Induction of ferroptosis promotes vascular smooth muscle cell phenotypic switching and aggravates neointimal hyperplasia in mice.

BACKGROUND: Stent implantation-induced neointima formation is a dominant culprit in coronary artery disease treatment failure after percutaneous coronary intervention. Ferroptosis, an iron-dependent regulated cell death, has been associated with various cardiovascular diseases. However, the effect of ferroptosis on neointima formation remains unclear. METHODS: The mouse common right carotid arteries were ligated for 16 or 30 days, and ligated tissues were collected for further analyses. Primary rat vascular smooth muscle cells (VSMCs) were isolated from the media of aortas of Sprague-Dawley (SD) rats and used for in vitro cell culture experiments. RESULTS: Ferroptosis was positively associated with neointima formation. In vivo, RAS-selective lethal 3 (RSL3), a ferroptosis activator, aggravated carotid artery ligation-induced neointima formation and promoted VSMC phenotypic conversion. In contrast, a ferroptosis inhibitor, ferrostatin-1 (Fer-1), showed the opposite effects in mice. In vitro, RSL3 promoted rat VSMC phenotypic switching from a contractile to a synthetic phenotype, evidenced by increased contractile markers (smooth muscle myosin heavy chain and calponin 1), and decreased synthetic marker osteopontin. The induction of ferroptosis by RSL3 was confirmed by the increased expression level of ferroptosis-associated gene prostaglandin-endoperoxide synthase 2 (Ptgs2). The effect of RSL3 on rat VSMC phenotypic switching was abolished by Fer-1. Moreover, N-acetyl-L-cysteine (NAC), the reactive oxygen species inhibitor, counteracted the effect of RSL3 on the phenotypic conversion of rat VSMCs. CONCLUSIONS: Ferroptosis induces VSMC phenotypic switching and accelerates ligation-induced neointimal hyperplasia in mice. Our findings suggest inhibition of ferroptosis as an attractive strategy for limiting vascular restenosis.

Insect Sf9 cells are suitable for functional expression of insect, but not vertebrate, striated muscle myosin.

Insect Sf9 cells are widely used for producing recombinant proteins, including myosin. It is expected that the protein folding machinery in Sf9 cells can meet the requirement for the proper folding of exogenous myosin. Of interest is that not all class II myosins are expressed functionally in Sf9 cells. Among vertebrate class II myosins, non-muscle myosin and smooth muscle myosin, but not striated muscle myosin, are functionally expressed in Sf9 cells, presumably due to lacking vertebrate striated muscle myosin-specific chaperone Unc45b in Sf9 cells. Insects only express a generic myosin-specific chaperone Unc45, which is expected to be responsible for the folding of all insect myosins, including striated muscle myosin. This rationale promotes us to investigate the folding of recombinant insect striated muscle myosins in Sf9 cells. We expressed the heavy meromyosin version of the striated muscle myosins from three insect species (Locusta migratoria, Drosophila melanogaster and Plutella xylostella) in Sf9 cells. Similar to vertebrate smooth muscle myosin, but unlike vertebrate striated muscle myosin, the insect striated muscle myosin expressed in Sf9 cells are soluble. The purified recombinant insect striated muscle myosins display normal myosin functions, including ATP-dependent actin interaction, actin-activated ATPase activity, and in vitro actin-gliding activity, indicating that Sf9 cells are suitable for expressing insect striated muscle myosin. We therefore conclude that, unlike vertebrate striated muscle myosin requiring striated muscle-specific chaperones (such as Unc45b) for its folding, insect striated muscle myosin can be properly folded by the generic protein folding machinery in insect cells.

Smooth Muscle Myosin Localizes at the Leading Edge and Regulates the Redistribution of Actin-regulatory Proteins during Migration.

