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Colorectal cancer is a notorious disease, with almost half of the patients succumbing to the disease. The prevalence and incidence rates of colorectal cancer are increasing in many parts of the world, highlighting the need to discover new biomarkers for diagnosis and therapy. Caldesmon (CaD), an actin-binding protein that plays a significant role in controlling cell motility, has emerged as a promising biomarker. The CALD1 gene encodes CaD as multiple transcripts that mainly encode two protein isoforms: High-molecular-weight (h-CaD), expressed in smooth muscle, and low-molecular-weight (l-CaD), expressed in nonsmooth muscle cells. Most studies have suggested an oncogenic role of CaD in colorectal cancer, but the exact subcellular localization of the two CaD isoforms in tumor cells and stroma have not been clarified yet. Here, we analyzed tissue samples from 262 colorectal cancer patients by immunohistochemistry analysis using specific antibodies for l-CaD and h-CaD. The results showed elevated cytoplasmic expression levels of l-Cad in 187/262 (71.4%) cases. l-Cad was expressed at low levels in the normal colon mucosa and was also consistently expressed in the cancer-associated stroma of all cases, suggesting that it could play a role in modulating the tumor microenvironment. l-CaD expression in cancer cells was associated with preinvasive stages of cancer. Survival analysis indicated that patients with high l-CaD expression in tumor cells could respond poorly to selective chemotherapeutic 5FU, but not combination chemotherapy. h-CaD was expressed in colonic and vascular smooth muscle cells as expected and to a lesser extent in the tumor-associated stroma, but it was not expressed in the cancer cells or normal colon mucosal epithelial cells. Collectively, these data clarify how the expression patterns of CaD isoforms in colorectal cancer can have applications in the management of colorectal cancer patients. more...

Perivascular epithelioid cell tumors (PEComas) are mesenchymal neoplasms with characteristic epithelioid or spindled cytomorphology that typically grow around blood vessels. These tumors are phenotypically and immunohistochemically distinct, expressing markers of both melanocytic and smooth muscle differentiation. Herein, we describe a case of histopathologically malignant cutaneous PEComa without metastatic spread, with review of the pertinent literature. Telescoping punch biopsy demonstrated an epithelioid neoplasm with marked atypia, hypercellularity, and increased mitotic activity. Immunohistochemical stains for HMB-45, NK1-C3, PGP9.5, MiTF, CD10, and CD68 were positive within tumor cells. In addition, there was diffuse expression of caldesmon and focal cytoplasmic staining for smooth muscle actin on the excision specimen. The patient underwent treatment with surgical excision with adjuvant radiation and surveillance computed tomography (CT). The patient remains free of recurrence or metastatic disease after 10 months of follow-up. To our knowledge, this is only the third reported case of a malignant cutaneous PEComa reported in the literature to date. more...

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. more...

Primary leiomyosarcomas of the colon (PLC) are rare tumors, representing 0.12% of all colon malignancies. We report a 59-year-old man with weight loss, mild anemia, and rectal bleeding. Colonoscopy revealed a 3.2 × 2.6-cm mass at the ileocecal valve. Histopathological examination of the biopsy showed a spindle cell neoplasm that stained positive for smooth muscle actin, caldesmon, and desmin. A diagnosis of PLC was made. Subsequently, a laparoscopic right hemicolectomy was performed, and no local recurrence was noted 6 months after the resection. Given the rarity of PLC, more studies on the clinical features and treatments of this tumor are warranted. more...

We present a case of a 67 year old male with a cortical nodular tumour of the left kidney. During a year's follow-up with ultrasound and MRI, the tumour was seen to increase in size by 16-20 mm. The nodule was surgically removed. Microscopically it was well defined and unencapsulated, with a proliferation of typical fusiform cells of smooth muscle cell appearance, clumped around well vascularized areas. Immunohistochemically, the neoplasm was positive for muscle markers (smooth muscle actin, desmin and caldesmon) and melanocyte markers (HMB-45 and Melan A). Our case would appear to be a renal neoplasm with an angioleiomyomatous pattern, but with immunohistochemical characteristics of angiomyolipoma (PEComa), however, without either a lipomatous or lipid cell component. We found no previous reports of this type of tumour in the literature. more...

Stress urinary incontinence (SUI) is a life changing condition, affecting 20 million women worldwide. In this study, we developed a bioactive, injectable bulking agent that consists of Permacol™ (Medtronic, Switzerland) and recombinant insulin like growth factor-1 conjugated fibrin micro-beads (fib_rIGF-1) for its bulk stability and capacity to induce muscle regeneration . Therefore, Permacol™ formulations were injected in the submucosal space of rabbit bladders. The ability of a bulking material to form a stable and muscle-inducing bulk represents for us a promising therapeutic approach to achieve a long-lasting treatment for SUI. The fib_rIGF-1 showed no adverse effect on human smooth muscle cell metabolic activity and viability in vitro based on AlamarBlue assays and Live/Dead staining. Three months after injection of fib_rIGF-1 together with Permacol™ into the rabbit bladder wall , we observed a smooth muscle tissue like formation within the injected materials. Positive staining for alpha smooth muscle actin , calponin , and caldesmon demonstrated a contractile phenotype of the newly formed smooth muscle tissue. Moreover, the fib_rIGF-1 treated group also improved the neovascularization at the injection site, confirmed by CD31 positive staining compared to bulks made of Permacol TM only. The results of this study encourage us to further develop this injectable, bioactive bulking material towards a future therapeutic approach for a minimal invasive and long-lasting treatment of SUI. more...

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. more...

Canine oral malignant melanomas (OMMs) exhibit a variety of morphologic phenotypes, including a spindloid variant. The microscopic diagnosis of spindloid OMMs is based on junctional activity and/or the presence of melanin pigment. In the absence of these features, spindloid OMMs are difficult to differentiate from soft tissue sarcomas (STS). An antibody cocktail (MDX) that includes Melan-A, PNL2, and tyrosinase-related proteins 1 and 2 (TRP-1 and TRP-2) is the current gold standard for identifying amelanotic OMMs by immunohistochemistry (IHC). However, MDX is less sensitive for diagnosing spindloid amelanotic OMMs. This raises concern for biopsy specimens that lack overlying epithelium, making it potentially difficult to differentiate OMM from STS by IHC. The goal of this study was to identify additional markers to help differentiate between STS and OMMs that lack pigment and junctional activity. SOX-10 has recently been proposed as a sensitive marker for melanocytes in humans but has not been validated in dogs. Similarly, RNA expression for various genes has been analyzed in humans, but not in the context of diagnosing canine melanocytic neoplasms. For this retrospective study, formalin-fixed, paraffin-embedded tissues from 20 OMMs, 20 STS, and 20 oral spindle cell tumors (OSCTs) that lacked junctional activity and pigmentation were selected. IHC for MDX, SOX-10, and laminin, in parallel with RT-qPCR ofTYR, SOX10, CALD1, CD34, DES, andLAMA1, was performed in all cases.TYR, CD34, andCALD1were the most discriminatory genes in differentiating between OMM and STS, all having 100% specificity and 65, 95, and 60% sensitivity, respectively. While all 20 OMMs were immunohistochemically labeled for SOX-10, two STS were also labeled (100% sensitivity and 90% specificity). MDX IHC labeled all 20 OMMs and no STS. Surprisingly, none of the 20 OSCTs expressedTYRRNA above the cutoff, and 14/20 OSCTs expressedCALD1orCD34RNA above the cutoff, thereby confirming them as STS. Four OSCT were suspect STS, and no OSCTs were confirmed as OMMs based on IHC and RNA expression patterns. In conclusion, the RNA levels of TYR, CD34, and CALD1 should be evaluated in suspected amelanotic OMMs that are negative for MDX to accurately differentiate between OMM and STS. more...

