Micro-osmotic Pumps Research Articles

Magnoflorine attenuates Ang II-induced cardiac remodeling via promoting AMPK-regulated autophagy.

Heart failure (HF) remains one of the most common events in the progression of hypertension. Magnoflorine (MNF) has been shown beneficial effects on the cardiovascular system. However, the action of MNF on angiotensin (Ang) II-induced cardiac remodeling and its underlying mechanisms have not yet been characterised. Here, we assessed the action of MNF in the development of hypertension-related HF. C57BL/6 male mice were subjected to Ang II through a micro-osmotic pump infusion continuously for 4 weeks to induce hypertensive HF. MNF (10 and 20 mg/kg) was administered in the final 2 weeks. Ang II content was measured by enzyme-linked immunosorbent assay (ELISA) kit. Values of ejection fraction (EF) and fractional shortening (FS) were detected using an ultrasound diagnostic instrument. The mRNA levels of hypertrophic and fibrotic genes were determined by real-time quantitative polymerase chain reaction (RT-qPCR). Haematoxylin and eosin (H&E), wheat germ agglutinin (WGA), Masson trichrome, and Sirius Red staining were used to analyse pathologic changes in heart tissues. The expression levels of phosphorylated AMP-activated protein kinase (AMPK), light chain 3 microtubule associated protein II (LC3 II) to LC3 I, and p62 were detected by western blot assay. MNF significantly improved cardiac dysfunction and the content of creatine kinase-MB without altering blood pressure in Ang II-challenged mice. MNF obviously corrected the phenotypes of cardiac hypertrophy and fibrosis, including the high mRNA levels of atrial natriuretic peptide (Anp), brain natriuretic peptide (Bnp), collagen1a (Col1a1), transforming growth factor beta (Tgfb1), enlarged myocardial areas, and increased positive areas of Masson trichrome and Sirius Red staining. In addition, MNF alleviated oxidative injury, reflected by the upregulation of glutathione and the downregulation of reactive oxygen species and malondialdehyde. The activation of AMPK was elevated accompanied by an increased level of autophagy by MNF in hypertensive heart tissues. The therapeutic action of MNF was confirmed in Ang II-challenged H9c2 cells. Specifically, the AMPK inhibitor could eliminate the autophagy pathway in which MNF is involved. MNF has benefits in hypertension-induced cardiac remodeling, which was partially associated with the improvement of oxidative stress via the mediation of the AMPK/autophagy axis.

Puerarin Alleviates Blood Pressure via Inhibition of ROS/TLR4/NLRP3 Inflammasome Signaling Pathway in the Hypothalamic Paraventricular Nucleus of Salt-Induced Prehypertensive Rats.

Puerarin is an isoflavone compound isolated from the roots of a leguminous plant, the wild kudzu. Various functional activities of this compound in multiple diseases have been reported. However, the effect and mechanism of puerarin in improving blood pressure remain non-elucidated. The current study was designed to assess the preventive effects of puerarin on the onset and progression of hypertension and to verify the hypothesis that puerarin alleviates blood pressure by inhibiting the ROS/TLR4/NLRP3 inflammasome signaling pathway in the hypothalamic paraventricular nucleus (PVN) of salt-induced prehypertensive rats. Male Dahl salt-sensitive rats were fed low NaCl salt (3% in drinking water) for the control (NS) group or 8% (HS) to induce prehypertension. Each batch was divided into two group and treated by bilateral PVN microinjection with either artificial cerebrospinal fluid or puerarin through a micro-osmotic pump for 6 weeks. The mean arterial pressure (MAP) was recorded, and samples were collected and analyzed. We concluded that puerarin significantly prevented the elevation of blood pressure and effectively alleviated the increase in heart rate caused by high salt. Norepinephrine (NE) in the plasma of salt-induced prehypertensive rats also decreased upon puerarin chronic infusion. Additionally, analysis of the PVN sample revealed that puerarin pretreatment decreased the positive cells and gene level of TLR4 (Toll-like receptor 4), NLRP3, Caspase-1 p10, NOX2, MyD88, NOX4, and proinflammatory cytokines in the PVN. Puerarin pretreatment also decreased NF-κBp65 activity, inhibited oxidative stress, and alleviated inflammatory responses in the PVN. We conclude that puerarin alleviated blood pressure via inhibition of the ROS/TLR4/NLRP3 inflammasome signaling pathway in the PVN, suggesting the therapeutic potential of puerarin in the prevention of hypertension.

P03 Anti-ageing effects of oestetrol on the skin: protection from cellular senescence and restoration of dermal architecture

