Differences of Uric Acid Transporters Carrying Extracellular Vesicles in the Urine from Uric Acid and Calcium Stone Formers and Non-Stone Formers.

Background: Low urine pH and volume are established risk factors for uric acid (UA) stone disease (UASD). Renal tubular epithelial cells exposed to an acidic pH and/or UA crystals can shed extracellular vesicles (EVs) into the tubular fluid, and these EVs may be a pathogenic biomarker of UASD. Methods: Urinary EVs bearing UA transporters (SLC2A9, SLC17A3, SLC22A12, SLC5A8, ABCG2, and ZNF365) were quantified in urine from UA stone formers (UASFs), calcium stone formers (CSFs), and age-/sex-matched non-stone formers (NSFs) using a standardized and published method of digital flow cytometry. Results: Urinary pH was lower (p < 0.05) and serum and urinary UA were greater (p < 0.05) in UASFs compared with NSFs. Urinary EVs carrying SLC17A3 and SLC5A8 were lower (p < 0.05) in UASFs compared with NSFs. Urinary EVs bearing SLC2A9, SLC22A12, SLC5A8, ABCG2, and ZNF365 were lower (p < 0.05) in CSFs than UASFs, while excretion of SLC17A3-bearing EVs did not differ between groups. Conclusion: EVs bearing specific UA transporters might contribute to the pathogenesis of UASD and represent non-invasive pathogenic biomarkers for calcium and UA stone risk.

Excretion of urine extracellular vesicles bearing markers of activated immune cells and calcium/phosphorus physiology differ between calcium kidney stone formers and non-stone formers.

BACKGROUNDS: Previous studies have demonstrated that excretion of urinary extracellular vesicles (EVs) from different nephron segments differs between kidney stone formers and non-stone formers (NSFs), and could reflect pathogenic mechanisms of urinary stone disease. In this study we quantified selected populations of specific urinary EVs carrying protein markers of immune cells and calcium/phosphorus physiology in calcium oxalate stone formers (CSFs) compared to non-stone formers (NSFs). METHODS: Biobanked urine samples from CSFs (n = 24) undergoing stone removal surgery and age- and sex- matched NSFs (n = 21) were studied. Urinary EVs carrying proteins related to renal calcium/phosphorus physiology (phosphorus transporters (PiT1 and PiT2), Klotho, and fibroblast growth factor 23 (FGF23); markers associated with EV generation (anoctamin-4 (ANO4) and Huntington interacting protein 1 (HIP1)), and markers shed from activated immune cells were quantified by standardized and published method of digital flow cytometry. RESULTS: Urine excretion of calcium, oxalate, phosphorus, and calcium oxalate supersaturation (SS) were significantly higher in CSFs compared to NSFs (P < 0.05). Urinary excretion of EVs with markers of total leukocytes (CD45), neutrophils (CD15), macrophages (CD68), Klotho, FGF23, PiT1, PiT2, and ANO4 were each markedly lower in CSFs than NSFs (P < 0.05) whereas excretion of those with markers of monocytes (CD14), T-Lymphocytes (CD3), B-Lymphocytes (CD19), plasma cells (CD138 plus CD319 positive) were not different between the groups. Urinary excretion of EVs expressing PiT1 and PiT2 negatively (P < 0.05) correlated with urinary phosphorus excretion, whereas excretion of EVs expressing FGF23 negatively (P < 0.05) correlated with both urinary calcium and phosphorus excretion. Urinary EVs with markers of HIP1 and ANO4 correlated negatively (P < 0.05) with clinical stone events and basement membrane calcifications on papillary tip biopsies. CONCLUSIONS: Urinary excretion of EVs derived from specific types of activated immune cells and EVs with proteins related to calcium/phosphorus regulation differed between CSFs and NSFs. Further validation of these and other populations of urinary EVs in larger cohort could identify biomarkers that elucidate novel pathogenic mechanisms of calcium stone formation in specific subsets of patients.

