Alzheimer’s disease (AD) is characterized by brain amyloid-β plaque formation, neuroinflammation and neurodegeneration, which lead to cognitive impairment (CI) and disruption of circadian rhythm. Bioactive steroidal Na/K-ATPase inhibitor marinobufagenin (MBG) modulates neuroinflammation. In 16-mo old double transgenic APPswe/PS1dE9 AD mice with advanced AD, treatment with MBG reduced mRNA expression of inflammatory markers. Here, we investigated whether treatment with MBG at early-stage AD may impact AD manifestation in this AD mouse model. Five months old male AD mice (n = 15) and WT mice (n = 28) were administered MBG (100 µg/day/kg body weight) (AD-MBG, n = 8; WT-MBG, n = 14) or vehicle for control (AD-C, n = 7; WT-C, n = 14) via subcutaneous ALZET osmotic minipumps for 3 months. At 8-mo of age, the mice underwent turn-based discrimination learning in a water T-maze (WTM) to assess procedural learning and 72-hour home cage activity (HCA) analysis to assess circadian rhythm. The hippocampal inflammatory and AD mRNAs (by qPCR) and MBG levels (by immunoassay) were measured. MBG administration increased plasma MBG levels in WT and AD mice (Table 1). AD-C mice exhibited greater home cage activity disruption vs. WT-C (Fig.1a); in WTM, AD-C required more trials to learn the turn-based task than WT-C (Fig. 1b). Though statistically insignificant, trends showed that MBG numerically worsened activity disruption by HCA and decreased ability to learn turn-based task by WTM in WT mice, while numerically improving both measures in AD mice. During the reversal phase of WTM where response requirements were reversed, WT-C mice performed significantly better than AD-C mice, measured by the cumulative amount of time spent in the incorrect arm until reaching criterion performance (p = 0.038). Similar patterns of numerical improvement in AD-MBG compared to AD-C are shown (Fig. 1c). Hippocampal inflammatory and AD markers mRNAs were upregulated in AD-C vs. WT-C, and MBG treatment downregulated the expression of TNF and GFAP in AD mice (Table 1). Behavioral testing results illustrate CI and circadian impairment in 8-mo old AD mice compared to WT. Numerical trends show a potential protective effect of MBG on AD CI likely via downregulation of hippocampal TNF and GFAP genes. 0Supported by the NIH/NIA Intramural Research Program

Cardiovascular diseases (CVD), including increased central arterial stiffness (CAS), accompany chronic kidney disease (CKD) development. An increase in pulse wave velocity (PWV), an index for CAS, is implicated in age-associated changes in the brain and potentiates the development of CI in CVD and CKD in humans and animal models. The aim of this study was to determine whether inducing CKD accelerates the development of CI and CAS in aged male and female rats. Ten months-old male and female Sprague-Dawley rats were fed with 0.25% adenine diet to accelerate CKD (n = 10-12; CKD-male, CKD-female) or regular diet (n = 8-10; CNT-male, CNT-female) for 8weeks. Body weight (BW), blood pressure (BP), heart rate (HR), aortic-PWV (aPWV), behavioral tests, blood urea nitrogen (BUN), creatinine and hematocrit were assessed at the endpoint. The anxiety-like behavior was tested in open field test (OFT) and elevated plus maze (EPM). Morris water maze (MWM) and cross maze (CM) were used to test spatial memory. The data were analyzed using two-way ANOVA. CKD development in both sexes was accompanied by kidneys enlargement, increase in BUN and creatinine, and reduction in hematocrit. In CKD-male the kidney function was more compromised with higher levels of BUN and creatinine vs. CKD-female (Table 1). CKD-male had lower BW and SBP vs. CNT-male (Table 1). CKD induced a reduction in HR, increase in aPWV and aortic weight in both sexes vs. respective CNT-groups (Figure 1). Both CKD-male and CKD-female demonstrated a tendency of increase in path length to find the hidden platform in MWM, e.g., both sexes had numerically impaired spatial memory. CKD-male spent less time in the center of OFT, a measure of anxiety, and loss of spatial memory, with a reduction of spontaneous alternation task assessed by CM. CKD-female exhibited higher level of anxiety spending less time in open arm of EPM vs. CNT-female (Figure 2). CKD development in old male and female Sprague-Dawley rats was accompanied by an increase in CAS, cardiovascular remodeling, and cognitive dysfunction. In CKD, CI affected different cognitive domains in the old males than in the age-matched females. Supported by NIA/NIH/IRP

