Expression Of Caldesmon Research Articles

XTP8 Promotes Ovarian Cancer Progression by Activating AKT/AMPK/mTOR Pathway to Regulate EMT

Ovarian cancer (OC) ranks as the fifth leading cause of cancer-related death in women. The main contributors to the poor prognosis of ovarian cancer are the high rates of recurrence and metastasis. Studies have indicated a crucial role for hepatitis B virus X Ag-Transactivated Protein 8 (XTP8), a protein containing the DEP domain, in various cellular processes, including cell growth, movement, and differentiation, across several types of cancers. However, the role of XTP8 in ovarian cancer remains unclear. We observed elevated expression of XTP8 in ovarian cancer. Silencing XTP8 inhibited cell proliferation, promoted apoptosis, and yielded contrasting results in cells overexpressing XTP8. Furthermore, XTP8 facilitated ovarian cancer invasion and migration, triggering epithelial-mesenchymal transition (EMT). Mechanistically, XTP8 silencing led to reduced phosphorylation levels of AKT, increased p-AMPK levels, and decreased p-mTOR levels, while XTP8 overexpression exerted the opposite effects. Additionally, the activation of p-AMPK rescued the promoting effect of XTP8 on EMT in ovarian cancer cell lines, indicating that XTP8 acts as an oncogene by modulating the AKT/AMPK/mTOR pathway. Through transcriptome sequencing to identify downstream targets of XTP8, we found that XTP8 influences the expression of Caldesmon (CALD1) at both transcriptional and translational levels. CALD1 can be considered a downstream target of XTP8. The collaborative action of XTP8 and CALD1 activates the AKT/AMPK/mTOR pathway, regulating EMT to promote ovarian cancer progression. Inhibiting this signaling axis might represent a potential therapeutic target for ovarian cancer.

Expression of Matrix Metalloproteinases 7 and 9, Desmin, Alpha-Smooth Muscle Actin and Caldesmon, in Odontogenic Keratocyst Associated with NBCCS, Recurrent and Sporadic Keratocysts.

Nevoid basal cell carcinoma syndrome (NBCCS) associated odontogenic keratocysts (OKCs) show more aggressive behavior and it has a higher frequency of relapse than non-syndromic OKCs. Stromal myofibroblasts (MFs), characterized by α-smooth muscle actin (αSMA), desmin and caldesmon expression, and metalloproteinases (MMPs) have an essential role in the remodeling of the extracellular matrix (ECM). The aim of the study is to analyze the immunohistochemical expression of MMP-7, MMP-9, αSMA and other new markers in the study of OKCs MFs such as desmin and caldesmon in NBCCS-associated OKCs compared to recurrent and sporadic keratocysts. Fourty 40 patients (23 M and 17 F) underwent surgery to remove the OKCs. The histological sections in paraffin were incubated with markers antibodies and a semi-quantitative score was used to evaluate the immunoreactivity. Densitometric analysis showed a very significantly increased expression of αSMA, caldesmon, MMP-7 and MMP-9 in NBCCS-OKCs compared to non-syndromic OKCs (p < 0.001). However, desmin showed a not significant increased expression in non-syndromic OKC compared to NBCCS-OKCs specimens in which desmin was slightly or not at all expressed. NBCSS-OKCs showed a greater distribution of MFs compared to the other OKCs subtypes. Further studies will be needed to evaluate whether the different expressions of these markers can be correlated to a different clinical behavior.

Intravascular fasciitis involving the external jugular vein and subclavian vein: A case report

