Control of fibrotic changes through the synergistic effects of anti-fibronectin antibody and an RGDS-tagged form of the same antibody

Tiwari, Anil; Kumar, Rajendra; Ram, Jagat; Sharma, Mukut; Luthra-Guptasarma, Manni

doi:10.1038/srep30872

Cited by 21 publications

(17 citation statements)

References 30 publications

(35 reference statements)

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“…In one study, Fn52RGDS, a 30 K targeting anti-FN antibody conjugated with an integrin binding FN sequence, RGDS, was found to be effective in causing a decline of fibrotic features in a fibrotic posterior capsular opacification model, a model used to study cataract formation (8). This effect included a reduction in cell migration, fibronectin deposition, and collagen gel contraction.…”

Section: Introductionmentioning

confidence: 99%

Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic

et al. 2018

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Purpose To develop PEGylated variants of pUR4/FUD (FUD), a fibronectin assembly inhibitor, using 10 kDa, 20 kDa, and 40 kDa PEGs to evaluate their binding affinity and inhibitory potency. Methods The FUD peptide was recombinantly expressed, purified, and PEGylated at the N-terminus using 10 kDa, 20 kDa, and 40 kDa methoxy-PEG aldehyde. The PEGylates were purified and fractionated using ion-exchange chromatography. The molecular weight and degree of PEGylation of each conjugate was verified using MALDI-TOF. The binding affinity of each PEG-FUD conjugate was studied using isothermal titration colorimetry (ITC) and their inhibitory potency was characterized by a cell-based matrix assembly in vitro assay. Results The 10 kDa, 20 kDa, and 40 kDa PEG-FUD conjugates were synthesized and isolated in good purity as determined by HPLC analysis. Their molecular weight was consistent with attachment of a single PEG molecule to one FUD peptide. The binding affinity (Kd) and the fibronectin fibrillogenesis inhibitory potency (IC50) of all PEG-FUD conjugates remained nanomolar and unaffected by the addition of PEG. Conclusions Retention of FUD fibronectin binding activity following PEGylation with three different PEG sizes suggest that PEG-FUD holds promise as an effective anti-fibrotic with therapeutic potential and a candidate for further pharmacokinetic and biodistribution studies.

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Section: Introductionmentioning

confidence: 99%

Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic

et al. 2018

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“…Interestingly, phosphorylation of the Smad2 linker region, known to interact with ERK1/2 (Shi and Massague, 2003), occurred after the increase in ERK1/2 signaling at 60 minutes. TGFβ has been shown previously to activate ERK1/2 signaling in lens cell lines such as primary human lens cells (Tiwari et al , 2016), FHL124 (Dawes et al , 2009) and SRA01/04 (Chen et al , 2014). In SRA01/04 cells, TGFβ-induced ERK1/2 signaling was shown to be upstream of Smad2/3 (Chen et al , 2014).…”

Section: Discussionmentioning

confidence: 98%

“…Likewise, in human lens cells, pharmacological inhibition of ERK1/2 signaling was sufficient in reducing TGFβ-induced α-SMA expression (Tiwari et al , 2016). In FHL124 lens cells, TGFβ-induced upregulation of α-SMA was shown to be independent of Smad4 (Dawes et al , 2009).…”

Section: Discussionmentioning

confidence: 99%

“…TGFβ-induced EMT in rat lens epithelial explants was also recently shown to be repressed when cells were transfected with other RTK-inhibitors, including Spred and Sef (Zhao et al , 2015). TGFβ-induced EMT can be suppressed through the inhibition of ERK1/2 signaling in different cellular contexts, such as primary human lens epithelial cells, SRA01/04 human lens epithelial cells, NMuMG epithelial cells, mouse cortical tubule (MCT) epithelial cells and HaCaT cells (Zavadil et al , 2001; Xie et al , 2004; Chen et al , 2014; Tiwari et al , 2016). Taken together, these studies strongly suggest a requirement for ERK1/2 signaling in TGFβ-induced EMT.…”

Section: Introductionmentioning

confidence: 99%

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ERK1/2 signaling is required for the initiation but not progression of TGFβ-induced lens epithelial to mesenchymal transition (EMT)

