Miller, Virginia M.

Objective Blood thrombogenicity and vascular inflammation may influence development of white matter hyperintensities (WMH) in the brain. This study aimed to determine if menopausal hormone treatments (MHT) modified that association in recently menopausal women who were enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic. Methods Women (n=95) underwent MRI prior to and at 18, 36 and 48 months after randomization to either transdermal 17β‐estradiol (tE2, n=30), conjugated equine estrogen (oCEE, n=29) both with progesterone for 12 days per month or placebo pills and patch (PBO, n=36). Fasting blood was analyzed for a set of 14 activation markers of platelets and endothelial cells, inflammation, and thrombogenicity. Principal components (PC) analysis was used to reduce dimensionality of these markers, and the first 5 PC identified were then assessed for association with treatment and changes in WMH. Results WMH increased in all groups over the 48 months of treatment with greater increases in the oCEE compared to tE2 and PBO (P=0.015). The 5 PC explained 62% of the variation in cellular activation markers. A global test of these 5 PC revealed a borderline association with treatment group (P=0.059), with the PC reflecting total platelet count, platelet microaggregates and ATP secretion showing higher scores in the oCEE compared to the other groups (P=0.006). The change in WMH over the 48 months of MHT showed an overall association with the 5 PC (P=0.026), the most prominent of which was the first PC reflecting vascular inflammation by number of blood microveicles (MV) from activated monocytes and endothelial cells and MV positive for intercellular adhesion molecule 1 (P=0.001). There was no evidence that this association differed by MHT. Conclusion Markers of blood thrombogenicity and inflammation may influence development of WMH in the brain but this relationship may be influenced by factors other than the type of MHT used in the KEEPS study. Support or Funding Information NIH P50 AG44170 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Acute administration of estrogen induces vasodilation in several vascular beds including coronary artery smooth muscle (CSM). We tested the hypothesis that the response of CSM to estrogen is mediated via the binding of estrogen to estrogen receptors (ER) and that the response will be influenced by hormonal status. Coronary smooth muscle was prepared from gonadally intact (INT) and. ovariectomized (OVX) female pigs. Immunocytochemistry and receptor binding assays were performed to confirm presence vs. absence of ER. In INT CSM cells, but not OVX CSM, acute administration of 17β‐estradiol resulted in a concentration‐dependent decrease in [Ca 2+ ] i responses to endothelin‐1. [Ca 2+ ] i responses to 17β‐estradiol were comparable between groups. The effect of 17β‐estradiol was inhibited by the ER antagonist ICI 182,780. 17β‐estradiol decreased [Ca 2+ ] i in INT (but not OVX) CSM cells when sarcoplasmic reticulum (SR) Ca 2+ reuptake was inhibited, but to a lesser extent than when extracellular Ca 2+ was present. This effect was abolished by extracellular lanthanum. These data suggest that 17β‐estradiol modulates [Ca 2+ ] i in CSM via a likely non‐genomic and necessary ER signaling cascade that mediates both plasma membrane Ca 2+ influx and efflux, but does not involve SR Ca 2+ release. Supported by NIH grants UL1RR024150‐01(Mayo Clinic CTSA awards to YSP & CMP) and HL74309 (GCS)

White matter hyperintensities (WMHs) are associated with cerebral microvascular disease. Our objective was to investigate changes in volume of WMH in relationship to conventional cerebrovascular risk factors in recently menopausal women (within 6 months – 3 yrs of their last menses) enrolled in the Kronos Early Estrogen Prevention Study (KEEPS). Platelet activation, numbers of platelet‐derived microvesicles and conventional risk factors for cerebrovascular disease were determined in women (105) enrolled in KEEPS at Mayo Clinic. Each woman underwent MRI of the brain and WMH volumes were derived from the segmented Fluid Attenuated Inversion Recovery images at baseline and 36 months after randomization to treatment. Parametric, nonparametric and log transformation statistical analyses were conducted appropriately. Blood pressure and lipid profiles at baseline were not associated with changes in WMHs. However, baseline platelet micro‐aggregates, annexin V positive microvesicles and plasma activated protein C significantly correlated with increases in WMHs at 36 months. In conclusion, this correlation of increase in WMH with markers of platelet activation measured at baseline suggests that activated platelets and factors contributing to platelet activation may be causal to development of WMH. Supported by the Aurora Fnd, NIH HL90639, NS066147, HD65987 and the Mayo Fnd.

