Endothelial Sheets Research Articles

Expression of Hormones' Receptors in Human Corneal Endothelium from Fuchs' Dystrophy: A Possible Gender' Association.

Background: Age and sex are the most significant risk of factors for advanced Fuchs dystrophy. Nevertheless, few data are available on the hormone's receptor pattern expressed in adult and advanced fuchs endothelial corneal dystrophy (FECD). We investigated the impact of gender, growth factors and extracellular matrix (ECM) regulatory proteins expressed by the dystrophic endothelia. Methods: Ten dystrophic endothelial tissues and 10 normal endothelial sheets (corneoscleral specimens; Eye Bank) were used for this characterization study. Hormones' receptors (ERα, AR, PR, SHBG), few growth factors (VEGFA, βNGF, TGFβ1), some ECM regulators (MMP1, MMP7) and few inflammatory cytokines (IFNγ, IL10) were analyzed by real-time RT-PCR. Results: ERα transcripts were significantly increased, AR and SHBG transcripts were decreased in Fuchs endothelia from female patients, and no changes were detected for PR transcripts. VEGFA, βNGF and TGFβ1 transcripts were upregulated in Fuchs' endothelia, but not significantly linked to gender. High MMP1 and low MMP7 transcripts' expression were detected in Fuchs' specimens, mainly in males than females. An increased IFNγ (Th1) transcript expression was observed in females than males, and a trend to increase for IL10 (Th2) transcripts was detected in males than females. Conclusions: Our findings clearly indicate that hormone receptors, growth factors and matrix mediators as well as a Th1 pathway are predominant in Fuchs' dystrophy, displaying a pattern of expression specific for the female phenotype. The differential expression of hormones' receptors and the Th1/Th2 ratio might prompt to new theories to be tested in vitro and in vivo models, such as the use of hormonal substitute for counteracting this endothelial cell lost.

Tissue-Engineered Corneal Endothelial Sheets Using Ultrathin Acellular Porcine Corneal Stroma Substrates for Endothelial Keratoplasty.

Tissue-engineered cornea endothelial sheets (TECES), created using a biocompatible thin and transparent carrier with corneal endothelial cells, could alleviate the shortage of donor corneas and provide abundant functional endothelial cells. In our previous clinical trials, the effectiveness and safety of the acellular porcine corneal stroma (APCS) applied in lamellar keratoplasty have been confirmed. In this study, we optimized the method to cut APCS into multiple 20 μm ultrathin lamellae by a cryostat microtome and investigated the feasibility of TECES by seeding rabbit corneal endothelial cells (RCECs) on ultrathin APCS. Cell adhesion, proliferation, and functional gene expression of RCECs on tissue-culture plastic and APCS of different thicknesses were compared. The results indicated that ultrathin lamellae were superior in increasing cell viability and maintaining cell functions. Analyzing with histology, electron microscopy, and immunofluorescence, we found that RCECs cultured on 20 μm ultrathin APCS for 5 days grew into a confluent monolayer with a density of 3726 ± 223 cells/mm2 and expressed functional biomarkers Na+/K+-ATPase and zonula occludens. After 14 days, RCECs formed an early stage of Descemet's membrane-like structure by synthesizing collagen IV and laminin. Human corneal endothelial cells were also used to further validate the supportive effect of ultrathin APCS on cells. The resulting constructs were flexible and tough enough to implant into rabbits' anterior chambers through small incisions. TECES adhered to the posterior corneal stroma, and the thickness of cornea gradually reduced to normal after grafting. These results indicate that the ultrathin APCS can serve as a tissue engineering carrier and might be a suitable alternative for endothelial cells expansion in endothelial keratoplasty.

Cardiac forces regulate zebrafish heart valve delamination by modulating Nfat signaling.

In the clinic, most cases of congenital heart valve defects are thought to arise through errors that occur after the endothelial–mesenchymal transition (EndoMT) stage of valve development. Although mechanical forces caused by heartbeat are essential modulators of cardiovascular development, their role in these later developmental events is poorly understood. To address this question, we used the zebrafish superior atrioventricular valve (AV) as a model. We found that cellularized cushions of the superior atrioventricular canal (AVC) morph into valve leaflets via mesenchymal–endothelial transition (MEndoT) and tissue sheet delamination. Defects in delamination result in thickened, hyperplastic valves, and reduced heart function. Mechanical, chemical, and genetic perturbation of cardiac forces showed that mechanical stimuli are important regulators of valve delamination. Mechanistically, we show that forces modulate Nfatc activity to control delamination. Together, our results establish the cellular and molecular signature of cardiac valve delamination in vivo and demonstrate the continuous regulatory role of mechanical forces and blood flow during valve formation.

