Urokinase plasminogen activator released by alveolar epithelial cells modulates alveolar epithelial repair in vitro

Intra-alveolar fibrin is formed following lung injury and inflammation and may contribute to the development of pulmonary fibrosis. Fibrin turnover is altered in patients with pulmonary fibrosis, resulting in intra-alveolar fibrin accumulation, mainly due to decreased fibrinolysis. Alveolar type II epithelial cells (AEC) repair the injured alveolar epithelium by migrating over the provisional fibrin matrix. We hypothesized that repairing alveolar epithelial cells modulate the underlying fibrin matrix by release of fibrinolytic activity, and that the degree of fibrinolysis modulates alveolar epithelial repair on fibrin. To test this hypothesis we studied alveolar epithelial wound repair in vitro using a modified epithelial wound repair model with human A549 alveolar epithelial cells cultured on a fibrin matrix. In presence of the inflammatory cytokine interleukin-1beta, wounds increase by 800% in 24 hours mainly due to detachment of the cells, whereas in serum-free medium wound areas decreases by 22.4 +/- 5.2% (p < 0.01). Increased levels of D-dimer, FDP and uPA in the cell supernatant of IL-1beta-stimulated A549 epithelial cells indicate activation of fibrinolysis by activation of the plasmin system. In presence of low concentrations of fibrinolysis inhibitors, including specific blocking anti-uPA antibodies, alveolar epithelial repair in vitro was improved, whereas in presence of high concentrations of fibrinolysis inhibitors, a decrease was observed mainly due to decreased spreading and migration of cells. These findings suggest the existence of a fibrinolytic optimum at which alveolar epithelial repair in vitro is most efficient. In conclusion, uPA released by AEC alters alveolar epithelial repair in vitro by modulating the underlying fibrin matrix.

The effect of natural habituation on coagulation responses to acute mental stress and recovery in men

Blood coagulation activation might be one mechanism linking acute mental stress with coronary events. We investigated the natural habituation of coagulation responses and recovery to short-term mental stress. Three times with one-week intervals, 24 men (mean age 47 +/- 7 years) underwent the same 13-min stressor (preparation, job interview, mental arithmetic). During each visit venous blood was obtained four times (baseline, immediately post-stress, 45 min of recovery, 105 min of recovery). Eight blood coagulation parameters were measured at weeks one and three. Acute stress provoked increases in von Willebrand factor antigen, fibrinogen, clotting factor FVII activity (FVII:C), FVIII:C, FXII:C (p's < or = 0.019), and D-dimer (N.S.). All coagulation parameters experienced full recovery except FVIII:C (p = 0.022). Stress did not significantly affect activated partial thromboplastin time and prothrombin time. At all time points FVIII:C and FXII:C levels were significantly higher at week one compared to week three (p's < or = 0.041). Before catheter insertion, systolic blood pressure (p = 0.001) and heart rate (p = 0.026) were relatively higher at week one. Unlike the magnitude of systolic blood pressure response to stress (p = 0.007) and of cortisol recovery from stress (p = 0.002), the magnitude of all coagulation responses to stress and the recovery from stress were similar in week one and week three. Sympathetic activation with anticipatory stress best explained increased baseline activity in FVIII and FXII at week one. An incapacity of the coagulation system to adapt to stress repeats is perhaps a consequence of evolution, but might also contribute to increased coronary risk in some individuals, particularly in those with cardiovascular diseases.

Off-pump coronary artery bypass operation does not increase procoagulant and fibrinolytic activity: preliminary results