Cellular leiomyoma (CL) represents an uncommon variant of uterine leiomyoma with limited data concerning its immunohistochemical and molecular profile. We performed a comprehensive analysis of 52 CL cases all of which were analyzed immunohistochemically. Molecular analysis was possible in 32 cases with sufficient DNA, and 38 cases with sufficient RNA. The immunohistochemical results showed a high expression of smooth muscle markers (calponin (100%), desmin (100%), smooth muscle actin (98.1%), caldesmon (96.1%), transgelin (96.1%), smooth muscle myosin heavy chain (86.5%), and smoothelin (61.5%)). Concerning markers of endometrial stromal differentiation, the expression of CD10 was observed in 65.4% cases (42.2% with H-score > 50), and IFITM1 in 36.5% cases (1.9% with H-score > 50). 36.5% showed HMGA2 overexpression at the IHC level, associated with increased mRNA expression in 14/14 cases. The rearrangement of the HMGA2 gene was detected in 13.2%. Chromosome 1p deletion was found in 19.3%, while 9.4% of tumors showed a pathogenic mutation in the MED12 gene. In conclusion, CL is immunohistochemically characterized by a high expression of "smooth muscle" markers commonly associated with a co-expression of "endometrial stromal" markers, where IFITM1 shows superior performance compared to CD10 regarding its specificity for differentiation from endometrial stromal tumors. The sensitivity of smoothelin in CL seems rather low, but no data is available to assess its specificity. On a molecular level, the most common mutually exclusive aberration in CL affects HMGA2, followed by chromosome 1p deletions and MED12 mutations. more...

Background Caldesmon gene (CALD1) plays an important role in many cellular functions. Some researchers have found the correlation between CALD1 expression and prognosis of gastrointestinal cancer (GI), but the association with tumor-infiltrating lymphocytes (TILs) still unclear. Methods The expression of CALD1 in different human tumor was analyzed by Oncomine and Tumor Immune Estimation Resource (TIMER) databases. The correlations between CALD1 and prognosis in types cancer were explored by Kaplan–Meier plotter and Gene Expression Profiling Interactive Analysis (GEPIA) databases. The association between CALD1 expression and tumor immune cell infiltration was further analyzed via TIMER and GEPIA databases. Results The CALD1 expressions in types cancer between tumor tissues and adjacent normal tissues were significantly different. The high expression of CALD1 was related with poor overall survival (OS) of patients with gastric cancer, especially in gastric cancer patients at N1, N2 and N3 stages. The expression of CALD1 was positively associated with immune-infiltrated, such as CD8+T cells, CD4+T cells, macrophages, neutrophils, and dendritic cells (DCs) in gastric cancer. Conclusions CALD1 was considerably a key role in prognosis of patients with gastric cancer. The expression level of CALD1 is significantly associated with immune-infiltrated in gastric cancer. Furthermore, CALD1 expression may be involved in regulating tumor-associated macrophages (TAMs), dendritic cells, exhausted T cells and regulatory T cells in gastric cancer. These findings suggest that CALD1 could be utilized as a marker of prognosis and immune infiltration in gastric cancer., Highlights • CALD1 plays an important role in immune infiltration in gastric cancer. • CALD1 can affect the prognosis in gastric cancer patients with lymph node metastasis. • CALD1 plays a vital role in immune escape in the gastric cancer microenvironment., CALD1, immune infiltration, gastric cancer, microenvironment more...

Aims Coronary microvascular smooth muscle cells (SMCs) respond to luminal pressure by developing myogenic tone (MT), a process integral to the regulation of microvascular perfusion. The cellular mechanisms underlying poor myogenic reactivity in patients with heart valve disease are unknown and form the focus of this study. Methods and results Intramyocardial coronary micro-arteries (IMCAs) isolated from human and pig right atrial (RA) appendage and left ventricular (LV) biopsies were studied using pressure myography combined with confocal microscopy. All RA- and LV-IMCAs from organ donors and pigs developed circa 25% MT. In contrast, 44% of human RA-IMCAs from 88 patients with heart valve disease had poor ( Conclusion These data demonstrate the first use of atrial and ventricular biopsies from patients and pigs to reveal that impaired coronary MT reflects a switch of viable SMCs towards a synthetic phenotype, rather than a loss of SMC viability. These arteries represent a model for further studies of coronary microvascular contractile dysfunction. more...

The goals of this study were to examine the cellular signaling pathways associated with the phosphorylation of caldesmon, the phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17), and the 20-kDa regulatory light chain of myosin (MLC20) induced by levobupivacaine in isolated rat aortas. The effects of genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, 1-butanol, and ML-7 HCl on levobupivacaine-induced contraction were assessed. The effect of genistein on the simultaneous calcium-tension curves induced by levobupivacaine was examined. The effects of GF109203X, genistein, PD98059 and extracellular signal-regulated kinase (ERK) siRNA on levobupivacaine-induced caldesmon phosphorylation were investigated. The effect of genistein on the ERK and tyrosine phosphorylation induced by levobupivacaine was examined. The effect of GF109203X, PD98059, Y-27632, SP600125, and ML-7 HCl on the levobupivacaine-induced phosphorylation of CPI-17 and MLC20 were investigated. Genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, ML-7 HCl, and 1-butanol attenuated levobupivacaine-induced contraction. Genistein caused a right downward shift of the calcium-tension curves induced by levobupivacaine. Genistein attenuated levobupivacaine-induced phosphorylation of protein tyrosine, ERK and caldesmon. PD98059, ERK siRNA and GF109203X attenuated levobupivacaine-induced caldesmon phosphorylation. GF109203X, Y-27632, SP600125, ML-7 HCl and PD98059 attenuated CPI-17 phosphorylation and MLC20 phosphorylation induced by levobupivacaine. These results suggest that levobupivacaine-induced caldesmon phosphorylation contributing to levobupivacaine-induced contraction is mediated by a pathway involving ERK, which is activated by tyrosine kinase or protein kinase C (PKC). The phosphorylation of CPI-17 and MLC20 induced by levobupivacaine is mediated by cellular signaling pathways involving PKC, Rho-kinase, and c-Jun NH2-terminal kinase or PKC, Rho-kinase, ERK, and myosin light chain kinase. more...

Endothelial barrier dysfunction leads to increased endothelial permeability and is an early step in the development of vascular inflammatory diseases such as atherosclerosis. Interferon-γ (IFN-γ), a proinflammatory cytokine, is known to cause increased endothelial permeability. However, the mechanisms by which IFN-γ disrupts the endothelial barrier have not been clarified. This study aimed to investigate how IFN-γ impairs the endothelial barrier integrity by specifically examining the roles of caldesmon, adherens junctions (AJs) and p38 mitogen-activated protein (MAP) kinase in IFN-γ-induced endothelial barrier dysfunction. IFN-γ exhibited a biphasic effect on caldesmon localization and both the structural organization and protein expression of AJs. In the early phase (4-8 h), IFN-γ induced the formation of peripheral caldesmon bands and discontinuous AJs, while AJ protein expression was unchanged. Interestingly, IFN-γ also stimulated caldesmon phosphorylation, resulting in actin dissociation from caldesmon at 8 h. Conversely, changes seen in the late phase (16-24 h) included cytoplasmic caldesmon dispersal, AJ linearization and junctional area reduction, which were associated with reduced membrane, cytoskeletal and total AJ protein expression. In addition, IFN-γ enhanced myosin binding to caldesmon at 12 h and persisted up to 24 h. Furthermore, inhibition of p38 MAP kinase by SB203580 did not reverse either the early or late phase changes observed. These data suggest that IFN-γ may activate signaling molecules other than p38 MAP kinase. In conclusion, our findings enhance the current understanding of how IFN-γ disrupts endothelial barrier function and reveal potential therapeutic targets, such as caldesmon and AJs, for the treatment of IFN-γ-associated vascular inflammatory diseases. more...

