Expression Of Myosin Light Chain Research Articles

The Reduction of PSMB4 in T24 and J82 Bladder Cancer Cells Inhibits the Angiogenesis and Migration of Endothelial Cells.

Bladder cancer (BC) is a malignant tumor of the urinary system with high mortality and recurrence rates. Proteasome subunit type 4 (PSMB4) is highly expressed and has been identified as having oncogenic properties in a variety of cancer types. This study aimed to explore the effect of PSMB4 knockdown on the survival, migration, and angiogenesis of human bladder cancer cells with different degrees of malignancy. We analyzed the effects of PSMB4 knockdown in bladder cancer cells and endothelial cells in the tumor microenvironment. PSMB4 was highly expressed in patients with low- and high-grade urothelial carcinoma. Inhibition of PSMB4 reduced protein expression of focal adhesion kinase (FAK) and myosin light chain (MLC), leading to reduced migration. Furthermore, the suppression of PSMB4 decreased the levels of vascular endothelial factor B (VEGF-B), resulting in lower angiogenic abilities in human bladder cancer cells. PSMB4 inhibition affected the migratory ability of HUVECs and reduced VEGFR2 expression, consequently downregulating angiogenesis. In the metastatic animal model, PSMB4 knockdown reduced the relative volumes of lung tumors. Our findings suggest the role of PSMB4 as a potential target for therapeutic strategies against human bladder cancer.

Cardioprotection by the adiponectin receptor agonist ALY688 in a preclinical mouse model of heart failure with reduced ejection fraction (HFrEF)

AimsAdiponectin has been shown to mediate cardioprotective effects and levels are typically reduced in patients with cardiometabolic disease. Hence, there has been intense interest in developing adiponectin-based therapeutics. The aim of this translational research study was to examine the functional significance of targeting adiponectin signaling with the adiponectin receptor agonist ALY688 in a mouse model of heart failure with reduced ejection fraction (HFrEF), and the mechanisms of cardiac remodeling leading to cardioprotection. Methods and resultsWild-type mice were subjected to transverse aortic constriction (TAC) to induce left ventricular pressure overload (PO), or sham surgery, with or without daily subcutaneous ALY688-SR administration. Temporal analysis of cardiac function was conducted via weekly echocardiography for 5 weeks and we observed that ALY688 attenuated the PO-induced dysfunction. ALY688 also reduced cardiac hypertrophic remodeling, assessed via LV mass, heart weight to body weight ratio, cardiomyocyte cross sectional area, ANP and BNP levels. ALY688 also attenuated PO-induced changes in myosin light and heavy chain expression. Collagen content and myofibroblast profile indicated that fibrosis was attenuated by ALY688 with TIMP1 and scleraxis/periostin identified as potential mechanistic contributors. ALY688 reduced PO-induced elevation in circulating cytokines including IL-5, IL-13 and IL-17, and the chemoattractants MCP-1, MIP-1β, MIP-1alpha and MIP-3α. Assessment of myocardial transcript levels indicated that ALY688 suppressed PO-induced elevations in IL-6, TLR-4 and IL-1β, collectively indicating anti-inflammatory effects. Targeted metabolomic profiling indicated that ALY688 increased fatty acid mobilization and oxidation, increased betaine and putrescine plus decreased sphingomyelin and lysophospholipids, a profile indicative of improved insulin sensitivity. ConclusionThese results indicate that the adiponectin mimetic peptide ALY688 reduced PO-induced fibrosis, hypertrophy, inflammation and metabolic dysfunction and represents a promising therapeutic approach for treating HFrEF in a clinical setting.

