Jayachandran, Muthuvel

Self‐replicating, self‐calcifying nanosized particles (NPs) can be isolated from mammalian blood and calcified human tissue. Intravenous injection of these biologic NPs into rabbits accelerates occlusion of endothelium‐denuded carotid arteries. Since platelet activation is an initiating step in arterial repair, experiments were designed to determine if biologic NPs alter platelet functions. Platelet rich plasma was prepared from human citrate anti‐coagulated blood. Platelet aggregation (light transmission) and ATP secretion were monitored in response to sTRAP (10 μM) prior to and following incubation with either control solution, biologic NPs (50, 150, 500 NTUs) or hydroxyapatite (HA). NPs increased light transmission and ATP secretion when added immediately to the platelet solution. Both rate and maximal aggregation induced by sTRAP were reduced in the presence of NPs. NPs increased stimulated release of ATP at moderate doses but only after 15 minutes of incubation. HA crystals reduced rather than augmented ATP secretion. These results suggest that NPs cause a shape change in platelets and formation of microaggregates under basal conditions. NPs selectively augment agonist‐stimulated ATP secretion in a dose and time dependent fashion. Therefore, NP activation of platelets could contribute to the increased vascular response to injury in vivo.

Heterogeneous, phospholipid‐rich sub‐micron vesicles (“microvesicles”) shed by activated vascular and blood cells may reflect or influence the coagulant propensity of blood. To determine how the anticoagulant used to collect the blood affects microvesicle (Mv) recovery and characteristics, blood was collected from laboratory volunteers and participants of the Kronos Early Estrogen Prevention Study (KEEPS) into tubes containing either sodium citrate, EDTA or hirudin plus tick anticoagulant peptide (H&T). Microvesicles prepared by double centrifugation were analyzed by flow cytometry to define size and surface markers. There were 2–3 times the number of platelet‐derived (CD61 positive) Mv in preparations from citrate and H&T plasma, relative to numbers in EDTA plasmas. Procoagulant activity of Mv was determined by surface phosphatidylserine (PS) expression using annexin‐V‐fluorescein. Annexin‐V (PS) positive Mv did not differ, but total (PS positive and negative) Mv was 2 fold lower from EDTA plasma compared to citrate and H&T. These results suggest that anticoagulation of blood itself alters microvesicle characteristics. Therefore, standardization and specification of the anticoagulant is warranted to identify potential contribution of microvesicles to cardiovascular pathophysiology. Funded by, NIH HL78638, Kronos Research Institute and Mayo Foundation.

Estrogen receptor beta (βER) is present in platelets but little is known about how βER contributes to platelet physiology. Platelet mitochondrial function and circulating platelet‐derived microparticles were analyzed in aged (24 months old) female wild type (WT) and estrogen receptor beta knockout (βERKO) mice. In intact, unstimulated platelets, ERβ did not co‐localized with mitochondria. Platelet lactate production, mitochondrial membrane potential and superoxide generation were similar in both groups, but activities of cytochrome bc 1 and cytochrome c oxidase were significantly less (P<0.05) in platelets of βERKO mice. Total numbers of platelet‐derived microparticles and those positive for procoagulant phosphatidylserine (PS) were two to three times higher (P<0.05) in βERKO than WT mice. Greater than 40% of procoagulant microparticles were derived from cells other than platelets in both groups of mice. Thrombin generating capacity of microvesicles was also greater in βERKO mice. Thus, loss of βER leads to significant changes in activities of enzymes of the mitochondrial electron transport system, and to increases in circulating procoagulant microparticles which may contribute to an increased thrombotic risk with aging in persons with βER gene polymorphisms. This study is supported by HL78638, AHA30503Z and Mayo Foundation.