Background We sought to compare the sensitivity and specificity of 2 different caldesmon antibodies in differentiating leiomyosarcoma from other cutaneous spindle cell neoplasms. Methods Representative cutaneous spindle cell neoplasms were identified, including leiomyosarcoma, atypical fibroxanthoma, dermatomyofibroma and spindle cell squamous cell carcinoma. Immunohistochemistry was performed with antibodies directed toward caldesmon, smooth-muscle actin (SMA) and desmin. Sensitivity and specificity were calculated using grades from 3 independent observers. Results The sensitivity of caldesmon (Ventana) was 100% (95% CI 78.2%-100%) and the specificity was 8.3% (2.8%-18.4%). Because this stain appeared to be non-specific, additional testing was performed on the same set of specimens using a second caldesmon clone (H-caldesmon, Dako), which had a sensitivity of 53.9% (25.1%-80.8%) and specificity of 96.6% (88.1%-99.6%). The sensitivity and specificity of SMA were 85.7% (57.2%-98.2%) and 84.5% (72.6%-92.7%), respectively. The sensitivity of desmin was 53.3% (26.6%-78.7%) with a specificity of 100% (94.0%-100%). Conclusions The Ventana caldesmon clone is not specific to smooth muscle, a potential pitfall to laboratories using this clone. The staining pattern, sensitivity and specificity of the Dako H-caldesmon antibody clone are similar to results from prior studies. The sensitivity and specificity of the Dako clone support its use in smooth muscle identification as an additional marker in challenging cases. more...

Primary synovial sarcoma of the breast is very rare. We report a case of a 33-year-old woman, who had previously undergone a radical mastectomy, having been diagnosed with fusocellular breast carcinoma. Histopathology revealed a hypercellular lesion formed by spindle cells with storiform and herringbone patterns. Immunohistochemistry showed strong expression of vimentin and CD99, and focal bcl2, EMA, CK AE1-AE3, actin and desmin, with negativity for S100, CD34, CK7, CK14, CK19, hormone receptors, caldesmon and myosin. Molecular biology revealed the expression of the fusion product of the SS18 and SSX genes, indicative of the translocation t(X;18)(p11.2;q11.2), which confirmed the diagnosis of synovial sarcoma. more...

Non-muscle caldesmon (l-CaD) is involved in the regulation of actin cytoskeletal remodeling in the podosome formation, but its function in osteoclastogenesis remains to be determined. In this study, RANKL-induced differentiation of RAW264.7 murine macrophages to osteoclast-like cells (OCs) was used as a model to determine the physiological role of l-CaD and its phosphorylation in osteoclastogenesis. Upon RANKL treatment, RAW264.7 cells undergo cell-cell fusion into multinucleate, and TRAP-positive large OCs with a concomitant increase of l-CaD expression. Using gain- and loss-of-function in OC precursor cells followed by RANKL induction, we showed that the expression of l-CaD in response to RANKL activation is an important event for osteoclastogenesis, and bone resorption. To determine the effect of l-CaD phosphorylation in osteoclastogenesis, three decoy peptides of l-CaD were used with, respectively, Ser-to-Ala mutations at the Erk- and Pak1-mediated phosphorylation sites, and Ser-to-Asp mutation at the Erk-mediated phosphorylation sites. Both the former two peptides competed with the C-terminal segment of l-CaD for F-actin binding and accelerated formation of podosome-like structures in RANKL-induced OCs, while the third peptide did not significantly affect the F-actin binding of l-CaD, and decreased the formation of podosome-like structures in OCs. With the experiments using dephosphorylated and phosphorylated l-CaD mutants, we further showed that dephosphorylated l-CaD mutant facilitated RANKL-induced TRAP activity with an increased cell fusion index, whereas phosphorylated l-CaD decreased the TRAP activity and cell fusion. Our findings suggested that both the level of l-CaD expression and the extent of l-CaD phosphorylation play a role in RANKL-induced osteoclast differentiation. more...

Outflow resistance in the aqueous drainage tract distal to trabecular meshwork is potentially an important determinant of intraocular pressure and success of trabecular bypass glaucoma surgeries. It is unclear how distal resistance is modulated. We sought to establish: (a) multimodal 2-photon deep tissue imaging and 3-dimensional analysis of the distal aqueous drainage tract (DT) in transgenic mice in vivo and ex vivo; (b) criteria for distinguishing the DT from blood and lymphatic vessels; and (c) presence of a DT wall organization capable of contractility. DT lumen appeared as scleral collagen second harmonic generation signal voids that could be traced back to Schlemm’s canal. DT endothelium was Prox1-positive, CD31-positive and LYVE-1-negative, bearing a different molecular signature from blood and true lymphatic vessels. DT walls showed prominent filamentous actin (F-actin) labeling reflecting cells in a contracted state. F-actin co-localized with mesenchymal smooth muscle epitopes of alpha-smooth muscle actin, caldesmon and calponin, which localized adjacent and external to the endothelium. Our findings support a DT wall organization resembling that of blood vessels. This reflects a capacity to contract and support dynamic alteration of DT caliber and resistance analogous to the role of blood vessel tone in regulating blood flow. more...

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.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.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).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. more...

Based on an electron microscopic immunocytochemical study of the body wall musculature in a Gordian worm, the three regulatory muscle proteins troponin, calponin and caldesmon appear to be present. Given that no caldesmon homolog has been identified in the genome of invertebrates to date, the result seemed surprising. Moreover, troponin and calponin/caldesmon should exclude each other. However, in view of the fact that troponin has recently also been found in non-striated invertebrate muscle and the smooth muscle proteins calponin and closely related proteins can occur in invertebrate oblique muscle types, it seems possible that in the body wall of Gordian worms a muscle type occurs that combines the features of smooth and striated muscle. Similarities to the Caenorhabditis elegans muscle underscore the phylogenetic relationship between the ecdysozoan phyla Nematoda and Nematomorpha. more...

Introduction/Objective Inflammatory myofibroblastic tumor (IMT) is an uncommon spindle cell lesion that can involve various organs and occurs in multiple body sites. While older terminology (i.e. inflammatory pseudotumor) suggested otherwise, recent molecular studies point toward a neoplastic pathogenesis for IMTs. Herein, we report a case of an abdominal IMT and discuss the morphologic and immunohistochemical pitfalls pertaining to this entity. Methods A 75-year-old woman presented with complaints of generalized abdominal pain and distention. An abdominal CT scan showed multiple peritoneal masses, the largest of which measured 23 cm. Biopsy revealed compact fascicles of bland spindle cells exhibiting diffuse actin and caldesmon immunoreactivity, consistent with a spindle cell tumor with smooth muscle differentiation. Mitotic activity was low-to-unapparent. Surgical excision was performed. The cut surface of the tumor was tan-white with hemorrhagic foci. Histopathologic examination of the tumor showed elongated spindle cells set in a loose myxoid stroma rich in blood vessels and a mixed inflammatory infiltrate. Deeper sections of the tumor were more cellular, showing a similar morphology to that seen in the original biopsy, which was virtually devoid of inflammatory cells. Immunohistochemistry showed diffuse staining for desmin, caldesmon, smooth muscle actin, and ALK. FISH analysis showed ALK gene rearrangement in 52% of tumor cells, confirming the diagnosis of IMT. Results Studies in the literature show that IMTs express smooth muscle markers such as SMA (90%) and desmin (50%). However, immunoreactivity for caldesmon is rarely reported. ALK immunoreactivity is seen in about 35–60% of cases, and when gene rearrangement involving ALK is detected the diagnosis can be confirmed. Conclusion Historically, several terms have been used to describe IMTs, including inflammatory pseudotumor and inflammatory pseudosarcomatous fibromyxoid tumor. While the nomenclature consistently implies an inflammatory infiltrate, this tumor could have various morphological patterns with some areas showing very little to absent inflammation, as demonstrated in our case. Hence, making an accurate diagnosis could be challenging on a limited biopsy. Therefore, ALK testing should be included as part of the diagnostic workup of spindle cell neoplasms with smooth muscle differentiation. more...

