Muscle Actin-positive Cells Research Articles

Expression of apoptotic and proliferation factors in gastric mucosa of patients with systemic sclerosis correlates with form of the disease

Despite high prevalence of patients with gastric disease in systemic sclerosis (SSc), its pathogenesis is still poorly understood. We immunohistochemically analysed biopsies of gastric mucosa (GM) in 5 controls and 15 patients with different forms of SSc: limited cutaneous (lc), diffuse cutaneous moderate (sys1) and severe (sys2). The number of positive cells was analysed by a Kruskall-Wallis test, P < 0.05 was considered statistically significant. Percentage of proliferating (Ki-67 positive) cells was highest in sys1 (3% in superficial and 4,6% in deeper parts of GM), which dropped to 1% in sys2. Percentage of α-smooth muscle actin (α-SMA) positive cells was 5% in controls, 9% in superficial GM, while in deeper GM rose from 7% to 19% in sys1 and sys2, thus indicating increased myofibroblast population. Caspase-3 positive apoptotic cells characterized 1,5–2% of controls, 8% of superficial and 6% of deeper GM cells in sys1. In sys2, apoptosis affected 50% of surface epithelial and gland cells and 30% of deeper glands, and correlated with increased fibrosis and decreased syndecan-1 expression. Our data demonstrate that sys1 is the most „active” proliferating form of SSc. Sys2 characterize collagen deposition, surface epithelium defects, extensive apoptosis and low proliferation, GM atrophy and loss of function.

Fate tracing of hepatocytes in mouse liver

Hepatocytes perform most of the functions of the liver and are considered terminally differentiated cells. Recently, it has been suggested that hepatocytes might have the potential to transdifferentiate or dedifferentiate under physiological or pathological conditions in vivo. Epithelial-mesenchymal transition of hepatocytes in liver fibrosis has also been proposed. However, these findings have not been fully confirmed. In this study, hepatocytes were genetically labelled for cell fate tracing using lacZ via the tamoxifen-induced CreERT/loxP system. After induction with tamoxifen, alb + cells were permanently marked by lacZ expression, and all progeny lacZ + cells were derived from a single source with no interference. We did not observe transdifferentiation or dedifferentiation of hepatocytes into cholangiocytes or hepatic progenitor cells under conditions of liver homeostasis or following a 2/3 partial hepatectomy. Meanwhile, lacZ/OPN-positive cells were observed in livers of 3,5-diethoxycarbonyl-1,4-dihydrocollidine-fed mice, and lacZ/alpha-smooth muscle actin-positive cells were detected in carbon tetrachloride-induced chronic liver injury models. These results suggested that some existing differentiated alb + cells might have the potential of transdifferentiation/dedifferentiation or epithelial-to-mesenchymal transition in vivo in some liver injury models, but the proportion of these alb + cells in liver was very low, and their significance and actual function during the pathological process remains to be elucidated.

Abstract 75: Ischemic Heart Failure is Exacerbated in CD39-null Mice

Background: CD39 (ectonucleoside triphosphate diphosphohydrolase) is a nucleotidase expressed on endothelial cells, vascular smooth muscles cells, and leukocytes. CD39 plays a key role in vascular homeostasis, hydrolyzing extracellular ATP and ADP. CD39 has been shown to be important in models of ischemic preconditioning and cardiac ischemia reperfusion. However, the effect of CD39 activity on functional recovery of heart after myocardial infarction (MI) has not been evaluated. Hypothesis: Genetic ablation of CD39 expression exacerbates post-myocardial infarction cardiac function and fibrosis. Methods: Wild-type (WT) and CD39-null mice were subjected to coronary artery ligation. Cardiac function and protein evaluation of fibrotic markers was performed at day 28 post-MI. Results: Evaluation at Day 28 post-MI revealed that while mice of both genotypes had similarly reduced ejection fraction and equally compromised contractile function (dP/dtmax), there was a more pronounced negative effect on lusitropy (dP/dtmin) and increased left ventricular end-diastolic pressure in CD39-null mice. Therefore, cd39 gene ablation associates with the development of worsening cardiac performance. Histological analysis revealed increased collagen deposition and abundance of alpha-smooth muscle actin (αSMA) positive interstitial cells in the CD39-null hearts compared to WT hearts. To quantify these findings immunoblot analysis for collagen and αSMA was performed. We found that collagen and αSMA were increased at Day 28 post-MI, in CD39-null hearts compared to WT hearts. Conclusion: CD39 ablation has detrimental effects on post-MI recovery, resulting in diminished cardiac performance and increased fibrosis.