Photobiological modulation (PBM) therapy, a form of low-dose light therapy, is beneficial in treating various disease conditions including Alzheimer’s disease (AD). Double-transgenic mice (APPswe/PS1dE9) develop progressive accumulation of amyloid-β (Aβ) plaques, cognitive impairment, and alterations in cardiovascular structure and function, and represent a model of AD. Prior studies have shown that PBM therapy reduces the size and number of Aβ plaques in the neocortex and hippocampus in the mouse models of AD. The aim of this study was to investigate the effects of PBM on cardiovascular structure and function and cognitive function in the aged AD mice. Six-month old female AD (APPswe/PS1dE9; AD-PBM, n = 8) and wild type mice (WT-PBM, n = 8) were exposed to near-infrared light (wavelength 850 nm, 4.5 J/cm 2 , 3min/day, 5 days/week) for 6 months. Control mice (AD-CNT, n = 6; WT-CNT, n = 6) were placed under the PBM apparatus without turning the light on for 3min/day, 5 days/week. Cardiac parameters were measured by echocardiography. Gait analysis (DigiGait), home cage activity (HCA), and spatial working memory (indicated by alternation rate in cross maze test, CM) were assessed after 6 months of treatment. Group differences were identified by two-way ANOVA. In HCA test, the AD-CNT mice exhibited circadian locomotor hyperactivity at night-time relative to WT-CNT mice (Figure 1A, B), which was reduced by PBM. AD-CNT mice had lower alternation rates in CM, indicating spatial working memory impairment. PBM had minimal effect on this parameter (Table 1). The AD-CNT vs. WT-CNT mice manifested differences in several gait parameters, specifically, they demonstrated a lower fore propel time than WT-CNT. PBM normalized this parameter in AD-PBM mice to the level of WT-CNT (Figure 1C). Both WT-PBM and AD-PBM mice had improved cardiovascular structure, which was demonstrated by lower relative wall thickness of left ventricle (LV) and LV mass, and higher LV volume at the end of diastole in PBM-treated vs. correspondent CNT groups (Table 1, Figure 2). PBM improved cardiovascular structure and AD-associated cognitive impairment in the APP/PS1 mouse model. It is possible that more effective cardiovascular structure and function contributes to Aβ removal from the brain after PBM treatment. Supported by NIA/NIH/IRP

Introduction: Blood pressure (BP) begins to increase early in life in Dahl salt-sensitive rats (DSS) that consume a normal salt (NS) diet. Epidemiologic studies have shown that early vascular aging (EVA) is associated with central arterial stiffening (CAS) and hypertension, which develop earlier in life than expected in general population and contribute to cognitive impairment later in life. Hypotheses: (i) DSS rats will develop EVA earlier in life on NS diet, and with advancing age will exhibit early CV aging; (ii) In DSS, CV remodeling and CAS are implicated in memory decline. We tested these hypotheses using a longitudinal repeated measure design in DSS and their parental breed Sprague-Dawley rats (SD) at 3- & 12-mo of age. Methods: Male SD and DSS were kept on NS diet (0.5% NaCl; n=8/group) for entire experiment. Repeated measures of systolic BP (SBP), pulse wave velocity (PWV; an index of CAS), echocardiography, and non-repeated measures of Morris water maze (MWM) to test spatial memory, aortic collagen, elastin (histochemistry), and left ventricle (LV) mRNA expression (qPCR) were assessed at 3- & 12-mo. Data analyses: linear mixed-effect 2-way ANOVA, t-test & linear regression (LR) modeling. A 2-sided p<0.05 was considered significant. Results: SBP, PWV, aortic weight, wall thickness and collagen/elastin ratio were higher in DSS-3 vs. SD-3, and in DSS-12 vs. DSS-3. RWT and LV expression of pro-fibrotic, inflammatory and senescence genes were higher in DSS-12 vs. DSS-3 and vs. SD-12 (Table). DSS-12 spent more time to find a hidden platform in MWM vs. SD-12; their impaired spatial hippocampal memory was positively associated with PWV and LV mass by LR analysis. Conclusions: EVA, indexed as higher BP, PWV and aortic remodeling was associated with early CV aging, manifested by activation of LV senescence, inflammatory and pro-fibrotic genes and by higher rates of age-associated changes in BP, RWT and LV mass in DSS vs. SD. In DSS, early CV aging was associated with memory decline.

The prevalence of cognitive impairment (CI) and dementia in end-stage chronic kidney disease (CKD) has been estimated at 30-60%. Cardiovascular dysfunction accompanies CKD and contributes to CI in humans and animal models. Since CKD occurs more often in females in clinical studies, the aim of this study was to determine whether cardiovascular and renal remodeling is associated with cognitive performance in the female rats with CKD. Four-month-old female Sprague-Dawley rats were fed with 0.25% adenine diet to induce CKD (n = 19) or a regular control diet (n = 16; CTRL) for 8weeks. Body weight (BW), blood pressure (BP), heart rate (HR), blood urea nitrogen (BUN), sodium, potassium, creatinine, hematocrit, estradiol, and behavioral tests were assessed at the end of the study. The level of anxiety was tested in open field test (OFT) and elevated plus maze (EPM). Spatial memory was tested by the ability to find a hidden platform in Morris water maze (MWM). The data were analyzed by two-tailed unpaired t-test and linear regression analysis (LRA). CKD was associated with higher creatinine and BUN, lower hematocrit, and enlarged hearts, aortae and kidneys vs. CTRL (Table 1). There was no difference in BP, estradiol, sodium and potassium, MWM, OFT and EPM performance between the groups. However, the LRA revealed the association of higher heart weights with a reduced spatial memory and higher anxiety level in both CTRL and CKD (Table 2; Figure 1A,B). The association of higher aortic weight with a reduced spatial memory, and higher kidney weight with higher anxiety levels was demonstrated in CKD only (Table 2; Figure 1C,D). Estradiol was associated with better spatial memory in CKD (LRA: R 2 = 0.264; p = 0.04). Anxiety-like behavior and spatial memory were associated with cardiovascular remodeling in young female rats with CKD. We suggest that (1) CKD stimulates development of cardiovascular remodeling, which may affect blood supply of cortex and hippocampus, responsible for anxiety and spatial memory, and (2) estradiol may contribute to a better cognitive performance in the young CKD female rats. The future direction is to investigate whether CKD influences the trajectory of neurocognitive and cardiovascular aging. Supported by NIA/NIH/IRP