Abstract Introduction and aims Skin is a major endocrine organ, undergoing profound structural and functional alterations in response to hormonal changes. Postmenopause, the rapid decline in circulating 17β-oestradiol (E2) leads to skin ageing, while exogenous E2 administration restores skin function, promoting the regenerative capacity of aged skin. Nevertheless, the risk of off-target effects limits the clinical use of E2 for skin indications. Oestetrol (E4), a natural oestrogenic steroid currently undergoing clinical trials as a hormone replacement therapy, presents a favourable safety profile and could be a suitable alternative to E2. However, as no studies have investigated the effects E4 on skin ageing, the aim of this study was to evaluate the potential of E4 to overcome effects of skin ageing. Methods Micro-osmotic pumps were subcutaneously implanted into 14-month-old female mice, allowing sustained systemic delivery of E2 or E4 over 3 weeks. Reversal of skin ageing was assessed via noninvasive skin parameter testing, histological analysis and biomechanics. Aged human dermal fibroblasts were treated with E4 to measure effects on the extracellular matrix deposition (ECM) in vitro. Finally, human keratinocytes were treated with E4 prior to inducing senescence to assess potential protective effects against skin ageing. Readouts included senescence-associated beta galactosidase staining, immunofluorescence and quantitative polymerase chain reaction. Results Systemic administration of E4 reversed multiple characteristics of skin ageing. E4 treatment significantly increased skin hydration, stimulated collagen deposition and elevated elastic fibre synthesis. These structural changes improved the biomechanical function of skin, increasing breaking strength and elasticity. In vitro studies confirmed direct anti-ageing activity on multiple cell types. E4 substantially increased ECM deposition in aged human dermal fibroblasts and protected against senescence induction in human keratinocytes. Conclusions Collectively, these preclinical data demonstrate significant beneficial effects of E4 on aged skin, strongly supporting future clinical evaluation of E4 to alleviate the effects of skin ageing following menopause.

Esaxerenone Inhibits Interferon-γ Induced Pyroptosis of Macrophages in the Lungs of Aldosterone-treated Mice.

Lung immune cells such as lymphocytes and macrophages can induce an inflammatory response due to the activation of mineralocorticoid receptor (MR), which is manifested by the infiltration of inflammatory cells and the secretion of inflammatory cytokines and subsequent apoptosis, pyroptosis and necrosis of intrinsic lung cells and immune cells. Macrophages are immune cells that are abundant in the lung and act as the first line of defense against pathogens but are also aggravating factors of infection. The activation of the renin-angiotensin-aldosterone system (RAAS), especially aldosterone-stimulated MR activation, can induce macrophage and CD8+T cell aggregation and the secretion of cytokines such as tumor necrosis factor-α (TNF-α) and interferon-gamma (IFN-γ). Increased IFN-γ secretion can induce macrophage pyroptosis and the release of interleukin 1-β (IL-1β), aggravating lung injury. In this study, lung injury in C57BL/6 mice was induced by subcutaneous micro-osmotic pump infusion of aldosterone. After 12weeks of administration, the kidney, heart, blood vessels and lungs all showed obvious inflammatory injury, which manifested as rapid accumulation of macrophages. The overexpression of IFN-γ in the lungs of aldosterone-treated mice and the stimulation of MH-S and RAW264.7 alveolar macrophages (AMs) with aldosterone in vitro showed that IFN-γ induced pyroptosis of macrophages via the activation of the inflammasome, and the MR blocker esaxerenone effectively inhibited this effect and alleviated lung injury. In addition, IFN-γ secreted by CD8+T cells is associated with macrophage pyroptosis. In conclusion, the inhibition of macrophage pyroptosis can effectively alleviate lung injury.

Prohibitin 1 deletion from activated cardiac fibroblasts in vivo attenuates cardiac fibrosis

Cardiac fibroblasts (CF) are responsible for extracellular matrix (ECM) deposition with cardiac injury. While fibrosis is a typical response to injury, excessive or uncontrolled fibrotic remodeling by CFs underlies the progression to heart failure. Cardiac injury-induced activated CFs are characteristically identified as periostin expressing cells that are proliferative and ECM secreting. This activated secretory phenotype is supported by altered mitochondrial mechanisms. Prohibitin 1 (PHB1) is known to play a role in cell survival via its impact on mitochondrial integrity, and we have established that it is enriched in activated CFs. This study aims to investigate the impact of conditional PHB1 deletion in vivo from activated CFs, and it is expected that PHB1 deletion will reduce or prevent isoproterenol (ISO)-induced fibrotic remodeling. Male and female Periostin-Cre (PC) Phb1fl/fl and WT Phb1fl/fl mice were infused with ISO (75 mg/kg/day) or vehicle (0.003% ascorbic acid in saline) for 1 or 2 weeks via osmotic micro-pump. Cre recombinase was induced by tamoxifen injection (75 mg/kg, i.p.). Hearts were collected, a mid-myocardial section was fixed and stained for fibrosis (Sirius red), and remaining tissue was frozen for protein and RNA extraction. Two weeks of ISO tended to increase hypertrophy in male (p=0.06) and female (p=0.08) mice compared to vehicle. Periostin protein expression was modulated similarly in male and female hearts, which exhibited a significant 7-fold increase following 2 weeks of ISO infusion (p=0.003 vs. vehicle). CF PHB1 deletion significantly attenuated this increase (p=0.009 vs. WT ISO). PHB1 deletion in activated CFs resulted in reduced Col1a1 gene expression following 2 weeks of ISO in males (38.6%) and in females (68.0%) relative to WT ISO. Further, both male and female PC mice exhibited ~55% reduction in Col3a1 gene expression after ISO infusion compared to WT. Taken together, these findings indicate that PHB1 deletion prevents ISO-induced CF activation in both sexes. Left ventricular fibrosis via Sirius red stain was more pronounced in mice treated for 2 weeks compared to 1 week, with males having markedly more fibrosis than females. The accumulation of ISO-induced fibrosis in males was prevented by PHB1 deletion. Interestingly, the extent of cardiac fibrosis with and without PHB1 deletion correlated with periostin protein expression in males, but not in females. Thus, the measured reduction in CF activation by conditional PHB1 deletion may limit ECM deposition in males. The lack of fibrotic tissue in the presence of elevated collagen gene expression and periostin in the ISO-treated females may reflect sex-specific CF phenotypes or fibrotic remodeling. In this model of fibrosis, it is unclear how PHB1 deletion is impacting the female heart structurally. Future studies will aim to elucidate altered mitochondrial mechanisms after PHB1 deletion in models of fibrosis, as well as sex-specific mechanisms of fibrotic remodeling. ECU REDE startup program, NIH T32HL007249-44, UArizona Sarver Heart Center. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lycorine inhibits Ang II-induced heart remodeling and inflammation by suppressing the PI3K-AKT/NF-κB pathway