BACKGROUNDIntravascular fasciitis (IVF) is a rare nodular fasciitis that often involves the layers and lumens of blood vessels; therefore, it is easily misdiagnosed as a malignant tumor with invasion into blood vessels.CASE SUMMARYA 13-year-old boy was admitted due to a mass on the left side of his neck. Duplex ultrasonography revealed a circular solid hypoechoic mass in the external jugular vein, and magnetic resonance imaging revealed an enhanced longitudinal mass-like lesion in the left supraclavicular fossa. Surgical treatment was arranged and completed, histopathological analysis showed a large amount of spindle cell proliferation, and immunohistochemistry showed that the spindle cells were positive for the expression of vimentin, caldesmon, and smooth muscle actin and negative for the expression of S-100 protein, desmin, CD34, and c-kit; Ki-67 staining revealed a low proliferative index (5%-10%), which confirmed the differentiation characteristics of myofibroblasts. Fluorescence in situ hybridization detected the rearrangement of USP6. IVF was subsequently diagnosed.CONCLUSIONIVF is characterized by intraluminal, intramural and extramural involvement of small to large arteries or veins. Unless the doctor has a deep understanding of the disease or suspects that there is an initial indicator, IVF may be confused with other intravascular malignancies, leading to unnecessary radical surgery. Imaging examination combined with histopathological examination can improve the diagnostic accuracy of this disease.

Effects of dietary calcium, phosphorus and microbial phytase on intestinal morphologyin laying hens

Different challenges are being applied in the poultry industry in order to protect animal health and to increase immunity and production. The supplementation of microbial phytase is essential in terms of both reducing the inorganic phytase rate and contributing to the absorption of other minerals. In this study, a newly isolated microbial phytase was added at different concentrations to the diet together with calcium (Ca$^{2+}$) and available phosphorus (AP), and the effects of this supplementation on intestinal absorption capacity and Ca$^{2+}$ binding capacity were investigated via morphological measurements and immunohistochemical examination of the duodenum and ileum. For this purpose, 90 Lohmann LSL-White laying hens were divided into three main diet groups: 1. Standard Ca$^{2+}$ and AP (Ca+AP), 2. Standard Ca$^{2+}$ and low AP (Ca+low AP), and 3. Low Ca2+ and low AP (low Ca+low AP). These three groups were further divided into three phytase subgroups each (without phytase [Phy-], commercial phytase [CP] and microbial phytase [MP]). At the end of the experiment, animals were euthanized, and duodenum and ileum samples were fixed and processed for histological examination. Villus height, crypt depth, total mucosa thickness, and villus width were measured and villus height: crypt depth ratio and villus absorption area were calculated. Caldesmon expression in the duodenum and ileum was also investigated immunohistochemically. The results indicated that villus height, total mucosa thickness, and villus absorption area increased (p ≤ 0.05) in birds fed with Ca+AP+MP. Stronger caldesmon expression was observed in the MP treated groups. We concluded that MP produced from Bacillus megaterium EBD 9-1 bacterium increases the utilization of Ca$^{2+}$ and AP and, thus, can have a beneficial role when these macrominerals are used insufficiently. Ca$^{2+}$, AP, and MP may have positive effects on the intestinal morphology and absorption area when used at optimum amounts.

Caldesmon: Biochemical and Clinical Implications in Cancer

Caldesmon, an actin-binding protein, can inhibit myosin binding to actin and regulate smooth muscle contraction and relaxation. However, caldesmon has recently attracted attention due to its importance in cancer. The upregulation of caldesmon in several solid cancer tissues has been reported. Caldesmon, as well as its two isoforms, is considered as a biomarker for cancer and a potent suppressor of cancer cell invasion by regulating podosome/invadopodium formation. Therefore, caldesmon may be a promising therapeutic target for diseases such as cancer. Here, we review new studies on the gene transcription, isoform structure, expression, and phosphorylation regulation of caldesmon and discuss its clinical implications in cancer.

Abnormal Aortic Wall Properties in Women with Turner Syndrome.

