Smooth Actin Research Articles

MicroRNA-503 Suppresses Oral Mucosal Fibroblast Differentiation by Regulating RAS/RAF/MEK/ERK Signaling Pathway.

The abnormal proliferation and differentiation of oral mucosal fibroblasts (FBs) is the key to the progression of oral submucosal fibrosis. To clarify the mechanism of platelet-derived growth factor (PDGF-BB)-induced FBs fibrosis in oral mucosa, real-time quantitative polymerase chain reaction and Western blot were used in this study to detect the expression of miR-503 and the expression of p-MEK, p-ERK, miR-503, RAF, smooth actin and type I collagen under different time and concentration stimulation of PDGF-BB. The effects of overexpression of miR-503 or RAF on the proliferation and migration of FBs were detected by cell counting kit 8 and cell scratch assay, respectively. A dual luciferase reporter gene assay was used to verify the targeting effect of miR-503 on RAF. The results showed that miR-503 was downregulated in a dose- and time-dependent manner in PDGF-BB-induced FBs. In addition, RAF is a direct target of miR-503 and can be negatively regulated. Overexpression of RAF can promote FB proliferation, migration, differentiation, collagen synthesis, and activation of downstream molecules (MEK/ERK), while overexpression of miR-503 can partially reverse the effects of RAF. Therefore, miR-503 regulates the biological behavior of PDGF-BB-induced oral mucosal FBs by influencing the activation of the RAS/RAF/MEK/ERK signaling pathway.

The tongue of the red-eared slider (Trachemys scripta elegans): morphological characterization through gross, light, scanning electron, and immunofluorescence microscopic examination

The red-eared slider (Trachemys scripta elegans) is renowned for its remarkable adaptations, yet much of its complex biology remains unknown. In this pioneering study, we utilized a combination of gross anatomy, scanning electron microscopy (SEM), light microscopy, and immunofluorescence techniques to examine the tongue’s omnivorous adaptation in this species. This research bridges a critical knowledge gap, enhancing our understanding of this intriguing reptile. Gross examination revealed a unique arrowhead-shaped tongue with a median lingual fissure and puzzle-piece-shaped tongue papillae. SEM unveiled rectangular filiform, conical, and fungiform papillae, with taste pores predominantly on the dorsal surface and mucous cells on the lateral surface of the papillae. Histologically, the tongue’s apex featured short rectangular filiform and fungiform papillae, while the body exhibited varying filiform shapes and multiple taste buds on fungiform papillae. The tongue’s root contained lymphatic tissue with numerous lymphocytes surrounding the central crypt, alongside lingual skeletal musculature, blood and lymph vessels, and Raffin corpuscles in the submucosa. The lingual striated muscle bundles had different orientations, and the lingual hyaline cartilage displayed a bluish coloration of the ground substance, along with a characteristic isogenous group of chondrocytes. Our research represents the first comprehensive application of immunofluorescence techniques to investigate the cellular intricacies of the red-eared slider’s tongue by employing seven distinct antibodies, revealing a wide array of compelling and significant findings. Vimentin revealed the presence of taste bud cells, while synaptophysin provided insights into taste bud and nerve bundle characteristics. CD34 and PDGFRα illuminated lingual stromal cells, and SOX9 and PDGFRα shed light on chondrocytes within the tongue’s cartilage. CD20 mapped B-cell lymphocyte distribution in the lingual tonsil, while alpha smooth actin (α-SMA) exposed the intricate myofibroblast and smooth muscle network surrounding the lingual blood vessels and salivary glands. In conclusion, our comprehensive study advances our knowledge of the red-eared slider’s tongue anatomy and physiology, addressing a significant research gap. These findings not only contribute to the field of turtle biology but also deepen our appreciation for the species’ remarkable adaptations in their specific ecological niches.

Aromatic Residues Dictate the Transcriptional Repressor and Single-Stranded DNA Binding Activities of Purine-Rich Element Binding Protein B.

Purine-rich element binding protein B (Purβ) is a single-stranded DNA (ssDNA) and RNA-binding protein that functions as a transcriptional repressor of genes encoding certain muscle-restricted contractile proteins in the setting of cellular stress or tissue injury. A prior report from our laboratory implicated specific basic amino acid residues in the physical and functional interaction of Purβ with the smooth muscle-α actin gene (Acta2) promoter. Independent structural analysis of fruit fly Purα uncovered a role for several aromatic residues in the binding of this related protein to ssDNA. Herein, we examine the functional importance of a comparable set of hydrophobic residues that are positionally conserved in the repeat I (Y59), II (F155), and III (F256) domains of murine Purβ. Site-directed Y/F to alanine substitutions were engineered, and the resultant Purβ point mutants were tested in various biochemical and cell-based assays. None of the mutations affected the cellular expression, structural stability, or dimerization capacity of Purβ. However, the Y59A and F155A mutants demonstrated weaker Acta2 repressor activity in transfected fibroblasts and reduced binding affinity for the purine-rich strand of an Acta2 cis-regulatory element in vitro. Mutation of Y59 and F155 also altered the multisite binding properties of Purβ for ssDNA and diminished the interaction of Purβ with Y-box binding protein 1, a co-repressor of Acta2. Collectively, these findings suggest that some of the same aromatic residues, which govern the specific and high-affinity binding of Purβ to ssDNA, also mediate certain heterotypic protein interactions underlying the Acta2 repressor function of Purβ.