Urinary extracellular vesicle-associated MCP-1 and NGAL derived from specific nephron segments differ between calcium oxalate stone formers and controls.

Randall's plaque (RP; subepithelial calcification) appears to be an important precursor of kidney stone disease. However, RP cannot be noninvasively detected. The present study investigated candidate biomarkers associated with extracellular vesicles (EVs) in the urine of calcium stone formers (CSFs) with low (<5% papillary surface area) and high (≥5% papillary surface area) percentages of RP and a group of nonstone formers. RPs were quantitated via videotaping and image processing in consecutive CSFs undergoing percutaneous surgery for stone removal. Urinary EVs derived from cells of different nephron segments of CSFs (n = 64) and nonstone formers (n = 40) were quantified in biobanked cell-free urine by standardized and validated digital flow cytometer using fluorophore-conjugated antibodies. Overall, the number of EVs carrying surface monocyte chemoattractant protein (MCP)-1 and neutrophil gelatinase-associated lipocalin (NGAL) were significantly lower in CSFs compared with nonstone former controls (P < 0.05) but did not differ statistically between CSFs with low and high RPs. The number of EVs associated with osteopontin did not differ between any groups. Thus, EVs carrying MCP-1 and NGAL may directly or indirectly contribute to stone pathogenesis as evidenced by the lower of these populations of EVs in stone formers compared with nonstone formers. Validation of EV-associated MCP-1 and NGAL as noninvasive biomarkers of kidney stone pathogenesis in larger populations is warranted.

Urinary Extracellular Vesicles of Podocyte Origin and Renal Injury in Preeclampsia.

Renal histologic expression of the podocyte-specific protein, nephrin, but not podocin, is reduced in preeclamptic compared with normotensive pregnancies. We hypothesized that renal expression of podocyte-specific proteins would be reflected in urinary extracellular vesicles (EVs) of podocyte origin and accompanied by increased urinary soluble nephrin levels (nephrinuria) in preeclampsia. We further postulated that podocyte injury and attendant formation of EVs are related mechanistically to cellfree fetal hemoglobin (HbF) in maternal plasma. Our study population included preeclamptic (n=49) and normotensive (n=42) pregnant women recruited at delivery. Plasma measurements included HbF concentrations and concentrations of the endogenous chelators haptoglobin, hemopexin, and α1- microglobulin. We assessed concentrations of urinary EVs containing immunologically detectable podocyte-specific proteins by digital flow cytometry and measured nephrinuria by ELISA. The mechanistic role of HbF in podocyte injury was studied in pregnant rabbits. Compared with urine from women with normotensive pregnancies, urine from women with preeclamptic pregnancies contained a high ratio of podocin-positive to nephrin-positive urinary EVs (podocin+ EVs-to-nephrin+ EVs ratio) and increased nephrinuria, both of which correlated with proteinuria. Plasma levels of hemopexin, which were decreased in women with preeclampsia, negatively correlated with proteinuria, urinary podocin+ EVs-to-nephrin+ EVs ratio, and nephrinuria. Administration of HbF to pregnant rabbits increased the number of urinary EVs of podocyte origin. These findings provide evidence that urinary EVs are reflective of preeclampsia-related altered podocyte protein expression. Furthermore, renal injury in preeclampsia associated with an elevated urinary podocin+ EVs-to-nephrin+ EVs ratio and may be mediated by prolonged exposure to cellfree HbF.

Preeclampsia and cognitive impairment later in life.

