast-derived mesenchymal stem cells abrogates airway fibrosis and airway hyperresponsiveness associated with chronic allergic airways disease.

Structural changes known as airway remodeling (AWR) characterize chronic/severe asthma and contribute to lung dysfunction. Thus, we assessed the in vivo efficacy of induced pluripotent stem cell and mesenchymoangioblast-derived mesenchymal stem cells (MCA-MSCs) on AWR in a murine model of chronic allergic airways disease (AAD)/asthma. Female Balb/c mice were subjected to a 9-wk model of ovalbumin (Ova)-induced chronic AAD and treated intravenously or intranasally with MCA-MSCs from weeks 9 to 11. Changes in airway inflammation (AI), AWR, and airway hyperresponsiveness (AHR) were assessed. Ova-injured mice presented with AI, goblet cell metaplasia, epithelial thickening, increased airway TGF-ß1 levels, subepithelial myofibroblast and collagen accumulation, total lung collagen concentration, and AHR (all P < 0.001 vs. uninjured control group). Apart from epithelial thickness, all other parameters measured were significantly, although not totally, decreased by intravenous delivery of MCA-MSCs to Ova-injured mice. In comparison, intranasal delivery of MCA-MSCs to Ova-injured mice significantly decreased all parameters measured (all P < 0.05 vs. Ova group) and, most notably, normalized aberrant airway TGF-ß1 levels, airway/lung fibrosis, and AHR to values measured in uninjured animals. MCA-MSCs also increased collagen-degrading gelatinase levels. Hence, direct delivery of MCA-MSCs offers great therapeutic benefit for the AWR and AHR associated with chronic AAD.-Royce, S. G., Rele, S., Broughton, B. R. S., Kelly, K., Samuel, C. S. Intranasal administration of mesenchymoangioblast-derived mesenchymal stem cells abrogates airway fibrosis and airway hyperresponsiveness associated with chronic allergic airways disease.

Evidence That Ly6C(hi) Monocytes are Protective in Acute Ischemic Stroke by Promoting M2 Macrophage Polarization.

BACKGROUND AND PURPOSE: Ly6C(hi) monocytes are generally thought to exert a proinflammatory role in acute tissue injury, although their impact after injuries to the central nervous system is poorly defined. CC chemokine receptor 2 is expressed on Ly6C(hi) monocytes and plays an essential role in their extravasation and transmigration into the brain after cerebral ischemia. We used a selective CC chemokine receptor 2 antagonist, INCB3344, to assess the effect of Ly6C(hi) monocytes recruited into the brain early after ischemic stroke. METHODS: Male C57Bl/6J mice underwent occlusion of the middle cerebral artery for 1 hour followed by 23 hours of reperfusion. Mice were administered either vehicle (dimethyl sulfoxide/carboxymethylcellulose) or INCB3344 (10, 30 or 100 mg/kg IP) 1 hour before ischemia and at 2 and 6 hours after ischemia. At 24 hours, we assessed functional outcomes, infarct volume, and quantified the immune cells in blood and brain by flow cytometry or immunofluorescence. Gene expression of selected inflammatory markers was assessed by quantitative polymerase chain reaction. RESULTS: Ly6C(hi) monocytes were increased 3-fold in the blood and 10-fold in the brain after stroke, and these increases were selectively prevented by INCB3344 in a dose-dependent manner. Mice treated with INCB3344 exhibited markedly worse functional outcomes and larger infarct volumes, in association with reduced M2 polarization and increased peroxynitrite production in macrophages, compared with vehicle-treated mice. CONCLUSIONS: Our data suggest that Ly6C(hi) monocytes exert an acute protective effect after ischemic stroke to limit brain injury and functional deficit that involves promotion of M2 macrophage polarization.

PI3Kδ inhibition reduces TNF secretion and neuroinflammation in a mouse cerebral stroke model.

