Showing papers on "Smoothelin published in 2020"

Complete Myogenic Differentiation of Adipogenic Stem Cells Requires Both Biochemical and Mechanical Stimulation

Abstract: Vascular tissue engineering of the middle layer of natural arteries requires contractile smooth muscle cells (SMC) which can be differentiated from adipose-derived mesenchymal stem cells (ASC) by treatment with transforming growth factor-β, sphingosylphosphorylcholine and bone morphogenetic protein-4 (TSB). Since mechanical stimulation may support or replace TSB-driven differentiation, we investigated its effect plus TSB-treatment on SMC orientation and contractile protein expression. Tubular fibrin scaffolds with incorporated ASC or pre-differentiated SMC were exposed to pulsatile perfusion for 10 days with or without TSB. Statically incubated scaffolds served as controls. Pulsatile incubation resulted in collagen-I expression and orientation of either cell type circumferentially around the lumen as shown by alpha smooth muscle actin (αSMA), calponin and smoothelin staining as early, intermediate and late marker proteins. Semi-quantitative Westernblot analyses revealed strongly increased αSMA and calponin expression by either pulsatile (12.48-fold; p < 0.01 and 38.15-fold; p = 0.07) or static incubation plus TSB pre-treatment (8.91-fold; p < 0.05 and 37.69-fold; p < 0.05). In contrast, contractility and smoothelin expression required both mechanical and TSB stimulation since it was 2.57-fold increased (p < 0.05) only by combining pulsatile perfusion and TSB. Moreover, pre-differentiation of ASC prior to pulsatile perfusion was not necessary since it could not further increase the expression level of any marker.

Inflammatory Drivers of Cardiovascular Disease: Molecular Characterization of Senescent Coronary Vascular Smooth Muscle Cells.

Abstract: The senescence of vascular smooth muscle cells (VSMCs) has been implicated as a causal pro-inflammatory mechanism for cardiovascular disease development and progression of atherosclerosis, the instigator of ischemic heart disease. Contemporary limitations related to studying this cellular population and senescence-related therapeutics are caused by a lack of specific markers enabling their detection. Therefore, we aimed to profile a phenotypical and molecular signature of senescent VSMCs to allow reliable identification. To achieve this goal, we have compared non-senescent and senescent VSMCs from two in vitro models of senescence, replicative senescence (RS) and DNA-damage induced senescence (DS), by analyzing the expressions of established senescence markers: cell cycle inhibitors- p16 INK4a, p14 ARF, p21 and p53; pro-inflammatory factors-Interleukin 1β (IL-1β), IL-6 and high mobility group box-1 (HMGB-1); contractile proteins-smooth muscle heavy chain- (MYH11), smoothelin and transgelin (TAGLN), as well as structural features (nuclear morphology and LMNB1 (Lamin B1) expression). The different senescence-inducing modalities resulted in a lack of the proliferative activity. Nucleomegaly was seen in senescent VSMC as compared to freshly isolated VSMC Phenotypically, senescent VSMC appeared with a significantly larger cell size and polygonal, non-spindle-shaped cell morphology. In line with the supposed switch to a pro-inflammatory phenotype known as the senescence associated secretory phenotype (SASP), we found that both RS and DS upregulated IL-1β and released HMGB-1 from the nucleus, while RS also showed IL-6 upregulation. In regard to cell cycle-regulating molecules, we detected modestly increased p16 levels in both RS and DS, but largely inconsistent p21, p14ARF, and p53 expressions in senescent VSMCs. Since these classical markers of senescence showed insufficient deregulation to warrant senescent VSMC detection, we have conducted a non-biased proteomics and in silico analysis of RS VSMC demonstrating altered RNA biology as the central molecular feature of senescence in this cell type. Therefore, key proteins involved with RNA functionality, HMGB-1 release, LMNB-1 downregulation, in junction with nuclear enlargement, can be used as markers of VSMC senescence, enabling the detection of these pathogenic pro-inflammatory cells in future therapeutic studies in ischemic heart disease and atherosclerosis.

Aldosterone enhances high phosphate-induced vascular calcification through inhibition of AMPK-mediated autophagy.

