scholarly journals Design and Evaluation of a Polypeptide that Mimics the Integrin Binding Site for EDA Fibronectin to Block Profibrotic Cell Activity

2021 ◽  
Vol 22 (4) ◽  
pp. 1575
Author(s):  
Lin Zhang ◽  
Hongyu Yan ◽  
Yifan Tai ◽  
Yueming Xue ◽  
Yongzhen Wei ◽  
...  
Keyword(s):  
Specific Binding ◽  
Smooth Muscle Actin ◽  
Cell Activity ◽  
Myofibroblast Differentiation ◽  
Integrin Receptors ◽  
Extracellular Signal ◽  
Cytotoxic Treatment ◽  
Integrin Binding Site ◽  
Binding Cleft

Fibrosis is characterized by excessive production of disorganized collagen- and fibronectin-rich extracellular matrices (ECMs) and is driven by the persistence of myofibroblasts within tissues. A key protein contributing to myofibroblast differentiation is extra domain A fibronectin (EDA-FN). We sought to target and interfere with interactions between EDA-FN and its integrin receptors to effectively inhibit profibrotic activity and myofibroblast formation. Molecular docking was used to assist in the design of a blocking polypeptide (antifibrotic 38-amino-acid polypeptide, AF38Pep) for specific inhibition of EDA-FN associations with the fibroblast-expressed integrins α4β1 and α4β7. Blocking peptides were designed and evaluated in silico before synthesis, confirmation of binding specificity, and evaluation in vitro. We identified the high-affinity EDA-FN C-C′ loop binding cleft within integrins α4β1 and α4β7. The polypeptide with the highest predicted binding affinity, AF38Pep, was synthesized and could achieve specific binding to myofibroblast fibronectin-rich ECM and EDA-FN C-C′ loop peptides. AF38Pep demonstrated potent myofibroblast inhibitory activity at 10 µg/mL and was not cytotoxic. Treatment with AF38Pep prevented integrin α4β1-mediated focal adhesion kinase (FAK) activation and early signaling through extracellular-signal-regulated kinases 1 and 2 (ERK1/2), attenuated the expression of pro-matrix metalloproteinase 9 (MMP9) and pro-MMP2, and inhibited collagen synthesis and deposition. Immunocytochemistry staining revealed an inhibition of α-smooth muscle actin (α-SMA) incorporation into actin stress fibers and attenuated cell contraction. Increases in the expression of mRNA associated with fibrosis and downstream from integrin signaling were inhibited by treatment with AF38Pep. Our study suggested that AF38Pep could successfully interfere with EDA-FN C-C′ loop-specific integrin interactions and could act as an effective inhibitor of fibroblast of myofibroblast differentiation.

scholarly journals Radiosynthesis, Biological Evaluation, and Preclinical Study of a 68Ga-Labeled Cyclic RGD Peptide as an Early Diagnostic Agent for Overexpressed αvβ3 Integrin Receptors in Non-Small-Cell Lung Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-11 ◽  
Author(s):  
Nazanin Pirooznia ◽  
Khosrou Abdi ◽  
Davood Beiki ◽  
Farshad Emami ◽  
Seyed Shahriar Arab ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Specific Binding ◽  
Small Cell ◽  
Integrin Receptors ◽  
Small Cell Lung ◽  
Early Diagnostic

The αvβ3 integrin receptors have high expression on proliferating growing tumor cells of different origins including non-small-cell lung cancer. RGD-containing peptides target the extracellular domain of integrin receptors. This specific targeting makes these short sequences a suitable nominee for theranostic application. DOTA-E(cRGDfK)2 was radiolabeled with 68Ga efficiently. The in vivo and in vitro stability was examined in different buffer systems. Metabolic stability was assessed in mice urine. In vitro specific binding, cellular uptake, and internalization were determined. The tumor-targeting potential of [68Ga]Ga-DOTA-E(cRGDfK)2 in a lung cancer mouse model was studied. Besides, the very early diagnostic potential of the 68Ga-labeled RGD peptide was evaluated. The acquisition and reconstruction of the PET-CT image data were also carried out. Radiochemical and radionuclide purity for [68Ga]Ga-DOTA-E(cRGDfK)2 was >%98 and >%99, respectively. Radiotracer showed high in vivo, in vitro, and metabolic stability which was determined by ITLC. The dissociation constant (Kd) of [68Ga]Ga-DOTA-E(cRGDfK)2 was 15.28 nM. On average, more than 95% of the radioactivity was specific binding (internalized + surface-bound) to A549 cells. Biodistribution data showed that radiolabeled peptides were accumulated significantly in A549 tumor and excreted rapidly by the renal system. Tumor uptake peaks were at 1-hour postinjection for [68Ga]Ga-DOTA-E(cRGDfK)2. The tumor was clearly visualized in all images. [68Ga]Ga-DOTA-E(cRGDfK)2 can be used as a peptide-based imaging agent allowing very early detection of different cancers overexpressing αvβ3 integrin receptors and can be a potential candidate in clinical peptide-based imaging for lung cancer.