Stroke is a major cause of death worldwide and the leading cause of permanent disability. Although reperfusion is currently used as treatment, the restoration of blood flow following ischaemia elicits a profound inflammatory response mediated by proinflammatory cytokines such as tumour necrosis factor (TNF), exacerbating tissue damage and worsening the outcomes for stroke patients. Phosphoinositide 3-kinase delta (PI3Kδ) controls intracellular TNF trafficking in macrophages and therefore represents a prospective target to limit neuroinflammation. Here we show that PI3Kδ inhibition confers protection in ischaemia/reperfusion models of stroke. In vitro, restoration of glucose supply following an episode of glucose deprivation potentiates TNF secretion from primary microglia-an effect that is sensitive to PI3Kδ inhibition. In vivo, transient middle cerebral artery occlusion and reperfusion in kinase-dead PI3Kδ (p110δ(D910A/D910A)) or wild-type mice pre- or post-treated with the PI3Kδ inhibitor CAL-101, leads to reduced TNF levels, decreased leukocyte infiltration, reduced infarct size and improved functional outcome. These data identify PI3Kδ as a potential therapeutic target in ischaemic stroke.

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

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.

Immune cell infiltration in malignant middle cerebral artery infarction: comparison with transient cerebral ischemia.

We tested whether significant leukocyte infiltration occurs in a mouse model of permanent cerebral ischemia. C57BL6/J male mice underwent either permanent (3 or 24 hours) or transient (1 or 2 hours+22- to 23-hour reperfusion) middle cerebral artery occlusion (MCAO). Using flow cytometry, we observed ∼15,000 leukocytes (CD45(+high) cells) in the ischemic hemisphere as early as 3 hours after permanent MCAO (pMCAO), comprising ∼40% lymphoid cells and ∼60% myeloid cells. Neutrophils were the predominant cell type entering the brain, and were increased to ∼5,000 as early as 3 hours after pMCAO. Several cell types (monocytes, macrophages, B lymphocytes, CD8(+) T lymphocytes, and natural killer cells) were also increased at 3 hours to levels sustained for 24 hours, whereas others (CD4(+) T cells, natural killer T cells, and dendritic cells) were unchanged at 3 hours, but were increased by 24 hours after pMCAO. Immunohistochemical analysis revealed that leukocytes typically had entered and widely dispersed throughout the parenchyma of the infarct within 3 hours. Moreover, compared with pMCAO, there were ∼50% fewer infiltrating leukocytes at 24 hours after transient MCAO (tMCAO), independent of infarct size. Microglial cell numbers were bilaterally increased in both models. These findings indicate that a profound infiltration of inflammatory cells occurs in the brain early after focal ischemia, especially without reperfusion.

Enhanced depolarization-induced pulmonary vasoconstriction following chronic hypoxia requires EGFR-dependent activation of NAD(P)H oxidase 2.

AIMS: Chronic hypoxia (CH) enhances depolarization-induced myofilament Ca(2+) sensitization and resultant pulmonary arterial constriction through superoxide (O(2)(-))-dependent stimulation of RhoA. Because NAD(P)H oxidase (NOX) has been implicated in the development of pulmonary hypertension, we hypothesized that vascular smooth muscle (VSM) depolarization increases NOX-derived O(2)(-) production leading to myofilament Ca(2+) sensitization and augmented vasoconstrictor reactivity following CH. As epidermal growth factor receptor (EGFR) mediates Rac1-dependent NOX activation in renal mesangial cells, we further sought to examine the role EGFR plays in this response. RESULTS: Vasoconstrictor responses to depolarizing concentrations of KCl were greater in lungs isolated from CH (4 wk, 0.5 atm) rats compared to normoxic controls, and this effect of CH was abolished by the general NOX inhibitor, apocynin. CH similarly augmented KCl-induced vasoconstriction and O(2)(-) generation (assessed using the fluorescent indicator, dihydroethidium) in Ca(2+)-permeabilized, pressurized small pulmonary arteries. These latter responses to CH were prevented by general inhibition of NOX isoforms (apocynin, diphenylene iodonium), and by selective inhibition of NOX 2 (gp91ds-tat), Rac1 (NSC 23766), and EGFR (AG 1478). Consistent with these observations, CH increased KCl-induced EGFR phosphorylation, and augmented depolarization-induced Rac1 activation in an EGFR-dependent manner. INNOVATION: This study establishes a novel signaling axis in VSM linking membrane depolarization to contraction that is independent of Ca(2+) influx, and which mediates myofilament Ca(2+) sensitization in the hypertensive pulmonary circulation. CONCLUSION: CH augments membrane depolarization-induced pulmonary VSM Ca(2+) sensitization and vasoconstriction through EGFR-dependent stimulation of Rac1 and NOX 2.