Airway smooth muscle cell migration plays an essential role in airway development, repair, and remodeling. Smooth muscle myosin II has been traditionally thought to localize in the cytoplasm solely and regulates cell migration by affecting stress fiber formation and focal adhesion assembly. In this study, we unexpectedly found that 20-kDa myosin light chain (MLC20) and myosin-11 (MYH11), important components of smooth muscle myosin, were present at the edge of lamellipodia. The knockdown of MLC20 or MYH11 attenuated the recruitment of c-Abl, cortactinProfilin-1 (Pfn-1), and Abi1 to the cell edge. Moreover, myosin light chain kinase (MLCK) colocalized with integrin ß1 at the tip of protrusion. The inhibition of MLCK attenuated the recruitment of c-Abl, cortactin, Pfn-1, and Abi1 to the cell edge. Furthermore, MLCK localization at the leading edge was reduced by integrin ß1 knockdown. Taken together, our results demonstrate that smooth muscle myosin localizes at the leading edge and orchestrates the recruitment of actin-regulatory proteins to the tip of lamellipodia. Mechanistically, integrin ß1 recruits MLCK to the leading edge, which catalyzes MLC20 phosphorylation. Activated myosin regulates the recruitment of actin-regulatory proteins to the leading edge, and promotes lamellipodial formation and migration.

Molecular-level evidence of force maintenance by smooth muscle myosin during LC20 dephosphorylation.

Smooth muscle (SM) is found in most hollow organs of the body. Phasic SM, as found in the gut, contracts to propel content, whereas tonic SM, as found in most blood vessels, maintains tension. This force maintenance is referred to as the latch state and occurs at low levels of myosin activation (myosin light chain [LC20] phosphorylation). Molecular mechanisms have been proposed to explain the latch state but have been studied only at the whole-muscle level because of technological limitations. In the current study, an assay chamber was devised to allow injection of myosin light chain phosphatase (MLCP) during laser trap and in vitro motility assays, without creating bulk flow, to reproduce latch state conditions at the molecular level. Using the laser trap in a single-beam mode, an actin filament was brought in contact with several myosin molecules on a pedestal. Myosin pulled on the actin filament until a plateau force was reached, at which point, MLCP was injected. Force maintenance was observed during LC20 dephosphorylation, the level of which was assessed in a parallel in vitro motility assay performed in the same conditions. Force was maintained longer for myosin purified from tonic SM than from phasic SM. These data support the longstanding dogma of strong bonds caused by dephosphorylated, noncycling cross-bridges. Furthermore, MLCP injection in an in vitro motility mixture assay performed with SM and skeletal muscle myosin suggests that the maintenance of these strong bonds is possible only if no energy is provided by surrounding actively cycling myosin molecules.

Intrauterine neuromuscular and stromal dysplasia of the bladder in retinoic acid-induced myelomeningocele fetal rats.

OBJECTIVES: To observe the changes in the bladder of fetal rats with myelomeningocele (MMC) induced by all-trans retinoic acid (atRA) during the embryonic development stages. METHODS: The fetal rat model of MMC was induced by intragastric administration of atRA to pregnant rats on embryonic day 10 (E10). Fetal rats were harvested at E16, E18, E20, and E21 for observation and further testing. Those with MMC were classified as the MMC group, while those without MMC as the RA group. The areas of MMC skin defect, the crown-rump length (CRL), and body weight in different groups were compared. The histopathological changes in the bladder were compared. The expression levels of alpha-smooth muscle actin (αSMA), smooth muscle myosin heavy chain (SMMHC), connexin 43 (Cx43), desmin, ß3 tubulin, and vesicular acetylcholine transporter (VAChT) in the bladder were investigated by immunohistochemical staining and Western blotting. Pregnant rats given intragastric administration with olive oil (OIL group) at E10 were set as the blank control group. RESULTS: A total of 415 fetal rats of different gestational ages were harvested with an MMC incidence of 56.05 % (139/248). The incidence rate increased with embryonic days (p < 0.001). Compared with the other two control groups, the CRL and bodyweight of MMC fetal rats were significantly delayed at E21 (p < 0.001). The expression levels of αSMA, SMMHC, Cx43, desmin, ß3 tubulin and VAChT in the bladder of MMC fetal rats were significantly decreased at E21 (p < 0.05). CONCLUSIONS: In atRA-induced MMC fetal rats, there is neural, muscular, and stromal dysplasia in the bladder at an early gestational age. Further investigations on neurogenic bladder secondary to MMC are applicable using this animal model.

Smooth Muscle Cell Notch2 Is Not Required for Atherosclerotic Plaque Formation in ApoE Null Mice.

