Electrospun fibre diameter and its effects on vascular smooth muscle cells

Bypass grafting is a technique used in the treatment of vascular disease, which is currently the leading cause of mortality worldwide. While technology has moved forward over the years, synthetic grafts still show significantly lower rates of patency in small diameter bypass operations compared to the gold standard (autologous vessel grafts). Scaffold morphology plays an important role in vascular smooth muscle cell (VSMC) performance, with studies showing how fibre alignment and surface roughness can modulate phenotypic and genotypic changes. Herein, this study has looked at how the fibre diameter of electrospun polymer scaffolds can affect the performance of seeded VSMCs. Four different scaffolds were electrospun with increasing fibre sizes ranging from 0.75 to 6 µm. Culturing VSMCs on the smallest fibre diameter (0.75 µm) lead to a significant increase in cell viability after 12 days of culture. Furthermore, interesting trends were noted in the expression of two key phenotypic genes associated with mature smooth muscle cell contractility (myocardin and smooth muscle alpha-actin 1), whereby reducing the fibre diameter lead to relative upregulations compared to the larger fibre diameters. These results showed that the smallest (0.75 µm) fibre diameter may be best suited for the culture of VSMCs with the aim of increasing cell proliferation and aiding cell maturity.

UBE2L3, a Partner of MuRF1/TRIM63, Is Involved in the Degradation of Myofibrillar Actin and Myosin

The ubiquitin proteasome system (UPS) is the main player of skeletal muscle wasting, a common characteristic of many diseases (cancer, etc.) that negatively impacts treatment and life prognosis. Within the UPS, the E3 ligase MuRF1/TRIM63 targets for degradation several myofibrillar proteins, including the main contractile proteins alpha-actin and myosin heavy chain (MHC). We previously identified five E2 ubiquitin-conjugating enzymes interacting with MuRF1, including UBE2L3/UbcH7, that exhibited a high affinity for MuRF1 (KD = 50 nM). Here, we report a main effect of UBE2L3 on alpha-actin and MHC degradation in catabolic C2C12 myotubes. Consistently UBE2L3 knockdown in Tibialis anterior induced hypertrophy in dexamethasone (Dex)-treated mice, whereas overexpression worsened the muscle atrophy of Dex-treated mice. Using combined interactomic approaches, we also characterized the interactions between MuRF1 and its substrates alpha-actin and MHC and found that MuRF1 preferentially binds to filamentous F-actin (KD = 46.7 nM) over monomeric G-actin (KD = 450 nM). By contrast with actin that did not alter MuRF1–UBE2L3 affinity, binding of MHC to MuRF1 (KD = 8 nM) impeded UBE2L3 binding, suggesting that differential interactions prevail with MuRF1 depending on both the substrate and the E2. Our data suggest that UBE2L3 regulates contractile proteins levels and skeletal muscle atrophy.

Plaque Vulnerability Index Predicts Cardiovascular Events: A Histological Study of an Endarterectomy Cohort

Background The balance between stabilizing and destabilizing atherosclerotic plaque components is used in experimental studies and in imaging studies to identify rupture prone plaques. However, we lack the evidence that this balance predicts future cardiovascular events. Here we explore whether a calculated histological ratio, referred to as vulnerability index (VI), can predict patients at higher risk to suffer from future cardiovascular events. Methods and Results Carotid plaques and clinical information from 194 patients were studied. Tissue sections were used for histological analysis to calculate the VI (CD68 [cluster of differentiation 68], alpha‐actin, Oil red O, Movat pentachrome, and glycophorin A). Postoperative cardiovascular events were identified through the Swedish National Inpatient Health Register (2005–2013). During the follow‐up (60 months) 45 postoperative cardiovascular events were registered. Patients with a plaque VI in the fourth quartile compared with the first to third quartiles had significantly higher risk to suffer from a future cardiovascular event ( P =0.0002). The VI was an independent predictor and none of the 5 histological variables analyzed separately predicted events. In the 13 patients who underwent bilateral carotid endarterectomy, the VI of the right plaque correlated with the VI of the left plaque and vice versa ( r =0.7, P =0.01). Conclusions Our findings demonstrate that subjects with a high plaque VI have an increased risk of future cardiovascular events, independently of symptoms and other known cardiovascular risk factors . This strongly supports that techniques which image such plaques can facilitate risk stratification for subjects in need of more intense treatment.

