Alpha-smooth Actin Research Articles

A comprehensive exploration of diverse skin cell types in the limb of the desert tortoise (Testudo graeca) through light, transmission, scanning electron microscopy, and immunofluorescence techniques

The Greek tortoise, inhabiting harsh desert environments, provides a compelling case for investigating skin adaptations to extreme conditions. We have utilized light microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and immunofluorescence analysis to describe the structure of the arid-adapted limb skin in the Greek tortoise. Our aim was to identify the cell types that reflect the skin adaptation of this tortoise to arid conditions. Utilizing seven antibodies, we localized and elucidated the functions of various skin cells, shedding light on how the tortoise adapts to adverse environmental conditions. Our findings unveiled numerous scales on the limbs, varying in size and color, acting as protective armor against abrasions, bites, and other potential threats in their rocky habitats. The epidermis comprises four layers: stratum basalis, stratum spinosum, peri-corneous layer, and stratum corneum. Cytokeratin 14 (CK14) was explicitly detected in the basal layer of the epidermis, suggesting a role in maintaining epidermal integrity and cellular function. Langerhans cells were observed between epidermal cells filled with ribosomes and Birbeck granules. Numerous dendritic-shaped Langerhans cells revealed through E-Cadherin signify strong immunity in tortoises' skin. Melanophores were identified using the Melan-A antibody, labeling the cytoplasm, and the SOX10 antibody, labeling the nucleus, providing comprehensive insights into melanophores morphology and distribution. Two types of melanophores were found: dendritic below the stratum basalis of the epidermis and clustered oval melanophores in the deep dermal layer. Varied melanophores distribution resulted in a spotted skin pattern, potentially offering adaptive camouflage and protection against environmental challenges. Numerous myofibroblasts were discerned through alpha-smooth actin (α-SMA) expression, indicating that the Greek tortoise's skin possesses a robust tissue repair and remodeling capacity. B-cell lymphocytes detected via CD20 immunostaining exhibited sporadic distribution in the dermis, concentrating in lymphoid aggregates and around vessels, implying potential roles in local immune responses and inflammation modulation. Employing Tom20 to identify skin cells with abundant mitochondria revealed a notable presence in melanophores and the basal layer of the epidermis, suggesting high metabolic activity in these cell types and potentially influencing cellular functions. These findings contribute to our comprehension of tortoise skin anatomy and physiology, offering insights into the remarkable adaptations of this species finely tuned to their specific environmental habitats.

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.

Senescent epithelial cells remodel the microenvironment for the progression of oral submucous fibrosis through secreting TGF-β1.

Cellular senescence is strongly associated with fibrosis and tumorigenesis. However, whether the epithelium of oral submucous fibrosis (OSF) undergoes premature senescence remains unclear. This study investigates the roles of senescent epithelial cells in OSF. The immunohistochemistry and Sudan black B staining were performed to identify epithelium senescence in OSF tissues. Arecoline was used to induce human oral keratinocytes (HOKs) senescence. The cell morphology, senescence-associated β galactosidase activity, cell counting Kit 8, immunofluorescence, quantitative real-time PCR, and western blot assay were used to identification of senescent HOKs. The enzyme-linked immunosorbent assay was exerted to evaluate the levels of transforming growth factor β1 (TGF-β1) in the supernatants of HOKs treated with or without arecoline. The senescence-associated markers, p16 and p21, were overexpressed in OSF epithelium. These expressions were correlated with alpha-smooth actin (α-SMA) positively and proliferating cell nuclear antigen (PCNA) negatively. Moreover, Sudan black staining showed that there was more lipofuscin in OSF epithelium. In vitro, HOKs treated with arecoline showed senescence-associated characteristics including enlarged and flattened morphology, senescence-associated β galactosidase staining, cell growth arrest, γH2A.X foci, upregulation of p53, p21, and TGF-β1 protein levels. Moreover, senescent HOKs secreted more TGF-β1. Senescent epithelial cells are involved in OSF progression and may become a promising target for OSF treatment.

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.

