Limor Raz

Estrogen attenuates ischemic oxidative damage via an estrogen receptor alpha-mediated inhibition of NADPH oxidase activation.

The goal of this study was to elucidate the mechanisms of 17β-estradiol (E2) antioxidant and neuroprotective actions in stroke. The results reveal a novel extranuclear receptor-mediated antioxidant mechanism for E2 during stroke, as well as a hypersensitivity of the CA3/CA4 region to ischemic injury after prolonged hypoestrogenicity. E2 neuroprotection was shown to involve a profound attenuation of NADPH oxidase activation and superoxide production in hippocampal CA1 pyramidal neurons after stroke, an effect mediated by extranuclear estrogen receptor α (ERα)-mediated nongenomic signaling, involving Akt activation and subsequent phosphorylation/inactivation of Rac1, a factor critical for activation of NOX2 NADPH oxidase. Intriguingly, E2 nongenomic signaling, antioxidant action, and neuroprotection in the CA1 region were lost after long-term E2 deprivation, and this loss was tissue specific because the uterus remained responsive to E2. Correspondingly, a remarkable loss of ERα, but not ERβ, was observed in the CA1 after long-term E2 deprivation, with no change observed in the uterus. As a whole, the study reveals a novel, membrane-mediated antioxidant mechanism in neurons by E2 provides support and mechanistic insights for a “critical period” of E2 replacement in the hippocampus and demonstrates a heretofore unknown hypersensitivity of the CA3/CA4 to ischemic injury after prolonged hypoestrogenicity.

The neuropathology and cerebrovascular mechanisms of dementia

The prevalence of dementia is increasing in our aging population at an alarming rate. Because of the heterogeneity of clinical presentation and complexity of disease neuropathology, dementia classifications remain controversial. Recently, the National Plan to address Alzheimer’s Disease prioritized Alzheimer’s disease-related dementias to include: Alzheimer’s disease, dementia with Lewy bodies, frontotemporal dementia, vascular dementia, and mixed dementias. While each of these dementing conditions has their unique pathologic signature, one common etiology shared among all these conditions is cerebrovascular dysfunction at some point during the disease process. The goal of this comprehensive review is to summarize the current findings in the field and address the important contributions of cerebrovascular, physiologic, and cellular alterations to cognitive impairment in these human dementias. Specifically, evidence will be presented in support of small-vessel disease as an underlying neuropathologic hallmark of various dementias, while controversial findings will also be highlighted. Finally, the molecular mechanisms shared among all dementia types including hypoxia, oxidative stress, mitochondrial bioenergetics, neuroinflammation, neurodegeneration, and blood–brain barrier permeability responsible for disease etiology and progression will be discussed.

Thrombogenic microvesicles and white matter hyperintensities in postmenopausal women

Objective: To determine the association of conventional cardiovascular risk factors, markers of platelet activation, and thrombogenic blood-borne microvesicles with white matter hyperintensity (WMH) load and progression in recently menopausal women. Methods: Women (n = 95) enrolled in the Mayo Clinic Kronos Early Estrogen Prevention Study underwent MRI at baseline and at 18, 36, and 48 months after randomization to hormone treatments. Conventional cardiovascular risk factors, carotid intima-medial thickness, coronary arterial calcification, plasma lipids, markers of platelet activation, and thrombogenic microvesicles were measured at baseline. WMH volumes were calculated using a semiautomated segmentation algorithm based on fluid-attenuated inversion recovery MRI. Correlations of those parameters with baseline WMH and longitudinal change in WMH were adjusted for age, months past menopause, and APOE ε4 status in linear regression analysis. Results: At baseline, WMH were present in all women. The WMH to white matter volume fraction at baseline was 0.88% (0.69%, 1.16%). WMH volume increased by 122.1 mm3 (95% confidence interval: −164.3, 539.5) at 36 months (p = 0.003) and 155.4 mm3 (95% confidence interval: −92.13, 599.4) at 48 months (p < 0.001). These increases correlated with numbers of platelet-derived and total thrombogenic microvesicles at baseline (p = 0.03). Conclusion: Associations of platelet-derived, thrombogenic microvesicles at baseline and increases in WMH suggest that in vivo platelet activation may contribute to a cascade of events leading to development of WMH in recently menopausal women.

Considerations of Sex and Gender Differences in Preclinical and Clinical Trials

Women continue to be underrepresented in clinical trials, particularly in Phases I and II of experimental drug studies in spite of legislative guidelines in the USA, Canada, the European Union, Australia, and Japan requiring the inclusion of women in clinical trials. As such, women remain a vulnerable population subject to the adverse effects of pharmacological therapies. Thus, women experience higher rates of adverse drug reactions than do men and for women of reproductive age or who may be pregnant, therapeutic options may be limited. This chapter provides a brief history of inclusion of sex and gender as variables in clinical trials, summarizes governmental legislation for consideration of sex and gender in clinical trials and provides specific examples of drugs which have been withdrawn from the market because of side effects in women. Additional information related to sex and gender in preclinical testing, trial design, challenges to recruitment of women for clinical trials and statistical methods for analysis of data also is considered.

