Human Aortic Adventitial Fibroblasts Research Articles

Developing Biomimetic Hydrogels of the Arterial Wall as a Prothrombotic Substrate for In Vitro Human Thrombosis Models.

Current in vitro thrombosis models utilise simplistic 2D surfaces coated with purified components of the subendothelial matrix. The lack of a realistic humanised model has led to greater study of thrombus formation in in vivo tests in animals. Here we aimed to develop 3D hydrogel-based replicas of the medial and adventitial layers of the human artery to produce a surface that can optimally support thrombus formation under physiological flow conditions. These tissue-engineered medial- (TEML) and adventitial-layer (TEAL) hydrogels were developed by culturing human coronary artery smooth muscle cells and human aortic adventitial fibroblasts within collagen hydrogels, both individually and in co-culture. Platelet aggregation upon these hydrogels was studied using a custom-made parallel flow chamber. When cultured in the presence of ascorbic acid, the medial-layer hydrogels were able to produce sufficient neo-collagen to support effective platelet aggregation under arterial flow conditions. Both TEML and TEAL hydrogels possessed measurable tissue factor activity and could trigger coagulation of platelet-poor plasma in a factor VII-dependent manner. Biomimetic hydrogel replicas of the subendothelial layers of the human artery are effective substrates for a humanised in vitro thrombosis model that could reduce animal experimentation by replacing current in vivo models.

TGFβ1 induces myofibroblast transdifferentiation via increasing Smad-mediated RhoGDI-RhoGTPase signaling.

This study serves to investigate the effects of the Smad pathway on TGFβ1-mediated RhoGDI expression and its binding to RhoGTPases in myofibroblast transdifferentiation. Myofibroblast transdifferentiation was induced by TGFβ1 in vitro. Cells were pretreated with different siRNAs or inhibitors. Myofibroblast transdifferentiation was detected by immunohistochemistry. Immunofluorescence was used to observe the nuclear translocation of Smad4, and PSR (Picrositius Red) staining was used to measure collagen concentration. TGFβ1 induced the phosphorylation of Smad2/3 and the nuclear translocation of Smad4 in human aortic adventitial fibroblasts (HAAFs). Furthermore, TGFβ1 increased the expression of RhoGDI and its binding to RhoGTPases. Nevertheless, inhibition of Smad2/3 phosphorylation decreased TGFβ1-induced RhoGDI1/2 expressions and RhoGDI2-RhoGTPases interactions. These data suggested that the inhibition of Smad phosphorylation attenuates myofibroblast transdifferentiation by inhibiting TGFβ1-induced RhoGDI1/2 expressions and RhoGDI-RhoGTPases signaling.

Inflammatory, Thrombotic, and Diabetic Responses in Human Arterial Fibroblasts and Endothelial Cells are Regulated by SARS‐CoV‐2 Structural Proteins

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is responsible for many pathological processes, such as altered vascular disease development, dysfunctional thrombosis, and a heightened inflammatory state. Although many clinical reports support these observations, there has been limited experimental work to determine the underlying mechanisms and cellular processes induced by exposure to SARS‐CoV‐2 structural proteins. Importantly, these structural proteins are conserved amongst all current known SARS‐CoVs. Our objective was to investigate the effects of the Spike (S), Nucleocapsid (N), and Membrane‐Envelope (M) SARS‐CoV‐2 structural proteins on inflammation, thrombosis, and diabetic disease markers of human aortic adventitial fibroblasts and human umbilical vein endothelial cells. We hypothesized that short‐term exposure to SARS‐CoV‐2 structural proteins would result in increased expression of inflammatory, thrombotic, and diabetic proteins in both cell types, which would support a mechanism for altered vascular disease progression. To test this, the cells were incubated independently with the three SARS‐CoV‐2 proteins for one hour, after which we analyzed the expression of gC1qR, ICAM‐1, tissue factor, RAGE, GLUT‐4, thrombomodulin, PECAM‐1, and Connexin‐43 (in their respective cell types), using an ELISA approach. All cells were monitored for maintenance of typical culture parameters using a live/dead cell cytotoxicity assay and the MTT assay (for metabolic activity). We observed that each of these markers were significantly up‐regulated after exposure to SARS‐CoV‐2 structural proteins as compared to fibroblasts or endothelial cells that were not exposed to these proteins. Interestingly, the extent of the expression of these markers was sometimes significantly different for each of the SARS‐CoV‐2 structural proteins. This suggests that each of the SARS‐CoV‐2 proteins interacts with these cells through different mechanisms, however, more work will need to be undertaken to determine the mechanisms by which these proteins interact with cells. Thus, these results indicate that the cellular response of vascular cells towards SARS‐CoV‐2 structural proteins promotes inflammatory, thrombotic, and vascular dysfunction. However, these interactions are regulated by complex and possibly different cellular receptors/signal transduction pathways that should be explored further.

