Myosin Light Chain Protein Research Articles

101 5-HT4 receptor-mediated vasorelaxation occurs through a cyclic mononucleotide-dependent mechanism in the isolated bovine lateral saphenous vein

Abstract Serotonin (5-HT)-mediated vasorelaxation of the isolated bovine lateral saphenous vein occurs primarily through 5-HT4 receptor activation. Vasorelaxation responses mediated through 5-HT4 have only been documented to occur in cattle and sheep and thus, may represent a novel vasorelaxation mechanism that is specific to ruminants. In tissues from other species, the 5-HT4 receptor and its associated G protein couple with adenylate cyclase. However, the mechanisms contributing to 5-HT4-mediated vasorelaxation in vascular tissue remain undefined. To better understand the mechanisms of 5-HT4-mediated vasorelaxation, lateral saphenous veins from cattle (n = 4) were collected and assessed for vasoactivity in response to increasing concentrations of a selective 5-HT4 receptor agonist (BIMU-8) in the absence and presence of selective inhibitors of downstream proteins involved with cyclic mononucleotide-mediated signaling. Isolated vessel segments were suspended in chambers of a multi-myograph containing 5 mL of continuously oxygenated Krebs-Henseleit buffer and equilibrated to 1 g tension for 90 min. After the equilibration period, vessels were exposed to dimethyl sulfoxide (DMSO; vehicle control) or selective inhibitors of downstream protein targets dissolved in DMSO (1×10-5 M) for 5 min. Vessels were pre-contracted with 1×10-4 M phenylephrine for 10 min to achieve a sustained increase in tension. After the 10-min pre-contraction period, vessels were exposed to increasing concentrations (1×10-9 to 1×10-4) of BIMU-8 non-cumulatively. Vasoactive response data were normalized as a percentage of the maximum contractile response induced by the phenylephrine pre-contraction. In the absence of selective inhibitors (control), BIMU-8 induced (P < 0.01) dose-dependent relaxations (mean -log EC50 = 6.30 M) with a 112% increase in maximal vasorelaxation activity occurring at 1×10-4 M. In the presence of the selective inhibitors for adenylate cyclase (NKY80) or guanylate cyclase (NS2028), 66% and 86% of the vasorelaxation responses to BIMU-8 were attenuated (P < 0.01), respectively. In the presence of the selective inhibitors for protein kinase A [protein kinase A inhibitor fragment (6-22) amide] or protein kinase G (Rp-8-bromo-PET-Cyclic GMPS), 66% and 97% of the maximal vasorelaxation responses to BIMU-8 were attenuated (P < 0.01), respectively. In the presence of non-selective inhibitors for Ca2+ channels and K+ channels, vasorelaxation responses to BIMU-8 were attenuated (P < 0.01). In the presence of selective inhibitors for the protein phosphatase 1 and 2A subunits of myosin light chain (calyculin A) and heat shock protein synthesis (quercetin), vasorelaxation responses to BIMU-8 were attenuated (P < 0.01). The findings of the current study demonstrate that 5-HT4-receptor mediated vasorelaxation occurs through a cyclic mononucleotide-dependent mechanism involving cross-activation of protein kinase A and G signaling. Downstream proteins involved in the signaling cascade leading to vasorelaxation include Ca2+ channels, K+ channels, myosin light chain phosphatase, and heat shock proteins. More research is needed to fully describe the cellular and molecular mechanisms responsible for 5-HT4 receptor-mediated vasorelaxation.

Activation of the ROCK/MYLK Pathway Affects Complex Molecular and Morphological Changes of the Trabecular Meshwork Associated With Ocular Hypertension.

The Rho-associated protein kinase and myosin light chain kinase (ROCK/MYLK) pathway undeniably plays a pivotal role in the pathophysiology of primary open-angle glaucoma (POAG). In our study, we utilized both ocular hypertension (OHT) rabbit models and clinical investigations to gain invaluable insights that propel the development of novel treatments targeting proteins and genes associated with the trabecular meshwork (TM), thereby offering promising avenues for the management of POAG. Following microbead injections into the anterior chamber of the ocular cavity of rabbits, we observed elevated histiocyte numbers and immune scores for MYLK-4/ pMLC-2, alongside a reduction in the void space within the TM. Notably, treatment was performed with 0.1% ITRI-E-(S)-4046, a compound with dual kinase inhibitor (highly specific inhibitor of ROCK1/2 and MYLK4), significantly reduced intraocular pressure (IOP; P < 0.05) and expanded the void space within the TM (P < 0.0001) compared with OHT rabbits. In clinical investigations, we utilized whole transcriptome sequencing to analyze gene expression specifically related to the TM, obtained from patients (5 early-onset and 5 late-onset) undergoing trabeculectomy. Our findings revealed 103 differential expression genes (DEGs) out of 265 molecules associated with the Rho family GTPase pathway, exhibiting a P value of 1.25E-10 and a z-score of -2.524. These results underscore significant differences between the early-onset and late-onset POAG and highlight the involvement of the ROCK/MYLK pathway. These findings underscore the critical involvement of the ROCK/MYLK pathway in both OHT-related and different onsets of POAG, providing valuable insights into the TM-related molecular mechanisms underlying the disease.