BackgroundAng II induces hypertensive heart failure (HF) via hemodynamic and non-hemodynamic actions. Lycorine (LYC) is an alkaloid derived from Lycoris bulbs, and it possesses anti-cardiovascular disease-related activities. Herein, we explored the potential LYC-mediated regulation of Ang II-induced HF. MethodsOver 4 weeks, we established a hypertensive HF mouse model by infusing Ang II into C57BL/6 mice using a micro-osmotic pump. For the final two weeks, mice were administered LYC via intraperitoneal injection. The LYC signaling network was then deduced using RNA sequencing. ResultsLYC administration strongly suppressed hypertrophy, myocardial fibrosis, and cardiac inflammation. As a result, it minimized heart dysfunction while causing no changes in blood pressure. The Nuclear Factor kappa B (NF-κB) network/phosphoinositol-3-kinase (PI3K)-protein kinase B (AKT) was found to be a major modulator of LYC-based cardioprotection using RNA sequencing study. We further confirmed that in cultured cardiomyocytes and mouse hearts, LYC reduced the inflammatory response and downregulated the Ang II-induced PI3K-AKT/NF-κB network. Moreover, PI3K-AKT or NF-κB axis depletion in cardiomyocytes completely abrogated the anti-inflammatory activities of LYC. ConclusionHerein, we demonstrated that LYC safeguarded hearts in Ang II -stimulated mice by suppressing the PI3K-AKT/NF-κB-induced inflammatory responses. Given the evidence mentioned above, LYC is a robust therapeutic agent for hypertensive HF.

Irisin attenuates vascular remodeling in hypertensive mice induced by Ang II by suppressing Ca2+-dependent endoplasmic reticulum stress in VSMCs.

Vascular remodeling plays a vital role in hypertensive diseases and is an important target for hypertension treatment. Irisin, a newly discovered myokine and adipokine, has been found to have beneficial effects on various cardiovascular diseases. However, the pharmacological effect of irisin in antagonizing hypertension-induced vascular remodeling is not well understood. In the present study, we investigated the protection and mechanisms of irisin against hypertension and vascular remodeling induced by angiotensin II (Ang II). Adult male mice of wild-type, FNDC5 (irisin-precursor) knockout, and FNDC5 overexpression were used to develop hypertension by challenging them with Ang II subcutaneously in the back using a microosmotic pump for 4 weeks. Similar to the attenuation of irisin on Ang II-induced VSMCs remodeling, endogenous FNDC5 ablation exacerbated, and exogenous FNDC5 overexpression alleviated Ang II-induced hypertension and vascular remodeling. Aortic RNA sequencing showed that irisin deficiency exacerbated intracellular calcium imbalance and increased vasoconstriction, which was parallel to the deterioration in both ER calcium dysmetabolism and ER stress. FNDC5 overexpression/exogenous irisin supplementation protected VSMCs from Ang II-induced remodeling by improving endoplasmic reticulum (ER) homeostasis. This improvement includes inhibiting Ca2+ release from the ER and promoting the re-absorption of Ca2+ into the ER, thus relieving Ca2+-dependent ER stress. Furthermore, irisin was confirmed to bind to its receptors, αV/β5 integrins, to further activate the AMPK pathway and inhibit the p38 pathway, leading to vasoprotection in Ang II-insulted VSMCs. These results indicate that irisin protects against hypertension and vascular remodeling in Ang II-challenged mice by restoring calcium homeostasis and attenuating ER stress in VSMCs via activating AMPK and suppressing p38 signaling.

OTUD1 promotes hypertensive kidney fibrosis and injury by deubiquitinating CDK9 in renal epithelial cells.

Hypertensive renal disease (HRD) contributes to the progression of kidney dysfunction and ultimately leads to end-stage renal disease. Understanding the mechanisms underlying HRD is critical for the development of therapeutic strategies. Deubiquitinating enzymes (DUBs) have been recently highlighted in renal pathophysiology. In this study, we investigated the role of a DUB, OTU Domain-Containing Protein 1 (OTUD1), in HRD models. HRD was induced in wild-type or Otud1 knockout mice by chronic infusion of angiotensin II (Ang II, 1 μg/kg per min) through a micro-osmotic pump for 4 weeks. We found that OTUD1 expression levels were significantly elevated in the kidney tissues of Ang II-treated mice. Otud1 knockout significantly ameliorated Ang II-induced HRD, whereas OTUD1 overexpression exacerbated Ang II-induced kidney damage and fibrosis. Similar results were observed in TCMK-1 cells but not in SV40 MES-13 cells following Ang II (1 μM) treatment. In Ang II-challenged TCMK-1 cells, we demonstrated that OTUD1 bound to CDK9 and induced CDK9 deubiquitination: OTUD1 catalyzed K63 deubiquitination on CDK9 with its Cys320 playing a critical role, promoting CDK9 phosphorylation and activation to induce inflammatory responses and fibrosis in kidney epithelial cells. Administration of a CDK9 inhibitor NVP-2 significantly ameliorated Ang II-induced HRD in mice. This study demonstrates that OTUD1 mediates HRD by targeting CDK9 in kidney epithelial cells, suggesting OTUD1 is a potential target in treating this disease.