scholarly journals FAK Inhibition Attenuates Corneal Fibroblast Differentiation In Vitro

Biomolecules ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1682
Author(s):  
Vincent Yeung ◽  
Sriniwas Sriram ◽  
Jennifer A. Tran ◽  
Xiaoqing Guo ◽  
Audrey E. K. Hutcheon ◽  
...  
Keyword(s):  
Wound Healing ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Myofibroblast Differentiation ◽  
Corneal Scarring ◽  
Corneal Fibroblast ◽  
Corneal Fibroblasts ◽  
Corneal Wound ◽  
Tgf Β1

Corneal fibrosis (or scarring) occurs in response to ocular trauma or infection, and by reducing corneal transparency, it can lead to visual impairment and blindness. Studies highlight important roles for transforming growth factor (TGF)-β1 and -β3 as modulators in corneal wound healing and fibrosis, leading to increased extracellular matrix (ECM) components and expression of α-smooth muscle actin (αSMA), a myofibroblast marker. In this study, human corneal fibroblasts (hCF) were cultured as a monolayer culture (2D) or on poly-transwell membranes to generate corneal stromal constructs (3D) that were treated with TGF-β1, TGF-β3, or TGF-β1 + FAK inhibitor (FAKi). Results show that hCF 3D constructs treated with TGF-β1 or TGF-β3 impart distinct effects on genes involved in wound healing and fibrosis—ITGAV, ITGB1, SRC and ACTA2. Notably, in the 3D construct model, TGF-β1 enhanced αSMA and focal adhesion kinase (FAK) protein expression, whereas TGF-β3 did not. In addition, in both the hCF 2D cell and 3D construct models, we found that TGF-β1 + FAKi attenuated TGF-β1-mediated myofibroblast differentiation, as shown by abrogated αSMA expression. This study concludes that FAK signaling is important for the onset of TGF-β1-mediated myofibroblast differentiation, and FAK inhibition may provide a novel beneficial therapeutic avenue to reduce corneal scarring.

Cofilin is a marker of myofibroblast differentiation in cells from porcine aortic cardiac valves

2008 ◽  
Vol 294 (4) ◽  
pp. H1767-H1778 ◽  
Author(s):  
M. Pho ◽  
W. Lee ◽  
D. R. Watt ◽  
C. Laschinger ◽  
C. A. Simmons ◽  
...  
Keyword(s):  
Stem Cells ◽  
Side Population ◽  
Smooth Muscle Actin ◽  
Tensile Force ◽  
Type I ◽  
Myofibroblast Differentiation ◽  
Side Population Cells ◽  
Isolated Cells ◽  
Color Flow

The formation of myofibroblasts in valve interstitial cell (VIC) populations contributes to fibrotic valvular disease. We examined myofibroblast differentiation in VICs from porcine aortic valves. In normal valves, cells immunostained for α-smooth muscle actin (α-SMA, a myofibroblast marker) were rare (0.69 ± 0.48%), but in sclerotic valves of animals fed an atherogenic diet, myofibroblasts were spatially clustered and abundant (31.2 ± 6.3%). In cultured VIC populations from normal valves, SMA-positive myofibroblasts were also spatially clustered, abundant (21% positive cells after 1 passage), and stained for collagen type I and vimentin but not desmin. For an analysis of stem cells, two-color flow cytometry of isolated cells stained with Hoechst 33342 demonstrated that 0.5% of VICs were side population cells; none stained for SMA. Upon culture, sorted side population cells generated ∼85% SMA-positive cells, indicating that some myofibroblasts originate from a rare population with stem cell characteristics. Plating cells on rigid collagen substrates enabled the formation of myofibroblasts after 5 days in culture, which was completely blocked by culture of cells on compliant collagen substrates. Exogenous tensile force also significantly increased SMA expression in VICs. Isotope-coded affinity tags and mass spectrometry were used to identify differentially expressed proteins in myofibroblast differentiation of VICs. Of the nine proteins that were identified, cofilin expression and phospho-cofilin were strongly increased by conditions favoring myofibroblast differentiation. Knockdown of cofilin with small-interfering RNA inhibited collagen gel contraction and reduced myofibroblast differentiation as assessed by the SMA incorporation into stress fibers. When compared with normal valves, diseased valves showed strong immunostaining for cofilin that colocalized with SMA in clustered cells. We conclude that in VICs, cofilin is a marker for myofibroblasts in vivo and in vitro that arise from a rare population of stem cells and require a rigid matrix for formation.