INTRODUCTION: We previously identified Notch2 in smooth muscle cells (SMC) in human atherosclerosis and found that signaling via Notch2 suppressed human SMC proliferation. Thus, we tested whether loss of Notch2 in SMC would alter atherosclerotic plaque progression using a mouse model. METHODS: Atherogenesis was examined at the brachiocephalic artery and aortic root in a vascular SMC null (inducible smooth muscle myosin heavy chain Cre) Notch2 strain on the ApoE-/- background. We measured plaque morphology and size, as well as lipid, inflammation, and smooth muscle actin content after Western diet. RESULTS: We generated an inducible SMC Notch2 null on the ApoE-/- background. We observed ∼90% recombination efficiency with no detectable Notch2 in the SMC. Loss of SMC Notch2 did not significantly change plaque size, lipid content, necrotic core, or medial area. However, loss of SMC Notch2 reduced the contractile SMC in brachiocephalic artery lesions and increased inflammatory content in aortic root lesions after 6 weeks of Western diet. These changes were not present with loss of SMC Notch2 after 14 weeks of Western diet. CONCLUSIONS: Our data show that loss of SMC Notch2 does not significantly reduce atherosclerotic lesion formation, although in early stages of plaque formation there are changes in SMC and inflammation.

Self-assembly of smooth muscle myosin filaments: adaptation of filament length by telokin and Mg·ATP.

The contractile apparatus of smooth muscle is malleable to accommodate stress and strain exerted on the muscle cell and to maintain optimal contractility. Structural lability of smooth muscle myosin filaments is believed to play an important role in the cell's malleability. However, the mechanism and regulation of myosin filament formation is still poorly understood. In the present in vitro study, using a static light scattering method, length distributions were obtained from suspensions of short myosin filaments (SFs) formed by rapid dilution or long ones (LFs) formed by slow dialysis. The distributions indicated the presence of dynamic equilibriums between soluble myosin and the SFs; i.e.: trimers, hexamers and mini filaments, covering the range up to 0.75 µm. The LFs were more stable, exhibiting favorable sizes at about 1.25, 2.4 and 4.5 µm. More distinct distributions were obtained from filaments adsorbed to a glass surface, by evanescent wave scattering and local electric field enhancement. Addition of telokin (TL) to the suspensions of unphosphorylated SFs resulted in widening of the soluble range, while in the case of the LFs this shift was larger, and accompanied by reduced contribution of the soluble myosin species. Such changes were largely absent in the case of phosphorylated myosin. In contrast, the presence of Mg·ATP resulted in elongation of the filaments and clear separation of filaments from soluble myosin species. Thus, TL and Mg·ATP appeared to modify the distribution of myosin filament lengths, i.e., increasing the lengths in preparing for phosphorylation, or reducing it to aid dephosphorylation.

Neutralization of S100A4 induces stabilization of atherosclerotic plaques: role of smooth muscle cells.

AIMS: During atherosclerosis, smooth muscle cells (SMCs) accumulate in the intima where they switch from a contractile to a synthetic phenotype. From porcine coronary artery, we isolated spindle-shaped (S) SMCs exhibiting features of the contractile phenotype and rhomboid (R) SMCs typical of the synthetic phenotype. S100A4 was identified as a marker of R-SMCs in vitro and intimal SMCs, in pig and man. S100A4 exhibits intra- and extracellular functions. In this study, we investigated the role of extracellular S100A4 in SMC phenotypic transition. METHODS AND RESULTS: S-SMCs were treated with oligomeric recombinant S100A4 (oS100A4), which induced nuclear factor (NF)-κB activation. Treatment of S-SMCs with oS100A4 in combination with platelet-derived growth factor (PDGF)-BB induced a complete SMC transition towards a pro-inflammatory R-phenotype associated with NF-κB activation, through toll-like receptor-4. RNA sequencing of cells treated with oS100A4/PDGF-BB revealed a strong up-regulation of pro-inflammatory genes and enrichment of transcription factor binding sites essential for SMC phenotypic transition. In a mouse model of established atherosclerosis, neutralization of extracellular S100A4 decreased area of atherosclerotic lesions, necrotic core, and CD68 expression and increased α-smooth muscle actin and smooth muscle myosin heavy chain expression. CONCLUSION: We suggest that the neutralization of extracellular S100A4 promotes the stabilization of atherosclerotic plaques. Extracellular S100A4 could be a new target to influence the evolution of atherosclerotic plaques.

