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Premenopausal females are protected from angiotensin II (ANG II)-induced hypertension following the adoptive transfer of T cells from normotensive donors. For the present study, we hypothesized that the transfer of hypertensive T cells (HT) or splenocytes (HS) from hypertensive donors would eliminate premenopausal protection from hypertension. Premenopausal recombination-activating gene-1 (Rag-1)(−/−) females received either normotensive (NT) or hypertensive cells 3 wk before ANG II infusion (14 days, 490 ng/kg/min). Contrary to our hypothesis, no increase in ANG II-induced blood pressure was observed in the NT/ANG or HT/ANG groups. Flow cytometry demonstrated that renal FoxP3(+) T regulatory cells were significantly decreased, and immunohistochemistry showed an increase in renal F4/80(+) macrophages in the HT/ANG group, suggesting a shift in the renal inflammatory environment despite no change in blood pressure. Renal mRNA expression of macrophage chemoattractant protein-1 (MCP-1), endothelin-1 (ET-1), and G protein-coupled estrogen receptor-1 (GPER-1) was significantly decreased in the HT/ANG group. The adoptive transfer of hypertensive splenocytes before ANG II infusion (HS/ANG) eliminated premenopausal protection from hypertension and significantly decreased splenic FoxP3(+) T regulatory cells compared with females that received normotensive splenocytes (NS/ANG). Expression of macrophage inflammatory protein 1α/chemokine (C-C motif) ligand 3 (MCP-1/CCL3), a potent macrophage chemokine, was elevated in the HS/ANG group; however, no increase in renal macrophage infiltration occurred. Together, these data show that in premenopausal females, T cells from hypertensive donors are not sufficient to induce robust ANG II-mediated hypertension; in contrast, transfer of hypertensive splenocytes (consisting of T/B lymphocytes, dendritic cells, and macrophages) is sufficient. Further work is needed to understand how innate and adaptive immune cells and estrogen signaling coordinate to cause differential hypertensive outcomes in premenopausal females. NEW & NOTEWORTHY Our study is the first to explore the role of hypertensive T cells versus hypertensive splenocytes in premenopausal protection from ANG II-induced hypertension. We show that the hypertensive status of T cell donors does not impact blood pressure in the recipient female. However, splenocytes, when transferred from hypertensive donors, significantly increased premenopausal recipient blood pressure following ANG II infusion, highlighting the importance of further investigation into estrogen signaling and immune cell activation in females.

The transition from lean to obese states involves systemic metabolic remodeling that impacts insulin sensitivity, lipid partitioning, inflammation, and glycemic control. Here, we have taken a pharmacological approach to test the role of a nutrient-regulated chromatin modifier, lysine-specific demethylase (LSD1), in obesity-associated metabolic reprogramming. We show that systemic administration of an LSD1 inhibitor (GSK-LSD1) reduces food intake and body weight, ameliorates non-alcoholic fatty liver disease (NAFLD), and improves insulin sensitivity and glycemic control in mouse models of obesity. GSK-LSD1 has little effect on systemic metabolism of lean mice, suggesting LSD1 has a context-dependent role in promoting maladaptive changes in obesity. Analysis of insulin target tissues identified white adipose tissue as the major site of insulin sensitization by GSK-LSD1, where it reduces adipocyte inflammation and lipolysis. We demonstrate that GSK-LSD1 reverses NAFLD in a non-hepatocyte-autonomous manner, suggesting an indirect mechanism via inhibition of adipocyte lipolysis and subsequent effects on lipid partitioning. Pair-feeding experiments further revealed that effects of GSK-LSD1 on hyperglycemia and NAFLD are not a consequence of reduced food intake and weight loss. These findings suggest that targeting LSD1 could be a strategy for treatment of obesity and its associated complications including type 2 diabetes and NAFLD. O_FIG O_LINKSMALLFIG WIDTH=189 HEIGHT=200 SRC="FIGDIR/small/469928v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@64cf24org.highwire.dtl.DTLVardef@5b6aa4org.highwire.dtl.DTLVardef@1b64594org.highwire.dtl.DTLVardef@1922175_HPS_FORMAT_FIGEXP M_FIG C_FIG