Immunohistochemical Analysis of Tight Junction Proteins.

Tight junction proteins are integral membrane proteins located apically on epithelial and endothelial cells. They form a selective paracellular barrier restricting the passage of solutes and ions across epithelial and endothelial sheets. In brain endothelial cells, the enrichment of tight junction proteins is one of the unique features of the blood-brain barrier, the physiological boundary that separates the blood from the parenchyma. The predominant tight junction family proteins are the claudins, but several others have been described in recent years including the marvel family, occludin, and lipolysis-stimulated lipoprotein receptor. Together, the tight junctions create a highly electrical-resistant, impermeable paracellular channel that strictly restricts the movement of material from the blood to the parenchyma and vice versa. In this chapter, we will discuss immunohistochemical methods to assess tight junction expression and localization and an ImageJ-based method for quantifying tight junction staining in healthy and diseased states.

Mechanical Forces Regulate Asymmetric Vascular Cell Alignment

Mechanical forces between cells and their microenvironment critically regulate the asymmetric morphogenesis and physiological functions in vascular systems. Here, we investigated the asymmetric cell alignment and cellular forces simultaneously in micropatterned endothelial cell ring-shaped sheets and studied how the traction and intercellular forces are involved in the asymmetric vascular morphogenesis. Tuning the traction and intercellular forces using different topographic geometries of symmetric and asymmetric ring-shaped patterns regulated the vascular asymmetric morphogenesis in vitro. Moreover, pharmacologically suppressing the cell traction force and intercellular force disturbed the force-dependent asymmetric cell alignment. We further studied this phenomenon by modeling the vascular sheets with a mechanical force-propelled active particle model and confirmed that mechanical forces synergistically drive the asymmetric endothelial cell alignments in different tissue geometries. Further study using mouse diabetic aortic endothelial cells indicated that diseased endothelial cells exhibited abnormal cell alignments, traction, and intercellular forces, indicating the importance of mechanical forces in physiological vascular morphogenesis and functions. Overall, we have established a controllable micromechanical platform to study the force-dependent vascular asymmetric morphogenesis and thus provide a direct link between single-cell mechanical processes and collective behaviors in a multicellular environment.

Evaluation of reconstructed human corneal endothelium sheets made with porcine Descemet's membrane in vitro and in vivo

PurposeTo identify the feasibility of reconstructing corneal endothelial sheets by seeding non-infected monoclonal human corneal endothelial cells (HCECs) onto porcine Descemet's membrane (DM) and verifying the function in vitro and in vivo. MethodsDenuded porcine DM was decellularized for haematoxylin and eosin staining, and DNA was removed via incubation with ethylene glycol diglycidyl ether (EGDE). The physical properties of the incubated DMs were evaluated and compared to those of unincubated DMs. The non-infected monoclonal HCECs were examined by chromosome analysis and the cell proliferation was evaluated by BrdU-labelling. Then HCECs at passage 30 were then seeded on the DM and cultured for approximately 5 days. The cell growth, density and expression of the sodium-potassium adenosine triphosphatase (Na+/K+-ATPase), the tight-junction-associated protein zonula occludens (ZO-1) and acetylated alpha tubulin were examined by electron microscopy and immunocytochemistry to compare HCECs cultivated on porcine DM and those cultured in vitro. Cells on the reconstructed HCEC sheets were labeled with DiI, and the sheets were subsequently transplanted into cat eyes via DM endothelial keratoplasty (DMEK). The corneal transparency, thickness, anterior segment, and HCEC density were monitored in vivo, and the corneal endothelial cell morphology and histological structure were examined ex vivo 98 days after surgery. ResultsNo significant differences were observed in the elongation at break of the DMs and the thickness of the DMs incubated with EGDE compared to those of the unincubated DMs (P > 0.05). Results of chromosome analysis shown the number of the HCEC cell line was still 46 and no abnormal chromosome structure was found. BrdU-labelling shown the HCECs stopped proliferating after 5 days and the cells formed a single layer. The cells transferred to porcine DM formed tight connections with the substrate and generated layers of hexagonal cells on day 5. Adjacent cells cultivated on DM were closely attached to each other, tightly adhered to the porcine DM and expressed the Na+/K+-ATPase, ZO-1 protein and acetylated alpha tubulin, as did HCECs cultured in vitro. In addition, the HCEC density on DMs was 3020.14 ± 52.30 cells/mm2. After surgery, the corneas gradually became transparent, and the thickness decreased to 525.33 ± 56.23 μm at day 98 after the transplantation, while the control corneas showed consistent oedema during the monitoring period. The HCEC density was 2521.60 ± 78.24 cells/mm2 in vivo 98 days after transplantation. The histological results showed that the DiI-labeled cells were dense in the transplanted area and had a hexagonal or polygonal morphology and a normal ultrastructure; adjacent cells were closely attached to each other and tightly adhered to the porcine DM. ConclusionsSeeding non-infected monoclonal HCECs on porcine DM could reconstruct functional corneal endothelial sheets. These results may help uncover new applications for tissue-engineered endothelium in endothelial keratoplasty.