Neonatal Streptococcus Pneumoniae pneumonia induces airway SMMHC expression through HMGB1/TLR4/ERK.

BACKGROUND: Our previous study showed that neonatal S. pneumoniae pneumonia promoted airway smooth muscle myosin heavy chain (SMMHC) expression and AHR development. Researches demonstrated HMGB1, TLR4 and ERK are involved in smooth muscle contractile protein expression, so we hypothesis that HMGB1/TLR4/ERK pathway participated in airway SMMHC overexpression in neonatal S. pneumoniae pneumonia model. METHOD: Neonatal (1-week-old) BALB/c mice were intranasal inoculated with D39 to establish non-lethal S. pneumoniae pneumonia model. TLR4 was inhibited 2 weeks after infection with TLR4 specific inhibitor (TAK-242). Five weeks after infection, the bronchoalveolar lavage fluid (BALF) and lungs of neonatal S. pneumoniae pneumonia and mock infection mice with or without TLR4 inhibition were collected to assess the expressions of HMGB1, TLR4 and p-ERK1/2. Airway Hyperresponsiveness (AHR) of the three groups was determined by whole-body plethysmograph. RESULTS: Our results demonstrated that neonatal S. pneumoniae pneumonia promoted HMGB1/TLR4 production, SMMHC expression and AHR development significantly, with ERK1/2 phosphorylation decreased remarkably. TLR4 inhibition after pneumonia significantly increased ERK1/2 phosphorylation, reversed airway SMMHC overexpression and alleviated AHR. CONCLUSION: Neonatal S. pneumoniae pneumonia promotes airway SMMHC expression and AHR through HMGB1/TLR4/ERK.

Decreased vascular smooth muscle contractility in Hutchinson-Gilford Progeria Syndrome linked to defective smooth muscle myosin heavy chain expression.

Children with Hutchinson-Gilford Progeria Syndrome (HGPS) suffer from multiple cardiovascular pathologies due to the expression of progerin, a mutant form of the nuclear envelope protein Lamin A. Progerin expression has a dramatic effect on arterial smooth muscle cells (SMCs) and results in decreased viability and increased arterial stiffness. However, very little is known about how progerin affects SMC contractility. Here, we studied the LaminAG609G/G609G mouse model of HGPS and found reduced arterial contractility at an early age that correlates with a decrease in smooth muscle myosin heavy chain (SM-MHC) mRNA and protein expression. Traction force microscopy on isolated SMCs from these mice revealed reduced force generation compared to wild-type controls; this effect was phenocopied by depletion of SM-MHC in WT SMCs and overcome by ectopic expression of SM-MHC in HGPS SMCs. Arterial SM-MHC levels are also reduced with age in wild-type mice and humans, suggesting a common defect in arterial contractility in HGPS and normal aging.

Evidence for S2 flexibility by direct visualization of quantum dot-labeled myosin heads and rods within smooth muscle myosin filaments moving on actin in vitro.

Myosins in muscle assemble into filaments by interactions between the C-terminal light meromyosin (LMM) subdomains of the coiled-coil rod domain. The two head domains are connected to LMM by the subfragment-2 (S2) subdomain of the rod. Our mixed kinetic model predicts that the flexibility and length of S2 that can be pulled away from the filament affects the maximum distance working heads can move a filament unimpeded by actin-attached heads. It also suggests that it should be possible to observe a head remain stationary relative to the filament backbone while bound to actin (dwell), followed immediately by a measurable jump upon detachment to regain the backbone trajectory. We tested these predictions by observing filaments moving along actin at varying ATP using TIRF microscopy. We simultaneously tracked two different color quantum dots (QDs), one attached to a regulatory light chain on the lever arm and the other attached to an LMM in the filament backbone. We identified events (dwells followed by jumps) by comparing the trajectories of the QDs. The average dwell times were consistent with known kinetics of the actomyosin system, and the distribution of the waiting time between observed events was consistent with a Poisson process and the expected ATPase rate. Geometric constraints suggest a maximum of ∼26 nm of S2 can be unzipped from the filament, presumably involving disruption in the coiled-coil S2, a result consistent with observations by others of S2 protruding from the filament in muscle. We propose that sufficient force is available from the working heads in the filament to overcome the stiffness imposed by filament-S2 interactions.