Estrogen and cerebrovascular regulation in menopause.

Estrogen (E2), classically viewed as a reproductive steroid hormone, has non-reproductive functions throughout the body including in the brain and vasculature. Studies report diminished neuroprotection with declining E2 levels, corresponding with higher incidence of cerebrovascular and neurological disease. However, the effects of menopausal hormone therapy (MHT) on the cerebral vasculature and brain function remain controversial. This review will focus on evidence of 17β-estradiol actions in the cerebral vasculature, with a particular emphasis on the vasoactive, anti-inflammatory, anti-oxidant, metabolic and molecular properties. Controversies surrounding MHT in relation to cerebrovascular disease and stroke risk will be discussed, particularly the emerging evidence from clinical trials supporting the critical period hypothesis of estrogen protection.

Differential effects of oral and transdermal menopausal hormone therapy on prostacyclin and thromboxane in platelets

Menopausal hormone therapies (MHT) may increase thrombotic risk but modulate endothelial function and reduce development of vascular lesions. This study compared effects of MHT on prostanoid‐modulated adenosine triphosphate (ATP) secretion from platelets in relationship with endothelial reactive hyperemia (RH) index and carotid intima medial thickness (CIMT). Participants were healthy, recently menopausal women of the Kronos Early Estrogen Prevention Study (KEEPS) randomized to one of three treatments: oral conjugated equine estrogen (oCEE, 0.45 mg/day), transdermal 17β‐estradiol (tE2, 50 μg/day) each with intermittent oral progesterone or placebo pills and patch (PL). Prostacyclin and thromboxane A2 were assessed by quantification of their stable metabolites (6‐keto‐prostaglandin F1α, 6‐k‐PGF1α; thromboxane B2, TXB2), using ELISA. Dense granule ATP secretion from activated platelets was determined by bioluminescence; RH and CIMT were determined by fingertip tonometry and ultrasound, respectively. After 48 months of treatment, platelet content of 6‐k‐PGF1α and TXB2 was significantly lower in oCEE compared to the PL. Inhibition of ATP secretion by exogenous activation of cAMP associated with platelet 6‐k‐PGF1α (r = −0.41, P = 0.04) and TXB2 (r = 0.71, P = 0.0005) only in the oCEE group. Serum and platelet content of 6‐k‐PGF1α and TXB2 associated positively in the PL and tE2 groups. Serum 6‐k‐PGF1α positively associated with RH in the oCEE group (r = 0.73, P = 0.02), while serum TXB2 positively associated with CIMT in the tE2 group (r = 0.64, P = 0.01). Thus, oCEE and tE2 differentially affect prostanoid‐mediated platelet secretory pathways but alone would not account for an increased thrombotic risk for oral MHT. Furthermore, platelet‐derived prostanoids may contribute to RH and vascular remodeling in healthy menopausal women.

Differential effects of hormone therapy on serotonin, vascular function and mood in the KEEPS

Abstract Background Serotonin (5-hydroxytryptamine, 5-HT) is modulated by sex steroid hormones and affects vascular function and mood. In the Kronos Early Estrogen Prevention Cognitive and Affective Ancillary Study (KEEPS-Cog), women randomized to oral conjugated equine estrogens (oCEE) showed greater benefit on affective mood states than women randomized to transdermal 17β-estradiol (tE2) or placebo (PL). This study examined the effect of these treatments on the platelet content of 5-HT as a surrogate measure of 5-HT synthesis and uptake in the brain. Methods The following were measured in a subset (n = 79) of women enrolled in KEEPS-Cog: 5-HT by ELISA, carotid intima-medial thickness (CIMT) by ultrasound, endothelial function by reactive hyperemic index (RHI), and self-reported symptoms of affective mood states by the Profile of Mood States (POMS) questionnaire. Results Mean platelet content of 5-HT increased by 107.0%, 84.5% and 39.8%, in tE2, oCEE and PL groups, respectively. Platelet 5-HT positively correlated with estrone in the oCEE group and with 17β- estradiol in the tE2 group. Platelet 5-HT showed a positive association with RHI, but not CIMT, in the PL and oCEE groups. Reduction in mood scores for depression-dejection and anger-hostility was associated with elevations in platelet 5-HT only in the oCEE group (r = −0.5, p = 0.02). Conclusions Effects of oCEE compared to tE2 on RHI and mood may be related to mechanisms involving platelet, and perhaps neuronal, uptake and release of 5-HT and reflect conversion of estrone to bioavailable 17β-estradiol in platelets and the brain.