A 41-year-old male patient refers to a painful tumor on the lateral side of the right leg. Ultrasonography visualized a predominantly hypoechoic well-delineated large heterogeneous mass in the subcutaneous tissue, showing an internal hyperechoic area. Magnetic resonance imaging (MRI) demonstrated an oval and lobulated tumor with well-defined margins at the anterolateral part of the leg, with contrast enhancement. The anatomopathological evaluation revealed a neoplasm with an extensive chondromyxoid matrix. The immunohistochemical study demonstrated positive vimentin (V9 clone), positive S-100 protein and negative desmin (D33 clone), smooth muscle actin (1A4), CD68 (KP1 clone) and caldesmon. The set of findings is compatible with myxoid liposarcoma. more...

Microgravity exposure results in vascular remodeling and cardiovascular dysfunction. Here, the effects of mitochondrial oxidative stress on vascular smooth muscle cells (VSMCs) in rat cerebral arteries under microgravity simulated by hindlimb unweighting (HU) was studied. Endoplasmic reticulum (ER)-resident transmembrane sensor proteins and phenotypic markers of rat cerebral VSMCs were examined. In HU rats, CHOP expression was increased gradually, and the upregulation of the PERK-eIF2α-ATF4 pathway was the most pronounced in cerebral arteries. Furthermore, PERK/p-PERK signaling, CHOP, GRP78 and reactive oxygen species were augmented by PERK overexpression but attenuated by the mitochondria-targeting antioxidant MitoTEMPO. Meanwhile, p-PI3K, p-Akt and p-mTOR protein levels in VSMCs were increased in HU rat cerebral arteries. Compared with the control, HU rats exhibited lower α-SMA, calponin, SM-MHC and caldesmon protein levels but higher OPN and elastin levels in cerebral VSMCs. The cerebral VSMC phenotype transition from a contractile to synthetic phenotype in HU rats was augmented by PERK overexpression and 740Y-P but reversed by MitoTEMPO and the ER stress inhibitors tauroursodeoxycholic acid (TUDCA) and 4-phenylbutyric acid (4-PBA). In summary, mitochondrial oxidative stress and ER stress induced by simulated microgravity contribute to phenotype transition of cerebral VSMCs through the PERK-eIF2a-ATF4-CHOP pathway in a rat model. more...

Bionanotehnologija je obsežno področje, ki dobiva v družbi zaradi številnih gospodarskih, okoljskih, zdravstvenih in socialnih razlogov vedno več pozornosti. Pomemben del bionanotehnologije predstavlja razvoj novih materialov, ki bi jih lahko uporabili za različne namene. S sintezno biologijo, s katero uvajamo v biotehnologijo inženirske pristope, lahko na osnovi naravnih proteinskih gradnikov in takšnih, ki ne obstajajo v naravi, načrtujemo in pripravljamo popolnoma nove bionanomateriale. Če želimo pripraviti bionanostrukture, ki se same sestavijo v načrtovano strukturo, je potrebno najprej natančno načrtovati gradnike, ki bodo sestavljali načrtovano nanostrukturo ali material, nato pa te osnovne gradnike pridobiti in okarakterizirati, na kar smo se osredotočili v tem magistrskem delu. Polipeptidne gradnike 34cald56, različico 34cald56 s fluorescenčnim proteinom in 3APH4 smo pripravili za namene gradnje dveh načrtovanih dvodimenzionalnih proteinskih mrež, ki imata različno geometrijo in se samosestavita iz enega polipeptidnega gradnika. Vse tri proteine smo uspešno pripravili v bakteriji E. coli, izolirali in očistili ter okarakterizirali z biokemijskimi metodami. Potrdili smo, da je sekundarna struktura polipeptidnih gradnikov 34cald56 in 3APH4 v skladu z načrtovano. Iz podatkov meritev cirkularnega dikroizma smo zaključili tudi, da je temperaturna stabilnost proteina 34cald56 višja od temperaturne stabilnosti 3APH4. Z meritvami dinamičnega sipanja svetlobe smo ugotovili, da se zvita polipeptida 34cald56 in 3APH4 v raztopini samosestavita v dimere in večje nanostrukture, ki postanejo netopne in se oborijo iz raztopine, lahko pa se sestavita tudi v topne nanostrukture, ki so v primeru 34cald56 velike približno 120 nm, v primeru 3APH4 pa 60 nm. Bionanotechnology is a broad field that is receiving increasing attention in society for many economic, environmental, health and social reasons. An important part of bionanotechnology is the development of new materials that could be used for various purposes. With synthetic biology, which introduces engineering approaches to biotechnology, we can design and prepare completely new bionanomaterials based on natural protein building blocks and those that do not exist in nature. In order to prepare bionanostructures that self-assemble into the planned structure, it is necessary to first carefully design the building blocks for the planned nanostructure or material, and then to acquire and characterize these basic building blocks, which is the focus of this master's thesis. The 34cald56, the 34cald56 version with fluorescence protein and the 3APH4 polypeptide building blocks were prepared for the purpose of constructing two designed two-dimensional protein lattices having different geometries and self-assembling from one polypeptide building block. All three proteins were successfully produced in bacteria E. coli, isolated and characterized with biochemical methods. We have confirmed that the secondary structure of the 34cald56 and the 3APH4 polypeptide building blocks is in accordance with the designed. From the data of the circular dichroism measurements, we concluded that the temperature stability of the protein 34cald56 is higher than the temperature stability of the 3APH4. Measurements of the dynamic light scattering have revealed that the folded polypeptides 34cald56 and 3APH4 in solution self-assemble into dimers and larger nanostructures that become insoluble and precipitate out of solution, but can also be assembled into soluble nanostructures, which in the case of 34cald56 are large around 120 nm, and in the case of 3APH4 about 60 nm. more...

In early studies on smooth muscle, I described a crude myosin fraction (CMF) in which self-assembly of myosin filaments was observed. For the first time, the 14-nm periodicity stemming from regular arrangement of myosin heads on the filament surface was observed (Sobieszek in J Mol Biol 70:741-744, 1972). In this fraction, we also observed formation of long ribbon-shaped aggregates exhibiting a 5.6-nm periodicity, characteristic of tropomyosin (TM) paracrystals (Sobieszek and Small in Phil Trans R Soc Lond B 265:203-212, 1973). We therefore concluded that these ribbons were made of TM and they might be related to the myosin ribbons observed in electron micrographs (EM) of intact smooth muscle (Lowy and Small in Nature 227:46-51, 1970; Small and Squire in Mol Biol 67:117-149, 1972). Subsequently, Small (J Cell Sci 24:327-349, 1977) concluded that the ribbons observed in the EM sections were an artifact, but their observation in the CMF was not addressed. I have now revisited two aspects of the above studies. Firstly, based on my new multi-angle laser-scattering data and considering the length and stability of the building unit for the filament, a myosin trimer fit better to the previously proposed helical structure. Secondly, after two decades of systematic examinations of protein compositions in multiple smooth muscle extracts and isolated filaments, I concluded that the ribbons were made of caldesmon and not TM. Thirdly, actin-activated ATPase activity measurements indicated that modulation of this activity (by CaD and TM) was synergistic, cooperative and depended on myosin to actin ratio. more...