Abstract B11: Understanding how the Hedgehog pathway mediates stromal desmoplasia in Pancreatic Ductal Adencarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal cancers partly due to its profound primary chemoresistance. Previous work from this laboratory found that the prominent desmoplastic stroma observed in PDA is a significant factor in the tumor chemoresistance. We reported that, in established tumors, inhibition of the Hedgehog (Hh) pathway in tumor fibroblasts caused a loss of tumor stroma including α-smooth muscle actin positive (α-SMA) cells. This resulted in an increase in drug delivery to the tumor and subsequent reduction in tumor volume, leading to prolonged survival in a clinically relevant mouse model of PDA. However, the mechanisms by which Hh signaling contributes to the desmoplastic stroma in PDA remain unknown. In this study we have attempted to identify the target genes of the Hh pathway in the stroma of pancreatic tumors. Using a novel tumor biopsy procedure coupled with RNA-SEQ analysis we have identified genes acutely sensitive to Hh pathway inhibition in the KPC mouse model of PDA. Following 2 days of post biopsy treatment with the Smoothened inhibitor IPI926 the Hh pathway genes Gli1 and Ptch2 were significantly down-regulated. In addition, the Hh target gene, wnt inhibitory factor 1 (Wif1) and other novel genes which may be promoting stromal desmoplasia were significantly down-regulated due to Hh pathway inhibition. In addition to studying the target genes of the Hh-pathway in the stroma we have utilized a Gli1-GFP reporter mouse to study the Hh target cells in the stroma. Previous work has suggested that Hh target cells in pancreatic tumors are myofibroblasts and thus α-SMA positive. However, we observed that while some Gli1 positive stromal cells are α-SMA positive a significant proportion are α-SMA negative. In addition, costaining KPC-Gli1-GFP tumors for other stromal markers, including Fibroblast specific protein-1 (FSP-1), Glial Fibrillic Acidic Protein (GFAP), zinc finger E-box binding homeobox 1 (Zeb1) and Neuron-Glial Antigen 2 (NG-2) reveals subsets of Hh target cells in the tumor stroma. These data suggest a complex mechanism of Hh-regulated development of the desmoplastic stroma. To date the mechanisms of Hh-mediated stromal desmoplasia remain refractory. Taken together, these studies attempt to uncover the Hh target genes and cell types in the stroma of pancreatic tumors and provide an explanation for Hh-mediated stromal desmoplasia in PDA. Citation Format: Dafydd H. Thomas, Albert K. Lee, Stephen A. Sastra, Carmine F. Palermo, Raul Rabadan, P Olive Kenneth. Understanding how the Hedgehog pathway mediates stromal desmoplasia in Pancreatic Ductal Adencarcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B11.

Hepatic myofibroblasts and fibrogenic progression of chronic liver diseases.

