Associations of Sex Hormones and Hormonal Status With Arterial Stiffness in a Female Sample From Reproductive Years to Menopause.

Objective: Loss of sex hormones has been suggested to underlie menopause-associated increment in cardiovascular risk. We investigated associations of sex hormones with arterial stiffness in 19-58-years-old women. We also studied associations of specific hormonal stages, including natural menstrual cycle, cycle with combined oral contraceptives (COC) and menopausal status with or without hormone therapy (HT), with arterial stiffness. Methods: This study includes repeated measurements of 65 healthy women representing reproductive (n=16 natural, n=10 COC-users) and menopause (n=5 perimenopausal, n=26 postmenopausal, n=8 HT-users) stages. Arterial stiffness outcomes were aortic pulse wave velocity (PWVao) and augmentation index (AIx%) assessed using Arteriograph-device. Generalized estimating equation models were constructed to investigate associations of each hormone (wide age-range models) or hormonal stage (age-group focused models) with arterial stiffness. PWVao models with cross-sectional approach, were adjusted for age, relative fitness, fat mass and mean arterial pressure, while models with longitudinal approach were adjusted for mean arterial pressure. AIx% models used the same approach for adjustments and were also adjusted for heart rate. Results: Negative and positive associations with arterial stiffness variables were observed for estradiol and follicle-stimulating hormone, respectively, until adjustment for confounding effect of age. In naturally menstruating women, AIx% was higher at ovulation (B=3.63, p<0.001) compared to the early follicular phase. In COC-users, PWVao was lower during active (B=-0.33 - -0.57, p<0.05) than inactive pills. In menopausal women, HT-users had higher PWVao (B=1.43, p=0.03) than postmenopausal non-HT-users. Conclusions: When using wide age-range assessments covering reproductive to menopausal lifespan it is difficult to differentiate age- and hormone-mediated associations, because age-mediated influence on arterial stiffness seemed to overrule potential hormone-mediated influences. However, hormonal status associated differentially with arterial stiffness in age-group focused analyses. Thus, the role of sex hormones cannot be excluded. Further research is warranted to resolve potential hormone-mediated mechanisms affecting arterial elasticity.

Macrophage Infiltration in the Saccular Intracranial Aneurysm Wall as a Response to Locally Lysed Erythrocytes That Promote Degeneration.

Saccular intracranial aneurysm (sIA) rupture is often fatal. Rupture-prone sIA walls are infiltrated by macrophages expressing hemoglobin-receptor CD163, suggesting a role for erythrocyte lysis in the degenerative remodeling predisposing to rupture. We therefore studied erythrocyte remnants in 16 unruptured and 20 ruptured sIA walls using histology and immunohistochemistry. Glycophorin A (GPA), an erythrocyte membrane protein, was present in 34/36 (94%) sIA walls and correlated with loss of αSMA+ cells, reflecting loss of mural smooth muscle cells ([SMCs]; r = -0.592, p < 0.001), wall degeneration (p = 0.008), and rupture (p = 0.005). GPA correlated with high numbers of CD163+ and CD68+ phagocytes (r = 0.65 and r = 0.54, p ≤ 0.001 for both). CD163+ phagocytes were mostly HLA-DR-. Interestingly, single SMCs expressed HLA-DR and also CD163 was expressed in sporadic SMCs, which may reflect their response to hemoglobin accumulation. GPA associated with iron (p = 0.014) was detectable by MRI. An additional 11 sIAs were therefore imaged ex vivo with a 4.7 T MRI prior to histology. In the sIA walls, high GPA and iron accumulation associated with signal intensity in T1-weighted gradient echo MRI. We conclude that accumulation of lysed erythrocytes is a potential driver of inflammatory response in the sIA walls and is associated with the degenerative wall remodeling, thereby predisposing to rupture.

Extracellular Lipids Accumulate in Human Carotid Arteries as Distinct Three-Dimensional Structures and Have Proinflammatory Properties.