Revisiting Existing Evidence of Corneal Endothelial Progenitors and Their Potential Therapeutic Applications in Corneal Endothelial Dysfunction.

A recent successful clinical trial demonstrated that a less invasive cell-injection procedure is a viable medical modality for treating corneal endothelial dystrophy. This medical advance still relies on human corneal endothelial cell (HCEC) sources derived from rare cornea donations. The progenitor of the corneal endothelium, which has the characteristics of active proliferation and lineage restriction, will be an ideal cell source for expansion ex vivo. However, the distribution of progenitor-like cells in the corneal endothelial sheet has been under debate for more than a decade. This paper re-examines the scientific evidence of the existence of human corneal endothelial progenitors (HCEPs) from the aspects of (1) the origin of cornea formation during ocular development, (2) manifestations from clinical studies, and (3) the distinctive properties of ex vivo-cultured subpopulations. The discrepancies regarding different types of progenitor-like cells in various locations of the cornea are based on the fact that the corneal endothelium is derived from different cell types with multiple origins during corneal formation. Resolving this long-standing issue in corneal biology will enable various types of progenitors to be isolated and their potencies regarding the formation of functional endothelial cells to be examined. Additionally, an effective niche system for quantitatively producing therapeutic cells can be formulated to satisfy the burning need associated with corneal endothelial dystrophy in the future.

Improving endothelial cell migration by novel in vitro 3D culture of corneal grafts

AbstractPurposeStudying in vitro cell migration of endothelial sheets is challenging since capacity for cell migration needs to be maintained while the tissue must remain fixed on a rigid substrate. Here, a 3D culture technique is designed to maintain cellular viability and reduce tissue handling in order to analyze in vitro endothelial cell migration from corneal grafts.MethodsTwelve Quarter‐Descemet membrane endothelial keratoplasty (Q‐DMEK) grafts obtained from four corneas with intact and viable endothelial cells but ineligible for transplantation were included for explant culture. Graft quadrants were embedded in temperature‐reversible hydrogel and cultured over 2–3 weeks. Cell movement was assessed by light microscopy at fixed standardized time intervals. After gel removal, immunohistochemistry analysis was performed to characterize the phenotype of the migrating cells. Cell viability was assessed by Trypan Blue and Calcein‐AM.ResultsAll grafts embedded in the 3D hydrogel system maintained cell viability and displayed a capacity for cell migration. The migration pattern was asymmetric as endothelial cells moved as a sheet only from the radial cut graft edges, while no activity was seen in the peripheral graft area. Immunostaining performed on the samples after removing the gel showed the presence of proliferation and migration markers in the endothelial cell monolayers expanded from the radial graft edges. The endothelial cells maintained the expression patterns of phenotypical markers.ConclusionsTemperature‐reversible hydrogel proved to be a very useful matrix for studying in vitro endothelial cell migration from explant grafts and allowed for subsequent immunohistochemistry analysis after gel removal. Studying in vitro endothelial cell migration from customized endothelial grafts should result in a better understanding of cellular migration mechanisms which play an important role in achieving faster corneal clearance in patients after corneal transplantation.

Endothelial repair is dependent on CD11c+ leukocytes to establish regrowing endothelial sheets with high cellular density.