Alzheimer’s disease (AD) is characterized by brain amyloid beta plaque formation, neuroinflammation and neuronal degradation, which lead to cognitive impairment and decline in normal circadian rhythm (CR). Bioactive steroid marinobufagenin (MBG) modulates neuroinflammation. In 16‐mo old double transgenic APPswe/PS1dE9 AD mice with advanced AD, amyloid precursor protein (APP) and interleukin 6 (IL6) mRNAs were upregulated vs. wild type (WT) control, and treatment with MBG reduced mRNA expression. Here, we investigated whether treatment with MBG at early‐stage AD may impact AD development in this AD mouse model.

The prevalence of cognitive impairment (CI) and dementia in end-stage chronic kidney disease (CKD) has been estimated at 30-60%. Cardiovascular diseases (CVD) including increased central arterial stiffness (CAS) accompany CKD development and contribute to CI. Elevated pulse wave velocity (PWV) is associated with higher mortality in CVD and CKD independently of blood pressure (BP). Marinobufagenin (MBG) is a pro-fibrotic factor, which increases in CKD and contributes to CAS. The aim of this study was to determine whether CKD potentiates CI development via CAS and cardiovascular remodeling in a rat model of CKD. Four months old male Sprague-Dawley rats were fed with 0.25% adenine (n=19; CKD group) diet to induce CKD or regular diet (n=16; CTRL group) for 8 weeks. Body weight (BW), BP, heart rate (HR), aortic PWV (aPWV), and behavioral tests were conducted at the end of the study. The level of anxiety was tested in an open field test (OFT). Morris water maze (MWM) was used to test spatial memory. Blood was collected for measurements of plasma sodium, potassium, blood urea nitrogen (BUN), hematocrit and MBG. The data were analyzed using two-tailed unpaired t-test and linear regression. Animals in the CKD group had higher levels of plasma creatinine and BUN, which indicate the development of CKD, higher plasma sodium, potassium, and MBG, and higher aPWV, lower hemoglobin, hematocrit, BW, HR, enlarged hearts, aortae, and kidneys, vs. CTRL group (Table 1). BP was similar in both groups. .Rats in CKD group spent less time in the center of open field compared to their CTRL counterparts, indicating increased anxiety-like behavior (Figure 1 A, B). Although there was no difference in performance in MWM between the groups, there was a positive correlation between aortic weight and ability to find a hidden platform in the CKD group (Figure 1C). We demonstrated that CKD development in young Sprague-Dawley rats was accompanied by increased CAS, estimated as aPWV, which developed in blood pressure-independent manner. Higher PWV was associated with a higher level of anxiety, and aortic tissue remodeling was associated with spatial memory in CKD model. Supported by NIH/NIA IRP

Double-mutant mice (APPswe/PS1dE9; 2xTg-AD) represent a model of Alzheimer’s disease (AD). 2xTg-AD mice develop progressive accumulation of amyloid plaques and cognitive impairment (CI) with age in >12 months old animals. The aim of the present study was to investigate the associations of CI and cardiovascular function in 2xTg-AD mice. Aortic pulse wave velocity (aPWV), an estimation of aortic stiffness, and cardiac parameters were measured by echocardiography in 16-mo old male 2xTg-AD (n=11) and wild type (WT; n=17) mice. Spatial memory (using Morris water maze; MWM) and the anxiety level (using open field test; OFT) were assessed. The relationship between cardiovascular and behavioral parameters were modeled using a linear regression analysis (LRA) with a backward elimination. P-values <0.05 were considered significant, and 0.05≤P<0.1 were considered near significant and were included in the models. 2xTg-AD mice exhibited an increase in aPWV and had a thicker left ventricle (LV) posterior wall in diastole (LVPWd) and narrower LV internal diameter (LVIDd) vs. WT mice (Table 1). 2xTg-AD mice spent less time in the center of OF vs. WT mice (Table 1). LRA revealed a negative association between time spent in the center of OF and LVPWd, and a positive association with LV volume in diastole (LVvold) in 2xTg-AD mice (Table 2). In MWM, 2xTg-AD mice exhibited impaired spatial memory estimated as prolonged average escape latency during their training vs. WT counterparts (Table 1). Although there was no difference between performance in the MWM probe trial, LRA showed a negative association between LVPWd and the mean distance from the platform in the probe trial in 2xTg-AD mice, and a positive association of LVPWd with number of entries to the platform zone (Table 2). There was a positive association between aPWV and mean distance from the platform in the probe trial in both 2xTg-AD and WT mice. LRA results indicate that LV remodeling is associated with anxiety-like behavior in 2xTg-AD mice, but not with spatial memory impairment. Positive association of aPWV with spatial memory impairment in both groups suggests that aortic stiffness contributes to the age-associated CI. Supported by the NIH/NIA IRP.

Cardiovascular diseases and hypertension are the major contributors to the pathogenesis of vascular dementia. The Dahl salt‐sensitive (DSS) rat model is characterized by age‐associated cardiovascular remodeling and the development of central arterial stiffness (CAS) in the presence of renin‐angiotensin system disbalance, accompanied by cognitive decline. We hypothesized, that the anti‐hypertensive treatment, antagonist for angiotensin II receptors losartan (LOS), via reducing CAS, will improve cardiovascular parameters and cognitive function in aged DSS male rats.