L-Thyroxine induces left ventricular remodeling and fibrosis in rats by upregulating miR-21 in a reactive oxygen-dependent mechanism: a protective role of N-acetylcysteine

miR-21 is the most studied pro-fibrotic marker in the majority of mammalian tissues. The precise mechanism by which hyperthyroidism induces left ventricular LV fibrosis and remodeling remains unclear. In this study, we have investigated the role of miR-21 on l-thyroxine (l-Thy)-induced cardiac fibrosis in rats. Adult male Sprague–Dawley rats were divided into four groups as control, l-Thy, l-Thy + miR antagomir (inhibitor), and l-Thy + N-acetylcysteine (NAC/glutathione (GSH) precursor). Administration of l-Thy significantly increased mRNA levels of miR-21 in the LVs of the treated rats. Also, it impaired the LV systolic and diastolic function and increased the production of reactive oxygen species (ROS), the transactivation of NF-κB p65, the expression of NRLP3 inflammasome, and levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in these LVs. Concomitantly, l-Thy increased the ventricular collagen deposition, and stimulated the expression of collagen 1/3, alpha-smooth actin (α-SMA), transforming growth factor-β1, and Smad3/p-Smad3 but suppressed the expression of Smad7. All these effects were reversed by pre-treatment with miR-21 antagomir or co-administration of NAC. In conclusion, l-Thy-induced LV remodeling and fibrosis include a ROS-dependent upregulation of miR-21 which in turns activates NF-κB/NRLP3 inflammasome and suppresses SMad7.

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.

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.

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.

The Effect of Radiation on Acellular Dermal Matrix and Capsule Formation in Breast Reconstruction

The authors compared clinical outcomes to determine whether acellular dermal matrix altered the capsular tissue architecture in irradiated and nonirradiated breasts following matrix-assisted expander reconstruction. Part I included all 27 patients who underwent bilateral tissue expander reconstruction with acellular dermal matrix between 2007 and 2012 and subsequent unilateral radiation therapy. Part II included a subset of patients with capsular biopsy specimens taken at the time of implant exchange for histologic analysis. Specimens included irradiated and nonirradiated acellular dermal matrix and irradiated and nonirradiated native capsule. Clinical outcomes were analyzed in relation to capsule architecture and acellular dermal matrix performance. In part I, mean follow-up was 28 months. Grade III/IV contractures were identified in nine patients (all on the irradiated side), and 12 developed noncontracture complications (75 percent on the irradiated side). Nine patients were unable to continue with implant reconstruction and required salvage with autologous tissue. In part II, postirradiation biopsy specimens were taken of the peri-implant capsule in six patients at the time of secondary surgery. Elastin content and the total cellular infiltrate were significantly greater in the irradiated versus nonirradiated native capsules (p = 0.0015). Conversely, the irradiated matrix capsule was composed of similar amounts of cellular infiltrate and collagen as the nonirradiated matrix capsules and nonirradiated native capsules. Irradiated acellular dermal matrix showed the least amount of alpha-smooth actin staining but a similar number of blood vessels. Acellular dermal matrix appears to limit the elastosis and chronic inflammation seen in irradiated implant reconstructions and is potentially beneficial in these patients. Therapeutic, III.

PKCε regulates vessel formation by peri-vascular adipose tissue (PVAT) cells

Vessel formation is crucial in tumour growth and tissue regeneration. Protein kinase C (PKC) e has a well-known role on hematopoietic and mesenchymal progenitor cell differentiation and proliferation (Gobbi et al. 2013). Although PKCe has a demonstrated role in vascular restenosis, data on PKCe and vascular progenitor differentiation are still lacking. The aim of this work was to study the role of PKCe in vessel formation by adult adipose tissue cell progenitors. We, first, isolated the vessel progenitors from the adipose tissue localized between aortic arch and pulmonary artery of adult mice by collagenase/elastase digestion followed by magnetic immunoselection of Sca1+ cells (Passmann et al. 2008). We, then, tested their capability to form vessels in collagen gels and to differentiate to endothelial and smooth muscle lineage after treatment with PKCe specific activator and inhibitor peptides. The functional experiments showed that the pharmacological activation of endogenous PKCe abrogated tubule formation with a concomitant decrease of smooth alpha-actin (SMA) and platelet endothelial cell adhesion molecule (PECAM) together with the up-regulation of p-PAK1 expression. In vivo transient over-expression of PKCe significantly reduced SMA and PECAM expression levels in vessel wall cells. Together our data suggests that PKCe may affect vessel wall remodelling balancing the “phenotypic switching” (Salmon et al. 2013) between the proliferative and the differentiated state of smooth muscle and endothelial progenitor mesenchymal cells.