Network pharmacology combined with an animal model to reveal the material basis and mechanism of Amomum villosum in alleviating constipation in mice

Constipation is a prevalent gastrointestinal disorder, with its prevalence showing an annual upward trend. There are many factors involved in the occurrence of constipation, such as abnormal smooth muscle contraction and disorders of gastrointestinal hormone secretion. Amomum villosum (A. villosum) has been proven to be effective in improving digestive system diseases, but there is no report on improving constipation. Therefore, we used network pharmacology prediction combined with animal experiments to explore the key active components of A. villosum and their pharmacological mechanisms. The results of network pharmacological prediction showed that β-sitosterol was the key laxative compound of A. villosum, which may play a laxative role by activating the adrenoceptor alpha 1 A-myosin light chain (ADRA1A-MLC) pathway. Further animal experiments showed that β-sitosterol could significantly shorten the time to first black stool; increase faecal weight, faecal number, and faecal water content; and promote gastrointestinal motility. β-sitosterol may promote intestinal motility by upregulating the expression of ADRA1A and myosin light chain 9 (Myl9) mRNA and protein in the colon, thereby activating the ADRA1A-MLC signalling pathway. In addition, it is possible to improve constipation symptoms by regulating serum neurotransmitters and gastrointestinal motility-related factors, such as the serum content of 5-hydroxytryptamine (5-HT) and acetylcholinesterase (AchE) and the mRNA expression of 5-hydroxytryptamine receptor 4 (5-HT4), stem cell factor (SCF), stem cell factor receptor (c-Kit) and smooth muscle myosin light chain kinase (smMLCK) in the colon. These results lay a foundation for the application of A. villosum and β-sitosterol in constipation.

MYL9 promotes squamous cervical cancer migration and invasion by enhancing aerobic glycolysis.

This study explored the mechanism of squamous cervical cancer (SCC) progression. Reverse transcription-quantitative polymerase chain reaction and western blotting were used to evaluate the expression of myosin light chain 9 (MYL9) in SCC tissues and cell lines. Furthermore, Transwell and Boyden assays were used to assess the function of MYL9 in SCC progression. In addition, the levels of lactate and aerobic glycolysis were used to explore the detailed mechanism of MYL9 in SCC. The mRNA and protein levels of MYL9 were elevated in SCC tissues, and MYL9 knockdown inhibited the migration and invasion of SCC cell lines. A mechanistic study demonstrated that MYL9 promotes SCC migration and invasion by enhancing aerobic glycolysis and increasing the activity of the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. MYL9 was upregulated in SCC, and it enhanced JAK2/STAT3 pathway activity and promoted metastasis and glycolysis in SCC.

Abstract P1025: Hsp70 Co-chaperones Regulate Sarcomeric Proteostasis In Hypertrophic Cardiomyopathy

Significance: Recent Genome Wide Association Studies (GWAS) identified non-sarcomeric variants in hypertrophic cardiomyopathy (HCM) with three of the top 10 variant loci in or near genes that encode for co-chaperones of HSP70: BAG3, DNAJC18 and HSPB7. These variants are also present in dilated cardiomyopathy (DCM), but have an opposing effect on cardiac function, leading us to hypothesize that GWAS variants in HSP70 co-chaperones can affect the proteostasis of contractile proteins in the HCM heart. As a first step, we determined the effects each HSP70 co-chaperone knockdown (KD) had on sarcomeric and z-disc protein expression and the poly-ubiquitinated state of cardiomyocyte-specific proteins. Methods and Results: Transduction of human iPSC-CMs (iPSC-CMs) with adenoviral vectors driving shRNA expression against BAG3, DNAJC18, HSPB7, or scramble (control) yielded transduction efficiencies > 75% and >80% reduction in target transcript 4 days post-transduction. BAG3 KD increased poly-ubiquitinated proteins by 38+/-9% (p<0.001) and reduced steady state expression of sarcomeric and z-disc proteins, including: MyBP-C (p<0.0001), actin (p<0.0001), cardiac troponin I (p<0.05), tropomyosin (TPM1) (p<0.01), myosin light chain 3 (MYL3) (p<0.0001), filamin C (p<0.05), and alpha-actinin-2 (p<0.0001). DNAJC18 KD increased poly-ubiquitinated protein by 66+/-17% (p<0.01) but did not impact sarcomere protein expression. Conversely, HSPB7 KD reduced poly-ubiquitinated proteins by 21+/-7% (p<0.01) and increased protein expression of MyBP-C (p<0.01), TPM1 (p<0.05), and myosin light chain 2 (p<0.05). DNAJC18 KD increased BAG3 and HSPB7 protein expression levels by ~2-fold (p<0.001) without increasing transcript levels, suggesting BAG3 and HSPB7 may be protein clients of DNAJC18. Conclusions: HSP70 co-chaperones that have an established genetic association with human HCM play an important role in cardiomyocyte proteostasis. Multiple sarcomeric and z-disc proteins appear to be clients of BAG3, while BAG3 and HSPB7 themselves may be clients of DNAJC18. Targeting this axis could be therapeutic for patients with HCM that possess genetic variants in sarcomeric proteins.