Liver fibrogenesis is a dynamic and highly integrated molecular, tissue and cellular process that during the course of a chronic liver disease (CLD) leads progressively to an excess deposition of extracellular matrix (ECM) components in an attempt to limit the consequences of chronic parenchymal injury. Irrespective of etiology, liver fibrogenesis is sustained and modulated by an intense cross talk occurring between different hepatic cell populations that involves the synthesis and release of several mediators, including growth factors, cytokines, chemokines, reactive oxygen species, adipokines, vasoactive agents and plasma proteins. In this scenario a major pro-fibrogenic role is played by a heterogeneous population of α-smooth muscle actin (α-SMA) positive cells defined as hepatic myofibroblasts (MFs). Hepatic MFs are highly proliferative and contractile cells, primarily responsible for excess deposition of ECM components and involved in ECM altered remodeling observed in CLDs. MFs also represent a unique and critical cellular crossroad able to integrate incoming paracrine or autocrine signals, released from all hepatic cell populations involved or available in the microenvironment, as well as to synthetize and release mediators which sustain and perpetuate fibrogenesis, chronic inflammatory response and neo-angiogenesis. This review has been designed to offer critical knowledge on hepatic MFs, including terminology, essential definitions and characterization of MFs, with a focus on the origin of these cells (mainly from hepatic stellate cells and portal fibroblasts or, to a lesser extent, bone marrow-derived cells), the process of activation and the functional responses that these cells can operate in the fibrogenic progression of CLDs.

Abstract 15037: Novel Role of Kelch-like ECH-associated Protein 1/NF-E2-related Factor 2 System in Vascular Smooth Muscle Cell Apoptosis Following Vascular Injury

Abnormal increases in vascular smooth muscle cells (VSMCs) in the intimal region after vascular injury are a key event in the neointimal hyperplasia followed by vascular occlusive diseases. To maintain vascular functions, the number of VSMCs is tightly controlled by those proliferation and apoptosis during vascular remodeling. Kelch-like ECH-associated protein 1 (Keap1)-NF-E2-related factor 2 (Nrf2) system plays a critical role in the oxidative stress response. While Keap1 ubiquitinates Nrf2 for degradation under unstressed conditions, this Keap1 function is abrogated in response to oxidative stress, leading to Nrf2 stabilization and coordinated up-regulation of antioxidant genes. We have previously found that Nrf2 plays an important role in neointimal hyperplasia after vascular injury via regulating platelet-derived growth factor-induced reactive oxygen species-dependent VSMC migration; however, the role of Keap1-Nrf2 system in VSMC apoptosis has not been established. Here we show that TUNEL-positive cells are detected in both the layers of neointima and media, both of which observe alpha-smooth muscle actin positive and high Nrf2-expressed cells, 14 days after transluminal arterial injury in mice. Nrf2 deficient mice show decreased TUNEL-positive cells in neointimal and medial areas (60%) and enhanced neointimal formation (I/M ratio: 152%) 14 days after vascular injury compared with the wild-type mice. In VSMCs isolated from the thoracic aorta of rats, depletion of Keap1 with siRNA increases nuclear Nrf2 (685%) and induces its target genes, including NAD(P)H: quinone oxidoreductase-1 (664%) and heme oxygenase-1 (230%). Functionally, Keap1 depletion increase apoptotic morphological features such as cell shrinkage and nuclear condensation (4114%), annexin V binding (512%), and positive TUNEL staining in VSMCs, which is associated with caspase-3/7 activation (576%). Pretransfection of VSMCs with Nrf2 siRNA inhibits apotosis mediated by Keap1 siRNA. In summary, Keap1-Nrf2 system regulates VSMC apoptosis in the process of neointimal formation, thereby inhibiting VSMC hyperproliferation, which may contribute to the development of neointimal hyperplasia after vascular injury.

