Desmin-positive Cells Research Articles

Leptin, pre-existing vascular disease, and increased arteriovenous fistula maturation failure in dialysis patients

The adipocytokine leptin is an independent cardiovascular risk factor and exerts proatherogenic effect. Pre-existing vascular disease is an important cause of arteriovenous fistula (AVF) maturation failure. We explored the association between serum leptin, pre-existing vascular disease, and AVF maturation failure in incident hemodialysis patients. Vein samples from 62 patients were collected at the time of AVF creation. Pre-existing vascular disease was evaluated with histologic changes and immunohistochemical characteristics of cellular phenotypes in intima. AVF maturation failure was defined as an AVF that could not be used successfully by the third month after its creation. The prevalence of body mass index ≥30kg/m2 was 17%, and AVF maturation failure occurred in 28 (45%) patients. Patients within the highest leptin tertile showed significantly higher maturation failure rate, independent of age, gender, diabetes, and body mass index. On histologic examination, significant differences in intimal hyperplasia (13.3± 4.5 vs 18.2± 5.2 vs 30.3± 14.3μm) and medial thickening (76.8± 23.7 vs 103.9± 33.6 vs 109.3± 36.5μm) were observed across leptin tertiles. Similarly, medial fibrosis was most severe in the highest tertile. According to the immunohistochemical staining, most intimal cells were α-smooth muscle actin-positive, vimentin-positive, desmin-negative myofibroblasts. However, in the lowest tertile, desmin-positive contractile smooth muscle cells were also frequently observed, suggesting relatively slow phenotypic changes in this group. Furthermore, as leptin tertiles increased, the expression of leptin receptor in the luminal border of intima was significantly decreased. Obesity-related higher fistula maturation failure rate may be partly mediated by higher leptin level-associated pre-existing vascular diseases in end-stage renal disease patients. Decreased expression of leptin receptor may be related to this association.

An immortalized rat pancreatic stellate cell line RP-2 as a new cell model for evaluating pancreatic fibrosis, inflammation and immunity

Pancreatic stellate cells (PSCs) play a critical role in the pathogenesis of pancreatic fibrosis and have emerging functions as progenitor cells, immune cells or intermediaries in pancreatic exocrine secretion. Increasing evidence has shown that desmin as an exclusive cytoskeleton marker of PSC is only expressed in part of these cells. This study was to establish a desmin-positive PSC cell line and evaluate its actions on pancreatic fibrosis, inflammation and immunity. The presence of cytoskeletal proteins, integrin α5β1 or TLR4, was determined by immunocytochemistry while the production of desmin, collagen I, MMP-1, MMP-2, TIMP-2, or CD14 was evaluated by Western blotting. The levels of desmin, collagen I, IL-1 and IL-6 mRNA were determined by real-time quantitative PCR. The secretion of cytokines was detected by ELISA. Cell function was assessed using adhesion, migration, or proliferation assays. A stable activated rat PSC cell line (designated as RP-2) was established by RSV promoter/enhancer-driven SV40 large T antigen expression. RP-2 cells retained typical PSC properties, exhibited a myofibroblast-like phenotype and persistently produced desmin. The cells produced collagen I protein, matrix metalloproteinases and inhibitors thereof. RP-2 cells demonstrated typical PSC functions, including proliferation, adherence, and migration, the latter two of which occurred in response to fibronectin and were mediated by integrin α5β1. TLR4 and its response genes including proinflammatory cytokines (IL-1, IL-6, TNF-alpha) and chemotactic cytokines (MCP-1, MIP-1α, Rantes) were produced by RP-2 cells and activated by LPS. LPS-induced IL-1 or IL-6 mRNA expression in this cell line was fully blocked with MyD88 inhibitor. RP-2 cells provide a novel tool for analyzing the properties and functions of PSCs in the pathogenesis of fibrosis, inflammation and immunity in the pancreas.