Histone Modification of Osteogenesis Related Genes Triggered by Substrate Topography Promotes Human Mesenchymal Stem Cell Differentiation.

The clinical success of orthopedic implants is closely related to their integration in the bone tissue promoted by rough device surfaces. The biological response of precursor cells to their artificial microenvironments plays a critical role in this process. In this study, we elucidated the relation between cell instructivity and surface microstructure of polycarbonate (PC)-based model substrates. The rough surface structure (hPC) with an average peak spacing (Sm) similar to the trabecular spacing of trabecular bone improved osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs), as compared to the smooth surface (sPC) and the surface with a moderate Sm value (mPC). The hPC substrate promoted the cell adhesion and assembling of F-actin and enhanced cell contractile force by upregulating phosphorylated myosin light chain (pMLC) expression. The increased cell contractile force led to YAP nuclear translocation and the elongation of cell nuclei, presenting higher levels of active form of Lamin A/C. The nuclear deformation alternated the histone modification profile, particularly the decrease of H3K27me3 and increase of H3K9ac on the promoter region of osteogenesis related genes (ALPL, RUNX2, and OCN). Mechanism study using inhibitors and siRNAs elucidated the role of YAP, integrin, F-actin, myosin, and nuclear membrane proteins in such a regulatory process of surface topography on stem cell fate. These mechanistical insights on the epigenetic level give a new perspective in understanding of the interaction of substrate and stem cells as well as provide valuable criteria for designing bioinstructive orthopedic implants.

The Use of Collagen Methacrylate in Actuating Polyethylene Glycol Diacrylate-Acrylic Acid Scaffolds for Muscle Regeneration.

After muscle loss or injury, skeletal muscle tissue has the ability to regenerate and return its function. However, large volume defects in skeletal muscle tissue pose a challenge to regenerate due to the absence of regenerative elements such as biophysical and biochemical cues, making the development of new treatments necessary. One potential solution is to utilize electroactive polymers that can change size or shape in response to an external electric field. Poly(ethylene glycol) diacrylate (PEGDA) is one such polymer, which holds great potential as a scaffold for muscle tissue regeneration due to its mechanical properties. In addition, the versatile chemistry of this polymer allows for the conjugation of new functional groups to enhance its electroactive properties and biocompatibility. Herein, we have developed an electroactive copolymer of PEGDA and acrylic acid (AA) in combination with collagen methacrylate (CMA) to promote cell adhesion and proliferation. The electroactive properties of the CMA + PEGDA:AA constructs were investigated through actuation studies. Furthermore, the biological properties of the hydrogel were investigated in a 14-day in vitro study to evaluate myosin light chain (MLC) expression and metabolic activity of C2C12 mouse myoblast cells. The addition of CMA improved some aspects of material bioactivity, such as MLC expression in C2C12 mouse myoblast cells. However, the incorporation of CMA in the PEGDA:AA hydrogels reduced the sample movement when placed under an electric field, possibly due to steric hindrance from the CMA. Further research is needed to optimize the use of CMA in combination with PEGDA:AA as a potential scaffold for skeletal muscle tissue engineering.

Identification and validation of critical genes with prognostic value in gastric cancer.