Endothelial injury makes the vessel wall vulnerable to cardiovascular diseases. Injured endothelium regenerates by collective sheet migration, that is, the endothelial cells coordinate their motion and regrow as a sheet of cells with retained cell-cell contacts into the wounded area. Leukocytes appear to be involved in endothelial repair in vivo; however, little is known about their identity and role in the reparative sheet migration process. To address these questions, we developed a high-quality en face technique that enables visualizing of leukocytes and endothelial cells simultaneously following an endoluminal scratch wound injury of the mouse carotid artery. We discovered that regrowing endothelium forms a broad proliferative front accompanied by CD11c+ leukocytes. Functionally, the leukocytes were dispensable for the initial migratory response of the regrowing endothelial sheet, but critical for the subsequent formation and maintenance of a front zone with high cellular density. Marker expression analyses, genetic fate mapping, phagocyte targeting experiments, and mouse knock-out experiments indicate that the CD11c+ leukocytes were mononuclear phagocytes with an origin from both Ly6Chigh and Ly6Clow monocytes. In conclusion, CD11c+ mononuclear phagocytes are essential for a proper endothelial regrowth following arterial endoluminal scratch injury. Promoting the endothelial-preserving function of CD11c+ leukocytes may be a strategy to enhance endothelial repair following surgical and endovascular procedures.

Vav3-induced cytoskeletal dynamics contribute to heterotypic properties of endothelial barriers.

Through multiple cell-cell and cell-matrix interactions, epithelial and endothelial sheets form tight barriers. Modulators of the cytoskeleton contribute to barrier stability and act as rheostats of vascular permeability. In this study, we sought to identify cytoskeletal regulators that underlie barrier diversity across vessels. To achieve this, we correlated functional and structural barrier features to gene expression of endothelial cells (ECs) derived from different vascular beds. Within a subset of identified candidates, we found that the guanosine nucleotide exchange factor Vav3 was exclusively expressed by microvascular ECs and was closely associated with a high-resistance barrier phenotype. Ectopic expression of Vav3 in large artery and brain ECs significantly enhanced barrier resistance and cortical rearrangement of the actin cytoskeleton. Mechanistically, we found that the barrier effect of Vav3 is dependent on its Dbl homology domain and downstream activation of Rap1. Importantly, inactivation of Vav3 in vivo resulted in increased vascular leakage, highlighting its function as a key regulator of barrier stability.

A comprehensive molecular and morphological study of the effects of space flight on human capillary endothelial cells: sample quality assessment and preliminary results.

ESA (ESA ILSRA-2009-1026); ASI (contract no. 5681); Regione Toscana (POR FSE 2007-2013-FORTEC); Kayser Italia; Lions Club International, District 108LA, Toscana, Italy.

Evaluation of BMMSCs-EPCs sheets for repairing alveolar bone defects in ovariectomized rats

The aim of this paper is to investigate the effect that bone marrow mesenchymal stem cells (BMMSCs) - endothelial progenitor cells (EPCs), BMMSCs and EPCs sheets have on repairing maxillary alveolar defects in ovariectomized (OVX) rats. In this study, after identification using multi-lineage differentiation and flow cytometry, BMMSCs and EPCs were isolated from female rats. The BMMSCs-EPCs, BMMSCs and EPCs sheets were detected by hematoxylin-eosin (H&E) staining, alkaline phosphatase (ALP) staining and qRT-PCR. Defects were created in maxillary alveoli and repaired with BMMSCs-EPCs, BMMSCs and EPCs sheets in OVX rats. The repair effects were determined by histological staining and micro-CT analysis at 2, 4 and 8 weeks after implantation. We aim to clarify whether BMMSCs-EPCs sheets are more effective in repairing alveolar bone defects than are BMMSCs and EPCs sheets in OVX rats. The results show that the osteogenic potential and the effect of bone repair are greater in the BMMSCs-EPCs sheet group and that this group has a higher ability to repair alveolar bone defects in OVX rats. These results suggest that BMMSCs-EPCs sheets have potential in clinical applications for treating humans with osteoporosis.

D(-) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression.

Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(−) lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(−) lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(−) lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET) production (NETosis) in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(−) lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(−) lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1). d(−) lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(−) lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(−) lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis.

β1‐Adrenoceptor stimulation suppresses endothelial IKCa‐channel hyperpolarization and associated dilatation in resistance arteries

