Female Wistar-Kyoto Rats Research Articles

Maternal Diet High in Linoleic Acid Alters Renal Branching Morphogenesis and mTOR/AKT Signalling Genes in Rat Fetal Kidneys.

Linoleic acid (LA), an n-6 polyunsaturated fatty acid (PUFA), is obtained from the maternal diet during pregnancy, and is essential for normal fetal growth and development. A maternal high-LA (HLA) diet alters maternal and offspring fatty acids, maternal leptin and male/female ratio at embryonic (E) day 20 (E20). We investigated the effects of an HLA diet on embryonic offspring renal branching morphogenesis, leptin signalling, megalin signalling and angiogenesis gene expression. Female Wistar Kyoto rats were fed low-LA (LLA; 1.44% energy from LA) or high-LA (HLA; 6.21% energy from LA) diets during pregnancy and gestation/lactation. Offspring were sacrificed and mRNA from kidneys was analysed by real-time PCR. Maternal HLA decreased the targets involved in branching morphogenesis Ret and Gdnf in offspring, independent of sex. Furthermore, downstream targets of megalin, namely mTOR, Akt3 and Prkab2, were reduced in offspring from mothers consuming an HLA diet, independent of sex. There was a trend of an increase in the branching morphogenesis target Gfra1 in females (p = 0.0517). These findings suggest that an HLA diet during pregnancy may lead to altered renal function in offspring. Future research should investigate the effects an HLA diet has on offspring kidney function in adolescence and adulthood.

Maternal Diet High in Linoleic Acid Alters Offspring Lipids and Hepatic Regulators of Lipid Metabolism in an Adolescent Rat Model.

Linoleic acid (LA), an n-6 polyunsaturated fatty acid (PUFA), is essential for fetal growth and development. A maternal high LA (HLA) diet alters cardiovascular development in adolescent rats and hepatic function in adult rats in a sex-specific manner. We investigated the effects of an HLA diet on adolescent offspring hepatic lipids and hepatic lipid metabolism gene expression, and the ability of the postnatal diet to alter these effects. Female Wistar Kyoto rats were fed low LA (LLA; 1.44% energy from LA) or high LA (HLA; 6.21% energy from LA) diets during pregnancy and gestation/lactation. Offspring, weaned at postnatal day (PN) 25, were fed LLA or HLA and euthanised at PN40 (n = 6-8). Maternal HLA increased circulating uric acid, decreased hepatic cholesterol and increased hepatic Pparg in males, whereas only hepatic Srebf1 and Hmgcr increased in females. Postnatal (post-weaning) HLA decreased liver weight (% body weight) and increased hepatic Hmgcr in males, and decreased hepatic triglycerides in females. Maternal and postnatal HLA had an interaction effect on Lpl, Cpt1a and Pparg in females. These findings suggest that an HLA diet both during and after pregnancy should be avoided to improve offspring disease risk.

Differential transcriptomic alterations in nasal versus lung tissue of acrolein-exposed rats

Introduction: Acrolein is a significant component of anthropogenic and wildfire emissions, as well as cigarette smoke. Although acrolein primarily deposits in the upper respiratory tract upon inhalation, patterns of site-specific injury in nasal versus pulmonary tissues are not well characterized. This assessment is critical in the design of in vitro and in vivo studies performed for assessing health risk of irritant air pollutants.Methods: In this study, male and female Wistar-Kyoto rats were exposed nose-only to air or acrolein. Rats in the acrolein exposure group were exposed to incremental concentrations of acrolein (0, 0.1, 0.316, 1 ppm) for the first 30 min, followed by a 3.5 h exposure at 3.16 ppm. In the first cohort of male and female rats, nasal and bronchoalveolar lavage fluids were analyzed for markers of inflammation, and in a second cohort of males, nasal airway and left lung tissues were used for mRNA sequencing.Results: Protein leakage in nasal airways of acrolein-exposed rats was similar in both sexes; however, inflammatory cells and cytokine increases were more pronounced in males when compared to females. No consistent changes were noted in bronchoalveolar lavage fluid of males or females except for increases in total cells and IL-6. Acrolein-exposed male rats had 452 differentially expressed genes (DEGs) in nasal tissue versus only 95 in the lung. Pathway analysis of DEGs in the nose indicated acute phase response signaling, Nrf2-mediated oxidative stress, unfolded protein response, and other inflammatory pathways, whereas in the lung, xenobiotic metabolism pathways were changed. Genes associated with glucocorticoid and GPCR signaling were also changed in the nose but not in the lung.Discussion: These data provide insights into inhaled acrolein-mediated sex-specific injury/inflammation in the nasal and pulmonary airways. The transcriptional response in the nose reflects acrolein-induced acute oxidative and cytokine signaling changes, which might have implications for upper airway inflammatory disease susceptibility.