Stroke increases g protein-coupled estrogen receptor expression in the brain of male but not female mice.

The novel estrogen receptor, G protein-coupled estrogen receptor (GPER, previously named GPR30), is widely distributed throughout the male and female brain and, thus, could potentially play a role in estrogen-mediated neuroprotective effects in diseases such as stroke. We hypothesized that GPER distribution and expression in the brain of male, intact female, and ovariectomized (OVX) mice is increased after 0.5 h middle cerebral artery occlusion. Using immunohistochemistry, we found that ischemia reperfusion increased GPER distribution in the peri-infarct brain regions of male mice, but surprisingly not in intact females or OVX mice. Similar differences were observed in the male and female human brain after stroke. In contrast, GPER distribution was decreased in the infarct core of all mice examined. Furthermore, GPER immunofluorescence was co-localized with the endothelial cell marker, von Willebrand factor, and the neuronal marker, NeuN. Consistent with the immunohistochemical findings, Western blot analysis showed GPER expression is only elevated in the ischemic hemisphere of male mice. Moreover, GPER mRNA expression in males was elevated at 4 h but had returned to baseline by 24 h. In conclusion, these findings indicate that GPER may be a potential therapeutic target after stroke, especially in males, in whom estrogen therapy is not feasible.

Chronic treatment with tempol does not significantly ameliorate renal tissue hypoxia or disease progression in a rodent model of polycystic kidney disease.

In the present study, we tested whether polycystic kidney disease (PKD) is associated with renal tissue hypoxia and oxidative stress, which, in turn, contribute to the progression of cystic disease and hypertension. Lewis polycystic kidney (LPK) rats and Lewis control (Lewis) rats were treated with tempol (1 mmol/L in drinking water) from 3 to 13 weeks of age or remained untreated. The LPK rats developed polyuria, uraemia and proteinuria. At 13 weeks of age, LPK rats had greater mean arterial pressure (1.5-fold), kidney weight (sixfold) and plasma creatinine (3.5-fold) than Lewis rats. Kidneys from LPK rats were cystic and fibrotic. Renal hypoxia was evidenced by staining for pimonidazole adducts and hypoxia-inducible factor (HIF)-1α in cells lining renal cysts and upregulation of HIF-1α and its downstream targets vascular endothelial growth factor (VEGF), glucose transporter-1 (Glut-1) and heme oxygenase 1 (HO-1). However, total HO activity did not differ greatly between kidney tissue from LPK compared with Lewis rats. Renal oxidative and/or nitrosative stress was evidenced by ninefold greater immunofluorescence for 3-nitrotyrosine in kidney tissue from LPK compared with Lewis rats and a > 10-fold upregulation of mRNA for p47phox and gp91phox. Total renal superoxide dismutase (SOD) activity was sevenfold less and expression of SOD1 mRNA was 70% less in kidney tissue from LPK compared with Lewis rats. In LPK rats, tempol treatment reduced immunofluorescence for 3-nitrotyrosine and HIF1A mRNA while upregulating VEGF and p47phox mRNA expression, but otherwise had little impact on disease progression, renal tissue hypoxia or hypertension. Our findings do not support the hypothesis that oxidative stress drives hypoxia and disease progression in PKD.

Post-stroke inflammation and the potential efficacy of novel stem cell therapies: focus on amnion epithelial cells.