Skin Progenitor Cells Contribute to Bleomycin‐Induced Skin Fibrosis

The origin of the cells that contribute to skin fibrosis is unclear. We undertook the present study to assess the contribution of Sox2-expressing skin progenitor cells to bleomycin-induced scleroderma. Scleroderma was induced, by bleomycin administration, in wild-type mice and in mice in which CCN2 was deleted from Sox2-expressing cells. Lineage tracing analysis was performed to assess whether cells expressing Sox2 are recruited to fibrotic lesions in response to bleomycin-induced scleroderma. In response to bleomycin, Sox2-positive/α-smooth muscle actin-positive cells were recruited to fibrotic tissue. CCN2-conditional knockout mice in which CCN2 was deleted from Sox2-expressing cells exhibited resistance to bleomycin-induced skin fibrosis. Collectively, these results indicate that CCN2 is required for the recruitment of progenitor cells and that CCN2-expressing progenitor cells are essential for bleomycin-induced skin fibrosis. Lineage tracing analysis using mice in which a tamoxifen-dependent Cre recombinase was expressed under the control of the Sox2 promoter confirmed that progenitor cells were recruited to the fibrotic lesion in response to bleomycin, and that this did not occur in CCN2-knockout mice. The ability of serum to induce α-smooth muscle actin expression in skin progenitor cells required the presence of CCN2. Sox2-positive skin progenitor cells are required in order for bleomycin-induced skin fibrosis to occur, and CCN2 is required for the recruitment of these cells to the fibrotic lesion. Targeting stem cell recruitment or CCN2 may therefore represent a useful therapeutic approach in combating fibrotic skin disease.

Characterization of α‐smooth muscle actin positive cells during multilineage differentiation of dental pulp stem cells

Dental pulp tissue contains stem cells that can differentiate into multiple lineages under specific culture conditions; the origin of these dental pulp stem cells, however, is still unknown. Here we have utilized an α-SMA-GFP transgenic mouse model to characterize expression of a-smooth muscle actin (SMA)-GFP in subpassages of pulp-tissue-derived dental pulp cells, as perivascular cells express α-SMA. During subculturing, percentages of cells expressing a-SMA increased significantly from passage 1 to 3. α-SMA-GFP-positive cells expanded faster than α-SMA-GFP-negative cells. The dental pulp cells at passage 3 were induced towards osteogenic, adipogenic or chondrogenic differentiation. All three differentiated cell lines expressed high levels of α-SMA (mineralized nodules, lipid droplets and chondrocyte pellets). GFP expression colocalized with differentiated osteoblasts, adipocytes and chondrocytes. Co-culturing the α-SMA-GFP-positive cells with human endothelial cells promoted formation of tube-like structures and robust vascular networks, in 3-D culture. Taken together, the a-SMA-GFP-positive cells were shown to have multilieange differentiation ability and to promote vascularization in a co-culture system with endothelial cells.

A convenient method for producing the bleomycin-induced mouse model of scleroderma by weekly injections using a methylcellulose gel

Systemic sclerosis (SSc) is a connective tissue disease characterized by vasculopathy, excessive accumulation of extracellular matrix, and fibrosis of the skin and internal organs. An animal model of SSc, the bleomycin-induced mouse model, has been established and used extensively to investigate the pathogenesis of SSc and to seek novel therapeutic agents. We recently developed thermo-reversible combination gels that can be injected subcutaneously and are made in aqueous solution by forming a complex coacervate with the substance of interest and cationic macromolecules, followed by co-formulation with methylcellulose (MC) as a negative thermosensitive polysaccharide. The objective of this study was to demonstrate whether weekly injections of bleomycin using combination gels loaded with bleomycin can induce the skin fibrosis model of SSc in susceptible mouse strains. A low molecular weight MC (4%) gel with 4.5% ammonium sulfate was made in aqueous solution, and mixed with bleomycin. This was injected subcutaneously into female C3H/He mice at weekly intervals. Control mice were injected with the gel made with phosphate-buffered saline. After 4 weeks, histological examination and gene expression assays of cytokines were performed. Examination in vitro showed that more than 80% of the bleomycin was released from the gel by the 4th day. Histological examination showed that dermal thickness increased in the MC-bleomycin-injected group compared with the control, and semi-quantitative analysis indicated that the extent of inflammation did not differ between the groups. In the MC-bleomycin-injected group, dermal fibrosis assessed with the Masson-Trichrome stain and numbers of alpha-smooth muscle actin-positive fibroblastic cells also increased. The procedure for inducing scleroderma in which bleomycin is injected weekly as an easily-made gel system using methylcellulose, can induce dermal fibrosis in susceptible mice without causing inflammation. We believe this system represents a time- saving and convenient procedure that should facilitate research on SSc.