Dementia is characterized by progressive cognitive impairment (CI), which is accompanied by decline in reasoning, planning and memory. Cardiovascular diseases are the major contributors to the pathogenesis of vascular dementia and affect mainly the aging population. Even on a normal salt diet, Dahl salt-sensitive (DSS) rats exhibit age-associated hypertension and CI. We hypothesized, that anti-hypertensive treatment with the angiotensin II receptor blocker losartan (LOS) would improve cardiovascular and cognitive function in aged DSS rats. Male DSS rats (n=30) were kept on a normal salt diet (0.5% NaCl) for the duration of the study. Baseline measurements were taken at 6-mo of age. After that, treatment with 30mg/kg/day of LOS, added to the drinking water (n=14), or control (n=16) was started and continued for 6-mo. Systolic blood pressure (SBP), aortic pulse wave velocity (aPWV), a measure of aortic stiffness, and behavioral testing in a visually cued reaction task to assess attention and inhibitory control were assessed every 3-mo. Elevated plus maze (EPM) and Morris Water Maze (MWM) tests were performed at 12-mo of age to measure anxiety-like behavior and to evaluate spatial memory, respectively. Data were analyzed by 2-way repeated measures ANOVA or t-tests as applicable. Prior to the treatment, LOS and control groups did not display differences in any parameter analyzed. Control rats exhibited gradual increase in SBP and aPWV from 6 to 12-mo of age. Following 3- and 6-mo of treatment, LOS rats exhibited lower SBP and aPWV vs. controls (Table 1). At 12-mo of age, LOS rats had improved performance in the reaction task (Figure 1), and exhibited lower anxiety levels, spending less time on the closed arm in EPM and 1.5-fold more time in the open arm vs. control rats. LOS rats also spent less time to find a hidden platform in the MWM vs. controls, indicating that LOS treatment improved spatial memory (Table 1). Improvement of cardiovascular function and reduction in aortic stiffness by LOS were associated with better attentional control and memory and lower anxiety levels, demonstrating an association of CI with cardiovascular changes in the DSS rat model of vascular dementia.

Previous studies implicated cardiotonic steroids, including Na/K-ATPase inhibitor marinobufagenin (MBG), in the pathogenesis of preeclampsia (PE). Recently, we demonstrated that (i) MBG induces fibrosis in rat tissues via a mechanism involving Fli1, a negative regulator of collagen-1 synthesis, and (ii) MBG sensitive Na/K-ATPase inhibition is reversed by mineralocorticoid antagonists. We hypothesized that in human PE elevated MBG level is associated with the development of fibrosis of the umbilical arteries and that this fibrosis can be attenuated by canrenone. Fifteen patients with PE (mean BP = 118 ± 4 mmHg; 34 ± 2 years; 38 ± 0.3 weeks gest. age) and twelve gestational age-matched normal pregnant subjects (mean BP = 92 ± 2 mmHg; 34 ± 1 years; 39 ± 0.2 weeks gest. age) were enrolled in the study. PE was associated with a higher plasma MBG level, with a four-fold decrease in Fli1 level and a three-fold increase in collagen-1 level in the PE umbilical arteries vs. those from the normal subjects (p < 0.01). Isolated rings of umbilical arteries from the subjects with PE exhibited impaired responses to the relaxant effect of sodium nitroprusside vs. control vessels (EC50 = 141 nmol/L vs. EC50 = 0.9 nmol/L; p < 0.001). The effects of PE on Fli1 and collagen-1 were blocked by the in vitro treatment of umbilical arteries by 10 μmol/L canrenone. Similar results were obtained for umbilical arteries pretreated with MBG. These data demonstrate that elevated MBG level is implicated in the development of the fibrosis of umbilical arteries in PE, and that this could be blocked by mineralocorticoid antagonists.

Objective: Cardiovascular diseases are the major contributors to the pathogenesis of vascular dementia. The Dahl salt sensitive (Dahl-S) rat model is characterized by the development of central arterial stiffness (CAS), hypertension and cognitive decline with age on a normal salt diet. We hypothesized that treatment with losartan (LOS), angiotensin II type 1 receptor (AT1R) blocker, via reduction of blood pressure (BP) and CAS, will improve cognitive function in aged Dahl-S rats. Design and method: Male Dahl-S rats (n = 30) were kept on a normal salt diet (0.5% NaCl) for the duration of the study. Baseline measurements were taken at 3- and/or 6-mo of age following by the treatment with LOS in drinking water (30 mg/kg/day, n = 14) or control treatment (n = 16) for 6-mo. Measurements were taken every 3 months and included systolic and diastolic BP (SBP, DBP), pulse wave velocity (PWV), a measure of CAS, left ventricular posterior wall thickness in diastole (LVPWd), fractional shortening (FS) and LV mass (by echocardiography). At 12-mo of age the rats were tested in operant chambers using a simple reaction task to assess attention and impulsivity, and in elevated plus maze (EPM) to assess anxiety-like behavior. Statistical analyses were performed using 2-way ANOVA and t-test. Data are presented as mean ± SEM. Results: Prior to the treatment, LOS and control groups did not display differences in any parameter analyzed. Following the treatment, LOS rats had lower SBP, DBP, PWV, thinner LVPWd, higher FS and smaller LV mass at 9- and 12-mo of age vs. control group (Fig.1A-F). In the reaction task, LOS rats demonstrated more correct responses and fewer premature responses. The LOS rats exhibited lower anxiety level, because they spent more time in the open arm of EPM vs. control rats (Fig.1G,H). Conclusions: Beneficial effect of LOS on cognitive function in aged male Dahl-S rats was associated with the improvement of cardiovascular function and remodeling. The CAS stabilizing and BP reduction by LOS treatment was associated with improved attentional performance, lower impulsivity and lower anxiety. The mechanistic basis of these effects of LOS on cognition via cerebrovascular and brain changes will be further investigated.