Effects of prenatal nicotine exposure on vascular endothelial function and structure in offspring rats

To clarify the effect of prenatal nicotine exposure (PNE) on vascular endothelial function (VEF) in offspring rats, and to explore whether these effects are long-lasting and sex-dependent. Pregnant Sprague-Dawley rats were randomized into two groups and exposed to either 102 mg/mL nicotine (experimental group) or normal saline (control group) through a subcutaneously implanted osmotic micropump. Vascular rings from the thoracic aorta were collected from offspring rats at 1, 4, and 12 months of age. The specimens were used to determine VEF using functional tests and to observe morphologic and pathologic changes of blood vessels in hematoxylin and eosin-stained samples with light microscopy. The percentage of acetylcholine-induced endothelium-dependent vasorelaxation (EDV) of the thoracic aorta was significantly higher in the experimental group than in the control group in 1-, 4-, and 12-month-old female offspring rats (18.68±2.32 vs. 13.85±6.10, 17.44±3.91 vs. 10.77±5.77, and 30.96±22.26 vs. 18.94±13.61, respectively; P <0.05). The percentage of acetylcholine-induced EDV was significantly lower in the experimental group than in the control group in 1-, 4-, and 12-month-old male offspring rats (10.31±4.05 vs. 16.05±14.80, 5.57±2.81 vs. 12.12±5.62, and 11.98±7.24 vs. 58.87±32.43, respectively; P <0.05). The hematoxylin & eosin-stained vascular rings of the offspring rats displayed an uneven lumen, scattered intimal thickening, partial shedding and vacuolar degeneration of endothelial cells, and inflammatory cell infiltration and phagocytosis in the experimental group. In addition, overt smooth muscle atrophy in tunica media, disordered cell arrangement, and unclear structure of the elastic fiber layer was observed. None of these histopathologic changes were found in the control group. In adult offspring rats, PNE not only affected the VEF of the thoracic aorta but also led to pathologic changes in the vascular structure. Additionally, the effects of PNE on VEF were sex-specific, manifesting primarily as significantly improved VEF in female offspring rats and significantly impaired VEF in male offspring rats, lasting into adulthood.

Interferon gamma (IFN-g) continuous infusion affects engraftment in a murine transplant model

IntroductionEffects of Interferon gamma (IFN-g) on hematopoiesis have been reported as both stimulatory and suppressive. In humans, IFN-g appears to be deleterious in conditions such as chronic neutropenia, anemia of chronic disease, and aplastic anemia, suggesting some level of bone marrow suppression. Inborn errors of immunity often have an inflammatory component as part of their phenotype, and this inflammation may impact on their transplant outcomes leading to increased graft failure. We thus developed a murine model using implanted pumps to assess the impact of exogenous IFN-g administered by continuous infusion during transplantation. MethodsMurine model and IFN-g administration: Bone marrow nucleated cells (BMNCs) were collected from femurs and tibias of 8- to 10-week-old B6. SJL-Ptprca-Pep3b-/BoyJ donors (CD45.1+) and 5 million cells were injected via tail vein into 10- to 12-week-old recipient C57BL6 mice (CD45.2+) after 400cGy irradiation. Micro-osmotic pumps (Alzet®1007D/2001) were surgically implanted 2 days before transplant to provide continuous infusion of recombinant mouse IFN-g at 5ug/day for 7 days. Donor chimerism%CD45.1 + engraftment inT, B, NK, and myeloid cells was determined in peripheral blood every 2 weeks (from 4 to 16 weeks) post-transplant using cell specific monoclonal antibodies. 16 weeks after transplant, mice were euthanized, and %CD45.1+ engraftment was measured in spleen and bone marrow. ResultsAlthough the absolute peripheral blood numbers were similar (Fig.1A), the infusion of IFN-g in this non myeloablative transplant resulted in lower donor engraftment as compared to control transplants (Fig.1B). Myeloid engraftment appeared to be most affected, with a statistically significant reduction of engraftment (p < 0.005) in monocytes and granulocytes. The quantitative reduction was higher than 70% as compared to control transplants worsening over time, suggesting that the long-term repopulating cells were most affected (Fig.1C). Bone marrow was affected in a similar pattern (Fig.1D). [Display omitted] ConclusionsWe established a mixed-chimera model to assess the impact of exogenous IFN-g on engraftment. Our results provide evidence that continuously elevated IFN-g levels are deleterious for engraftment and may help explain the clinical phenomena of graft loss in this patients. Further analysis of other inflammatory pathways and IFN-g blockers are ongoing.