Salmonella manipulates macrophage migration via SteC-mediated myosin light chain activation to penetrate the gut-vascular barrier

The intestinal pathogen Salmonella enterica rapidly enters the bloodstream after the invasion of intestinal epithelial cells, but how Salmonella breaks through the gut-vascular barrier is largely unknown. Here, we report that Salmonella enters the bloodstream through intestinal CX3CR1+ macrophages during early infection. Mechanistically, Salmonella induces the migration/invasion properties of macrophages in a manner dependent on host cell actin and on the pathogen effector SteC. SteC recruits host myosin light chain protein Myl12a and phosphorylates its Ser19 and Thr20 residues. Myl12a phosphorylation results in actin rearrangement, and enhanced migration and invasion of macrophages. SteC is able to utilize a wide range of NTPs other than ATP to phosphorylate Myl12a. We further solved the crystal structure of SteC, which suggests an atypical dimerization-mediated catalytic mechanism. Finally, in vivo data show that SteC-mediated cytoskeleton manipulation is crucial for Salmonella breaching the gut vascular barrier and spreading to target organs.

Dietary Cannabidiol Activates PKA/AMPK Signaling and Attenuates Chronic Inflammation and Leaky Gut in DSS-Induced Colitis Mice.

Inflammatory bowel disease (IBD) is characterized by chronic inflammation in the gut, accompanied by impaired epithelial integrity, increased macrophage infiltration, and enhanced colon cancer risk. Cannabidiol (CBD), a phytocannabinoid isolated from cannabis plants, is supplemented into mice diet, and its beneficial effects against dextran sulfate sodium (DSS)-induced experimental colitis is evaluated. Eight-week-old mice were fed a standard diet supplemented with or without CBD (200mg kg-1 ) for 5 weeks. In the 4th week of dietary treatment, mice were subjected to 2.5% DSS induction for 7 days, followed by 7 days of recovery, to induce colitis. CBD supplementation reduced body weight loss, gross bleeding, fecal consistency, and disease activity index. In addition, CBD supplementation protected the colonic structure, promoted tissue recovery, and ameliorated macrophage infiltration in the colonic tissue, which was associated with the activation of cyclic AMP-protein kinase A, extracellular signal-regulated kinase ½, and AMP-activated protein kinase signaling pathways. CBD supplementation also suppressed NLRP3 inflammasome activation and related pro-inflammatory marker secretion. Consistently, CBD feeding reduced tight junction protein claudin2 and myosin light chain kinase in DSS-treated mice. Dietary CBD protects against inflammation and colitis symptoms induced by DSS, providing an alternative approach to IBD management.

The Cdc42/Rac1 pathway: a molecular mechanism behind iron-deficiency-driven aortic medial degeneration.

To elucidate the underlying mechanism of iron deficiency augmented Angiotensin II-induced aortic medial degeneration. ApoE-/- mice were randomly divided into four groups: normal control group (NC group), Angiotensin II (Ang II) subcutaneous pumped alone Group (Ang II group), iron deficiency (ID) group (ID group) and ID+Ang II group. The survival time, systolic blood pressure (SBP), and aortic medial degeneration (AMD) formation were monitored. Iron deposition in the aortas was assessed using Prussian blue iron staining. The expression of iron metabolism indicators, aortopathies and the cytoskeleton of vascular smooth muscle cells (VSMCs) were analyzed. In an in vitro setting, deferoxamine (DFO) was employed to mimic ID to examine the effects of Ang II on the cytoskeletal and contractile function of VSMCs during ID. Ras-related C3 botulinum toxin substrate 1 (Rac-1) expression was inhibited with EHT1864 to verify the role of Cdc42/Rac1 pathway in this pathological process. Blood samples were collected from 150 patients with aortic dissection (AD) and 60 patients with hypertension who were admitted to the Department of Cardiovascular Surgery at Renmin Hospital of Wuhan University between June 2018 and September 2019. The aortic tissues were obtained during the surgical treatment of Stanford type A AD patients and the heart donor. The iron metabolism status in plasma and aortic tissue was analyzed. In vivo experiments revealed that, in comparison to the NC and ID groups, mice in the Ang II and ID+Ang II groups exhibited increased SBP, significantly reduced survival time, and an expanded range of aortic dissection (P < 0.05). ID feeding augmented the Ang II-induced aortopathies. Both in vitro and in vivo results indicated that ID led to diminished expression of phosphorylated myosin light chain (p-MLC) and recombinant Cell Division Cycle Protein 42 (Cdc42) in VSMCs, while Rac-1 expression increased. The clinical sample testing data further confirmed the discovery that individuals diagnosed with AD display ID in both the plasma and the diseased aortas. The Cdc42/Rac1 pathway plays a crucial role in disrupting the cytoskeleton of vascular smooth muscle cells during iron deficiency, which leads to aortic medial degeneration both in vivo and in vitro.

