The effect of short-term aerobic training on coagulation and fibrinolytic factors in sedentary healthy postmenopausal women

Objective

The purpose of this study was to assess the effect of short-term aerobic training on the fibrinolytic and coagulative factors in postmenopausal women.

Study design

Twenty volunteer sedentary healthy postmenopausal women (48–53 years), who entered the menopause naturally, were divided randomly into two groups: training (n = 10) and control (n = 10).

Methods and main outcome measures

Training consisted of 10 sessions of submaximal aerobic cycling, 35 min for each session (5 min warm-up, 25 min aerobic training with 70% HRmax, 5 min active and 15 min passive recovery), 3 times a week. Coagulation and fibrinolytic factors were assessed both before and after aerobic program in both groups.

Results

Fibrinogen, von Willbrand factor (vWF-Ag) antigen, plasminogen activator inhibitor-1 activity (PAI-1:Ac) and antigen (PAI-1:Ag) showed significant reduction after 10 sessions in the training group (P < 0.05). Also after training, prothrombin time (PT), partial thromboplastin time (PTT), tissue plasminogen activator activity (tPA:Ac) and antigen (tPA:Ag) increased (P < 0.05).

Conclusion

It was concluded that fibrinolytic activity on postmenopausal women could be improved by a 3-week regular submaximal training program. These changes on the hemostatic factors suggest that short-term aerobic training may prevent the decline in fibrinolytic function observed in sedentary postmenopausal women.     

Reduced monocyte adhesion to aortae of diabetic plasminogen activator inhibitor-1 knockout mice

Objective and design

To determine the requirement of plasminogen activator inhibitor-1-knockout (PAI-1) for monocyte adhesion in animals and cells under diabetic conditions.

Methods and subjects

Monocyte adhesion assay, enzyme-linked immunosorbent assay, and Western blotting were used in analyzing samples from PAI-1-knockout (PAI-1-KO) mice or cultured human umbilical vein endothelial cells (HUVEC).

Treatments

Diabetes in PAI-1-KO and wild-type mice was induced by intraperitoneal injection of streptozotocin (STZ). HUVEC was transfected with short interference RNA (siRNA) against PAI-1, tumor necrosis factor-α (TNFα), or toll-like receptor (TLR4), and then was treated with glycated low-density lipoproteins (glyLDL).

Results

The adhesion of monocytes to aortic intima was reduced in PAI-1-KO mice, which was associated with decreased levels of TNFα and monocyte chemotactic protein-1 (MCP-1) in plasma and cardiovascular tissue, and increased abundances of urokinase plasminogen activator (uPA) and uPA receptor (uPAR) in cardiovascular tissue compared to wild-type mice. Significant reductions in monocyte adhesion, inflammatory, and fibrinolytic regulators were detected in cardiovascular tissue or plasma in diabetic PAI-1-KO mice compared to wild-type diabetic mice. Transfection of PAI-1, TNFα or TLR4 siRNA to HUVEC inhibited glyLDL-induced monocyte adhesion to EC. PAI-1 siRNA inhibited the abundances of TLR4 and TNFα in EC.

Conclusion

The findings suggest that PAI-1 is required for diabetes-induced monocyte adhesion via interactions with uPA/uPAR, and it also regulates TLR4 and TNFα expression in vascular EC. Inhibition of PAI-1 potentially reduces vascular inflammation under diabetic condition.

     

Relationship between plasminogen activator inhibitor type-1 (PAI-1) gene polymorphisms and osteoporosis in Turkish women

OBJECTIVE:

The development of osteoporosis is associated with several risk factors, such as genetic structures that affect bone turnover and bone mass. The impact of genetic structures on osteoporosis is not known. Plasminogen activator inhibitor type-1 regulates the bone matrix and bone balance. This study assessed the correlation between plasminogen activator inhibitor type-1 gene 4G/5G polymorphisms and osteoporosis in a population of Turkish women.

METHODS:

A total of 195 postmenopausal female patients who were diagnosed with osteoporosis (Group I) based on bone mineral density measurements via dual-energy x-ray absorptiometry and 90 females with no osteoporosis (Group II) were included in this study. Correlations between PAI-1 gene 4G/5G polymorphisms and osteoporosis were investigated through the identification of PAI-1 gene 4G/5G polymorphism genotypes using the polymerase chain reaction.