Activated platelets release mitogenic factors, vascular remodeling enzymes, inflammatory and cellular adhesive molecules into the vascular microenvironment to alter chemotactic and adhesive properties of vascular endothelium. Platelet‐derived growth factor‐BB (PDGF‐BB) and matrix metalloproteinase‐2 (MMP‐2) increased in the platelets following ovariectomy but decreased with estrogen treatment in pigs. Experiments were designed to identify changes in these factors using custom antibody arrays in lysate of platelets collected from women enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) at Mayo Clinic. Washed platelets were prepared from anticoagulated blood before (baseline) and 48 months after randomization into either placebo or oral or transdermal MHT. Preliminary analysis shows after 48 months compared to baseline, platelet content of C‐reactive protein, intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), PDGF‐BB, MMP‐2 and transforming growth factor‐beta (TGF‐beta) were lower (20–60%) in two groups but higher (20–74%) in one group. Based on our studies in pigs, it is expected that the two groups showing decreases in platelet proteins would be from women randomized to MHT. However, KEEPS will not be unblinded until February 2012. (Supported by grants from Aurora Foundation, NIH HL90639, 1UL1 RR024150).

Biologic nanoparticles (NPs) ‐ protein/mineral complexes isolatable from human calcified, but not uncalcified arteries and from plasma ‐ propagate and accumulate calcium phosphate when in cell culture conditions. IV injection of NPs into animals results in vascular calcification at sites of endothelial injury. Our aim was to determine if NPs promote mineralization by localizing to vascular smooth muscle cells (VSMCs), then sequestering calcium from their environment. Porcine VSMCs were cultured in 24‐well plates to sub‐confluency (day 0). 45 Ca (1 μCi/well) to label deposited mineral was added to media containing vehicle (control), NPs (10 or 100 nephelometric turbidity units; NTU; average diameter 210 nm), or 2 mM Pi (positive‐control). After 15 days, VSMCs formed multicellular nodules. VSMCs in control media were negative for calcification by von Kossa staining; whereas micron‐sized to smaller particles stained positive for calcium in nodules from NP‐treated and Pi‐treated cells. Cells treated identically were demineralized with 0.6N HCl and the liberated 45 Ca was quantified by scintillation counting. CPM/well (x10 3 ) were 1.5 in controls and 4.6, 28.2 and 64.7 in cells treated with 10 NTU NPs, Pi, and 100 NTU NPs, respectively. NPs localize to VSMCs in vitro and promote mineralization through sequestration of Ca from the environment. (Supported by NIH grant HL88988, and Mayo Clinic).

Estrogen modulates the prostanoids prostacyclin (PGI 2 ) and thromboxane A 2 (TXA 2 ) which contribute to vascular hemostasis in endothelium and platelets. Estrogen decreases in women at menopause, and menopausal hormone therapy (MHT) may increase risk of thrombosis. This study aimed to determine effects of MHT on serum prostanoid levels and on prostanoid‐related platelet functions in healthy, recently‐menopausal women. Women (age 52.3±2.3 yrs; n=115) enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) were randomized to either: 1) oral conjugated equine estrogen, 2) transdermal 17β estradiol each with intermittent oral progesterone or 3) placebo, for 48 months. Serum PGI 2 and TXA 2 levels were assessed by quantification (ng/ml) of their stable metabolites 6‐k‐PGF 1α and TXB 2 , respectively. Initial TXB 2 levels (46.9±7.5) were unchanged (P>; 0.05) after 48 months in each treatment group. However, 6‐k‐PGF 1α decreased from 2.1±0.2 to 1.4±0.1 at 48 months in combined groups, but was statistically significant (1.5±0.3) only in the transdermal group. Inhibition of platelet ATP secretion by PGE 1 did not correlate with serum 6‐k‐PGF 1α levels. These results suggest that serum PGI 2 levels decrease with age in menopausal women and are unaffected by MHT. Factors other than changes in serum prostanoids may impose thrombotic risk associated with MHT. (Supported by the Aurora Fnd, NIH HL88988 and Mayo Clinic).