Role of protein kinase D1 in vasoconstriction and haemodynamics in rats

AimsProtein kinase D (PKD), once considered an effector of protein kinase C (PKC), now plays many pathophysiological roles in various tissues. However, little is known about role of PKD in vascular function. We investigated the role of PKD in contraction of rat aorta and human aortic smooth muscle cells (HASMCs) and in haemodynamics in rats. Methods and resultsIsometric tension of rat aortic was measured to examine norepinephrine-induced contraction in the presence of PKD, PKC and Rho-kinase inhibitors. Phosphorylation of PKD1, myosin targeting subunit-1 (MYPT1), myosin light chain (MLC), CPI-17 and heat-shock protein 27 (HSP27), and actin polymerization were measured in the aorta. Phosphorylation of MYPT1 and MLC was also measured in HASMCs knocked down with specific siRNAs of PKD 1, 2 and 3. Intracellular calcium concentrations and cell shortening were measured in HASMCs.Norepinephrine-induced aortic contraction was accompanied by increased phosphorylation of PKD1, MYPT1 and MLC and actin polymerization, all of which were attenuated with PKD inhibitor CRT0066101. PKD1 phosphorylation was not inhibited by PKC inhibitor, chelerythrine or Rho kinase inhibitor, fasudil. In HASMCs, the phosphorylation of MYPT1 and MLC was attenuated by PKD1, but not PKD2, 3 knockdown. In HASMCs, CRT0066101 inhibited norepinephrine-induced cell shortening without affecting calcium concentration. Administration of CRT0066101 decreased systemic vascular resistance and blood pressure without affecting cardiac output in rats. ConclusionsPKD1 may play roles in aorta contraction and haemodynamics via phosphorylation of MYPT1 and actin polymerization in a calcium-independent manner.

Opportunities and Challenges for the Development of MRCK Kinases Inhibitors as Potential Cancer Chemotherapeutics.

Cytoskeleton organization and dynamics are rapidly regulated by post-translational modifications of key target proteins. Acting downstream of the Cdc42 GTPase, the myotonic dystrophy-related Cdc42-binding kinases MRCKα, MRCKβ, and MRCKγ have recently emerged as important players in cytoskeleton regulation through the phosphorylation of proteins such as the regulatory myosin light chain proteins. Compared with the closely related Rho-associated coiled-coil kinases 1 and 2 (ROCK1 and ROCK2), the contributions of the MRCK kinases are less well characterized, one reason for this being that the discovery of potent and selective MRCK pharmacological inhibitors occurred many years after the discovery of ROCK inhibitors. The disclosure of inhibitors, such as BDP5290 and BDP9066, that have marked selectivity for MRCK over ROCK, as well as the dual ROCK + MRCK inhibitor DJ4, has expanded the repertoire of chemical biology tools to study MRCK function in normal and pathological conditions. Recent research has used these novel inhibitors to establish the role of MRCK signalling in epithelial polarization, phagocytosis, cytoskeleton organization, cell motility, and cancer cell invasiveness. Furthermore, pharmacological MRCK inhibition has been shown to elicit therapeutically beneficial effects in cell-based and in vivo studies of glioma, skin, and ovarian cancers.

Impaired intestinal physical barrier of on-growing grass carp by deficient iron under Aeromonas hydrophila infection