Uterine expression of smooth muscle alpha- and gamma-actin and smooth muscle myosin in bitches diagnosed with uterine inertia and obstructive dystocia.

Primary uterine inertia (PUI) is the most common type of dystocia in dogs. We hypothesized that PUI develops because of lower than normal expression of the basic contractile elements in the uterus, i.e., smooth muscle (SM) α- and γ-actin and SM-myosin, and that the expression of these proteins is influenced by the number of fetuses present in utero. Full-thickness inter-placental uterine biopsies were collected during Cesarean sections from dogs with PUI (n = 11), and from bitches with obstructive dystocia (OD) still presenting strong labor contractions (designated as the control group, n = 7). Relative gene expression was determined by semi-quantitative real-time (TaqMan) PCR, and protein localization by immunohistochemistry. Gene expression between PUI and OD bitches, and between PUI bitches carrying small, large, or average number of fetuses according to their breed, were compared. Uterine SM-γ-actin and SM-myosin mRNA levels were significantly higher in PUI than in OD dogs, while SM-α-actin did not differ. PUI bitches carrying large litters had lower uterine SM-γ-actin gene expression than those with small litters (P = 0.008). Immunostaining for SM-actin isoforms and SM-myosin was present in the myometrium, and localization pattern and staining intensity appeared similar in the PUI and OD groups. All proteins stained in blood vessels, and SM-γ-actin was also present in endometrial luminal and glandular epithelium. In conclusion, higher uterine SM-γ-actin and SM-myosin gene expression in PUI bitches, compared with OD dogs, might be an indication of abnormal progression with labor. Whether this is the cause of PUI due to an intrinsic error of the myometrium not becoming committed to labor, or the consequence of inadequate endocrine or mechanical stimuli, is not clear. Litter size was previously shown to be one of the risk factors for the development of uterine inertia in dogs, and our findings suggest possible differing uterine pathophysiology of PUI with respect to litter size.

Muscle myosins form folded monomers, dimers, and tetramers during filament polymerization in vitro.

Muscle contraction depends on the cyclical interaction of myosin and actin filaments. Therefore, it is important to understand the mechanisms of polymerization and depolymerization of muscle myosins. Muscle myosin 2 monomers exist in two states: one with a folded tail that interacts with the heads (10S) and one with an unfolded tail (6S). It has been thought that only unfolded monomers assemble into bipolar and side-polar (smooth muscle myosin) filaments. We now show by electron microscopy that, after 4 s of polymerization in vitro in both the presence (smooth muscle myosin) and absence of ATP, skeletal, cardiac, and smooth muscle myosins form tail-folded monomers without tail-head interaction, tail-folded antiparallel dimers, tail-folded antiparallel tetramers, unfolded bipolar tetramers, and small filaments. After 4 h, the myosins form thick bipolar and, for smooth muscle myosin, side-polar filaments. Nonphosphorylated smooth muscle myosin polymerizes in the presence of ATP but with a higher critical concentration than in the absence of ATP and forms only bipolar filaments with bare zones. Partial depolymerization in vitro of nonphosphorylated smooth muscle myosin filaments by the addition of MgATP is the reverse of polymerization.

Phenotype transitions induced by mechanical stimuli in airway smooth muscle are regulated by differential interactions of parvin isoforms with paxillin and Akt.

Mechanical tension and humoral stimuli can induce transitions in airway smooth muscle phenotype between a synthetic inflammatory state that promotes cytokine secretion and a differentiated state that promotes the expression of smooth muscle phenotype-specific proteins. When tissues are maintained under high tension, Akt activation and eotaxin secretion are suppressed, but expression of the differentiation marker protein, smooth muscle myosin heavy chain (SmMHC), is promoted. When tissues are maintained under low tension, Akt activation and eotaxin secretion are stimulated, and the differentiated phenotype is suppressed. We hypothesized that mechanical stimuli are differentially transduced to Akt-mediated signaling pathways that regulate phenotype expression by α-parvin and ß-parvin integrin-linked kinase/PINCH/parvin (IPP) signaling complexes within integrin adhesomes. High tension or ACh triggered paxillin phosphorylation and the binding of phospho-paxillin to ß-parvin IPP complexes. This inhibited Akt activation and promoted SmMHC expression. Low tension or IL-4 did not elicit paxillin phosphorylation and triggered the binding of unphosphorylated paxillin to α-parvin IPP complexes, which promoted Akt activation and eotaxin secretion and suppressed SmMHC expression. Expression of a nonphosphorylatable paxillin mutant or ß-parvin depletion by siRNA promoted the inflammatory phenotype, whereas the depletion of α-parvin promoted the differentiated phenotype. Results demonstrate that phenotype expression is regulated by the differential interaction of phosphorylated and unphosphorylated paxillin with α-parvin and ß-parvin IPP complexes and that these complexes have opposite effects on the activation of Akt. Our results describe a novel molecular mechanism for transduction of mechanical and humoral stimuli within integrin signaling complexes to regulate phenotype expression in airway smooth muscle.