Scleraxis Alters Arterial Smooth Muscle Phenotypic Switching and Exacerbates Arterial Stiffness

Arterial stiffness (AS) is an impairment in arterial relaxation and function in blood pressure regulation and is associated with the development of multiple cardiovascular diseases including hypertension, atherosclerosis, and adverse outcomes such as stroke, heart failure, and death. AS comprises an increase in vascular wall components (extracellular matrix proteins or vascular smooth muscle cell (VSMC) composition) or cellular malfunction, or a combination of these factors. Preliminary data from our lab showed that using the Transverse Aortic Constriction (TAC) model, the transcription factor scleraxis is upregulated in the aortic wall on the pre-TAC/high pressure side versus post-TAC/low pressure side. Scleraxis also induces cellular phenotype activation of fibroblasts to myofibroblasts in cardiac fibrosis and mediates epithelial to mesenchymal transition in development. Angiotensin II (AngII) induces hypertension and increases stiffness in arteries, and we found that it also up-regulates scleraxis expression, suggesting the possibility that scleraxis may alter arterial wall composition. In this study, we investigated the role of scleraxis expression in VSMC, and its effect when over-expressed along with AngII treatment in small resistance arteries and aorta. In vivo, we upregulated scleraxis in VSMC using a Cre/LoxP approach and the tamoxifen-inducible Myh11cre mouse, with or without AngII delivery by micro-osmotic pump, then isolated 3rd order mesenteric arteries and assessed arterial function and stiffness by pressure myography. We harvested aortas for immunohistology staining and gene expression analysis. In vitro, we upregulated scleraxis in VSMC with or without AngII treatment and measured their proliferation by flow cytometry. Our findings show that scleraxis induces stiffness in mesenteric arteries, but not aorta by induction of VSMC proliferation. Scleraxis upregulation with AngII infusion exacerbates vascular stiffness in mesenteric arteries due to significant changes in arterial wall components, reducing internal lumen diameter and smooth muscle relaxation. At the molecular level, extracellular matrix protein expression is reduced in aortas. In in vitro studies, scleraxis-AngII treated VSMC exhibit lower proliferative capacity, distinct morphological changes and higher contractile gene expression, indicative of a hypertrophic contractile phenotype altering overall arterial stiffness. Our study shows that scleraxis alone can induce AS by regulating VSMC phenotype toward proliferation. In contrast, arterial stiffness is exacerbated in scleraxis overexpression and AngII-infused vessels by inducing VSMC towards a hypertrophic contractile phenotype. Therefore, scleraxis alters vascular smooth muscle phenotypic switching and arterial stiffness through different mechanisms, depending on the presence of AngII, which may have different pathological impacts depending on the specific underlying conditions. This work is supported by funding from Heart &amp; Stroke Foundation and BMO Studentship. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sexual Dimorphism in the Closure of the Hippocampal Postnatal Critical Period of Synaptic Plasticity after Intrauterine Growth Restriction: Link to Oligodendrocyte and Glial Dysregulation

Intrauterine growth restriction (IUGR) resulting from hypertensive disease of pregnancy (HDP) leads to sexually dimorphic hippocampal-dependent cognitive and memory impairment in humans. In our translationally relevant mouse model of IUGR incited by HDP, we have previously shown that the synaptic development in the dorsal hippocampus including GABAergic development, NPTX2⁺ excitatory synaptic formation, axonal myelination, and perineural net (PNN) formation were perturbed by IUGR at adolescent equivalence in humans (P40). The persistence of these disturbances through early adulthood and the potential upstream mechanisms are currently unknown. Thus, we hypothesized that NPTX2⁺ expression, PNN formation, axonal myelination, all events closing synaptic development in the hippocampus, will be persistently perturbed, particularly affecting IUGR female mice through P60 given the fact that they had worse short-term recognition memory in this model. We additionally hypothesized that such sexual dimorphism is linked to persistent glial dysregulation. We induced IUGR by a micro-osmotic pump infusion of a potent vasoconstrictor U-46619, a thromboxane A₂-analog, in the last week of the C57BL/6 mouse gestation to precipitate HDP. Sham-operated mice were used as controls. At P60, we assessed hippocampal and hemispheric volumes, NPTX2 expression, PNN formation, as well as myelin basic protein (MBP), Olig2, APC/CC1, and M-NF expression. We also evaluated P60 astrocytic (GFAP) reactivity and microglial (Iba1 and TMEM119) activation using immunofluorescent-immunohistochemistry and Imaris morphological analysis plus cytokine profiling using Meso Scale Discovery platform. IUGR offspring continued to have smaller hippocampal volumes at P60 not related to changes in hemisphere volume. NPTX2⁺ puncta counts and volumes were decreased in IUGR hippocampal CA subregions of female mice compared to sex-matched shams. Intriguingly, NPTX2⁺ counts and volumes were concurrently increased in the dentate gyrus (DG) subregion. PNN volumes were smaller in CA1 and CA3 of IUGR female mice along with PNN intensity in CA3 but they had larger volumes in the CA3 of IUGR male mice. The myelinated axon (MBP⁺) areas, volumes, and lengths were all decreased in the CA1 of IUGR female mice compared to sex-matched shams, which correlated with a decrease in Olig2 nuclear expression. No decrease in the number of APC/CC1⁺ mature oligodendrocytes was identified. We noted an increase in M-NF expression in the mossy fibers connecting DG to CA3 only in IUGR female mice. Reactive astrocytes denoted by GFAP areas, volumes, lengths, and numbers of branching were increased in IUGR female CA1 but not in IUGR male CA3 compared to sex-matched shams. Lastly, activated microglia were only detected in IUGR female CA1 and CA3 subregions. We detected no difference in the cytokine profile between sham and IUGR adult mice of either sex. Collectively, our data support a sexually dimorphic impaired closure of postnatal critical period of synaptic plasticity in the hippocampus of young adult IUGR mice with greater effects on females. A potential mechanism supporting such dimorphism may include oligodendrocyte dysfunction in IUGR females limiting myelination, allowing axonal overgrowth followed by a reactive glial-mediated synaptic pruning.