This research discovered the impacts of dietary iron on intestine health of on-growing grass carp (Ctenopharyngodon idella). Six hundred and thirty grass carp (242.32 ± 0.58 g) were fed different diets containing 12.15, 35.38, 63.47, 86.43, 111.09, 136.37 and 73.50 mg iron/kg, where the iron source first six groups was ferrous fumarate, and the last group was ferrous sulfate. Feeding trial lasted for sixty days. Thereafter, a fourteen days' challenge test was carried out through Aeromonas hydrophila infection. Previous study showed that the optimum iron level of on-growing grass carp based on per weight gain was estimated to be 75.65 mg/kg. Results from the present study displayed that in contrast to optimum iron level, deficient iron downregulated gene expression of tight junction complexes, Apoptosis regulator Bcl-2 (Bcl-2), MCL1 apoptosis regulator BCL2 family member (Mcl-1), inhibitor of apoptosis protein (IAP) and NF-E2-related factor 2 (Nrf2). It also reduced activities and gene expression of antioxidant enzymes, and upregulated gene expression of apoptosis-related cysteine peptidase CASP (caspase) -2, -7, -8, -9, apoptotic protease activating factor 1 (Apaf-1), Apoptosis regulator Bax (Bax) (in MI and DI), Fas ligand (FasL), p38 mitogen activated protein kinase (p38MAPK), Kelch like ECH associated protein 1a (Keap1a), Keap1b, claudin-12 and myosin light chain kinase (MLCK). In addition, deficient iron raised malondialdehyde (MDA), protein carbonyl (PC) and reactive oxygen species (ROS) generation in intestine of grass carp (P < 0.05). These data demonstrated that deficient iron is harmful to intestinal physical barrier of fish. Besides, the efficiency of ferrous fumarate on intestinal physical barrier function was better than ferrous sulfate in fish. At last, based on MDA contents in MI and DI, the iron requirements were 83.40 and 80.20 mg/kg diet (ferrous fumarate as sources), respectively.

Formation of human cardiomyocytes is impaired by fibroblastic paracrine signalling: breaking down the mechanisms involved in rebuilding the heart

Abstract To date (pre-)clinical effects of inducing cardiac regeneration by cardiac cell therapy have been disappointing. This lack of success may result from the fact that it remains largely unclear how the receiving pathological microenvironment affects this process of cardiac differentiation of implanted cells, and thereby may (negatively) influence the therapeutic outcome. We have recently generated lines of conditionally immortalized human CMCs (ciCMCs) through doxycycline-dependent expression of SV40 large T antigen. In these cells, proliferation and differentiation can be tightly controlled. The aim of this study is to improve our understanding of cardiac differentiation of transplanted cells in the context of the receiving microenvironment from a paracrine perspective. To investigate the role of fibroblastic paracrine signalling on cardiomyogenic differentiation, transwell experiments were conducted. Cardiac differentiation was induced in human ciCMCs while cardiac fibroblasts were cultured in the top inserts in different ratios (human ciCMCs:cardiac fibroblasts 1:1, 2:1 and 3:1). Cardiomyogenic differentiation was determined by immunostaining for cardiac specific markers, quantitative RT-PCR, and electrophysiological analysis. After 12 days of differentiation, under the influence of the fibroblastic paracrine signaling, the amount of ciCMCs that expressed the sarcomeric protein α-actinin and myosin light chain 2a was significantly and increasingly reduced (P&amp;lt;0.01) in the groups cultured under the influence of the cardiac fibroblasts (4.8±0.6, 8.1±1.9, 22.4±4.2 for 1:1, 2:1 and 3:1 human ciCMCs:cardiac fibrolasts respectively [%, mean±SD]) while in absence of the cardiac fibroblasts (control group) 41% (±9.8) human ciCMCs were formed. In corroboration with these results, quantitative RT-PCR showed significant reduction (P&amp;lt;0.05) of all cardiac specific markers on a mRNA level in 1:1 human ciCMC: cardiac fibroblasts cultures compared to the control group. Electrophysiological analysis showed a significantly reduced (P&amp;lt;0.01) conduction velocity in the group cultured under the influence of the highest amount of cardiac fibroblasts compared to the control group (13.1±1.0 vs 10.6±0.9 control group vs 1:1 human ciCMCs:cardiac fibroblasts [cm/s, mean±SD]). Additionally, action potential duration was significantly prolonged (P&amp;lt;0.05) in the 1:1 human ciCMC:cardiac fibroblast cultures compared to the control group (93.6±4.8 vs 129.9±3.5 control group vs 1:1 human ciCMCs:cardiac fibroblasts [ms, mean±SD]). In conclusion, fibroblastic paracrine signalling has a negative effect on the structural and electrical differentiation of human cardiomyocytes. This not only emphasizes the need to consider the interaction between the transplanted cells and the receiving microenvironment in cardiac regenerative medicine, but also offers new leads to increase the therapeutic potential of this strategy. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Leiden University Fund

Curcumin Mitigates TNFα-Induced Caco-2 Cell Monolayer Permeabilization Through Modulation of NF-κB, ERK1/2, and JNK Pathways.