Development of a clonal equine myoblast cell line capable of terminal differentiation into mature myotubes in vitro.

To produce a clonal equine myoblast cell line that retains the ability to divide for multiple passages and differentiate into multinucleated myotubes during specific conditions. Cultured primary equine skeletal muscle-derived cells from a healthy Thoroughbred. Cell cultures were transfected by electroporation with a plasmid (pNIT) that expresses the temperature-sensitive simian vacuolating virus 40 large T antigen (TAg), which can be controlled by a doxycycline-responsive promoter. Cells that stably integrated the TAg were selected and expanded to passage 25. For each passage, differentiation and fusion properties of the cells were determined and immunocytochemical analyses were performed to evaluate expression of TAg and other muscle-specific proteins. Optimum conditions that led to cell differentiation into myotubes were also determined. Compared with nontransfected control cells, myogenic, desmin-positive cells expressed the TAg when incubated at 33°C and could be maintained in culture for numerous passages. Reduced expression of TAg was identified in cells incubated at 37°C or when incubated with doxycycline at 33°C. Expression of TAg was not detected when cells were incubated with doxycycline at 37°C, and when serum was withdrawn from the culture medium, those clones differentiated into a pure population of multinucleated myotubes. Results indicated that production of an immortalized clonal equine skeletal muscle cell line was possible. A clonal equine skeletal muscle cell line will be a valuable in vitro tool for use in equine physiology and disease research.

Comparative Pathology of Canine Soft Tissue Sarcomas: Possible Models of Human Non-rhabdomyosarcoma Soft Tissue Sarcomas

Comparative analyses of canine and human soft tissue sarcomas (STSs) are lacking. This study compared the histological and immunohistochemical (labelling for desmin, smooth muscle actin [SMA], CD31, pancytokeratin, S100 and CD34) appearance of 32 archived, formalin-fixed, paraffin wax-embedded canine STS tumour specimens by board-certified veterinary and medical pathologists, both blinded to the other's interpretations. Comparison between the veterinary and human diagnoses revealed a generally consistent pattern of interpretation with few notable variations. Most tumours (13/32) were judged to display similar histomorphological appearance to human low-grade spindle cell sarcomas, appearing non-distinctive and morphologically of a fibroblastic/myofibroblastic type. Five canine cases resembled human liposarcoma, but with atypical desmin-positive epithelioid cells present. Five canine cases resembled human spindle cell sarcoma with myxoid features and two additional cases resembled human myxofibrosarcoma. Seven canine cases were noted to resemble human undifferentiated sarcoma. Findings in the present study demonstrate that canine STSs display histological and immunohistochemical features similar to their human equivalents. Because of these cross-species similarities, a particular opportunity exists to understand the biology and treatment of human STS by potentially including dogs as clinical models.