Memory deficits and hippocampal cytokine expression in a rat model of ADHD

Attention-deficit/hyperactivity disorder (ADHD) is a complex behavioral disorder characterized by hyperactivity, impulsivity, inattention, and deficits in working memory and time perception. While animal models have advanced our neurobiological understanding of this condition, there are limited and inconsistent data on working and elapsed time memory function. Inflammatory signaling has been identified as a key factor in memory and cognitive impairments, but its role in ADHD remains unclear. Additionally, the disproportionate investigation of male subjects in ADHD research has contributed to a poor understanding of the disorder in females. This study sought to investigate the potential connections between memory, neuroimmunology, and ADHD in both male and female animals. Specifically, we utilized the spontaneously hypertensive rat (SHR), one of the most extensively studied animal models of ADHD. Compared to their control, the Wistar-Kyoto (WKY) rat, male SHR are reported to exhibit several behavioral phenotypes associated with ADHD, including hyperactivity, impulsivity, and poor sustained attention, along with impairments in learning and memory. As the hippocampus is a key brain region for learning and memory, we examined the behavior of male and female SHR and WKY rats in two hippocampal-dependent memory tasks. Our findings revealed that SHR have delay-dependent working memory deficits that were similar to, albeit less severe than, those seen in hippocampal-lesioned rats. We also observed impairments in elapsed time processing in female SHR, particularly in the discrimination of longer time durations. To investigate the impact of inflammatory signaling on memory in these rats, we analyzed the levels of several cytokines in the dorsal and ventral hippocampus of SHR and WKY. Although we found some sex and genotype differences, concentrations were generally similar between groups. Taken together, our results indicate that SHR exhibit deficits in spatial working memory and memory for elapsed time, as well as some differences in hippocampal cytokine concentrations. These findings contribute to a better understanding of the neurobiological basis of ADHD in both sexes and may inform future research aimed at developing effective treatments for the disorder. Nonetheless, the potential mediating role of neuroinflammation in the memory symptomatology of SHR requires further investigation.

Comparative Effects of Intra-Articular versus Intravenous Mesenchymal Stromal Cells Therapy in a Rat Model of Osteoarthritis by Destabilization of Medial Meniscus.

Transplanted mesenchymal stromal cells (MSCs) exhibit a robust anti-inflammatory and homing capacity in response to high inflammatory signals, as observed in studies focused on rheumatic diseases that target articular cartilage (AC) health. However, AC degradation in osteoarthritis (OA) does not necessarily coincide with a highly inflammatory joint profile. Often, by the time patients seek medical attention, they already have damaged AC. In this study, we examined the therapeutic potential of a single bone marrow MSC transplant (2 × 106 cells/kgbw) through two different routes: intra-articular (MSCs-IAt) and intravenous (MSCs-IVt) in a preclinical model of low-grade inflammatory OA with an established AC degeneration. OA was induced through the destabilization of the medial meniscus (DMM) in female Wistar Kyoto rats. The animals received MSCs 9 weeks after surgery and were euthanized 4 and 12 weeks post-transplant. In vivo and ex vivo tracking of MSCs were analyzed via bioluminescence and imaging flow cytometry, respectively. Cytokine/chemokine modulation in serum and synovial fluid was measured using a multiplex panel. AC degeneration was quantified through histology, and hindlimb muscle balance was assessed with precision weighing. To our knowledge, we are the first group to show the in vivo (8 h) and ex vivo (12 h) homing of cells to the DMM-OA joint following MSCs-IVt. In the case of MSCs-IAt, the detection of cellular bioluminescence at the knee joint persisted for up to 1 week. Intriguingly, intra-articular saline injection (placebo-IAt) resulted in a worse prognosis of OA when compared to a non-invasive control (placebo-IVt) without joint injection. The systemic cytokines/chemokines profile exhibited a time-dependent variation between transplant routes, displaying a transient anti-inflammatory systemic response for both MSCs-IVt and MSCs-IAt. A single injection of MSCs, whether administered via the intra-articular or intravenous route, performed 9 weeks after DMM surgery, did not effectively inhibit AC degeneration when compared to a non-invasive control.