The hypertensive response in Dahl salt-sensitive (DSS) rats on a high-salt (HS) diet is accompanied by central arterial stiffening (CAS), a risk factor for dementia, and heightened levels of a prohypertensive and profibrotic factor, the endogenous Na/K-ATPase inhibitor marinobufagenin (MBG). We studied the effect of the in vivo administration of MBG or HS diet on blood pressure (BP), CAS, and behavioral function in young DSS rats and normotensive Sprague–Dawley rats (SD), the genetic background for DSS rats. Eight-week-old male SD and DSS rats were given an HS diet (8% NaCl, n = 18/group) or a low-salt diet (LS; 0.1% NaCl, n = 14–18/group) for 8 weeks or MBG (50 µg/kg/day, n = 15–18/group) administered via osmotic minipumps for 4 weeks in the presence of the LS diet. The MBG-treated groups received the LS diet. The systolic BP (SBP); the aortic pulse wave velocity (aPWV), a marker of CAS; MBG levels; spatial memory, measured by a water maze task; and tissue collection for the histochemical analysis were assessed at the end of the experiment. DSS-LS rats had higher SBP, higher aPWV, and poorer spatial memory than SD-LS rats. The administration of stressors HS and MBG increased aPWV, SBP, and aortic wall collagen abundance in both strains vs. their LS controls. In SD rats, HS or MBG administration did not affect heart parameters, as assessed by ECHO vs. the SD-LS control. In DSS rats, impaired whole-heart structure and function were observed after HS diet administration in DSS-HS vs. DSS-LS rats. MBG treatment did not affect the ECHO parameters in DSS-MBG vs. DSS-LS rats. The HS diet led to an increase in endogenous plasma and urine MBG levels in both SD and DSS groups. Thus, the prohypertensive and profibrotic effect of HS diet might be partially attributed to an increase in MBG. The prohypertensive and profibrotic functions of MBG were pronounced in both DSS and SD rats, although quantitative PCR revealed that different profiles of profibrotic genes in DSS and SD rats was activated after MBG or HS administration. Spatial memory was not affected by HS diet or MBG treatment in either SD or DSS rats. Impaired cognitive function was associated with higher BP, CAS, and cardiovascular remodeling in young DSS-LS rats, as compared to young SD-LS rats. MBG and HS had similar effects on the cardiovascular system and its function in DSS and SD rats, although the rate of change in SD rats was lower than in DSS rats. The absence of a cumulative effect of increased aPWV and BP on spatial memory can be explained by the cerebrovascular and brain plasticity in young rats, which help the animals to tolerate CAS elevated by HS and MBG and to counterbalance the profibrotic effect of heightened MBG.

In end-stage chronic kidney disease (CKD) the prevalence of cognitive impairment (CI) has been estimated at 30-60%. Cardiovascular diseases (CVD) including increased central arterial stiffness (CAS) accompany CKD development. CAS drives pulse pressure (PP) and pulsatile component index (PCI) increases and cerebral microvascular damage resulting in a reduction of blood supply to the brain hereby contributing to CI. Elevated PP and PCI are associated with higher mortality in CVD and CKD independently of BP. The aim of the study was to determine whether CKD potentiates CI development due to CAS and the resultant increase in the PCI, a major contributor to brain and renal microvasculature damage.Measurements of systolic and diastolic blood pressure (SBP, DBP), echocardiography (ECHO), plasma amyloid beta-40 (Aβ-40), BUN, creatinine, aspartate aminotransferase (AST), brain natriuretic peptide (BNP), and mini-mental status examination (MMSE) were performed in 25 patients with stage IV CKD and in 22 age- and sex-matched healthy controls (Table 1). PCI was calculated as a function of SBP and DBP. The data were analyzed by unpaired two-tailed t-test, Pearson correlation with P values adjusted for false discovery rate, and are presented as mean±SEM.CKD patients had higher SBP, PP, PCI, LV systolic and diastolic volume, lower LV ejection fraction, higher plasma BNP and AST (CVD markers), plasma Aβ-40 (Alzheimer's disease marker), BUN and creatinine (CKD markers) and lower score in MMSE vs. healthy control (Table 1). Cognitive test results positively correlated with LV stroke volume, negatively correlated with PCI, a measurement of pulsatility of BP, and negatively correlated with plasma ASP, a marker of CVD (Table 2). SBP and DBP did not correlate with cognitive parameters. Further investigation of the possible metabolic role of Aβ-40 in the development of CI in CKD patients, and whether plasma AST can be a marker of CI in CKD, will be assessed in the future studies.Higher PP, PCI and lower score in MMSE in CKD patients supports our hypothesis that cardiovascular remodeling in CKD is a key component of CI progression. Supported by the NIH Grant 1R15HL150721 (K.S.), and in part by NIH/NIA Intramural Research Program.