Wojciechowski

Mahmutovic

Shu

et al. 2017

Experimental Eye Research

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Transforming Growth Factor Beta (TGFβ) potently induces lens epithelial to mesenchymal transition (EMT). The resultant mesenchymal cells resemble those found in plaques of human forms of subcapsular cataract. Smad signaling has long been implicated as the sole driving force of TGFβ-mediated activity. Rat lens epithelial explants were used to examine the role of the Smad-independent signaling, namely the MAPK/ERK1/2 signaling pathway, in the initiation and progression of TGFβ-induced EMT. Phase contrast microscopy was used to observe the morphological changes associated with TGFβ-induced EMT in this model, including cell elongation, cell membrane blebbing, cell loss as indicated by the area of bare capsule and capsular wrinkling. The levels of Smad2, Smad2/3 and ERK1/2 phosphorylation measured using western blotting confirmed that the addition of UO126 was sufficient in blocking all TGFβ-induced ERK1/2 activation, as well as reducing Smad signaling at 18 hours. Immunofluorescent labeling and further western blotting confirmed that TGFβ-induced EMT was associated with an increase in α-smooth muscle actin (α-SMA) and a reduction of E-cadherin at cell borders. Pre-treatment with UO126 was effective at blocking the TGFβ-induced EMT, as evidenced by a reduction of α-SMA expression and protein labeling, E-cadherin labeling at cell borders, and a reduction of cell loss, cell elongation and capsular wrinkling. Post-treatment with UO126 at 2 and 6 hours after TGFβ addition was also effective at blocking EMT while post-treatment with UO126 at 24 and 48 hours was not sufficient in hampering TGFβ-induced EMT. Our data implicates ERK1/2 signaling in the initiation but not the progression of TGFβ-induced EMT in rat lens epithelial cells. The tight regulation of intracellular signaling pathways such as ERK1/2 are required for the maintenance of lens epithelial cell integrity and hence tissue transparency. A greater understanding of the molecular mechanisms that drive the induction and progression of EMT in the lens will provide the basis for potential therapeutics for human cataract.

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“…In addition to studying such behavior under ordinary culture conditions, we also use protein-engineered versions of ECM components, and examine the effects of these in comparison with non-protein-engineered components. One aspect that we commonly examine is the presence and activation status of metalloproteases such as MMP-2 and MMP-9, as a function of cell type and the presence of different factors including protein-engineered factors (Sharma et al, 2013;Tiwari et al, 2015;Tiwari et al, 2016;Mehta et al, 2018).…”

Section: Supplementarymentioning

confidence: 99%

Sustained exposure to trypsin causes cells to transition into a state of reversible stemness that is amenable to transdifferentiation

Sharma

Kumar

Sharma

et al. 2019

Preprint

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During cell culture, trypsin, a serine protease, is applied to cells for 5-10 minutes to separate them from each other and from the underlying substratum so that they can be transferred to a different vessel, for re-plating, after growth medium containing 10 % serum has been added to the cells, in a well-known technique known as 'passaging'. The serum in the growth medium contains alpha-1 antitrypsin, which is a potent inhibitor of trypsin, elastase and other serine proteases.Although what is used is bovine serum in which levels of proteins could be different from levels seen in humans, normal human serum contains A1AT (> 1 mg/ml; > ~18 µmol/L) as well as trypsin itself (< 460 ng/ml, or ~0.02 µmol/L), with the former in a ~900-fold molar excess over the latter. Thus, it may be assumed there is also enough A1AT in the bovine serum added during passaging, to neutralize the trypsin (~100 μM) present in the small volume of trypsin-EDTA solution used to separate cells. What are the consequences of not adding serum, when growth medium is added, or of maintaining cells for a few tens of hours in the presence of trypsin, in a serum-free growth medium? What does such sustained exposure to trypsin during cell culture do to cells? More generally, what are the responses of cells within an organism to the balance of trypsin and A1AT in the serum that bathes them constantly? We know that excesses and deficiencies in the levels of either trypsin or A1AT are associated with disease. We know that cellular metabolism can be influenced through signaling involving protease activated membrane GPCR receptors (PAR1-4). In particular, we know of a receptor called PAR2, which is specifically activated by trypsin, expressed by cells at baseline levels, and upregulated through some feedback involving trypsin-activation. We also know that cells at sites of injury or inflammation produce and secrete trypsin, and that this trypsin can act locally upon cells in a variety of ways, all of which have probably not yet been elucidated. Here, we show that sustained exposure to trypsin induces cells to de-differentiate into a stem-like state. We show that if serum is either not added at all, or added and then washed away (after confluency is attained), during cell culture, all cells exposed to exogenously-added trypsin undergo changes in morphology, transcriptome, secretome, and developmental potential, and transition into a state of stemness, in minimal essential medium (MEM). Regardless of their origins, i.e., independent of whether they are derived from primary cultures, cell lines or cancer cell lines, and regardless of the original cell type used, exposure to trypsin (~10 µM; ~250 µg/ml) at a concentration 10-fold lower than that used to separate cells during passaging (~100 μM), over a period of 24-48 hours, causes cells to (1) become rounded, (2) cluster together, (3) get arrested in the G0/G1 stage of the cell cycle, (4) display increased presence of 5-hydroxymethyl cytosine in their nuclei (indicative of reprogramming), (5) display increased...

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Control of fibrotic changes through the synergistic effects of anti-fibronectin antibody and an RGDS-tagged form of the same antibody

Cited by 21 publications

References 30 publications

Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic

Characterization of the PEGylated Functional Upstream Domain Peptide (PEG-FUD): a Potent Fibronectin Assembly Inhibitor with Potential as an Anti-Fibrotic Therapeutic

ERK1/2 signaling is required for the initiation but not progression of TGFβ-induced lens epithelial to mesenchymal transition (EMT)

Sustained exposure to trypsin causes cells to transition into a state of reversible stemness that is amenable to transdifferentiation

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