333 Intraventricular Ziconotide Improves Cognitive Outcomes following Lateral Fluid Percussion Injury in a Rat Model

INTRODUCTION: Traumatic brain injury (TBI) is a major source of morbidity and mortality worldwide. In previous studies, intravenous ziconotide, a selective N-type voltage-gated calcium channel blocker, reduced brain calcium accumulation, improved brain mitochondrial respiration, and improved cognitive and motor outcomes in rodent models of TBI. Attempts to translate intravenous ziconotide therapy to humans were halted due to peripheral side-effects. However, ziconotide is now FDA-approved for treatment of severe pain syndromes when administered intrathecally instead of intravenously. We hypothesize that following TBI intraventricular ziconotide will improve cognitive function in the absence of significant side effects. METHODS: Seventy-seven Sprague-Dawley rats underwent sham (n = 11 males, n = 11 females) or lateral fluid percussion (LFP) injury (n = 21 males, n = 23 females). One-hour post-injury, rats received either an intraventricular injection of saline (n = 22 sham, n = 23 LFP) or ziconotide (n = 21 LFP). A catheter was implanted in the left lateral ventricle and connected to an Alzet micro-osmotic pump to allow for 72 hours of continuous infusion. Cognitive outcomes were evaluated using Morris water maze (MWM; days 10-15). RESULTS: TBI-saline rats performed significantly worse than shams on MWM (p &lt; 0.01). Ziconotide treated rats had an intermediate phenotype on MWM. TBI-saline rats swam a less efficient path than sham animals (p &lt; 0.01); ziconotide path efficiency was not significantly different from shams. Performance on the MWM was likely not related to motor deficits as there was no significant effect of group on mean swimming speed in either males or females. Female sham animals performed significantly better on probe trials compared to TBI-saline (p &lt; 0.05); ziconotide animals were not different from shams. CONCLUSIONS: These findings support centrally delivered Ziconotide as a potential neuroprotective agent to improve cognitive performance following TBI. Further studies are required to characterize the neuroprotective effects of Ziconotide therapy.

Protective Effect and Mechanism of Kuntai Capsule on Angiotensin II -Induced Hypertension in Ovariectomized Rats.

To explore the protective effect and mechanism of Kuntai (KT) Capsule on angiotensin II (Ang II)-induced hypertension in ovariectomized (OVX) rats. Fifty-four rats were randomly divided into 6 groups according to a random number table, 9 in each group: control, OVX sham+Ang II, OVX, OVX+Ang II, OVX+Ang II +E2, and OVX+Ang II +KT. OVX rats model was constructed by retroperitoneal bilateral ovariectomy. After 4 weeks of pretreatment with KT Capsule [0.8 g/(kg·d) and 17- β -estradiol (E2, 1.2 mg/(kg·d)] respectively, Ang II was injected into a micro-osmotic pump with a syringe to establish a hypertensive rat model. Blood pressure of rat tail artery was measured in a wake state of rats using a non-invasive sphygmomanometer. Blood pressure changes were compared between the intervention groups (OVX+Ang II +KT, OVX+Ang II +E2) and the negative control group (OVX+Ang II). Serum malondialdehyde (MDA) level and superoxide dismutase (SOD) activity were detected respectively. The expressions of oxidative stress-related protein superoxide dismutase2 (SOD2) and anti-thioredoxin (TRX), autophagy marker protein [beclin1, light chain (LC) 3 II/I ratio and autophagy canonical pathway protein phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (AKT)-mammalian target of rapamycin (mTOR)] were evaluated by Western blotting. Compared with the OVX+Ang II group, the systolic blood pressure of OVX+Ang II +KT group was significantly lowered (P<0.05) but not the diastolic blood pressure. Besides, SOD2 and TRX protein levels in mycardial tissues were significantly reduced in the OVX+Ang II +KT group compared with the OVX+Ang II group (P<0.05). Oxidative stress serum markers MDA and SOD were down- and up-regulated in the OVX+Ang II +KT group, respectively (P<0.05). Compared with OVX+Ang II group, the levels of cardiac proteins beclin-1 and LC3II/LC3 I in OVX+Ang II +KT group were also up-regulated (P<0.05), and the expression levels of p-PI3K, p-AKT and mTOR protein were down-regulated (P<0.05). KT could protect blood pressure of Ang II-induced OVX rats by inhibiting oxidative stress and up-regulating protective autophagy.

Shenxian-Shengmai Oral Liquid Evoke Autophagy of Fibroblast to Attenuate Sinoatrial Node Fibrosis in Sick Sinus Syndrome Mice via the AKT/mTOR Pathway.