Autoantibodies against laminin-521 are pathogenic in anti-glomerular basement membrane disease

Anti-glomerular basement membrane (anti-GBM) disease is an organ-specific autoimmune disorder characterized by autoantibodies against GBM components. Evidence from human inherited kidney diseases and animal models suggests that the α, β, and γ chains of laminin-521 are all essential for maintaining the glomerular filtration barrier. We previously demonstrated that laminin-521 is a novel autoantigen within the GBM and that autoantibodies to laminin-521 are present in about one-third of patients. In the present study, we investigated the pathogenicity of autoantibodies against laminin-521 with clinical and animal studies. Herein, a rare case of anti-GBM disease was reported with circulating autoantibodies binding to laminin-521 but not to the NC1 domains of α1-α5(IV) collagen. Immunoblot identified circulating IgG from this patient bound laminin α5 and γ1 chains. A decrease in antibody levels was associated with improved clinical presentation after plasmapheresis and immunosuppressive treatments. Furthermore, immunization with laminin-521 in female Wistar-Kyoto rats induced crescentic glomerulonephritis with linear IgG deposits along the GBM, complement activation along with infiltration of T cells and macrophages. Lung hemorrhage occurred in 75.0% of the rats and was identified by the presence of erythrocyte infiltrates and hemosiderin-laden macrophages in the lung tissue. Sera and kidney-eluted antibodies from rats immunized with laminin-521 demonstrated specific IgG binding to laminin-521 but not to human α3(IV)NC1, while the opposite was observed in human α3(IV)NC1-immunized rats. Thus, our patient data and animal studies imply a possible independent pathogenic role of autoantibodies against laminin-521 in the development of anti-GBM disease.

Acute nitric oxide synthase inhibition induces greater increases in blood pressure in female versus male Wistar Kyoto rats.

Nitric oxide (NO) contributes to blood pressure (BP) regulation via its vasodilatory and anti-inflammatory properties. We and others previously reported sex differences in BP in normotensive and hypertensive rat models where females have lower BP than age-matched males. As females are known to have greater NO bioavailability than age-matched males, the current study was designed to test the hypothesis that anesthetized female normotensive Wistar Kyoto rats (WKY) are more responsive to acute NOS inhibition-induced increases in BP compared to male WKY. Twelve-week-old male and female WKY were randomized to infusion of the nonspecific NOS inhibitor NG -nitro-L-arginine methyl ester (L-NAME, 1 mg/kg/min) or selective NOS1 inhibition with vinyl-L-NIO (VNIO, 0.5 mg/kg/min) for 60 min. Mean arterial BP, glomerular filtration rate (GFR), urine volume, and electrolyte excretion were assessed before, and during L-NAME or VNIO infusion. L-NAME and VNIO significantly increased BP in both sexes; however, the increase in BP with L-NAME infusion was greater in females versus males compared to baseline BP values. Acute infusion of neither L-NAME nor VNIO for 60 min altered GFR in either sex. However, urine volume, sodium, chloride and potassium excretion levels increased comparably in male and female WKY with L-NAME and VNIO infusion. Our findings suggest sex differences in BP responses to acute non-isoform-specific NOS inhibition in WKY, with females being more responsive to L-NAME-induced elevations in BP relative to male WKY. However, sex differences in the BP response did not coincide with sex differences in renal hemodynamic responses to acute NOS inhibition.