Background: Gastric cancer (GC) is a digestive system tumor with high morbidity and mortality rates. Molecular targeted therapies, including those targeting human epidermal factor receptor 2 (HER2), have proven to be effective in clinical treatment. However, better identification and description of tumor-promoting genes in GC is still necessary for antitumor therapy. Methods: Gene expression and clinical data of GC patients were downloaded from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. Last absolute shrinkage and selection operator (LASSO) Cox regression were applied to build a prognostic model, the Prognosis Score. Functional enrichment and single-sample gene set enrichment analysis (ssGSEA) were used to explore potential mechanisms. Western blotting, RNA interference, cell migration, and wound healing assays were used to detect the expression and function of myosin light chain 9 (MYL9) in GC. Results: A four-gene prognostic model was constructed and GC patients from TCGA and meta-GEO cohorts were stratified into high-prognosis score groups or low-prognosis score groups. GC patients in the high-prognosis score group had significantly poorer overall survival (OS) than those in the low-prognosis score groups. The GC prognostic model was formulated as PrognosisScore = (0.06 × expression of BGN) - (0.008 × expression of ATP4A) + (0.12 × expression of MYL9) - (0.01 × expression of ALDH3A1). The prognosis score was identified as an independent predictor of OS. High expression of MYL9, the highest weighted gene in the prognosis score, was correlated with worse clinical outcomes. Functional analysis revealed that MYL9 is mainly associated with the biological function of epithelial-mesenchymal transition (EMT). Knockdown of MYL9 expression inhibits migration of GCcells in vitro. Conclusion: We found that PrognosisScore is potential reliable prognostic marker and verified that MYL9 promotes the migration and metastasis of GC cells.

Single-cell transcriptomics provides insights into hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic heart disease that is characterized by unexplained segmental hypertrophy that is usually most pronounced in the septum. While sarcomeric gene mutations are often the genetic basis for HCM, the mechanistic origin for the heterogeneous remodeling remains largely unknown. A better understanding of the gene networks driving the cardiomyocyte (CM) hypertrophy is required to improve therapeutic strategies. Patients suffering from HCM often receive a septal myectomy surgery to relieve outflow tract obstruction due to hypertrophy. Using single-cell RNA sequencing (scRNA-seq) on septal myectomy samples from patients with HCM, we identify functional links between genes, transcription factors, and cell size relevant for HCM. The data show the utility of using scRNA-seq on the human hypertrophic heart, highlight CM heterogeneity, and provide a wealth of insights into molecular events involved in HCM that can eventually contribute to the development of enhanced therapies.

Bridging the muscle genome to phenome across multiple biological scales.

Muscle is highly hierarchically organized, with functions shaped by genetically controlled expression of protein ensembles with different isoform profiles at the sarcomere scale. However, it remains unclear how isoform profiles shape whole-muscle performance. We compared two mouse hindlimb muscles, the slow, relatively parallel-fibered soleus and the faster, more pennate-fibered tibialis anterior (TA), across scales: from gene regulation, isoform expression and translation speed, to force-length-velocity-power for intact muscles. Expression of myosin heavy-chain (MHC) isoforms directly corresponded with contraction velocity. The fast-twitch TA with fast MHC isoforms had faster unloaded velocities (actin sliding velocity, Vactin; peak fiber velocity, Vmax) than the slow-twitch soleus. For the soleus, Vactin was biased towards Vactin for purely slow MHC I, despite this muscle's even fast and slow MHC isoform composition. Our multi-scale results clearly identified a consistent and significant dampening in fiber shortening velocities for both muscles, underscoring an indirect correlation between Vactin and fiber Vmax that may be influenced by differences in fiber architecture, along with internal loading due to both passive and active effects. These influences correlate with the increased peak force and power in the slightly more pennate TA, leading to a broader length range of near-optimal force production. Conversely, a greater force-velocity curvature in the near-parallel fibered soleus highlights the fine-tuning by molecular-scale influences including myosin heavy and light chain expression along with whole-muscle characteristics. Our results demonstrate that the individual gene, protein and whole-fiber characteristics do not directly reflect overall muscle performance but that intricate fine-tuning across scales shapes specialized muscle function.

Effect of Rho-Kinase and Autophagy on Remote Ischemic Conditioning-Induced Cardioprotection in Rat Myocardial Ischemia/Reperfusion Injury Model.