Ischemic stroke is a debilitating disease for which there are currently no effective treatments besides the clot-buster, tissue plasminogen activator (t-PA), which is administered to less than 10% of patients due to a limited (4.5 h) time window of efficacy. Thus, there is an urgent need for novel therapies that can prevent or reverse the effects of stroke-induced brain injury. Recent encouraging reports have revealed that stem cells derived from human tissue, including embryonic, induced pluripotent, neural, and mesenchymal cells, can rescue injured brain tissue and improve functional recovery in experimental models of stroke. However, there are potentially major limitations to each of these types of stem cells that may ultimately prevent or restrict their use as viable mainstream treatment options for stroke patients. Conversely, stem cells derived from the placenta, such as human amnion epithelial cells (hAECs), appear to have several important advantages over other stem cell lineages, in particular their non-tumorigenic and non-immunogenic characteristics. Surprisingly, so far hAECs have received little attention as a potential stroke therapy. This brief review will firstly describe the inflammatory response and immune cell involvement following stroke, and then consider the potential for hAECs to improve stroke outcome given their unique characteristics. These actions of hAECs may involve a reduction of local inflammation and modulation of the immune response, promotion of neural recovery, differentiation into neural tissue, re-innervation of lost connections, and secretion of necessary cytokines, growth factors, hormones and/or neurotransmitters to restore cellular function.

Inhibition of Nox2 oxidase activity ameliorates influenza A virus-induced lung inflammation.

Influenza A virus pandemics and emerging anti-viral resistance highlight the urgent need for novel generic pharmacological strategies that reduce both viral replication and lung inflammation. We investigated whether the primary enzymatic source of inflammatory cell ROS (reactive oxygen species), Nox2-containing NADPH oxidase, is a novel pharmacological target against the lung inflammation caused by influenza A viruses. Male WT (C57BL/6) and Nox2(-/y) mice were infected intranasally with low pathogenicity (X-31, H3N2) or higher pathogenicity (PR8, H1N1) influenza A virus. Viral titer, airways inflammation, superoxide and peroxynitrite production, lung histopathology, pro-inflammatory (MCP-1) and antiviral (IL-1ß) cytokines/chemokines, CD8(+) T cell effector function and alveolar epithelial cell apoptosis were assessed. Infection of Nox2(-/y) mice with X-31 virus resulted in a significant reduction in viral titers, BALF macrophages, peri-bronchial inflammation, BALF inflammatory cell superoxide and lung tissue peroxynitrite production, MCP-1 levels and alveolar epithelial cell apoptosis when compared to WT control mice. Lung levels of IL-1ß were ∼3-fold higher in Nox2(-/y) mice. The numbers of influenza-specific CD8+D(b)NP(366)+ and D(b)PA(224)+ T cells in the BALF and spleen were comparable in WT and Nox2(-/y) mice. In vivo administration of the Nox2 inhibitor apocynin significantly suppressed viral titer, airways inflammation and inflammatory cell superoxide production following infection with X-31 or PR8. In conclusion, these findings indicate that Nox2 inhibitors have therapeutic potential for control of lung inflammation and damage in an influenza strain-independent manner.

Mechanisms contributing to cerebral infarct size after stroke: gender, reperfusion, T lymphocytes, and Nox2-derived superoxide.

Cerebral infarct volume is typically smaller in premenopausal females than in age-matched males after ischemic stroke, but the underlying mechanisms are poorly understood. In this study we provide evidence in mice that this gender difference only occurs when the ischemic brain is reperfused. The limited tissue salvage achieved by reperfusion in male mice is associated with increased expression of proinflammatory proteins, including cyclooxygenase-2 (Cox-2), Nox2, and vascular cell adhesion molecule-1 (VCAM-1), and infiltration of Nox2-containing T lymphocytes into the infarcted brain, whereas such changes are minimal in female mice after ischemia-reperfusion (I-R). Infarct volume after I-R was no greater at 72 h than at 24 h in either gender. Infarct development was Nox2 dependent in male but not in female mice, and Nox2 within the infarct was predominantly localized in T lymphocytes. Stroke resulted in an approximately 15-fold increase in Nox2-dependent superoxide production by circulating, but not spleen-derived, T lymphocytes in male mice, and this was approximately sevenfold greater than in female mice. These circulating immune cells may thus represent a major and previously unrecognized source of superoxide in the acutely ischemic and reperfused brain of males (and potentially in postmenopausal females). Our findings provide novel insights into mechanisms that could be therapeutically targeted in acute ischemic stroke patients who receive thrombolysis therapy to induce cerebral reperfusion.