Background Turner syndrome (TS) is associated with aortic dilatation and dissection, but the underlying process is unclear. The aim of this study was to investigate the elastic properties and composition of the aortic wall in women with TS. Methods In this cross-sectional study, 52 women with TS aged 35 ± 13 years (50% monosomy, 12 with bicuspid aortic valve [BAV] and 4 with coarctation) were investigated using carotid-femoral pulse wave velocity (CF-PWV) by echocardiography and ascending aortic distensibility (AAD) and aortic arch pulse wave velocity (AA-PWV) by magnetic resonance imaging (MRI). As control group, 13 women with BAV without TS and 48 healthy patients were included. Results Women with TS showed a higher AA-PWV (β = 1.08, confidence interval [CI]: 0.54–1.62) after correcting for age and comorbidities compared with controls. We found no significant difference in AAD and CF-PWV. In women with TS, the presence of BAV, coarctation of the aorta, or monosomy (45, X) was not associated with aortic stiffness. In addition, aortic tissue samples were investigated with routine and immunohistochemical stains in five additional women with TS who were operated. The tissue showed more compact smooth muscle cell layers with abnormal deposition and structure of elastin and diminished or absent expression of contractile proteins desmin, actin, and caldesmon, as well as the progesterone receptor. Conclusion Both aortic arch stiffness measurements on MRI and histomorphological changes point toward an inherent abnormal thoracic aortic wall in women with TS.

Decreased smooth muscle function, peristaltic activity, and gastrointestinal transit in dystrophic (mdx) mice.

Duchenne muscular dystrophy (DMD) is characterized by the lack of dystrophin in skeletal, cardiac, and smooth muscle. Slow colonic transit and constipation are common in DMD patients and animal models of DMD. However, the cause of this hypocontractility and the expression of contractile proteins in smooth muscle are unknown. The aim of the study was to investigate the expression of contractile proteins in the colonic smooth muscle and the function of the colon in control and mdx mice. Muscle contraction was measured in muscle strips and isolated muscle cells. Peristaltic activity was measured in ex vivo preparations by spatiotemporal mapping, and gastrointestinal (GI) transit in vivo was measured by the distribution of fluorescent marker along the intestine and colon. mRNA expression of contractile proteins smoothelin, caldesmon, calponin, and tropomyosin was measured by qRT-PCR. Expression of mRNA for contractile proteins was decreased in colonic smooth muscle of mdx mice compared with control. Contraction in response to acetylcholine and KCl was decreased in colonic muscle strips and in isolated muscle cells of mdx mice. Distension of ex vivo colons with Krebs buffer induced peristalsis in both control and mdx mice; however, significantly fewer full peristaltic waves were recorded in the colons of mdx mice. GI transit was also inhibited in mdx mice. The data indicate that the lack of dystrophin causes decrease in colonic smooth muscle contractility, peristalsis, and GI transit and provides the basis for analysis of mechanisms involved in smooth muscle dysfunction in DMD.

The Use of Smoothelin and Other Antibodies in the Diagnosis of Uterine and Soft Tissue Smooth Muscle Tumors.

Smoothelin is a cytoplasmic protein expressed in differentiated smooth muscle cells. Immunohistochemical evaluation of smoothelin has previously been reported in gastrointestinal (GI) smooth muscle tumors, but has yet to be studied in smooth muscle tumors of uterine and other soft tissue origin. DOG1 expression is reported to be specific for GI stromal tumors; however, variable expression has been reported in leiomyosarcomas (LMS) depending on site of origin. Overexpression of p16 is common in LMS of uterine and other sites of origin, but has not been correlated with tumor grade. This study explores the differential expression of these markers, as well as caldesmon, in LMS cases to assess diagnostic utility. Using tissue microarrays and cases from Tulane Medical Center and Medical College of Wisconsin, expression of smoothelin, DOG1, caldesmon, and p16 was evaluated by immunohistochemistry in 87 cases of LMS. The cases were subdivided by location of origin into uterine (N=31) and nonuterine (N=56) with 10 of the nonuterine of GI origin, as well as by grade into low grade (N=27) and intermediate and high grade (N=60). Differential expression among different grades and locations was evaluated. The same markers were evaluated in atypical leiomyoma cases (N=4) and 1 smooth muscle tumor of uncertain malignant potential case (N=1). Smoothelin expression was also assessed in 20 benign uterine leiomyomas. Weak DOG1 expression is rare but possible in extrauterine LMS. Expression of p16 is common in both uterine and extrauterine LMS, and more frequent in higher grades. Expression of smoothelin in this study differed depending on tumor type, grade, and site of origin. All leiomyomas and most atypical leiomyomas showed cytoplasmic positivity for smoothelin, whereas only 5% of LMS had cytoplasmic expression. The study suggests smoothelin may be downregulated in the cytoplasm of malignant smooth muscle tumor cells and may serve as a supportive aid in the distinction of LMS from benign smooth muscle tumors in cases where it is difficult by morphology alone.