RESULTS:

No significant differences in the genotype and allele frequency of 4G/5G plasminogen activator inhibitor type-1 polymorphisms were observed between the two groups, and both groups exhibited the most frequently observed 4G5G genotype.

CONCLUSION:

No correlation between the development of osteoporosis in the female Turkish population and 4G/5G plasminogen activator inhibitor type-1 gene polymorphisms was observed.     

Safety of herbal medicinal products in women with breast cancer

Objective

To review and summarise current evidence on the efficacy and safety of herbal medicinal products for the relief of hot flushes in women with previous breast cancer.

Methods

A literature search was conducted in the databases of Medline, EMBASE, The Cochrane Central Register of Controlled Trials (CENTRAL), PSYCHINFO, AMED (Allied and Complementary Medicine), NCCAM (The National Centre for Complementary and Alternative Medicine).

Results

Black cohosh and phytoestrogens have received the most research attention but there is currently insufficient evidence to recommend either for relief of flushes. Black cohosh use appears safe in women with previous breast cancer. Opposing advice has been given regarding the safety of dietary phytoestrogen use for women with previous breast cancer, but there is emerging data that soyfood phytoestrogen intake may have a beneficial effect on tumour recurrence.

Conclusions

The majority of studies, regarding the efficacy of herbal treatments for hot flushes, have not been conducted in women with breast cancer and many are of short duration. Increased pharmacovigilance practices for herbal medicines are required with initiatives to stimulate reporting of suspected adverse reactions.     

Mechanism of hepatotoxicity due to black cohosh (Cimicifuga racemosa): Histological,immunohistochemical and electron microscopy analysis of two liver biopsies with clinical correlation

Background

Consumption of herbal supplements in the developed world remains high. Cimicifuga racemosa (C. racemosa) extract, or black cohosh, is widely used as a hormone replacing and an anti-inflammatory agent, and has been shown to cause idiosyncratic hepatitis. The mechanism of acute liver injury in those cases is unclear. To date, hepatotoxic effects of C. racemosa have been studied mostly in vitro and in animal models. Data on human tissue is extremely limited, and mostly confined to histological findings of explanted livers.

Methods

We evaluated clinical data and examined surgical diagnostic liver biopsy specimens obtained from two female patients, who developed acute submassive liver necrosis, following consumption of C. racemosa. Both patients presented with acute elevation of liver enzymes, cholestasis, absence of reactivity to hepatitis A, B and C antibodies, and weak non-specific positivity for autoimmune serological markers. Initial histological interpretation of the biopsies, with focus on hepatic parenchyma and portal tracts, was done by light microscopy, followed by special stain series and immunohistochemical studies, including Cam 5.2, AE1/AE3, reticulin, α-actin, sirius red, and PAS with diastase. Areas of prominent lymphocytic infiltration of the periportal liver plate, observed microscopically, were further evaluated by electron microscopy (EM). 4HNE adduction study, an immunofluorescent assay, was performed to detect products of the oxidative damage and their localization in the liver parenchyma.

Results

Oxidative damage was evident by accumulation of 4HNE protein adducts in the cytoplasm of hepatocytes, secondary lysosomes and macrophages. We hypothesize that the adducted proteins, accumulated in the liver parenchyma, serve as autoantigens, which provoke an autoimmune response, and cause migration of lymphocytes to the affected regions. The formation of immunological synapses between hepatocytes and lymphocytes, predominantly T-lymphocytes, is demonstrated by electron microscopy. The autoimmune response induces piecemeal, or troxis necrosis of hepatocytes, a well described biological phenomenon, where lymphocytes gradually remove hepatocytes in a piecemeal fashion, slowly consuming them and leaving fragments of liver cells, or nubbins of anuclear cytoplasm of liver cell, at the interface between lymphocytes and hepatocytes.

Conclusion

The pattern of pathological injury of liver cells in both patients, following consumption of black cohosh, is identical to troxis necrosis, seen during autoimmune hepatitis. Recognition of the possibility of the acute hepatic injury by the herbal supplement black cohosh is essential for early accurate diagnosis, and timely patient management.     