Congenital diaphragmatic hernia: focus on abnormal muscle formation

BackgroundCDH is a major birth defect, characterized by high mortality. How the initial defective mesenchymal substructures affects muscle malformation is unclear. Defects of genes involved in diaphragmatic development, such as friend-of-GATA2 (Fog2), may play an important role in its pathogenesis. We investigated the expression of Fog2 and proteins of myogenesis in a series of CDH and in diaphragms at different fetal ages, in order to clarify the role of muscular components during diaphragmatic development in cases with CDH. Material and methodsSpecimen were obtained from seven diaphragms of CDH cases undergoing surgery, 3 entire diaphragms from non repaired CDH, 5 control diaphragms at different gestational ages (16, 17, 22, 32, and 40g.w.), and 3 biopsy samples of normal voluntary muscle. The thickness of diaphragms at the edge of the defect in CDH and in developing diaphragms was measured. All samples were processed for HE staining and immunohistochemistry. Immunohistochemical expression of MyoD, Myf4, Pax7, Mib1 and Fog2 was evaluated. ResultsMean thickness at the edge of the defect was 4.14mm. Contralateral hemi-diaphragm in 3 autopsies and in controls at 32 and 40weeks measured 2.25mm; histology showed a higher density of desmin-positive muscular cells at the edge of defect. CDH displayed scattered Myf4-positive cells (range 0%–10%, mean 2.4%), numerous Pax7-positive cells (range 0%–24%, mean 12.1%) and less than 1% Mib1-positive cells. Controls showed a reduction of positive cell with the progression of gestational age for Myf4 (30% at 16weeks, 20% at 17weeks, 5% at 22weeks, 1% at 32 and 40weeks), Pax7 (85% at 16weeks and 17weeks, 35% at 22weeks, 11% at 32weeks) and Mib1 (20% at 16weeks, 8% at 17weeks, 7% at 22weeks, 2% at 32weeks). Fog-2 was diffusely positive in mesenchymal, mesothelial and muscular cells, in diaphragms from 16 to 22weeks, decreasing to 20% of positive muscular cells in 32-week diaphragm. In CDH only mesothelial and mesenchymal cells were positive. Stem cell markers were negative in cases and controls. CommentCDH shows a thick muscular border, with high number of mature muscle cells and significant increase of quiescent satellite cells (PAX7+, Mib1−). Abnormal architecture may affect the normal process of myogenesis and thus signaling and cell-cell interactions of myocytes. The expression of Fog2 in mesothelial and mesenchymal cells in CDH demonstrates the absence of a genetic defect involving Fog2 in our cases. Being Fog2 expressed in muscle cells at early stage supports the hypothesis that the altered diaphragmatic genesis may undermine also the muscular component instead of the only mesenchymal one.

Perspectives of using stem cells in diabetes mellitus treatment

Using stem cells is one of the most perspective methods of diabetes mellitus treatment. Different stem cells populations are used for this purpose. Pancreatic stem cells are considered to be the most appropriate. This review is devoted to new methods of diabetes mellitus treatment by using stem cells and perspectives of using C-kit- positive pancreas cells and desmin-positive stellate pancreas cells as the main candidate to the role of pancreatic stem cells.

The effect of age and medical comorbidities on in vitro myoblast expansion in women with and without pelvic organ prolapse.

This is an observational study is designed to assess the influence of age, prolapse and medical co-morbidities on myogenic stem cells growth in-vitro. A biopsy of the rectus abdominus muscle was obtained during surgery in patients with and without pelvic organ prolapse (POP). Nuclei number and fiber count were correlated with patient's age, presence of POP, and medical comorbidities. Efficiency of expansion of myogenic stem cells in vitro was calculated. The percentage of Pax7-, MyoD-, and desmin-positive cells was correlated with age, POP status, and medical comorbidities. A total of 17 specimens were obtained; 13 specimens were available for histologic analysis. There was no correlation between patient's age, POP status or medical comorbidities and nuclei or fiber count, growth rate, or the percentage of Pax7- and MyoD-positive cells. Patients with 2 to 4 medical comorbidities were noted to have a significantly lower percentage of desmin-positive cells. Specimens with a higher nuclear count had significantly better cellular expansion. Data were analyzed using Kruskal-Wallis or Wilcoxon rank sum statistics. Multiple medical comorbidities but not patient's age or POP status influenced in vitro myogenic stem cell growth. These data suggest that patients with advancing age or POP may be acceptable autologous donors if treatment of urinary or anal incontinence requires myoblast transplantation.