Caldesmon Expression in Metastatic and Non-Metastatic Oral Squamous Cell Carcinoma—A Mediator of Epithelial Mesenchymal Transition

Introduction: For the past decades long-term survival of oral squamous cell carcinoma (OSCC) patients remain unchanged which provides a wide platform for research. Caldesmon (CaD), an actin-myosin binding protein, has a contributing role in cytoskeleton modulation and cell motility. The aim of this study is to evaluate the expression of CaD in metastatic and non-metastatic OSCC and to discuss the possible role of CaD in epithelial mesenchymal transition. Materials and Methods: Archival blocks of 25 metastatic and 25 non-metastatic OSCC patients are included where CaD expression is evaluated immunohistochemically. Results: Overall expression and staining intensity of CaD were statistically significant in different grades of metastatic and non-metastatic OSCC. In summary, high expression of CaD is observed in increasing grades of OSCC but the metastatic potential of the tumour doesn’t seem to have any relation with CaD.

TGFβ1-induced expression of caldesmon mediates epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is an important process that mediates organ development and wound healing, and in pathological contexts, it can contribute to the progression of fibrosis and cancer. During EMT, cells exhibit marked changes in cytoskeletal organization and increased expression of a variety of actin associated proteins. Here, we sought to determine the role of caldesmon in mediating EMT in response to transforming growth factor (TGF)-β1. We find that the expression level and phosphorylation state of caldesmon increase as a function of time following induction of EMT by TGFβ1 and these changes in caldesmon correlate with increased focal adhesion number and size and increased cell contractility. Knockdown and forced expression of caldesmon in epithelial cells reveals that caldesmon expression plays an important role in regulating the expression of the myofibroblast marker alpha smooth muscle actin. Results from these studies provide insight into the role of cytoskeletal associated proteins in the regulation of EMT and may suggest ways to target the cell cytoskeleton for regulating EMT processes.

Fibroblasts play a potential role in bone destruction via osteopontin related caldesmon expression and polymerization in human non-functioning pituitary adenomas

Non-functioning pituitary adenomas (NFPAs) are the most frequent pituitary tumors. The elucidation of the mechanisms of aggressive NFPAs in bone destruction is required in order to guide the clinical diagnosis and treatment of NFPAs. In the present study, we investigated the differential proteomics of fibroblasts isolated from clinical specimens of NFPAs with or without bone destruction. Proteomic analysis revealed a group of molecules associated with cytoskeleton organization, including caldesmon, were differentially expressed between fibroblasts isolated from bone destruction NFPAs (BD-NFPAs) and fibroblasts isolated from non-bone destruction NFPAs (NBD-NFPAs). The secreted proteins analysis found that osteopontin was significantly upregulated in BD-NFPAs fibroblasts. Furthermore, immunohistochemical staining of the NFPAs clinical samples showed that the expression of caldesmon in stromal cells and the expression of osteopontin in both tumor cells and stroma were significantly increased in BD-NFPAs. Taken together, our results indicate a possible way that osteopontin secreted from both NFPA cells and surrounding fibroblasts modify caldesmon expression and polymerization in fibroblasts, which may contribute to bone destruction in NFPA patients.