Lipid accumulation is a key characteristic of advancing atherosclerotic lesions. Herein, we analyzed the ultrastructure of the accumulated lipids in endarterectomized human carotid atherosclerotic plaques using three-dimensional (3D) electron microscopy, a method never used in this context before. 3D electron microscopy revealed intracellular lipid droplets and extracellular lipoprotein particles. Most of the particles were aggregated, and some connected to needle-shaped or sheet-like cholesterol crystals. Proteomic analysis of isolated extracellular lipoprotein particles revealed that apolipoprotein B is their main protein component, indicating their origin from low-density lipoprotein, intermediate-density lipoprotein, very-low-density lipoprotein, lipoprotein (a), or chylomicron remnants. The particles also contained small exchangeable apolipoproteins, complement components, and immunoglobulins. Lipidomic analysis revealed differences between plasma lipoproteins and the particles, thereby indicating involvement of lipolytic enzymes in their generation. Incubation of human monocyte-derived macrophages with the isolated extracellular lipoprotein particles or with plasma lipoproteins that had been lipolytically modified in vitro induced intracellular lipid accumulation and triggered inflammasome activation in them. Taken together, extracellular lipids accumulate in human carotid plaques as distinct 3D structures that include aggregated and fused lipoprotein particles and cholesterol crystals. The particles originate from plasma lipoproteins, show signs of lipolytic modifications, and associate with cholesterol crystals. By inducing intracellular cholesterol accumulation (ie, foam cell formation) and inflammasome activation, the extracellular lipoprotein particles may actively enhance atherogenesis.

Smooth Muscle Cell Foam Cell Formation, Apolipoproteins, and ABCA1 in Intracranial Aneurysms: Implications for Lipid Accumulation as a Promoter of Aneurysm Wall Rupture.

Saccular intracranial aneurysm (sIA) aneurysm causes intracranial hemorrhages that are associated with high mortality. Lipid accumulation and chronic inflammation occur in the sIA wall. A major mechanism for lipid clearance from arteries is adenosine triphosphate-binding cassette A1 (ABCA1)-mediated lipid efflux from foam cells to apolipoprotein A-I (apoA-I). We investigated the association of wall degeneration, inflammation, and lipid-related parameters in tissue samples of 16 unruptured and 20 ruptured sIAs using histology and immunohistochemistry. Intracellular lipid accumulation was associated with wall remodeling (p = 0.005) and rupture (p = 0.020). Foam cell formation was observed in smooth muscle cells, in addition to CD68- and CD163-positive macrophages. Macrophage infiltration correlated with intracellular lipid accumulation and apolipoproteins, including apoA-I. ApoA-I correlated with markers of lipid accumulation and wall degeneration (p = 0.01). ApoA-I-positive staining colocalized with ABCA1-positive cells particularly in sIAs with high number of smooth muscle cells (p = 0.003); absence of such colocalization was associated with wall degeneration (p = 0.017). Known clinical risk factors for sIA rupture correlated inversely with apoA-I. We conclude that lipid accumulation associates with sIA wall degeneration and risk of rupture, possibly via formation of foam cells and subsequent loss of mural cells. Reduced removal of lipids from the sIA wall via ABCA1-apoA-I pathway may contribute to this process.

Vascular cell kinetics in response to intimal injury ex vivo.