There is a sharp rise in cardiovascular disease (CVD) risk and progression with the onset of menopause. The 4-vinylcyclohexene diepoxide (VCD) model of menopause recapitulates the natural, physiological transition through perimenopause to menopause. We hypothesized that menopausal female mice were more susceptible to CVD than pre- or perimenopausal females. Female mice were treated with VCD or vehicle for 20 consecutive days. Premenopausal, perimenopausal, and menopausal mice were administered angiotensin II (ANG II) or subjected to ischemia-reperfusion (I/R). Menopausal females were more susceptible to pathological ANG II-induced cardiac remodeling and cardiac injury from a myocardial infarction (MI), while perimenopausal, like premenopausal, females remained protected. Specifically, ANG II significantly elevated diastolic (130.9 ± 6.0 vs. 114.7 ± 6.2 mmHg) and systolic (156.9 ± 4.8 vs. 141.7 ± 5.0 mmHg) blood pressure and normalized cardiac mass (15.9 ± 1.0 vs. 7.7 ± 1.5%) to a greater extent in menopausal females compared with controls, whereas perimenopausal females demonstrated a similar elevation of diastolic (93.7 ± 2.9 vs. 100.5 ± 4.1 mmHg) and systolic (155.9 ± 7.3 vs. 152.3 ± 6.5 mmHg) blood pressure and normalized cardiac mass (8.3 ± 2.1 vs. 7.5 ± 1.4%) compared with controls. Similarly, menopausal females demonstrated a threefold increase in fibrosis measured by Picrosirus red staining. Finally, hearts of menopausal females (41 ± 5%) showed larger infarct sizes following I/R injury than perimenopausal (18.0 ± 5.6%) and premenopausal (16.2 ± 3.3, 20.1 ± 4.8%) groups. Using the VCD model of menopause, we provide evidence that menopausal females were more susceptible to pathological cardiac remodeling. We suggest that the VCD model of menopause may be critical to better elucidate cellular and molecular mechanisms underlying the transition to CVD susceptibility in menopausal women. NEW & NOTEWORTHY Before menopause, women are protected against cardiovascular disease (CVD) compared with age-matched men; this protection is gradually lost after menopause. We present the first evidence that demonstrates menopausal females are more susceptible to pathological cardiac remodeling while perimenopausal and cycling females are not. The VCD model permits appropriate examination of how increased susceptibility to the pathological process of cardiac remodeling accelerates from pre- to perimenopause to menopause.

Although it is known that the prevalence and severity of hypertension increases in women after menopause, the contribution of T cells to this process has not been explored. Although the immune system is both necessary and required for the development of angiotensin II (ANG II) hypertension in men, we have demonstrated that premenopausal women are protected from T cell-mediated hypertension. The goal of the current study was to test the hypotheses that 1) female protection against T cell-mediated ANG II hypertension is eliminated following progression into menopause and 2) T regulatory cells (Tregs) provide premenopausal protection against ANG II-induced hypertension. Menopause was induced in Rag-1−/− mice (via 4-vinylcyclohexene diepoxide), and all mice received a 14-day ANG II infusion. Donor CD3+ T cells were adoptively transferred 3 wk before ANG II infusion. In the absence of T cells, systolic blood pressure responses to ANG II were similar to those seen in premenopausal mice (Δ12 mmHg). After adoptive transfer of T cells, ANG II significantly increased systolic blood pressure in postmenopausal females (Δ28 mmHg). A significant increase in F4/80 positive renal macrophages, an increase in renal inflammatory gene expression, along with a reduction in renal expression of mannose receptor C-type 1, a marker for M2 macrophages, accompanied the increase in systolic blood pressure (SBP). Flow cytometric analysis identified that Tregs were significantly decreased in the spleen and kidneys of Rag-1−/− menopausal mice versus premenopausal females, following ANG II infusion. In a validation study, an anti-CD25 antibody was used to deplete Tregs in premenopausal mice, which induced a significant increase in SBP. These results demonstrate that premenopausal protection against T cell-mediated ANG II hypertension is eliminated once females enter menopause, suggesting that a change in hormonal status upregulates macrophage-induced proinflammatory and T cell-dependent responses. Furthermore, we are the first to report that the presence of Tregs are required to suppress ANG II hypertension in premenopausal females. NEW & NOTEWORTHY Whether progression into menopause eliminated female protection against T cell-mediated hypertension was examined. Menopausal mice without T cells remained protected against angiotensin II (ANG II) hypertension; however, in the presence of T cells, blood pressure responses to ANG II increased significantly in menopause. Underlying mechanisms examined were anti-inflammatory protection provided by T regulatory cells in premenopausal females and renal inflammatory processes involving macrophage infiltration and cytokine activation.