Background Osteoclasts (OCs) are motile multinucleated cells derived from differentiation and fusion of hematopoietic progenitors of the monocyte-macrophage lineage that undergo a multistep process called osteoclastogenesis. The biological function of OCs is to resorb bone matrix for controlling bone strength and integrity, which is essential for bone development. The bone resorption function is based on the remodelling of the actin cytoskeleton into an F-actin-rich structure known as the sealing zone for bone anchoring and matrix degradation. Non-muscle caldesmon (l-CaD) is known to participate in the regulation of actin cytoskeletal remodeling, but its function in osteoclastogenesis remains unclear. Methods/results In this study, gain and loss of the l-CaD level in RAW264.7 murine macrophages followed by RANKL induction was used as an experimental approach to examine the involvement of l-CaD in the control of cell fusion into multinucleated OCs in osteoclastogenesis. In comparison with controls, l-CaD overexpression significantly increased TRAP activity, actin ring structure and mineral substrate resorption in RANKL-induced cells. In contrast, gene silencing against l-CaD decreased the potential for RANKL-induced osteoclastogenesis and mineral substrate resorption. In addition, OC precursor cells with l-CaD overexpression and gene silencing followed by RANKL induction caused 13% increase and 24% decrease, respectively, in cell fusion index. To further understand the mechanistic action of l-CaD in the modulation of OC fusion, atomic force microscopy was used to resolve the mechanical changes of cell spreading and adhesion force in RANKL-induced cells with and without l-CaD overexpression or gene silencing. Conclusions l-CaD plays a key role in the regulation of actin cytoskeletal remodeling for the formation of actin ring structure at the cell periphery, which may in turn alter the mechanical property of cell-spreading and cell surface adhesion force, thereby facilitating cell-cell fusion into multinucleated OCs during osteoclastogenesis. Electronic supplementary material The online version of this article (10.1186/s12929-019-0505-1) contains supplementary material, which is available to authorized users. more...

The testes of sexually mature males of six mammalian species (men, bulls, boars, rats, mice, guinea pigs) have been studied using biochemical as well as light and electron microscopical techniques, in particular immunolocalizations. In these tissues, the peritubular walls represent lamellar encasement structures wrapped around the seminiferous tubules as a bandage system of extracellular matrix layers, alternating with monolayers of very flat polyhedral “lamellar smooth muscle cells” (LSMCs), the number of which varies in different species from 1 to 5 or 6. These LSMCs are complete SMCs containing smooth muscle α-actin (SMA), myosin light and heavy chains, α-actinin, tropomyosin, smoothelin, intermediate-sized filament proteins desmin and/or vimentin, filamin, talin, dystrophin, caldesmon, calponin, and protein SM22α, often also cytokeratins 8 and 18. In the monolayers, the LSMCs are connected by adherens junctions (AJs) based on cadherin-11, in some species also with P-cadherin and/or E-cadherin, which are anchored in cytoplasmic plaques containing β-catenin and other armadillo proteins, in some species also striatin family proteins, protein myozap and/or LUMA. The LSMC cytoplasm is rich in myofilament bundles, which in many regions are packed in paracrystalline arrays, as well as in “dense bodies,” “focal adhesions,” and caveolae. In addition to some AJ-like end-on-end contacts, the LSMCs are laterally connected by numerous vertical AJ-like junctions located in variously sized and variously shaped, overlapping (alter super alterum) lamelliform cell protrusions. Consequently, the LSMCs of the peritubular wall monolayers are SMCs sensu stricto which are laterally connected by a novel architectonic system of arrays of vertical AJs located in overlapping cell protrusions. more...

Sonic hedgehog ( Shh) is crucial for organogenesis in the foregut. This study investigated the function of Shh at the late-gestational stage; during which, the esophagus continues to differentiate. We established cytokeratin 14 ( CK14)-Cre;Shhfl/fl mice in which the down-regulation of Shh in the epithelium occurred at approximately the same time as esophageal muscle conversion. Hematoxylin and eosin and immunohistochemical staining, with antibodies against keratin 14, Shh, patched 1 (Ptch1), Gli1, proliferating cell nuclear antigen (PCNA), α-smooth muscle actin (αSMA), high-molecular-weight caldesmon (hCD), myogenin, paired box 7 (Pax7), β3-tubulin, and protein gene product 9.5 (PGP9.5), was performed to detect specific tissue dysplasia. Organ culture was conducted in vitro, and total mRNA was extracted to determine the transcriptional dysregulation. The esophagus of CK14-Cre;Shhfl/fl mice developed into an independent tube with an obvious dilatation at postnatal d 0.5. The number of cell layers and the expression of PCNA were decreased in mutant mice, compared with those in wild-type mice. The expression of hCD declined progressively in the middle, distal, and lower esophageal sphincter levels of the mutant esophagus from embryonic d 17.5, compared with the expression in wild-type littermates. Pax7 accumulation and myogenin reduction in mutant mice indicated that esophageal skeletal-myoblast progression was blocked. RNA sequencing analysis revealed a significant down-regulation of genes involved in proliferation and muscular motivation in CK14-Cre;Shhfl/fl mice. Thus, loss of Shh at the late-gestational stage leads to megaesophagus with reduced proliferation and a muscle development disorder in mice.-Jia, X., Min, L., Zhu, S., Zhang, S., Huang, X. Loss of sonic hedgehog gene leads to muscle development disorder and megaesophagus in mice. more...

Migration of vascular smooth muscle cells (VSMCs) from the media to intima constitutes a critical step in the development of proliferative vascular diseases. To elucidate the regulatory mechanism of VSMCs motility, the roles of caldesmon (CaD) were investigated previously. CaD is an actin-binding protein dynamically regulating cytoskeleton structure. In this study, the potential role of CaD in mediating proliferation and motility of VSMCs were discussed. First, structural effect of CaD on cytoskeleton integrity was analyzed with CaD knock-down; second, the proliferation of VSMCs was measured in CaD knock-down and control cells; third, the specific role of CaD on VSMCs motilities was evaluated with in vitro migration and invasion assays. We found that CaD is an integral component to maintain cytoskeleton integrity of VSMCs. Our data indicated that CaD suppression does not show significant influence on VSMCs proliferation, but negatively modulates the motilities of VSMCs, and CaD depletion would significantly facilitate migration and invasion of VSMCs. more...

Muscles of bivalve molluscs have double calcium regulation — myosin-linked and actin-linked. While the mechanism of myosin-linked regulation is sufficiently studied, there is still no consensus on the mechanism of actin-linked regulation. Earlier we showed a high degree of Ca 2 + -sensitivity of thin filaments from the adductor muscle of the mussel Crenomytilus grayanus (Mytiloida). In order to elucidate the nature of this regulation, we isolated the fraction of minor proteins from the mussel thin filaments, which confers Ca 2 + -sensitivity to reconstituted actomyosin–tropomyosin. Proteins of this fraction, ABP-19, ABP-20, and ABP-28, were chromatographically purified and identified. According to the results of mass spectrometry and Western blot analysis, as well as by their functional properties, these mussel actin-binding proteins appeared to correspond to the troponin components from the skeletal muscles of vertebrates (TnC, TnI and TnT). The reconstituted mussel troponin complex confers to actomyosin–tropomyosin more than 80% Ca 2 + -sensitivity. The in vivo molar ratio of actin/tropomyosin/troponin was calculated to be 7:1:0.5, i.e., the content of troponin in mussel thin filaments is two times lower than in thin filaments of skeletal muscles of vertebrates. These data demonstrate that troponin-like regulation found in the catch muscle of the mussel C. grayanus is present at least in two suborders of bivalves: Pectinoida and Mytiloida. more...

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. more...

A low-grade sinonasal sarcoma with neural and myogenic features has recently been defined and characterized. We present a case of this morphologic entity and discuss the differential diagnostic considerations, immunophenotypic character, electron microscopy (EM) findings and positron emission tomography (PET) appearance. We propose an alternative hypothesis of its origin on the basis of immunophenotypic and EM features. A 59-year-old Caucasian male was found to have a mass filling the right ethmoid sinus. On PET, the lesion had a maximum standardized uptake value of 2.9, which is of borderline intensity for sarcoma. Pathologic examination showed morphologic features similar to those reported for low-grade sinonasal sarcoma with neural and myogenic features. The tumor was positive for S100, β-catenin, caldesmon, and vimentin and negative for smooth muscle actin, muscle-specific actin, desmin, myogenin, and pankeratin. Positivity for β-catenin raises the alternative possibility of fibroblastic differentiation instead of the proposed myogenic differentiation. EM findings were also consistent with fibroblastic cells. more...