In small arteries, small conductance Ca²⁺-activated K⁺ channels (SK(Ca)) and intermediate conductance Ca²⁺-activated K⁺ channels (IK(Ca)) restricted to the vascular endothelium generate hyperpolarization that underpins the NO- and PGI₂-independent, endothelium-derived hyperpolarizing factor response that is the predominate endothelial mechanism for vasodilatation. As neuronal IK(Ca) channels can be negatively regulated by PKA, we investigated whether β-adrenoceptor stimulation, which signals through cAMP/PKA, might influence endothelial cell hyperpolarization and as a result modify the associated vasodilatation. Rat isolated small mesenteric arteries were pressurized to measure vasodilatation and endothelial cell [Ca²⁺]i , mounted in a wire myograph to measure smooth muscle membrane potential or dispersed into endothelial cell sheets for membrane potential recording. Intraluminal perfusion of β-adrenoceptor agonists inhibited endothelium-dependent dilatation to ACh (1 nM-10 μM) without modifying the associated changes in endothelial cell [Ca²⁺]i . The inhibitory effect of β-adrenoceptor agonists was mimicked by direct activation of adenylyl cyclase with forskolin, blocked by the β-adrenoceptor antagonists propranolol (non-selective), atenolol (β₁) or the PKA inhibitor KT-5720, but remained unaffected by ICI 118 551 (β₂) or glibenclamide (ATP-sensitive K⁺ channels channel blocker). Endothelium-dependent hyperpolarization to ACh was also inhibited by β-adrenoceptor stimulation in both intact arteries and in endothelial cells sheets. Blocking IK(Ca) {with 1 μM 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34)}, but not SK(Ca) (50 nM apamin) channels prevented β-adrenoceptor agonists from suppressing either hyperpolarization or vasodilatation to ACh. In resistance arteries, endothelial cell β₁-adrenoceptors link to inhibit endothelium-dependent hyperpolarization and the resulting vasodilatation to ACh. This effect appears to reflect inhibition of endothelial IK(Ca) channels and may be one consequence of raised circulating catecholamines.

Monolayer Stress Microscopy: limitations, artifacts, and accuracy of recovered intercellular stresses

In wound healing, tissue growth, and certain cancers, the epithelial or the endothelial monolayer sheet expands. Within the expanding monolayer sheet, migration of the individual cell is strongly guided by physical forces imposed by adjacent cells. This process is called plithotaxis and was discovered using Monolayer Stress Microscopy (MSM). MSM rests upon certain simplifying assumptions, however, concerning boundary conditions, cell material properties and system dimensionality. To assess the validity of these assumptions and to quantify associated errors, here we report new analytical, numerical, and experimental investigations. For several commonly used experimental monolayer systems, the simplifying assumptions used previously lead to errors that are shown to be quite small. Out‐of‐plane components of displacement and traction fields can be safely neglected, and characteristic features of intercellular stresses that underlie plithotaxis remain largely unaffected. Taken together, these findings validate Monolayer Stress Microscopy within broad but well‐defined limits of applicability. This research was supported by the Spanish Ministry for Science and Innovation (BFU2009–07595), the European Research Council (Grant Agreement 242993) and the National Institutes of Health (R01HL102373, R01HL107561).

Monolayer Stress Microscopy: Limitations, Artifacts, and Accuracy of Recovered Intercellular Stresses

In wound healing, tissue growth, and certain cancers, the epithelial or the endothelial monolayer sheet expands. Within the expanding monolayer sheet, migration of the individual cell is strongly guided by physical forces imposed by adjacent cells. This process is called plithotaxis and was discovered using Monolayer Stress Microscopy (MSM). MSM rests upon certain simplifying assumptions, however, concerning boundary conditions, cell material properties and system dimensionality. To assess the validity of these assumptions and to quantify associated errors, here we report new analytical, numerical, and experimental investigations. For several commonly used experimental monolayer systems, the simplifying assumptions used previously lead to errors that are shown to be quite small. Out-of-plane components of displacement and traction fields can be safely neglected, and characteristic features of intercellular stresses that underlie plithotaxis remain largely unaffected. Taken together, these findings validate Monolayer Stress Microscopy within broad but well-defined limits of applicability.

Programmed Cell-to-Cell Variability in Ras Activity Triggers Emergent Behaviors during Mammary Epithelial Morphogenesis

Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

A coronary artery disease-associated gene product, JCAD/KIAA1462, is a novel component of endothelial cell–cell junctions

Cell–cell junctions play crucial roles in the organization and function of epithelial and endothelial cellular sheets. Here, we have identified the protein product for KIAA1462 gene, whose single nucleotide polymorphisms (SNPs) have recently reported to be associated with coronary artery disease, as a novel component of cell–cell junctions. We propose the name of KIAA1462 protein junctional protein associated with coronary artery disease (JCAD). JCAD is a ∼145kDa protein without any known domains but contains a proline-rich region. Immunolocalization studies revealed that JCAD is specifically localized at cell–cell junctions in endothelial cells but not in epithelial cells. The accumulation of JCAD at cell–cell junctions in cultured endothelial cells was impaired by RNAi-mediated suppression of VE-cadherin expression. In cell adhesion-deficient mouse L fibroblasts, JCAD was recruited to cell–cell contacts when cadherin-mediated cell–cell adhesion was induced. These results indicate that JCAD is a component of VE-cadherin-based cell–cell junctions in endothelial cells. This study also suggests the implication of endothelial cell–cell adhesion in coronary artery disease.