BACKGROUND: This study analyzes the effects on coagulation and fibrinolysis comparing off-pump coronary artery bypass (OPCAB) and on-pump CABG operations. METHODS: In a prospective, nonrandomized, comparative evaluation, patients scheduled for elective myocardial revascularization were studied. Due to possible confounding factors patients with postoperative retransfusion of mediastinal shed blood were excluded. Nine patients underwent OPCAB operation and 16 underwent on-pump CABG. Activated clotting time (ACT) was adjusted to 250 seconds in OPCAB (81 +/- 18 [mean +/- SD] IU/kg heparin) and to more than 480 seconds in on-pump CABG (400 IU/kg heparin, additional 10,000 IU in pump prime). Perioperatively blood samples were collected and hematologic and hemostatic variables including fibrinopeptide A (FPA), fibrin monomer (FM), thrombin-antithrombin complex (TAT), and D-dimer were analyzed. RESULTS: Both groups showed comparable demographic variables. Number of grafts per patient was slightly higher in the on-pump group (3.6 +/- 0.6 versus 3.0 +/- 1.1, p = 0.23). The FPA levels did not differ significantly between the groups. The FM, TAT, and D-dimer values were significantly higher in on-pump CABG (p < 0.0001, p < 0.01, and p < 0.0001, respectively), reflecting increased coagulant and fibrinolytic activity. This was also the case when values were corrected for hemodilution. CONCLUSIONS: Despite lower systemic anticoagulation activation of coagulation and fibrinolysis is reduced in OPCAB compared with on-pump CABG. Reduced thrombin generation and reduced fibrinolytic activity in OPCAB indicates better preservation of hemostasis. We suggest the term "preserved hemostasis" instead of "hypercoagulant activity" with respect to OPCAB.

Independent association between lower level of social support and higher coagulation activity before and after acute psychosocial stress

OBJECTIVE: To investigate the relationship between social support and coagulation parameter reactivity to mental stress in men and to determine if norepinephrine is involved. Lower social support is associated with higher basal coagulation activity and greater norepinephrine stress reactivity, which in turn, is linked with hypercoagulability. However, it is not known if low social support interacts with stress to further increase coagulation reactivity or if norepinephrine affects this association. These findings may be important for determining if low social support influences thrombosis and possible acute coronary events in response to acute stress. We investigated the relationship between social support and coagulation parameter reactivity to mental stress in men and determined if norepinephrine is involved. METHODS: We measured perceived social support in 63 medication-free nonsmoking men (age (mean +/- standard error of the mean) = 36.7 +/- 1.7 years) who underwent an acute standardized psychosocial stress task combining public speaking and mental arithmetic in front of an audience. We measured plasma D-dimer, fibrinogen, clotting Factor VII activity (FVII:C), and plasma norepinephrine at rest as well as immediately after stress and 20 minutes after stress. RESULTS: Independent of body mass index, mean arterial pressure, and age, lower social support was associated with higher D-dimer and fibrinogen levels at baseline (p < .012) and with greater increases in fibrinogen (beta = -0.36, p = .001; DeltaR(2) = .12), and D-dimer (beta = -0.21, p = .017; DeltaR(2) = .04), but not in FVII:C (p = .83) from baseline to 20 minutes after stress. General linear models revealed significant main effects of social support and stress on fibrinogen, D-dimer, and norepinephrine (p < .035). Controlling for norepinephrine did not change the significance of the reported associations between social support and the coagulation measures D-dimer and fibrinogen. CONCLUSIONS: Our results suggest that lower social support is associated with greater coagulation activity before and after acute stress, which was unrelated to norepinephrine reactivity.

Overcommitment but not effort-reward imbalance relates to stress-induced coagulation changes in teachers

BACKGROUND: Stress-related hypercoagulability might link job stress with atherosclerosis. PURPOSE: This paper aims to study whether overcommitment, effort-reward imbalance, and the overcommitment by effort-reward imbalance interaction relate to an exaggerated procoagulant stress response. METHODS: We assessed job stress in 52 healthy teachers (49 +/- 8 years, 63% women) at study entry and, after a mean follow-up of 21 +/- 4 months, when they underwent an acute psychosocial stressor and had coagulation measures determined in plasma. In order to increase the reliability of job stress measures, entry and follow-up scores of overcommitment and of effort-reward imbalance were added up to total scores. RESULTS: During recovery from stress, elevated overcommitment correlated with D-dimer increase and with smaller fibrinogen decrease. In contrast, overcommitment was not associated with coagulation changes from pre-stress to immediately post-stress. Effort-reward imbalance and the interaction between overcommitment and effort-reward imbalance did not correlate with stress-induced changes in coagulation measures. CONCLUSIONS: Overcommitment predicted acute stress-induced hypercoagulability, particularly during the recovery period.