Abstract IA27: The FAP+ stromal cell and escape from immunosurveillance

Abstract IA27: The FAP+ stromal cell and escape from immunosurveillance

Periostin Facilitates Skin Sclerosis via PI3K/Akt Dependent Mechanism in a Mouse Model of Scleroderma

ObjectivePeriostin, a novel matricellular protein, is recently reported to play a crucial role in tissue remodeling and is highly expressed under fibrotic conditions. This study was undertaken to assess the role of periostin in scleroderma.MethodsUsing skin from patients and healthy donors, the expression of periostin was assessed by immunohistochemistry and immunoblotting analyses. Furthermore, we investigated periostin−/− (PN−/−) and wild-type (WT) mice to elucidate the role of periostin in scleroderma. To induce murine cutaneous sclerosis, mice were subcutaneously injected with bleomycin, while untreated control groups were injected with phosphate-buffered saline. Bleomycin-induced fibrotic changes were compared in PN−/− and WT mice by histological analysis as well as by measurements of profibrotic cytokine and extracellular matrix protein expression levels in vivo and in vitro. To determine the downstream pathway involved in periostin signaling, receptor neutralizing antibody and signal transduction inhibitors were used in vitro.ResultsElevated expression of periostin was observed in the lesional skin of patients with scleroderma compared with healthy donors. Although WT mice showed marked cutaneous sclerosis with increased expression of periostin and increased numbers of myofibroblasts after bleomycin treatment, PN−/− mice showed resistance to these changes. In vitro, dermal fibroblasts from PN−/− mice showed reduced transcript expression of alpha smooth actin and procollagen type-I alpha 1 (Col1α1) induced by transforming growth factor beta 1 (TGFβ1). Furthermore, recombinant mouse periostin directly induced Col1α1 expression in vitro, and this effect was inhibited by blocking the αv integrin-mediated PI3K/Akt signaling either with anti-αv functional blocking antibody or with the PI3K/Akt kinase inhibitor LY294002.ConclusionPeriostin plays an essential role in the pathogenesis of Bleomycin-induced scleroderma in mice. Periostin may represent a potential therapeutic target for human scleroderma.

Matrix metalloproteinase 14 overexpression reduces corneal scarring

Abstract Purpose Corneal wound healing is an everyday preoccupation for ophthalmologists.Corneal transparency depends on the scarring quality after a traumatic corneal wound, but also after refractive corneal surgery. Cicatrisation and fibrosis formation involve epithelial/fibroblast interactions via paracrin signals inducing extracellular matrix (ECM) remodelling. The major event is fibroblast activation and differentiation into myofibroblasts. These cells have a key role in the fibrotic response. They acquire contractile properties, and synthetise a new ECM, mainly composed of type III collagen. This scar tissue is less organised than the regular stroma, thus explaining corneal opacity. ECM remodelling is a critical step which aims to digest the excess of ECM by proteolysis of type III collagen. MMP14 is a membrane‐bound fibrillar collagenase from the Matrix Metalloprotease family. We hypothesised that its overexpression in the corneal stroma during wound repair will increase ECM remodelling and thus prevent collagen deposition in the scar tissue. Methods We developed an adeno‐associated virus‐based vector expressing murine MMP14 under the control of the CMV promoter. We evaluated MMP14 overexpression after viral transfection in a murine model of corneal wound healing. We characterised several parameters: clinical observation, histology, and wound healing markers. Results Our preliminary results showed a decreased in oedema and corneal scar formation, associated with a decreased expression of alpha smooth actin and type III collagen. Conclusion These results represent proof of concept that gene transfer of MMP14 can reduce scar formation, which could have therapeutic applications after corneal trauma.

Characterization of electroconvulsive seizure-induced TIMP-1 and MMP-9 in hippocampal vasculature

Degradation of the vascular basement membrane stimulates angiogenesis and is tightly controlled by balancing the actions of metalloproteases and their inhibitors. Previous work demonstrated that electroconvulsive seizure (ECS) elevates angiogenic factors and endothelial proliferation in the hippocampus. The robust induction of tissue inhibitor of matrix metalloprotease 1 (TIMP-1) in the stratum lacunosum moleculare (SLM) corresponds to sites of increased vascular density. This led us to examine the spatial and cellular expression of TIMP-1 and its substrate, matrix metalloprotease 9 (MMP-9). Chronic ECS increased TIMP-1 by 12-fold and MMP-9 by 3-fold in discrete SLM cells. We then characterized the expression of TIMP-1 mRNA in relation to vasculature in the SLM and glial-limiting membrane (GLM). Employing laser microdissection we identified the cell types associated with SLM vasculature and also phenotyped the cells expressing TIMP-1 and MMP-9. We concluded that TIMP-1 is produced by perivascular cells positive for alpha smooth actin and that MMP-9 is expressed by GFAP-positive astrocytes. These studies suggest that ECS-induced remodelling occurs at the vascular basement membrane and facilitates neovascularization.

