Fibroblasts regulate monocyte response to ECM-derived matrix: The effects on monocyte adhesion and the production of inflammatory, matrix remodeling, and growth factor proteins

Monocytes/macrophages and fibroblasts are recruited to the injury site and orchestrate the host response and tissue repair. We have previously shown that polyethylene glycol (PEG)-ylated arginine-glycine-aspartic acid (RGD) sequence grafted onto an extracellular matrix (ECM)-based semi-interpenetrating network (sIPN) enhances monocyte adhesion, and modulates subsequent gene expression and release of inflammatory and matrix remodeling factors. In this study, we investigate the direct influence of fibroblasts on monocyte response to this ECM mimic. Key wound-healing factors in inflammation, matrix remodeling, and regeneration were analyzed to gain insight into the interrelated role of regulation in fibroblast-monocyte interaction. Interleukin-lalpha/-lbeta (IL-lα/-iβ), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte inflammatory protein-lalpha/-lbeta (MIP-lα/- iβ), transforming growth factor-alpha (TGF-α), monocyte chemoattractant factor (MCP-1), matrix metalloproteinase-2/-9 (MMP-2/-9), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM- CSF) were analyzed. Fibroblasts decreased monocyte adhe sion onto the RGD-grafted sIPN while increasing monocyte GM-CSF on all surfaces over time except for on RGD and PHSRN-grafted sIPN at 96 h. Monocytes decreased initial fibroblast IL-lα and TGF-α, but drastically increased fibroblast MMP-2 and GM-CSF. Monocyte IL-iβ, TNF-α, MIP- lp, MCP-1, MMP-9, and GM-CSF expression was increased over time in the presence of all sIPNs, and when the sIPNs were immobilized with ligands, a down-regulation of fibroblast IL-iβ, MlP-lα, MIP-iβ compared with unmodified sIPN was observed. When the ligand immobilized was RGD, monocyte TGF-α, MIP-iβ, and VEGF expression was increased while monocyte GM-CSF was decreased at selected time points. These results showed a dynamic monocyte response to selected ECM components in the presence of fibroblasts. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res 89A: 841-853, 2009. © 2009 Wiley Periodicals, Inc.Link_to_subscribed_fulltex

Fibroblasts regulate monocyte response to ECM-derived matrix: The effects on monocyte adhesion and the production of inflammatory, matrix remodeling, and growth factor proteins

Monocytes/macrophages and fibroblasts are recruited to the injury site and orchestrate the host response and tissue repair. We have previously shown that polyethylene glycol (PEG)-ylated arginine-glycine-aspartic acid (RGD) sequence grafted onto an extracellular matrix (ECM)-based semi-interpenetrating network (sIPN) enhances monocyte adhesion, and modulates subsequent gene expression and release of inflammatory and matrix remodeling factors. In this study, we investigate the direct influence of fibroblasts on monocyte response to this ECM mimic. Key wound-healing factors in inflammation, matrix remodeling, and regeneration were analyzed to gain insight into the interrelated role of regulation in fibroblast-monocyte interaction. Interleukin-lalpha/-lbeta (IL-lα/-iβ), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte inflammatory protein-lalpha/-lbeta (MIP-lα/- iβ), transforming growth factor-alpha (TGF-α), monocyte chemoattractant factor (MCP-1), matrix metalloproteinase-2/-9 (MMP-2/-9), vascular endothelial growth factor (VEGF), granulocyte-macrophage colony-stimulating factor (GM- CSF) were analyzed. Fibroblasts decreased monocyte adhe sion onto the RGD-grafted sIPN while increasing monocyte GM-CSF on all surfaces over time except for on RGD and PHSRN-grafted sIPN at 96 h. Monocytes decreased initial fibroblast IL-lα and TGF-α, but drastically increased fibroblast MMP-2 and GM-CSF. Monocyte IL-iβ, TNF-α, MIP- lp, MCP-1, MMP-9, and GM-CSF expression was increased over time in the presence of all sIPNs, and when the sIPNs were immobilized with ligands, a down-regulation of fibroblast IL-iβ, MlP-lα, MIP-iβ compared with unmodified sIPN was observed. When the ligand immobilized was RGD, monocyte TGF-α, MIP-iβ, and VEGF expression was increased while monocyte GM-CSF was decreased at selected time points. These results showed a dynamic monocyte response to selected ECM components in the presence of fibroblasts. © 2009 Wiley Periodicals, Inc. J Biomed Mater Res 89A: 841-853, 2009. © 2009 Wiley Periodicals, Inc.Link_to_subscribed_fulltex