This work studies the capacity of curcumin to inhibit tumor necrosis alpha (TNFα)-induced inflammation, oxidative stress, and loss of intestinal barrier integrity, characterizing the underlying mechanisms. Caco-2 cell monolayers are incubated with TNFα (10ng mL-1 ), in the absence or presence of curcumin. TNFα causes an increase in interleukin (IL)-6 and IL-8 release, which is inhibited by curcumin in a dose-dependent manner (half-maximal inhibitory concentration (IC50 ) = 3.4 µM for IL-6). Moreover, TNFα leads to: i) increased intercellular adhesion molecule 1 (ICAM-1) and NLRP3 inflammasome expression; ii) increased cell monolayer permeability and decreased levels of tight junction proteins; iii) increased cellular and mitochondrial oxidant production; iv) decreased mitochondrial membrane potential and complex I-III activity; v) activation of redox-sensitive pathways, i.e., nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase 1/2 (ERK1/2), and c-Jun N-terminal kinases (JNK); and vi) increased myosin light-chain kinase (MLCK) expression and phosphorylation levels of myosin light-chain protein MLC. Curcumin (2-8 µM) inhibits all these TNFα-triggered undesirable outcomes, mostly showing dose-dependent effects. The inhibition of NF-κB, ERK1/2, and JNK activation could be in part involved in the capacity of curcumin to mitigate intestinal inflammation, oxidant production, activation of redox-sensitive pathways, and prevention of monolayer permeabilization. These results support an action of dietary curcumin in sustaining gastrointestinal tract physiology.

Major shrimp allergen peptidomics signatures and potential biomarkers of heat processing

It is important to develop tools that can be used to understand the effects of processing on allergenic foods in order to achieve personalized food labeling. To evaluate the effect of heating on the allergy-relevant structural properties of tropomyosin (TM), arginine kinase (AK), myosin light chain (MLC) and sarcoplasmic calcium-binding protein (SCP) shrimp allergens, trypsin digests of raw, fried and baked shrimp extracts were analyzed by peptidomics and epitope correlations. Processing altered the number of peptides released from the distinct allergens, and each treatment generated a specific epitope-matched peptide allergen fingerprint. Among the four allergens, TM led to a number of released peptides and epitope changes being detected, and AK provided the epitope-matched 331MGLTEFQAVK340 sequence as a common differentiating peptide for heat processing. These results provide new views on the structural effects of processing on major shrimp allergens and peptide candidates as processing biomarkers.

Abstract 14286: Crosstalk Between Methylglyoxal and Acetylation Modifications on Actin, Myosin, and Myosin Light Chain Proteins Affects Sarcomere Functionality

Diabetes affects ten percent of Americans. Risk of developing heart failure in these patients is doubled, independent of coronary artery disease and hypertension. However, the mechanistic connection between these two diseases is poorly understood. We have shown one connection is likely due to methylglyoxal (MGO), a highly reactive glycolysis product that can irreversibly modify lysine and arginine residues, a process called glycation. Moreover, we found MGO glycation of the myofilament decreased both calcium sensitivity and maximum calcium activated force (Fmax). Here, we hypothesized that MGO may also negatively impact function by competing for lysine residues that would otherwise be targets for acetylation, which has been shown to be beneficial to cardiac function. Using non-failing human left ventricular (LV) myocardial tissue that had been exposed to acute treatment of MGO (500 μM) or vehicle and subsequently treated with a high dose of acetic anhydride in acetonitrile (300μM) overnight, we found that compared to the vehicle treated samples, MGO inhibited overall acetylation, suggesting that these two PTMs do compete for a subset of lysine residues in the heart. Next, we compared mass spectrometry data sets from diabetic humans, MGO treated mouse myocytes, and acetylated myocytes to identify the specific overlapping residues that acetylation and glycation compete for. This analysis identified multiple sites of crosstalk on actin, myosin, and myosin light chains - proteins essential for normal contractile function. Our preliminary data support a contractile consequence to this crosstalk. Incubation of mouse LV isolated skinned myocytes with acetic anhydride in acetonitrile (300μM, overnight) increased both calcium sensitivity and Fmax. However, exposure to MGO first (500μM) blocked these beneficial functions acetylation (300μM, overnight) in mouse myocytes. Together, we believe these data demonstrate that glycation and acetylation do compete for residues in the myofilament, and that glycation is capable of blocking the beneficial functional effects of protein acetylation. If these results are confirmed and extended in vivo, they could have important consequences in diabetes and in the utility of using HDAC inhibitors clinically.