Smooth Muscle-Alpha Actin R149C Pathogenic Variant Downregulates Integrin Recruitment at Cell-Matrix Adhesions and Decreases Cellular Contractility.

Thoracic aortic aneurysm is found in patients with ACTA2 pathogenic variants. ACTA2 missense variants are associated with impaired aortic smooth muscle cell (SMC) contraction. This study tested the hypothesis that the Acta2R149C/+ variant alters actin isoform expression and decreases integrin recruitment, thus, reducing aortic contractility. Stress relaxation measurements in thoracic aortic rings showed two functional regimes with a reduction of stress relaxation in the aorta from Acta2R149C/+ mice at low tension, but not at high tension values. Contractile responses to phenylephrine and potassium chloride were 50% lower in Acta2R149C/+ mice than in wild-type (WT) mice. Additionally, SMC were immunofluorescently labeled for specific proteins and imaged by confocal or total internal reflection fluorescence microscopy. The quantification of protein fluorescence of Acta2R149C/+ SMC showed a downregulation in smooth muscle α-actin (SMα-actin) and a compensatory upregulation of smooth muscle γ-actin (SMγ-actin) compared to WT cells. These results suggest that downregulation of SMα-actin leads to reduced SMC contractility, while upregulation of SMγ-actin may lead to increased SMC stiffness. Decreased α5β1 and α2β1 integrin recruitment at cell-matrix adhesions further reduce the ability of mutant cells to participate in cell-matrix crosstalk. Collectively, the results suggest that mutant Acta2R149C/+ aortic SMC have reduced contractility and interaction with the matrix, which are potential long-term contributing factors to thoracic aortic aneurysms.

Immunohistochemical Expression of Alpha Smooth Muscle Actin Positive Myofibroblast and Laminin 5 gamma 2 in Squamous Cell Carcinoma and Verrucous Carcinoma of the Oral Cavity

Introduction: Squamous cell carcinoma is the most common type of oral cancer that shows certain tissue changes. Laminin-5γ2 is a protein that plays a central role in migration of the neoplastic cells during tumor invasion. Expression of this protein in cells and stroma adjacent to tumor can confirm the cellular invasion. The aim of this study was to evaluate stroma in verrucous carcinoma and squamous cell carcinoma of the mouth using two markers α SMA and laminin 5 gamma 2.Materials & Methods: In this retrospective descriptive-analytical study, 60 samples were selected with the diagnosis of squamous cell carcinoma and verrucous oral carcinoma. Then, two markers of α SMA and laminin 5 gamma 2 were examined in epithelium and mesenchymal stroma by immunohistochemistry. The vasculature was used as a positive immunoreactivity control for alpha-smooth muscle and the normal mucosa was used to control the positive immunoreactivity of laminin 5 gamma 2. The results were analyzed using Chi-square and Spearman correlation tests (α = 0.05).Results: Out of 31 SCC samples, 80.6% showed laminin 5 gamma 2 and 90.3% showed alpha smooth and out of 9 samples of varocus carcinoma, 66.7% showed laminin 5 gamma 2 and 77.8% showed alpha smooth actin. The higher the positive percentage of laminin, the higher the percentage of positive α SMA.Conclusion: Expression of alpha smooth muscle actin and lamminin 5 gamma 2 was significantly higher in oral squamous cell carcinoma and there exists a correlation between expression of alpha smooth muscle actin and the increase in myofibroblasts takes place during the carcinogenesis process.

Expression of Pannexin 1 in the Human Kidney during Embryonal, Early Fetal and Postnatal Development and Its Prognostic Significance in Diabetic Nephropathy