Neurotransmitter noradrenaline downregulate cytoskeletal protein expression of VSMCs

Objective

This study investigates the effects of noradrenaline (NA) on cytoskeletal protein expression of vascular smooth muscle cells (VSMCs).

Methods

VSMCs were isolated from rat aortic tissue and cultured. The cultured VSMCs were divided into 4 experimental groups: (1) control group, (2) NA treatment group, (3) starvation group, and (4) NA treatment + starvation group. The expression of cytoskeletal protein (smooth muscle α-actin, β-tubulin and desmin) was evaluated by (i) Coomassie blue staining, (ii) immunofluorescent staining, and (iii) RT-PCR and Western Blot.

Results

NA treatment significantly downregulated the expression of SM α-actin, β-tubulin and desmin (P < 0.05). The serum starvation did not affect the expression of cytoskeletal protein (SM α-actin, β-tubulin and desmin), but when the cells were treated with the combination of NA and serum starvation, the expression of SM α-actin, β-tubulin and desmin were down-regulated than those of the serum starvation group (P < 0.05).

Conclusion

These results suggested that NA might play a key role in regulating the cytoskeletal protein expression of VSMCs.     

Metformin, but not glimepiride, improves carotid artery diameter and blood flow in patients with type 2 diabetes mellitus

OBJECTIVE:

To compare the effects of glimepiride and metformin on vascular reactivity, hemostatic factors and glucose and lipid profiles in patients with type 2 diabetes.

METHODS:

A prospective study was performed in 16 uncontrolled patients with diabetes previously treated with dietary intervention. The participants were randomized into metformin or glimepiride therapy groups. After four months, the patients were crossed over with no washout period to the alternative treatment for an additional four-month period on similar dosage schedules. The following variables were assessed before and after four months of each treatment: 1) fasting glycemia, insulin, catecholamines, lipid profiles and HbA₁ levels; 2) t-PA and PAI-1 (antigen and activity), platelet aggregation and fibrinogen and plasminogen levels; and 3) the flow indices of the carotid and brachial arteries. In addition, at the end of each period, a 12-hour metabolic profile was obtained after fasting and every 2 hours thereafter.

RESULTS:

Both therapies resulted in similar decreases in fasting glucose, triglyceride and norepinephrine levels, and they increased the fibrinolytic factor plasminogen but decreased t-PA activity. Metformin caused lower insulin and pro-insulin levels and higher glucagon levels and increased systolic carotid diameter and blood flow. Neither metformin nor glimepiride affected endothelial-dependent or endothelial-independent vasodilation of the brachial artery.

CONCLUSIONS:

Glimepiride and metformin were effective in improving glucose and lipid profiles and norepinephrine levels. Metformin afforded more protection against macrovascular diabetes complications, increased systolic carotid artery diameter and total and systolic blood flow, and decreased insulin levels. As both therapies increased plasminogen levels but reduced t-PA activity, a coagulation process was likely still ongoing.     

Premature differentiation of vascular smooth muscle cells in human congenital diaphragmatic hernia

Background

Congenital diaphragmatic hernia (CDH) is a rare congenital anomaly characterized by the herniation of abdominal organs into the chest cavity. The high mortality and morbidity of CDH patients are primarily caused by the associated pulmonary hypertension (PH), characterized by the thickening of the vascular media and adventitia. The media consist of heterogeneous populations of vascular smooth muscle cells (VSMC), ranging from synthetic to the characteristic contractile cells. VSMCs are influenced by developmental and environmental cues and may play a role in the development of the structural changes observed in CDH patients. Therefore, we hypothesized that the distribution of the VSMC populations may already be different at the origin of CDH development.

Methodology

We analyzed the protein expression of specific markers associated with synthetic and contractile VSMC phenotypes in human lungs at different developmental stages. Next, we compared lungs of premature and term CDH patients, as well as patients with lung hypoplasia due to renal agenesis or PROM, with age-matched controls.

Results

Synthetic and contractile VSMCs are distributed in a temporal and spatial specific pattern along the proximodistal axis of the lung. CDH patients have more abundant contractile VSMCs which are also more distally distributed. This different distribution pattern is already observed from 19 weeks of gestation onwards.

Conclusion

Our data suggest that the more extensive distribution of contractile VSMCs is associated with an early maturation of the pulmonary vasculature, contrasting the concept that CDH might be the result of delayed maturation of the epithelium.     