Abstract MP17: Neuropilin-1 Is A Novel Regulator Of Vascular Tone And Blood Pressure

Introduction: Neuropilin-1 (NRP1) is a transmembrane receptor present in vascular smooth muscle cells (VSMC) that mediates the inhibition of Rho signaling by binding the Class 3 Semaphorin (SEMA) ligand SEMA3A. Hypothesis: We hypothesize that loss of NRP1 in VSMC mitigates SEMA3A-induced Rho inhibition, thereby increasing vascular tone and blood pressure in vivo . Methods: Male and female adult mice (8-12 weeks) with inducible, smooth muscle cell-specific deletion of NRP1 (SM22a-Cre ERT2 X Nrp1 flox/flox ) were examined. Following recombination using 4-hydroxy tamoxifen (SM- NRP1 KO), systolic blood pressure (SBP) was measured using a tail cuff and compared to age- and sex-matched mice that did not receive tamoxifen (control). Aortic vascular reactivity and expression of key proteins in the Rho signaling cascade were measured using ex vivo tension myography and western blotting, respectively. Results: SBP was significantly increased in SM- NRP1 KO mice following recombination compared to control mice (SBP: 136.5 ± 10.9 vs 112.9 ± 5.6 mmHg; p=0.0006). Contractile responses in aortas of SM- NRP1 KO mice to phenylephrine (p=0.025), KCl (p=0.012), and the thromboxane agonist U44619 (p=0.019) were significantly enhanced compared to controls. Expression of total myosin light chain and LIMK-2 proteins were increased in SM- NRP1 KO compared to control aortas. In vitro , treatment of murine primary VSMC expressing NRP1 with SEMA3A decreased angiotensin II-induced Rho-GTP activation. Additionally, control and SM- NRP1 KO mice (starting at 2 weeks post-recombination) were administered angiotensin II (490 ng/kg/day) for 4 weeks. While there was no significant difference in SBP at weeks 1 and 2, SM- NRP1 KO mice had significantly lower SBP at weeks 3 and 4 following angiotensin II infusion compared to controls (Week 4 SBP: 150 ± 1.4 vs 130.5 ± 2.5 mmHg; p=0.02), suggesting a low ejection fraction and cardiac dysfunction in these mice. In support of this observation, mRNA expression of atrial natriuretic peptide was increased in hearts of angiotensin II-infused SM- NRP1 KO mice. Conclusion: Our data suggest that VSMC NRP1 regulates basal tone and blood pressure, and that loss of NRP1 causes hypertension and exacerbates cardiac dysfunction.

Effect of acupoint thread-embedding on tight junction of intestinal mucosa epithelium in rats with ulcerative colitis

To observe the effect of acupoint thread-embedding on tight junction of intestinal mucosal epithelial barrier in rats with ulcerative colitis (UC) under the state of "deficiency and stasis", and to explore its mechanism. Sixty male SD rats were randomly divided into a control group (n=12) and a UC model group (n=48). The rats were gavaged with adenine and folium sennae by stages to prepare the "deficiency and stasis" state, and then 5% 2,4,6-trinitrobenzene sulfonic acid (TNBS, 100 mg/kg) and 0.25 mL 50% ethanol were used to induce UC model. Thirty rats with successful model establishment were selected and randomly divided into a model group, a medication group and a thread-embedding group, 10 rats in each group. The rats in the thread-embedding group were intervened with thread-embedding at "Zusanli" (ST 36), "Tianshu" (ST 25), "Geshu" (BL 17), "Pishu" (BL 20), "Shenshu" (BL 23) and "Dachangshu" (BL 25), once every 14 days, totaling 3 times. The rats in the medication group were gavaged with sulfasalazine, once a day. The rats in the control group, model group and thread-embedding group were gavaged with the same amount of 0.9% sodium chloride solution at the same time for 42 days. The general condition and body weight of rats in each group were observed. The appearance and morphological changes of colonic tissue were observed by naked eye and HE staining. The serum levels of calmodulin-dependent protein kinaseⅡ(CaMKⅡ) and myosin light chain kinase (MLCK) were detected by ELISA. The mRNA levels of colonic tight junction (TJ) proteins including occludin, claudin1, claudin2 and ZO-1were detected by quantitative real-time PCR. The protein expressions of occludin, claudin1, claudin2, ZO-1 and CaMKⅡ, MLCK were detected by Western blot. Compared with the control group, in the model group the body weight was decreased (P<0.01), colon tissue was significantly swelled with bleeding and a large number of inflammatory cells infiltration, and the colon tissues damage index score was increased (P<0.01), serum CaMKⅡ and MLCK levels were increased (P<0.01), and the mRNA and protein expression levels of occludin, claudin1 and ZO-1 in colon tissue were decreased (P<0.01), the mRNA and protein expression levels of claudin2 were increased (P<0.01), and the protein expression levels of CaMKⅡ and MLCK were increased (P<0.01). Compared with the model group, in the medication group and thread-embedding group the weight was increased (P<0.01), the colonic lesions were significantly alleviated, the tissues damage index scores were decreased (P<0.01), the serum CaMKⅡ and MLCK contents were decreased (P<0.01), the mRNA and protein expression levels of occludin, claudin1 and ZO-1 in colonic tissue were increased (P<0.01, P<0.05), the mRNA and protein expression levels of claudin2 were decreased (P<0.01), and the protein expression levels of CaMKⅡ and MLCK were decreased (P<0.01, P<0.05). There was no significant difference of each index between the medication group and thread-embedding group (P>0.05). The thread-embedding could repair the tight junction of intestinal mucosa epithelium and reduce the permeability of intestinal mucosa epithelium, which may be related to the decrease of the expression of CaMKⅡ, MLCK and other protein kinases.