Notch signaling is required for the formation of mesangial cells from a stromal mesenchyme precursor during kidney development

Mesangial cells are specialized pericyte/smooth muscle cells that surround and constrain the vascular network within the glomerulus of the kidney. They are derived from the stromal mesenchyme, a progenitor population distinct from nephron stem cells. Whether mesangial cells have a distinct origin from vascular smooth muscle cells (VSMCs) and the pathways that govern their specification are unknown. Here we show that Notch signaling in stromal progenitors is essential for mesangial cell formation but is dispensable for the smooth muscle and interstitial cell lineages. Deletion of RBPjk, the common DNA-binding partner of all active Notch receptors, with Foxd1(tgCre) results in glomerular aneurysm and perinatal death from kidney failure. This defect occurs early in glomerular development as stromal-derived, desmin-positive cells fail to coalesce near forming nephrons and thus do not invade the vascular cleft of the S-shaped body. This is in contrast to other mutants in which the loss of the mesangium was due to migration defects, and suggests that loss of Notch signaling results in a failure to specify this population from the stroma. Interestingly, Pdgfrb-positive VSMCs do not enter the vascular cleft and cannot rescue the mesangial deficiency. Notch1 and Notch2 act redundantly through γ-secretase and RBPjk in this process, as individual mutants have mesangial cells at birth. Together, these data demonstrate a unique origin of mesangial cells and demonstrate a novel, redundant function for Notch receptors in mesangial cell specification, proliferation or survival during kidney development.

C-kit and desmin-positive cells in expression in islets of pancreas during alloxan diabetes in rats

C-kit and desmin-positive cells in expression in islets of pancreas during alloxan diabetes in rats

Significance of Serum TGF-β1 Levels for Estimating Bilirubin Decrease Following Biliary Drainage in Jaundiced Patients Caused by Biliary Tract Carcinoma

Purpose: Biliary drainage is generally performed in patients with obstructive jaundice (OJ) before major hepatectomy for biliary tract carcinomas. However, estimation of bilirubin decrease rate after biliary drainage may be often difficult. In this study, we investigated useful biochemical marker for estimating a period for bilirubin decrease, and also studied mechanisms responsible for delayed bilirubin reduction. Methods: Peripheral blood samples were taken in 22 patients with OJ caused by biliary tract carcinoma before and after biliary drainage. Serum transforming growth factor-β1 (TGF- β1) and platelet-derived growth factor BB (PDGF-BB), and bilirubin decrease rates (“b”value) were investigated. Expression of desmin and α-smooth muscle actin (α-SMA) in surgically resected liver specimens were also investigated in 12 patients who underwent radical surgery, using immunohistochemical staining, to evaluate hepatic stellate cell (HSC) status. Results: Serum TGF- β1 and PDGF-BB levels were significantly higher in patients with OJ than in controls (p<0.01). Serum levels of total bilirubin and TGF- β1 decreased significantly following biliary drainage. However, bilirubin reduction in patients with high TGF- β1 was significantly slower than those with low TGF- β1, despite of the height of the serum bilirubin levels before drainage. Immunohistochemical studies revealed increased number of HSCs (desmin-positive cells) and activated HSCs (α-SMA-positive cells) in portal areas of the liver in patients with the high TGF- β1 at surgery. Conclusion: Serum TGF- β1 mostly produced by activated HSCs, reflecting hepatic damage by cholestasis, may be a useful biochemical marker for predicting the period of bilirubin reduction in patients with OJ.

Midkine-deficient mice delayed degeneration and regeneration after skeletal muscle injury

Midkine (MK), a heparin-binding growth factor, was previously found to be expressed in the rat myotube-forming stage. We investigated MK gene-deficient (Mdk−/−) mice in terms of skeletal muscle degeneration and regeneration after injury by bupivacaine injection into the tibialis anterior muscle. Injured muscles showed intense inflammatory cell infiltration. Myotubes, myofibers with centrally located nuclei in their cytoplasm, were significantly smaller in Mdk−/− mice than in wild type (Mdk+/+) mice 7 days after injury (p=0.02). The distribution of myotube sizes showed quantitative differences between the two groups at 5 and 7 days, but not at 14 days. Many small myotubes were found in the regenerative area of Mdk−/− mice compared with that of Mdk+/+mice 5 and 7 days after injury. The expression of Iba1, a macrophage marker, was significantly lower in Mdk−/− mice 3 days after injury (p=0.01). The number of desmin-positive cells like myoblasts in Mdk−/− mice was significantly fewer than that in Mdk+/+ mice 3 days after injury. Our results suggested that deletion of MK results in a delay in regeneration, preceded by decelerated migration of macrophages to the damaged area, and that MK has a role in cell differentiation and maturation after skeletal muscle injury.