尼古丁对血管内皮细胞释放t-PA及PAI-1的影响

目的: 研究尼古丁对人脐静脉内皮细胞(HUVECs)释放组织型纤溶酶原激活物(t-PA)和纤溶酶原激活物抑制物-1(PAI-1)的影响。方法: HUVECs培养后接种于24孔培养板中,随机分为对照组及实验组,分别进行以下实验。(1)以0.1、1、10、100 μmol/L 尼古丁孵育HUVECs,12 h后收集各组上清液;(2)以100 μmol/L尼古丁与HUVECs孵育0、4、6、8、12 及24 h,收集各组上清液。采用ELISA法测定各组t-PA和PAI-1的浓度。结果: HUVECs与不同浓度尼古丁孵育12 h后,100 μmol/L尼古丁组PAI-1蛋白较对照组明显增加(P<0.01);0.1、1及10 μmol/L尼古丁组PAI-1蛋白与对照组比较,均无显著差异(均P>0.05);各浓度组t-PA蛋白与对照组比较,均无显著差异(均P>0.05)。HUVECs 与100 μmol/L的尼古丁分别孵育4 、6 、8 、12 及24 h,各组PAI-1蛋白均较对照组明显升高(P<0.05),且其升高呈时间依赖性;各组t-PA与对照组比较,均无显著差异(均P>0.05)。结论: 尼古丁可抑制HUVECs的纤溶活性,对内皮细胞具有损伤作用。     

Applying qualitative methods in developing a culturally tailored workbook for black patients with hypertension

Objective

To apply qualitative research methods in developing a culturally tailored, educational workbook for hypertensive black patients.

Methods

The workbook was developed using formative qualitative data from 60 black primary care patients with hypertension. Participants were interviewed using qualitative methods and data were analyzed through sequential steps of open coding, axial coding, and selective coding. From these analyses, themes describing patients’ cultural beliefs about hypertension were derived and used to develop the workbook.

Results

The workbook, “Living With Hypertension: Taking Control” is a 37-page illustrated workbook with 11 chapters based on patients’ perceptions of hypertension. These chapters focus on strengthening participants’ ability to take control and manage hypertension and on providing knowledge and health behavior techniques.

Conclusion

Qualitative research methods were used to inform the development of a culturally tailored educational workbook.

Practice implications

The workbook developed in this study may offer a practical and effective means of educating patients about blood pressure control in primary care settings.     

Circulating levels of vascular endothelial markers in obstructive sleep apnoea syndrome. Effects of nasal continuous positive airway pressure

Introduction

Obstructive sleep apnoea syndrome (OSAS) is an important risk factor in cardiovascular disorders. Although the exact mechanism remains to be elucidated, the endothelial dysfunction process seems to be implicated.

Material and methods

In order to test this hypothesis, blood circulating levels of endothelial markers were measured at baseline and 1 year after treatment with continuous positive airway pressure (CPAP). We studied 37 males using polysomnography: 20 subjects with OSAS and a 17-subject control group. An OSAS-validated sleep questionnaire covering the most important cardiovascular risk factors was applied to all subjects. Furthermore, patients received a complete general physical examination and biochemistry test with lipid profile. The specific markers measured were intercellular cell adhesion molecule-1 (ICAM-1), E-selectin, endothelin-1, von Willebrand factor (vWF) and plasminogen activator inhibitor-1 (PAI-1).

Results

Obstructive sleep apnoea syndrome patients presented higher circulating levels of ICAM-1, endothelin-1 and PAI-1 than the control group. On the other hand, no differences were found in E-selectin and vWF. After 1 year of CPAP treatment, there was a significant decrease in circulating levels of ICAM-1 and PAI-1. On the other hand, no differences were found in endothelin-1, E-selectin and vWF.

Conclusions

Obstructive sleep apnoea syndrome is associated with elevated levels of ICAM-1 and PAI-1 and these levels normalize after treatment with CPAP.     

Digitized assessment of mammographic breast density--effects of continuous combined hormone therapy, tibolone and black cohosh compared to placebo

Objectives

To determine the effects of continuous combined hormone therapy, tibolone, black cohosh, and placebo on digitized mammographic breast density in postmenopausal women.