Pannexins are transmembrane glycoproteins that constitute channels involved in purinergic signaling through ATP release from cells in various physiological and pathological processes. In this study, the distribution of Panx1 expression in different cell populations of healthy postnatal human kidneys and during human embryonic and early fetal development was investigated by double immunohistochemistry. In addition, the glomerular and tubular expression of Panx1 was examined in patients with type 2 diabetes mellitus (DM2) and the control group, and renal Panx1 expression was correlated with serum creatinine. In the 6th week of embryonic development (DW), Panx1 expression was found in mesonephric glomeruli and mesonephric tubules. At the transition from 6th to 7th DW, Panx1 immunoreactivity was found in the mesonephric tubules and mesonephric duct, as well as in the metanephric ureteric bud and ampullae. In the 7th DW, strong Panx1 immunoreactivity was observed in the developing ureteric bud in the metanephros, whereas no Panx1 immunoreactivity was found in the metanephric cup. In the 8th DW, Panx1 expression was also found in the ureteric bud of the metanephros, the renal vesicle and comma-shaped nephron, and the epithelial cells of Bowman’s capsule. Expression of Panx1 was found at an early stage in both the paramesonephric duct and the mesonephric duct and diminished toward the 8th DW. During the 6th–10th DW, colocalization of Panx1 with alpha smooth actin (aSMA) was found in developing blood vessels. In the postnatal kidney, strong Panx1 immunoreactivity was present in medullary and cortical collecting duct cells, renin-producing cells, and proximal tubules. Very weak Panx1 immunoreactivity was found in certain distal tubule cells and the thin descending limbs of the loop of Henle. Panx1 immunoreactivity was also found in nephrin-immunoreactive podocytes. Panx1 was not colocalized with aSMA immunoreactivity in the vessels of the postnatal human kidney, but it was present in the endothelium. A significant positive correlation was found between Panx1 expression in glomeruli and serum creatinine only in diabetic patients and was not found in the nondiabetic group. The spatiotemporal expression of Panx1 during the early stages of human kidney development supports its possible role in cellular differentiation, migration, and positioning in the developing human kidney. In addition, our data suggest that glomerular Panx1 expression is a potential indicator of worsening renal function in patients with type 2 diabetes.

Studies of efficacy of Achillea species ethanol extract and honey on full thickness wound healing in diabetic albino rats

Wound healing is deemed a clinical issue in diabetic patients. This study aimed to investigate the efficiency of Achillea species ethanolic extract and honey on wound healing enhancement in streptozotocin-induced diabetic rats. Thirty adult male Albino rats (180- 230 gm) were used for creating full thickness skin wounds. Animals were grouped to control (GI) and diabetics (GII- GVI). Diabetes was induced by streptozotocin (45 mg/kg) and all animals received metformin (50 mg/kg orally for 14 days to control hyperglycemia). Full thickness wound (2X2 cm) was created on the back skin of all animals. Wounded diabetic animals were divided into 5 subgroups based on wound treatment into diabetic wounded untreated (GII), mebo cream (GШ), Achillea extract preparation (GIV), honey (GV), Achillea + honey (GVI). One ml of each treatment was used for daily wound painting by fine brush for 14 days. Wounds were observed daily for healing criteria or complications and photographed with a well-known scale to measure contraction rate. Smears were taken from wounds for microbiological study. Wounded area was dissected at 14 days, processed for paraffin sectioning and staining by hematoxylin and eosin, alpha –smooth actin (α-SA) and proliferating cell nuclear antigen (PCNA). Achillea extract, Achillea + honey, honey and mebo enhanced wound healing in diabetic rats. Most treatments modified microbial contents, blood profile and histological healing process. Achillea extracts, Achillea + honey and honey showed marked influence in enhancing wound healing in diabetic rats even better than mebo. So, it is recommended to use Achillea alone or mixed with honey for enhancing large a wound defects in diabetic patients.

Fetal Megacystis: A New Morphologic, Immunohistological and Embriogenetic Approach

Congenital anomalies of the kidney and urinary tract (CAKUT) include isolated kidney malformations and urinary tract malformations. They have also been reported in Prune-Belly syndrome (PBS) and associated genetic syndromes, mainly 13, 18 and 21 trisomy. The AA focuses on bladder and urethral malformations, evaluating the structural and histological differences between two different cases of megacystis. Both bladders were examined by routine prenatal ultrasound screening and immunohistochemistry, comparing the different expression of smooth muscular actin (SMA), S100 protein and WT1c in megacystis and bladders of normal control from fetuses of XXI gestational age. Considering the relationship between the enteric nervous system and urinary tract development, the AA evaluated S100 and WT1c expression both in bladder and bowel muscular layers. Both markers were not expressed in the bladder and bowel of PBS associated with anencephaly. In conclusion, megacystis could be considered only a macroscopic definition, concerning the size of the fetal bladder rather than the embryologic origin; it may be a single or multiple malformation; the possible association with the bowel and/or encephalic malformations will decide the outcome and prognosis in fetal megacystis.

Functional comparison of beating cardiomyocytes differentiated from umbilical cord-derived mesenchymal/stromal stem cells and human foreskin-derived induced pluripotent stem cells.