Desmin‐positivity in spindle cells: Under‐recognized immunophenotype of lipoblastoma

Lipoblastoma is a distinct benign fatty tumor composed of adipocytes, lipoblasts, and primitive mesenchymal cells with a myxoid stroma. Lipoblastoma harbors characteristic fusion genes involving the PLAG1, resulting in aberrant expression of PLAG1. However, the nature of the primitive mesenchymal cells remains obscure. In our routine pathology practice, we noticed desmin-positive spindle mesenchymal cells in lipoblastomas, which is a hitherto poorly described phenomenon. Thus, we examined the expression of several myogenic markers including desmin in a variety of 95 mesenchymal tumors with fatty elements. Fourteen of the 15 lipoblastomas examined contained desmin-positive spindle cells, which also showed nuclear expression of PLAG1, whereas α-smooth muscle actin, muscle specific actin, h-caldesmon, and myogenin were negative. Some spindle cells in subsets of atypical lipomatous tumors/well differentiated liposarcomas (6/20), dedifferentiated liposarcomas (11/31) and pleomorphic liposarcomas (2/10) were positive for actins and/or desmin, supporting focal myofibroblastic or smooth muscle differentiation. The other tumors, including 11 myxoid/round cell liposarcomas, four spindle cell lipomas, and four lipofibromatoses, were negative for all of the myogenic markers assessed. The almost consistent desmin expression in spindle mesenchymal cells suggests a potential diagnostic utility of this marker and myofibroblastic phenotype of fractions in lipoblastoma cells.

Imaging and therapy of liver fibrosis using bioreducible polyethylenimine/siRNA complexes conjugated with N-acetylglucosamine as a targeting moiety

Diagnosis and therapy of early stage liver fibrosis is very important for the treatment of fatal liver diseases. Here, we report on the targeted imaging and therapy of activated hepatic stellate cells (HSCs) and fibrotic liver tissue using N-acetylglucosamine (GlcNAc)- and indocyanine green (ICG)-conjugated PEI/siRNA complexes. The conjugation of a disulfide bond to PEI (PEI-D) was achieved by Michael addition. We modified PEI with N-acetylglucosamine (PEI-D-GlcNAc), which can specifically interact with desmin on activated HSCs, using the EDC coupling method. Confocal microscopic analysis showed that the PEI-D-GlcNAc/siRNA was internalized by HSCs upon interaction with surface desmin. In vitro western blot analysis confirmed that PEI-D-GlcNAc provided strong protein knock-down after transfection with TGFβ1siRNA into HSCs. After a tail vein injection of ICG-conjugated complexes, the PEI-D-GlcNAc-ICG/siRNA complex accumulated to a greater extent in the livers of fibrotic mice than in normal mice over an extended duration. Moreover, immunohistofluorescence analysis confirmed that the PEI-D-GlcNAc-ICG/siRNA complex specifically colocalized with HSCs, which are desmin-positive cells, in fibrotic liver tissues. In vivo TGFβ1siRNA delivery also resulted in superior protein knock-down when using the PEI-D-GlcNAc complex. These results demonstrate that the PEI-D-GlcNAc-ICG/TGFβ1siRNA complex is a useful tool for imaging and treatment of liver fibrosis.