Sick sinus syndrome (SSS) is closely associated with cardiac syncope and sudden death, wherein sinoatrial node (SAN) fibrosis is one of the main pathological changes that occur. Shenxian-Shengmai oral liquid (SXSM) has been clinically proven to significantly improve the heart rate of SSS patients. In this study, we aimed to explore the mechanism of SXSM in reducing the SAN fibrosis by combining in vitro and in vivo experiments. Accordingly, the SSS model was constructed by slowly pumping angiotensin II (AngII) with a micro-osmotic pump. The degree of fibrosis was evaluated by Masson's trichrome staining and immunofluorescence staining of the fibrosis marker protein. In addition, NIH-3T3 mouse fibroblasts were used to simulate SAN fibroblasts to further explore the mechanism, with AngII used as the cellular fibrosis inducer. Monodansylcadaverine (MDC) staining and transmission electron microscopy were employed to assay the autophagy content, whereas immunofluorescence staining and Western blotting were employed to elucidate the related protein expression. Finally, fibroblasts were given the AKT phosphorylation agonist SC79 to reversely verify the effects of SXSM. The results showed that SXSM could significantly increase the heart rate of SSS mice by reducing the deposition of extracellular matrix (ECM) in SAN induced by AngII. According to in vivo experiments, when compared with the model group, SSS mice treated with SXSM developed less fibrosis in the SAN area. In vitro experiments revealed that AngII could restrain autophagy by activating the phosphorylation of the AKT/mTOR pathway, thereby increasing the deposition of ECM. Moreover, SXSM pretreatment prevented this upregulation. After the intervention of SC79, the protective effect of SXSM was weakened. In conclusion, SXSM activated autophagy through the AKT/mTOR pathway, which in turn reduced the deposition of the ECM in SAN induced by AngII, attenuated the fibrosis of SAN, and improved the decreased heart rate in the SSS mice.

Inhibition of MyD88 attenuates angiotensin II-induced hypertensive kidney disease via regulating renal inflammation

BackgroundKidney damage is a frequent event in the course of hypertension. Recent researches highlighted a critical role of non-hemodynamic activities of angiotensin II (Ang II) in hypertension-associated kidney fibrosis and inflammation. These activities are mediated through toll-like receptors (TLRs) but the mechanisms by which Ang II links TLRs to downstream inflammatory and fibrogenic responses is not fully known. In this study, we investigated the role of TLR adapter protein called myeloid differentiation primary-response protein-88 (MyD88) as the potential link. MethodsC57BL/6 mice were administered Ang II by micro-osmotic pump infusion for 4 weeks to develop nephropathy. Mice were treated with small-molecule MyD88 inhibitor LM8. In vitro, MyD88 was blocked using siRNA or LM8 in Ang II-challenged renal tubular epithelial cells. ResultsWe show that MyD88 is mainly located in tubular epithelial cells and Ang II increases the interaction between TLR4 and MyD88. This interaction activates MAPKs and nuclear factor-κB (NF-κB), leading to increased production of inflammatory and fibrogenic factors. Inhibition of MyD88 by siRNA or selective inhibitor LM8 supresses MyD88-TLR4 interaction, NF-κB activation, and elaboration of inflammatory cytokines and fibrosis-associated factors. These protective actions resulted in decreased renal pathological changes and preserved renal function in LM8-treated hypertensive mice, without affecting hypertension. ConclusionThese results demonstrate that Ang II induces inflammation and fibrosis in renal tubular epithelial cells through MyD88 and present MyD88 as a potential point of intervention for hypertension-associated kidney disease.

Diacerein alleviates Ang II-induced cardiac inflammation and remodeling by inhibiting the MAPKs/c-Myc pathway

BackgroundHeart failure is a common event in the course of hypertension. Recent studies have highlighted the key role of the non-hemodynamic activity of angiotensin II (Ang II) in hypertension-related cardiac inflammation and remodeling. A naturally occurring compound, diacerein, exhibits anti-inflammatory activities in various systems. Hypothesis/PurposeIn this study, we have examined the potential effects of diacerein on Ang II-induced heart failure. MethodsC57BL/6 mice were administered Ang II by micro-osmotic pump infusion for 4 weeks to develop hypertensive heart failure. Mice were treated with diacerein by gavage for final 2 weeks. RNA-sequencing analysis was performed to explore the potential mechanism of diacerein. ResultsWe found that diacerein could inhibit inflammation, myocardial fibrosis, and hypertrophy to prevent heart dysfunction, without the alteration of blood pressure. To explore the potential mechanism of diacerein, RNA-sequencing analysis was performed, indicating that MAPKs/c-Myc pathway is involved in that cardioprotective effects of Diacerein. We further confirmed that diacerein inhibits Ang II-activated MAPKs/c-Myc pathway to reduce inflammatory response in mouse hearts and cultured cardiomyocytes. Deficiency of MAPKs or c-Myc in cardiomyocytes abolished the anti-inflammatory effects of diacerein. ConclusionOur results indicate that diacerein protects hearts in Ang II-induced mice through inhibiting MAPKs/c-Myc-mediated inflammatory responses, rendering diacerein a potential therapeutic candidate agent for hypertensive heart failure.

