Expression and function of endothelin and its receptors in vascular adventitial fibroblasts

Objective: The adventitia has been recognized to play important roles in vascular oxidative stress, remodelling and contraction. We recently demonstrated that adventitial fibroblasts are able to express endothelin-1 (ET-1) in response to angiotensin II (ANG II). However, the mechanisms by which ANG II induces ET-1 expression are unknown. It is also unclear whether the ET-1 receptors are expressed in the adventitia. We therefore examined the role of oxidative stress in the regulation of ET-1. We also investigated the expression of both the ETA and ETB receptors and the roles of these two types of receptors in collagen synthesis and ET-1 clearance in adventitial fibroblasts. Methods and Results: Adventitial fibroblasts were isolated and cultured from the thoracic mouse aorta. Cells were treated with ANG II (lOOnM), ET-1 (lOpM), NADPH oxidase inhibitor apocynin (lOOfiM), the superoxide anion scavenger tempol (lOOfiM), the ANG II receptor antagonists (100[aM), losartan (AT| receptor) and PD 1233 19 (AT2 receptor), the ET-1 receptor antagonists (lOOuM), BQ123 (ETA receptor) and BQ788 (ETB receptor), and the ETB receptor agonist (lOOnM) Sarafotoxin 6C. ET-1 peptide levels were determined by ELISA, while ETA ,ETB and collagen levels were determined by Western blot. ANG II increased ET-1 peptide levels in a time-dependent manner reaching significance when incubated for 24 hours. NAD(P)H oxidase inhibitor, apocynin, as well as the superoxide scanverger, tempol, significantly reduced ANG Il-induced ET-1 peptide levels while over-expression of SOD1 (endogenous antioxidant enzyme) significantly decreased ANG Il-induced collagen I expression, therefore implicating reactive oxygen species in the mediation of ET-1. ANG II increased ETA receptor protein as well as collagen in a similar fashion, reaching significance after 4, 6, and 24 hours treatment. ANG II induced collagen was reduced while in the presence of the ETA receptor antagonist suggesting the role of the ETa receptor in the regulation of the extracellular matrix. ANG II treatment also increased ETB receptor protein levels in a time-dependent manner. ANG II treatment in the presence of the ETB receptor antagonist significantly increased ET-1 peptide levels. On another hand, the ETB receptor agonist, Sarafotoxin 6C, significantly decreased ET-1 peptide levels. These data implicate the role of the ETb receptor in the clearance of the ET-1 peptide. Conclusion: ANG II-induced increases of ET-1 peptide appears to be mediated by reactive oxygen species derived from NAD(P)H oxidase. Both the ETA and ETB receptors are expressed in adventitial fibroblasts. The ETA receptor subtype mediates collagen I expression, while the ETB receptor may play a protective role through increasing the clearance of the ET- 1 peptide.

Mechanisms of endothelin-1 induced reactive oxygen species production in vascular adventitial fibroblasts

With the relationship between endothelin-1 (ET-1) stimulation and reactive oxygen species (ROS) production unknown in adventitial fibroblasts, I examined the ROS response to ET-1 and angiotensin (Ang II). ET-1 -induced ROS peaked following 4 hrs of ET-1 stimulation and was inhibited by an ETA receptor antagonist (BQ 123, 1 uM) an extracellular signal-regulated kinase (ERK) 1/2 inhibitor (PD98059, 10 uM), and by both a specific, apocynin (10 uM), and non-specific, diphenyleneiodonium (10 uM), NAD(P)H oxidase inhibitor. NOX2 knockout fibroblasts did not produce an ET-1 induced change in ROS levels. Ang II treatment increased ROS levels in a biphasic manner, with the second peak occurring 6 hrs following stimulation. The secondary phase of Ang II induced ROS was inhibited by an ATi receptor antagonist, Losartan (100 uM) and BQ 123. In conclusion, ET-1 induces ROS production primarily through an ETA-ERKl/2 NOX2 pathway, additionally, Ang II-induced ROS production also involves an ETa pathway.