In postmenopausal women, decreases in estrogen disrupt the normal balance of bone turnover, increasing bone resorption relative to bone formation. This study investigated the effects of menopausal hormone treatment (MHT) on volumetric bone mineral density (vBMD) and markers of bone turnover in plasma of healthy, recently‐menopausal women. Women (age 52.3±2.3 yrs; n=115) enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) were randomized to either: oral conjugated equine estrogen (oCEE), transdermal 17β estradiol (tE2) each with intermittent oral micronized progesterone or placebo (Pl), for 48 months. vBMD was assessed by quantitative computed tomography of the wrist, and plasma levels of osteocalcin (OCN; a bone turnover marker), and of Fetuin‐A, a hepatocyte‐derived modulator of osteogenesis, were quantified by ELISA. At 48 months, cortical vBMD at the distal radius had decreased by 2.9% in the Pl group, correspondent with an increase above initial plasma OCN (15.0 ng/ml) of 38.5%. MHT attenuated the vBMD decrease (0.9% in both treatment groups combined; P=0.015), and the OCN increase (8.8 and 4.3% in oCEE and tE2, respectively). Plasma Fetuin‐A levels did not differ significantly among groups at 48 mo. Unexpectedly, Fetuin‐A was negatively associated (r=‐0.50 to ‐0.62; P <0.05) with changes in trabecular bone microarchitectural parameters at the distal radius in the Pl group, whereas both oCEE and tE2 attenuated this effect (all P‐values >0.05). As expected, MHT attenuated bone turnover in postmenopausal women. However, the relationship between processes regulated by OCN and Fetuin‐A require further evaluation. Grant Funding Source : Supported by Aurora Foundation, HL90639 and AG44170

Experiments were designed to investigate isolatable factors that could either promote or inhibit formation of hydroxyapatite (HA) crystals associated with mineralization of the fibrous capsule surrounding silicone breast implants. Explanted capsular tissue was minced, filtered (0.2μm), inoculated into culture plates containing DMEM/10% FBS and kept at 37°C, 95%O 2 . Within 14 days, biofilm‐like and suspending (planktonic) aggregates containing HA formed to a greater extent in plates inoculated with sample from calcified (CC) than non‐calcified (NC) capsules (158 vs 4 nephelometric turbidity units, respectively). Confocal microscopy of formalin‐fixed sections of NC showed the major cell types to be macrophages and fibroblasts. These cells were immunopositive for fetuin, a blood protein. In contrast, CC were largely fibrous; fetuin labeling was sparse except for the calcific plaque itself on the surface of the capsule adjacent to the implant. Thus, a factor which affects HA formation, possibly a HA nucleator, or a modifier of nucleator activity, can be isolated from mineralized fibrous breast capsules. Localization of fetuin within NC but not CC cells is consistent with its known role in inhibiting HA crystal growth. Supported by grants from the Fetzer Foundation, NIH HL88988 and Division of Experimental Surgery, Mayo Clinic.

Little is known about mechanisms by which bacterial infection contributes to thrombotic risk. Experiments were designed to identify the relationship between toll‐like receptor 4 (TLR4) activation by bacterial lipopolysaccharide (LPS) and circulating procoagulant (phosphatidylserine (PS) positive) microvesicles (MV). Blood from wild type (WT), and TLR4‐ gene deleted (dTLR4) female mice was collected prior to and following exposure to either saline or LPS 20ng/g body weight administered as a single intravenous injection (acute exposure) or after administration by subcutaneous osmotic pump (chronic exposure). MV were isolated from blood by differential centrifugation and identified by flow cytometry. MV thrombin generating capacity was measured by solid phase fluorescent assay. Procoagulant MV were significantly lower in dTLR4 compared to WT mice (P<0.007). Platelet derived MV were the most prominent MV in saline treated mice. Following LPS‐treatment, counts of procoagulant MV increased (P<0.006) significantly only in WT mice. The increase was greater after chronic compared to acute exposure. Thrombin generating capacity positively correlated (r 2 =0.9) with PS expression. These results suggest that increased procoagulant MV through TLR4 activation and thus explain in part increased thrombotic risk with bacterial infection. This work is supported by AHA‐SDG 30503Z and Mayo Foundation.

Risk of CAD increases at menopause and platelets contribute to the etiology of CAD. We hypothesized that individual variation in platelet procoagulant activity at menopause correlates with the extent of CAD as reflected by coronary artery calcification ( CAC ). We developed several novel and global assays of platelet procoagulant activity: dense (ATP release) and alpha (P‐selectin expression) granule secretion and GPα IIb β 3 fibrinogen‐binding (PAC1 binding) to ADP stimulation, and prostaglandin E 1 (PEG 1 ) sensitivity as a surrogate for prostacyclin sensitivity. Platelets were prepared from venous blood collected from women screened for the Kronos Early Estrogen Prevention Study (n=125; mean±SD age=52.5±2.4 years) within 19.4±9.2 months of last menses. Baseline CAC score was determined. Of conventional CAD risk factors, only systolic blood pressure correlated with positive CAC score (n=23). ATP secretion tended to be less in women with positive CAC scores and was negatively correlated with blood glucose (P<0.001). P‐selectin expression and PAC1 binding increased with total cholesterol (P<0.05). PEG 1 sensitivity increased with increasing age and waist circumference (P<0.05). These results indicate that platelet functions change with age and in association with conventional risk factors for the disease in women at menopause. Supported by the Kronos Longevity Research Institute and NIH HL90639.