BACKGROUND: Hypertension is a risk factor for cerebrovascular disease and cognitive impairment. Women with hypertensive episodes during pregnancy report variable neurocognitive changes within the first decade following the affected pregnancy. However, long-term follow-up of these women into their postmenopausal years has not been conducted. OBJECTIVE: The aim of this study was to examine whether women with a history of preeclampsia were at increased risk of cognitive decline 35-40 years after the affected pregnancy. STUDY DESIGN: Women were identified and recruited through the medical linkage, population-based Rochester Epidemiologic Project. Forty women with a history of preeclampsia were age- and parity-matched to 40 women with a history of normotensive pregnancy. All women underwent comprehensive neuropsychological assessment and completed self-report inventories measuring mood, ie, depression, anxiety, and other symptoms related to emotional state. Scores were compared between groups. In addition, individual cognitive scores were examined by neuropsychologists and a neurologist blinded to pregnancy status, and a clinical consensus diagnosis of normal, mild cognitive impairment, or dementia for each participant was conferred. RESULTS: Age at time of consent did not differ between preeclampsia (59.2 [range 50.9-71.5] years) and normotensive (59.6 [range 52.1-72.2] years) groups, nor did time from index pregnancy (34.9 [range 32.0-47.2] vs 34.5 [range 32.0-46.4] years, respectively). There were no statistically significant differences in raw scores on tests of cognition and mood between women with histories of preeclampsia compared to women with histories of normotensive pregnancy. However, a consensus diagnosis of mild cognitive impairment or dementia trended toward greater frequency in women with histories of preeclampsia compared to those with normotensive pregnancies (20% vs 8%, P = .10) and affected more domains among the preeclampsia group (P = .03), most strongly related to executive dysfunction (d = 1.96) and verbal list learning impairment (d = 1.93). CONCLUSION: These findings suggest a trend for women with a history of preeclampsia to exhibit more cognitive impairment later in life than those with a history of normotensive pregnancy. Furthermore, the pattern of cognitive changes is consistent with that observed with vascular disease/white matter pathology.

EVpedia: a community web portal for extracellular vesicles research.

MOTIVATION: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Tubulointerstitial Fibrosis of Living Donor Kidneys Associates with Urinary Monocyte Chemoattractant Protein 1.

BACKGROUND: Non-invasive biomarkers that detect occult pathology in patients with normal glomerular filtration rate (GFR) and normal urine albumin excretion may help identify patients at risk for chronic kidney diseases. METHODS: Two promising biomarkers of interstitial fibrosis, urinary monocyte chemoattractant protein 1 (MCP-1) and collagen IV, were assayed among 634 living kidney donors from 2005 to 2011, who had both a frozen pre-donation spot urine sample and a core needle biopsy of their donated kidney at transplantation ('time zero biopsy'). The association of urine MCP-1 and collagen IV with kidney function (GFR and urine albumin excretion), kidney volume on computed tomographic imaging and histological findings was assessed. RESULTS: The mean ± SD age was 45 ± 12 years, 24-hour urine albumin was 4 ± 7 mg and measured GFR (mGFR) was 102 ± 18 ml/min/1.73 m2. The median (25th-75th percentile) urine level of MCP-1 was 146 (54-258) pg/ml and of collagen IV was 2.0 (1.0-3.5) µg/l. Higher urine MCP-1 associated with higher 24-hour urine albumin excretion; higher urine collagen IV associated with male gender. On kidney biopsy, any interstitial fibrosis was present in 22% and fibrosis >5% in 4% of donors. The mean MCP-1/Cr ratio was 1.49 pg/mg for 0% fibrosis, 1.80 pg/mg for 1-5% fibrosis, 2.33 pg/mg for 6-10% fibrosis and 4.33 pg/mg for >10% fibrosis. After adjustment for age, sex, mGFR and 24-hour urine albumin, higher urine MCP-1 but not collagen IV associated with interstitial fibrosis and tubular atrophy. CONCLUSION: Urine MCP-1 may detect early tubulointerstitial fibrosis in adults with normal kidney function.

A history of preeclampsia is associated with a risk for coronary artery calcification 3 decades later.