Nodular fasciitis is a pseudosarcomatous reactive process composed of fibroblasts and myofibroblasts, and it is most common in the upper extremities. Nodular fasciitis of the external auditory canal is rare. To the best of our knowledge, less than 20 cases have been reported to date. We present a case of nodular fasciitis arising in the cartilaginous part of the external auditory canal. A 19-year-old man complained of an auricular mass with pruritus. Computed tomography showed a 1.7 cm sized soft tissue mass in the right external auditory canal, and total excision was performed. Histologic examination revealed spindle or stellate cells proliferation in a fascicular and storiform pattern. Lymphoid cells and erythrocytes were intermixed with tumor cells. The stroma was myxoid to hyalinized with a few microcysts. The tumor cells were immunoreactive for smooth muscle actin, but not for desmin, caldesmon, CD34, S-100, anaplastic lymphoma kinase, and cytokeratin. The patient has been doing well during the 1 year follow-up period. more...

We describe a primary leiomyosarcoma of bone located in the distal fibula in a 67- year-old man. Plain radiographs and computer tomography scan revealed a lytic destructive lesion in the distal metaepiphyseal region of the left fibula with little involvement of the surrounding soft tissues. The lesion was composed of proliferating spindle-shaped cells with very slim cytoplasm and narrow oval cigar shaped nuclei. Immunohistochemistry studies demonstrated a strong positivity for actin and desmin, and weak positivity for caldesmon. more...

Smooth muscle differentiated adipose tissue-derived stem cells are a valuable resource for regeneration of gastrointestinal tissues, such as the gut and sphincters. Hypoxia has been shown to promote adipose tissue-derived stem cells proliferation and maintenance of pluripotency, but the influence of hypoxia on their smooth myogenic differentiation remains unexplored. This study investigated the phenotype and contractility of adipose-derived stem cells differentiated toward the smooth myogenic lineage under hypoxic conditions. Oxygen concentrations of 2%, 5%, 10%, and 20% were used during differentiation of adipose tissue-derived stem cells. Real time reverse transcription polymerase chain reaction and immunofluorescence staining were used to detect the expression of smooth muscle cells-specific markers, including early marker smooth muscle alpha actin, middle markers calponin, caldesmon, and late marker smooth muscle myosin heavy chain. The specific contractile properties of cells were verified with both a single cell contraction assay and a gel contraction assay. Five percent oxygen concentration significantly increased the expression levels of α-smooth muscle actin, calponin, and myosin heavy chain in adipose-derived stem cell cultures after 2 weeks of induction (p more...

Malign tümörlerde damar invazyonu varlığı, prognostik öneminin olması yanısıra adjuvan KT ve/veya RT kararını yönlendirmesi açısından da oldukça önemlidir. Bu çalışmada intrahepatik kolanjiokarsinomlarda prognostik açıdan ve evreleme açısından büyük önem arz eden damar invazyonunun h-CD ve CK19 ikili immünohistokimyası ile daha kolay fark edilmesi amaçlanmış olup, bu yöntemin kullanışlı bulunması sonucunda rutin pratiğe dâhil olması öngörülmüştür.Gazi Üniversitesi Tıbbi Patoloji Anabilim Dalı'nda, 2009-2018 yılları arasında intrahepatik kolanjiokarsinom tanısı alan 21 hastanın 23 parsiyel hepatektomi materyaline ait 82 blokta bulunan 226 odak ele alınmıştır. Yirmi bir hastaya ait yaş, cinsiyet, tümör yerleşim yeri, tümör çapı, tanının ilk raporlandığı tarihte görülen vasküler invazyon, perinöral invazyon ve lenf nodu metastazı varlığı, T, N, M değerleri ve zemindeki viral hepatit varlığı incelenmiştir. Olası vasküler invazyon odaklarının hepatektomi materyalindeki lokalizasyonu, histolojik özellikleri gözden geçirilmiş ve bu odaklar 3 tipe ayrılmıştır: Tip 1, tümörün bir damar lümeninde olduğunu yüksek olasılıkla düşündüren odaklar, Tip 2, tümör odağının etrafında, damar duvarı olduğundan şüphelenilen, tümör odağını `manşonvari örüntüde tamamıyle veya kısmen sarmalayan`, fakat boş lümen veya eritrosit varlığının görülmediği odaklar, Tip3, uygulanan immünohistokimyasal yöntem sonrası ortaya çıkan odaklar. Ayrıca h-CD'nin spesifitesini araştırma amacıyla tümör dışı karaciğer dokularından hazırlanan kesitlere, h-CD tekli olarak boyanmış olup bu kesitlerdeki vasküler elemanların ve safra duktuslarının boyanma durumları değerlendirilmiştir. Saptanan odakların çapları mikroskopik olarak ölçülmüş ve kaydedilmiştir.Çalışmamızda 226 odağın %41,3'ü Tip 3, %39,3'ü Tip 2 ve %19,4'ü Tip 1 odak tipindedir. Odakların çoğu (%63,7) intrahepatik yerleşimlidir. İkili İHK uygulaması sonrası vasküler invazyonun görüldüğü vaka sayısı toplam 14 olup, bunların 3'ü ilk raporlamada vasküler invazyon varlığı görülmemiş vakalardır. İlk raporlamada vasküler invazyon görülmeyen vakalarda ikili İHK uygulaması sonrası vasküler invazyonun görüldüğü odak tipleri incelenmiş, bu odakların %66,1'inin Tip 3, %25'inin Tip 2 ve %8,9'unun Tip 1 tipinde oldukları saptanmıştır. İlk raporlamada vasküler invazyon görülmemesi nedeniyle histolojik evresi pT1 ve prognostik evresi I olan 3 olgunun 2'sinde uygulama sonrası vasküler invazyon saptanması nedeniyle histolojik evreleri pT2'ye, prognostik evreleri ise II'ye yükselmiştir.h-CD'nin tekli olarak boyandığı tümör dışı normal karaciğer dokusunda görülen safra duktuslarının hiçbirinde h-CD ile boyanma saptanmamıştır. Hepatik arterlerin ve portal venlerin tamamında h-CD ile boyanma mevcuttur. Hepatik venlerin %13,2'sinde h-CD ile boyanma gözlenirken, %86,8'inde boyanma saptanmamıştır. Çalışmamızın sonucunda, H&E boyalı lamlarda kuşkulanılamayacak, ancak yardımcı teknikler ile ortaya çıkarılabildiğini gösterdiğimiz damar invazyonu odakları fark edilmiştir. H&E boyalı lamlarda ilk raporlamada fark edilmeyen, ikili İHK uygulaması ile vasküler invazyon varlığının gösterildiği, bunun sonucunda histolojik ve prognostik evrenin yükseltildiği hastalar mevcuttur. Vasküler invazyonun ikili İHK kullanımı sonrası fark edilip histolojik ve prognostik evrenin yükseltildiği hastalarda en fazla görülen odak tipi `Tip 3`tür. İkili İHK ile fark edilen `Tip 3` odaklar H&E boyalı lamlara dönülüp tekrar bakıldığında, bu odakların bir kısmının, tümör grubu çevresinde eozinofilik bir sınır bulundurduğu izlenmiş olup bu alan `çevre dokudan mesafeli bir sınırla ayrılma (distant circumscribed stroma)` olarak isimlendirilmiştir. h-CD portal ven ve hepatik arterin tüm dallarında pozitif boyanırken, hepatik ven dallarında aynı etkiyi göstermemektedir. h-CD, hepatik venlerde aranan vasküler invazyon için uygun bir belirteç olmadığı düşünülmüştür. h-CD safra duktuslarını hiçbir şekilde boyamamaktadır.Anahtar kelimeler: Vasküler invazyon, h-Caldesmon, intrahepatik kolanjiokarsinom The presence of vascular invasion in malignant tumors is very important in directing the decision of adjuvant chemotherapy and / or radiotherapy besides being a prognostic precursor.It was aimed to make the vascular invasion, which is a great prognostically and staging aspect of intrahepatic cholangiocarcinomas, easier to recognize with h-CD and CK19 dual immunohistochemistry and it is guessed that this method will be included in routine practice as a result of its usefulness in this study.Eighty-two slides from 21 patients' 23 partial hepatectomy specimens which are diagnosed intrahepatic cholangiocarcinoma, diagnosed from 2009 to 2018 at the Pathology Department of Gazi University, were reviewed and examined which revealed 226 probable vascular invasion foci. Age, gender, tumor site, tumor diameter, TNM stage, vascular invasion, perineural invasion and metastatic lymph nodes at the first report date and the viral hepatitis condition for these 21 patients were obtained. Additionally, anatomic site and histologic features of vascular invasion foci were reviewed and divided into 3 types: Type 1, foci that would likely suggest that the tumor focus is in a vessel lumen, Type 2, foci in which there is an outer wall that can not be foreseeable without an ancillary technique suspected of having a vessel wall around the tumor center,and, Type 3, a focus which could be detected only with the immunohistochemical procedures mentioned. In addition, to investigate the specificity of h-CD, h-CD was stained in sections of non-tumor liver tissue, and the staining status of vascular elements and bile ducts in these sections were evaluated. Diameters of the detected foci were measured and recorded microscopically.In our study, out of 226 foci, 41,3% were type 3, 39,3% were type 2 and 19,4% were type 1. 79,6% of Type 3 foci, and 63,6% of Type 1 foci are intrahepatic. Of the type 2 foci, 47,2% are intrahepatic. The number of cases in which vascular invasion has been observed after double IHC is 14, of which 3 have no vascular invasion in the first report. All of the 11 cases which vascular invasion was noted in the first report, the presence of vascular invasion after double IHC was confirmed. When the focus types in which vascular invasion is observed after double IHC stain, 66.1% of these foci were found to be Type 3, 25% were Type 2 and 8.9% were Type 1. Two cases which their histologic stages were pT1 and prognostic stage I, were histologically evolved pT2 because of presence of vascular invasion after dual immunohistochemistry.No h-CD staining was detected in any of the bile ducts seen in the non-tumoral liver tissue when h-CD stained singly. All of the hepatic arteries and portal veins are stained with h-CD. h-CD staining was observed in 13,2% of the hepatic venules, whereas 86,8% staining was not observed.As a result of our study, we have noticed foci of vessel invasion that can not be suspected in H&E painted slides but which can be revealed by ancillary techniques. There are patients in which the histological and prognostic stage are elevated by demonstrating the presence of vascular invasion with dual IHC stain. Type 3 is the most common type of vascular invasion foci seen in patients with histologic and prognostic elevation after dual IHC. When we turn back to H&E stained slides for `Type 3` foci recognized by dual IHC, some of these foci were observed to have an eosinophilic border around the tumor group, and this area was named `distant circumscribed stroma`. While h-CD is positive in all branches of portal vein and hepatic artery, it does not show the same effect in hepatic vein branches. h-CD is thought not to be a suitable marker for vascular invasion in hepatic venules. h-CD is not positive in bile ducts any way.Key words: Vascular invasion, h-Caldesmon, intrahepatic cholangiocarcinoma 91 more...

