Hunter, Larry W.

Objective Activation of brain microvascular endothelium by inflammatory and procoagulant stimuli increases expression of surface cell‐adhesion molecules (CAMs), releases endothelium‐derived microvesicles (MV) into the circulation, and increases permeability of the blood‐brain barrier (BBB). This study evaluated characteristics of MV released from human brain microvascular endothelial cells (HBMEC) following stimulation with proinflammatory or procoagulant stimuli and examined their effect on monolayer permeability. Methods Cultured HBMECs from age‐matched male and female donors were treated for 24 hours with medium supplemented with tumor necrosis factor alpha (TNF α , 20 ng/mL), thrombin (THR, 2 U/mL), or vehicle (control). MVs released into the medium were isolated by differential centrifugation, labeled with annexin V together with antibodies against PECAM‐1, ICAM‐1, E‐selectin, integrin aV, or MCAM, and then analyzed by digital flow cytometry. Permeability (mg/mL FITC‐dextran) of the HBMEC monolayer in response to MV treatment was quantified by fluorescence microplate assay of passage of FITC‐dextran across monolayer cells cultured on microporous supports. Results Fewer specific antigen‐positive MV were released from unstimulated male compared to female HBMECs. TNF a , but not THR induced early‐stage apoptosis; determined by fluorogenic Caspase‐3 assay in the HBMEC monolayers. TNF a induced release of all MV subtypes from male cells, whereas only integrin aV, ICAM‐1 and E‐selectin positive MVs were released from female cells. THR increased release of PECAM‐1, ICAM‐1, and MCAM positive MVs from male cells, but did not increase release of MV in female cells. After 24 hours exposure to MV (2,500 MV/mL) derived from TNF a ‐treated female cells, permeability increased by 17%. Permeabiity was not increased when female cells were exposed to THR‐generated MV. Conclusions These results indicate that: 1) inflammatory and procoagulant stimuli release a different array of MV subtypes from male and female HBMECs; 2) specific stimuli‐derived MVs can modify HMBMEC permeability. Support or Funding Information (Supported by NIH P50 AG44170 and the Mayo Clinic Fnd.).

The disruption of the cerebral microvascular endothelium and increases in capillary permeability are characteristic of many brain pathologies, including Alzheimer's disease, small vessel disease, ischemia/hypoxia, and hemorrhagic stroke. This study was designed to investigate whether microvesicles (MVs) released from activated human brain microvascular endothelial cells (HBMECs) alter permeability of endothelial monolayers. Male donor HBMECs were cultured to confluence in flasks, then treated for 24 hrs with medium alone (control) or with medium plus either tumor necrosis factor (TNF a , 20 ng/ml), thrombin (THR, 2 U/ml), angiotensin II (AngII, 10 nM), or hydrogen peroxide (H 2 O 2 , 0.8 mM). MVs released into the medium were isolated by differential centrifugation, labeled with annex in V plus antibodies against endothelial adhesion molecules which have modified expression during inflammation (PECAM‐1, ICAM‐1, E‐select in, integrin aV, or MCAM); and then analyzed by FACS. Total number of MVs released (MVs/ml) by each of the agonists (TNF a , 615.8±13.9 MV/μL; THR, 113.8±4.8 MV/μL; and H 2 O 2, 152.8±23.5 MV/μL) were significantly greater (P<0.05) compared the number released from cells treated medium alone (82.2±5.4 MV/μL). Each agonist treatment released distinct types of MV identified by FACS. Permeability (% control) of the HBMEC monolayer assessed using FITC‐dextran transport across the monolayers increased after 90 minute exposure to all agonist treatments: 199.5%, 234.1%, 126.8%, and 588.3% for TNF a , THR, AngII, and H 2 O 2 , respectively. MV derived from the treated cells were labeled with PKH67 and applied to monolayers of (control) HBMECs cultures. Within 30 minutes of exposure punctate structures with PKH67 labeling were observed within the cytoplasm, and after 60 min, similar structures also associated with nuclei and some of which were co‐labeled with the lysosome marker lysotracker red. Co‐localization of labeled MVs with early endosomes, (labeled with anti‐EEA‐1 Ab/Alexa Fluor 647) was absent at these time points. Within these parameters, no differences in vesicle‐trafficking were observed between MVs generated from cells exposed to either medium, TNF a , or H 2 O 2 . These results suggest that treatment of HBMECs with agonists associated with inflammation and thrombosis increase release of distinct MVs which in turn increase paracellular permeability. Capillary endothelial permeability may be disturbed by populations of MVs released from proximal activated cells or MV derived from activated cells and carried in the blood. Support or Funding Information Supported by the Aurora Fnd, NIH HL88988 and Mayo Clinic

