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.

The validation of heart rate variability in individuals with spinal cord injury

The current classification system for spinal cord injury (SCI) considers only somatic information and neglects autonomic damage after injiuy. Heart rate variability (HRV) has the potential to be a valuable measure of cardiac autonomic control after (SCI). Five individuals with tetraplegia and four able-bodied controls underwent 1 min continuous ECG recordings during rest, after Metoprolol administration (max dose=3x5mg) and after Atropine administration (0.02mg/kg) in both supine and 40° head-up tilt. After Metoprolol administration there was a 61.8% decrease in the LF:HF ratio in the SCI participants suggesting that the LF:HF ratio is a reflection of cardiac sympathetic outflow. After Atropine administration there was a 99.1% decrease in the HF power in the SCI participants suggesting that HF power is highly representative of cardiac parasympathetic outflow. There were no significant differences between the SCI and able-bodied participants. Thus, HRV measures are a valid index of cardiac autonomic control after SCI.

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.

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

A possible role for chemokine signalling through CXCR4 as a downstream effector of retinoic acid signalling in the regenerating tail and spinal cord of Notophthalmus viridescens

The newt, Notopthalmus viridescens is one of the few tet rapod vertebrates capable of extensive regeneration of the central nervous system, however, the factors involved in this process are still unknown. Chemokine signalling through the receptor CXCR4, has been found to be involved in the development of the central nervous system of mammals and more recently in epimorphic fin regeneration in zebrafish. We have hypothesized that the CXCR4 signalling pathway is involved in spinal cord and tail regeneration in the adul t newt , possibly as a downstream target of retinoic acid signalling. We found that CXCR4 mRNA expression was observed in the brain, spinal cord, heart, gut, liver and regenerating tail blastemas. CXCR4 expression increased over the f i rst 12 days of tail regeneration and returned to basal expression levels at day 21 of regeneration. Inhibition of CXCR4 wi th AMD3100, a specific receptor antagonist, led to a decrease in CXCR4 mRNA in the regenerating tail 14 days post amputation. Histological analysis suggests a delay in the early stages of tail and spinal cord regeneration. Spinal cord explants t reated wi th CXCL12, the ligand to CXCR4, displayed enhanced neurite outgrowth in vitro. Explants t reated wi th AMD3100 abolished any retinoic acid enhanced neurite outgrowth effects suggesting a link between these signalling pathways.

The Effect of Functional Electrically Stimulated Ambulation Training on Locomotor Function and Quality of Life in Individuals With Incomplete Spinal Cord Injury

The purpose of this study was to investigate the effects of a 12-week FES-ambulation program on locomotor function and quality of life after incomplete spinal cord injury. Six individuals with incomplete SCI participated in the study. Over-ground walking endurance (6MWT), speed (10MWT), independence (WISCI II) and body-weight support were assessed. Quality of life was assessed via the SF-36, WHOQOL-BREF, Perceived Stress Scale, Center of Epidemiological Studies for Depression scale, and task self-efficacy. Participants experienced significant improvements in walking endurance (223.6±141.5m to 297.3±164.5m; p=0.03), body-weight support (55.3±12.6% to 14.7±23.2%; p= 0.005) and four of the six participants showed improvements on the WISCI II scale (1-4 points). In addition, there was a significant reduction in reported bodily pain (6.5±1.2 to 5.0±1.7; p=0.04). Therefore, FES-ambulation is an effective means for enhancing over-ground locomotor function in individuals with incomplete SCI. It may also be an effective method for reducing pain in individuals with SCI.