In recent years, stem cell-based therapies shown to have promising effects on the clinical management of ischemic heart disease. Moreover, stem cells differentiation into cardiomyocytes (CMs) can overcome the cell source limitations. The current research involves the isolation and expansion of mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), their differentiation into CMs and subsequent construction of tissue-engineered myocardium supported by random and aligned polycaprolactone (PCL) nanofibrous matrices (av. dia: 350-850 nm). Umbilical cord matrix (UCM)-derived MSCs were isolated successfully by routine enzymatic digestion and a nonenzymatic explant culture method and characterized by their morphology, differentiation into different lineages, and surface marker expression. Treatment of UCM-derived MSCs with 5-azacytidine (5 μM) induced their differentiation into putative cardiac cells, as revealed by the expression of cardiac-specific troponin T (cTnT), smooth muscles actin, myogenin (MYOG), smoothelin, cardiac α-actin genes and cTnT, α-actinin proteins by RT-PCR and immunocytochemistry, respectively. However, no beating cells were observed in differentiated MSCs. On the other hand, adult human foreskin-derived iPSCs cultured on Matrigel™-coated aligned PCL nanofibrous matrices showed anisotropic behavior along the PCL nanofibers and, upon differentiation, expressed cardiac-specific cTnT (23.34 vs. 32.55%) proteins and showed more synchronized beating than those differentiated on Matrigel™-coated tissue culture coated polystyrene surfaces. Moreover, aligned PCL nanofibers are able to promote cells orientation parallel to the fibers, thus providing an effective way to control anisotropic nature under in vitro condition.

Integrin \u03b17 expression is increased in asthmatic patients and its inhibition reduces Kras protein abundance in airway smooth muscle cells

Airway smooth muscle (ASM) cells exhibit plastic phenotypic behavior marked by reversible modulation and maturation between contractile and proliferative phenotypic states. Integrins are a class of transmembrane proteins that have been implicated as novel therapeutic targets for asthma treatment. We previously showed that integrin α7 is a novel marker of the contractile ASM phenotype suggesting that targeting this protein may offer new avenues to counter the increase in ASM cell mass that underlies airways hyperresponsiveness (AHR) in asthma. We now determine whether inhibition of integrin α7 expression would revert ASM cells back to a proliferative phenotype to cause an increase in ASM cell mass. This would be detrimental to asthmatic patients who already exhibit increased ASM mass in their airways. Using immunohistochemical analysis of the Melbourne Epidemiological Study of Childhood Asthma (MESCA) cohort, we show for the first time that integrin α7 expression in patients with severe asthma is increased, supporting a clinically relevant role for this protein in asthma pathophysiology. Moreover, inhibition of the laminin-integrin α7 signaling axis results in a reduction in smooth muscle-alpha actin abundance and does not revert ASM cells back to a proliferative phenotype. We determined that integrin α7-induced Kras isoform of p21 Ras acts as a point of convergence between contractile and proliferative ASM phenotypic states. Our study provides further support for targeting integrin α7 for the development of novel anti-asthma therapies.

Effective generation of autologous corneal keratocytes from mesenchymal stem cells by cell revTM mSC diffKera for clinical use

Background & Aim Autologous corneal tissue regeneration is an ideal source of cornea transplants as it minimizes the risks of inflammation and immune rejection. Corneal blindness is one of the two leading causes of blindness worldwide. Currently, cornea transplantation is at the mercy of donated corneas which limited numbers in many countries result in more than 5 years waiting-list for such operative therapy. Because mesenchymal Stem Cells(mSC) are armed with key features of multipotent differentiation potentials, and they are also a feasible source to obtain from patients, here by using our Cell RevTM msc diffKera recipe, we effectively generate autologous corneal keratocytes, the critical corneal stromal cells, needed to maintain corneal structure and function. Methods, Results & Conclusion Our formulation includes FDA approved biochemical components essential to direct and maintain corneal keratocyte cell fate. Within 7 days of culturing mSCs in our diffKera media, we were able to achieve 5 folds proliferation as well as efficient differentiation such that we observed more than 90% dendritic star-like morphology similar to keratocytes but not mSCs. Like keratocytes, the newly differentiated cells were positive to proteoglycans production specific to corneal stroma, lumican, as well as keratocyte biomarkers such as ALDH1A1 and CD34. The control media, dubbed Keratocyte media (KM), commonly used in maintaining keratocytes as well as differentiating mSCs to keratocytes, showed only 20% cell growth and only a few dendritic shaped cells. Our generated keratocytes also expressed key components to corneal function i.e., collagen transcripts, and importantly, they did not express alpha smooth actin nor high levels of TGF-beta as these proteins are parts of inflammation signaling. These results suggest that our mSC diffKera formulation provides potential solution to effective autologous corneal keratocytes, and thus bringing us one step closer to autologous cornea generation in the near future.

Rhu-Epo down-regulates pro-tumorigenic activity of cancer-associated fibroblasts in multiple myeloma.