Abstract: It remains unclear whether the necessity of calcified mellitus induced by high inorganic phosphate (Pi) is required and the roles of autophagy plays in aldosterone (Aldo)-enhanced vascular calcification (VC) and vascular smooth muscle cell (VSMC) osteogenic differentiation. In the present study, we found that Aldo enhanced VC both in vivo and in vitro only in the presence of high Pi, alongside with increased expression of VSMC osteogenic proteins (BMP2, Runx2 and OCN) and decreased expression of VSMC contractile proteins (α-SMA, SM22α and smoothelin). However, these effects were blocked by mineralocorticoid receptor inhibitor, spironolactone. In addition, the stimulatory effects of Aldo on VSMC calcification were further accelerated by the autophagy inhibitor, 3-MA, and were counteracted by the autophagy inducer, rapamycin. Moreover, inhibiting adenosine monophosphate-activated protein kinase (AMPK) by Compound C attenuated Aldo/MR-enhanced VC. These results suggested that Aldo facilitates high Pi-induced VSMC osteogenic phenotypic switch and calcification through MR-mediated signalling pathways that involve AMPK-dependent autophagy, which provided new insights into Aldo excess-associated VC in various settings.

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

Abstract: 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.

Incorporation of Smooth Muscle Cells Derived from Human Adipose Stem Cells on Poly(Lactic-co-Glycolic Acid) Scaffold for the Reconstruction of Subtotally Resected Urinary Bladder in Athymic Rats.

Abstract: The urinary tract can be affected by both congenital abnormalities as well as acquired disorders, such as cancer, trauma, infection, inflammation, and iatrogenic injuries, all of which may lead to organ damage requiring eventual reconstruction As a gold standard, gastrointestinal segment is used for urinary bladder reconstruction However, one major problem is that while bladder tissue prevents reabsorption of specific solutes, gastrointestinal tissue actually absorbs them Therefore, tissue engineering approach had been attempted to provide an alternative tissue graft for urinary bladder reconstruction Human adipose-derived stem cells isolated from fat tissues were differentiated into smooth muscle cells and then seeded onto a triple-layered PLGA sheet to form a bladder construct Adult athymic rats underwent subtotal urinary bladder resection and were divided into three treatment groups (n = 3): Group 1 (“sham”) underwent anastomosis of the remaining basal region, Group 2 underwent reconstruction with the cell-free scaffold, and Group 3 underwent reconstruction with the tissue-engineered bladder construct Animals were monitored on a daily basis and euthanisation was performed whenever a decline in animal health was detected All animals in Groups 1, 2 and 3 survived for at least 7 days and were followed up to a maximum of 12 weeks post-operation It was found that by Day 14, substantial ingrowth of smooth muscle and urothelial cells had occurred in Group 2 and 3 In the long-term follow up of group 3 (tissue-engineered bladder construct group), it was found that the urinary bladder wall was completely regenerated and bladder function was fully restored Urodynamic and radiological evaluations of the reconstructed bladder showed a return to normal bladder volume and functionHistological analysis revealed the presence of three muscular layers and a urothelium similar to that of a normal bladder Immunohistochemical staining using human-specific myocyte markers (myosin heavy chain and smoothelin) confirmed the incorporation of the seeded cells in the newly regenerated muscular layers Implantation of PLGA construct seeded with smooth muscle cells derived from human adipose stem cells can lead to regeneration of the muscular layers and urothelial ingrowth, leading to formation of a completely functional urinary bladder

Myostatin in the Arterial Wall of Patients with End-Stage Renal Disease.