Objective Remote ischemic conditioning (RIC) is a cardioprotective method in ischemia/reperfusion (I/R) injury. This study investigated the mechanism of Rho-kinase-mediated autophagy in RIC. Methods Sixty male Sprague–Dawley rats were randomly divided into six groups: sham, I/R, RIC, I/R+fasudil, RIC+wortmannin, and RIC+fasudil+wortmannin. Throughout the experiment, mean arterial pressure and heart rate were continuously monitored. Histopathology and ultrastructure and myocardial enzymes' expression were evaluated to determine the degree of cardiac injury. The protein expression of the Rho-kinase substrates myosin light chain (MLC) and myosin phosphatase target subunit 1 (MYPT1), autophagy-related protein light chain 3-II (LC3-II) and Beclin 1, and protein kinase B (AKT) was measured in the myocardial tissue. Results Compared with the sham group, the mean arterial pressure and heart rate were decreased, myocardial enzyme levels were increased, and myocardial damage was aggravated in the I/R group; however, RIC improved these alterations. The expression of phosphorylated MLC and MYPT1 was lower, while LC3-II, Beclin 1, and phospho-AKT expression levels were higher in the RIC group compared with the I/R group. Obviously, treatment of the I/R group rats with fasudil, a Rho-kinase inhibitor, significantly ameliorated the I/R effects, whereas treatment of the RIC group rats with wortmannin, a phosphatidylinositol-3 kinase (PI3K) inhibitor, inhibited the RIC protective effects. Moreover, the rats in the RIC+fasudil+wortmannin group showed similar changes to those in the RIC+wortmannin group. Conclusion These results showed that RIC protected the myocardium from I/R injury by suppressing Rho-kinase and the underlying mechanism may be related to enhancing autophagy via the PI3K/AKT pathway.

Myosin light chain 9 promotes the proliferation, invasion, migration and angiogenesis of colorectal cancer cells by binding to Yes-associated protein 1 and regulating Hippo signaling

ABSTRACT Colorectal cancer is a common type of cancer with high incidence and poor prognosis. Increased expression of myosin light chain 9 (MYL9) has been reported in early-stage and recurrent colorectal cancer tissues. This study aimed to investigate the precise role of MYL9 on the progression of colorectal cancer. MYL9 expression in several colorectal cancer cell lines was detected by Western blotting and RT-qPCR. Following MYL9 overexpression or knockdown, MYL9 expression was determined via RT-qPCR. Cell proliferation was detected with Cell Counting Kit-8 assay. Cell invasion, migration and angiogenesis were, respectively, examined with transwell, wound healing and tube formation assays. The binding between MYL9 and Yes-associated protein 1 (YAP1) was verified by a co-immunoprecipitation assay. The expression of YAP1, connective tissue growth factor and cysteine-rich angiogenic inducer 61 was examined by Western blotting. Subsequently, YAP1 silencing or Hippo antagonist was performed to clarify the regulatory mechanisms of MYL9 in colorectal cancer progression. Experimental results showed that MYL9 expression was elevated in colorectal cancer cell lines. MYL9 overexpression promoted cell proliferation, invasion, migration and angiogenesis, while silencing of MYL9 exerted the opposite effects. Results of co-immunoprecipitation assay indicated that MYL9 could bind to YAP1. Further experiments revealed that MYL9 affected the expression of YAP1 and its downstream signaling proteins. Afterward, YAP1 knockdown or the addition of Hippo antagonist inhibited the proliferation, invasion, migration and angiogenesis of colorectal cancer cells. Overall, MYL9 promotes the proliferation, invasion, migration and angiogenesis of colorectal cancer cells by binding to YAP1 and thereby activating Hippo signaling.

Nitric oxide could promote development of Barrett's esophagus by S-nitrosylation-induced inhibition of Rho-ROCK signaling in esophageal fibroblasts.