We have previously demonstrated that recombinant human erythropoietin (rHuEpo) is involved in the regulation of the angiogenic response in multiple myeloma (MM) through a direct effect on macrophages and endothelial cells isolated from the bone marrow of patients with MM. The aim of the present study was designed to determine the effects of rHuEpo on cancer-associated fibroblasts (CAFs) from monoclonal gammopathy of undetermined significance (MGUS) and MM patients by means of in vitro and in vivo assays. rHuEpo treatment reduces the expression of mRNA levels of fibroblast activation markers, namely alpha smooth actin (αSMA) and fibroblast activation protein (FAP) in MGUS and MM CAFs, and of pro-inflammatory and pro-angiogenic cytokines, including interleukin (IL)-6 and IL-8, vascular endothelial growth factor-A (VEGF-A), fibroblast growth factor-2 (FGF-2), and hepatocyte growth factor (HGF) in MM CAFs. Moreover, rHuEpo inhibits the proliferative activity of MM CAFs and increased the apoptosis of MGUS and MM CAFs. Overall, these data suggest that rHu-Epo down-regulates CAFs pro-tumorigenic activity. Moreover, these results are not suggestive for a pro-angiogenic activity of rHuEpo on CAFs. In fact, rHuEpo pre-treatment induces a low angiogenic response in vivo in the chorioallantoic membrane (CAM) assay of MGUS and MM CAFs conditioned medium, not comparable to that of a well-known angiogenic cytokine, VEGF-A, tested in the same assay.

Viscoelastic properties of multi-layered cellularized vascular tissues fabricated from collagen gel.

Since collagen is one of the major extracellular matrix components in vascular tissues, its use for vascular tissue engineering has several advantages. However, collagen extraction and processing for tissue engineering application alters its structure. As a result, collagen-based vascular constructs show poor mechanical properties compared to native tissues. In this work, multi-layer (single, double, and triple) vascular tissue constructs were engineered from porcine smooth muscle cells (PSMCs) entrapped in collagen gel by concentrically and sequentially layering after compaction of the previous layer(s). The engineered tissues were matured for either 14 or 21 days to allow the collagen gel to remodel before viscoelasticity, compliance, histological, and protein expression studies were conducted. While there was no significant difference upon addition of the different layers on the elastic modulus (p > .05), the viscous modulus of the single layer construct was significantly lower than the double and triple layer constructs (p < .05). Increasing the number of layers of the cellularized collagen construct increased the wall thickness and the viscous modulus of the construct. Furthermore, the cellularized single-layer construct had a relatively high compliance, but the double and triple layer constructs had compliance values comparable to both engineered vessels and native vessels. PSMCs were uniformly distributed throughout the cross-section and expressed the anticipated marker proteins smooth muscle-α actin, calponin, and smooth muscle myosin heavy chain. Taken together, this study demonstrated the viscoelastic responsiveness of multi-layer collagen-gel based vascular tissues.

Elevated levels of β6 integrin and alpha smooth actin positive microparticles in nasal lavage fluid in chronic rhinosinusitis with nasal polyps: a potential biomarker of epithelial-mesenchymal transition in the pathophysiology of polyp recurrence

Although features of epithelial-mesenchymal transition (EMT) have been reported in chronic rhinosinusitis (CRS), the extent of EMT involvement in the pathophysiology of CRS remains uncertain. Microparticles (MPs) are extracellular vesicles released from cell membrane during activation. We reported that MPs in nasal lavage fluid (NLFs) may serve as non-invasive biomarkers to reflect structural and immune cell activation in CRS (Takahashi T,et al. JACI 2017). β6 integrin (ITGB6) is a specific epithelial cell surface protein up-regulated during repair and alpha smooth actin (αSMA) is an intracellular marker of EMT. We evaluated ITGB6 and αSMA positive MPs in CRS. We collected NLFs from CRS subjects without polyps (CRSsNP,n=33), CRS with polyps (CRSwNP,n=71) and controls (n=26), and measured ITGB6(+)αSMA(+)MPs in NLFs using flow cytometry. We permeabilized MPs to detect αSMA in ITGB6(+)MPs during the analysis. ITGB6(+)MPs were elevated in CRSwNP (1.5-fold vs. control, p<0.001), but were not elevated in CRSsNP (1.3-fold,p=0.10) when compared with control. Compared with control, αSMA(+)ITGB6(+)MPs were elevated in both CRSwNP (2.8-fold,p<0.001) and CRSsNP (2.0-fold,p<0.02). Among CRSwNP subjects, αSMA(+)ITGB6(+)MPs were elevated compared to controls by 7.3-fold in those with asthma and prior surgery (revision surgery patients, n=15,p<0.001), but were elevated by only 3.6-fold in those with revision surgery without asthma (n=8,p<0.001) and only 2.1-fold in those with asthma but without a history of revision surgery (n=15,p<0.08). MPs positive for both αSMA and ITGB6 were significantly elevated in NLFs from CRSwNP, especially in subjects with asthma and recurrence of polyps, indicating that EMT is associated with the pathophysiology of nasal polyposis.