Macrophage matrix metalloproteinase-2/-9 gene and protein expression following adhesion to ECM-derived multifunctional matrices via integrin complexation

Macrophages are commonly observed at the biomaterial-tissue interface and interact with the extracellular matrix (ECM) mainly by integrin receptors to play a critical role in ECM turnover by secreting matrix metalloproteinases (MMPs). To investigate β1 and β3 containing integrin-mediated adhesion and subsequent MMP-2/-9 protein and gene expression in human blood-derived monocytes, biofunctional peptides immobilized onto flexible polyethylene glycol (PEG) arms were grafted onto a gelatin-based interpenetrating network (IPN). Adherent monocyte density was dramatically greater in the presence of RGD immobilized onto flexible PEG arms of the gelatin-based IPN. Pretreatment of monocytes with either anti-integrin β1 or β3 led to a significant decrease in adherent cell density on RGD-PEG-grafted IPNs. MMP-2 and MMP-9 protein and MMP-9 mRNA expression increased in the presence of IPNs initially, independent of ligand identity. Anti-integrin β1 or β3 antibody pretreatment of monocytes led to a general decrease in MMP-2/-9 protein expression. These results demonstrate the importance of β1 and β3 containing integrins in mediating monocyte adhesion onto RGD immobilized onto flexible PEG arms of the IPN. The results also reveal that MMP-2/-9 protein and gene expression is influenced by the presence of gelatin and not the ligands immobilized on the PEG arms of the IPN. © 2006 Elsevier Ltd. All rights reserved.Link_to_subscribed_fulltex

Either integrin subunit β1 or β3 is involved in mediating monocyte adhesion, IL-1β protein and mRNA expression in response to surfaces functionalized with fibronectin-derived peptides

We synthesized gelatin-based, interpenetrating network (IPN) scaffolds immobilized with fibronectin (FN)-derived peptides to assess monocyte-biomaterial interaction. Human primary monocytes were seeded onto peptide-grafted IPN or tissue-culture polystyrene (TCPS) pre-adsorbed with FN or FN-derived peptides. Monocyte cell density on both TCPS and IPN surfaces was higher in the presence of the arginine-glycine-aspartic acid (RGD) peptide. Pretreatment with anti-integrin β1 or β3 antibody decreased monocyte density on all ligand-modified TCPS and IPN. Interleukin-1 β (IL-1β) protein levels of cells on modified TCPS decreased over time. IL-1β expression of monocytes in the presence of IPNs peaked at 24 h and then decreased through 168 h. Ligand identity did not affect IL-1β expression in either TCPS or IPN samples. Pretreatment with anti-integrin β1 or β3 antibody reduced IL-1β levels from both TCPS and IPN samples in a ligand-independent manner, particularly at 24 h. Monocytic IL-1β mRNA expression in IPN samples without antibody pretreatment was highest at 2 h and decreased over time. IL-1β mRNA expression in cells with anti-integrin β1 or β3 antibody pretreatment was similar to those without antibody pretreatment, except for methoxygrafted IPN samples. The change in IL-1β mRNA expression did not correlate with changes in protein expression. The results indicate that monocyte adhesion was affected by the substrate and the RGD sequence and β1 or β3 containing integrin receptors. β1- or β3-containing integrin receptors were also involved in IL-1β gene and protein expression in monocytes adhered to gelatin-based biomaterial surfaces. © 2007 VSP.Link_to_subscribed_fulltex