Background Na/K-ATPase is a keystone enzyme in all living cells. Steroidal inhibitor of Na/K-ATPase, marinobufagenin (MBG), participates in regulating cardiac function, vessel architecture, intracellular signaling transduction, and anti-neuroinflammatory response. The aged double-transgene APPswe/PS1dE9 mice, a model of Alzheimer's disease (AD), exhibit lower levels of urine MBG compared to wild-type (WT) mice, impairments in cognitive function and amyloid β (Aβ) pathologies. Compromised blood supply to the brain may contribute to AD development and Aβ pathologies. Here we examined Aβ pathology in the cerebral and central vasculature of aged AD mice and whether MBG treatment affects neuroinflammation, neurodegeneration, and cognitive function. Methods To aged male AD (n=8; 15-mo old) and age-matched WT mice (n=4) MBG (100 µg/day/kg body weight) was administered via subcutaneous ALZET minipumps for 3-mo. Age-matched control mice received saline (AD-C, n=8; WT-C, n=4). Systolic blood pressure (SBP; by tail cuff plethysmography), cognitive flexibility (reversal learning in a water T-maze), plasma (for MBG measurement), brain and tissue samples (for qPCR, 3D green light laser microscopy and immunohistochemistry analysis) were collected at 18-mo. Data was analyzed by 2-way ANOVA and presented as mean±SE. Results In aged AD mice, we observed granulated Aβ plaques and Aβ localization around small cerebral blood vessels (Fig 1B; white arrows). Aβ protein was also found in significantly larger abundance in aortae vessel wall of old AD mice vs. WT mice (Fig 2; blue stain). SBP was not affected by treatment and did not differ between AD and WT mice. Endogenous plasma MBG was lower in AD-C vs. WT-C (125±21 vs. 223±41 pmol/L; p<0.05). Plasma MBG was elevated in both WT-MBG and AD-MBG (483±116 and 657±176 pmol/L; p<0.05 vs. WT-C and AD-C, correspondently). Hippocampal mRNA expression of inflammatory marker IL6 was upregulated 1.5-fold in AD-C vs. WT-C (p<0.05); MBG reduced this expression in AD-MBG vs. AD-C (p<0.01). Hippocampal mRNA expression of amyloid precursor protein (APP) was upregulated 2.6-fold in AD-C vs. WT-C (p<0.01); MBG reduced this expression in AD-MBG vs. AD-C (p<0.01). AD mice showed normal learning for a turn-based discrimination rule but were impaired during the reversal phase vs. WT mice. MBG treatment did not affect the amount of Aβ plaque load in the brains of AD-MBG vs. AD-C. Conclusion Old AD mice exhibited lower endogenous plasma MBG, impaired cognitive flexibility, higher hippocampal IL6 and APP mRNAs, and Aβ accumulation in cerebral vessels and aortae relative to WT counterparts. MBG treatment significantly reduced hippocampal APP and inflammatory mRNA markers; however, it had no effect on existing Aβ load. In future experiments, we will examine how MBG treatment affects the formation of brain Aβ plaques in younger AD mice.

Background Aged Dahl Salt-Sensitive (DSS) rats develop central arterial stiffness (CAS) and hypertension on a normal salt (NS) diet. These changes have effects on cognition via brain structural changes associated with CAS. Previous results showed that ACE inhibitor lisinopril (Lis) improved cardiovascular and cognitive function in male DSS rats. Here we determined whether these effects of Lis on physiology and behavior could be achieved in age-matched female DSS rats. Methods Female DSS rats (n=23) were kept on a NS diet (0.5% NaCl) for the duration of the study. Baseline (BL) measurements were taken at 3- and 6-mo of age. After 6-mo measurements, rats were given either Lis in drinking water (15mg/kg/day, n=12) or control treatment (n=11). Final measurements were taken after 7-mo of Lis treatment, before tissues were collected for histochemistry using Verhoeff's stain for collagen and elastin. Systolic blood pressure (SBP) was measured using tail-cuff plethysmography. Pulse wave velocity (PWV), a measure of CAS, was measured by echocardiography. Open field test (OFT) was performed to assess anxiety-like behavior, and Morris water maze (MWM) was performed to assess spatial memory function. Rats were tested in an operant chambers attention challenge to assess attention and impulsivity. Statistical analyses were performed using Pearson correlation and 2-way ANOVA mixed effects model. Data are presented as mean ± SEM. Results At 3 and 6-mo of age, the treatment groups did not display differences in SBP or PWV. Following treatment, the Lis treated rats had lower SBP vs. controls (116 ± 3.7 vs. 187 ± 9.5 mmHg; p<0.01). Similarly, PWV was lower at 13-mo in treated vs. control rats (4.4 ± 0.3 vs. 5.7 ± 1.0 m/s; p<0.05) (Fig. 1A-B). Histochemical analysis revealed that the aortic media collagen abundance, a measure of fibrosis, was lower (18.5 ± 0.9% vs. 21.5 ± 0.8%, p<0.05) and the elastin/collagen ratio in the aortic media of treated rats was higher (2.5 ± 0.1 vs. 2.0 ± 0.1, p<0.05) than that of controls (Fig. 1C-D). Performance on the OFT at 13-mo compared to 6-mo BL indicated that the treated group had an increase in activity in the center of the field with age (+25% vs. BL) i.e., lower anxiety levels, while in control rats there was an opposite effect (-32% vs. BL) (Fig. 2A). PWV was negatively correlated with distance traveled in the center of the OFT at 13-mo (R=-0.672, P<0.05). MWM results showed that treated rats traveled a shorter distance to a hidden platform compared to control rats (6.5 ± 0.7 vs. 8.8 ± 0.9 m; p=0.039), indicating better spatial memory in the treated group (Fig. 2B). In the attention test at 13-mo of age, treated rats had more correct and fewer premature responses vs. control rats (Fig. 2C-D). Conclusion Female DSS rats show beneficial effects of Lis on cardiovascular and cognitive function. Lower SBP, reduced aortic fibrosis, and stabilized PWV in Lis treated female DSS rats were associated with improved behavioral test performance. The mechanistic basis of the effects of Lis on cognition via cerebrovascular and brain changes is being further investigated.