Effect of chronic unpredictable stress in female Wistar-Kyoto rats subjected to progesterone withdrawal: Relevance for Premenstrual Dysphoric Disorder neurobiology

Premenstrual Dysphoric Disorder (PMDD) is related to an abrupt drop in progesterone and impairments in the HPA axis that cause anxiety. Suffering persons report higher daily-life stress and anxiety proneness that may contribute to developing PMDD, considered a chronic stress-related disorder. Here, we explored the effect of chronic unpredictable stress (CUS) in rats subjected to progesterone withdrawal (PW) and evaluated gene expression of HPA axis activation in the stress-vulnerable Wistar-Kyoto (WKY) rat strain that is prone to anxiety. Ovariectomized WKY rats were randomly assigned to CUS or Standard-housed conditions (SHC) for 30 days. To induce PW, animals received 2 mg/kg of progesterone on day 25th for 5 days; 24 h later, they were tested using the anxiety-like burying behavior test (BBT). After behavioral completion, rats were euthanized, and brains were extracted to measure Crh (PVN) and Nr3c1 (hippocampus) mRNA. Blood corticosterone and vasopressin levels were determined. Results showed that PW exacerbated anxiety-like behaviors through passive coping in CUS-WKY. PW decreased Crh-PVN mRNA and the Nr3c1-hippocampal mRNA expression in SHC. CUS decreased Crh-PVN mRNA compared to SHC, and no further changes were observed by PW or BBT exposure. CUS reduced Nr3c1-hippocampal gene expression compared to SHC animals, and lower Nr3c1 mRNA was detected due to BBT. The PW increased corticosterone in SHC and CUS rats; however, CUS blunted corticosterone when combined with PW+BBT and similarly occurred in vasopressin concentrations. Chronic stress blunts the response of components of the HPA axis regulation when PW and BBT (systemic and psychogenic stressors, respectively) are presented. This response may facilitate less adaptive behaviors through passive coping in stress-vulnerable subjects in a preclinical model of premenstrual anxiety.

Sex-specific respiratory and systemic endocrine effects of acute acrolein and trichloroethylene inhalation

Acrolein and trichloroethylene (TCE) are priority hazardous air pollutants due to environmental prevalence and adverse health effects; however, neuroendocrine stress-related systemic effects are not characterized. Comparing acrolein, an airway irritant, and TCE with low irritancy, we hypothesized that airway injury would be linked to neuroendocrine-mediated systemic alterations. Male and female Wistar-Kyoto rats were exposed nose-only to air, acrolein or TCE in incremental concentrations over 30min, followed by 3.5-hr exposure to the highest concentration (acrolein - 0.0, 0.1, 0.316, 1, 3.16ppm; TCE - 0.0, 3.16, 10, 31.6, 100ppm). Real-time head-out plethysmography revealed acrolein decreased minute volume and increased inspiratory-time (males>females), while TCE reduced tidal-volume. Acrolein, but not TCE, inhalation increased nasal-lavage-fluid protein, lactate-dehydrogenase activity, and inflammatory cell influx (males>females). Neither acrolein nor TCE increased bronchoalveolar-lavage-fluid injury markers, although macrophages and neutrophils increased in acrolein-exposed males and females. Systemic neuroendocrine stress response assessment indicated acrolein, but not TCE, increased circulating adrenocorticotrophic hormone, and consequently corticosterone, and caused lymphopenia, but only in males. Acrolein also reduced circulating thyroid-stimulating hormone, prolactin, and testosterone in males. In conclusion, acute acrolein inhalation resulted in sex-specific upper respiratory irritation/inflammation and systemic neuroendocrine alterations linked to hypothalamic-pituitary-adrenal axes activation, which is critical in mediating extra-respiratory effects.

Noninvasive Monitoring of Blood Pressure and Heart Rate during Estrous Cycle Phases in Normotensive Wistar-Kyoto and Spontaneously Hypertensive Female Rats.

Since 2015, the National Institutes of Health has called for its funded preclinical research to include both male and female subjects. However, much of the basic animal research that has studied heart rate and blood pressure in the past has used male rats. Male rats have been preferred for these studies to avoid the possible complicating effects of the female estrous cycle. The aim of the current study was to determine whether blood pressure and heart rates vary as a function of the estrous cycle phase of young normotensive Wistar-Kyoto (WKY) and Spontaneously Hypertensive (SHR) female rats. Blood pressure and heart rate were measured at the same time of day throughout the estrous cycle by using a noninvasive tail cuff sphygmomanometric technique. As expected, 16-wk-old female SHR rats had higher blood pressure and heart rates than did age-matched female WKY rats. However, no significant differences in mean, systolic, or diastolic arterial blood pressure or heart rate were detected across the different stages of the estrous cycle in either strain of female rats. Consistent with previous reports, heart rates were higher and showed less variation in the hypertensive SHR female rats as compared with the normotensive WKY female rats. These results indicate that studies measuring blood pressure and heart rate can include young female SHR and WKY rats with no effect of estrous cycle stage.