The roles of ERK1/2 and PI3K in abnormal vascular functions in angiotensin II-infused hypertensive rats

Background: Ang II plays a major role in cardiovascular regulation. Recently, it has become apparent that vascular superoxide anion may play an important role in hypertension development. Treatment with antisense NAD(P)H oxidase or SOD decreased BP in Ang II-infused rats. Wang et al recently reported mice which lack one of the subunits of NAD(P)H oxidase developed hypertension at a much lower extent when compared to the wild type animals infused with Ang II, indicating that superoxide anion contributes to elevation in BP in the Ang II-infused hypertensive model. In the Ang II-infused hypertensive model, altered reactivity of blood vessels is often associated with the elevation of systolic blood pressure. We have observed abnormal tension development and impaired endothelium-dependent relaxation in the isolated aorta of Ang II-infused and DOCA-salt hypertensive rats. Recently, several other cellular signal molecules, including ERK1I2 and PI3K, have been determined to play important roles in the regulation of smooth muscle contraction and relaxation. ERKl/2 and PI3K pathways are also reported to contribute to Ang II induced cell growth, hypertrophy, remodeling and contraction. Moreover, these signaling pathways have shown ROS-sensitive properties. Therefore, the aim of the present study is to investigate the roles of ERKl12 and PI3K in vascular oxidative stress, spontaneous tone and impaired endothelium relaxation in Ang II-infused hypertensive model. Hypothesis: We hypothesize that the activation of ERKl12 and PI3K are elevated in response to an Ang II infusion for 6 days. The elevated activation of phospho-ERKl/2 and PI3K mediated the increased level of vascular superoxide anion, the abnormal vascular contraction and impaired endothelium-dependent vascular relaxation in Ang II-infused hypertensive rats. Methods: Vascular superoxide anion level is measured by lucigenin chemiluminescence. Spontaneous tone and ACh-induced endothelium-dependent relaxation was measured by isometric tension recording in organ chamber. The activity of ERK pathway will be measured by its Western blot of phosphorylation of ERK. PI3K activity was evaluated indirectly by Western blot of the phosphorylation of PDKl, a downstream protein of PI3K signaling pathway. The role of each pathway was also addressed via comparing the responses to the specific inhibitors. Results: Superoxide anion was markedly increased in the isolated thoracic aorta from Ang II-infused rats. There was spontaneous tone developed in rings from Ang II-induced hypertensive but not sham-operated normotensive rats. ACh-induced endothelium-dependent relaxation function is impaired in Ang II-infused hypertensive rats. Superoxide dismutase and NAD(P)H oxidase inhibitor, apocynin, inhibited the abnormal spontaneous tone and ameliorated impaired endothelium-dependent relaxation. The expression of phopho-ERKII2 was enhanced in Ang II-infused rats, indicating the activity of ERK1I2 could be increased. MEK1I2 inhibitors, PD98059 and U126, but not their inactive analogues, SB203580 and U124, significantly reduced the vascular superoxide anion in aortas from Ang II-infused rats. The MEK1I2 inhibitors reduced the spontaneous tone and improved the impaired endothelium-dependent relaxation in aorta of hypertension. These findings supported the role of ERKII2 signaling pathway in vascular oxidative stress, spontaneous tone and impaired endothelium-dependent relaxation in Ang II-infused hypertensive rats. The amount of phospho-PDK, a downstream protein of PI3K was increased in Ang II rats indicating the activity of PI3K activity was elevated. Strikingly, PI3K significantly inhibited the increase of superoxide anion level, abnormal spontaneous tone and restored endothelium-dependent relaxation in Ang II-infused hypertensive rats. These findings indicated the important role of PI3K in Ang II-infused hypertensive rats. Conclusion: ERKII2 and PI3K signaling pathways are sustained activated in Ang II-infused hypertensive rats. The activated ERKII2 and PI3K mediate the increase of vascular superoxide anion level, vascular abnormal spontaneous tone and impaired endothelium-dependent relaxation.