Leiomyosarcoma of the Skin and Subcutaneous Tissue in a Nellore Cow

Background: Leyomiosarcomas are uncommon malignant smooth muscle tumors of hollow organs. Bovine leyomiosarcomas have been described in several sites, but smooth muscle tumors that arise from the skin are rare in domestic animals. These neoplasms may show variable histologic features depending on their grade of differentiation; therefore, diagnostic tools as special stain, electron microscopy and immunohistochemistry are essential for the definitive diagnosis. Due to the lack of reports of skin leyomiosarcomas in cattle, we described the occurrence of a leyomiosarcoma in the skin and subcutaneous tissue of a Nellore cow.Case: A 10-year-old Zebu Nellore cow was admitted at the Veterinary Diagnosis Center (CEDIVET) of Pará Federal University (UFPA), Brazil, to investigate a tumor on the hip. At clinical examination the cow showed a decreased appetite and weight loss. Due to the bad prognosis the veterinarian recommended euthanasia. Representative tissue samples were submitted for histopathological and immunohistochemistry evaluation at the Department of Pathology of the Federal Rural University of Rio de Janeiro (UFRRJ). The slides were stained by Hematoxylin and Eosin (HE), Masson’s trichrome and Van Gieson stains and submitted for immunohistochemistry with primary antibodies vimentin, troponin C, smooth muscle alpha actin and desmin. Grossly, the tumor appeared as a multilobular, ulcerated and firm 30 cm diameter mass with black and hemorrhagic areas on the surface. The mass was scattered throughout the subcutaneous tissue and infiltrated the adjacent muscle. On light microscopy, the neoplasm consisted of a non-encapsulated proliferation into the derma, composed of highly pleomorphic spindle to round cells arranged in broad interlacing fascicles and small islands. Metastatic lesions were found in the lungs and local lymph nodes. Masson’s trichrome stain revealed collagen production and Van Gieson stains was inconsistent. Immunohistochemically, the neoplastic cells expressed vimentin diffusely and smooth Alfa actin in 10% of the cells. Desmin and troponin C failed to express immunoreactivity.Discussion: The histological, histochemical and immunohistochemical features of the neoplasm were consistent with a leiyomiosarcoma of the skin and subcutaneous tissue. Leiomyosarcomas are uncommon in farm animals and most of them arise from the female genital tract, especially in the vagina and within the uterus. The majority of smooth muscle tumors of the skin are benign; however, we described here a neoplasm that displayed clear signs of malignancy. Due to the high grade of differentiation, Van-Gieson stain failed to determine the origin of the neoplasm. Many sarcomas have resembling histologic features and busually display interlacing bundles of spindle shaped cells, similar to the pattern described in the present tumor. Therefore, immunohistochemistry stain with vimetin and smooth alpha actin was essential for the definitive diagnosis. We identified metastatic lesions in the local lymph nodes and lungs. This tumor tends to develop slowly and metastasize late. These findings underline the importance of diagnostic tools, such as immunohistochemistry and special stains to guide the pathologist and to establish the diagnosis of bovine smooth muscle tumors. Consequently, an association of histochemical and immunohistochemical procedures is indispensable to guide the pathologist for the establishment of the diagnosis of bovine smooth muscle tumors.

The effects of inner vertical outer spiral complex scaffold in repairing long segment of urethral defect

Objective To explore the possibility of the inner vertical outer spiral complex tubular urethra scaffold vascularization in repairing long segment of urethral defect. Methods From August 2014 to October 2015, 27 clean male New Zealand white rabbits were divided into 3 groups, S1 group was transfected recombinant vascular endothelial growth factor(VEGF) gene lentiviral vector group. S2 group was vascular pedicle transfer tube group. C group was simple stent group. A 3.0 cm inner vertical outer spiral complex scaffold was constructed by using the small intestine acellular matrix (SIS) and polylactic acid copolymer (PLGA) modified by type Ⅰ collagen surface, and adipose-derived mesenchymal stem cells (ADSC) and smooth muscle cells after transformation from New Zealand white rabbits. In S1 group, the seed cells were transfected by recombinant vascular endothelial growth factor (VEGF) gene lentivirus, which express VEGF protein.The complex scaffold was used to repair 3.0 cm rabbit urethral defect In S2 group, the un-transfected cells were seeded into the scaffold and embedded in the skin near the groin artery 3 weeks for repairing urethral defect with vascular pedicle transfer tube. In group C, the unseeded scaffold was used to repair the urethral defect alone.Postoperative observation and urethrography were followed 4, 8 and 24 weeks after implantation. The HE staining, fluorescence tracing, immunohistochemical and scanning electron microscopy were evaluated at the same phase. Results In S1 group, there were one urinary fistula and one urethral stricture-related death, respectively. The urethra was smooth and patent, histological examination showed active hyperplasia of urethral capillary. In S2 group, there were one urinary fistula and two urethral stricture-related deaths, respectively. The urethral was rough, local thinning or dilated. Fat accumulation and mucosal contraction were found in the urethral submucosal, respctively. In C group, there were one urinary fistula, three hypospadias, and three urethral stricture-related deaths. The thickness of the urethra was uneven and stricture bending. The urethral mucosa was poorly repaired and the scar was narrow. HE and CD31 staining showed that S1 and S2 groups were active in the proliferation of urethral capillaries, and the angiogenesis was abundant. VEGF staining showed that the cytoplasm of endothelial cell layer, smooth muscle layer of vascular wall and the urothelial epithelial cell layer were fully expressed at 24 weeks, especially in epithelial cell layer. CKpan staining showed that the epithelium of S1 and S2 group developed to stratified epithelium, and the morphology of urethra was similar to normal urethra at 24 weeks. The urethral epithelial in C group of grew poor as single-level, irregular arrangement, 24 weeks is still a lack of effective stratified epithelium. HE and α-SMA staining showed that the smooth muscle and actin gradually increased in group S1 and S2, α-SMA staining in group C was scarce and increased at 24 weeks. PLGA was encapsulated by the surrounding tissue and the structure of electrospinning was clear after 4 weeks, absorbed and degraded after 8 weeks and absorbed after 24 weeks. Conclusions The inner vertical outer spiral complex tubular urethra scaffold maybe a reasonable method in repairing long segment urethral defects, and the methods of tubular urethra scaffold vascularization by transfected VEGF gene recombinant lentiviral vector and vascular flap deserve more research. Key words: Tissue engineering; Scaffold; Urethra defect; Vascularization