Human Adventitial Fibroblast Phenotype Depends on the Progression of Changes in Substrate Stiffness.

Adventitial fibroblasts (AFs) are major contributors to vascular remodeling and maladaptive cascades associated with arterial disease, where AFs both contribute to and respond to alterations in their surrounding matrix. The relationships between matrix modulus and human aortic AF (AoAF) function are investigated using poly(ethylene glycol)-based hydrogels designed with matrix metalloproteinase (MMP)-sensitive and integrin-binding peptides. Initial equilibrium shear storage moduli for the substrates examined are 0.33, 1.42, and 2.90 kPa; after 42 days of culture, all hydrogels exhibit similar storage moduli (0.3-0.7 kPa) regardless of initial modulus, with encapsulated AoAFs spreading and proliferating. In 10 and 7.5 wt% hydrogels, modulus decreases monotonically throughout culture; however, in 5 wt% hydrogels, modulus increases after an initial 7 days of culture, accompanied by an increase in myofibroblast transdifferentiation and expression of collagen I and III through day 28. Thereafter, significant reductions in both collagens occur, with increased MMP-9 and decreased tissue inhibitor of metalloproteinase-1/-2 production. Releasing cytoskeletal tension or inhibiting cellular protein secretion in 5 wt% hydrogels block the stiffening of the polymer matrix. Results indicate that encapsulated AoAFs initiate cell-mediated matrix remodeling and demonstrate the utility of dynamic 3D systems to elucidate the complex interactions between cell behavior and substrate properties.

Three-Layered Silk Fibroin Tubular Scaffold for the Repair and Regeneration of Small Caliber Blood Vessels: From Design to in vivo Pilot Tests.

Silk fibroin (SF) is an eligible biomaterial for the development of small caliber vascular grafts for substitution, repair, and regeneration of blood vessels. This study presents the properties of a newly designed multi-layered SF tubular scaffold for vascular grafting (SilkGraf). The wall architecture consists of two electrospun layers (inner and outer) and an intermediate textile layer. The latter was designed to confer high mechanical performance and resistance on the device, while electrospun layers allow enhancing its biomimicry properties and host's tissues integration. In vitro cell interaction studies performed with adult Human Coronary Artery Endothelial Cells (HCAECs), Human Aortic Smooth Muscle Cells (HASMCs), and Human Aortic Adventitial Fibroblasts (HAAFs) demonstrated that the electrospun layers favor cell adhesion, survival, and growth. Once cultured in vitro on the SF scaffold the three cell types showed an active metabolism (consumption of glucose and glutamine, release of lactate), and proliferation for up to 20 days. HAAF cells grown on SF showed a significantly lower synthesis of type I procollagen than on polystyrene, meaning a lower fibrotic effect of the SF substrate. The cytokine and chemokine expression patterns were investigated to evaluate the cells' proliferative and pro-inflammatory attitude. Interestingly, no significant amounts of truly pro-inflammatory cytokines were secreted by any of the three cell types which exhibited a clearly proliferative profile. Good hemocompatibility was observed by complement activation, hemolysis, and hematology assays. Finally, the results of an in vivo preliminary pilot trial on minipig and sheep to assess the functional behavior of implanted SF-based vascular graft identified the sheep as the more apt animal model for next medium-to-long term preclinical trials.