Prothrombotic changes with acute psychological stress: combined effect of hemoconcentration and genuine coagulation activation

INTRODUCTION: Acute psychosocial stress accelerates blood coagulation and elicits hemoconcentration which mechanisms are implicated in acute coronary thrombotic events. We investigated the extent to which the change in prothrombotic measures with acute stress reflects hemoconcentration and genuine activation of coagulation. MATERIAL AND METHODS: Twenty-one middle-aged healthy men underwent three sessions of a combined speech and mental arithmetic task with one-week intervals. Coagulation and plasma volume were assessed at baseline, immediately post-stress, and 45 min post-stress at sessions one and three. Measures of both visits were aggregated to enhance robustness of individual biological stress responses. Changes in eight coagulation measures with and without adjustment for simultaneous plasma volume shift were compared. RESULTS: From baseline to immediately post-stress, unadjusted levels of fibrinogen (p=0.028), clotting factor VII activity (FVII:C) (p=0.001), FVIII:C (p<0.001), FXII:C (p<0.001), and von Willebrand factor (VWF) (p=0.008) all increased. Taking into account hemoconcentration, fibrinogen (p=0.020) and FVII:C levels (p=0.001) decreased, activated partial prothrombin time (APPT) shortened (p<0.001) and prothrombin time (PT) was prolonged (p<0.001). Between baseline and 45 min post-stress, unadjusted (p=0.050) and adjusted (p=0.001) FVIII:C levels increased, adjusted APTT was prolonged (p=0.017), and adjusted PT was shortened (p=0.033). D-dimer levels did not significantly change over time. CONCLUSIONS: Adjustment for stress-hemoconcentration altered the course of unadjusted levels of several prothrombotic factors. After adjustment for hemoconcentration, APPT was shortened immediately post-stress, whereas 45 min post-stress, FVIII:C was increased and PT was shortened. Procoagulant changes to acute stress may reflect both hemoconcentration and genuine activation of coagulation molecules and pathways.

A bHLH Complex Controls Embryonic Vascular Tissue Establishment and Indeterminate Growth in Arabidopsis

Plants have a remarkable potential for sustained (indeterminate) postembryonic growth. Following their specification in the early embryo, tissue-specific precursor cells first establish tissues and later maintain them postembryonically. The mechanisms underlying these processes are largely unknown. Here we define local control of oriented, periclinal cell division as the mechanism underlying both the establishment and maintenance of vascular tissue. We identify an auxin-regulated basic helix-loop-helix (bHLH) transcription factor dimer as a critical regulator of vascular development. Due to a loss of periclinal divisions, vascular tissue gradually disappears in bHLH-deficient mutants; conversely, ectopic expression is sufficient for triggering periclinal divisions. We show that this dimer operates independently of tissue identity but is restricted to a small vascular domain by integrating overlapping transcription patterns of the interacting bHLH proteins. Our work reveals a common mechanism for tissue establishment and indeterminate vascular development and provides a conceptual framework for developmental control of local cell divisions.

J- vs. H-type assembly: pentamethine cyanine (Cy5) as a near-IR chiroptical reporter

The DNA-enabled dimerization of pentamethine cyanine (Cy5) dyes was studied by optical methods. The value of cyanine as a chiroptical reporter using a monomer-to-dimer switch is demonstrated. The specific shape of the CD signal and its high intensity are a result of J-type assembly.

Structural rearrangements of the central region of the morbillivirus attachment protein stalk domain trigger F protein refolding for membrane fusion

It is unknown how receptor binding by the paramyxovirus attachment proteins (HN, H, or G) triggers the fusion (F) protein to fuse with the plasma membrane for cell entry. H-proteins of the morbillivirus genus consist of a stalk ectodomain supporting a cuboidal head; physiological oligomers consist of non-covalent dimer-of-dimers. We report here the successful engineering of intermolecular disulfide bonds within the central region (residues 91-115) of the morbillivirus H-stalk; a sub-domain that also encompasses the putative F-contacting section (residues 111-118). Remarkably, several intersubunit crosslinks abrogated membrane fusion, but bioactivity was restored under reducing conditions. This phenotype extended equally to H proteins derived from virulent and attenuated morbillivirus strains and was independent of the nature of the contacted receptor. Our data reveal that the morbillivirus H-stalk domain is composed of four tightly-packed subunits. Upon receptor binding, these subunits structurally rearrange, possibly inducing conformational changes within the central region of the stalk, which, in turn, promote fusion. Given that the fundamental architecture appears conserved among paramyxovirus attachment protein stalk domains, we predict that these motions may act as a universal paramyxovirus F-triggering mechanism.