Utilizing a Phenomenological Approach to Examine the Experience of Sexuality for Women Concerned about Urinary Incontinence following Spinal Cord Injury

Sexuality after spinal cord injury (SCI) is a complex issue that is influenced by a number of social, psychological and physiological factors, one of which is urinary incontinence (UI). Using a phenomenological approach, seven mixed methods interviews combining both the interview guide and standardized open-ended approaches were conducted to examine the experience of sexuality for women who are concerned about UI following SCI. Sexual function was one of the top priorities for the women after SCI, and UI was one of the main concerns the women had regarding sexuality. The findings of this study demonstrate that various dimensions of intimacy and the sexual experience as a whole were affected by UI, and the women discussed both physical and psychological concerns. The main issues regarding sexuality included concerns related to relationships, frustrations with limited sexual activities and the difficulty of being sexually satisfied, the number of unanswered questions and concerns, and a fear of being hurt or injured while participating in sexual activities. The main concerns regarding UI were embarrassment, the work and inconvenience involved with the clean-up of UI, bladder infections, the lack of accessible washrooms, and the negative effects of UI medications. When examining sexuality and UI together, the major issues were the constant comparison to the way things were before SCI, as well as the new concerns that the women did not have to worry about previously, worrying about how their partner would react if UI were to occur during sexual activity, and the impact of their own feelings toward UI on sexuality, a connection between pleasurable sexual sensations and UI as well as difficulty differentiating between the sensation of UI with the sensation of UI, dealing with infected urine during sexual activity, having to discuss UI with a new potential sexual partner, and a fear of rejection. Other identified issues included those related to body image, a lack of resources, Doctors who were inadequately educated regarding SCI, and issues related to both having and raising children. There is a significant shortage of information available for women with SCI to use as a resource regarding sexual function in general, and sexual function as it relates to UI. It is necessary that future work focus on creating resources to assist in this area, and that the dissemination of those resources becomes both appropriate and effective. Addressing sexual function and UI which are among the top concerns for this population has the opportunity to greatly improve quality of life (QOL) for these individuals.

Exploring Body-Related Experiences among Individuals with Spinal Cord Injury

Using modified constructivist grounded theory, the purpose of the present study was to explore body-related experiences, specifically body image, in people with spinal cord injury. A total of nine participants (five women, four men) who had a broad range of body image experiences (from very negative to very positive) were interviewed. Most participants explained experiencing a fluctuating body image that varied from day-to-day. Negative body image experiences were represented by appearance, weight concerns, and function with all body image experiences encompassed by self-presentational concerns and tactics (an unanticipated finding). Positive body image was represented by acceptance, appreciation and gratitude of the body. Interestingly, negative body image experiences were not found to be represented by the opposite of positive body image experiences as they were each distinct. These findings have direct implications for medical professionals in hospital and rehabilitation settings to understand the importance of body image after spinal cord injury.

The role of microRNAs and retinoid signaling during spinal cord regeneration in the adult newt.

The molecular events after spinal cord injury that lead to the establishment of a permissive environment and epimorphic regeneration remain unclear. Two molecular pathway regulators that may converge to create a spinal cord regeneration-permissive environment in the urodele are retinoic acid (RA) and microRNAs (miRNAs). Recent evidence suggests that RARβ-mediated signaling is necessary for tail and caudal spinal cord regeneration in the adult newt. MicroRNAs are attractive candidates as mediators of retinoid signaling during regeneration, as their pleiotropic effects are vital in situations where global changes in gene expression are required. Thus, the overall aim of this thesis was to determine if miRNAs are involved in tail and caudal spinal cord regeneration in the adult newt, and if they act as regulators and/or effectors of retinoid signaling during this process. I have demonstrated here, for the first time, that multiple miRNAs are dysregulated in response to spinal cord injury in the adult newt, as well as in response to inhibition of retinoid signaling. Two of these miRNAs, miR-133a and miR-1, appear to target RARβ2 transcripts both in vivo and in vitro. Inhibition of RA signaling via RARβ with a selective antagonist, LE135, alters the pattern of expression of these miRNAs, which leads to an inhibition of tail regeneration. These data are indicative of a negative feed back loop, albeit potentially an indirect one. I also aimed to examine which miRNAs are affected by inhibiting RA synthesis during regeneration, and provided a long list of miRNAs that are dysregulated. These data provide the foundation for future studies on the putative roles of these miRNAs, as well as their function in retinoid signaling. Overall, these studies provide the first evidence for a role for miRNAs as mediators of retinoid signaling during caudal spinal cord regeneration in any system.