Localization of the mechanism of pH-dependent non- photochemical fluorescence quenching in thylakoid membranes

Higher plants have evolved a well-conserved set of photoprotective mechanisms, collectively designated Non-Photochemical Quenching of chlorophyll fluorescence (qN), to deal with the inhibitory absorption of excess light energy by the photosystems. Their main contribution originates from safe thermal deactivation of excited states promoted by a highly-energized thylakoid membrane, detected via lumen acidification. The precise origins of this energy- or LlpH-dependent quenching (qE), arising from either decreased energy transfer efficiency in PSII antennae (~ Young & Frank, 1996; Gilmore & Yamamoto, 1992; Ruban et aI., 1992), from alternative electron transfer pathways in PSII reaction centres (~ Schreiber & Neubauer, 1990; Thompson &Brudvig, 1988; Klimov et aI., 1977), or from both (Wagner et aI., 1996; Walters & Horton, 1993), are a source of considerable controversy. In this study, the origins of qE were investigated in spinach thylakoids using a combination of fluorescence spectroscopic techniques: Pulse Amplitude Modulated (PAM) fluorimetry, pump-probe fluorimetry for the measurement of PSII absorption crosssections, and picosecond fluorescence decay curves fit to a kinetic model for PSII. Quenching by qE (,..,600/0 of maximal fluorescence, Fm) was light-induced in circulating samples and the resulting pH gradient maintained during a dark delay by the lumenacidifying capabilities of thylakoid membrane H+ ATPases. Results for qE were compared to those for the addition of a known antenna quencher, 5-hydroxy-1,4naphthoquinone (5-0H-NQ), titrated to achieve the same degree of Fm quenching as for qE. Quenching of the minimal fluorescence yield, F0' was clear (8 to 130/0) during formation of qE, indicative of classical antenna quenching (Butler, 1984), although the degree was significantly less than that achieved by addition of 5-0H-NQ. Although qE induction resulted in an overall increase in absorption cross-section, unlike the decrease expected for antenna quenchers like the quinone, a larger increase in crosssection was observed when qE induction was attempted in thylakoids with collapsed pH gradients (uncoupled by nigericin), in the absence of xanthophyll cycle operation (inhibited by DTT), or in the absence of quenching (LlpH not maintained in the dark due to omission of ATP). Fluorescence decay curves exhibited a similar disparity between qE-quenched and 5-0H-NQ-quenched thylakoids, although both sets showed accelerated kinetics in the fastest decay components at both F0 and Fm. In addition, the kinetics of dark-adapted thylakoids were nearly identical to those in qEquenched samples at F0' both accelerated in comparison with thylakoids in which the redox poise of the Oxygen-Evolving Complex was randomized by exposure to low levels of background light (which allowed appropriate comparison with F0 yields from quenched samples). When modelled with the Reversible Radical Pair model for PSII (Schatz et aI., 1988), quinone quenching could be sufficiently described by increasing only the rate constant for decay in the antenna (as in Vasil'ev et aI., 1998), whereas modelling of data from qE-quenched thylakoids required changes in both the antenna rate constant and in rate constants for the reaction centre. The clear differences between qE and 5-0H-NQ quenching demonstrated that qE could not have its origins in the antenna alone, but is rather accompanied by reaction centre quenching. Defined mechanisms of reaction centre quenching are discussed, also in relation to the observed post-quenching depression in Fm associated with photoinhibition.

Weight-preoccupation, personality and depression in university students : an interactionist perspective