Barrett's esophagus arises in the process of wound healing in distal esophageal epithelium damaged by gastroesophageal reflux disease. Differentiation of fibroblast into myofibroblasts, a smooth muscle cell-like phenotype and tissue contraction are crucial processes in wound healing. No study has evaluated mechanism by which luminal esophageal nitric oxide (NO) affect Rho-associated coiled coil-forming protein kinase (Rho-ROCK) signaling pathway, a key factor of tissue contraction, in stromal fibroblasts to develop Barrett's esophagus. Using esophageal fibroblasts, we performed collagen-based cell contraction assays and evaluated influence of Rho-ROCK signaling in the exposure to acidic bile salts and NOC-9, which is an NO donor. We found that enhanced cell contraction induced by acidic bile salts was inhibited by NO, accompanied by decrease in phosphorylated myosin light chain expression and stress fiber formation. NO directly S-nitrosylated GTP-RhoA and consequently blocked Rho-ROCK signaling. Moreover, exposure to NO and Y27632, a Rho-ROCK signaling inhibitor, decreased α-SMA expression and increased bone morphogenetic protein-4 (BMP4) expression and secretion. These findings could account for the increased expression of BMP4 in the columnar epithelial cells and stromal fibroblasts in human Barrett's esophagus. NO could impair wound contraction by blocking the Rho-ROCK signaling pathway and promote the development of Barrett's esophagus.NEW & NOTEWORTHY Barrett's esophagus is the condition where esophageal epithelium damaged by gastroesophageal reflux disease (GERD) is abnormally healed via replacing of metaplastic columnar epithelium, but very few studies have conducted focusing wound healing in the development of Barrett's esophagus. Esophageal luminal nitric oxide inhibits Rho-ROCK signaling pathway in esophageal fibroblasts, which leads to delay tissue contraction, a pivotal step in proper wound healing. Moreover, this inhibition increases tissue BMP4 expression. Impaired wound healing could be related to Barrett's esophagus.

Expression of atrial‑fetal light chains in cultured human cardiomyocytes after chemical ischemia‑reperfusion injury.

Atrial light chains (ALC1) are naturally present in adult heart atria, while ventricular light chains (VLC1) are predominant in ventricles. Degradation of VLC1 and re-expression of ALC1 in heart ventricles are associated with heart disorders in response to pressure overload. The aim of the current study was to investigate changes in myosin light chain expression after simulated ischemia and simulated reperfusion (sI/sR). Human cardiomyocytes (HCM) isolated from adult heart ventricles were subjected to chemical ischemia. The control group was maintained under aerobic conditions. Myocyte injury was determined by testing lactate dehydrogenase (LDH) activity. The gene expression of ALC1, VLC1 and MMP-2 were assessed by reverse transcription-quatitive PCR. Additionally, protein synthesis was measured using ELISA kits and MMP-2 activity was measured by zymography. The results revealed that LDH activity was increased in sI/sR cell-conditioned medium (P=0.02), confirming the ischemic damage of HCM. ALC1 gene expression and content in HCM were also increased in the sI/sR group (P=0.03 and P<0.001, respectively), while VLC1 gene expression after sI/sR was decreased (P=0.008). Furthermore, MMP-2 gene expression and synthesis were lower in the sI/sR group when compared with the aerobic control group (P<0.001 and P=0.03, respectively). MMP-2 activity was also increased in sI/sR cell-conditioned medium (P=0.006). In conclusion, sI/sR treatment led to increased ALC1 and decreased VLC1 expression in ventricular cardiomyocytes, which may constitute an adaptive mechanism to altered conditions and contribute to the improvement of heart function.

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.

Cyclic Adenosine Monophosphate, Inositol 1,4,5-trisphosphate, Calcium, and Phosphorylated Myosin Light Chain Regulation Through M2 and M3 Muscarinic Receptors of Scleral Fibroblast Cells in Rat Myopia Model