Cutaneous myopericytoma is a rarely reported mesenchymal neoplasm with a benign biologic behavior. It is seen more commonly in males and typically occurs in adults on the distal extremities. To the best of our knowledge, there are only 13 reports describing 45 cases of cutaneous myopericytoma in the literature. The 3 cases in this report expand the clinical presentation and reinforce the histopathologic features of cutaneous myopericytoma. While the clinical presentation in 2 cases (located on the scalp and heel) was in keeping with that reported previously of a slow-growing painless firm nodule, the third case, located on the dorsal wrist, presented as a scaly keratotic nodule. Histopathologic examination of all 3 cases revealed an unencapsulated dermal nodule with concentric perivascular arrangement of plump, spindle-shaped myoid cells admixed with thin-walled blood vessels. Immunohistochemical staining revealed the lesional cells to be actin- (3/3) and caldesmon- (2/3) positive and negative for other smooth muscle markers, compatible with perivascular myopericytic differentiation. more...

Contraction of human pulmonary artery smooth muscle cells (HPASMC) isolated from pulmonary arterial hypertensive (PAH) and normal (non-PAH) subject lungs was determined and measured with real-time electrical impedance. Treatment of HPASMC with vasoactive peptides, endothelin-1 (ET-1) and bradykinin (BK) but not angiotensin II, induced a temporal decrease in the electrical impedance profile mirroring constrictive morphological change of the cells which typically was more robust in PAH as opposed to non-PAH cells. Inhibition with LIMKi3 and a cofilin targeted motif mimicking cell permeable peptide (MMCPP) had no effect on ET-1 induced HPASMC contraction indicating a negligible role for these actin regulatory proteins. On the other hand, a MMCPP blocking the activity of caldesmon reduced ET-1 promoted contraction pointing to a regulatory role of this protein and its activation pathway in HPASMC contraction. Inhibition of this MEK/ERK/p90RSK pathway, which is an upstream regulator of caldesmon phosphorylation, reduced ET-1 induced cell contraction. While the regulation of ET-1 induced cell contraction was found to be similar in PAH and non-PAH cells, a key difference was the response to pharmacological inhibitors and to siRNA knockdown of Rho kinases (ROCK1/ROCK2). The PAH cells required much higher concentrations of inhibitors to abrogate ET-1 induced contractions and their contraction was not affected by siRNA against either ROCK1 or ROCK2. Lastly, blocking of L-type and T-type Ca2+ channels had no effect on ET-1 or BK induced contraction. However, inhibiting the activity of the sarcoplasmic reticulum Ca2+ ATPase blunted ET-1 and BK induced HPASMC contraction in both PAH and non-PAH derived HPASMC. In summary, our findings here together with previous communications illustrate similarities and differences in the regulation PAH and non-PAH smooth muscle cell contraction relating to calcium translocation, RhoA/ROCK signaling and the activity of caldesmon. These findings may provide useful tools in achieving the regulation of the vascular hypercontractility taking place in PAH. more...

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. more...

High molecular weight caldesmon (H-caldesmon) is the protein that binds to actin and tropomyosin regulating smooth muscle contraction function.1 It is mainly expressed in smooth muscle cells and the research data showed the specificity of smooth muscle is higher than that of desmin and actin.2 In clinical diagnosis, H-caldesmon can be used to identify myofibroblastic tumors from true leiomyoma and to differentiate uterine smooth muscle tumors from endometrial stromal sarcoma combined with CD10. Resently, we encountered a case of ovarian adult granulosa cell tumor. This article is protected by copyright. All rights reserved. more...

Solitary fibrous tumor (SFT) is uncommon in the oral cavity, and there are no reports in children. We report a case of a large SFT affecting the upper lip of a one year-old boy. The tumor was characterized by proliferation of spindle and multinucleated stellate-shaped cells that were intermingled between thin and thick collagen fibers. The tumor cells were diffusely positive for CD34, Bcl-2, CD99, and negative for specific muscle actin, smooth muscle actin, calponin, and caldesmon. Ki-67 labeling was less than 1%. The patient remains well after tumor excision. more...

A 3-year-old male black-tailed prairie dog (Cynomys iudovicianus) was presented with a dome-shaped cutaneous mass over the left femur. Microscopically, the mass was encapsulated and composed of proliferating spindle cells arranged in haphazard, interlacing bundles. There were vascular structures within the mass and some spindle cells had transitioned from the peripheral regions of the vascular wall. Immunohistochemically, the cells expressed vimentin, α-smooth muscle actin, desmin and heavy caldesmon. Based on these findings, the mass was diagnosed as a cutaneous angioleiomyoma. To the best of our knowledge, this is the first case of cutaneous angioleiomyoma in a black-tailed prairie dog. more...