Acceleration of autologous in vivo recellularization of decellularized aortic conduits by fibronectin surface coating

Decellularization is a promising option to diminish immune and inflammatory response against donor grafts. In order to accelerate the autologous in vivo recellularization of aortic conduits for an enhanced biocompatibility, we tested fibronectin surface coating in a standardized rat implantation model. Detergent-decellularized rat aortic conduits (n = 36) were surface-coated with covalently Alexa488-labeled fibronectin (50 μg/ml, 24 h) and implanted into the systemic circulation of Wistar rats for up to 8 weeks (group FN; n = 18). Uncoated implants served as controls (group C; n = 18). Fibronectin-bound fluorescence on both surfaces of the aortic conduits was persistent for at least 8 weeks. Cellular repopulation was examined by histology and immunofluorescence (n = 24). Luminal endothelialization was significantly accelerated in group FN (p = 0.006 after 8 weeks), however, local myofibroblast hyperplasia with significantly increased ratio of intima-to-media thickness occurred (p = 0.0002 after 8 weeks). Originating from the adventitial surface, alpha-smooth muscle actin and desmin positive cell invasion into the media of fibronectin-coated conduits was significantly increased as compared to group C (p < 0.0001). In these medial areas, in situ zymography revealed enhanced matrix metalloproteinase activity. In both groups, inflammatory cell markers (CD3 and CD68) and signs of thrombosis proved negative. With regard to several markers of cell adhesion, inflammation and calcification, quantitative real-time PCR (n = 12) revealed no significant inter-group differences. Fibronectin surface coating of decellularized cardiovascular implants proved feasible and persistent for at least 8 weeks in the systemic circulation. Biofunctional protein coating accelerated the autologous in vivo endothelialization and induced a significantly increased medial recellularization. Therefore, this strategy may contribute to the improvement of current clinically applied bioprostheses.

Enhanced Effectivity of an ALK5-Inhibitor after Cell-Specific Delivery to Hepatic Stellate Cells in Mice with Liver Injury

Transforming growth factor-β (TGF-β) is a major pro-fibrotic cytokine, causing the overproduction of extracellular matrix molecules in many fibrotic diseases. Inhibition of its type-I receptor (ALK5) has been shown to effectively inhibit fibrosis in animal models. However, apart from its pro-fibrotic effects, TGF-β also has a regulatory role in the immune system and influences tumorigenesis, which limits the use of inhibitors. We therefore explored the cell-specific delivery of an ALK5-inhibitor to hepatic stellate cells, a key cell in the development of liver fibrosis. We synthesized a conjugate of the ALK5-inhibitor LY-364947 coupled to mannose-6-phosphate human serum albumin (M6PHSA), which binds to the insulin-like growth factor II receptor on activated HSC. The effectivity of the conjugate was evaluated in primary HSC and in an acute liver injury model in mice. In vitro, the free drug and the conjugate significantly inhibited fibrotic markers in HSC. In hepatocytes, TGF-β-dependent signaling was inhibited by free drug, but not by the conjugate, thus showing its cell-specificity. In vivo, the conjugate localized in desmin-positive cells in the liver and not in hepatocytes or immune cells. In the acute liver injury model in mice, the conjugate reduced fibrogenic markers and collagen deposition more effectively than free drug. We conclude that we can specifically deliver an ALK5-inhibitor to HSC using the M6PHSA carrier and that this targeted drug reduces fibrogenic parameters in vivo, without affecting other cell-types.

A novel murine model to deplete hepatic stellate cells uncovers their role in amplifying liver damage in mice