Expression of von Willebrand factor and caldesmon in the placental tissues of pregnancies complicated with intrauterine growth restriction

Purpose: The decreased placental perfusion is the underlying reason for intrauterine growth restriction that in turn leads to reduced placental perfusion and ischemia. However, there are several issues to be understood in the pathophysiology of intrauterine growth restriction. We aimed to study whether any compensatory response in placental vascular bed occur in pregnancies complicated with intrauterine growth restriction by the immunohistochemical staining of von Willebrand factor and caldesmon in placental tissues.Materials and methods: A total of 103 pregnant women was enrolled in the study including 50 patients who were complicated with IUGR and 50 uncomplicated control patients. The study was designed in a prospective manner. All placentas were also stained with von Willebrand factor and caldesmon monoclonal kits.Results: The immunohistochemical staining of von Willebrand factor and caldesmon expressions in placental tissues were different between normal and intrauterine growth restriction group. The percentages of 2+ and 3+ von Willebrand factor expression were higher in the intrauterine growth restriction group comparing with the normal group, although the difference was not statistically significant. The intensity of caldesmon expression was significantly lower in the intrauterine growth restriction group in comparison with the normal group (p < .001).Conclusion: Angiogenesis occurs as a placental response to intrauterine growth restriction which is a hypoxic condition. But newly formed vessels are immature and not strong enough. Our study is important to clarify the pathophysiology and placental compensatory responses in intrauterine growth restriction.

Cystic Nephroma in Adults: A Clinicopathologic Study of 46 Cases.

Cystic nephroma in adults is an uncommon multicystic benign tumor of kidney and, over the last century, only a few small series or case reports have studied it. We analyzed 46 tumors from adults (41 female, median age: 55 y), collecting data on gross, microscopic, and immunohistochemical findings. All of the tumors were composed entirely of cysts separated by septa; the majority were surrounded by a pseudocapsule. Hypocellular fibrous and hypercellular spindle cell stroma were the most common components of the septa. Hypocellular collagenized fibrous stroma predominated in older patients (P=0.027), whereas hypercellular stroma (including wavy spindle cell stroma) prevailed in younger patients (P=0.042). Small foci of stroma of moderate cellularity in which epithelial elements ranging from a handful of cells with no lumen, to tiny cysts with pinpoint lumens to slightly larger cysts were found in 18 tumors. The cysts were lined by flat, cuboidal, or hobnail cells. Rarely, foci of clear cells, delicate papillae, or focal nuclear atypia were observed in the cyst linings. The stroma of all the tumors was positive for smooth muscle actin. Desmin and caldesmon expression were variable. The stroma stained positive for progesterone and estrogen receptors in 85% and 50% of the tumors. Staining for inhibin was present in small clusters of polygonal or rarely spindle cells in the stroma in 41% of the tumors. A positive nuclear reaction for SF1 was detected in the same clusters of cells. On the basis of our findings, we hypothesize that cystic nephroma originates as a stromal proliferation with the epithelial component arising from a stromal-epithelial transition and that the stroma matures with decreasing cellularity over time, ultimately becoming hypocellular and collagenized.

Mixed Epithelial and Stromal Tumor of the Kidney: A Clinicopathologic Study of 53 Cases.

Mixed epithelial and stromal tumor of the kidney is an uncommon complex neoplasm, and previous studies have included only a modest number of cases and have left gaps in our understanding of its morphology. We analyzed 53 tumors (46 F, median age: 49 y), collecting data on gross, histologic, and immunohistochemical features. The most common gross appearance was solid and cystic (47%). Hypocellular fibrous and cellular spindle cell stroma were the most common stromal types, followed by smooth muscle differentiation, edematous stroma, and adipose tissue. Hypocellular fibrous stroma and adipose tissue were more common in larger tumors (P=0.003 and 0.04, respectively) and cellular spindle cell stroma in smaller tumors (P=0.0009). Combinations of diverse stromal elements were common: roughly 50% of tumors contained >4 types of stroma. With regard to epithelium, tiny crowded and branching glands were present in 60% of tumors. Round glands lined by tall cuboidal epithelium, reminiscent of thyroid follicles, spatulate papillae reminiscent of phyllodes tumor, glands reminiscent of nephrogenic adenoma, and complex papillae were also frequently found. Combinations of diverse epithelial elements were common: 64% of tumors contained >4 types of epithelium. All of the tumors except 1 were positive for smooth muscle actin in the stroma. Desmin and caldesmon expression were more variable. Stains for progesterone and estrogen receptors showed positivity in the stromal component in 85% and 73% of tumors, respectively. CD10 and CD34 immunolabeling were restricted to pericystic spindle cells. No tumor expressed inhibin. In summary, this study demonstrates a strong tendency for mixed epithelial and stromal tumors to contain multiple types of stroma and epithelium and comprehensively analyzes the immunohistochemical profile.