Motor Unit Force Potentiation and Calcium Handling Protein Concentration in Rat Fast Muscle After Resistance Training.

Post-tetanic potentiation (PTP) of force depends on intramuscular Ca2+ levels and sensitivity and may be affected by fatigue. The aim of this study was to determine the ability of isolated fast fatigue-resistant (FR) and fast-fatigable (FF) motor units (MUs) to potentiate force evoked with single and 40-Hz electrical stimulation after 5 weeks of voluntary weight-lifting training. Tetanic contractions evoked by gradually increasing (10–150 Hz) stimulation frequency served as conditioning stimulation. Additionally, the concentration of myosin light chain kinase and proteins engaged in calcium handling was measured in rat fast medial gastrocnemius muscle. After the training, the potentiation of twitch force and peak rate of force development was increased in FF but not FR MUs. Force potentiation of 40-Hz tetanic contractions was increased in both fast MU types. After the training, the twitch duration of FR MUs was decreased, and FF MUs were less prone to high-frequency fatigue during conditioning stimulation. Muscle concentration of triadin was increased, whereas concentrations of ryanodine receptor 1, junctin, FKBP12, sarcoplasmic reticulum calcium ATPase 1, parvalbumin, myosin light chain kinase, and actomyosin adenosine triphosphatase content were not modified. After short-term resistance training, the twitch contraction time and twitch:tetanus force ratio of FR MUs are decreased, and PTP ability is not changed. However, PTP capacity is increased in response to submaximal activation. In FF MUs increase in PTP ability coexists with lesser fatigability. Further work is required to find out if the increase in triadin concentration has any impact on the observed contractile response.

Cartiotonic steroids affect monolayer permeability in lymphatic endothelial cells.

Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100nM. Some LECs were pretreated with 1μM L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100nM MBG or cinobufotalin (CINO). Expression of β-catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure β-catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (≥ 1nM) and CINO (≥ 10nM) caused an increase (p < 0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1μM L-NAME attenuated (p < 0.05) the MPLEC. The β-catenin expression in LECs was downregulated (p < 0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p < 0.05) by CINO, and MLC20 phosphorylation was upregulated (p < 0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.

Activating Sphingosine-1-phospahte signaling in endothelial cells increases myosin light chain phosphorylation to decrease endothelial permeability thereby inhibiting cancer metastasis.

Targeting the metastatic process to prevent disease dissemination in cancer remains challenging. One step in the metastatic cascade involves cancer cells transiting through the vascular endothelium after inflammation has increased the permeability of this cellular layer. Reducing inflammation-mediated gaps in the vascular endothelium could potentially be used to retard metastasis. This study describes the development of a novel ASR396-containing nanoparticle designed to activate the Sphingosine-1-Phosphate Receptor 1 (S1PR1) in order to tighten the junctions between the endothelial cells lining the vascular endothelium thereby inhibiting metastasis. ASR396 was derived from the S1PR1 agonist SEW2871 through chemical modification enabling the new compound to be loaded into a nanoliposome. ASR396 retained S1PR1 binding activity and the nanoliposomal formulation (nanoASR396) made it systemically bioavailable upon intravenous injection. Studies conducted in microvessels demonstrated that nanoASR396 significantly attenuated inflammatory mediator-induced permeability increase through the S1PR1 activation. Similarly, nanoASR396 inhibited gap formation mediated by inflammatory agents on an endothelial cell monolayer by decreasing levels of phosphorylated myosin light chain protein thereby inhibiting cellular contractility. In animal models, nanoASR396 inhibited lung metastasis by up to 80%, indicating its potential for retarding melanoma metastasis. Thus, a novel bioavailable nanoparticle-based S1PR1 agonist has been developed to negate the effects of inflammatory mediators on the vascular endothelium in order to reduce the metastatic dissemination of cancer cells.

High concentrations of H7 human embryonic stem cells at the point of care for acute myocardial infarction.