T cells are required for the development of hypertension in male and postmenopausal female mice while premenopausal females are protected from T cell mediated hypertension. To better understand sex differences in immune-cell mediated hypertensive responses, we sought to determine if there were any significant differences in the immune cell profiles of premenopausal female (F), VCD-treated postmenopausal female (PMF), and male (M) mice. Spleens were collected from all mice and processed for flow cytometric analysis of T cell populations (n=8/group). Analysis of splenic T cell populations revealed no significant difference in the frequency of CD3+ or CD4+ T cells between groups (CD3+: F 33.4%, PMF 30.3%, M 30.2% of total lymphocytes, CD4+: F 64.8%, PMF 70.7%, M 67.5% of CD3+ cells). However, postmenopausal females had a significantly lower frequency of splenic CD8+ T cells compared to both males and premenopausal females (CD8+: F 27.9%, PMF 19.5%*, M 25.3% of CD3+ cells *p

Although it is known that the prevalence and severity of hypertension increases in women after menopause, the contribution of T cells to this process has not been explored. Although the immune system is both necessary and required for the development of angiotensin II (ANG II) hypertension in men, we have demonstrated that premenopausal women are protected from T cell-mediated hypertension. The goal of the current study was to test the hypotheses that 1) female protection against T cell-mediated ANG II hypertension is eliminated following progression into menopause and 2) T regulatory cells (Tregs) provide premenopausal protection against ANG II-induced hypertension. Menopause was induced in Rag-1(−/−) mice (via 4-vinylcyclohexene diepoxide), and all mice received a 14-day ANG II infusion. Donor CD3(+) T cells were adoptively transferred 3 wk before ANG II infusion. In the absence of T cells, systolic blood pressure responses to ANG II were similar to those seen in premenopausal mice (Δ12 mmHg). After adoptive transfer of T cells, ANG II significantly increased systolic blood pressure in postmenopausal females (Δ28 mmHg). A significant increase in F4/80 positive renal macrophages, an increase in renal inflammatory gene expression, along with a reduction in renal expression of mannose receptor C-type 1, a marker for M2 macrophages, accompanied the increase in systolic blood pressure (SBP). Flow cytometric analysis identified that Tregs were significantly decreased in the spleen and kidneys of Rag-1(−/−) menopausal mice versus premenopausal females, following ANG II infusion. In a validation study, an anti-CD25 antibody was used to deplete Tregs in premenopausal mice, which induced a significant increase in SBP. These results demonstrate that premenopausal protection against T cell-mediated ANG II hypertension is eliminated once females enter menopause, suggesting that a change in hormonal status upregulates macrophage-induced proinflammatory and T cell-dependent responses. Furthermore, we are the first to report that the presence of Tregs are required to suppress ANG II hypertension in premenopausal females. NEW & NOTEWORTHY Whether progression into menopause eliminated female protection against T cell-mediated hypertension was examined. Menopausal mice without T cells remained protected against angiotensin II (ANG II) hypertension; however, in the presence of T cells, blood pressure responses to ANG II increased significantly in menopause. Underlying mechanisms examined were anti-inflammatory protection provided by T regulatory cells in premenopausal females and renal inflammatory processes involving macrophage infiltration and cytokine activation.