Study design

A prospective, double-blind, placebo-controlled study of 154 postmenopausal women randomized to estradiol 2 mg/norethisterone acetate 1 mg (E2/NETA), tibolone 2.5 mg or placebo and a prospective, open, uncontrolled drug safety study, of which 65 postmenopausal women were treated with black cohosh. Mammograms, at baseline and after six months of treatment, were previously classified according to visual quantification scales.

Main outcome measures

Reanalysis of assessable mammograms by digitized quantification of breast density.

Results

Treatment groups were comparable at baseline. During treatment, both E2/NETA and tibolone significantly increased breast density (mean increase 14.3%, p < 0.001 and 2.3%, p < 0.001, respectively), while black cohosh and placebo did not. Twenty-four out of the 43 women on E2/NETA had an increase in density exceeding 10% and 6 women had an increase of 30% or more. In the tibolone group, only one woman had an increase in density of more than 10%. The difference in increase in breast density between E2/NETA on the one hand and tibolone, black cohosh and placebo on the other was highly significant (p < 0.0001).

Conclusions

Digitized mammographic breast density is a highly sensitive method confirming significant increase in density by standard E2/NETA treatment and to a lesser extent by tibolone, whereas black cohosh does not influence mammographic breast density during six months treatment. Digitized assessment also yields data on individual variation and small increases left undetectable by visual classification.     

Age and aerobic training status effects on plasma and skeletal muscle tPA and PAI-1

Introduction

Reductions in fibrinolytic potential occur with both aging and physical inactivity and are associated with an increased cardiovascular disease risk. Plasmin, the enzyme responsible for the enzymatic degradation of fibrin clots, is activated by tissue plasminogen activator (tPA), while plasminogen activator inhibitor-1 (PAI-1) inhibits its activation. Currently, fibrinolysis research focuses almost exclusively on changes within the plasma. However, tPA and PAI-1 are expressed by human skeletal muscle (SM). Currently, no studies have focused on changes in SM fibrinolytic activity with regard to aging and aerobic fitness.

Purpose

The purpose of this study was to cross-sectionally evaluate effects of age and aerobic fitness on tPA and PAI-1 expressions and activity in SM.

Methods

Twenty-six male subjects were categorized into the following groups: (1) young aerobically trained (n = 8); (2) older aerobically trained (n = 6); (3) young aerobically untrained (n = 7); and (4) older aerobically untrained (n = 5). Muscle biopsies were obtained from each subject. SM tPA activity was assessed using gel zymography and SM tPA and PAI-1 expressions were assessed using RT-PCR.

Results

Trained subjects had higher SM tPA activity compared to untrained (25.3 ± 2.4 × 10³ vs. 21.5 ± 5.6 × 10³ pixels, respectively; p = 0.03) with no effect observed for age. VO_{2 max} and SM tPA activity were also significantly correlated (r = 0.42; p < 0.04). SM tPA expression was higher in older participants, but no effect of fitness level was observed. No differences were observed for PAI-1 expression in SM.

Conclusions

Higher levels of aerobic fitness are associated with increased fibrinolytic activity in SM.     

Day/Night Rhythm of Hemostatic Factors in Obstructive Sleep Apnea

Study Objectives:

To investigate the hypothesis that day/night patterns of prothrombotic activity differ between patients with obstructive sleep apnea (OSA) and individuals with no OSA.

Design:

Prothrombotic markers'' day/night rhythms recorded over one 24-h period.

Setting:

General clinical research center.

Patients:

38 untreated OSA patients as verified by polysomnography (apnea-hypopnea index ≥10/h sleep) and 22 non-OSA controls.

Measurements and Results:

Blood samples were collected every 2 h to measure plasma levels of fibrinolysis-inhibiting plasminogen activator inhibitor (PAI)-1 and the primary fibrin degradation product D-dimer. Day/night variation in hemostasis factors was examined using a cosinor analysis. Mesor (mean) PAI-1 over the 24-h period was higher (P = 0.015), and mesor of D-dimer was lower (P = 0.001) in patients with OSA than in the non-OSA controls. These group differences stayed significant when controlling for age and gender. After further adjustment for body mass index, mean arterial pressure, and smoking, the relationship between OSA and PAI-1 became non-significant, but the relationship between OSA and D-dimer continued to be significant (P = 0.006). In the fully adjusted analysis, the amplitude (peak) for D-dimer was lower in OSA patients than in non-OSA controls (P = 0.048). The acrophase (time of the peak) for PAI-1 and D-dimer did not significantly differ between groups.