Tabersonine attenuates Angiotensin II-induced cardiac remodeling and dysfunction through targeting TAK1 and inhibiting TAK1-mediated cardiac inflammation

BackgroundAngiotensin II (Ang II)-induced cardiac inflammation contribute to pathological cardiac remodeling and hypertensive heart failure (HF). Tabersonine (Tab) is an indole alkaloid mainly isolated from Catharanthus roseus and exhibits anti-inflammatory activity in various systems. However, the role of Tab in hypertensive HF and its molecular targets remains unknown. Hypothesis/purposeWe aimed to investigate potential cardioprotective effects and mechanism of Tab against Ang II-induced cardiac injuries. MethodsC57BL/6 mice were administered Ang II (at 1000 ng/kg/min) by micro-osmotic pump infusion for 30 days to develop hypertensive HF. Tab at 20 and 40 mg/kg/day was administered during the last 2 weeks to elucidate the cardioprotective properties. Cultured cardiomyocyte-like H9c2 cells and rat primary cardiomyocytes were used for mechanistic studies of Tab. ResultsWe demonstrate for the first time that Tab provides protection against Ang II–induced cardiac dysfunction in mice, associated with reduced cardiac inflammation and fibrosis. Mechanistically, we show that Tab may interacts with TAK1 to inhibit Ang II-induced TAK1 ubiquitination and phosphorylation. Disruption of TAK1 activation by Tab blocked downstream NF-κB and JNK/P38 MAPK signaling activation and decreased cardiac inflammation and fibrosis both in vitro and in vivo. TAK1 knockdown also blocked Ang II-induced cardiomyocytes injuries and prevented the innately pharmacological effects of Tab. ConclusionOur results indicate that Tab protects hearts against Ang II-mediated injuries through targeting TAK1 and inhibiting TAK1-mediated inflammatory cascade and response. Thus, Tab may be a potential therapeutic candidate for hypertensive HF.

Corynoline protects ang II-induced hypertensive heart failure by increasing PPARα and Inhibiting NF-κB pathway.

Heart failure is a fairly common outcome of hypertension. Recent studies have highlighted the key role of the non-hemodynamic activity of angiotensin II (Ang II) in hypertensive heart failure via inducing cardiac inflammation. Drugs that disrupt Ang II-induced cardiac inflammation may have clinical utility in the treatment of hypertensive heart failure. A naturally occurring compound, corynoline, exhibit anti-inflammatory activities in other systems. C57BL/6 mice were injected with Ang II via a micro-osmotic pump for four weeks to develop hypertensive heart failure. The mice were treated with corynoline by gavage for two weeks. RNA-sequencing analysis was performed to explore the potential mechanism of corynoline. We found that corynoline could inhibit inflammation, myocardial fibrosis, and hypertrophy to prevent heart dysfunction, without the alteration of blood pressure. RNA-sequencing analysis indicates that the PPARα pathway is involved Ang II-induced cardiac fibrosis and cardiac remodeling. Corynoline reversed Ang II-induced PPARα inhibition both in vitro and in vivo. We further found that corynoline increases the interaction between PPARα and P65 to inhibit the NF-κB pro-inflammatory pathway in H9c2 cells. Our studies show that corynoline relieves Ang II-induced hypertensive heart failure by increasing the interaction between PPARα and P65 to inhibit the NF-κB pathway.

Long‐Term Cardioprotective Potential of Exogenous Ubiquitin in Myocardial Ischemia/Reperfusion Injury

BackgroundCoronary heart disease (CHD) is a leading cause of death worldwide. Myocardial ischemia/reperfusion (I/R) injury is a clinical manifestation of CHD. Timely reperfusion of coronary vessels in an ischemic injury is essential to limit the infarct size. However, reperfusion itself can induce cardiac cell death, and lead to increased adverse remodeling after an I/R injury. Previously, our lab has shown that pretreatment with ubiquitin (UB; a highly evolutionarily conserved protein) decreases inflammatory response and preserves heart function 3 days post‐I/R. The objective of this study is to investigate the long‐term cardioprotective potential of UB post‐I/R. To enhance the clinical relevance, UB treatment was started at the time of reperfusion. It is hypothesized that UB treatment has long‐term cardioprotective potential, improves cardiac function, and attenuates cardiac remodeling post injury.MethodsC57BL/6 male mice underwent ligation of the left anterior descending coronary artery (LCA) for 45 min. Mice were then treated with UB (1 µg/g body weight, i.p. injection) or saline at time of reperfusion of LCA. Sham animals underwent the same surgery without the ligation of LCA. Mice were treated with UB or saline twice/day (i.p. injections) for 3 days followed by treatment with UB (1 μg/g/h)or saline using micro‐osmotic pumps for 28 days. Heart function was measured at 3, 7, 14 and 28 days post‐I/R using echocardiography. Mice were sacrificed at 28 days post‐I/R. Left ventricular (LV) tissue was used to measure remodeling parameters and serum samples were used to measure a panel of 23 cytokines/chemokines using multiplex assay.ResultsI/R+saline group exhibited a significant reduction in heart function as observed by decreased percent fractional shortening (%FS) and percent ejection fraction (%EF) vs sham (p&lt;0.05) at 3, 7, 14 and 28 days post‐I/R. However, I/R+UB group showed significantly higher %FS and %EF vs I/R+saline (p&lt;0.05) throughout the observation period. Increases in fibrosis (measured by trichrome staining), myocyte apoptosis (measured using TUNEL assay), and hypertrophy (measured using WGA staining) were significantly lower in I/R+UB vs I/R+saline (p&lt;0.05). Expression of MMP‐2 and TIMP‐4 was lower, while expression of MMP‐9 (active) was significantly higher in I/R+UB vs I/R+saline (p&lt;0.05). Serum levels of IL‐6, IL‐2 and G‐CSF were significantly lower in I/R+UB vs I/R+saline (p&lt;0.05).ConclusionPost‐ischemic UB treatment improves heart function, and associates with decreased myocardial remodeling parameters (fibrosis, apoptosis, and hypertrophy) as well as decreased serum cytokines/chemokines levels.