Cannabinoid Receptor 2 Blockade Prevents Anti-Depressive-like Effect of Cannabidiol Acid Methyl Ester in Female WKY Rats.

The pathophysiology of major depressive disorder (MDD) is diverse and multi-factorial, yet treatment strategies remain limited. While women are twice as likely to develop the disorder as men, many animal model studies of antidepressant response rely solely on male subjects. The endocannabinoid system has been linked to depression in clinical and pre-clinical studies. Cannabidiolic Acid-Methyl Ester (CBDA-ME, EPM-301) demonstrated anti-depressive-like effects in male rats. Here, we explored acute effects of CBDA-ME and some possible mediating mechanisms, using a depressive-like genetic animal model, the Wistar-Kyoto (WKY) rat. In Experiment 1, Female WKY rats underwent the Forced swim test (FST) following acute CBDA-ME oral ingestion (1/5/10 mg/kg). In Experiment 2, Male and female WKY rats underwent the FST after injection of CB1 (AM-251) and CB2 (AM-630) receptor antagonists 30 min before acute CBDA-ME ingestion (1 mg/kg, males; 5 mg/kg, females). Serum levels of Brain-Derived Neurotrophic Factor (BDNF), numerous endocannabinoids and hippocampal Fatty Acid Amide Hydrolase (FAAH) levels were assessed. Results indicate that females required higher doses of CBDA-ME (5 and 10 mg/kg) to induce an anti-depressive-like effect in the FST. AM-630 blocked the antidepressant-like effect in females, but not in males. The effect of CBDA-ME in females was accompanied by elevated serum BDNF and some endocannabinoids and low hippocampal expression of FAAH. This study shows a sexually diverse behavioral anti-depressive response to CBDA-ME and possible underlying mechanisms in females, supporting its potential use for treating MDD and related disorders.

COVID-19: Understanding the impact of anti-hypertensive drugs and hydroxychloroquine on the ACE1 and ACE2 in lung and adipose tissue in SHR and WKY rats.

Hypertensive individuals taking anti-hypertensive drugs from renin-angiotensin system inhibitors may exhibit a more severe evolution of the disease when contracting the SARS-CoV-2 virus (COVID-19 disease) due to potential increases in ACE2 expression. The study investigated ACE1 and ACE2 axes and hydroxychloroquine in the lungs and adipose tissue of male and female normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHRs). SHRs were treated with losartan (10mg/kg/day) or captopril (10mg/kg/day) for 14 days or 7 days with hydroxychloroquine (200 mg/kg/day) in drinking water. WKY rats were also treated for 7 days with hydroxychloroquine. Blood pressure (BP), protein, and mRNA expression of ACE1 and ACE2 were analyzed in serum, adipose, and lung tissues. Losartan and captopril reduced BP in both sexes in SHR, whereas hydroxychloroquine increased BP in WKY rats. Losartan reduced ACE2 in serum and lungs in both sexes and in adipose tissue of male SHRs. Captopril decreased ACE2 protein in the lung of females and in adipose tissue in both sexes of SHRs. Hydroxychloroquine decreased ACE1 and ACE2 proteins in the lungs in both sexes and adipose tissue in male SHRs. In female WKY rats, ACE2 protein was lower only in the lungs and adipose tissue. Losartan effectively inhibited ACE2 in male and captopril in female SHRs. Hydroxychloroquine inhibited ACE2 in male SHRs and female WKY rats. These results further our understanding of the ACE2 mechanism in patients under renin-angiotensin anti-hypertensive therapy and in many trials using hydroxychloroquine for COVID-19 treatment and potential sex differences in response to drug treatment.

Dysregulation of BDNF and PI3K/Akt signaling in the brain of female Wistar-Kyoto rats exposed to chronic mild stress

Dysregulation of BDNF and PI3K/Akt signaling in the brain of female Wistar-Kyoto rats exposed to chronic mild stress

Stress-Induced Changes in Trophic Factor Expression in the Rodent Urinary Bladder: Possible Links With Angiogenesis.