Effect of botulinum toxin type A injection on the expression of substance P, TGF-β1 and α-SMA in rabbit ear model of hypertrophic scar

Objective To investigate the effect of botulinum toxin type A injection on the expres-sion of substance P, TGF-β1 and α-SMA in rabbit ear model of hypertrophic scar. Methods The hyper-trophic sear model was established in 24 Japanese rabbits'ears. The wounds in ventral surface of ear were divided in Group I (lateral wounds) and Group S (medial wounds), 3 wounds each side per ear, totally 72 wounds each group. The wound-healing time and the growth of scar were observed and recorded. On post-wounding day 28, the wounds were created in another 6 rabbits in the same way and the normal skin were harvested as Group C. Likewise, the scar samples in Groups I and S were harvested. The mRNA expression of substance P, TGF-β1 and α-SMA were detected quantitatively by using real-time PCR and α-SMA was also detected with Western blot. Results No difference between the ratio of healed or infec-tious wounds on post-wounding day 14. The mRNA expression of SP, TGF-β1 and α-SMA in Group I was significantly lower than Group S, but higher than those in Group C (P<0. 01 and <0. 05, respec-tively). The blot density of α-SMA and the ratio of blot density of a-SMA to β-actin in Group I were be-tween that of the other two groups. Conclusion Without delaying the wound-healing, botulinum toxin type A injection significantly decreases the expression of SP, TGF-β1 and α-SMA in rabbit ear model of hypertrophic scar. Key words: Botulinum toxin type A; Hypertrophic scar; Substance P; Transforming growth factor beta-1; Alpha smooth music actin A

Distribution of chondrocytes containing alpha-smooth muscle actin in human normal, osteoarthrotic, and transplanted articular cartilage

The aim of our study was to evaluate the occurrence of chondrocytes containing alpha-smooth muscle actin in human normal and diseased cartilage. Immunohistochemistry using monoclonal antibodies for alpha-smooth actin, muscle-specific actin, S-100 protein, CD 34, and desmin was performed on samples of human articular cartilage obtained at autopsy following sudden death, during total hip and knee replacement for osteoarthritis, or after femoral neck fracture in patients without symptoms of osteoarthritis. Moreover, the layers of residual cartilage from chondral posttraumatic defects obtained during preoperative arthroscopy and of newly formed cartilage after autologous-chondrocyte transplantation (Hyalograft ® C) obtained during second-look arthroscopy were also examined by immunohistochemistry and RT PCR. Our study showed that a significant percentage of articular chondrocytes express alpha-smooth muscle actin in healthy, diseased, and regenerated articular cartilage. Alpha-actin positive chondrocytes (18%) were observed predominantly in the upper zone of normal articular cartilage. By contrast, only approximately 10% of cartilage cells in the deep region stained for this contractile actin isoform. Actin-positive chondrocytes (myochondrocytes) are formed predominantly in response to injury to the osteoarthrotic cartilage, at sites of defective healing, and in newly formed cartilage after autologous chondrocyte transplantation. Fibrocartilage is present in some of these conditions, and it is known that this tissue contains chondrocytes with actin. The presence of myochondrocytes in the surface layer of normal articular cartilage indicates that this region probably plays an important role in maintaining cartilage integrity. Myochondrocytes may utilize the contractile actin isoform in manipulating the extracellular matrix of articular cartilage. It is also possible that actin-containing chondrocytes have a higher potential for regeneration in contrast to chondrocytes that do not contain this contractile material in their cytoplasm.

Atypical teratoid/rhabdoid tumour: 7-year event-free survival with gross total resection and radiotherapy in a 7-year-old boy

We report the case of a 7-year-old boy who presented in 1998 a tumour of the left frontal lobe. Initially diagnosed as anaplastic ependymoma, the boy was treated by gross total resection followed by radiotherapy at the operated site. In July 2005, an orbital tumour was discovered and resected. The tumour was composed of sheets of rhabdoid cells which diffusely expressed vimentin and focally epithelial membrane antigen (EMA) and alpha-smooth actin by immunohistochemistry. The first tumour was re-examined. Small foci of rhabdoid cells were found. Immunohistochemistry anti-INI1 performed on both tumours was negative. Molecular techniques performed on frozen specimen of the orbital tumour confirmed the diagnosis of atypical teratoid/rhabdoid tumour (ATRT). We discuss the pathological criteria for diagnosis of ATRT and the usefulness of early radiotherapy in the light of the recent literature.