A Subhepatic Calcified Cyst

Question: A 68-year-old woman, asymptomatic, with no medical history, underwent an abdominal computed tomography scan in a traumatic context. An enhanced series (Figure A) revealed a 4-cm cystic mass with tissue and calcified rim components located under the left liver (Figure A, arrow) inside the ligamentum teres. The lesion was supplied by a left hepatic artery branch. The frontal view showed the mass drainage into the left external iliac vein through a long pedicle (the umbilicus vein connected to the left inferior epigastric vein) (v). A laparoscopic resection of this mass was performed because of the risk of spontaneous hemorrhage linked to the dense tumoral vasculature and the lack of formal histologic diagnosis. During the procedure, the surgeon observed a cystic mass attached to ligamentum teres between the liver and the umbilicus. At pathologic examination (Figure B), a well-circumscribed largely cystic mass, with a fibrous and calcified shell and hemorrhagic modifications (arrow) was observed. Histologically (Figure C), the fibrous wall contained many large vessels and a small cellular area (star). This area consisted of small nests of moderate-sized monotonous clear cells with normochromatic ovoid nuclei. There was no nuclear atypia and no mitosis. The tumor exhibited an elaborate network of small capillaries. Tumor cells expressed the melanocytic marker HMB45 and smooth muscular actin. What is your diagnosis? Look on page 489 for the answer and see the Gastroenterology website (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and images in GI. PEComas are tumors derived from epithelioid perivascular cells that typically coexpress smooth muscle and melanocytic markers. The family of PEComas includes angiomyolipoma, clear cell “sugar” tumor, and lymphangioleiomyomatosis. Some of these tumors may be associated with tuberous sclerosis complex. PEComas of the ligamentum teres (also called in this location “clear cell myomelanocytic tumors”) are rare, but the ligamentum teres location is the most classic in children. Thirteen cases have been reported in the literature, within or in the immediate vicinity of falciform ligament/ligamentum teres.1Folpe A.L. Goodman Z.D. Ishak K.G. et al.Clear cell myomelanocytic tumor of the falciform ligament/ligamentum teres: a novel member of the perivascular epithelioid clear cell family of tumors with a predilection for children and young adults.Am J Surg Pathol. 2000; 24: 1239-1246Crossref PubMed Scopus (201) Google Scholar, 2Folpe A.L. Mentzel T. Lehr H.A. et al.Perivascular epithelioid cell neoplasms of soft tissue and gynecologic origin.Am J Surg Pathol. 2005; 29: 1558-1575Crossref PubMed Scopus (679) Google Scholar, 3Alaggio R. Cecchetto G. Martignoni G. et al.Malignant perivascular epithelioid cell tumor in children: description of a case and review of literature.J Pediatr Surg. 2012; 47: 31-40Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar There was a marked female predominance with a mean age of 20 years (range, 3-54), a mean size of 8 cm (range, 5-20), and a significant risk of metastasis (3 of 13 cases). Many of the lesions were calcified and had hemorrhagic and cystic alterations. In the absence of established malignancy criteria, PEComas must be considered to have an uncertain malignant potential, requiring surgical resection and long-term monitoring. A mass of the ligamentum teres should always lead to consider the diagnosis of PEComa, even in adults, and even with cystic presentation. Moreover, most frequently, other tumors of the ligamentum teres are malignant (local extension of hepatocellular carcinoma, metastatic adenocarcinoma of gastrointestinal or gynecological origin). The patient was free of disease at 6 months follow-up.