Vascular tight junction disruption and angiogenesis in spontaneously hypertensive rat with neuroinflammatory white matter injury

Vascular cognitive impairment is a major cause of dementia caused by chronic hypoxia, producing progressive damage to white matter (WM) secondary to blood-brain barrier (BBB) opening and vascular dysfunction. Tight junction proteins (TJPs), which maintain BBB integrity, are lost in acute ischemia. Although angiogenesis is critical for neurovascular remodeling, less is known about its role in chronic hypoxia. To study the impact of TJP degradation and angiogenesis during pathological progression of WM damage, we used the spontaneously hypertensive/stroke prone rats with unilateral carotid artery occlusion and Japanese permissive diet to model WM damage. MRI and IgG immunostaining showed regions with BBB damage, which corresponded with decreased endothelial TJPs, claudin-5, occludin, and ZO-1. Affected WM had increased expression of angiogenic factors, Ki67, NG2, VEGF-A, and MMP-3 in vascular endothelial cells and pericytes. To facilitate the study of angiogenesis, we treated rats with minocycline to block BBB disruption, reduce WM lesion size, and extend survival. Minocycline-treated rats showed increased VEGF-A protein, TJP formation, and oligodendrocyte proliferation. We propose that chronic hypoxia disrupts TJPs, increasing vascular permeability, and initiating angiogenesis in WM. Minocycline facilitated WM repair by reducing BBB damage and enhancing expression of TJPs and angiogenesis, ultimately preserving oligodendrocytes.

Hypoxia promotes tau hyperphosphorylation with associated neuropathology in vascular dysfunction

BACKGROUND Hypertension-induced microvascular brain injury is a major vascular contributor to cognitive impairment and dementia. We hypothesized that chronic hypoxia promotes the hyperphosphorylation of tau and cell death in an accelerated spontaneously hypertensive stroke prone rat model of vascular cognitive impairment. METHODS Hypertensive male rats (n = 13) were fed a high salt, low protein Japanese permissive diet and were compared to Wistar Kyoto control rats (n = 5). RESULTS Using electron paramagnetic resonance oximetry to measure in vivo tissue oxygen levels and magnetic resonance imaging to assess structural brain damage, we found compromised gray (dorsolateral cortex: p = .018) and white matter (corpus callosum: p = .016; external capsule: p = .049) structural integrity, reduced cerebral blood flow (dorsolateral cortex: p = .005; hippocampus: p < .001; corpus callosum: p = .001; external capsule: p < .001) and a significant drop in cortical oxygen levels (p < .05). Consistently, we found reduced oxygen carrying neuronal neuroglobin (p = .008), suggestive of chronic cerebral hypoperfusion in high salt-fed rats. We also observed a corresponding increase in free radicals (NADPH oxidase: p = .013), p-Tau (pThr231) in dorsolateral cortex (p = .011) and hippocampus (p = .003), active interleukin-1β (p < .001) and neurodegeneration (dorsolateral cortex: p = .043, hippocampus: p = .044). Human patients with subcortical ischemic vascular disease, a type of vascular dementia (n = 38; mean age = 68; male/female ratio = 23/15) showed reduced hippocampal volumes and cortical shrinking (p < .05) consistent with the neuronal cell death observed in our hypertensive rat model as compared to healthy controls (n = 47; mean age = 63; male/female ratio = 18/29). CONCLUSIONS Our data support an association between hypertension-induced vascular dysfunction and the sporadic occurrence of phosphorylated tau and cell death in the rat model, correlating with patient brain atrophy, which is relevant to vascular disease.

Estrogen Attenuates Ischemic Oxidative Damage via an ERα-Mediated Inhibition of NADPH Oxidase Activation

The goal of this study was to elucidate the mechanisms of 17β-estradiol (E2) antioxidant and neuroprotective actions in stroke. The results reveal a novel extranuclear receptor-mediated antioxidant mechanism for E2 during stroke, as well as a hypersensitivity of the CA3/CA4 region to ischemic injury after prolonged hypoestrogenicity. E2 neuroprotection was shown to involve a profound attenuation of NADPH oxidase activation and superoxide production in hippocampal CA1 pyramidal neurons after stroke, an effect mediated by extranuclear estrogen receptor α (ERα)-mediated nongenomic signaling, involving Akt activation and subsequent phosphorylation/inactivation of Rac1, a factor critical for activation of NOX2 NADPH oxidase. Intriguingly, E2 nongenomic signaling, antioxidant action, and neuroprotection in the CA1 region were lost after long-term E2 deprivation, and this loss was tissue specific because the uterus remained responsive to E2. Correspondingly, a remarkable loss of ERα, but not ERβ, was observed in the CA1 after long-term E2 deprivation, with no change observed in the uterus. As a whole, the study reveals a novel, membrane-mediated antioxidant mechanism in neurons by E2 provides support and mechanistic insights for a “critical period” of E2 replacement in the hippocampus and demonstrates a heretofore unknown hypersensitivity of the CA3/CA4 to ischemic injury after prolonged hypoestrogenicity.