BACKGROUND: Recent studies indicate that the smooth muscle-like cells contributing to neointimal hyperplasia after vascular injury derive from circulating precursor cells. Here, we define the time course of precursor cell influx, the roles of separate vascular layers, and the relative role of migration versus proliferation to intimal hyperplasia. METHODS AND RESULTS: After rat aortic denudation injury the neointimal cell number increased several 100-fold between days 4 and 28, preceded by a 5-fold increase in the number of adventitial cells and a 4-fold increase in the number of adventitial microvessels. The influx, migration, and maturation of neointimal cells were quantitated by culturing whole vessel explants at different time points after injury. Explant outgrowth increased 14-fold, and cell migration 3.5-fold on days 2-14 after injury. Cell proliferation increased less than 2-fold. The frequency of precursors to outgrowing cells, determined using limiting dilution analysis, increased 8-fold between days 2 and 4 after injury. Many outgrowing cells displayed characteristics of undifferentiated cells. CONCLUSIONS: Adventitial activation precedes development of the neointima, and precursor cell influx occurs on days 2-14 after injury. Cell migration, more than proliferation, contributes to fibrointimal dysplasia. These findings underline the importance of early therapeutic intervention with antimigratory compounds to prevent neointimal hyperplasia.

Caveolar transport through nasal epithelium of birch pollen allergen Bet v 1 in allergic patients.

BACKGROUND: Previous work in type I pollen allergies has focused on aberrant immunoresponses. OBJECTIVE: Our systems-level analyses explore the role of epithelium in early pathogenesis of type I allergic reactions. METHODS: We began top-down analyses of differences in human nasal epithelial cells and biopsy specimens obtained from patients with birch allergy and healthy control subjects in the resting state and after intranasal in vivo birch pollen challenges. Immunohistochemistry, immunotransmission electron microscopy, mass spectrometry, transcriptomics, and integration of data to a pathway were conducted. RESULTS: Bet v 1 allergen bound to epithelium immediately after in vivo birch pollen challenge during winter only in allergic individuals. It also travelled through epithelium with caveolae to mast cells. Sixteen unique proteins were found to bind to the Bet v 1 column only in lysates from allergic epithelial cells; 6 of these were caveolar and 6 were cytoskeletal proteins. The nasal epithelial transcriptome analysis from allergic and healthy subjects differed during the winter season, and these subjects also responded differentially to birch pollen challenge. Within this pollen-induced response, the gene ontology categories of cytoskeleton and actin cytoskeleton were decreased in allergic patients, whereas the actin-binding category was enriched in healthy subjects. Integration of microscopic, mass spectrometric, and transcriptomic data to a common protein-protein binding network showed how these were connected to each other. CONCLUSION: We propose a hypothesis of caveolae-dependent uptake and transport of birch pollen allergen in the epithelium of allergic patients only. Application of discovery-driven methodologies can provide new hypotheses worth further analysis of complex multifactorial diseases, such as type I allergy.

Mechanisms behind the synergistic effect of sirolimus and imatinib in preventing restenosis after intimal injury.

BACKGROUND: We have shown that the combination of sirolimus and imatinib synergistically inhibits denudation-induced neointimal hyperplasia in rats. We have now dissected the mechanisms behind this synergy and evaluated its long-term efficacy. METHODS: After aortic denudation injury, rats received established submaximal doses of sirolimus (1.0 mg/kg/day), imatinib (10.0 mg/kg/day), the combination of these, or vehicle per os from 3 days before the operation until 14 days after injury. Vessel histology and complete blood counts were monitored until 90 days after injury. Neointimal cell outgrowth, migration and proliferation were evaluated in ex vivo vessel cultures. Quantitative real-time polymerase chain reaction and immunohistochemistry were used for gene and protein expression analysis. RESULTS: The combination therapy caused a synergistic decrease in the number of neointimal nuclei and area throughout the observation period. It also prevented postinjury thrombocytosis and leukocytosis, and almost abolished neointimal cell outgrowth and migration. Furthermore, the combination therapy resulted in upregulation of smooth muscle cell (SMC) markers SM22alpha and cysteine and glycine-rich protein 2, and of the anti-apoptotic BCL2 mRNA. CONCLUSIONS: Combination therapy confers superior long-term vasculoprotection, possibly by inhibition of postoperative thrombocytosis and leukocytosis, inhibition of neointimal cell migration to the injury site and maintenance of cell integrity by inhibition of apoptosis and SMC dedifferentiation.