Collecting duct epithelium and injury: Not all cells are created equal

Studies by Butt et al. in the developing fetus provide new and timely insights into the regulation of repair and fibrosis in the injured kidney. Using a clinically relevant model, they have examined the response of the medullary collecting duct to ureteral obstruction, with some unexpected findings.

The Role of Fibroblasts in Skin Substitutes

Skin substitutes are slowly finding a position in the treatment of burns, scar reconstructions and chronic wounds. Some of the substitutes consist of extracellular matrix replacement material only, such as Integra and Alloderm; some include allogeneic cells (Dermagraft, Appligraf). The ideal skin substitute has not been developed yet, since none of the presently available products can ultimately prevent scar formation.Study of the role of autologous fibroblasts in the healing process might give further insight into the scarring process and eventually lead to improved skin substitutes.We compared wound reepithelialization of experimental wounds treated with proliferating keratinocytes and a dermal substitute with either dermal fibroblasts, adipose tissue derived fibroblasts or no fibroblasts. We also investigated the rate of keratinocyte migration of human skin equivalents cultured in vitro in the presence of dermal or adipose tissue derived fibroblasts.We reached successful wound closure in 8 days with transfer of proliferating keratinocytes on a dermal substitute seeded with dermal fibroblasts. However, the wounds treated with substitutes which contained adipose tissue derived fibroblasts or no fibroblasts at all were not closed even after 21 days.Keratinocytes seeded onto collagen lattices populated with either dermal or fat‐derived fibroblasts showed similar findings: a retarded migration and/or proliferation of keratinocytes on the collagen lattices with fat‐derived fibroblasts. The collagen lattices populated with fat‐derived fibroblasts also showed a marked contraction, up till 50% of the original area.In both models, more alpha‐smooth muscle actin positive cells were found in the fibroblast population from adipose origin.We conclude that epidermal regeneration is negatively influenced by the presence of fat‐derived fibroblasts in a dermal matrix; possibly, myofibroblasts play a role in this.

Tissue Engineering Designs for the Future: New Logics, Old Molecules

Tissue Engineering Designs for the Future: New Logics, Old Molecules

In situ detection of matrix metalloproteinase-2 (MMP2) and the metalloproteinase inhibitor TIMP2 transcripts in human primary hepatocellular carcinoma and in liver metastasis

Background/Aims: Metalloproteinase (MMP)-2 and the metalloproteinase inhibitor TIMP2, play a critical role in tumor invasion. We have investigated the cellular sources of MMP2 and TIMP2 in primary and secondary human liver cancers. Methods: Using in situ hybridization and zymography, we analyzed surgical biopsies from matching pairs of tumoral and non-tumoral liver from six hepatocellular carcinomas and seven liver metastases and from four liver donors. The cellular sources of MMP2 and TIMP2 were further characterized using an anti-α-smooth muscle actin antibody on contiguous sections. Results: In hepatocellular carcinoma and liver metastases, in situ hybridization showed that MMP2 and TIMP2 mRNA were expressed y anti-α-smooth muscle actin-positive cells at the invasive front. Slender fibroblasts embedded in a denser matrix were MMP2(+)/TIMP2(+)/anti-α-smooth muscle actin(+). Intratumor microvessels showed a strong labeling for MMP2 but weak for TIMP2 mRNA. In contrast, the endothelial lining of the central veins was MMP2(+)/TIMP2(+) in non-tumoral areas with signs of blood-flow obstruction. In control livers, MMP2 and TIMP2 mRNA distribution was restricted to fibroblasts and endothelial cells within portal tracts and scattered sinusoidal cells. Direct zymography of samples comprising the invasive front revealed variable amounts of both proMMP2 and its active form in hepatocellular carcinoma, whereas strong bands corresponding to both active and latent forms of MMP2 were detected liver metastases. Conclusions: The striking density of MMP2(+)/TIMP(+)/anti-αSM(+) stellate-shaped cells in the perisinusoidal space adjacent to liver tumors suggests that hepatic stellate cells, upon differentiation to myofibroblasts, may contribute to the dissemination of liver metastases through the sinusoidal network.