scholarly journals Myofibroblasts in Nasal Polyposis: Regulation by Topical Steroids

1997 ◽  
Vol 4 (4) ◽  
pp. 205-210 ◽  
Author(s):  
Guy M Tremblay ◽  
Manabu Nonaka ◽  
Bengt Särnstrand ◽  
Jerry Dolovich ◽  
Jack Gauldie ◽  
...  
Keyword(s):  
Nasal Polyp ◽  
Nasal Polyposis ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Topical Steroids ◽  
Myofibroblast Differentiation ◽  
Dependent Manner ◽  
Tgf Beta ◽  
Alpha Smooth Muscle Actin

OBJECTIVES: To compare numbers of myofibroblasts, a fibroblast phenotype associated with chronically inflamed tissue, in nasal polyp tissues from untreated and corticosteroid-treated subjects. To study whether corticosteroids can directly affect myofibroblast differentiation in vitro.PATIENTS AND METHODS: Immunolocalization of alpha-smooth muscle actin (alpha-SMA) as a marker of myofibroblast differentiation, vimentin and transforming growth factor (TGF)-beta in nasal polyp tissues from nine patients who had taken no steroids for at least a month before polypectomy and from eight patients who had received intranasal budesonide or beclomethasone dipropionate continuously for four to 12 months before polypectomy. Cultured nasal polyp fibroblasts were exposed to budesonide for six days and alpha-SMA expression was determined by immunocytochemistry.RESULTS: Nasal polyp tissues from untreated subjects were characterized by the presence of a substantial number of myofibroblasts compared with tissues from corticosteroid-treated subjects. The median percentages of alpha-SMA positive cell areas per total area for the two groups were 6.4% (0.5 to 11.5) and 1.3% (0.1 to 4.7), respectively. This difference in alpha-SMA staining between the two groups was not due to a decrease in fibroblast numbers (vimentin-positive spindle-shape cells). Numbers of TGF-beta positive cells were similar in the two groups of subjects. In vitro, budesonide treatment decreased the number of alpha-SMA positive fibroblasts in primary lines in a dose dependent manner.CONCLUSIONS: The difference in myofibroblast numbers between nasal polyp tissues from untreated and corticosteroid-treated subjects, as shown by immunolocalization of alpha-SMA, suggests a new therapeutic effect of nasal topical corticosteroids in nasal polyposis. This could be a direct effect of the drug on fibroblast differentiation and/or modulation of cytokine production.

scholarly journals Cub domain-containing protein 1 negatively regulates TGF-β signaling and myofibroblast differentiation

2018 ◽  
Vol 314 (5) ◽  
pp. L695-L707 ◽  
Author(s):  
Nina Noskovičová ◽  
Katharina Heinzelmann ◽  
Gerald Burgstaller ◽  
Jürgen Behr ◽  
Oliver Eickelberg
Keyword(s):  
Cell Surface ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Proteome Analysis ◽  
Lung Fibroblasts ◽  
Myofibroblast Differentiation ◽  
Dependent Manner ◽  
Cub Domain ◽  
Tgf Β1