Experiments were designed to determine if short‐term exposure to LPS affected transdifferentiation of arterial smooth muscle cells to express bone‐related proteins. Smooth muscle cells were grown to 90% confluency (passage 4 to 5), in M199 medium (control solution). Either LPS (100pg/ml, low; 100ng/ml, mid; 10μg/ml, high) or equal volume of vehicle was added to the media for 3 days at which time the media was exchanged for control solution. Cells were collected at 3, 6, and 12 days after exposure to LPS or vehicle. Expression of smooth muscle phenotypic proteins (desmin, vimentin, αSM‐actin) and bone‐associated phenotypic proteins (matrix gla protein, MGP; osteoprotegrin, OPG, and bone sialoprotein, BSP) were determined by Western blot. Intracellular calcium was measured by colorimetric method and alkaline phosphatase activity was measured by pNPP phosphatase assay. Exposure to mid‐concentration of LPS for 3 days transiently increased then decreased only desmin expression. LPS at mid‐ and high concentrations significantly increased expression of MGP, BSP and OPG, an increase which was sustained for 9 days after removal of LPS. Intracellular calcium concentration showed a time dependent decrease in all groups. Exposure to LPS augmented the decrease 3 days after removal of LPS. Alkaline phosphatase activity decreased at 6 days in the mid and high LPS groups and in all groups at day 12. These results indicate that short‐term exposure (3 days) of smooth muscle cells to LPS specifically modulates a SMC‐phenotypic protein and dose‐dependently facilitates expression of bone‐associated proteins. These findings suggest that infection may contribute to arterial calcification by facilitating transdifferentiation of smooth muscle cells.

Infection increases the risk of thrombotic disease. Platelets contribute to defense against infection and to thrombosis. Gram negative bacteria produce lipopolysaccharides (LPS) that activate Toll‐like‐receptor‐4 (TLR4) in the host and initiate inflammatory responses to infection. Platelets express TLR4, but whether or how LPS affects platelet functions that contribute to thrombotic disease is not understood. To determine if TLR4 signaling influences platelet functions that contribute to thrombotic disease, we studied responses of platelets from normal mice (C57BL/10SnJ; n=10) and mice that do not express TLR4 (C57BL/10ScN; dTLR4, n=10). Prior to LPS exposure, the number of circulating platelets, platelet RNA content and expression of P‐selectin following stimulation with thrombin were diminished in mice that lack TLR4 compared to wild type mice, while platelet aggregation and secretion of ATP were unchanged. These results suggest that loss of TLR4 function affects platelet production and membrane binding characteristics of the circulating platelet pool. One week following a single intravenous injection of LPS (200μg/kg/iv), the time it takes for the circulating platelet pool to turnover, both wild‐type and dTLR4 mice had similar numbers of circulating platelets, and thrombin‐induced expression of P‐selectin and platelet aggregation to collagen increased comparably in both groups of mice. These results indicate that TLR4 function is required for normal platelet homeostasis. Exposure to LPS stimulates a TLR4‐independent shift of the platelet pool toward a reactive phenotype, which could contribute to increased thrombotic risk during or following infection.

Objective Activated platelets serve as a catalyst for thrombin generation. Furthermore, oral MHT may carry greater thrombotic risk than transdermal products. This study compared effects of oral and transdermal MHT on platelet characteristics including platelet‐derived microvesicles (MV) in recently menopausal women. Methods 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 4 years of treatment with either oral conjugated equine estrogen (0.45 mg, oCEE), transdermal 17β estradiol (50 µg/day, tE2), each with intermittent progesterone (200 mg/day for 12 days a month), or placebo pills and patch. Results Platelet count, and activated platelet P‐selectin and fibrinogen‐binding receptor expression were similar across groups. Numbers of tissue factor positive and platelet‐derived MV increased significantly in the tE2 compared to placebo. Conclusions Four years of MHT did not increase thrombogenic characteristics of platelets. Thrombotic risk associated with oral MHT most likely involves liver‐derived proteins than platelets per se. Differential effects of oCEE and tE2 on circulating MV may reflect the hormonal composition of the two formulations on non‐genomic pathways in circulating platelets. Funding This work was supported by grants from the Aurora Foundation to the Kronos Longevity Research Institute, NIH HL90639, UL1TR00013 1 , American Heart Association‐ Scientist Development Grant, AHA 08‐30503Z, American Heart Association, Grant‐in‐Aid, 12GRNT12050147, and the Mayo Foundation.