Abstract B33: Plasticity of the Ewing sarcoma invasive program: How microenvironmental stresses alter the drivers and repressors of metastasis

Abstract Purpose of the Study: Metastatic Ewing sarcoma is notoriously difficult to treat and patient survival rates are still dismal. Therefore, better understanding the modulators of Ewing Sarcoma metastasis is required. The purpose of this study was to define the contribution of tumor cell plasticity to dynamic changes in cell migration, invasion, and invadopodia formation, in vitro proxies of the metastatic phenotype in vivo. We hypothesized that microenvironmental stress (i.e. nutrient deprivation and hypoxia) may promote transition of Ewing sarcoma cells from less motile to more migratory and invasive states by repressing proteins known to inhibit invadopodia formation, such as caldesmon, while also activating drivers of invasion, such as Src kinase. Experimental Procedures: The Ewing Sarcoma cell lines A673, CHLA10 and CHLA25 were stressed with either nutrient deprivation (serum withdrawal) or hypoxia (1% oxygen). Cell migration and invasion assays with transwells and x-Celligence real time cell monitoring assays were utilized. Genome-wide RNA synthesis was analyzed by Bru-Seq technology, where bromouridine labeled RNA is used to examine newly synthesized RNA in an unbiased manner. To specifically focus on invadopodia (actin based cell invasive structures) formation, we performed matrix degradation assays by plating cells on FITC-labeled substrate and analyzing matrix degradation via confocal microscopy. Protein expression and phosphorylation status was confirmed by western blot. The commercially available Src inhibitor dasatinib was used to disrupt stress induced Src activation. Data Summary: Nutrient deprivation enhanced Ewing Sarcoma migration and invasion. Bru-Seq analysis was performed on cells deprived of serum for 24 hours to determine RNA that was newly synthesized under this specific stress. Geneset enrichment analysis of this data revealed significant overlap with gene signatures of hypoxic stress. Given this result, we examined cell motility and invasive behaviors under hypoxic stress. We found that hypoxia also significantly enhanced migration and invasion compared to normoxic controls. Invadopodia are actin-rich cellular structures that harbor matrix metalloproteinases and are “lead-points” to matrix degradation and cancer cell invasion. Src kinase is a driver of invadopodia formation, while the actin modulating protein caldesmon is a reported inhibitor of invadopodia formation. Western blot analysis of cells exposed to serum deprivation and/or hypoxic conditions revealed changes in Src phosphorylation and/or caldesmon expression in each of the cell lines examined. Using FITC-labeled gelatin based matrix assays, we examined invadopodia formation and matrix degradation in Ewing sarcoma cells under stress. While single stresses (i.e. nutrient withdrawal or hypoxia in isolation) led to increases in cell matrix degradation, combining these two stresses dramatically enhanced the ability of these cells to form invadopodia and degrade underlying matrix. Current studies are underway to modify stress-induced changes in Src activity with the commercially available drug dasatinib, a known potent inhibitor of Src at nanomolar concentrations. Conclusion: The combination of nutrient deprivation and hypoxia commonly occur simultaneously in tumor microenvironments. We have shown that this stress combination promotes invadopodia formation and Ewing sarcoma cell invasion. Further, changes in expression and phosphorylation of key regulators of invadopodia formation (i.e. Src and caldesmon) were noted in stress conditions. Our current and future work aims to further dissect the role of Src and caldesmon in metastatic Ewing sarcoma. We will attempt to use drugs already in clinical use, such as dasatinib, to block invadopodia matrix degradation under conditions of stress. Citation Format: Kelly M. Bailey, Merlin Airkik, Melanie Krook, Brian Magnuson, Mats Ljungman, Elizabeth Lawlor. Plasticity of the Ewing sarcoma invasive program: How microenvironmental stresses alter the drivers and repressors of metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B33.