An exuberant corneal pannus usually develops in adults with a history of surgery or trauma in the anterior central stroma and appears as a glistening, vascularized, moderately elevated, well circumscribed white nodule. We describe a 78-year-old woman with such a pannus, which in the past has typically been referred to as keloidal or hypertrophic. The involved eye had only light perception, and she underwent a penetrating keratoplasty that improved her vision to 20/100. Histopathologic and immunohistochemical evaluations of a the specimen disclosed a reactive spindle cell stromal proliferation of myofibroblasts that were smooth muscle actin positive with a low Ki67 proliferation index. Desmin, caldesmon, and calponin were negative, in keeping with the incomplete myofilamentary differentiation of a myofibroblast. There was a generous admixture of CD68/163-positive histiocytes and dispersed C3/5-positive T-lymphocytes. An absence of CD138- and IgG4-positive plasma cells ruled out an IgG4-related disease. For a lesion to be keloidal, the collagen must have a thick hyaline character, sharp edges, and a sparsity of intervening cells and vessels. A hypertrophic pannus would be composed of large swollen cells not necessarily increased in number. We therefore recommend adoption of the term hyperplastic for lesions like that described here because of the obvious increase in cellularity from proliferating myofibroblasts and the lack of true keloidal collagen. more...

Myopericytoma is a benign mesenchymal neoplasm thought to comprise part of a spectrum of perivascular myoid cell neoplasms with myofibroma, angioleiomyoma, and glomus tumor. We describe 2 such neoplasms involving the urinary tract: 1 incidentally identified in the kidney of a 59-year-old woman and 1 in the urinary bladder of a 52-year-old woman who presented with urinary frequency and dysuria. Histologically, the bladder tumor was composed of numerous blood vessels surrounded by plump perivascular myoid cells, as in subcutaneous myopericytoma. The renal tumor showed similar morphology centrally and a symplastic glomus tumor-like growth pattern at the periphery. Immunohistochemically, both tumors were reactive for markers of smooth muscle differentiation, such as smooth muscle actin and caldesmon/calponin but negative for CD34, cathepsin K, and S100 protein. Both patients are free of disease 14 and 39 months after resection, respectively. Our findings broaden the morphologic spectrum of myopericytoma. more...

A 7-year-old boy was presented with a long-standing slowly growing mass of the left supraorbital area. A biopsy specimen revealed a bland spindle cell proliferation with scattered polygonal cells with acidophilic cytoplasm and cross-striations. Our differential diagnosis included rhabdomyoma of fetal type, leiomyoma with trapping of regenerating skeletal muscle elements, and rhabdomyomatous mesenchymal hamartoma of the skin. Immunohistochemistry demonstrated strong positivity of myoglobin and desmin as well as negativity of caldesmon, suggesting skeletal muscle lineage. The excisional specimen confirmed our diagnosis of cutaneous fetal rhabdomyoma of intermediate type. Additional immunostaining performed on the excisional specimen showed strong Wilms Tumor 1 but only a very faint and focal p63 expression. more...

Background Studies have shown that shock-induced vascular hyporeactivity is associated with the decrease in 20-kDa myosin light chain (MLC20) phosphorylation. Whether and how a non-MLC20 phosphorylation pathway participates in the regulation of vascular reactivity after shock is not known. Methods With superior mesentery artery (SMA) obtained from rats in hemorrhagic shock and hypoxia-treated SMA, the regulatory effect of platelet-derived growth factor (PDGF) on vascular reactivity and the roles of caldesmon, 27-kDa heat shock protein (HSP27), extracellular signal–regulated protein kinase (Erk), and p38 mitogen–activated protein kinase (MAPK), the main molecules that are involved in the non-MLC20 phosphorylation pathway of the regulation of smooth-muscle contraction, were investigated. Results PDGF (40–100 ng/mL) increased the vascular reactivity after shock in a dose-dependent manner, whereas it did not increase the MLC20 phosphorylation in a dose-dependent manner. PDGF with concentration more than 60 ng/mL did not further increase the MLC20 phosphorylation, whereas upregulated the phosphorylation of HSP27, Erk, and p38MAPK, and the activity of myosin adenosine triphosphatase in SMAs, and downregulated the phosphorylation of caldesmon. p38MAPK antagonist, SB203580, not only antagonized PDGF-induced increase in the phosphorylation of HSP27, but also antagonized PDGF-induced decrease in the phosphorylation of caldesmon, whereas Erk antagonist, PD98059, only antagonized PDGF-induced decrease in the phosphorylation of caldesmon. Conclusions These findings suggested that a non-MLC20 phosphorylation pathway participated in the regulation of vascular reactivity after shock. Caldesmon- and HSP27-mediated change in myosin adenosine triphosphatase activity and Erk and p38MAPK played an important role in this process. These findings may provide some potential targets for the treatment of vascular hyporeactivity after shock. more...

Remodeling of arterioles is a pivotal event in the manifestation of many inflammation-based cardio-vasculopathologies, such as hypertension. During these remodeling events, vascular smooth muscle cells (VSMCs) switch from a contractile to a synthetic phenotype. The latter is characterized by increased proliferation, migration, and invasion. Compounds with anti-inflammatory actions have been successful in attenuating this phenotypic switch. While the vast majority of studies investigating phenotypic modulation were undertaken in VSMCs isolated from large vessels, little is known about the effect of such compounds on phenotypic switch in VSMCs of microvessels (microVSMCs). We have recently characterized a novel homoisoflavonoid that we called 7-O-methylpunctatin (MP). In this study, we show that MP decreased FBS-induced cell proliferation, migration, invasion, and adhesion. MP also attenuated adhesion of THP-1 monocytes to microVSMCs, abolished FBS-induced expression of MMP-2, MMP-9, and NF-&kappa, B, as well as reduced activation of ERK1/2 and FAK. Furthermore, MP-treated VSMCs showed an increase in early (myocardin, SM-22&alpha, SM-&alpha, ) and mid-term (calponin and caldesmon) differentiation markers and a decrease in osteopontin, a protein highly expressed in synthetic VSMCs. MP also reduced transcription of cyclin D1, CDK4 but increased protein levels of p21 and p27. Taken together, these results corroborate an anti-inflammatory action of MP on human microVSMCs. Therefore, by inhibiting the synthetic phenotype of microVSMCs, MP may be a promising modulator for inflammation-induced arteriolar pathophysiology. more...

Thin filament-associated proteins such as calponin, caldesmon, and smoothelin are believed to regulate acto-myosin interaction and thus, muscle contraction. Oxidative stress has been found to affect the normal contractile behavior of smooth muscle and is involved in the pathogenesis of a number of human diseases such as diabetes mellitus, hypertension, and atherosclerosis. However, very little is known about the effect of oxidative stress on the expression of smooth muscle contractile proteins. The aim of the current study is to investigate the effect of oxidative stress on the expression of thin filament-associated proteins in rat gastric smooth muscle. Single smooth muscle cells of the stomach obtained from Sprague-Dawley rats were used. Muscle cells were treated with hydrogen peroxide (H2O2) (500 μM) for 30 min or the peroxynitrite donor 3-morpholinosydnonimine (SIN-1) (1 mM) for 90 min to induce oxidative stress. Calponin, caldesmon, and smoothelin expressions were measured via specifically designed enzyme-linked immunosorbent assay. We found that exposure to exogenous H2O2 or incubation of dispersed gastric muscle cells with SIN-1 significantly increased the expression of calponin, caldesmon, and smoothelin proteins. In conclusion: oxidative stress increases the expression of thin filament-associated proteins in gastric smooth muscle, suggesting an important role in gastrointestinal motility disorders associated with oxidative stress. more...