Abstract: Aim Myostatin (Mstn) has been described as a trigger for the progression of atherosclerosis. In this study, we evaluated the role of Mstn in arterial remodeling in patients with end-stage renal disease (ESRD). Methods Vascular specimens were collected from 16 ESRD patients (56.4±7.9 years) undergoing renal transplant (recipients) and 15 deceased kidney non-uremic donors (55.4±12.1 years). We studied gene and protein expression of Mstn, ubiquitin ligases, Atrogin-1, and muscle ring finger protein-1 (MuRF-1), inflammatory marker CCL2, cytoskeleton components, and Klotho by reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Moreover, we assessed vascular calcification and collagen deposition. Finally, we studied the effects of recombinant Mstn on rat vascular smooth muscle cells (VSMCs, A7r5) and evaluated the effects of uremic serum (US) on primary human VSMCs. Results Myostatin mRNA was upregulated in the arterial vascular wall of recipients compared with donors (~15- folds, p＜0.05). This response was accompanied by the upregulation of gene expression of Atrogin-1 and MuRF-1 (＋2.5- and ＋10-fold) and CCL2 (＋3-fold). Conversely, we found downregulation of protein expression of Smoothelin, α-smooth muscle actin (α-SMA), vimentin, and Klotho (-85%, -50%, -70%, and -80%, respectively; p＜0.05) and gene expression of vimentin and Klotho. Exposition of A7r5 to Mstn induced a time-dependent SMAD 2/SMAD 3 phosphorylation and expression of collagen-1 and transforming growth factor β (TGFβ) mRNA, while US induced overexpression of Mstn and Atrogin-1 and downregulation of Smoothelin and Klotho. Conclusions Our data suggest that uremia might induce vascular Mstn gene expression together with a complex pathway of molecular and structural changes in the vascular wall. Myostatin, in turn, can translate the metabolic alterations of uremia into profibrotic and stiffness inducing signals.

Encapsulated three-dimensional bioprinted structure seeded with urothelial cells: a new construction technique for tissue-engineered urinary tract patch.

Abstract: Background: Traditional tissue engineering methods to fabricate urinary tract patch have some drawbacks such as compromised cell viability and uneven cell distribution within scaffold. In this study, we combined three-dimensional (3D) bioprinting and tissue engineering method to form a tissue-engineered urinary tract patch, which could be employed for the application on Beagles urinary tract defect mode to verify its effectiveness on urinary tract reconstruction. Methods: Human adipose-derived stem cells (hADSCs) were dropped into smooth muscle differentiation medium to generate induced microtissues (ID-MTs), flow cytometry was utilized to detect the positive percentage for CD44, CD105, CD45, and CD34 of hADSCs. Expression of vascular endothelial growth factor A (VEGFA) and tumor necrosis factor-stimulated gene-6 (TSG-6) in hADSCs and MTs were identified by Western blotting. Then the ID-MTs were employed for 3D bioprinting. The bioprinted structure was encapsulated by transplantation into the subcutaneous tissue of nude mice for 1 week. After retrieval of the encapsulated structure, hematoxylin and eosin and Masson’s trichrome staining were performed to demonstrate the morphology and reveal collagen and smooth muscle fibers, integral optical density (IOD) and area of interest were calculated for further semiquantitative analysis. Immunofluorescent double staining of CD31 and α-smooth muscle actin (α-SMA) were used to reveal vascularization of the encapsulated structure. Immunohistochemistry was performed to evaluate the expression of interleukin-2 (IL-2), α-SMA, and smoothelin of the MTs in the implanted structure. Afterward, the encapsulated structure was seeded with human urothelial cells. Immunofluorescent staining of cytokeratins AE1/AE3 was applied to inspect the morphology of seeded encapsulated structure. Results: The semi-quantitative assay showed that the relative protein expression of VEGFA was 0.355 ± 0.038 in the hADSCs vs. 0.649 ± 0.150 in the MTs (t = 3.291, P = 0.030), while TSG-6 expression was 0.492 ± 0.092 in the hADSCs vs. 1.256 ± 0.401 in the MTs (t = 3.216, P = 0.032). The semi-quantitative analysis showed that the mean IOD of IL-2 in the MT group was 7.67 ± 1.26, while 12.6 ± 4.79 in the hADSCs group, but semi-quantitative analysis showed that there was no statistical significance in the difference between the two groups (t = 1.724, P = 0.16). The semi-quantitative analysis showed that IOD was 71.7 ± 14.2 in non-induced MTs (NI-MTs) vs. 35.7 ± 11.4 in ID-MTs for collagen fibers (t = 3.428, P = 0.027) and 12.8 ± 1.9 in NI-MTs vs. 30.6 ± 8.9 in ID-MTs for smooth muscle fibers (t= 3.369, P = 0.028); furthermore, the mean IOD was 0.0613 ± 0.0172 in ID-MTs vs. 0.0017 ± 0.0009 in NI-MTs for α-SMA (t = 5.994, P = 0.027), while 0.0355 ± 0.0128 in ID-MTs vs. 0.0035 ± 0.0022 in NI-MTs for smoothelin (t= 4.268, P = 0.013), which indicate that 3D bioprinted structure containing ID-MTs could mimic the smooth muscle layer of native urinary tract. After encapsulation of the urinary tract patch for additional cell adhesion, urothelial cells were seeded onto the encapsulated structures, and a monolayer urothelial cell was observed. Conclusion: Through 3D bioprinting and tissue engineering methods, we provided a promising way to fabricate tissue-engineered urinary tract patch for further investigation. Key words: Human adipose-derived stem cells; Urinary tract patch; Microtissues; Tissue engineering; Three-dimensional bioprinting