Substantive evidence supports a role of chronic stress in the development, maintenance, and even enhancement of functional bladder disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). Increased urinary frequency and bladder hyperalgesia have been reported in rodents exposed to a chronic stress paradigm. Here, we utilized a water avoidance stress (WAS) model in rodents to investigate the effect of chronic stress on vascular perfusion and angiogenesis. Female Wistar-Kyoto rats were exposed to WAS for 10 consecutive days. Bladder neck tissues were analyzed by western immunoblot for vascular endothelial growth factor (VEGF) and nerve growth factor precursor (proNGF). Vascular perfusion was assessed by fluorescent microangiography followed by Hypoxyprobe testing to identify regions of tissue hypoxia. The expression of VEGF and proNGF in the bladder neck mucosa was significantly higher in the WAS rats than in the controls. There was a trend toward increased vascular perfusion, but without a statistically significant difference from the control group. The WAS rats displayed a 1.6-fold increase in perfusion. Additionally, a greater abundance of vessels was observed in the WAS rats, most notably in the microvasculature. These findings show that chronic psychological stress induces factors that can lead to increased microvasculature formation, especially around the bladder neck, the region that contains most nociceptive bladder afferents. These findings may indicate a link between angiogenesis and other inflammatory factors that contribute to structural changes and pain in IC/BPS.

SACUBITRIL/VALSARTAN IMPROVES SEXUAL FUNCTION AND FIBROSIS OF THE CLITORAL AND VAGINAL TISSUES IN FEMALE SPONTANEOUSLY HYPERTENSIVE RATS

Objective: Female sexual dysfunction is common in hypertension. The effects of sacubitril/valsartan (SAC/VAL) as a potential therapy for hypertension and heart failure have not been studied in relation to sexual function and genital fibrosis in female spontaneously hypertensive rats (SHRs). To evaluate the effect of SAC/VAL on sexual function and genital fibrosis in female SHRs. Design and method: Thirty female SHRs were administered VAL (30 mg/kg/day), SAC/VAL (60 mg/kg/day) or saline. Ten normotensive female Wistar-Kyoto (WKY) rats were included in the control group. We assessed estrous cyclicity and sexual behavior in the female rats. In addition, the morphology of clitoral and vaginal tissues was evaluated by histological analyses. Western blotting and enzyme-linked immunosorbent assays were used to assess the levels of fibrotic markers in vaginal and clitoral tissues. Furthermore, the protein levels of phosphatase and tensin homolog deleted from chromosome 10 (PTEN), phosphoinositide-3-kinase (PI3K) and AKT expression were measured by western blotting. We investigated the effect of SAC/VAL on the fibrosis of vaginal and clitoral tissues, estrus cyclicity and sexual behavior, including sexual receptivity (lordosis quotient and intensity), plus the number of proceptive and aggressive events. Results: SAC/VAL treatment improved hypertension-induced sexual dysfunction, exhibited as a prolonged estrus phase, increased receptivity and proceptive events, and decreased aggressive events, compared to those of VAL treatment and control SHRs without treatments. In addition, SAC/VAL-treated SHRs had lower levels of fibrotic markers than the levels of VAL-treated SHRs or SHRs without treatment. Moreover, SAC/VAL decreased p-PTEN expression and increased p-PI3K and p-AKT expression at the protein level compared to those in VAL treatment alone. Conclusions: SAC/VAL treatment is superior to VAL treatment in attenuating sexual dysfunction and fibrosis of vaginal and clitoral tissues, which may be related to the PTEN/PI3K/AKT pathway.

RHO KINASE BLOCKADE ALLEVIATE BLOOD PRESSURE ELEVATION AND INHIBIT VASCULAR REMODELING IN APATINIB-INDUCED HYPERTENSION TO BE CONFERMED