The present research was designed to examine Beck's (1983; 1987) cognitive diathesis-stress model of depression in a new context. Specifically, this cross-sectional study investigated whether Beck's cognitive-personality traits of sociotropy and autonomy interacted with a specific daily hassle/stressor, weightpreoccupation, in the prediction of depressed mood in women and men. Weightpreoccupation was chosen as the particular daily stressor, because, according to Kanner, Coyne, Schaeffer, and Lazarus (1981). concern about weight and physical appearance were two of the most troublesome daily hassles among university students. ContraIy to current stereotypes, men also express concerns with weight and appearance (Cash, Winstead, &, landi, 1986), in both the direction of weight loss and weight/muscle gain (Drewnowski &, Yee, 1987). Thus, unique to this study was the examination of weight-preoccupation in men, not only in the direction of weight loss, but also in the direction of weight and muscle gain. Two hundred and fifty-one undergraduates were administered the revised SociotropyAutonomy Scale (SAS), the Beck Depression Inventory (BDij, the Hassles and Uplifts Scale, the Eating Disorders Inventory (EDI), and the Restraint Scale (RS). Through a process of model building, a final model was devised in which depression (ie., BDI total scores) was regressed on personality (Le., SAS Sociotropy) and weight-preoccupation (i.e., Drive for Thinness, Body Dissatisfaction, and the Restraint Scale). Separate hierarchical multiple regression analyses, with BDI as the dependent variable, were carried out for each of the 2 3 weight-preoccupation variables. Results revealed a specific congruent interaction between weight,..preoccupation and personality. Specifically, drive for thinness and body dissatisfaction interacted with sociotropy to predict depressed mood in both women and men, but the interaction between sociotropy and the Restraint Scale was marginal. Findings suggested that weight-preoccupied women and men experienced depressed mood to the extent that they were characterized as more highly sociotropic. As predicted, personality (i.e., sociotropy), moderated the relationship between daily stress (i.e., weight-preoccupation) and depression. Thus, results revealed that the proposed diathesis-stress model seemed to operate in the same manner for women and men. Moreover, the examination of weightpreoccupation among men (i.e., in the direction of both weight loss and weight/muscle gain) further revealed that, when curvilinearity was taken into account, highly sociotropic men, who had a low desire to gain weight and muscle mass (i.e., most likely those who desired to lose weight), experienced higher levels of depressed mood than those who had a "medium" desire for weight and muscle gain. However, the highest levels of depressed mood was experienced by highly sociotropic men who were highly weight-preoccupied in the direction of wanting to gain weight and muscle mass. In addition, possible reasons for the present fmdings, as wen as iimitations and impiicalions for future research are discussed.

A constructivist grounded theory of firefighter perceptions of stress, coping and the relationship to health

This qualitative research was a constructivist grounded theory designed to develop an understanding of how firefighters perceive and cope with stressful situations and the impact this has on their perceptions of health. This study was framed in a social ecological perspective with the community of firefighting providing the environment within which to explore stress and coping. Of particular concern here are the stressors associated with firefighting. Prior research with firefighters has often been epidemiological and statistical in nature, focusing on measures of cardiovascular disease, cancer, and depression (Baker & Williams, 2001 ; Brown et al., 2002; Murphy et al.,1999; Regehr et al., 2002; Regehr et al., 2003). Qualitative research examining the perception of stress among firefighters that includes personal stories allows firefighters the opportunity to describe what it is like to be met with physically and mentally challenging situations on a daily basis. Twelve in-depth, semi-structured, face-to-face interviews with a brief questionnaire were conducted with firefighters from a Southern Ontario Fire Department. Four main themes emerged describing the persona of the firefighter, the stressors of firefighters, coping strategies of firefighters, and firefighters' perceptions of health. Stressors include requirements of the job, traumatic calls, tensions with co-workers, the struggle between the family at home and the family at work, political stressors with the City, and the inner struggle. Avoidance coping, approach coping, and gaining perspective emerged as the three coping styles of firefighters. Health was defined as including physical, mental, social and spiritual aspects. A model of the findings is provided that depicts the cyclical nature of the stress-coping-health relationship among firefighters.

The relationship between early age of first sexual intercourse and vulnerability to depression among adolescents

Past empirical literature has provided conflicted results regarding the association between adolescent coitus and depression. While some studies conclude that those youth who are sexually active may be at risk for depression, others provide contrary results, or findings that are only representative of high-risk sexual behaviors such as intercourse without a condom. Thus, the results are unclear as to whether depression results directly from coitus, or if this relationship is spurious; that is, there may be biological, psychological, or sociological variables that may predict both depression and early sexual intercourse. Using the Add Health restricted dataset, I analyzed the depressive symptomatology of adolescents over a seven-year time period. The final sample (n=6,51O) was comprised of 49.35% male (n=3,213) and 50.65% female (n=3,297) participants. Results indicated that the relationship between earlier adolescent sexual intercourse and later depressive symptomatology is spurious. Although an earlier age of first coitus is predictive of later depressive symptomatology, both variables appear to be concomitant outcomes of the biopsychosocial process. Thus, while one may be able to use early coitus as a marker for subsequent depressive symptomatology, it does not occur because of early coitus. Furthermore, the reverse relationship was not found to be significant in this study. That is, higher levels of depressive symptomatology do not predict an earlier age of first sexual intercourse in adolescents.