BACKGROUND: A history of preeclampsia is an independent risk factor for cardiac events and stroke. Changes in vasculature structure that contribute to these associations are not well understood. OBJECTIVE: The aim of this study was to quantify coronary artery calcification (CAC), a known risk factor for cardiac events, in a prospective cohort of women with and without histories of preeclampsia. STUDY DESIGN: Women without prior cardiovascular events (40 with and 40 without histories of preeclampsia, matched for parity and age at index birth) were recruited from a large population-based cohort of women who were residents of Olmsted County, Minnesota, and who delivered from 1976 through 1982. Computed tomography was performed to measure CAC in Agatston units. All pregnancy histories and covariates were confirmed by review of the medical records. Current clinical variables were assessed at the time of imaging. Differences between women with and without histories of preeclampsia were examined using χ(2) tests and tests; CAC, in particular, was compared as a categorical and ordinal variable, with a χ(2) test and with Wilcoxon 2-sample tests and ordinal logistic regression, as appropriate. RESULTS: Mean age (SD) at imaging was 59.5 (±4.6) years. Systolic and diastolic blood pressures, hyperlipidemia, and current diabetes status did not differ between women with and without histories of preeclampsia. However, the frequencies of having a current clinical diagnosis of hypertension (60% vs 20%, P < .001) and higher body mass index in kg/m(2) (expressed as median [25th-75th percentile], 29.8 [25.9-33.7] vs 25.3 [23.1-32.0], P = .023) were both greater in the women with histories of preeclampsia compared to those without. The frequency of a CAC score >50 Agatston units was also greater in the preeclampsia group (23% vs 0%, P = .001). Compared to women without preeclampsia, the odds of having a higher CAC score was 3.54 (confidence interval [CI], 1.39-9.02) times greater in women with prior preeclampsia without adjustment, and 2.61 (CI, 0.95-7.14) times greater after adjustment for current hypertension. After adjustment for body mass index alone, the odds of having a higher CAC based on a history of preeclampsia remained significant at 3.20 (CI, 1.21-8.49). CONCLUSION: In this first prospective cohort study with confirmation of preeclampsia by medical record review, a history of preeclampsia is associated with an increased risk of CAC >30 years after affected pregnancies, even after controlling individually for traditional risk factors. A history of preeclampsia should be considered in risk assessment when initiating primary prevention strategies to reduce cardiovascular disease in women. Among women with histories of preeclampsia, the presence of CAC may be able to identify those at a particularly high cardiovascular risk, and should be the subject of future studies.

Preeclampsia and Extracellular Vesicles.

Preeclampsia is a hypertensive pregnancy disorder characterized by development of hypertension and proteinuria after 20 weeks of gestation that remains a leading cause of maternal and neonatal morbidity and mortality. While preeclampsia is believed to result from complex interactions between maternal and placental factors, the proximate pathophysiology of this syndrome remains elusive. Cell-to-cell communication is a critical signaling mechanism for feto-placental development in normal pregnancies. One mechanism of cellular communication relates to activated cell-derived sealed membrane vesicles called extracellular vesicles (EVs). The concentrations and contents of EVs in biological fluids depend upon their cells of origin and the stimuli which trigger their production. Research on EVs in preeclampsia has focused on EVs derived from the maternal vasculature (endothelium, vascular smooth muscle) and blood (erythrocytes, leukocytes, and platelets), as well as placental syncytiotrophoblasts. Changes in the concentrations and contents of these EVs may contribute to the pathophysiology of preeclampsia by accentuating the pro-inflammatory and pro-coagulatory states of pregnancy. This review focuses on possible interactions among placental- and maternal-derived EVs and their contents in the initiation and progression of the pathogenesis of preeclampsia. Understanding the contributions of EVs in the pathogenesis of preeclampsia may facilitate their use as diagnostic and prognostic biomarkers.

Longitudinal effects of menopausal hormone treatments on platelet characteristics and cell-derived microvesicles.