Predifferentiated Smooth Muscle-Like Adipose-Derived Stem Cells for Bladder Engineering.

Abstract: Introduction: All organs of human body are a conglomerate of various cell types with multidirectional interplay between the different cells and the surrounding microenvironment, leading to a stable tissue formation, homeostasis, and function. To develop a functional smooth muscle tissue, we need to simulate and create a multicellular microenvironment. The multilineage adipose-derived stem cells (ADSCs), which can be easily harvested in large numbers, may provide an alternative cell source for the replacement of smooth muscle cells (SMCs) in cell-based detrusor bioengineering therapeutic approaches. The aim of this study was to investigate whether predifferentiated smooth muscle-like ADSC (pADSC) can support SMCs to generate stable smooth muscle tissue through remodeling of extracellular matrix (ECM) and factor secretion. Methods: Rat SMC and pADSC were mono- and cocultured in the cell ratios 1:1, 1:2, 1:3, and 1:5 (SMC-pADSC) and grown for up to 2 weeks in vitro. The expression of the SMC-specific markers alpha-smooth muscle actin, calponin, myosin heavy chain 11 (MyH11), and smoothelin was assessed, and cell proliferation and contractility were analyzed. Proteomic analysis of the secretome (cell-cell contact was compared with a noncontact transwell 1:1 coculture) and the cell pellets was performed, with the focus on ECM deposition and remodeling, integrin expression and growth factor secretion. Results: SMC and pADSC were strongly positive for all smooth muscle markers. After 1 and 2 weeks of culture, the 1:1 cell ratio developed a significantly higher number of smooth muscle organoids and improved contractility. These organoids were highly structured, consisting of an SMC core surrounded by a pADSC layer. The deposition of various EMC proteins, such as collagens 1a1, 1a2, 2a1, 3a1, 5a2, 6a2, 12a1, and fibrillin 1, was significantly increased. A decreased matrix metalloproteinase 3 (MMP3), MMP9 and MMP13 secretion, as well as increased tissue inhibitors of metalloproteinase 1 (TIMP1) and TIMP2 secretion were found in the contact coculture compared with the monoculture controls. Conclusion: SMC-pADSC 1:1 cocultures exhibit an improved cell proliferation, contractility, and organoid formation compared with all other ratios and monoculture, while retaining a stable phenotype that is comparable with the SMC monoculture. These effects are mediated by increased ECM deposition and tight ECM remodeling by the secreted MMP and TIMP. Impact statement Harvesting smooth muscle cells (SMCs) from diseased bladders represents a significant limitation for clinical translation of bladder Tissue Engineering. Our results suggest that autologous predifferentiated smooth muscle-like adipose-derived stem cell can substitute SMCs, and may be used in combination with SMCs to generate contractile detrusor muscle tissue for patients suffering from end-stage bladder diseases. We demonstrate a beneficial effect when using these cells in a 1:1 ratio with improved deposition of extracellular matrix (ECM) molecules and superior remodeling of the ECM by matrix metalloproteinases and decreased tissue inhibitors of metalloproteinase activity.

Generation of an enteric smooth muscle cell line from the pig ileum.