Background: Hypertension and cardiovascular disease may be significant contributors to dementia and Alzheimer’s disease. However, the mechanism of this process is poorly understood. Central arterial stiffness (CAS) is accelerated by systemic hypertension and may be accompanied by increased stiffness in the cerebral arteries. Methods & Results: We used Dahl salt sensitive rats (Dahl-S) which develop CAS and hippocampal memory decline as they develop variable degrees of hypertension on a 0.5% salt diet with age. At 22 weeks, systolic blood pressure (SBP), pulse wave velocity (PWV), open field tests (OFT), hippocampal N-acetylaspartate (NAA) concentrations and blood flow (BF) data were all collected. The rats displayed moderate hypertension as compared to a 3 months baseline (SBP: 165±16 vs. 142±5 mmHg; P<0.01, t-test). SBP and PWV are positively correlated (a). PWV was negatively correlated with hippocampal BF (b), and positively correlated with hippocampal NAA (c). Ratio of central rearing duration to total rearing duration in the OFT, a negative measure of anxiety, positively correlated with hippocampal BF (d). Conclusion: In the Dahl-S model of hypertension, greater PWV, a marker for CAS, was associated with decreased hippocampal perfusion. Relationship between CAS and hippocampal BF was as hypothesized. Likewise, hippocampal BF correlates with hippocampal NAA concentration as expected. The positive correlation between hippocampal BF and increased central rearing duration suggests that higher cerebral BF is associated with a lower anxiety level. Supported entirely by the Intramural research Program of the NIH, National institute on Aging.

AbstractBackgroundDahl salt‐sensitive rats (DSS) represent a model of vascular dementia, because they demonstrate an age‐associated increase in blood pressure (BP) and central arterial stiffness (CAS) accompanied by cognitive decline independent of a high salt intake. We hypothesized that treatment with an anti‐hypertensive drug ACE inhibitor Lisinopril will lower CAS and improve cognitive function in the adult hypertensive DSS.MethodMale DSS were kept on a normal salt diet for 12 months. Six‐month old rats received vehicle (control; DSS‐C; n = 20) or Lisinopril (15 mg/kg/day; DSS‐LIS; n = 20) for 6‐mo. Systolic BP (SBP), pulse wave velocity (PWV), a marker of CAS (by Doppler echocardiography), open field test (OFT) to assess anxiety‐like behavior and locomotion, and cued reaction time task (CRTT) to assess executive functioning were performed at BL (before treatment), after 3 and 6‐mo of treatment. Morris Water maze (MWM) test was performed to assess hippocampal spatial memory. Aortae were stained for elastin and collagen (immunohistochemistry). Statistical analysis: 2‐way ANOVA repeated measures and t‐test; data presented as mean ± SEM.ResultAll non‐treated DSS were hypertensive. DSS‐LIS have lower SBP vs. BL and vs. age‐matched DSS‐C. PWV was lower in 12‐mo DSS‐LIS vs. BL and vs. age‐matched DSS‐C (Table). In MWM, DSS‐LIS had a higher path efficiency and traveled shorter distance to find the invisible platform vs. DSS‐C. In CRTT, DSS‐LIS had less premature responses than DSS‐C 3‐mo post treatment (Table). There was no change in total distance and distance traveled in the center in OFT at all time points in DSS‐LIS vs. DSS‐C (Table). 12‐mo old DSS‐LIS had lower heart and aortic weight, less aortic medial wall collagen and greater elastin/collagen ratio vs. DSS‐C (Table).ConclusionChronic treatment with Lisinopril effectively lowered SBP, CAS and reduced aortic collagen in aged hypertensive DSS rats. Lisinopril resulted in improved hippocampal spatial memory and reduced impulsive behavior demonstrating a beneficial cognitive effect in the DSS model of vascular dementia. Further studies will need to elucidate the effect of Lisinopril on cerebral blood vessels and whether ACE inhibitor has a direct effect of the brain in addition to its effect on CAS. Supported by the Intramural Research Program of the NIA/NIH.