Hemozoin formation in malaria: a two-step process involving histidine-rich proteins and lipids

Major blood stage antimalarial drugs like chloroquine and artemisinin target the heme detoxification process of the malaria parasite. Hemozoin formation reactions in vitro using the Plasmodium falciparum histidine-rich protein-2 (Pfhrp-2), lipids, and auto-catalysis are slow and could not explain the speed of detoxification needed for parasite survival. Here, we show that malarial hemozoin formation is a coordinated two component process involving both lipids and histidine-rich proteins. Hemozoin formation efficiency in vitro is 1-2% with Pfhrp-2 and 0.25-0.5% with lipids. We added lipids after 9h in a 12h Pfhrp-2 mediated reaction that resulted in sixfold increase in hemozoin formation. However, a lipid mediated reaction in which Pfhrp-2 was added after 9h produced only twofold increase in hemozoin production compared to the reaction with Pfhrp-2 alone. Synthetic peptides corresponding to the Pfhrp-2 heme binding sequences, based on repeats of AHHAAD, neither alone nor in combination with lipids were able to generate hemozoin in vitro. These results indicate that hemozoin formation in malaria parasite involves both the lipids and the scaffolding proteins. Histidine-rich proteins might facilitate hemozoin formation by binding with a large number of heme molecules, and facilitating the dimer formation involving iron-carboxylate bond between two heme molecules, and lipids may then subsequently assist the mechanism of long chain formation, held together by hydrogen bonds or through extensive networking of hydrogen bonds.

Synthesis and Biological Activity of 7,8,9-Trideoxy- and 7 R DesTHP-Peloruside A

The stereoselective syntheses of 7,8,9-trideoxypeloruside A (4) and a monocyclic peloruside A analogue lacking the entire tetrahydropyran moiety (3) are described. The syntheses proceeded through the PMB-ether of an ω-hydroxy β-keto aldehyde as a common intermediate which was elaborated into a pair of diastereomeric 1,3-syn and -anti diols by stereoselective Duthaler–Hafner allylations and subsequent 1,3-syn or anti reduction. One of these isomers was further converted into a tetrahydropyran derivative in a high-yielding Prins reaction, to provide the precursor for bicyclic analogue 4. Downstream steps for both syntheses included the substrate-controlled addition of a vinyl lithium intermediate to an aldehyde, thus connecting the peloruside side chain to C15 (C13) of the macrocyclic core structure in a fully stereoselective fashion. In the case of monocyclic 3 macrocyclization was based on ring-closing olefin metathesis (RCM), while bicyclic 4 was cyclized through Yamaguchi-type macrolactonization. The macrolactonization step was surprisingly difficult and was accompanied by extensive cyclic dimer formation. Peloruside A analogues 3 and 4 inhibited the proliferation of human cancer cell lines in vitro with micromolar and sub-micromolar IC50 values, respectively. The higher potency of 4 highlights the importance of the bicyclic core structure of peloruside A for nM biological activity.

Dimerization of β-site β-amyloid precursor protein-cleaving enzyme

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

Testosterone and acute stress are associated with fibrinogen and von Willebrand factor in African men: the SABPA study.

BACKGROUND Low testosterone, acute and chronic stress and hypercoagulation are all associated with hypertension and hypertension-related diseases. The interaction between these factors and future risk for coronary artery disease in Africans has not been fully elucidated. In this study, associations of testosterone, acute cardiovascular and coagulation stress responses with fibrinogen and von Willebrand factor in African and Caucasian men in a South African cohort were investigated. METHODS Cardiovascular variables were studied by means of beat-to-beat and ambulatory blood pressure monitoring. Fasting serum-, salivary testosterone and citrate coagulation markers were obtained from venous blood samples. Acute mental stress responses were evoked with the Stroop test. RESULTS The African group demonstrated a higher cardiovascular risk compared to Caucasian men with elevated blood pressure, low-grade inflammation, chronic hyperglycemia (HbA1c), lower testosterone levels, and elevated von Willebrand factor (VWF) and fibrinogen levels. Blunted testosterone acute mental stress responses were demonstrated in African males. In multiple regression analyses, higher circulating levels of fibrinogen and VWF in Africans were associated with a low T environment (R(2) 0.24-0.28; p≤0.01), but only circulating fibrinogen in Caucasians. Regarding endothelial function, a low testosterone environment and a profile of augmented α-adrenergic acute mental stress responses (diastolic BP, D-dimer and testosterone) were associated with circulating VWF levels in Africans (Adj R(2) 0.24; p<0.05). CONCLUSIONS An interdependence between acute mental stress, salivary testosterone, D-dimer and vascular responses existed in African males in their association with circulating VWF but no interdependence of the independent variables occurred with fibrinogen levels.