Fibroblasts are thought to be the prime cell type for producing and secreting extracellular matrix (ECM) proteins in the connective tissue. The profibrotic cytokine transforming growth factor-β1 (TGF-β1) activates and transdifferentiates fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts, which exhibit increased ECM secretion, in particular collagens. Little information, however, exists about cell-surface molecules on fibroblasts that mediate this transdifferentiation process. We recently identified, using unbiased cell-surface proteome analysis, Cub domain-containing protein 1 (CDCP1) to be strongly downregulated by TGF-β1. CDCP1 is a transmembrane glycoprotein, the expression and role of which has not been investigated in lung fibroblasts to date. Here, we characterized, in detail, the effect of TGF-β1 on CDCP1 expression and function, using immunofluorescence, FACS, immunoblotting, and siRNA-mediated knockdown of CDCP1. CDCP1 is present on interstitial fibroblasts, but not myofibroblasts, in the normal and idiopathic pulmonary fibrosis lung. In vitro, TGF-β1 decreased CDCP1 expression in a time-dependent manner by impacting mRNA and protein levels. Knockdown of CDCP1 enhanced a TGF-β1-mediated cell adhesion of fibroblasts. Importantly, CDCP1-depleted cells displayed an enhanced expression of profibrotic markers, such as collagen V or α-SMA, which was found to be independent of TGF-β1. Our data show, for the very first time that loss of CDCP1 contributes to fibroblast to myofibroblast differentiation via a potential negative feedback loop between CDCP1 expression and TGF-β1 stimulation.

scholarly journals Extracellular Vesicles Secreted by Corneal Epithelial Cells Promote Myofibroblast Differentiation

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1080 ◽  
Author(s):  
Tina B. McKay ◽  
Audrey E. K. Hutcheon ◽  
James D. Zieske ◽  
Joseph B. Ciolino
Keyword(s):  
Epithelial Cells ◽  
Extracellular Vesicles ◽  
Corneal Epithelium ◽  
Smooth Muscle Actin ◽  
Initial Response ◽  
Protein Translation ◽  
Myofibroblast Differentiation ◽  
Corneal Epithelial Cells ◽  
Corneal Epithelial

The corneal epithelium mediates the initial response to injury of the ocular surface and secretes a number of profibrotic factors that promote corneal scar development within the stroma. Previous studies have shown that corneal epithelial cells also secrete small extracellular vesicles (EVs) in response to corneal wounding. In this paper, we hypothesized that EVs released from corneal epithelial cells in vitro contain protein cargo that promotes myofibroblast differentiation, the key cell responsible for scar development. We focused on the interplay between corneal epithelial-derived EVs and the stroma to determine if the corneal fibroblast phenotype, contraction, proliferation, or migration were promoted following vesicle uptake by corneal fibroblasts. Our results showed an increase in myofibroblast differentiation based on α-smooth muscle actin expression and elevated contractility following EV treatment compared to controls. Furthermore, we characterized the contents of epithelial cell-derived EVs using proteomic analysis and identified the presence of provisional matrix proteins, fibronectin and thrombospondin-1, as the dominant encapsulated protein cargo secreted by corneal epithelial cells in vitro. Proteins associated with the regulation of protein translation were also abundant in EVs. This paper reveals a novel role and function of EVs secreted by the corneal epithelium that may contribute to corneal scarring.

Abstract 271: Deletion of Delta-like 1 Homolog Accelerates Fibroblast-myofibroblast Differentiation and Induces Myocardial Fibrosis

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Luca Troncone ◽  
Patricia Rodriguez ◽  
Yassine Sassi ◽  
Ludovic Benard ◽  
Kiyotake Ishikawa ◽  
...  
Keyword(s):  
Myocardial Fibrosis ◽  
Cardiac Fibrosis ◽  
Cardiac Fibroblasts ◽  
Transmembrane Protein ◽  
Smooth Muscle Actin ◽  
Cardiac Fibroblast ◽  
Myofibroblast Differentiation ◽  
Potential Candidate ◽  
Smad 3

Myocardial fibrosis is associated with profound changes in ventricular architecture and geometry, resulting in diminished cardiac function. Here we uncover that Delta-like homologue 1 (Dlk1), a paternally imprinted gene encoding a transmembrane protein belonging to the Epidermal Growth Factor (EGF)-like family, orchestrates the process of cardiac fibroblast to myofibroblast differentiation and controls myocardial fibrosis. We first show that cardiomyocytes and cardiac fibroblasts express different Dlk1 mRNA spliced variants and its absence accelerates fibroblast differentiation into myofibroblasts in vitro. Overexpression of Dlk1 in cardiac fibroblasts resulted in inhibition of fibroblast proliferation and differentiation into myofibroblasts. This process appears to be regulated by TGFβ-1 signaling, since fibroblasts lacking Dlk1 exhibited a higher activation of the TGFβ-1/Smad-3 pathway at baseline, leading to an earlier acquisition of the myofibroblast phenotype. Dlk1-null mice myocardium displayed increased TGFβ-1/Smad3 profibrotic activity, resulting in infiltration/accumulation of myofibroblasts, and induction and deposition of the extracellular matrix fibronectin extra domain A isoform and collagen, supporting a role for Dlk1 in cardiac fibrosis. Furthermore, these profibrotic events were associated with reduced myofibril integrity, myocyte hypertrophy and cardiac dysfunction. Interestingly, Dlk1 expression was downregulated in ischemic heart tissue from human patients and in the border and scar-zones of infarcted pigs’ hearts. This phenotype was paralleled by increased expression of the profibrotic markers, collagen I, lysyl oxidase and α-smooth muscle actin. Mechanistically, the inhibitory action of Dlk1 on cardiac fibroblast-myofibroblast differentiation is mediated by miR-370 direct targeting of TGFβ-R2/Smad-3 signaling in the myocardium. Given the deleterious effects of continuous activation of this pathway, we propose Dlk1 as a new potential candidate for therapy in cases where aberrant TGFβ signaling leads to chronic fibrosis.