Monocytic U937 adhesion, tumor necrosis factor-alpha and interleukin-1 beta expression in response to gelatin-based networks grafted with arginine-glycine-aspartic acid and proline-histidine-serine-arginine-asparagine oligopeptides

In this study we synthesized gelatin-based, tissue-engineering, interpenetrating network (IPN) scaffolds immobilized with fibronectin (FN)-derived peptides to assess monocyte-biomaterial interaction. Human promonocytic U937 cells were seeded onto peptide-grafted IPN or tissue-culture polystyrene plate (TCPS) pre-adsorbed with FN or FN-derived peptides. The presence of RGD influenced U937 density on IPN. Interleuldn-1 beta (IL-1β) messenger ribonucleic acid (mRNA) expression in adherent U937 on treated TCPS was slightly upregulated at 4h. Tumor necrosis factor alpha (TNF-α) and IL-1β mRNA expression in adherent U937 on all IPNs was generally downregulated at 4 h. This downregulation of IL-1β mRNA apparently varied in IPNs grafted with different ligand and was still present at 24 h. TNF-α and IL-1β proteins released from U937 on treated TCPS were comparable with the control at 24 h, but TNF-α and IL-1β protein expression in U937 on IPNs was lower at 24 h than on the TCPS control. The results indicate that the tissue-engineering substrate and the bioactive peptides modulate the initial U937 adhesion and the subsequent inflammatory cytokine gene and protein expression. © Mary Ann Liebert, Inc.Link_to_subscribed_fulltex

Monocyte inflammatory and matrix remodeling response modulated by grafted ECM-derived ligand concentration

Ligands presented on biomaterials are a common method to facilitate and control the host response. In a gelatin and polyethylene glycol diacrylate (PEGdA) based semi-interpenetrating network (sIPN), the effects of extracellular matrix (ECM)-derived peptide amount on monocyte adhesion and subsequent protein and mRNA expression were examined. Peptide amount on the sIPN surface was controlled by varying the wt % ratio of the peptide-PEG grafted gelatin to PEGdA. We hypothesized that increasing bioactive peptide amount would modulate human blood-derived monocyte adhesion, cytokine expression, and gene regulation. Monocyte adhesion, release of gelatin degrading proteases matrix metalloprotease-2 (MMP-2), matrix metalloprotease-9 (MMP-9), and proinflammatory protein interleukin-1β (IL-1β), and mRNA expression of these proteins were evaluated. We found RGD-PEG grafted sIPNs with higher surface RGD concentrations showed increased adherent density. MMP-2 and IL-1β protein release was also influenced by the ligand concentration, as initial increase in protein concentration was observed at higher ligand concentrations. MMP-9 protein showed an initial increase that subsided then increased. A decreased IL-1β protein and mRNA expression was observed over time but MMP-2 mRNA was not detected at any time though MMP-2 protein concentrations showed an initial burst. Hence, monocyte behavior was modulated by surface ligand identity in tandem with ligand concentration. © 2008 Wiley Periodicals, Inc.Link_to_subscribed_fulltex

Thiol-ene-based biological/synthetic hybrid biomatrix for 3-D living cell culture