Objective: Hypertension is one of the major adverse effects of tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factors. However, the mechanism underlying TKIs-induced hypertension remains unclear. Here, we evaluated BP responses at different doses of apatinib, a TKI used for anti-tumor therapy, in normotensive Wistar–Kyoto rats. Furthermore, we explored the role of the RhoA/Rho kinase (ROCK) emodellin pathway in elevation of blood pressure (BP) induced by apatinib, a selective TKI approved in China for treatment of advanced or metastatic gastric cancer. A non-specific ROCK inhibitor, Y27632, was then combined with apatinib and its efficacy in alleviating apatinib-induced hypertension was evaluated. Design and method: Normotensive female Wistar–Kyoto rats were exposed to two different doses of apatinib, or apatinib combined with Y27632, or vehicle for two weeks. BP was monitored by a tail-cuff plethysmography system. The mRNA levels and protein expression in the RhoA/ROCK pathway were determined, and vascular emodelling assessed. Results: Administration of either a high or low dose of apatinib was associated with a rapid rise in BP, reaching a plateau after 12 days. Apatinib treatment mediated upregulation of RhoA and ROCK 2 in the mid-aorta, more significant in the high dose group. However, ROCK1 expression showed no statistically significant differences. Furthermore, the mRNA level of GRAF3 decreased dose-dependently. Apatinib administration was also associated with decreased levels of MLCP, and elevated endothelin-1 (ET-1) and collagen 1, which was accompanied with increased mid-aortic media. However, treatment with Y27632 attenuated the above changes. Conclusions: Results of the present study indicated that apatinib treatment increased BP and promoted vascular emodelling by activating the RhoA/ROCK emodellin pathway. However, Y27632, a non-specific ROCK inhibitor, reversed apatinib-induced increase in BP and vascular emodelling. Therefore, RhoA/ROCK activation could be the underlying mechanism of apatinib-induced hypertension, while ROCK inhibitors could be potential therapeutic drugs.

Estrogen Metabolite 2-Methoxyestradiol Attenuates Blood Pressure in Hypertensive Rats by Downregulating Angiotensin Type 1 Receptor.

The therapeutic potential of 2-Methoxyestradiol (2ME2) is evident in cardiovascular disease. Our laboratory has previously demonstrated the mechanism involved in the 2ME2 regulation of angiotensin type 1 receptor (AT1R) in vitro. However, 2ME2 regulation of angiotensin receptors and its effects on blood pressure (BP) and resting heart rate (RHR) are uncertain. In this study, male and female Wistar-Kyoto (WKY) rats infused with angiotensin II (65 ng/min) and male spontaneously hypertensive rats (SHR) were surgically implanted with telemetric probes to continuously assess arterial BP and RHR. In both male and female WKY rats, 2ME2 treatment (20 mg/kg/day for 2 weeks) resulted in a significant reduction of Ang II-induced systolic, diastolic, and mean arterial BP. Moreover, significant weight loss and RHR were indicated in all groups. In a separate set of experiments, prolonged 2ME2 exposure in male SHR (20 mg/kg/day for 5 weeks) displayed a significant reduction in diastolic and mean arterial BP along with RHR. We also found downregulation of angiotensin receptors and angiotensinogen (AGT) in the kidney and liver and a reduction of plasma Ang II levels. Collectively, we demonstrate that 2ME2 attenuated BP and RHR in hypertensive rats involves downregulation of angiotensin receptors and body weight loss.

Delayed Minocycline Treatment Ameliorates Hydrocephalus Development and Choroid Plexus Inflammation in Spontaneously Hypertensive Rats.

Hydrocephalus is a complicated disorder that affects both adult and pediatric populations. The mechanism of hydrocephalus development, especially when there is no mass lesion present causing an obstructive, is poorly understood. Prior studies have demonstrated that spontaneously hypertensive rats (SHRs) develop hydrocephalus by week 7, which was attenuated with minocycline. The aim of this study was to determine sex differences in hydrocephalus development and to examine the effect of minocycline administration after hydrocephalus onset. Male and female Wistar–Kyoto rats (WKYs) and SHRs underwent magnetic resonance imaging at weeks 7 and 9 to determine ventricular volume. Choroid plexus epiplexus cell activation, cognitive deficits, white matter atrophy, and hippocampal neuronal loss were examined at week 9. In the second phase of the experiment, male SHRs (7 weeks old) were treated with either saline or minocycline (20 mg/kg) for 14 days, and similar radiologic, histologic, and behavior tests were performed. Hydrocephalus was present at week 7 and increased at week 9 in both male and female SHRs, which was associated with greater epiplexus cell activation than WKYs. Male SHRs had greater ventricular volume and epiplexus cell activation compared to female SHRs. Minocycline administration improved cognitive function, white matter atrophy, and hippocampal neuronal cell loss. In conclusion, while both male and female SHRs developed hydrocephalus and epiplexus cell activation by week 9, it was more severe in males. Delayed minocycline treatment alleviated hydrocephalus, epiplexus macrophage activation, brain pathology, and cognitive impairment in male SHRs.