Activated platelets serve as a catalyst for thrombin generation and a source of vasoactive and mitogenic factors affecting vascular remodeling. Oral menopausal hormone treatments (MHT) may carry greater thrombotic risk than transdermal products. This study compared effects of oral and transdermal MHT on platelet characteristics, platelet proteins, and platelet-derived microvesicles (MV) in recently menopausal women. Platelets and MV were prepared from blood of a subset of women (n = 117) enrolled in the Kronos Early Estrogen Prevention Study prior to and after 48 months of treatment with either oral conjugated equine estrogen (0.45 mg/day), transdermal 17ß-estradiol (50 µg/day), each with intermittent progesterone (200 mg/day for 12 days a month), or placebo pills and patch. Platelet count and expression of platelet P-selectin and fibrinogen receptors were similar across groups. An aggregate measure of 4-year change in vasoactive and mitogenic factors in platelet lysate, by principle component analysis, indicated significantly lower values in both MHT groups compared to placebo. Increases in numbers of tissue factor positive and platelet-derived MV were significantly greater in the transdermal compared to placebo group. MHT was associated with significantly reduced platelet content of vasoactive and mitogenic factors representing a potential mechanism by which MHT may affect vascular remodeling. Various hormonal compositions and doses of MHT could differentially regulate nuclear transcription in bone marrow megakaryocytes and non-genomic pathways in circulating platelets thus determining numbers and characteristics of circulating MV. Thrombotic risk associated with oral MHT most likely involves liver-derived inflammatory/coagulation proteins rather than circulating platelets per se.

Women With a History of Preeclampsia Exhibit Accelerated Aging and Unfavorable Profiles of Senescence Markers.

BACKGROUND: Senescence, a mechanism of cellular aging, which is characterized by irreversible proliferation arrest and a proinflammatory secretory phenotype, has been documented in women with preeclampsia. As cellular senescence can persist and progress, we postulated that it is associated with accelerated aging phenotype and accumulation of comorbidities in women with a history of preeclampsia. METHODS: We included a cohort of women with a history of preeclampsia (n=40) age- and parity-matched to a group of referent women with normotensive pregnancies (n=40). Women with prior major cardiovascular events, neurological, or autoimmune conditions were excluded. We collected urine and blood samples to study markers of aging, data on multimorbidity at the time of enrollment, and prospectively followed them for events over the course of 6 years, on average. RESULTS: Women with a history of preeclampsia exhibited unfavorable aging profiles compared with referent women, including decreased urinary α-Klotho (P=0.018); increased leptin (P=0.016) and leptin/adiponectin ratio (P=0.027), and increased extracellular vesicles positive for tissue factor (P=0.025). Women with a history of preeclampsia likewise had a higher rate of comorbidities at the time of enrollment (P=0.003) and had a 4× higher risk of developing major cardiovascular events compared with referent women (P=0.003). CONCLUSIONS: Our data suggest that a history of preeclampsia is associated with accelerated aging as indicated by senescence marker differences and the accumulation of multimorbidity later in life. Targeting cellular senescence may offer novel, mechanism-based approaches for the diagnosis and treatment of adverse health outcomes in women with a history of preeclampsia.

Preeclampsia and the Kidney: Pathophysiology and Clinical Implications.

Preeclampsia and other hypertensive disorders of pregnancy are major contributors to maternal morbidity and mortality worldwide. This group of disorders includes chronic hypertension, gestational hypertension, preeclampsia, preeclampsia superimposed on chronic hypertension, and eclampsia. The body undergoes important physiological changes during pregnancy to allow for normal placental and fetal development. Several mechanisms have been proposed that may lead to preeclampsia, including abnormal placentation and placental hypoxia, impaired angiogenesis, excessive pro-inflammatory response, immune system imbalance, abnormalities of cellular senescence, alterations in regulation and activity of angiotensin II, and oxidative stress, ultimately resulting in upregulation of multiple mediators of endothelial cell dysfunction leading to maternal disease. The clinical implications of preeclampsia are significant as there are important short-term and long-term health consequences for those affected. Preeclampsia leads to increased risk of preterm delivery and increased morbidity and mortality of both the developing fetus and mother. Preeclampsia also commonly leads to acute kidney injury, and women who experience preeclampsia or another hypertensive disorder of pregnancy are at increased lifetime risk of chronic kidney disease and cardiovascular disease. An understanding of normal pregnancy physiology and the pathophysiology of preeclampsia is essential to develop novel treatment approaches and manage patients with preeclampsia and hypertensive disorders of pregnancy. © 2023 American Physiological Society. Compr Physiol 13:4231-4267, 2023.