We have developed a novel model for depleting mouse hepatic stellate cells (HSCs) that has allowed us to clarify their contributions to hepatic injury and fibrosis. Transgenic (Tg) mice expressing the herpes simplex virus thymidine kinase gene (HSV-Tk) driven by the mouse GFAP promoter were used to render proliferating HSCs susceptible to killing in response to ganciclovir (GCV). Effects of GCV were explored in primary HSCs and in vivo. Panlobular damage was provoked to maximize HSC depletion by combining CCl(4) (centrilobular injury) with allyl alcohol (AA) (periportal injury), as well as in a bile duct ligation (BDL) model. Cell depletion in situ was quantified using dual immunofluorescence (IF) for desmin and GFAP. In primary HSCs isolated from both untreated wild-type (WT) and Tg mice, GCV induced cell death in ≈ 50% of HSCs from Tg, but not WT, mice. In TG mice treated with CCl(4) +AA+GCV, there was a significant decrease in GFAP and desmin-positive cells, compared to WT mice (≈ 65% reduction; P < 0.01), which was accompanied by a decrease in the expression of HSC-activation markers (alpha smooth muscle actin, beta platelet-derived growth factor receptor, and collagen I). Similar results were observed after BDL. Associated with HSC depletion in both fibrosis models, there was marked attenuation of fibrosis and liver injury, as indicated by Sirius Red/Fast Green, hematoxylin and eosin quantification, and serum alanine/aspartate aminotransferase. Hepatic expression of interleukin-10 and interferon-gamma was increased after HSC depletion. No toxicity of GCV in either WT or Tg mice accounted for the differences in injury. Activated HSCs significantly amplify the response to liver injury, further expanding this cell type's repertoire in orchestrating hepatic injury and repair.

Immunohistochemical Characterization of Macrophages and Myofibroblasts in Fibrotic Liver Lesions Due to &lt;i&gt;Fasciola&lt;/i&gt; Infection in Cattle

Histopathologically, fibrosis in Fasciola-infected cattle livers was characterized by inflammatory cell infiltration, such as eosinophils and macrophages, pseudo-lobule, pseudo-bile ducts and fibrotic bridges separating pseudo-lobules; the fibrotic lesions were developed in the Glisson's sheath. Pseudo-bile ducts consisting of epithelial cells reacted clearly to cytokeratin (CK) 19, indicating cholangiocyte origin. Immunophenotypes of macrophages and myofibroblasts were investigated in the fibrotic livers. Macrophages positive for CD68 (reflecting phagocytosis) and CD163 (representing proinflammatory cytokine production) were increased, and those for CD204 (implying lipid metabolism) and Iba-1 (a calcium-binding protein playing role in chemotaxis) decreased in fibrotic livers compared to control livers. Spindle-shaped myofibroblasts positive for vimentin, desmin and α-smooth muscle actin (α-SMA) increased in the peribiliary connective tissues, although the desmin-positive cells were fewer. In addition to the usefulness of these antibodies for macrophage detection in cattle livers, this study shows that macrophages with different immunophenotypes participate in Fasciola-infected cattle livers, in relation to development of myofibroblasts expressing mainly vimentin and α-SMA.

Expression of small leucine-rich proteoglycans in rat anterior pituitary gland

Proteoglycans are components of the extracellular matrix and comprise a specific core protein substituted with covalently linked glycosaminoglycan chains. Small leucine-rich proteoglycans (SLRPs) are a major family of proteoglycans and have key roles as potent effectors in cellular signaling pathways. Research during the last two decades has shown that SLRPs regulate biological functions in many tissues such as skin, tendon, kidney, liver, and heart. However, little is known of the expression of SLRPs, or the characteristics of the cells that produce them, in the anterior pituitary gland. Therefore, we have determined whether SLRPs are present in rat anterior pituitary gland. We have used real-time reverse transcription with the polymerase chain reaction to analyze the expression of SLRP genes and have identified the cells that produce SLRPs by using in situ hybridization with a digoxigenin-labeled cRNA probe. We have clearly detected the mRNA expression of SLRP genes, and cells expressing decorin, biglycan, fibromodulin, lumican, proline/arginine-rich end leucine-rich repeat protein (PRELP), and osteoglycin are located in the anterior pituitary gland. We have also investigated the possible double-staining of SLRP mRNA and pituitary hormones, S100 protein (a marker of folliculostellate cells), desmin (a marker of capillary pericytes), and isolectin B4 (a marker of endothelial cells). Decorin, biglycan, fibromodulin, lumican, PRELP, and osteoglycin mRNA have been identified in S100-protein-positive and desmin-positive cells. Thus, we conclude that folliculostellate cells and pericytes produce SLRPs in rat anterior pituitary gland.