Comparison of the efficiency of transgelin, smooth muscle myosin, and CD31 antibodies for the assessment of vascular tumor invasion and free tumor deposits in gastric, pancreatic, and colorectal adenocarcinomas.

BACKGROUND: This study aimed to compare CD31, smooth muscle myosin (SMM), and transgelin antibodies for their efficiency in detecting venous invasion (VI) and the nature of free tumor deposits (TDs) in gastric, pancreatic, and colorectal adenocarcinomas. MATERIALS AND METHODS: Eleven Whipple, 5 gastrectomy, and 3 colectomy specimens and 1 low anterior resection specimen were reviewed and examined, revealing 254 probable foci. Foci were reviewed and divided into 3 types: Type A, the "orphan artery" pattern; Type F, free TDs in the periorgan adipose and connective tissue without an unaccompanied artery; and Type X, a focus that could be detected only with the immunohistochemical procedures mentioned. RESULTS: No foci were positive for CD31. Transgelin staining was more sensitive than SMM staining in all focus types, Type A only and Type F only (P < 0.001, P = 0.001, and P = 0.10, respectively). In free TDs (Type F), 35.7% of the samples were negative for all four stains, and 64.2% of the samples were positive for SMM and transgelin. We did not make the distinction between a metastatic lymph node and VI in positive foci. CONCLUSION: We conclude that hematoxylin and eosin (H and E) staining is inadequate and that smooth muscle markers, such as transgelin and/or SMM, are more effective than endothelial markers, such as CD31, in revealing VI and lymph node/large extramural invasion.

The central role of the tail in switching off 10S myosin II activity.

Myosin II is a motor protein with two heads and an extended tail that plays an essential role in cell motility. Its active form is a polymer (myosin filament) that pulls on actin to generate motion. Its inactive form is a monomer with a compact structure (10S sedimentation coefficient), in which the tail is folded and the two heads interact with each other, inhibiting activity. This conformation is thought to function in cells as an energy-conserving form of the molecule suitable for storage as well as transport to sites of filament assembly. The mechanism of inhibition of the compact molecule is not fully understood. We have performed a 3-D reconstruction of negatively stained 10S myosin from smooth muscle in the inhibited state using single-particle analysis. The reconstruction reveals multiple interactions between the tail and the two heads that appear to trap ATP hydrolysis products, block actin binding, hinder head phosphorylation, and prevent filament formation. Blocking these essential features of myosin function could explain the high degree of inhibition of the folded form of myosin thought to underlie its energy-conserving function in cells. The reconstruction also suggests a mechanism for unfolding when myosin is activated by phosphorylation.

Doxorubicin induces detrusor smooth muscle impairments through myosin dysregulation, leading to a risk of lower urinary tract dysfunction.