Impact of Aging and Cellular Senescence in the Pathophysiology of Preeclampsia.

The incidence of hypertensive disorders of pregnancy is increasing, which may be due to several factors, including an increased age at pregnancy and more comorbid health conditions during reproductive years. Preeclampsia, the most severe hypertensive disorder of pregnancy, has been associated with an increased risk of future disease, including cardiovascular and kidney diseases. Cellular senescence, the process of cell cycle arrest in response to many physiologic and maladaptive stimuli, may play an important role in the pathogenesis of preeclampsia and provide a mechanistic link to future disease. In this article, we will discuss the pathophysiology of preeclampsia, the many mechanisms of cellular senescence, evidence for the involvement of senescence in the development of preeclampsia, as well as evidence that cellular senescence may link preeclampsia to the risk of future disease. Lastly, we will explore how a better understanding of the role of cellular senescence in preeclampsia may lead to therapeutic trials. © 2023 American Physiological Society. Compr Physiol 13:5077-5114, 2023.

Microvesicles at the crossroads between infection and cardiovascular diseases.

Observational and experimental studies continue to support the association of infection and infection-stimulated inflammation with development of cardiovascular disease (CVD) including atherosclerosis and thrombosis. Microvesicles (MV) are heterogeneous populations of sealed membrane-derived vesicles shed into circulation by activated mammalian cells and/or pathogenic microbes that may represent an interface between bacterial/microbial infection and increased risk of CVD. This review evaluates how MV act to modulate and intersect immunological and inflammatory responses to infection with particular attention to progression of CVD. Although infection-related stimuli provoke release of MV from blood and vascular cells, MV express phosphatidylserine and other procoagulant factors on their surface, which initiate and amplify blood coagulation. In addition, MV mediate cell-cell adhesion, which may stimulate production of pro-inflammatory cytokines in vascular cells, which in turn aggravate progression of CVD and propagate atherothrombosis. MV transfer membrane receptors, RNA and proteins among cells, and present auto-antigens from their cells of origin to proximal or remote target cells. Because MV harbor cell surface proteins and contain cytoplasmic components of the parent cell, they mediate biological messages and play a pivotal role in the crossroad between infection-stimulated inflammation and CVDs.

Inflammatory Cells in Nephrectomy Tissue from Patients without and with a History of Urinary Stone Disease.

BACKGROUND AND OBJECTIVES: Urinary stone disease has been associated with inflammation, but the specific cell interactions that mediate events remain poorly defined. This study compared calcification and inflammatory cell patterns in kidney tissue from radical nephrectomy specimens of patients without and with a history of urinary stone disease. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: Nontumor parenchyma of biobanked radical nephrectomy specimens from age- and sex-matched stone formers (n=44) and nonstone formers (n=82) were compared. Calcification was detected by Yasue staining and inflammatory cell populations by immunohistochemistry for CD68 (proinflammatory M1 macrophages), CD163 and CD206 (anti-inflammatory M2 macrophages), CD3 (T lymphocytes), and tryptase (mast cells). Calcifications and inflammatory cells were quantified in cortex and medulla using Image-Pro analysis software. RESULTS: Calcification in the medulla of stone formers was higher than in nonstone formers (P<0.001). M1 macrophages in the cortex and medulla of stone formers were greater than in nonstone formers (P<0.001), and greater in stone former medulla than stone former cortex (P=0.02). There were no differences in age, sex, body mass index, tumor characteristics (size, stage, or thrombus), vascular disease status, or eGFR between the groups. M2 macrophages, T lymphocytes, and mast cells did not differ by stone former status. There was a correlation between M1 macrophages and calcification in the medulla of stone formers (rho=0.48; P=0.001) and between M2 macrophages and calcification in the medulla of nonstone formers (rho=0.35; P=0.001). T lymphocytes were correlated with calcification in the cortex of both nonstone formers (rho=0.27; P=0.01) and stone formers (rho=0.42; P=0.004), whereas mast cells and calcification were correlated only in the cortex of stone formers (rho=0.35; P=0.02). CONCLUSIONS: Higher medullary calcification stimulated accumulation of proinflammatory rather than anti-inflammatory macrophages in stone formers.