Distinct Redox Signalling following Macrophage Activation Influences Profibrotic Activity

Aims To date, the ROS-generating capacities of macrophages in different activation states have not been thoroughly compared. This study is aimed at determining the nature and levels of ROS generated following stimulation with common activators of M1 and M2 macrophages and investigating the potential for this to impact fibrosis. Results Human primary and THP-1 macrophages were treated with IFN-γ+LPS or IL-4-activating stimuli, and mRNA expression of established M1 (CXCL11, CCR7, IL-1β) and M2 (MRC-1, CCL18, CCL22) markers was used to confirm activation. Superoxide generation was assessed by L-012-enhanced chemiluminescence and was increased in both M(IFN-γ+LPS) and M(IL-4) macrophages, as compared to unpolarised macrophages (MΦ). This signal was attenuated with NOX2 siRNA. Increased expression of the p47phox and p67phox subunits of the NOX2 oxidase complex was evident in M(IFN-γ+LPS) and M(IL-4) macrophages, respectively. Amplex Red and DCF fluorescence assays detected increased hydrogen peroxide generation following stimulation with IL-4, but not IFN-γ+LPS. Coculture with human aortic adventitial fibroblasts revealed that M(IL-4), but not M(IFN-γ+LPS), enhanced fibroblast collagen 1 protein expression. Macrophage pretreatment with the hydrogen peroxide scavenger, PEG-catalase, attenuated this effect. Conclusion We show that superoxide generation is not only enhanced with stimuli associated with M1 macrophage activation but also with the M2 stimulus IL-4. Macrophages activated with IL-4 also exhibited enhanced hydrogen peroxide generation which in turn increased aortic fibroblast collagen production. Thus, M2 macrophage-derived ROS is identified as a potentially important contributor to aortic fibrosis.

T-614 inhibits human aortic adventitial fibroblast proliferation and promotes interleukin-8 production in vitro.

The effect and underlying mechanism of T-614 (iguratimod) on Takayasu's arteritis (TA) are unknown. Here, we report the effects of T-614 on cell proliferation and interleukin-8 (IL-8) production in human aortic adventitial fibroblasts (HAAFs) in vitro and explore its initial benefit in terms of vascular wall inflammation and remodeling for patients with TA. HAAFs were cultured with 0, 5, 50, 100, or 250 μg/ml T-614 in the absence or presence of tumor necrosis factor-α (TNF-α) in vitro. Cell viability was determined by a modified MTT assay. Supernatant IL-8 levels were measured by enzyme-linked immunosorbent assays. In the presence of TNF-α, compared to that in the control group, cell viability of HAAFs significantly decreased in the 50, 100, and 250 μg/ml T-614 treatment groups (OD value: P < 0.01, P < 0.001, P < 0.001, respectively; survival fraction (SF): P < 0.05, P < 0.001, P < 0.001, respectively). However, there was no significant difference in cell viability between TNF-α-stimulated and unstimulated groups at the same concentration of T-614. In the absence or presence of TNF-α, T-614 suppressed HAAF cell viability dose-dependently (OD value: r = -0.915, P = 0.000; r = -0.926, P = 0.000, respectively; SF: r = -0.897, P = 0.000; r = -0.885, P = 0.000, respectively). Compared to that in the control group, in the absence of TNF-α, IL-8 levels in the 5 and 100 μg/ml T-614-treated groups were significantly higher (P < 0.05); in the presence of TNF-α, IL-8 levels in the 5, 50, and 100 μg/ml T-614-treated groups were significantly higher (P < 0.001, P < 0.001, P < 0.01, respectively). Further, there was a negative correlation between supernatant IL-8 levels and T-614 concentration in groups stimulated with TNF-α (r = -0.670, P = 0.000), but there was no significant correlation between these parameters in groups that were not stimulated with TNF-α. In the absence or presence of TNF-α, T-614 can inhibit HAAF proliferation and promote IL-8 production in vitro; therefore, it could be used to prevent adventitial thickening of the aorta and improve vascular remodeling in inflammatory environments in vitro and might provide a new immunotherapeutic intervention for TA.