BackgroundNeurons rely on Na/K‐ATPase (NKA) to create the electrochemical gradient that allows them to fire action potentials. The NKA also acts as a receptor for cardiotonic steroids (CTS), which have been shown to participate in intracellular signaling transduction, cardiovascular function, and have potential neuroprotective effects. Despite the importance of the NKA and CTS in brain physiology and pathology, their potential roles in Alzheimer’s disease (AD) pathology are not well studied. Marinobufagenin (MBG) is a bufadienolide CTS, and its potential neuroprotective effect has never been studied. MBG levels were significantly lower in 15‐month old double‐mutant Alzheimer’s mice (APPswe/PS1dE9; 2xTg‐AD; AD) compared to age‐matched, non‐transgenic controls. The present study examined whether the treatment of AD mice by MBG affects cardiovascular and behavioral parameters.MethodsMale AD (n=16) and wild type (WT; n=8) mice (15‐mo old) were included in the experimental design. Mice were administered MBG (100 μg/day/kg body weight; AD‐MBG, n=8; WT‐MBG, n=4) or vehicle (control: AD‐C, n=8; WT‐C, n=4) treatment using subcutaneous ALZET osmotic minipumps over the course of 12 weeks before data (Systolic blood pressure (SBP; by tail cuff plethysmography), open field test behavior (OFT), and plasma, tissues samples) was collected. Data was analyzed by 2‐way ANOVA and t‐test and presented as mean±SEM.ResultsSBP did not differ between WT‐C and AD‐C mice. AD mice weighed more than WT (P<0.05). MBG did not affect either parameters (Table 1). WT‐MBG mice had higher heart rates than WT‐C mice (Table 1, P<0.05). Plasma MBG was lower in AD‐C mice vs. WT‐C (125±21 pmol/L vs. 223±41 pmol/L, P<0.05, t‐test). MBG increased plasma levels of this steroid in both WT‐MBG and AD‐MBG mice, which indicated the efficiency of the drug delivery (Table 1). AD‐C mice had 1.6‐fold higher brain cortical expression of inflammatory marker interleukin‐6 (IL6) mRNA vs. WT‐C; MBG significantly reduced cortical mRNA IL6 expression in AD‐MBG mice (Fig 1). No differences were found for total distance traveled, nor for time spent in the center during first 90 seconds of OFT between AD‐C and WT‐C, and between AD‐MBG and AD‐C. Distance traveled in the center showed an effect of strain (P<0.05), an effect of treatment (P<0.05), and a trend‐level treatment:strain interaction (P<0.1). WT traveled farther in the center of the field vs. AD mice. MBG treated mice traveled less in the center vs. non‐treated mice. MBG caused a greater reduction in distance traveled in the center for WT mice than it did for AD mice (Fig 1).ConclusionAD mice had lower endogenous plasma MBG and higher brain pro‐inflammatory mRNA marker IL6 vs. WT mice. Treatment with MBG significantly reduced levels of this inflammatory marker. WT mice exhibited more exploratory behavior than AD mice in the OFT; whereas, MBG decreased exploratory behavior. Exploratory behavior in WT‐MBG mice was more affected than AD‐MBG mice. The association of the brain inflammatory response, cognitive functions and MBG will be further explored in this AD mouse model.Support or Funding InformationSupported by the NIH/NIA Intramural Research ProgramEffect of MBG treatment on expression of IL6 mRNA and OFT performance during first 90 seconds of task. IL6 mRNA (a), time spent in center (b), distance traveled in center (c), and total distance traveled (d) in wild type (WT) and 2xTg‐AD (AD) mice. By unpaired T‐test (a) †P<0.05, WT vs. AD, #P<0.05 AD‐C vs AD‐MBG; by 2‐way ANOVA Linear Mixed Effects followed by Tukey post‐test (b–d): *P<0.05 WT vs. AD; ##P<0.05 Vehicle vs. MBG: drug x strain interaction: P=0.06.Figure 1 Clinical, physiological and behavioral parameters in 15 months old AD mice and the wild type control mice with and without 12‐week treatment with MBG WT AD Control (n=4) MBG (n=4) Control (n=8) MBG (n=8) Body weight, g 35.0 ± 1.3 34.5 ± 1.8 38.1 ± 1.6 * 38.1 ± 0.5 SBP, mm Hg 98.2 ± 4.3 100.0 ± 4.3 91.6 ± 1.0 103.6 ± 5.9 HR, beats/min 457 ± 11 594 ± 4 513 ± 29 557 ± 19 # Plasma MBG, pmol/L 223 ± 41 483 ± 116 125 ± 21 † 657 ± 176 # OFT distance center, m 0.44 ± 0.14 0.11 ± 0.04 0.17 ± 0.04 * 0.15 ± 0.03 # OFT distance total, m 3.66 ± 0.48 2.56 ± 0.47 2.37 ± 0.48 2.79 ± 1.09 OFT time center, s 4.48 ± 1.84 2.48 ± 1.44 3.01 ± 1.38 0.92 ± 0.38 Data presented as Mean ± SEM. By 2‐way ANOVA followed by Tukey post‐test: *P<0.05, AD vs. WT; #P<0.05. MBG vs. control. By unpaired t‐test: †P<0.05. AD vs. WT. For OFT distance center, effect of strain: P=0.03: effect of drug, P=0.02; drug x strain interaction: P=0.06. Post‐hoc: WT‐MBG HR higher than WT‐C HR (P<0.05). WT, wild type mice: AD, 2xTg‐AD mice; MBG, marinobufagenin; SBP, systolic blood pressure; HR, heart rate: OFT, openfield test.