Loss of Acta2 in cardiac fibroblasts does not affect myofibroblast differentiation or cardiac repair after myocardial infarction

In response to myocardial infarction (MI), quiescent cardiac fibroblasts differentiate into myofibroblasts mediating tissue repair in the infarcted area. One of the most widely accepted markers of myofibroblast differentiation is the expression of Acta2 which encodes smooth muscle alpha-actin (SMαA) that is assembled into stress fibers. However, the requirement of Acta2 in the myofibroblast differentiation of cardiac fibroblasts and its role in post-MI cardiac repair were still not known. To answer these questions, we generated a tamoxifen-inducible cardiac fibroblast-specific Acta2 knockout mouse line. Surprisingly, mice that lacked Acta2 in cardiac fibroblasts had a normal survival rate after MI. Moreover, Acta2 deletion did not affect the function or overall histology of infarcted hearts. No difference was detected in the proliferation, migration, or contractility between WT cardiac fibroblasts and Acta2-null cardiac myofibroblasts. Additional analysis identified that Acta2-null cardiac myofibroblasts had a normal total filamentous actin level and total actin level. Acta2 deletion caused a unique compensatory increase in the transcription level of Actg2 and a possible increase in the protein abundance of cytoplasmic actin isoforms. In conclusion, SMαA stress fibers are not required for myofibroblast differentiation of cardiac fibroblasts or the post-MI cardiac repair, and the increase in the expression of non-SMαA actin isoforms and the functional redundancy between actin isoforms are likely able to compensate for the loss of Acta2 in cardiac myofibroblasts.

Generation of Transgenic Medaka Oryzias curvinotus (Nichols & Pope, 1927) Carrying a Cyan Fluorescent Protein Gene Driven by Alpha Actin Promoter

The study aimed to produce fluorescent protein transgenic medaka Oryzias curvinotus (Nichols & Pope, 1927) as a novel strain of ornamental fish. These fish were produced by transferring a plasmid consisting of a fluorescent reporter gene and a strong promoter into one-cell stage embryos. For this purpose, myosin light chain 2, but not other promoters, was mainly used. The study also evaluated the stability of the transgenic medaka germline acquiring vivid fluorescent phenotypes via the transgenesis of the cyan fluorescent protein (CFP) gene under the control of O. curvinotus skeletal alpha-actin (OCacta) promoter. The pOCacta-CFP plasmid, containing a OCacta promoter and CFP reporter gene, was transferred into the one-cell stage of O. curvinotus embryos by a microinjection technique. As a result, 36 of 1386 microinjected O. curvinotus embryos exhibited CFP signals in their trunks. The expressed CFP signals in O. curvinotus embryos and adults were detected under a microscope using a green fluorescent protein (GFP) filter (450–490 nm wavelength), and blue LED light (400–450 nm wavelength). Five O. curvinotus founders showing clear CFP signals were selected and crossed with non-transgenic counterparts to produce subsequent generations. Among strains, the frequency of germline transmission from founder to F1 was highly variable. Only two of the five founders successfully pass the transgene to the F1 generation. At present, the progeny of subsequent generations is being produced and tested for the expression of CFP signals, and therefore, stable lines are ongoing.

Smooth muscle aortic alpha actin A2 (ACTA2) is differentially expressed in metastatic breast cancer, both in metastases to the brain and to the lymph nodes.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes to discover genes associated with brain metastasis in patients with metastatic breast cancer. We found that smooth muscle aortic alpha actin A2, encoded by ACTA2, was among the genes whose expression was most different in the metastatic tumor tissues of patients with metastatic breast cancer, both in metastases to brain and to the lymph nodes when compared to primary tumors of the breast or normal breast tissue, respectively. We observed significant down-regulation of ACTA2 in metastasis to the brain. Molecular functions and down-regulation of smooth muscle aortic alpha actin A2 may be important for metastasis of primary tumor-derived cancer cells to the lymph nodes and to the brain in humans with metastatic breast cancer and suggests some level of common origin for metastases that reside in the lymph nodes and colonize the brain.

Abstract MP102: Skeletal Muscle Alpha Actin Acetylation Enhances Myosin Binding and Increases Calcium Sensitivity in vitro

Actin and myosin are key proteins for muscle contraction/relaxation, while tropomyosin regulates actin-myosin interactions in the presence or absence of calcium. Mutations or dysregulation of any of these proteins results in cardiomyopathy. We previously showed that skeletal muscle alpha actin (ACTA1) was significantly deacetylated on lysine residues, K52, K317, and K328 in remodeled left ventricular tissue of obese mice. Computational modeling suggests that the positively charged lysine residues K328 and K317 of ACTA1 can interact electrostatically with the negatively charged glutamic acid residue E181 of tropomyosin and E286 of myosin. As acetylation is predicted to neutralize the positively charged lysine, ACTA1 acetylation would be postulated to decrease actin-myosin or actin-tropomyosin electrostatic interactions. To test this hypothesis, we used an in vitro actin motility assay to determine myosin sliding velocity, calcium sensitivity, and attachment/detachment kinetics of acetylated/deacetylated ACTA1. In addition, an actin binding protein spin-down assay was used to determine actin-myosin binding affinity using skeletal and cardiac myosin. In these assays, ACTA1 was chemically acetylated with acetic anhydride. In vitro actin motility analysis showed a significant decrease in sliding velocity with acetylated ACTA1 and skeletal myosin (1.709±0.210 μm/s) compared with deacetylated ACTA1 (4.427±0.275 μm/s). A similar significant decrease was also noted with cardiac myosin. Further analysis showed a significant increase in calcium sensitivity with ACTA1 acetylation (3.197x10-7 Kd compared to deacetylated ACTA1 1.191x10-6 Kd) and a loss of tropomyosin regulation with increasing ACTA1 acetylation. Lastly, ACTA1 acetylation enhanced actin binding affinity to cardiac and skeletal myosin. Investigation of attachment/detachment kinetics are currently underway. These data suggest that ACTA1 acetylation disrupts tropomyosin’s ability to inhibit myosin binding in the absence of calcium and further regulates actin-myosin interactions. Lastly, these data highlight acetylation as an additional post-translational modification outside of phosphorylation in the regulation of muscle contraction.