H19, a marker of developmental transition, is reexpressed in human atherosclerotic plaques and is regulated by the insulin family of growth factors in cultured rabbit smooth muscle cells.

H19 is a developmentally regulated gene with putative tumor suppressor activity, and loss of H19 expression may be involved in Wilms' tumorigenesis. In this report, we have performed in situ hybridization analysis of H19 expression during normal rabbit development and in human atherosclerotic plaques. We have also used cultured smooth muscle cells to identify H19 regulatory factors. Our data indicate that H19 expression in the developing skeletal and smooth muscles correlated with specific differentiation events in these tissues. Expression of H19 in the skeletal muscle correlated with nonproliferative, actin-positive muscle cells. In the prenatal blood vessel, H19 expression was both temporally and spatially regulated with initial loss of expression in the inner smooth muscle layers adjacent to the lumen. We also identified H19-positive cells within the adult atherosclerotic lesion and we suggest that these cells may recapitulate earlier developmental events. These results, along with the identification of the insulin family of growth factors as potent regulatory molecules for H19 expression, provide additional clues toward understanding the physiological regulation and function of H19.

Cytoskeletal characteristics of myofibroblasts in benign neoplastic and reactive fibroblastic lesions

The characteristics of the cytoskeleton of myofibroblasts were examined immunohistochemically in 10 extra-abdominal desmoid tumours, 3 palmar and 2 plantar fibromatoses and 5 nodular fasciitis; in the cultured cells of one desmoid tumour, and also ultrastructurally in 3 desmoid tumours. Polyclonal anti-desmin antibody reacted with the cells in 7 extra-abdominal desmoid tumours, 1 palmar fibromatosis, 1 plantar fibromatosis and 3 nodular fasciitis. Monoclonal antidesmin antibody reacted with cells in only 2 desmoid tumours. Desmin-positive spindle cells were scattered throughout these lesions. There were no marked ultrastructural differences between desmin-positive and desmin-negative desmoids. All specimens except one specimen of nodular fasciitis showed immunoreactivity for alpha-smooth muscle actin and vimentin. Muscle actin-positive cells were observed in all specimens. Cultured cells gave positive reactions with polyclonal desmin antibody as well as to vimentin antibodies and two preparations of actin antibodies, whereas the original tumour did not react with desmin antibody. The present studies suggested that the cytoskeleton of some myofibroblasts in both neoplastic and reactive lesions resembles that of smooth muscle cells.

Alveolar Soft Part Sarcoma: Immunohistochemical Evidence for Muscle Cell Differentiation

Seven cases of alveolar soft part sarcomas (ASPSs) were studied immunohistochemically for the presence of a number of differentiation markers in an attempt to define the cellular nature of this tumor. Desmin-positive tumor cells were found in three and muscle actin-positive cells in four cases when studied in formaldehyde-fixed and paraffin-embedded material. In one case studied in frozen sections, focal desmin positivity but no other intermediate filaments were found. Immunostaining for synaptophysin, a general neuroendocrine marker, was negative in all cases. All tumors were positive with a monoclonal antibody NK1C3, which consistently stains melanomas, and three cases showed significant numbers of S-100 protein-positive tumor cells, but immunostaining with HMB-45, a melanoma-specific monoclonal antibody, was negative in all cases. However, several rhabdomyosarcomas studied for NK1C3 and S-100 protein for comparison were also at least focally positive. Electron microscopic examination, performed in three cases, showed uniform paucity of all kinds of filaments in the tumor cells of ASPS, and it specifically failed to reveal any signs of smooth or striated muscle cell differentiation. Thus, the results of the present study do not unequivocally define the nature of ASPS but speak against its paraganglionic character and present evidence for muscle cell differentiation.