Menopause, the cessation of menstrual cycling, is associated with an increase in risk for cardiovascular diseases, including hypertension. Currently, the average age of menopause is 51 and studies have found that the risk factors for postmenopausal diseases begin to develop during perimenopause, the 5- to 10-year period preceding the onset of menopause. Clinical studies have demonstrated that only 36% of postmenopausal women with hypertension have their blood pressure under control, despite taking antihypertensives. Women taking a monotherapy of a beta-blocker, an Angiotensin-converting enzyme (ACE) inhibitor, or a calcium channel blocker are less likely to have their blood pressure under control than women who take a diuretic alone. Discrepancies in disease pathology between pre- and postmenopausal women, and the cause of the reduced effectiveness of the major antihypertensive therapeutics remain unclear. This chapter highlights recent developments in our understanding of postmenopausal hypertension, including potential mechanistic pathways that may provide new treatment paradigms, including the activation of immune cells and inflammatory pathways in the postmenopausal kidney and vasculature.

Activation of T cell-dependent pro-inflammatory responses are required for Ang II hypertension in male mice. However, females are protected from T cell-mediated hypertension and may suppress hypertension by directly preventing Ang II-induced pro-inflammatory T cell activation. Here we sought to determine whether transferring T cells from hypertensive donor mice eliminates female protection against T cell-mediated hypertension. Splenic CD3 + T cells were transferred from normotensive (NT) or Ang II-hypertensive (HT) C57BL/6J male donors to female Rag-1 -/- (NT T cell female-NTF; HT T cell female-HTF) or male Rag-1 -/- (HT T cell male-HTM) recipient mice. Blood pressure was monitored (tail cuff) for 5 weeks post-transfer. Ang II (490ng/kg/min) was infused into recipient mice for 14 days during weeks 4 and 5 post-transfer (NTFA; HTFA; HTMA). Ang II significantly increased MAP in donor male mice (NT 114 vs HT 157 mmHg, p+ 4 mmHg*, HTF 88 + 6 mmHg*, NTFA 101 + 5 mmHg*, HTFA 103 + 5 mmHg*, HTMA 138 + 3 mmHg, *p0.05). However, regulatory T cells were significantly reduced in male recipients compared to all female groups (Foxp3: NTF 21.6%*, NTFA 22.2%*, HTF 22.8%*, HTFA 22.6%*, HTMA 15.3%, *p

There is extensive evidence that activation of the immune system is both necessary and required for the development of angiotensin II (Ang II)–induced hypertension in males. The purpose of this study was to determine whether sex differences exist in the ability of the adaptive immune system to induce Ang II–dependent hypertension and whether central and renal T-cell infiltration during Ang II–induced hypertension is sex dependent. Recombinant activating gene-1 ( Rag-1 ) –/– mice, lacking both T and B cells, were used. Male and female Rag-1 –/– mice received adoptive transfer of male CD3 + T cells 3 weeks before 14-day Ang II infusion (490 ng/kg per minute). Blood pressure was monitored via tail cuff. In the absence of T cells, systolic blood pressure responses to Ang II were similar between sexes (Δ22.1 mm Hg males versus Δ18 mm Hg females). After adoptive transfer of male T cells, Ang II significantly increased systolic blood pressure in males (Δ37.7 mm Hg; P Hg). Flow cytometric analysis of total T cells and CD4 + , CD8 + , and regulatory Foxp3 + -CD4 + T-cell subsets identified that renal lymphocyte infiltration was significantly increased in males versus females in both control and Ang II–infused animals ( P + -positive T cells in the subfornical organ region of the brain was increased in males when compared with that in females. These results suggest that female Rag-1 –/– mice are protected from male T-cell–mediated increases in Ang II–induced hypertension when compared with their male counterparts, and this protection may involve sex differences in the magnitude of T-cell infiltration of the kidney and brain.