On the Role of Transferrin in 67Ga Uptake by Tumor Cells

1988 ◽  
Vol 27 (04) ◽  
pp. 151-153
Author(s):  
P. Thouvenot ◽  
F. Brunotte ◽  
J. Robert ◽  
L. J. Anghileri
Keyword(s):  
Specific Binding ◽  
Subcellular Fractionation ◽  
Animal Blood ◽  
Tumor Bearing ◽  
Cell Structures ◽  
The Difference ◽  
Sarcoma Cells ◽  
In Vitro Uptake

In vitro uptake of 67Ga-citrate and 59Fe-citrate by DS sarcoma cells in the presence of tumor-bearing animal blood plasma showed a dramatic inhibition of both 67Ga and 59Fe uptakes: about ii/io of 67Ga and 1/5o of the 59Fe are taken up by the cells. Subcellular fractionation appears to indicate no specific binding to cell structures, and the difference of binding seems to be related to the transferrin chelation and transmembrane transport differences

Interactions of Staphylokinase with Human Platelets

1995 ◽  
Vol 73 (03) ◽  
pp. 472-477 ◽  
Author(s):  
H R Lijnen ◽  
B Van Hoef ◽  
D Collen
Keyword(s):  
Platelet Aggregation ◽  
Platelet Function ◽  
Specific Binding ◽  
Platelet Rich Plasma ◽  
Human Platelets ◽  
Normal Plasma ◽  
Atp Secretion ◽  
Platelet Disaggregation ◽  
Activation Rate

SummaryThe interactions of recombinant staphylokinase (SakSTAR) with human platelets were investigated in a buffer milieu, in a human plasma milieu in vitro, and in plasma from patients with acute myocardial infarction (AMI) treated with SakSTAR.In a buffer milieu, the activation rate of plasminogen by SakSTAR or streptokinase (SK) was not significantly altered by addition of platelets. Specific binding of SakSTAR or SK to either resting or thrombin- activated platelets was very low. ADP-induced or collagen-induced platelet aggregation in platelet-rich plasma (PRP) was 94 ± 2.7% or 101 ± 1.7% of control in the presence of 0.1 to 20 μM SakSTAR, with corresponding values of 95 ± 2.8% or 90 ± 4.6% of control in the presence of 0.1 to 4 μM SK. No effects were observed on platelet disaggregation. ATP secretion following collagen-induced platelet aggregation was 4.3 ± 0.26 μM for SakSTAR (at concentrations of 0.1 to 20 μM) and 4.4 ± 0.35 μM for SK (at concentrations of 0.1 to 4 μM), as compared to 3.4 ± 0.70 μM in the absence of plasminogen activator.Fifty % lysis in 2 h (C50) of 60 μl 125I-fibrin labeled platelet-poor plasma (PPP) clots prepared from normal plasma or from plasma of patients with Glanzmann thrombasthenia and immersed in 0.5 ml normal plasma, was obtained with 12 or 16 nM SakSTAR and with 49 or 40 nM SK, respectively. C50 values for lysis of 60 μl PRP clots prepared from normal or patient plasma were also comparable for SakSTAR (19 or 21 nM), whereas SK was 2-fold more potent toward PRP clots prepared from Glanzmann plasma as compared to normal plasma (C50 of 130 versus 270 nM).No significant effect of SakSTAR on platelet function was observed in plasma from patients with AMI treated with SakSTAR, as revealed by unaltered platelet count, platelet aggregation and ATP secretion.Thus, no effects of high SakSTAR concentrations were observed on human platelets in vitro, nor of therapeutic SakSTAR concentrations on platelet function in plasma.

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