A Rare Case of Borderline Resectable Carcinosarcoma

Context Carcinosarcomas are neoplasms with mixed carcinomatous and sarcomatous elements, which rarely occur in the pancreas. Case report A 62-year-old man was admitted to our institute for the presence of intense abdominal pain, worsening of a type II diabetes and weight loss (10 kg in 3 months). The tumor markers (CEA, CA 19-9) were within normal range. A CT scan showed a large pancreatic head solid mass of about 5 cm in diameter, with central necrotic area with septa, peripheral contrast-enhancement and compression and dislocation of the superior mesenteric vein for a tract of 2.5 cm (G3). An US-guided percutaneous biopsy diagnosed a malignant, high grade spindle cells neoplasia with immunophenotypic pattern of sarcomas. Surgical approach was planned and a total pancreatectomy with vascular resection was performed. The vascular resection included a 8 cm tract of portal-mesenteric trunk and the reconstruction was performed using a donor’s femoral vein homograft. Pathological examination of the specimen showed a solid mass of 6 cm in diameter, with infiltration of the portal vein. Lymph nodes (n=48) were negative and the surgical margins were tumor-free. Microscopically the tumor was characterized by high grade, polymorphous, intensely desmin-positive spindle cells mixed with areas of moderately differentiated ductal adenocarcinoma. The remaining pancreatic gland was interested by a diffuse lympho-plasmacellular GEL-type acute pancreatitis. A final diagnosis of carcinosarcoma, pT3N0M0, was performed. The postoperative course was uneventful and the patient was discharged in post-operative day 14 th . He is dead, 4 months after surgery. Conclusion In literature only 10 cases of pancreatic carcinosarcomas have been described All cases were treated surgically and they showed a poor prognosis (average 6 months) and a rapid recurrence of the disease as in our case.

Mechanical preconditioning enables electrophysiologic coupling of skeletal myoblast cells to myocardium

The effect of mechanical preconditioning on skeletal myoblasts in engineered tissue constructs was investigated to resolve issues associated with conduction block between skeletal myoblast cells and cardiomyocytes. Murine skeletal myoblasts were used to generate engineered tissue constructs with or without application of mechanical strain. After in vitro myotube formation, engineered tissue constructs were co-cultured for 6 days with viable embryonic heart slices. With the use of sharp electrodes, electrical coupling between engineered tissue constructs and embryonic heart slices was assessed in the presence or absence of pharmacologic agents. The isolation and expansion procedure for skeletal myoblasts resulted in high yields of homogeneously desmin-positive (97.1% ± 0.1%) cells. Mechanical strain was exerted on myotubes within engineered tissue constructs during gelation of the matrix, generating preconditioned engineered tissue constructs. Electrical coupling between preconditioned engineered tissue constructs and embryonic heart slices was observed; however, no coupling was apparent when engineered tissue constructs were not subjected to mechanical strain. Coupling of cells from engineered tissue constructs to cells in embryonic heart slices showed slower conduction velocities than myocardial cells with the embryonic heart slices (preconditioned engineered tissue constructs vs embryonic heart slices: 0.04 ± 0.02 ms vs 0.10 ± 0.05 ms, P = .011), lower maximum stimulation frequencies (preconditioned engineered tissue constructs vs embryonic heart slices: 4.82 ± 1.42 Hz vs 10.58 ± 1.56 Hz; P = .0009), and higher sensitivities to the gap junction inhibitor (preconditioned engineered tissue constructs vs embryonic heart slices: 0.22 ± 0.07 mmol/L vs 0.93 ± 0.15 mmol/L; P = .0004). We have generated skeletal myoblast-based transplantable grafts that electrically couple to myocardium.