Overexpression of caldesmon is associated with tumor progression in patients with primary non-muscle-invasive bladder cancer.

The expression and function of caldesmon (CAD) in urothelial bladder carcinoma (BC) have not been reported. Here, we investigated the expression, prognostic value, and potential functional mechanism of CAD in primary non-muscle-invasive bladder cancer (NMIBC). Protein profiling of tissue samples using antibody microarrays showed significantly higher CAD expression in muscle-invasive BC tissues compared with NMIBC tissues. We then validated the CAD expression in BC cells by immunohistochemistry analysis using paraffin-embedded tissue blocks and western blots using BC cell lines. In addition, we examined the expression of CAD variants by reverse transcription-polymerase chain reaction, and confirmed the expression of low-molecular-weight isoforms (L-CAD), specifically encoded by WI-38 L-CAD II (transcript variant 2), in BC cells. Survival analysis in an independent primary NMIBC cohort comprising 132 patients showed that positive CAD expression was significantly associated with poorer prognosis than no CAD expression with regard to recurrence- and progression-free survival (p = 0.001 and 0.014, respectively). Multivariate analyses further indicated that positive CAD expression was an independent predictor of progression-free survival (p = 0.032; HR = 5.983). Data obtained from in vitro silencing and overexpression studies indicated that L-CAD promotes migration and invasiveness of BC cells. Immunofluorescence assays showed dramatic structural changes in the actin cytoskeleton of BC cells after L-CAD overexpression. Our findings collectively suggest that L-CAD overexpression in primary NMIBC is significantly associated with tumor progression and that a possible mechanism for L-CAD's activity is implicated in increased cell motility and invasive characteristics through morphological changes in BC cells.

MiR-133 modulates TGF-β1-induced bladder smooth muscle cell hypertrophic and fibrotic response: Implication for a role of microRNA in bladder wall remodeling caused by bladder outlet obstruction

Bladder outlet obstruction (BOO) evokes urinary bladder wall remodeling significantly, including the phenotype shift of bladder smooth muscle cells (BSMCs) where transforming growth factor-beta1 (TGF-β1) plays a pivotal role given the emerging function of modulating cellular phenotype. miR-133 plays a role in cardiac and muscle remodeling, however, little is known about its roles in TGF-β1-induced BSMC hypertrophic and fibrotic response. Here, we verified BOO induced bladder wall remodeling and TGF-β1 expression mainly located in bladder endothelium. Furthermore, we uncovered miR-133a/b expression profile in BOO rats, and then explored its regulated effects on BSMCs' phenotypic shift. Our study found that miR-133 became down-regulated during rat bladder remodeling. Next, we sought to examine whether the expression of miR-133 was down-regulated in primary BSMCs in response to TGF-β1 stimulation and whether forced overexpression of miR-133 could regulate profibrotic TGF-β signaling. We found that stimulation of BSMCs with exogenous TGF-β1 of increasing concentrations resulted in a dose-dependent decrease of miR-133a/b levels and transfection with miR-133 mimics attenuated TGF-β1-induced α-smooth muscle actin, extracellular matrix subtypes and fibrotic growth factor expression, whereas it upregulated high molecular weight caldesmon expression compared with the negative control. Also, downregulation of p-Smad3, not p-Smad2 by miR-133 was detected. Additionally, miR-133 overexpression suppressed TGF-β1-induced BSMC hypertrophy and proliferation through influencing cell cycle distribution. Bioinformatics analyses predicted that connective tissue growth factor (CTGF) was the potential target of miR-133, and then binding to the 3′-untranslated region of CTGF was validated by luciferase reporter assay. These results reveal a novel regulator for miR-133 to modulate TGF-β1-induced BSMC phenotypic changes by targeting CTGF through the TGF-β–Smad3 signaling pathway. A novel antifibrotic functional role for miR-133 is presented which may represent a potential target for diagnostic and therapeutic strategies in bladder fibrosis.