Although various cell encapsulation materials are available commercially for a wide range of potential therapeutic cells, their combined clinical impact remains inconsistent. Synthetic materials such as poly(ethylene glycol) (PEG) hydrogels are mechanically robust and have been extensively explored but lack natural biofunctionality. Naturally derived materials including collagen, fibrin and alginate-chitosan are often labile and mechanically weak. In this paper we report the development of a hybrid biomatrix based on the thiol-ene reaction of PEG diacrylate (PEGdA) and cysteine/PEG-modified gelatin (gel-PEG-Cys). We hypothesized that covalent crosslinking decreases gelatin dissolution thus increasing gelatin resident time within the matrix and the duration of its biofunctionality; at the same time the relative ratio of PEGdA to gel-PEG-Cys in the matrix formulation directly affects hydrogel bulk and local microenvironment properties. Bulk viscoelastic properties were highly dependent on PEGdA concentration and total water content, while gel-PEG-Cys concentration was more critical to swelling profiles. Microviscoelastic properties were related to polymer concentration. The covalently crosslinked gel-PEG-Cys with PEGdA decreased gelatin dissolution out of the matrix and collagenase-mediated degradation. Fibroblasts and keratinocyte increased adhesion density and formed intercellular connections on stiffer hydrogel surfaces, while cells exhibited more cytoplasmic spreading and proliferation when entrapped within softer hydrogels. Hence, this material system contains multiparametric factors that can easily be controlled to modulate the chemical, physical and biological properties of the biomatrix for soft tissue scaffolding and cell presentation to reconstruct lost tissue architecture and physical functionality. © 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.Link_to_subscribed_fulltex

Pomalidomide, dexamethasone, and daratumumab immediately after lenalidomide-based treatment in patients with multiple myeloma: updated efficacy, safety, and health-related quality of life results from the phase 2 MM-014 trial.

Patients with relapsed/refractory multiple myeloma (RRMM) need proven subsequent therapies after early-line lenalidomide treatment failure. The phase 2 MM-014 trial (NCT01946477) investigated pomalidomide, dexamethasone, and daratumumab after 1 to 2 prior treatment lines (62.5%, 1 prior line) in patients with RRMM and prior lenalidomide (75.0%, lenalidomide refractory). With a median follow-up of 28.4 months, overall response rate was 77.7% (52.7% achieved very good partial response or better) and median progression-free survival was 30.8 months. For patients with lenalidomide-refractory disease, these outcomes were 76.2%, 47.6%, and 23.7 months, respectively. No new safety signals were observed; 64.3% experienced grade 3/4 neutropenia. Health-related quality of life was preserved or trended toward improvement through 12 treatment cycles. Pomalidomide, dexamethasone, and daratumumab given immediately after early-line lenalidomide-based treatment continues to demonstrate safety and efficacy, supporting pomalidomide-dexamethasone as a foundation of combination therapy in RRMM and providing evidence that the immunomodulatory agent class delivers benefit after lenalidomide treatment failure

Pomalidomide plus low-dose dexamethasone in relapsed refractory multiple myeloma after lenalidomide treatment failure.

Patients with relapsed/refractory multiple myeloma (RRMM) for whom the benefits of lenalidomide have been exhausted in early treatment lines need effective therapies. In cohort A of the phase 2 MM-014 trial, we examined the safety and efficacy of pomalidomide plus low-dose dexamethasone immediately after lenalidomide-based treatment failure in patients with RRMM and two prior lines of therapy. Pomalidomide 4 mg was given on days 1 to 21 of 28-day cycles. Dexamethasone 40 mg (20 mg for patients aged >75 years) was given on days 1, 8, 15 and 22 of 28-day cycles. The primary endpoint was overall response rate (ORR), and secondary endpoints included progression-free survival (PFS), overall survival (OS) and safety. The intention-to-treat population comprised 56 patients; all received prior lenalidomide (87·5% lenalidomide refractory) and 39 (69·6%) received prior bortezomib. ORR was 32·1% (28·2% in the prior-bortezomib subgroup). Median PFS was 12·2 months (7·9 months in the prior-bortezomib subgroup). Median OS was 41·7 months (38·6 months in the prior-bortezomib subgroup). The most common grade 3/4 treatment-emergent adverse events were anaemia (25·0%), pneumonia (14·3%) and fatigue (14·3%). These findings support earlier sequencing of pomalidomide-based therapy in lenalidomide-pretreated patients with RRMM, including those who have become refractory to lenalidomide. Trial registration: www.ClinicalTrials.gov identifier NCT01946477