BackgroundEmbryonic stem cell (ESC)-derived cardiomyocytes have become one of the most attractive sources of cellular therapy for minimizing heart tissue damage following myocardial infarction (MI). In this study, we investigated the differentiation of BMS-189453-induced H7 human ESCs (hESCs) and purified ESCs in the treatment of induced acute MI.MethodsBMS-189453 was used to induce the differentiation of H7 hESCs into myocardial ESCs. We further purified ESCs cells. The expression levels of the myocardial-specific protein cardiac troponin T (cTnT) and the ventricular-specific protein Myosin Light Chain 2 (MLC-2V) were detected by western blot. Quantitative reverse transcription-polymerase chain reaction (QRT-PCR) was used to detect the expression of iroquois homeobox 4 (IRX4), an important transcription factor related to ventricular muscle development. Ultrasound, radionuclides, and Holter monitoring were used to evaluate the therapeutic effect of ESCs on acute MI induced in pigs.ResultsCompared with untreated myocardial tissue, myocardial ESCs and purified ESCs improved the outcome in pigs with MI. Treatment with non-purified ESCs and purified ESCs improved the myocardial perfusion grade and ventricular wall motion score index, increased the viable myocardial ratio (VMR), improved the ejection fraction and left ventricular end-diastolic diameter, and reduced the MI area. Further, compared with non-purified ESCs, purified ESCs resulted in fewer side effects and reduced the incidence of ventricular arrhythmias.ConclusionsIn the pig model of acute MI, treatment with ESCs significantly improved myocardial function, increased myocardial mass, and reduced scar tissue formation. Purified ESCs have a better treatment effect than non-purified ESCs and can reduce the incidence of ventricular arrhythmias. This study has unearthed new prospects for the clinical treatment of MI.

Role of protein kinase D1 in pulmonary hypertension in rats

Protein kinase D (PKD) is known to be implicated in a variety of physiological functions and pathological conditions including cardiovascular diseases, such as cardiac hypertrophy and ischemia reperfusion injury. PKD has been thought to be a new target of the diseases for treatments. Previously, we showed that PKD played a role in pulmonary arterial contraction through MYPT1 phosphorylation and actin polymerization and that inhibition of PKD activity decreased right cardiac ventricular pressure in monocrotaline‐induced pulmonary hypertension (PH) model,. In this study, we further investigated a precise role of PKD in PH and in human pulmonary smooth muscle cells (HPSMCs).Material and methodsEight‐week‐old male rats were given either 60 mg/kg MCT or saline 4 weeks before experiments. Isometric tension was measured using pulmonary arterial ring samples to examine the effect of PKD inhibitor, CRT0066101 on norepinephrine (NE)‐induced contraction and phosphorylation of PKD, myosin phosphatase target subunit 1 (MYPT1), regulatory myosin light chain (RLC) and other PKD substrate proteins. Expression of PKD and phosphorylated‐PKD in rats lungs were also examined with immunohistochemistry using anti‐phospho‐PKD antibodies. We also investigated precise roles of PKD isoforms using HPSMCs with RNA interference.ResultsIn pulmonary arterial rings, basal expression of PKD1 and phosphorylated PKD were increased in pulmonary arteries in PH rats. NE increased phosphorylation of PKD, MYPT1 and RLC in ring samples during contraction, which were attenuated by pretreatment of CRT0066101. In immunohistochemistry, increased expression of PKD1 was observed in pulmonary arteries in PH rats (Fig 1). In HASMCs, increased phosphorylation of MYPT1 and RLC with NE were significantly inhibited by PKD1 but not PKD2, 3 knock down.ConclusionsPKD1 may play role in contraction of pulmonary arteries and at least partially via MYPT1 phosphorylation. Increased PKD1 could be involved in PH development in rats.Support or Funding InformationGrant‐in‐Aid for Scientific Research (C) 17K11058Immunohistochemistry with anti‐PKD antibody in lung tissue from PH rats.Figure 1

Mechanism of α1-adrenergic receptor-induced increased contraction of rat mesenteric artery in aging and hypertension

When α1 adrenergic receptor (α1-AR) binds to the agonist, the G protein-coupled receptor signal is activated, leading to vasoconstriction, a process associated with increased intracellular calcium concentration and activation of the myosin light chain kinase (MLCK) and protein kinase C (PKC)-mediated signaling pathways. In our previous experiments, α1-AR activator phenylephrine(PE) resulted in a significant increase in maximal contraction of the mesenteric artery in elderly rats. However, the expression of α1-AR decreased in these tissues. Therefore, the purpose of this study is to further elucidate the changing mechanism of α1-AR activation leading to vasoconstriction in aging and hypertension.