The association between blood pressure and vascular characteristics in children

Hypertension is thought to exist in up to five percent of children. A select number of studies have investigated the role elevated blood pressure plays in pediatric atherosclerotic progression. However these studies contain significant methodological flaws and fail to recognize important confounding factors. Therefore, the influence of elevated blood pressure on arterial health in children remains to be clearly understood. The purpose of this study was to investigate the association between blood pressure (BP) and arterial thickness and stiffuess in children. Common carotid artery (CCA) intima-media thickness (IMT) and distensibility (Dist), as well as systemic pulse wave velocity (PWV) were measured in 21 elevated blood pressure (EBP; BP ~ 95th percentile) and 83 normal blood pressure (NBP; BP < 90th percentile) children 11-14 years of age. Both EBP and NBP groups demonstrated BP within the normal clinical range, but EBP showed significantly elevated BP as compared to the NBP group. Independent t-tests failed to show significant differences between the EBP and NBP groups for CCA IMT (0.43 ± 0.05 mm and 0.42 ± 0.06 mm, respectively) and Dist (0.0058 ± 0.0024 mmHg-1 and 0.0064 ± 0.0019 mmHil respectively). In contrast, a significantly elevated PWV (p

Does Education Setting Influence the Relationship between Substance use and Depression among Post Secondary Students

Background. This study examined whether experiences of and relationships between depressive symptoms and substance use differs for first year college and university students. Methods. A proportionate stratified random sample of 6,100 university students and a census sample of 7,300 college students were invited to anonymously complete the National College Health Assessment. The final sample included 444 young adult first year university (n = 298) and college (n = 146) students. Results. More college than university students used tobacco (26.7; 11.1%) and marijuana (26.7%; 20.8%). Similar proportions consumed alcohol (75.3%; 76.5%). Almost all students reported past-year depressive symptoms. Mean number of symptoms was 5.43. Tobacco, alcohol and marijuana use were each positively associated with depression after adjusting for age and gender. Educational setting moderated the relationship between depression and tobacco use, and depression and marijuana use, with the relationship being stronger for university students. Implications. University campus health professionals especially, need to assess depression among students using substances and vice versa. Differences between college and university students require further attention.

Social competence following mild head injury and moderate traumatic brain injury: Investigating the neuropsychological relationships between arousal, social decision-making and depression

Client-directed long-term rehabilitative goals and life satisfaction following head injury emphasize the importance of social inclusion, rather than cognitive or physical, outcomes. However, very little research has explored the socio-emotional factors that pose as barriers to social reintegration following injury. This study investigates social barriers following head injury (i.e., decision-making - Iowa Gambling Task [IGT] and mood – depression) and possible amelioration of those challenges (through treatment) in both highly functioning university students with and without mild head injury (MHI) and in individuals with moderate traumatic brain injury (TBI). An arousal manipulation using emotionally evocative stimuli was introduced to manipulate the subject’s physiological arousal state. Seventy-five university students (37.6% reporting a MHI) and 11 patients with documented moderate TBI were recruited to participate in this quasi-experimental study. Those with head injury were found to be physiologically underaroused (on measures of electrodermal activation [EDA] and pulse) and were less sensitive to the negative effects of punishment (i.e., losses) in the gambling task than those without head injury, with greater impairment being observed for the moderate TBI group. The arousal manipulation, while effective, was not able to maintain a higher state of arousal in the injury groups across trials (i.e., their arousal state returned to pre-manipulation levels more quickly than their non-injured cohort), and, subsequently, a performance improvement was not observed on the IGT. Lastly, head injury was found to contribute to the relationship between IGT performance and depressive symptom acknowledgment and mood status in persons with head injury. This study indicates the possible important role of physiological arousal on socio- emotional behaviours (decision-making, mood) in persons with even mild, non-complicated head injuries and across the injury severity continuum.