Rho kinase inhibition ameliorates vascular remodeling and blood pressure elevations in a rat model of apatinib-induced hypertension.

Objectives:Hypertension is one of the major adverse effects of tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factors. However, the mechanism underlying TKIs-induced hypertension remains unclear. Here, we explored the role of the RhoA/Rho kinase (ROCK) signaling pathway in elevation of blood pressure (BP) induced by apatinib, a selective TKI approved in China for treatment of advanced or metastatic gastric cancer. A nonspecific ROCK inhibitor, Y27632, was then combined with apatinib and its efficacy in alleviating apatinib-induced hypertension was evaluated.Methods:Normotensive female Wistar–Kyoto rats were exposed to two different doses of apatinib, or apatinib combined with Y27632, or vehicle for 2 weeks. BP was monitored by a tail-cuff plethysmography system. The mRNA levels and protein expression in the RhoA/ROCK pathway were determined, and vascular remodeling assessed.Results:Administration of either a high or low dose of apatinib was associated with a rapid rise in BP, reaching a plateau after 12 days. Apatinib treatment mediated upregulation of RhoA and ROCK II in the mid-aorta, more significant in the high-dose group. However, ROCK I expression showed no statistically significant differences. Furthermore, the mRNA level of GRAF3 decreased dose-dependently. Apatinib administration was also associated with decreased levels of MLCP, and elevated endothelin-1 (ET-1) and collagen I, which were accompanied with increased mid-aortic media. However, treatment with Y27632 attenuated the above changes.Conclusion:These findings suggest that activation of the RhoA/ROCK signaling pathway could be the underlying mechanism of apatinib-induced hypertension, while ROCK inhibitor have potential therapeutic value.

Abstract 11662: Extracellular Vesicle (EV)-Derived PiRNA, ImEX-Pi, is Protective in a Rat Model of Ischemia/Reperfusion Injury

Introduction: Cardiosphere-derived cells (CDCs ) are a population of heart-derived progenitors with demonstrated therapeutic efficacy in preclinical and clinical settings. CDCs exert their effect through the secretion of EVs, lipid-bilayer nanoparticles laden with bioactive molecules. Among the bioactive cargo identified in these immortalized CDC (imCDC)-derived EVs (imCDC-EV) are piwi RNAs (PiRNAs). PiRNAs bind Piwi proteins (members of the argonaute family) and regulate both the epigenome and transcriptome. The role of piRNAs (in particular EV-derived piRNAs) in modulating gene expression remains undescribed. Here we describe the role of a PiRNA highly enriched in imCDC-EVs (imEX-Pi). Methods: EVs were prepared by 24 hours hypoxic conditioning followed by normoxic recovery for three cycles. EVs were isolated from conditioned media using ultrafiltration. Fractions were analyzed for particle size, concentration and piRNA content. In vitro, bone marrow derived-macrophages (BMDM) were exposed to imCDC-EV, imEX-Pi and vehicle for transcriptomic analysis. In vivo, myocardial ischemia-reperfusion (I/R) injury was induced in 8-10-week-old Wistar-Kyoto female rats by LAD coronary ligation for 45 minutes followed by reperfusion. Twenty minutes later, imCDC-EV, imEX-Pi, scramble or vehicle were injected into the left ventricle with an aortic cross-clamp. Cardiac Troponin I levels, infarct size and circulating monocytes were assessed at 24 and 48 hrs post I/R. Results: RNA sequencing showed that piRNAs are enriched in both CDC-EV and imCDC-EV. ImEX-Pi was one of the most highly-expressed non-coding RNAs. In vitro , imEX-Pi altered the transcriptome of BMDMs compared to vehicle. This included upregulation of inflammatory and cell death pathways. In a rat model of I/R injury, imEX-Pi reduced infarct size and cardiac troponin levels compared to vehicle- and scrambled- treated animals. ImEX-Pi also significantly decreased circulating monocytes at 24 hours followed by a significant increase at 48hrs. Conclusions: We show that imEX-Pi is a key mediator of imCDC-EV therapeutic efficacy and that the principle target cells may be macrophages. This is further rationalized by the critical role that macrophages play in the injury and resolution response.