Prior to menopause, women are protected against cardiovascular disease (CVD) compared to age-matched men; this protection is gradually lost after menopause. Loss of estrogen is detrimental; yet, estrogen replacement as cardioprotective remains controversial. Gonadectomized rodents demonstrate benefit of estrogen replacement for CVD. Human studies that show benefit of hormone replacement therapy are challenged by studies that do not show benefit. A novel model of menopause utilizing 4-vinylcyclohexene diepoxide (VCD) induces gradual ovarian failure, preserving the “perimenopause” transitional period and androgen secreting capacity of residual ovarian tissue. We hypothesize that menopausal females are hypersensitive to CVD-induced pathological cardiac remodeling. To address this hypothesis, we instigated menopause in 2 month-old females by daily (i.p.) injections of VCD (160mg/kg, 20 consecutive days); control mice received sesame oil as vehicle. Vaginal cytology was used to monitor estrous cycles and determine when cycling ceased. Mice were considered menopausal after 15 consecutive days in persistent diestrus, non-cycling. Female mice from cycling, perimenopausal and menopausal groups received the hypertensive agent angiotensin II (Ang II, 800 ng/kg/min via alzet s.q. mini-pump, 14 days). Menopause did not impact systolic blood pressure (SBP) measured via tail cuff (control: 115.1±6.0 mmHg, n=7; menopause: 115.0±5.2 mmHg, n=10). Ang II infusion induced a significant exacerbation of hypertension (SBP) in menopausal females (156.9±4.8 mmHg; n=10) compared to Ang II controls (114.7±5.0 mmHg; n=8). This hypersensitivity to Ang II-induced hypertension was attenuated by estrogen supplementation started during perimenopause (0.1 mg, 60 day release). In addition, menopausal females demonstrated worsened pathological cardiac remodeling measured by functional (echocardiography), cellular (myocardial fibrosis) and molecular (fetal gene program) assessments. Using a novel model of menopause (VCD) combined with infusion of the hypertensive agent Ang II, we demonstrated that menopausal mice are more susceptible, or hypersensitive to pathological cardiac remodeling compared to cycling and perimenopausal mice.

Prior to menopause, women are protected against cardiovascular disease (CVD) compared to age-matched men; this protection is gradually lost after menopause. The mechanisms responsible for loss of protection and rise in CVD onset are unknown. A novel model of menopause utilizing 4-vinylcyclohexene diepoxide (VCD) induces gradual ovarian failure, preserving the “perimenopause” transitional period and androgen secreting capacity of residual ovarian tissue. Our central hypothesis states that menopausal females are hypersensitive to CVD-induced pathological cardiac remodeling. To address this hypothesis, we instigated menopause in 2 month-old females by daily (i.p.) injections of VCD (160mg/kg, 20 consecutive days); control mice received sesame oil as vehicle. Vaginal cytology was used to monitor estrous cycles and determine when cycling ceased. Mice were considered menopausal after 15 consecutive days in persistent diestrus, non-cycling. Female mice from cycling, perimenopausal and menopausal groups were subjected to 30 minutes of ischemia followed by 2 days of reperfusion (I/R). Following reperfusion, hearts were rapidly excised, sectioned and double-stained with Evans Blue and triphenyltetrazolium chloride to define the area at risk (AAR) and infarct size as area of necrosis (AON). On average, menopausal females displayed a significant (p

In females, menopause, the cessation of menstrual cycling, is associated with an increase in risk for several diseases such as cardiovascular disease, osteoporosis, diabetes, the metabolic syndrome, and ovarian cancer. The majority of women enter menopause via a gradual reduction of ovarian function over several years (perimenopause) and retain residual ovarian tissue. The VCD mouse model of menopause (ovarian failure in rodents) is a follicle-deplete, ovary-intact animal that more closely approximates the natural human progression through perimenopause and into the postmenopausal stage of life. In this review, we present the physiological parameters of how to use the VCD model and explore the VCD model and its application into the study of postmenopausal disease mechanisms, focusing on recent murine studies of diabetic kidney disease, the metabolic syndrome, and hypertension.