A randomized clinical trial of Behavioral Activation (BA) therapy for improving psychological and physical health in dementia caregivers: results of the Pleasant Events Program (PEP).

Dementia caregiving is associated with elevations in depressive symptoms and increased risk for cardiovascular diseases (CVD). This study evaluated the efficacy of the Pleasant Events Program (PEP), a 6-week Behavioral Activation intervention designed to reduce CVD risk and depressive symptoms in caregivers. One hundred dementia family caregivers were randomized to either the 6-week PEP intervention (N = 49) or a time-equivalent Information-Support (IS) control condition (N = 51). Assessments were completed pre- and post-intervention and at 1-year follow-up. Biological assessments included CVD risk markers Interleukin-6 (IL-6) and D-dimer. Psychosocial outcomes included depressive symptoms, positive affect, and negative affect. Participants receiving the PEP intervention had significantly greater reductions in IL-6 (p = .040), depressive symptoms (p = .039), and negative affect (p = .021) from pre- to post-treatment. For IL-6, clinically significant improvement was observed in 20.0% of PEP participants and 6.5% of IS participants. For depressive symptoms, clinically significant improvement was found for 32.7% of PEP vs 11.8% of IS participants. Group differences in change from baseline to 1-year follow-up were non-significant for all outcomes. The PEP program decreased depression and improved a measure of physiological health in older dementia caregivers. Future research should examine the efficacy of PEP for improving other CVD biomarkers and seek to sustain the intervention's effects.

AMPA RECEPTOR ALLOSTERISM: MEASUREMENT OF THE CONFORMATIONAL CHANGES IN THE LIGAND BINDING DOMAIN OF A FUNCTIONAL RECEPTOR

Ionotropic glutamate receptors are important excitatory neurotransmitter receptors in the mammalian central nervous system that have been implicated in a number of neuropathologies such as epilepsy, ischemia, and amyotrophic lateral sclerosis. Glutamate binding to an extracellular ligand binding domain initiates a series of structural changes that leads to the formation of a cation selective transmembrane channel, which consequently closes due to desensitization of the receptor. The crystal structures of the AMPA subtype of the glutamate receptor have been particularly useful in providing initial insight into the conformational changes in the ligand binding domain; however, these structures are limited by crystallographic constraint. To gain a clear picture of how agonist binding is coupled to channel activation and desensitization, it is essential to study changes in the ligand binding domain in a dynamic, physiological state. In this dissertation, a technique called Luminescence Resonance Energy Transfer was used to determine the conformational changes associated with activation and desensitization in a functional AMPA receptor (ÄN*-AMPA) that contains the ligand binding domain and transmembrane segments; ÄN*-AMPA has been modified such that fluorophores can be introduced at specific sites to serve as a readout of cleft closure or to establish intersubunit distances. Previous structural studies of cleft closure of the isolated ligand binding domain in conjunction with functional studies of the full receptor suggest that extent of cleft closure correlates with extent of activation. Here, LRET has been used to show that a similar relationship between cleft closure and activation is observed in the “full length” receptor showing that the isolated ligand binding domain is a good model of the domain in the full length receptor for changes within a subunit. Similar LRET investigations were used to study intersubunit distances specifically to probe conformational changes between subunits within a dimer in the tetrameric receptor. These studies show that the dimer interface is coupled in the open state, and decoupled in the desensitized state, similar to the isolated ligand binding domain crystal structure studies. However, we show that the apo state dimer interface is not pre-formed as in the crystal structure, hence suggesting a mechanism for functional transitions within the receptor based on LRET distances obtained.