Peripheral Markers of Neurovascular Unit Integrity and Amyloid-ß in the Brains of Menopausal Women.

BACKGROUND: The identification of blood-borne biomarkers for the diagnosis and prognosis of Alzheimer's disease and related dementias is more feasible at the population level than obtaining cerebrospinal fluid or neuroimaging markers. OBJECTIVE: This study determined the association of blood microvesicles, derived from cells of the neurovascular unit, with brain amyloid-ß deposition in menopausal women. METHODS: A subset of women from the Kronos Early Estrogen Prevention Study underwent brain amyloid-ß positron emission tomography three years following cessation of study treatment with placebo (PL, n = 29), transdermal 17ß-estradiol (tE2; n = 21), or oral conjugated equine estrogen (oCEE; n = 17). Isolated peripheral venous blood microvesicles were analyzed by digital flow cytometry using fluorophore conjugated antibodies directed toward total tau, amyloid-ß 1-42 (Aß1-42), neuron specific class III ß-tubulin (Tuj1), microglia ionized calcium -binding adaptor molecule 1(Iba1), glial fibrillary acid protein (GFAP), and low density lipoprotein receptor-related protein1 (LRP1). Principal components analysis reduced the dimensionality of these selected six markers to two principal components (PCs). Proportional odds ordinal logistic regression analysis was used with amyloid-ß deposition regressed on these PCs. RESULTS: Only the number of microvesicles positive for Aß1-42 differed statistically among prior treatment groups (median [IQR]: 6.06 [2.11, 12.55] in PL; 2.49 [0.73, 3.59] in tE2; and 4.96 [0.83, 10.31] in oCEE; p = 0.032). The joint association between the 2 PCs and brain amyloid-ß deposition was significant (p = 0.045). CONCLUSION: Six selected markers expressing peripheral blood microvesicles derived from cells of the neurovascular unit, when summarized into two principal components, were associated with brain amyloid-ß deposition.

Atherosclerosis and sex hormones: current concepts.

CVD (cardiovascular disease) is the leading cause of death for women. Considerable progress has been made in both our understanding of the complexities governing menopausal hormone therapy and our understanding of the cellular and molecular mechanisms underlying hormone and hormone receptor function. Understanding the interplay of atherosclerosis and sex steroid hormones and their cognate receptors at the level of the vessel wall has important ramifications for clinical practice. In the present review, we discuss the epidemiology of CVD in men and women, the clinical impact of sex hormones on CVD, and summarize our current understanding of the pathogenesis of atherosclerosis with a focus on gender differences in CVD, its clinical presentation and course, and pathobiology. The critical animal and human data that pertain to the role of oestrogens, androgens and progestins on the vessel wall is also reviewed, with particular attention to the actions of sex hormones on each of the three key cell types involved in atherogenesis: the endothelium, smooth muscle cells and macrophages. Where relevant, the systemic (metabolic) effects of sex hormones that influence atherogenesis, such as those involving vascular reactivity, inflammation and lipoprotein metabolism, are discussed. In addition, four key current concepts in the field are explored: (i) total hormone exposure time and coronary heart disease risk; (ii) the importance of tissue specificity of sex steroid hormones, critical timing and the stage of atherosclerosis in hormone action; (iii) biomarkers for atherosclerosis with regard to hormone therapy; and (iv) the complex role of sex steroids in inflammation. Future studies in this field will contribute to guiding clinical treatment recommendations for women and help define research priorities.