Smooth Muscle-Alpha Actin Inhibits Vascular Smooth Muscle Cell Proliferation and Migration by Inhibiting Rac1 Activity.

Smooth muscle alpha-actin (SMA) is a marker for the contractile, non-proliferative phenotype of adult smooth muscle cells (SMCs). Upon arterial injury, expression of SMA and other structural proteins decreases and SMCs acquire a pro-migratory and proliferative phenotype. To what extent SMA regulates migration and proliferation of SMCs is unclear and putative signaling pathways involved remain to be elucidated. Here, we used lentiviral-mediated gene transfer and siRNA technology to manipulate expression of SMA in carotid mouse SMCs and studied effects of SMA. Overexpression of SMA results in decreased proliferation and migration and blunts serum-induced activation of the small GTPase Rac, but not RhoA. All inhibitory effects of SMA are rescued by expression of a constitutively active Rac1 mutant (V12rac1). Moreover, reduction of SMA expression by siRNA technology results in an increased activation of Rac. Taken together, this study identifies Rac1 as a downstream target for SMA to inhibit SMC proliferation and migration.

Innervation of the Female Levator Ani Muscle and Consequences in Pelvic Floor Dysfunctions

IntroductionThe Levator Ani Muscle (LAM) and the Levator Ani Nerve (LAN) are distended and traumatized during pregnancy and vaginal delivery. Both mechanisms may sustainably impair the pelvis statics but the topography and functions of the nerve supply to the LAM remain anatomically controversial.ObjectiveTo characterize the nature and function of the LAM nerve pathways and to perform a comprehensive 3D reconstruction of the female pelvic innervation.MethodsThe Computer‐Assisted Anatomical dissection (CAAD) protocol was applied to pelvic blocks procured in 6 female human fetuses, in accordance with the National Biomedicine Agency. Specimens were serially sectioned and immunostained for: overall (S100), somatic (PMP22), adrenergic (TH), cholinergic (VaCHT) and nitrergic (nNOS) nerve fibers and for: smooth (smooth actin) and striated (myogenin) muscle fibers. Slides were digitized for 3D reconstructions on WinSurf®. A pelvic sample of an additional fetus was examined under electronic microscopy to analyze the ultrastructure of the LAM.ResultsThree main nerve pathways to the LAM were observed: the levator ani nerve (LAN), the pudendal nerve (PuN), the inferior hypogastric plexus (IHP). The PuN was both somatic and autonomic, located below the LAM (‘infralevator pathway’) and supplying innervation to the inferior aspect of the LAM. The LAN was solely somatic, located above the LAM (‘supralevator pathway’) and supplying innervation to the superior aspect of the LAM. The IHP nerve fibers were solely autonomic, located in between the LAM and the pelvic organs (‘endolevator pathway’) and supplying innervation to the medial portion of the LAM. Smooth muscle areas were identified with labelings and electronic microscopy: they 1) were located in the puboanal sling of the LAM 2) were bulging between rectum and vagina 3) were intermingled to the anococcygeal raphe 4) were innervated by the IHP and the PuN pathways.ConclusionHaving at once striated and smooth muscle fibers and being co‐innervated by the somatic and autonomic systems, the LAM represents a crucial anatomical structure involved in the pelvic floor function and its rehabilitation.Support or Funding Informationnone

Effect of angiotensin-converting enzyme inhibitor, lisinopril on morphological and biochemical aspects of fibrotic liver regeneration.

Background/Aims:Hepatic fibrosis results in defective liver regeneration following partial hepatectomy. Angiotensin converting enzyme (ACE) inhibitors can enhance liver regeneration and are also involved in the reduction of hepatic fibrosis. The present study has been conducted to evaluate the potential effect of an ACE inhibitor, lisinopril, on the morphological and biochemical aspects of fibrotic liver regeneration.Materials and Methods:Eight-week old female Sprague Dawley rats were made fibrotic by intragastric carbon tetrachloride treatment. Rats were given saline or lisinopril (1 mg/kg) orally for 1 week and were subjected to sham surgery or two-third partial hepatectomy. Liver regenerative and functional capacities were determined 48 hours post surgery.Results:Lisinopril administration did not affect the regeneration rate, proliferation cell nuclear antigen count, and hepatocellular area of fibrotic livers following partial hepatectomy. No statistically significant difference between treated and control rats regarding mitotic count, hepatocyte nuclear area, and binuclear hepatocyte frequency was observed. Serum biochemical analysis showed that lisinopril non-significantly decreased the partial hepatectomy induced elevated levels of alanine aminotransferase, aspartate transaminase, and alkaline phosphatase whereas lactate dehydrogenase and total bilirubin levels were significantly reduced. No marked reduction in hepatic collagen content and alpha smooth actin positive cells was observed by lisinopril treatment.Conclusion:ACE inhibitor lisinopril did not produce major histomorphological alterations in regenerating fibrotic liver following partial hepatectomy, however, it may improve its functional capability.