Abstract 119: Ccl18 as a Potential Mediator of the Pro-Fibrotic Actions of M2 Macrophages in the Vessel Wall During Hypertension

M2 macrophages contribute to vascular fibrosis and stiffening in hypertension and may mediate these actions via release of the pro-fibrotic chemokine, CCL18, which they release to a greater extent than TGF-β1. The role of CCL18 and its cognate receptor, CCR8 in hypertension and vascular fibrosis has not been investigated. This study aimed to identify vascular targets of CCL18 and explore its ability to promote fibrosis. Blood pressure (BP) of saline or angiotensin II (0.7 mg/kg/day, 28 days; s.c. )-treated male C57BL/6J mice was measured by tail cuff. Localisation and expression of CCR8 and the murine functional analogue of CCL18, CCL8, were assessed in the thoracic aorta by immunohistochemistry and qRT-PCR, respectively. Human aortic adventitial fibroblasts (AoAFs) and endothelial cells were treated with the pro-fibrotic agent TGF-β1 (10 ng/ml) or CCL18 (3-300 ng/ml) for 3-72h or 7d, respectively. In human AoAFs, expression of pro-collagen I, mature collagen I and α-SMA and were measured (qRT-PCR, Western blotting). Endothelial-mesenchymal transition was measured via VE-cadherin and α-SMA protein expression. Angiotensin II infusion significantly elevated systolic BP (151±5 mmHg, p&lt;0.05) as compared to saline treated mice (111±5 mmHg, n=8). In aortas from hypertensive mice, mRNA expression of CCL8 was elevated 3-fold (p&lt;0.05, n=4-8), and CCL8 expression in the aortic wall was co-localised with the M2 macrophage marker CD206. CCR8 expression was evident in the endothelium, adventitia and perivascular fat. In human AoAFs, TGF-β1 caused a 2-fold increase in protein expression of collagen I (24h, p&lt;0.01) and α-SMA (24-72h, p&lt;0.05). CCL18 (300 ng/ml) did not change α-SMA expression, but increased the protein expression of pro-collagen I by 2-fold (24h; p&lt;0.01), and elevated mature collagen I by 3.6-fold (72h; p&lt;0.05). In human aortic endothelial cells, CCL18 (10 ng/ml) induced a trend towards reduced VE-cadherin, while increasing α-SMA expression (1.5-fold, p&lt;0.05). CCL18 stimulates collagen synthesis from human AoAFs and may promote endothelial-to-mesenchymal cell transition. Expression of its receptor in the vascular wall suggests that the CCL18-CCR8 axis may serve as a potential target for the treatment of vascular fibrosis during hypertension.

ROCK inhibitors alleviate myofibroblast transdifferentiation and vascular remodeling via decreasing TGFβ1-mediated RhoGDI expression.

The aim of this study was to investigate the effects of the Rho GDP dissociation inhibitor (RhoGDI) on TGFβ1-mediated vascular adventitia myofibroblast transdifferentiation and on the inhibition of ROCK inhibitors. Myofibroblast transdifferentiation and vascular remodeling model were induced by TGFβ1 in vitro and by balloon injury in vivo. H&E (Hematoxylin & Eosin) and PSR (Picrosirius Red) staining were used to observe vascular morphology while immunofluorescence, immunohistochemistry, and Western blotting were used to measure protein expression. Fasudil treatment reduced the expression of TGFβ1, RhoGDI1, and RhoGDI2 in addition to vascular remodeling in the rat balloon injury model. TGFβ1 induced the expression of α-SMA, TGFβRI, phospho-TGFβRI, RhoGDI1, RhoGDI2, and collagen secretion in human aortic adventitial fibroblasts (HAAFs). These effects were diminished after treatment with Y27632. Suppressing both RhoGDI1 and RhoGDI2 expression also blocked TGFβ1-induced α-SMA expression and collagen secretion in HAAFs. Moreover, TGFβR inhibition blocked TGFβ1-mediated collagen secretion and the expression of α-SMA, RhoGDI1, and RhoGDI2. These data suggested that ROCK inhibitors alleviate myofibroblast transdifferentiation and vascular remodeling by decreasing TGFβ1-mediated expression of RhoGDI.