Biologic nanoparticles (NPs) can be isolated from bovine serum, human kidney stones, and human arterial calcifications. In addition, it is possible to detect both antigen and antibody for NPs in human blood, suggesting that perhaps NPs represent a novel infectious agent participating in human disease. However, it is not known when and how persons might become exposed to NPs and if exposure produces sensitization to subsequent exposures. Experiments were designed to determine whether exposure to nanoparticles would induce an immune response in rabbits that can be detected by a delayed‐type hypersensitivity test. Rabbits were injected intravenously with bovine‐derived NPs (1ml of 1000NTU solution). Plasma was collected prior to injection and on days, 3, 7, 14, 21, 28, 35 post‐injection. At this time, NPs (1ml of 1000NTU solution) were injected subcutaneously in the ear of exposed and naïve rabbits. Areas of injection were observed daily and after three days, blood and ears were collected for analysis. Injection sites showed leukocyte infiltration only in ears of rabbits previously exposed to NPs. These data indicate that biologic NPs induce immune sensitization in rabbits.

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.

Biologic nanoparticles (NPs) isolated from human arterial calcifications and kidney stones form an adherent film on the bottom of flasks when placed in standard cell culture conditions. Since NPs have certain features consistent with microorganisms, we hypothesized that antibiotics and/or RNAse might inhibit this biofilm formation. NPs isolated from calcified human arterial tissue were cultured onto glass coverslips in 24‐well plates containing DMEM with 10% fetal calf serum, plus either no treatment (control), RNase, tetracycline or gentamicin. At 1, 7 and 14 days after inoculation, coverslips were fixed, mounted, and analyzed using a CytoViva darkfield scope and KS‐400 imaging software. In control wells, the percent coverslip covered by biofilm increased by 120% from day 1 to day 14, compared to 47% and 35% in wells treated with RNase and gentamicin, respectively. Wells treated with tetracycline showed a 3% decrease in biofilm by day 14. Since RNase should not enter intact cells, these results indicate that extracellular RNA may play a role in biofilm formation by NPs. Although tetracycline can chelate calcium, it also inhibits ribosomal function, as does gentamicin. Therefore these results also suggest that intact ribosomal function is required for NP‐associated biofilm growth. These experiments suggest that biofilm formation by NPs consists of biological as well as physicochemical processes.

Biologic nanoparticles (NPs) isolated from human atheromas propagate and form an apatite shell when placed in cell‐culture conditions. These characteristics led us to hypothesize that NPs might derive from microorganisms. NPs isolated from human aortic aneurysms and maintained in DMEM, 10% fetal bovine serum at 37 o C increased both in density (measured in nephelometric turbidity units, NTU) from 1.6 to 107.1 and in diameter (from < 0.2 μm to 0.5‐1 μm) over the course of 12 days. Crystals present on the surface of NPs were found by electron microscopy/elemental analysis to be composed of Ca 2+ and P i . NPs adhered to the surface of the culture flasks, forming a biofilm strikingly similar to that seen in microbial communities. Confocal laser scanning microscopy showed that the biofilm stained positively with concanavalin‐A, a probe for mannose and glucose, both major constituents of microbial biofilm matrix exopolysaccharides. Further, the biofilm stained positively by Bac Light Green, a bacteria‐specific cell wall label, as well as the lipid membrane‐selective dye Vybrant CM‐DiI. These results reveal that calcifying NPs isolated from human atheromas share characteristics common to bacteria since they propagate when placed in cell culture conditions, form a biofilm containing polysaccharides, and stain with agents that recognize bacterial components. Supported by grants from the Fetzer Foundation and NIH HL88988.

Sepsis is associated with microvascular hyporeactivity to agonists and may be reflective of reduced Ca 2+ ‐sensitivity within smooth muscle cells. We hypothesize that impaired vasoreactivity during LPS‐mediated inflammation is associated with decreased Ca 2+ sensitivity in small mesenteric resistance arteries (SMRA). SMRAs (190–220 μm) isolated from LPS‐treated (15 mg/kg ip, 18 hours) and saline‐treated control mice were mounted on a pressure myograph, superfused, and loaded with fura‐2. Arteriolar diameter and global intracellular Ca 2+ were simultaneously measured. Smooth muscle Ca 2+ ‐sensitivity of SMRAs was assessed by stepwise increases in extracellular Ca 2+ (0 to 2 mM) under depolarizing conditions (120 mM K + ). SMRA isolated from mice treated with LPS demonstrated hyporesponsiveness to Ca 2+ . Extracellular mediated Ca 2+ vasoconstriction in intact vessels resulted in an increase in EC 50 (0.23 ± 0.01 mM vs 0.39 ± 0.03 mM∗) and a reduction in E max (42.8± 1.1% vs 26.6 ± 3.4%∗). Removal of the endothelium resulted in near normal response to Ca 2+ . In intact vessels, LPS treatment decreases vascular smooth muscle Ca 2+ sensitivity as compared to controls. NOS inhibition (L‐NNA, 1 μM) restores responsiveness to Ca 2+ without restoring Ca 2+ sensitivity. LPS‐induced inflammation results in NO‐independent decrease in Ca 2+ ‐sensitivity within vascular smooth muscle cells.

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).