Abstract: Smooth muscle cells (SMCs) play an important role in physiology and production in farm animals such as pigs. Here, we report the generation of a pig SMC line. Our original objective was to establish an enteroendocrine cell line from the pig ileum epithelium through lentiviral transduction of the Simian Virus (SV) 40 large T antigen. However, an initial expression analysis of marker genes in nine cell clones revealed that none of them were enteroendocrine cells or absorptive enterocytes, goblet cells, or Paneth cells, some of the major cell types existing in the ileum epithelium. A more detailed characterization of one clone named PIC7 by RNA-seq showed that these cells expressed many of the known smooth muscle-specific or -enriched genes, including smooth muscle actin alpha 2, calponin 1, calponin 3, myosin heavy chain 11, myosin light chain kinase, smoothelin, tenascin C, transgelin, tropomyosin 1, and tropomyosin 2. Both quantitative PCR and RNA-seq analyses showed that the PIC7 cells had a high expression of mRNA for smooth muscle actin gamma 2, also known as enteric smooth muscle actin. A Western blot analysis confirmed the expression of SV40 T antigen in the PIC7 cells. An immunohistochemical analysis demonstrated the expression of smooth muscle actin alpha 2 filaments in the PIC7 cells. A collagen gel contraction assay showed that the PIC7 cells were capable of both spontaneous contraction and contraction in response to serotonin stimulation. We conclude that the PIC7 cells are derived from an enteric SMC from the pig ileum. These cells may be a useful model for studying the cellular and molecular physiology of pig enteric SMCs. Because pigs are similar to humans in anatomy and physiology, the PIC7 cells may be also used as a model for human intestinal SMCs.

A case of hepatic leiomyosarcoma with osteosarcomatous differentiation (malignant mesenchymoma) in a dog.

Abstract: A rare spontaneous hepatic leiomyosarcoma with osteosarcomatous differentiation was observed in a female beagle dog and its morphological and immunohistochemical characteristics were examined. Upon necropsy, an endoceliac mass originating from the liver was detected, which was composed of hematoid fluid-filled cysts and white to grayish solid tissue. There were no macroscopic findings in other organ systems. Histopathologically, the hepatic mass consisted of two different mesenchymal components. One form was spindle cells arranged in interlacing fascicles immunohistochemically positive for smooth muscle actin (SMA) and smoothelin, indicating leiomyosarcomatous differentiation. The other form was composed of short spindle cells positive for S-100 and was producing various amounts of eosinophilic osteoid and trabecula-like matrices positive for osteocalcin, indicating osteosarcomatous differentiation. In addition, invasive growth in the hepatic parenchyma and cell atypia were observed. Based on these findings, the mass was diagnosed as hepatic leiomyosarcoma with osteosarcomatous differentiation (malignant mesenchymoma), which might be derived from undifferentiated mesenchymal cells.

Device and method for promoting rapid strut coverage and vascular endothelial coverage

Abstract: Myosin heavy chain 11 (MYH11)-siRNA-eluting, calponin 1 (CNN1)-siRNA-eluting, leiomodin 1 (LMOD)-siRNA-eluting, smoothelin (SMTN)-siRNA-eluting, tropomyosin (TPM)-siRNA-eluting, caldesmon 1 (CALD)-siRNA-eluting, actinin (ACTN)-siRNA-eluting, actin alpha (ACTA)-siRNA-eluting, and actin beta (ACTB)-siRNA-eluting medical device capable of selective and significant inhibition of vascular smooth muscle cells (VSMC) in the same or even higher order of magnitude of sirolimus or analogs and paclitaxel or analogs that are cell antiproliferative drugs currently used in drug-eluting stents (DES), whereas said MYH11-siFRNA-eluting, CNN1-siFRNA-eluting, LMOD-siRNA-eluting, SMTN-siRNA-eluting, TPM-siRNA-eluting, CALD-siRNA-eluting, ACTN-siRNA-eluting, ACTA-siRNA-eluting, and ACTB-siRNA-eluting medical devices promote early medical device struts coverage and/or vascular re-endothelialization compared to the current DES that elute cell antiproliferative drugs such as sirolimus or analogs and, paclitaxel or analogs.