Attenuation of Maladaptive Responses in Aortic Adventitial Fibroblasts through Stimuli-Triggered siRNA Release from Lipid-Polymer Nanocomplexes.

Lipid-siRNA assemblies are modified with photo-responsive polymers to enable spatiotemporally-controlled silencing of interleukin 1 beta (IL1β) and cadherin 11 (CDH11), two genes that are essential drivers of maladaptive responses in human aortic adventitial fibroblasts (AoAFs). These hybrid nanocomplexes address the critical challenge of locally mitigating fibrotic actions that lead to the high rates of vascular graft failures. In particular, the lipid-polymer formulations provide potent silencing of IL1β and CDH11 that is precisely modulated by a photo-release stimulus. Moreover, a dynamic modeling framework is used to design a multi-dose siRNA regimen that sustains knockdown of both genes over clinically-relevant timescales. Multi-dose suppression illuminates a cooperative role for IL1β and CDH11 in pathogenic adventitial remodeling and is directly linked to desirable functional outcomes. Specifically, myofibroblast differentiation and cellular proliferation, two of the primary hallmarks of fibrosis, are significantly attenuated by IL1β silencing. Meanwhile, the effects of CDH11 siRNA treatment on differentiation become more pronounced at higher cell densities characteristic of constrictive adventitial remodeling in vivo. Thus, this work offers a unique formulation design for photo-responsive gene suppression in human primary cells and establishes a new dosing method to satisfy the critical need for local attenuation of fibrotic responses in the adventitium surrounding vascular grafts.

Aortic adventitial fibroblast sensitivity to mitogen activated protein kinase inhibitors depends on substrate stiffness.

Adventitial fibroblasts (AFs) are key determinants of arterial function and critical mediators of arterial disease progression. The effects of altered stiffness, particularly those observed across individuals during normal vascular function, and the mechanisms by which AFs respond to altered stiffness, are not well understood. To study the effects of matrix stiffness on AF phenotype, cytokine production, and the regulatory pathways utilized to interpret basic cell-matrix interactions, human aortic AFs were grown in 5%, 7.5%, and 10% (w/v%) PEG-based hydrogels with Young's moduli of 1.2, 3.3, and 9.6kPa, respectively. In 5% gels, AFs had higher proliferation rates, elevated monocyte chemoattractant protein-1 secretion, and enhanced monocyte recruitment. Significantly more AFs were α-smooth muscle actin positive in 7.5% gels, indicating myofibroblast development. AFs in 10% gels had low proliferation rates but produced high levels of interleukin-6 and vascular endothelial growth factor-A. Importantly, these modulus-dependent changes in AF phenotype were accompanied by alterations in the mitogen-activated protein kinase (MAPK) pathways contributing to the production of cytokines. These data indicate that complex cell regulatory changes occur with altered tissue stiffness and suggest that therapeutics affecting MAPK pathways may have altered effects on AFs depending on substrate stiffness.

Collagen matrix stiffness influences fibroblast contraction force

Cell-embedded hydrogel has been widely used as engineered tissue equivalents in biomedical applications. In this study, contraction force in human aortic adventitial fibroblasts seeded within a 3D collagen matrix was quantified by a novel force sensing technique. We demonstrate that contraction forces in cells treated with histamine are regulated by the gel stiffness in a linear manner. These findings provide novel insights into the design of collagen-based biomaterials for tissue engineering and clinical applications.

A novel collagen gel-based measurement technique for quantitation of cell contraction force.

Cell contraction force plays an important role in wound healing, inflammation,angiogenesis and metastasis. This study describes a novel method to quantify single cell contraction force in vitro using human aortic adventitial fibroblasts embedded in a collagen gel. The technique is based on a depth sensing nano-indentation tester to measure the thickness and elasticity of collagen gels containing stimulated fibroblasts and a microscopy imaging system to estimate the gel area. In parallel, a simple theoretical model has been developed to calculate cell contraction force based on the measured parameters. Histamine (100 mM) was used to stimulate fibroblast contraction while the myosin light chain kinase inhibitor ML-7 (25 mM) was used to inhibit cell contraction. The collagen matrix used in the model provides a physiological environment for fibroblast contraction studies. Measurement of changes in collagen gel elasticity and thickness arising from histamine treatments provides a novel convenient technique to measure cell contraction force within a collagen matrix. This study demonstrates that histamine can elicit a significant increase in contraction force of fibroblasts embedded in collagen,while the Young's modulus of the gel decreases due to the gel degradation.