AIM: This study aims to investigate the concentration of cyclic adenosine monophosphate (cAMP), inositol 1,4,5-trisphosphate (IP3), calcium (Ca2+), and the expression phosphorylated myosin light chain (MLC) in Rattus norvegicus scleral fibroblast cells. METHODOLOGY: This study utilized an in vitro experimental study by applying Rattus norvegicus scleral fibroblast cell culture. The cultured cells were divided into control and lens-induced myopia (LIM) groups. The control and LIM culture groups were each divided into five groups, namely, negative control, 0.1 μM acetylcholine, 0.1 μM himbacine, 0.1 μM methoctramine, and 0.1 μM 4-DAMP group. The cAMP, IP3, and Ca2+ concentration were analyzed in the 0th, 5th, 10th, 20th, and 30th. The phosphorylated MLC expression was analyzed using confocal microscope. RESULTS: In the LIM group, the highest cAMP concentration is visible at the 10th min on the himbacine group (0.304 ± 0; p = 0.043) and on the 4-DAMP group (0.346 ± 0; p = 0.043). The highest IP3 concentration is found on the LIM group at the 20th min in comparison to the control group (2503.6 ± 11 vs. 2039.2 ± 2.1; p = 0.046). The highest Ca2+ concentration belongs to the 4-DAMP treatment group from the 5th to the 30th min. The highest average phosphorylated MLC expression value in the LIM group is shown by the 0.1μM 4-DAMP treatment (184.2 ± 37.9c au). CONCLUSION: The regulation of cAMP, IP3, Ca2+, and phosphorylated MLC on the M2 and M3 muscarinic receptor of the scleral fibroblast cells of myopia animal models differs from normal animal models which may be due to interactions of M2 and M3 muscarinic receptor as compensation reaction or crosstalk on myopia induction.

Tissue Expression of Atrial and Ventricular Myosin Light Chains in the Mechanism of Adaptation to Oxidative Stress

Ischemia/reperfusion (I/R) injury induces post-translational modifications of myosin light chains (MLCs), increasing their susceptibility to degradation by matrix metalloproteinase 2 (MMP-2). This results in the degradation of ventricular light chains (VLC1) in heart ventricles. The aim of the study was to investigate changes in MLCs content in the mechanism of adaptation to oxidative stress during I/R. Rat hearts, perfused using the Langendorff method, were subjected to I/R. The control group was maintained in oxygen conditions. Lactate dehydrogenase (LDH) activity and reactive oxygen/nitrogen species (ROS/RNS) content were measured in coronary effluents. Atrial light chains (ALC1) and ventricular light chains (VLC1) gene expression were examined using RQ-PCR. ALC1 and VLC1 protein content were measured using ELISA tests. MMP-2 activity was assessed by zymography. LDH activity as well as ROS/RNS content in coronary effluents was higher in the I/R group (p = 0.01, p = 0.04, respectively), confirming heart injury due to increased oxidative stress. MMP-2 activity in heart homogenates was also higher in the I/R group (p = 0.04). ALC1 gene expression and protein synthesis were significantly increased in I/R ventricles (p < 0.01, 0.04, respectively). VLC1 content in coronary effluents was increased in the I/R group (p = 0.02), confirming the increased degradation of VLC1 by MMP-2 and probably an adaptive production of ALC1 during I/R. This mechanism of adaptation to oxidative stress led to improved heart mechanical function.

Abstract B11: Collective extrusion initiates dissemination in organotypic model of high-grade serous carcinoma