Activated platelets contribute to atherogenic processes. Experiments were designed to examine the sex distribution of platelet‐associated cytokines in middle aged men and women in whom coronary arterial calcification was defined. Blood was obtained from 17 healthy individuals (11 female, 6 male) between the ages 48–55. Coronary arterial calcification (CAC) was evaluated by electron beam computed tomography. Plasma cholesterol, lipids, and hs‐CRP were analyzed by the Mayo Clinic Clinical Laboratory and were not different between males and females. Serum cytokines were determined using cytokine arrays (Human Atherosclerosis Antibody Array I, RayBiotech, Inc., GA,. USA). Marker intensity was measured using dot blot analysis with the UN‐SCAN‐IT gel 6.1. CAC was detected in only 2 women (scores of 62 and 84%) but in 4 men (scores of 34, 63, 83 and 95%). Correcting for all other risk factors, expression of platelet‐associated cytokines RANTES and PDGF‐BB was 50% greater in females than in males. In conclusion, platelet activation as indicated by increases in RANTES and PDGF‐BB may precede arterial calcification in woman and may be useful in evaluating cardiovascular risk in early menopausal women. (IRB approval 1299‐05 and funded by Dept. Surgery, Mayo Clinic and Fnd.)

Infection and oral estrogen treatments are risk factors for thrombosis. Experiments were designed to evaluate in vitro effects of priming doses of LPS and physiological doses of estrogen on platelets from healthy, newly menopausal women. Blood was collected from early postmenopausal women (42–56 years old) with a menopausal age between 6 months to 3 years into anticoagulants (citrate, hirudin and TAP). Platelet rich plasma was prepared and preincubated with LPS (ultrapure LPS from E. Coli: 10ng‐1000ng/mL) and/or 17β‐estradiol(10 −10 M/mL) Four groups were studied: control, LPS, 17β‐estradiol, 17β‐estradiol+LPS). Platelet aggregation was measured by lumiaggregometry. Thrombin receptor agonist peptide (TRAP 10μM) was used as an agonist. Platelet dense granules ATP secretion was measured by luciferin bioluminescence. Only the highest concentration of LPS (100ng/ml) decreased the maximum and rate of ATP secretion from dense granules and aggregation. At physiological concentrations, 17β‐estradiol did not affect platelet aggregation alone or in combination with LPS. These data suggest that LPS and estrogen may not increase thrombotic risk by direct activation of platelets; effects most likely require the interaction of the platelets with other cells in the circulation. (Supported by NIH HL78638, the Mayo Foundation, Kronos Longevity Institute and Japanese Menopause Society)