New species of Nectria and Thyronectria from Xinjiang, China

Nectria berberidis sp. nov. and Thyronectria berberidicola sp. nov. isolated from Berberis heteropoda in Xinjiang Uygur Autonomous Region, China, are described and illustrated. Nectria berberidis is characterized by clavate asci (50–87 × 8–12 μm) with ellipsoidal to fusiform, 1-septate ascospores. Thyronectria berberidicola is characterized by clavate asci (117–25.9 × 63.7–117.9 μm) with ellipsoidal to fusiform ascospores that have 5–8 transverse septa and 1(–2) longitudinal septum. Ascospores bud to produce hyaline, bacillar ascoconidia. Phylogenetic analyses based on alpha-actin (ACT), the internal transcribed spacer (ITS), the large nuclear ribosomal RNA subunit (LSU), translation elongation factor 1-alpha (TEF1) and the β-tubulin (TUB) sequence data revealed that isolates of N. berberidis and T. berberidicola form a distinct clade within Nectria and Thyronectria, respectively. In addition, Nectria nigrescens is reported for the first time in China.

Study On The Biomechanical Properties Of Rabbit Venous Arterialization

Objective : To investigate the mechanisms underlying restenosis following coronary artery bypass grafting using bridging veins.Method : We established a rabbit model of venous arterialisation, by transplanting veins into the arterial system as bridging vessels and investigated vessel tensile mechanical and histomorphological properties. Result : Control vein elasticity (k = 16.20) was less than that of the control artery (k = 58.04; P < 0.05), and vein walls were thinner. Following venous arterialisation, proliferating cell nuclear antigen and alpha-actin were upregulated and vein walls thickened (P < 0.05), with elasticity after venous arterialisation (k = 86.26) significantly higher than that of control veins (P < 0.05). Conclusion : This indicates that venous intima is damaged by high pressure following arterialisation, resulting in gradual restenosis, with thickening of the venous intima and an increase in vessel elasticity. Clinically, there is potential to repeat these experiments to determine the elastic extremum of the great saphenous vein and control the pressure in the lumen of this vessel, to ensure minimal damage to the intima before anastomosis, thereby facilitating improvement of long-term patency rates following vein bridge surgery. Whether the increase in venous bridge elasticity after venous arterialisation can be controlled, with the aim of preventing early-stage restenosis, warrants investigation.

Participation of the Immune System and Hedgehog Signaling in Neoangiogenesis Under Laser Photobiomodulation

Introduction: This study aimed to characterize immune and endothelial cells, myofibroblasts and pericytes, and positive cells for hedgehog proteins in late tissue repair of rats skin wounds treated with 670 nm photobiomodulation therapy (PBMT). Methods: A blind experimental study was conducted, in order to assess the effect of PBMT in later stages of healing, with emphasis on neoangiogenesis, immune cells and Hedgehog signaling. Forty Wistar rats were allocated randomly in two groups; control and treated with a diode GaAlAs laser (9 mW, 670 nm, 0.031 W/cm², spot size of 0.28 cm², fluence of 4 J/ cm2 applied every other day, until a total dose of 16 J/cm2 was achieved). Standardized skin wounds were performed and the animals were euthanized at 14, 21, 28 and 35 days. Tissue sections were subjected to hematoxylin-eosin and immunohistochemistry for CD31, NG2, smooth muscle alpha actin, CD8, CD68, Ptch, Gli-2 and Ihh. All histomorphometric data were statistically analyzed and significance level was at P<0.05. Results: At late stages of wound healing, neoangiogenesis persisted as revealed for the number of CD31+ cells (P=0.016) and NG2+ and smooth muscle alpha actin positive pericytes (P=0.025), for both experimental groups. By day 21, laser-treated group had decreased CD68+ cells (P=0.032) and increased CD8+ (P=0.038). At remodeling stage, there were positive cells for the hedgehog signaling pathway family which seemed to be activated. Conclusion: These data suggest that photobiomodulation therapy was able to modulate extracellular matrix remodelling even at the later stages of wound healing.??