Abstract Development of serous tubal intraepithelial carcinoma (STIC) from transformed fallopian tube secretory epithelial cells (FTSEC) is considered to be an initiating event for a significant proportion of high-grade serous carcinomas (HGSCs). Some regions of STIC lesions can form multicellular loosely cohesive outgrowths that apically extend out of the tubal epithelium. These outgrowths can detach and disseminate within the peritoneal cavity, causing organ obstruction. Immunohistochemical analysis of STICs suggests the possibility that molecular pathways associated with proliferation, DNA repair, cytoskeletal dynamics, and extracellular matrix adhesion contribute to outgrowth formation. Due to limited availability of transformed FTSEC in vitro culture models that faithfully capture outgrowth dynamics, the role of these pathways in STIC/HGSC outgrowth formation remains unknown. Here we present the development of an organotypic model of outgrowth formation. Long-term live-cell imaging of transformed human, and mouse FTSEC and STIC-like multicellular organotypic assemblies, have shown that outgrowths are initiated by collective apical extrusion of less-cohesive, loosely attached cell populations that can proliferate outside of the organotypic assembly. mRNA and immunofluorescence analysis of loosely attached cell populations revealed: (i) activation of Rho pathway, (ii) increased expression of several alpha integrin subunits that form heterodimers with beta 1 integrin, and (iii) nonpolarized apical and junctional localization of activated integrin beta 1. Pharmacologic or genetic inhibition of Rho-associated kinase (ROCK) blocked outgrowth formation without affecting cell proliferation. Conversely, activation of Rho significantly accelerated outgrowths, indicating that Rho-ROCK is a critical regulator of early steps of HGSC dissemination. Quantitative image-based evaluation of ROCK target phosphorylated myosin light chain expression in patient-derived xenograft cells revealed the presence of cell populations with high ROCK activation. Blocking integrin beta 1 activity resulted in the disintegration of already formed outgrowths. Our data indicate that cell populations with high Rho/ROCK activity remodel integrin beta 1-dependent adhesion to initiate apical extrusion and peritoneal dissemination. Citation Format: Sarah Alshehri, Sadaf Farsinejad, Douglas Kung, Estefan Santi, Yali Zhai, Thomas Cattabiani, Hongjun Wang, Kathleen Cho, Marcin Iwanicki. Collective extrusion initiates dissemination in organotypic model of high-grade serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B11.

Cytoprotective and cytofunctional effect of polyanionic polysaccharide alginate and gelatin microspheres on rat cardiac cells

This study investigated the cyto-functional effect of Alginate-Gelatin microspheres on rat cardiomyoblasts after 7 days. Rat cardiomyoblasts were encapsulated inside Alginate-Gelatin microspheres via application of high voltage rate and dropping in a stirring CaCl2 solution. The swelling rate, biodegradation, and mechanical features were measured. Cell viability was assessed using MTT. Cell membrane integrity was monitored via calculation supernatant SGOT, SGPT, CPK, and LDH. We also measured SOD, GPx, and anti-oxidant capacity. Protein levels of Nrf-2 and PCCG-1α were detected via western blotting. The cyto-functional activity of encapsulated cells was monitored using real-time PCR assay targeting the expression of Connexin-43, α-actinin, and myosin light chain.Data showed suitable biodegradation and swelling rate in Alginate-gelatin microspheres by time. 7-day incubation of rat cells inside microspheres did not exert cytotoxicity compared to control cells (p > 0.05). The release of SGPT, SGOT, CPK, and LDH in encapsulated cells was significantly decreased compared to the control group (p < 0.05). We also found enhanced anti-oxidant capacity and SOD and GPx activity in cells after being-encapsulated inside Alginate-Gelatin microspheres (p < 0.05) coincided with increased Nrf-2 synthesis (p < 0.05) compared to control cells. The expression of Connexin-43, α-actinin, and myosin light chain was significantly up-regulated, showing cyto-functional effect of Alginate-Gelatin microspheres after 7-days.

Cloning, Expression, and Epitope Identification of Myosin Light Chain 1: An Allergen in Mud Crab

Mud crab (Scylla paramamosain) is a commonly consumed seafood as a result of its high nutritional value; however, it is associated with food allergy. The current understanding of crab allergens remains insufficient. In the present study, an 18 kDa protein was purified from crab muscle and confirmed to be myosin light chain 1 (MLC1) by matrix-assisted laser desorption/ionization-tandem time-of-flight mass spectrometry. Total RNA was isolated and amplified to obtain a MLC1 open reading frame of 462 bp, encoding 154 amino acids. A structural analysis revealed that recombinant MLC1 (rMLC1) expressed in Escherichia coli contained α-helix and random coil. Moreover, rMLC1 displayed strong immunoactivity by dot blot and a basophil activation test. Furthermore, seven allergenic epitopes of MLC1 were predicted, and five critical epitope regions were identified by an inhibition enzyme-linked immunosorbent assay and human mast cell degranulation assay. This comprehensive research of an allergen helps to conduct component-resolved diagnoses and immunotherapies related to crab allergies.