Protease-Activated Receptor 2 Controls Vascular Smooth Muscle Cell Proliferation in Cyclic AMP-Dependent Protein Kinase/Mitogen-Activated Protein Kinase Kinase 1/2-Dependent Manner.

INTRODUCTION: Cardiovascular disorders are characterized by vascular smooth muscle (VSM) transition from a contractile to proliferative state. Protease-activated receptor 2 (PAR2) involvement in this phenotypic conversion remains unclear. We hypothesized that PAR2 controls VSM cell proliferation in phenotype-dependent manner and through specific protein kinases. METHODS: Rat clonal low (PLo; P3-P6) and high passage (PHi; P10-P15) VSM cells were established as respective models of quiescent and proliferative cells, based on reduced PKG-1 and VASP. Western blotting determined expression of cytoskeletal/contractile proteins, PAR2, and select protein kinases. DNA synthesis and cell proliferation were measured 24-72 h following PAR2 agonism (SLIGRL; 100 nM-10 µm) with/without PKA (PKI; 10 µm), MEK1/2 (PD98059; 10 µm), and PI3K (LY294002; 1 µm) blockade. RESULTS: PKG-1, VASP, SM22α, calponin, cofilin, and PAR2 were reduced in PHi versus PLo cells. Following PAR2 agonism, DNA synthesis and cell proliferation increased in PLo cells but decreased in PHi cells. Western analyses showed reduced PKA, MEK1/2, and PI3K in PHi versus PLo cells, and kinase blockade revealed PAR2 controls VSM cell proliferation through PKA/MEK1/2. DISCUSSION: Findings highlight PAR2 and PAR2-driven PKA/MEK1/2 in control of VSM cell growth and provide evidence for continued investigation of PAR2 in VSM pathology.

FoxO3 normalizes Smad3-induced arterial smooth muscle cell growth.

Transition of arterial smooth muscle (ASM) from a quiescent, contractile state to a growth-promoting state is a hallmark of cardiovascular disease (CVD), a leading cause of death and disability in the United States and worldwide. While many individual signals have been identified as important mechanisms in this phenotypic conversion, the combined impact of the transcription factors Smad3 and FoxO3 in ASM growth is not known. The purpose of this study was to determine that a coordinated, phosphorylation-specific relationship exists between Smad3 and FoxO3 in the control of ASM cell growth. Using a rat in vivo arterial injury model and rat primary ASM cell lysates and fractions, validated low and high serum in vitro models of respective quiescent and growth states, and adenoviral (Ad-) gene delivery for overexpression (OE) of individual and combined Smad3 and/or FoxO3, we hypothesized that FoxO3 can moderate Smad3-induced ASM cell growth. Key findings revealed unique cellular distribution of Smad3 and FoxO3 under growth conditions, with induction of both nuclear and cytosolic Smad3 yet primarily cytosolic FoxO3; Ad-Smad3 OE leading to cytosolic and nuclear expression of phosphorylated and total Smad3, with almost complete reversal of each with Ad-FoxO3 co-infection in quiescent and growth conditions; Ad-FoxO3 OE leading to enhanced cytosolic expression of phosphorylated and total FoxO3, both reduced with Ad-Smad3 co-infection in quiescent and growth conditions; Ad-FoxO3 inducing expression and activity of the ubiquitin ligase MuRF-1, which was reversed with concomitant Ad-Smad3 OE; and combined Smad3/FoxO3 OE reversing both the pro-growth impact of singular Smad3 and the cytostatic impact of singular FoxO3. A primary takeaway from these observations is the capacity of FoxO3 to reverse growth-promoting effects of Smad3 in ASM cells. Additional findings lend support for reciprocal antagonism of Smad3 on FoxO3-induced cytostasis, and these effects are dependent upon discrete phosphorylation states and cellular localization and involve MuRF-1 in the control of ASM cell growth. Lastly, results showing capacity of FoxO3 to normalize Smad3-induced ASM cell growth largely support our hypothesis, and overall findings provide evidence for utility of Smad3 and/or FoxO3 as potential therapeutic targets against abnormal ASM growth in the context of CVD.

Intraoperative ex vivo parathyroid aspiration: A point-of-care test to confirm parathyroid tissue.

BACKGROUND: Ex vivo aspiration of a parathyroid gland with intraoperative parathyroid hormone determination is a method for intraoperative confirmation of parathyroid tissue. The aim of this study was to describe the use and applicability of this technique at a single, high-volume institution. METHODS: This is a retrospective review of patients who underwent parathyroidectomy and ex vivo aspiration of suspected, abnormal parathyroid tissue for intraoperative parathyroid hormone level (pg/mL). Sensitivity and specificity were calculated for aspirate intraoperative parathyroid hormone levels which were compared with the baseline serum aspirate intraoperative parathyroid hormone obtained prior to parathyroid excision in each patient. RESULTS: Of 921 tissue aspirates, 847 (92%) were confirmed as parathyroid on histopathology, with a mean ± standard deviation aspirate intraoperative parathyroid hormone of 3,838 ± 1,615 pg/mL. The 847 aspirates included 833 (98%) with aspirate intraoperative parathyroid hormone levels greater than the serum aspirate intraoperative parathyroid hormone and 14 (2%) with aspirate intraoperative parathyroid hormone levels less than the serum aspirate intraoperative parathyroid hormone; 74 (8%) aspirates were not parathyroid tissue, with a mean aspirate intraoperative parathyroid hormone level of 25 ± 12.7 pg/mL. An aspirate intraoperative parathyroid hormone ≥1.5 times the serum aspirate intraoperative parathyroid hormone represented the optimal threshold for confirmation of parathyroid tissue. CONCLUSION: Intraoperative ex vivo aspiration of presumed parathyroid gland is a sensitive and specific point-of-care method to confirm the presence of parathyroid tissue. An aspirate intraoperative parathyroid hormone ≥1.5 times greater than the baseline serum aspirate intraoperative parathyroid hormone minimizes the likelihood of misidentifying parathyroid tissue.

Shoulder impingement: the effect of sitting posture on shoulder pain and range of motion.

The re-education of spinal posture is an integral part of shoulder impingement management yet supporting evidence is limited. The purpose of this study was to evaluate the effect of slouched versus erect sitting posture on shoulder pain intensity and range of motion (ROM) in subjects with impingement. A same-subject repeated-measures design was utilized. Maximum active shoulder flexion and associated pain intensity were measured in 28 subjects in slouched and erect sitting postures, using video-analysis and visual analogue scales, respectively. An intra-tester reliability study of the video-analysis system was completed and intra-class correlation coefficients calculated. Shoulder flexion differences between slouched and erect sitting posture were analysed using a repeated-measures analysis of variance (ANOVA). The intra-tester reliability of the video-analysis method was found to be 'excellent' (ICC = 0.99). Flexion ROM was significantly greater in the erect sitting posture (F = 100.3, P < 0.0001); the mean ROM difference between postures was 17.67 degrees (+/- 9.17 degrees). There was no significant difference in pain intensity between postures (F = 1.9, P = 0.179). An erect sitting posture appeared to increase active shoulder flexion in subjects with shoulder impingement, although there were no differences in reported pain intensity. Further research is required to investigate the long-term effects of postural re-education.