1127 HUMAN ADIPOSE TISSUE-DERIVED STEM CELLS DIFFERENTIATED INTO SMOOTH MUSCLE AND ENDOTHELIAL CELLS FOR TREATMENT OF ERECTILE DYSFUNCTION

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 20111127 HUMAN ADIPOSE TISSUE-DERIVED STEM CELLS DIFFERENTIATED INTO SMOOTH MUSCLE AND ENDOTHELIAL CELLS FOR TREATMENT OF ERECTILE DYSFUNCTION Hazem Orabi and Tom Lue Hazem OrabiHazem Orabi San Francisco, CA More articles by this author and Tom LueTom Lue San Francisco, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.724AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Adipose tissue-derived stem cells (ADSCs), being easily, repeatedly and efficiently harvested with considerably less donor morbidity, represent ideal source for cellular treatment for erectile dysfunction. The aims of this study were to investigate if human adipose derived stem cells can be differentiated into smooth muscle cells (SMC) and endothelial cells (EC) and if these differentiated cells can be constructed into cell sheets and cavernous tissue. METHODS Human ADSCs were isolated and expanded. They were induced into SMC and EC using human recombinant TGF-β1 and endothelial growth medium-2 (EGM-2) respectively. The growth and morphology of the cells were followed up for 4 weeks. The phenotype of induced cells were checked for SMC markers; actin, calponin and heavy chain myosin and for endothelial cell markers; CD31 and Von Willebrand factor (vWF) through immunocytochemistry and western blot. Then, the cells were used to construct SMC and EC sheets by culturing them in thermosensitive dishes for 7–10 days. Confluent cultured cells were harvested as a contiguous cell sheet only by lowering temperature. The cell sheets were stained with H&E and Masson Trichrome and verified for smooth muscle markers and for endothelial cell markers. The formation of extracellular matrix of the both cell sheets was tested using Picrosirius Red staining and collagen IV. Both smooth muscle and endothelial sheets were layered into 3D cell-dense tissues and cultured in vitro for 7 days. RESULTS The induced cells showed positive staining for smooth muscle markers and endothelial cell markers after 3 weeks for SMC cells and 2 weeks for EC. The western blot confirmed their phenotype conversion. The cells sheets could be detached easily by lowering the temperature in intact form and viable condition. The sheets were formed of 2–5 cell layers that showed positive staining for smooth muscle and endothelial cell markers. They also showed positive staining for Picrosirius Red and collagen IV indicating the formation of extracellular matrix. Cell viability was demonstrated for the monolayer and bilayered cell sheets. CONCLUSIONS The results showed the ability of human ADSCS to form SMC and EC and constitute renewable source for cellular therapy. The cell sheets made of ADSCs form new technology for improvement of erectile dysfunction. The smooth muscle and endothelial sheets can used to form cavernous tissue in vitro to be delivered to penis. In vivo studies are our next step to verify the therapeutic potential of this new treatment modality. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e453 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hazem Orabi San Francisco, CA More articles by this author Tom Lue San Francisco, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

A Steering Model of Endothelial Sheet Migration Recapitulates Monolayer Integrity and Directed Collective Migration

Cells in endothelial cell monolayers maintain a tight barrier between blood and tissue, but it is not well understood how endothelial cells move within monolayers, pass each other, migrate when stimulated with growth factor, and also retain monolayer integrity. Here, we develop a quantitative steering model based on functional classes of genes identified previously in a small interfering RNA (siRNA) screen to explain how cells locally coordinate their movement to maintain monolayer integrity and collectively migrate in response to growth factor. In the model, cells autonomously migrate within the monolayer and turn in response to mechanical cues resulting from adhesive, drag, repulsive, and directed steering interactions with neighboring cells. We show that lateral-drag steering explains the local coordination of cell movement and the maintenance of monolayer integrity by allowing closure of small lesions. We further demonstrate that directional steering of cells at monolayer boundaries, combined with adhesive steering of cells behind, can explain growth factor-triggered collective migration into open space. Together, this model provides a mechanistic explanation for the observed genetic modularity and a conceptual framework for how cells can dynamically maintain sheet integrity and undergo collective directed migration.