Conclusions:

The relatively higher average level of PAI-1 and lower average level of D-dimer across the 24-h in OSA patients might reflect decreased fibrinolytic capacity and fibrin degradation, respectively. The findings provide some evidence for a prothrombotic state in OSA, but were only partially independent of metabolic variables.

Citation:

von Käanel R; Natarajan L; Ancoli-Israel S; Mills PJ; Loredo JS; Dimsdale JE. Day/night rhythm of hemostatic factors in obstructive sleep apnea. SLEEP 2010;33(3):371-377.     

Neutralization of plasminogen activator inhibitor-1 (PAI-1) by activated protein C is species-dependent

The interaction of bovine and human activated protein C (APC) with type-1 plasminogen activator inhibitor (PAI-1) was studied in a cell-free system. Human plasma and a preparation of PAI-1 obtained from human endothelial cell cultures were used as sources of PAI-1. Bovine APC was able to neutralize PAI-1 inhibitory activity present in both sources in a dose-dependent manner; the concentration of bovine APC required to produce 50% (C₅₀) neutralization of endothelial PAI-1 (0.5nM) was 4 μg/ml (64nM). Moreover, when complexes between tissue plasminogen activator (t-PA, 60ng/ml, 0.84nM) and PAI-1(18.8ng/ml, 0.35nM) were incubated with bovine APC, the amount of such complexes decreased as a function of the concentration of added APC (C₅₀ = 2 μ/ml, 32 nM), and a concomitant increase in the amount of residual t-PA activity was observed. This effect was due to the formation of APC⊎PAI-1 complexes as detected by immunoblotting with monoclonal antibodies directed against PAI-1. By this mechanism bovine APC prevented the initial reaction between PAI-1 and t-PA and interfered with the stability of complexes between t-PA and PAI-1. The latter observation suggests that complexes between t-PA and PAI-1 may dissociate in the presence of bovine APC. In contrast with these findings, when the experiments were performed in an entirely human homologous cell-free system, human APC did not form a complex with PAI-1 and no effect either on PAI-1 or on the stability of preformed t-PA ⊎ PAI-1 complexes was observed. The results indicate that the neutralization of PAI-1 by APC is a phenomenon induced by interspecies molecular interactions.     

Potentiating effect of endothelial cells on astrocytic plasminogen activator inhibitor type-1 gene expression in an in vitro model of the blood–brain barrier

There is accumulating evidence of the importance of cellular communication between the cells that compose the blood-brain barrier (BBB). Astrocytes are known to affect the expression of tissue-type plasminogen activator (t-PA) and its inhibitor plasminogen activator inhibitor type-1 (PAI-1) in endothelial cells. We investigated the influence of endothelial cells on astrocytic gene expression of PAI-1, protease nexin-1 (PN-1) and t-PA using an in vitro model of the BBB. Primary rat astrocyte-enriched cultures were cocultured with primary adult rat brain microvascular endothelial cells on opposite sides of a transwell membrane. After coculturing for 9–11 days, the cultures were treated with lipopolysaccharide (LPS) for 8 h or 24 h. The levels of PAI-1, PN-1 and t-PA mRNA in untreated and treated monocultures and cocultures were analyzed by Real-Time RT-PCR. Cocultivation of astrocytes and endothelial cells increased astrocytic PAI-1 mRNA expression, and this response was further amplified by LPS treatment. The levels of PN-1 and t-PA mRNA expression in astrocytes were unaffected by cocultivation and/or LPS treatment. Analysis of endothelial PAI-1 and t-PA gene expression revealed increased PAI-1 mRNA levels in cocultured cells, whereas t-PA mRNA levels remained unchanged. These results demonstrate that the cocultivation of astrocytes and endothelial cells induces a pronounced increase in astrocytic PAI-1 gene expression, and that this effect is amplified by LPS treatment. These findings imply an important role for intercellular crosstalk in modulating PAI-1 gene expression within the BBB, under both physiologic and pathophysiologic conditions.