Background Break down of the blood brain barrier and activation of brain cells are implicated in progression of deposition of β‐amyloid that is associated with the pathophysiology of Alzheimer's disease. A previous study from our group indicated that deposition of β‐amyloid may be reduced in the brains of menopausal women treated with transdermal 17β‐estradiol. This study aimed to determine the association of peripheral microvesicles carrying markers of cells of the neurovascular unit with β‐amyloid in the brains of menopausal women and if the association was affected by prior menopausal hormone treatments (MHT). Methods Menopausal women previously enrolled in the Kronos Early Estrogen Prevention Study at Mayo Clinic underwent β‐amyloid positron emission tomography (PET) imaging of their brains 3 years following treatment with either placebo (PL, n=29), transdermal 17β‐estradiol (tE2; n=21), or oral conjugated equine estrogen (oCEE; n=17) for 4 years. Microvesicles (MV) were isolated from peripheral venous blood by differential centrifugation and analyzed by flow cytometry using fluorophore conjugated antibodies directed toward total tau, β‐amyloid 1‐42 (Ab1‐42), neuron specific class III β‐tubulin (Tuj1), microglia ionized calcium adaptor molecule 1(Iab‐1), glial fibrillar acid protein (GFAP), and low density lipoprotein receptor‐related protein 1. Principal components analysis was used to reduce the dimensionality of the 6 brain markers to 2 principal components (PCs). Proportional odds ordinal logistic regression analysis was used to regress deposition of β‐amyloid on the 2 PCs with and without adjustment for MHT group. Results Number of blood MV positive for Ab1‐42 differed significantly among MHT groups (median [IQR]: 6.06 [2.11, 12.55] in the PL; 2.49 [0.73, 3.59] in the tE2 and 4.96 [0.83, 10.31] in the oCEE; P=0.036). The first 2 PCs explain 58% of the variance of the 6 markers. PC#1 represented a contrast between MV‐Iab‐1 and the MV‐GFAP and MV‐Tuj1. In multivariable models, brain β‐amyloid load positively associated with the first PC after adjustment for MHT (P= 0.006). Conclusion MV derived from cells of the neurovascular unit can be detected in peripheral blood, and, when combined into summary scores, showed an association with deposition of β‐amyloid in the brain that is independent of MHT. Support or Funding Information Supported by grants from NIH P50 AG044170 and the Mayo Foundation This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Cerebrovascular reactivity to hypercapnia, an estimate of microvascular responses in the brain, is increased during menopausal hormone treatment (MHT) in postmenopausal women. It is unclear whether this effect of MHT continues beyond the treatment period. We hypothesized that prior MHT use would not affect cerebrovascular reactivity 3 years after treatment. In addition, we have previously shown that cerebrovascular reactivity is associated with vascular activation in postmenopausal women with elevated cardiovascular risk. We hypothesized that cerebrovascular reactivity is not associated with markers of intravascular cellular activation in postmenopausal women with low cardiovascular risk who are within 10 years of menopause. Cerebrovascular reactivity to hypercapnia was measured in 58 postmenopausal women from the Kronos Early Estrogen Prevention Study at Mayo Clinic 3 years after the cessation of MHT [oral conjugated equine estrogen (oCEE), n=15; transdermal 17β‐estradiol (tE2, n=22); or placebo (PLA, n=23)]. Cerebrovascular reactivity was estimated from the middle cerebral artery flow velocity (MCAv) response to hypercapnia. Mean arterial blood pressure (MAP) was measured continuously throughout the protocol. Fasting blood was analyzed for a set of 14 activation markers of platelet and endothelial cells, inflammation and thrombogenicity. Principal components (PC) analysis was used to reduce these measures to fewer dimensions on which the associations of intravascular cellular activation with cerebrovascular reactivity could be based. Women were 60±3 years of age with a body mass index of 28±4 Kg/m 2 . MAP (99±14 mmHg in PLA, 97±7 mmHg in tE2, 98±9 mmHg in oCEE; p>0.90) and MCAv (60±14 cm/s in PLA, 68±20 cm/s in tE2, 71±18cm/s in oCEE; p>0.16) did not differ among groups. Cerebrovascular reactivity did not differ among women based on previous MHT randomization (1.2±0.7 cm/s/mmHg in PLA, 1.4±1.0 cm/s/mmHg in tE2 and 1.7±1.0 cm/s/mmHg in oCEE; p>0.17). The 14 markers of intravascular cellular activation were reduced to five PC that did not differ based on prior MHT assignment. There was no overall association of cellular activation PC with cerebrovascular reactivity. Effects of MHT on cerebral microvascular reactivity do not extend beyond the time of menopausal hormone treatment. Intravascular cellular activation does not alter cerebrovascular reactivity to hypercapnia in postmenopausal women with low cardiovascular risk profiles 3 years after the cessation of MHT. Support or Funding Information NIH grant 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 .