A Novel Method for Differentiation of Human Mesenchymal Stem Cells into Smooth Muscle-Like Cells on Clinically Deliverable Thermally Induced Phase Separation Microspheres

Muscle degeneration is a prevalent disease, particularly in aging societies where it has a huge impact on quality of life and incurs colossal health costs. Suitable donor sources of smooth muscle cells are limited and minimally invasive therapeutic approaches are sought that will augment muscle volume by delivering cells to damaged or degenerated areas of muscle. For the first time, we report the use of highly porous microcarriers produced using thermally induced phase separation (TIPS) to expand and differentiate adipose-derived mesenchymal stem cells (AdMSCs) into smooth muscle-like cells in a format that requires minimal manipulation before clinical delivery. AdMSCs readily attached to the surface of TIPS microcarriers and proliferated while maintained in suspension culture for 12 days. Switching the incubation medium to a differentiation medium containing 2 ng/mL transforming growth factor beta-1 resulted in a significant increase in both the mRNA and protein expression of cell contractile apparatus components caldesmon, calponin, and myosin heavy chains, indicative of a smooth muscle cell-like phenotype. Growth of smooth muscle cells on the surface of the microcarriers caused no change to the integrity of the polymer microspheres making them suitable for a cell-delivery vehicle. Our results indicate that TIPS microspheres provide an ideal substrate for the expansion and differentiation of AdMSCs into smooth muscle-like cells as well as a microcarrier delivery vehicle for the attached cells ready for therapeutic applications.

Upregulation of cytoskeleton protein and extracellular matrix protein induced by stromal-derived nitric oxide promotes lung cancer invasion and metastasis.

Lung cancer commonly metastasizes to lymph nodes, brain and bones, which is the main cause of death. It is still a challenge to detect molecular biomarkers for early diagnosis and therapeutics of lung cancer. Our previous study found that bone marrow-derived stroma cells (BMSCs) under tumor microenvironment produced nitric oxide (NO), which was induced by inducible nitric oxide synthase (iNOS), and promoted invasion and metastasis of cancer cells by remodeling cytoskeleton. The aim of this study is to elucidate the relationship between the expressions of iNOS, cytoskeleton protein caldesmon, OPN, and clinical parameters especially the metastasis of lung cancer. We found that nitric oxide can remodel cytoskeleton and promoted the mobility of lung cancer cells. The expressions of iNOS, caldesmon, and OPN are closely correlated to metastasis of lung cancer. The intracranial metastatic tissue samples of lung cancer showed significantly higher expression of iNOS, caldesmon and OPN. A flow-cytometry analysis for peripheral blood of lung cancer patients showed increased EPCAM+/OPN+ cells in circulation of patients with bone metastasis compared to that of patients without metastasis, which is indicative of cancer circulating cells. The concentration of serum OPN was also positively related to the bone metastasis of lung cancer. Taken together, these results suggested that iNOS, caldesmon and OPN may work as biomarkers for metastasis of lung cancer.

In situ characterization of intrahepatic non-parenchymal cells in PSC reveals phenotypic patterns associated with disease severity.

Liver-infiltrating T cells have been implicated in the pathogenesis of primary sclerosing cholangitis (PSC), however little information is available about changes in other cellular compartments in the liver during PSC. This study aimed to characterize non-parenchymal intrahepatic cells in PSC livers and to find associations between phenotypes and disease severity. Using immunohistochemistry, followed by automated image analysis and quantification and a principal component analysis, we have studied non-parenchymal intrahepatic cells in PSC-patient livers (n = 17) and controls (n = 17). We observed a significant increase of T cells in the PSC patients, localized to the fibrotic areas. MAIT cells, normally present at high numbers in the liver, were not increased to the same extent. PSC patients had lower expression of MHC class I than controls. However, the levels of NKp46+ NK cells were similar between patients and controls, nevertheless, NKp46 was identified as a phenotypic marker that distinguished PSC patients with mild from those with severe fibrosis. Beyond that, a group of PSC patients had lost expression of Caldesmon and this was associated with more extensive bile duct proliferation and higher numbers of T cells. Our data reveals phenotypic patterns in PSC patients associated with disease severity.