Premenopausal females are resistant to the development of hypertension, and this protection is lost after the onset of menopause, resulting in a sharp increase in disease onset and severity. However, it is unknown how a fluctuating ovarian hormone environment during the transition from perimenopause to menopause impacts the onset of hypertension, and whether interventions during perimenopause prevent disease onset after menopause. A gradual transition to menopause was induced by repeated daily injections of 4-vinylcyclohexene diepoxide (VCD). ANG II (800 ng·kg−1·min−1) was infused into perimenopausal and menopausal female mice for 14 days. A separate cohort of mice received 17β-estradiol replacement during perimenopause. ANG II infusion produced significantly higher mean arterial pressure (MAP) in menopausal vs. cycling females, and 17β-estradiol replacement prevented this increase. In contrast, MAP was not significantly different when ANG II was infused into perimenopausal and cycling females, suggesting that female resistance to ANG II-induced hypertension is intact during perimenopause. ANG II infusion caused a significant glomerular hypertrophy, and hypertrophy was not impacted by hormonal status. Expression levels of aquaporin-2 (AQP2), a collecting duct protein, have been suggested to reflect blood pressure. AQP2 protein expression was significantly downregulated in the renal cortex of the ANG II-infused menopause group, where blood pressure was increased. AQP2 expression levels were restored to control levels with 17β-estradiol replacement. This study indicates that the changing hormonal environment in the VCD model of menopause impacts the severity of ANG II-induced hypertension. These data highlight the utility of the ovary-intact VCD model of menopause as a clinically relevant model to investigate the physiological mechanisms of hypertension that occur in women during the transition into menopause.

T lymphocytes are required for the development of Angiotensin II (Ang II) hypertension in male mice. Cycling female mice are protected against Ang II hypertension, and inducing ovarian failure (menopause) eliminates this protection. We have previously shown that female Rag-1 -/- mice with adoptively transferred T cells have significantly reduced renal T cell infiltration compared to males. Therefore, we hypothesized that an increase in renal T cell infiltration underlies the genesis of hypertension in menopausal female mice. 10 week old C57Bl/6 female mice received ip VCD injections for 20 consecutive days to induce ovarian failure. Cyclicity was monitored daily via vaginal cytology. Once in menopause, an osmotic minipump was implanted, releasing Ang II (800ng/kg/min) for 14 days. In a subset of mice, a 17-β estradiol pellet (0.1mg) was implanted subcutaneously. Upon harvest, T cell infiltration was measured via flow cytometry. Baseline blood pressure was similar among all groups. Ang II infusion elicited a significantly greater increase in MAP in menopausal mice compared to cycling control (Ang Δ11±10mmHg vs Meno/Ang Δ32±6, p2 Δ5±2mmHg). Despite elevated MAP, there was no significant increase in renal CD3 + , CD4 + or CD8 + T lymphocyte infiltration in Meno/Ang group vs control. However, 17-β estradiol replacement decreased IL-2 renal mRNA expression (0.45±0.13 fold), and increased IL-10 expression (2.41±0.73 fold) compared to Ang II-treated menopausal mice. Ang II induced glomerular hypertrophy in all groups, independent of hormonal status (Control 2156±54μm 2 vs. Ang 3970±383*μm 2 vs. Meno/Ang 3729±51*μm 2 vs. Meno/E 2 /Ang 3505±71*μm 2 , *p