Menopausal hormone therapy, blood thrombogenicity, and development of white matter hyperintensities in women of the Kronos Early Estrogen Prevention Study.

OBJECTIVE: Development of white matter hyperintensities (WMH) in the brain is associated with blood thrombogenicity in recently menopausal women. This study examined the influence of menopausal hormone treatments (MHTs) on this association. METHODS: Measures of blood thrombogenicity were examined in women of the Kronos Early Estrogen Prevention Study (n = 95) who had brain magnetic resonance imaging before and during the 48 months of randomization to transdermal 17ß-estradiol (n = 30), oral conjugated equine estrogen (n = 29) both with progesterone for 12 days per month or placebo pills and patch (n = 36). Principal components (PCs) analysis was used to reduce the dimensionality of 14 markers of platelet activation and blood thrombogenicity. The first 5 PCs were assessed for association with treatment and changes in WMH. Within-person slopes were obtained to capture the extent of WMH change for each woman. RESULTS: WMH increased in all groups over the 48 months (P = 0.044). The partial effect of PC1, representing an average of six thrombogenicity variables (microvesicles derived from endothelium, leukocytes, and monocytes, and positive for tissue factor and adhesion molecules) on WMH was significant (P = 0.003). PC3, reflecting a contrast of platelet microaggregates and adenosine triphosphate secretion versus total platelet count, differed across groups (P = 0.006) with higher scores in the oral conjugated equine estrogen group. The global association between PCs and WMH increase, however, did not differ significantly by MHT (P = 0.207 for interaction between MHT and PC's). CONCLUSION: In recently menopausal women, the type of MHT did not significantly influence the association of markers of blood thrombogenicity with development of WMH in the brain.

Risk factors of neurovascular ageing in women.

Biological sex and changes in sex hormones throughout life influence all aspects of health and disease. In women, changes in sex hormonal status reflect ovarian function, pregnancy and the use of exogenous hormonal treatments. Longitudinal data from defined cohorts of women will help to identify mechanisms by which the hormonal milieu contributes to cerebrovascular ageing, brain structure and ultimately cognition. This review summarises the phenotypes of three cohorts of women identified through the medical records-linkage system of the Rochester Epidemiology Project and the Mayo Clinic Specialized Center of Research Excellence (SCORE) on Sex Differences: (i) menopausal women with histories of normotensive or hypertensive pregnancies; (ii) women who had bilateral oophorectomy ≤45 years of age; and (iii) women who experienced natural menopause and used menopausal hormone treatments for 4 years. Data from these cohorts will influence the design of follow-up studies concerning how sex hormonal status affects neurovascular ageing in women.

COVID-19 and Sex Differences: Mechanisms and Biomarkers.

Men are consistently overrepresented in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and coronavirus disease 2019 (COVID-19) severe outcomes, including higher fatality rates. These differences are likely due to gender-specific behaviors, genetic and hormonal factors, and sex differences in biological pathways related to SARS-CoV-2 infection. Several social, behavioral, and comorbid factors are implicated in the generally worse outcomes in men compared with women. Underlying biological sex differences and their effects on COVID-19 outcomes, however, have received less attention. The present review summarizes the available literature regarding proposed molecular and cellular markers of COVID-19 infection, their associations with health outcomes, and any reported modification by sex. Biological sex differences characterized by such biomarkers exist within healthy populations and also differ with age- and sex-specific conditions, such as pregnancy and menopause. In the context of COVID-19, descriptive biomarker levels are often reported by sex, but data pertaining to the effect of patient sex on the relationship between biomarkers and COVID-19 disease severity/outcomes are scarce. Such biomarkers may offer plausible explanations for the worse COVID-19 outcomes seen in men. There is the need for larger studies with sex-specific reporting and robust analyses to elucidate how sex modifies cellular and molecular pathways associated with SARS-CoV-2. This will improve interpretation of biomarkers and clinical management of COVID-19 patients by facilitating a personalized medical approach to risk stratification, prevention, and treatment.