Cytotoxic chemotherapy is the foundation for the treatment of the wide variety of childhood malignancies; however, these therapies are known to have a variety of deleterious side effects. One common chemotherapy used in children, doxorubicin (DOX), is well known to cause cardiotoxicity and cardiomyopathy. Recent studies have revealed that DOX impairs skeletal and smooth muscle function and contributes to fatigue and abnormal intestinal motility in patients. In this study, we tested the hypothesis that systemic DOX administration also affects detrusor smooth muscle (DSM) function in the urinary bladder, especially when administered at a young age. The effects on the DSM and bladder function were assessed in BALB/cJ mice that received six weekly intravenous injections of DOX (3 mg·kg-1·wk-1) or saline for the control group. Systemic DOX administration resulted in DSM hypertrophy, increased voiding frequency, and a significant attenuation of DSM contractility, followed by a slower relaxation compared with the control group. Gene expression analyses revealed that unlike DOX-induced cardiotoxicity, the bladders from DOX-administered animals showed no changes in oxidative stress markers; instead, downregulation of large-conductance Ca2+-activated K+ channels and altered expression of myosin light-chain kinase coincided with reduced myosin light-chain phosphorylation. These results indicate that in vivo DOX exposure caused DSM dysfunction by dysregulation of molecules involved in the detrusor contractile-relaxation mechanisms. Collectively, our findings suggest that survivors of childhood cancer treated with DOX may be at increased risk of bladder dysfunction and benefit from followup surveillance of bladder function.

Using the SpyTag SpyCatcher system to label smooth muscle myosin II filaments with a quantum dot on the regulatory light chain.

The regulatory light chain (RLC) of myosin is commonly tagged to monitor myosin behavior in vitro, in muscle fibers, and in cells. The goal of this study was to prepare smooth muscle myosin (SMM) filaments containing a single head labeled with a quantum dot (QD) on the RLC. We show that when the RLC is coupled to a QD at Cys-108 and exchanged into SMM, subsequent filament assembly is severely disrupted. To address this, we used a novel approach for myosin by implementing the SpyTag002 SpyCatcher002 system to prepare SMM incorporated with RLC constructs fused to SpyTag or SpyCatcher. We show that filament assembly, actin-activated steady-state ATPase activities, ability to be phosphorylated, and selected enzymatic and mechanical properties were essentially unaffected if either SpyTag or SpyCatcher were fused to the C-terminus of the RLC. Crucially for our application, we also show that a QD coupled to SpyCatcher can be covalently attached to a RLC-Spy incorporated into a SMM filament without disrupting the filament, and that the filaments can move along actin in vitro.

A direct immunohistochemistry (IHC) method improves the intraoperative diagnosis of breast papillary lesions including breast cancer.

Tumors with the attributes of rapid growth, infiltration, and metastasis are the leading causes of death among cancer patients. Angiogenesis is essential to tumor nutrition support and tumor progression. Endoglin is a glycoprotein highly expressed on the endothelial cell membrane and is regarded as the most reliable marker of tumor vascular proliferation. In this review, we summarize recent advances in targeting endoglin for the imaging of cancer angiogenesis and the development of monoclonal antibodies and vaccines to inhibit cancer angiogenesis. In addition, we forecast the future promise of endoglin as a novel target for the diagnosis and treatment of malignant tumors.

RSK2 contributes to myogenic vasoconstriction of resistance arteries by activating smooth muscle myosin and the Na+/H+ exchanger.

Smooth muscle contraction is triggered when Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) phosphorylates the regulatory light chain of myosin (RLC20). However, blood vessels from Mlck-deficient mouse embryos retain the ability to contract, suggesting the existence of additional regulatory mechanisms. We showed that the p90 ribosomal S6 kinase 2 (RSK2) also phosphorylated RLC20 to promote smooth muscle contractility. Active, phosphorylated RSK2 was present in mouse resistance arteries under normal basal tone, and phosphorylation of RSK2 increased with myogenic vasoconstriction or agonist stimulation. Resistance arteries from Rsk2-deficient mice were dilated and showed reduced myogenic tone and RLC20 phosphorylation. RSK2 phosphorylated Ser19 in RLC in vitro. In addition, RSK2 phosphorylated an activating site in the Na+/H+ exchanger (NHE-1), resulting in cytosolic alkalinization and an increase in intracellular Ca2+ that promotes vasoconstriction. NHE-1 activity increased upon myogenic constriction, and the increase in intracellular pH was suppressed in Rsk2-deficient mice. In pressured arteries, RSK2-dependent activation of NHE-1 was associated with increased intracellular Ca2+ transients, which would be expected to increase MLCK activity, thereby contributing to basal tone and myogenic responses. Accordingly, Rsk2-deficient mice had lower blood pressure than normal littermates. Thus, RSK2 mediates a procontractile signaling pathway that contributes to the regulation of basal vascular tone, myogenic vasoconstriction, and blood pressure and may be a potential therapeutic target in smooth muscle contractility disorders.