Abstract 80: Modulating the Vascular Healing Response via SIRT1 Transgene Delivery

Bypass graft procedures are commonly performed to treat narrowing or blocked vessels. However, damage to the vascular wall from the surgical intervention can lead to pro-inflammatory events and oxidative stress that promote intimal hyperplasia and may eventually lead to restenosis. Therefore, strategies that seek to reduce inflammation and oxidative stress during the initial stages of wound healing at the graft anastomoses are expected to inhibit intimal hyperplasia and reduce graft failure rates. Sirtuin-1 (Sirt1), an NAD+-dependent lysine deacetylase, protects against inflammation and oxidative stress by deacetylating the RelA/p65 subunit of NF-kB and FOXO transcription factors. We hypothesize that the localized overexpression of Sirt1 at the perivascular wall via lentivirus entrapped in a thermoresponsive gel referred to as nanonets will lead to an improved vascular healing response at the graft anastomoses and attenuate intimal hyperplasia via reduced tissue inflammation and oxidative stress. Human aortic adventitial fibroblasts (AoAFs) were transduced with lentiviruses encoding for Sirt1 and assessed for NF-kB activity, cytokine expression, senescence, and upregulation of antioxidant genes. To assess perivascular transgene delivery, nanonets containing lentiviruses encoding GFP were applied perivascularly around the aortas of rats. After one week of in vivo transduction, aortas were explanted and probed for GFP positive cells. Upon TNFα stimulation, NF-kB activity and cytokine expression levels were decreased when compared to non-transduced and empty vector-transduced AoAFs. Furthermore, Sirt1-overexpression prevented cellular senescence and protected against oxidative stress. Nanonets containing lentiviruses effectively transduced adventitial fibroblasts in vivo. In conclusion, Sirt1 overexpression in AoAFs may be protective against inflammation and oxidative stress.

Effects of heparin and heparin‐binding growth factor on human aortic adventitial fibroblasts (1012.4)

Cardiovascular diseases are primary treated using bypass graft surgery and angioplasty; unfortunately, these procedures often fail due to maladaptive tissue remodeling leading to stenosis, fibrosis, and vessel failure. Methods to decrease failure rates are needed. We are developing instructive biomaterials for placement along the abluminal surface of at‐risk vessels to provide mechanical support and to deliver bioactive molecules and/or stem cells to help attenuate maladaptive responses, encourage healing, and improve clinical outcomes. The present study examines the effects of critical biomaterial components including basic fibroblast growth factor (FGF2), low‐molecular weight heparin (LMWH) and desulfated LMWH on vascular cells.Cell morphology, proliferation, and viability are assessed using fluorescence microscopy. Image Pro and SPSS software packages are used to acquire and analyze data. Data indicate that FGF2 and LMWH affect human aortic adventitial fibroblast (AoAF) phenotype. The presence of FGF2 significantly increased proliferation rates and the appearance of filopodia; whereas, the absence of FGF2 was associated with an increase in the prevalence of lamellipodia and stress fibers. Increasing LMWH doses correlated with decreased AoAF proliferation and alterations in cell morphology. Desulfated LMWH did not alter FGF2‐mediated effects on cell phenotype.These data suggest that the biomaterial components LMWH and FGF2 can influence the phenotype of cardiovascular cells like AoAFs. Although in vivo, LMWH is thought to potentiate FGF2‐induced effects, in our system, LMWH significantly decreased AoAF proliferation, particularly in the absence of FGF2. The sulfation state of LMWH was critical to these effects. Further studies to determine the mechanisms accounting for these effects and to establish combined effects on cell phenotype will be needed to enable development of injectable formulations that may be useful in improving clinical outcome.Grant Funding Source: supported by the Delaware INBRE program with a grant from the National Institute of General Medical

Oncostatin M and TLR-4 Ligand Synergize to Induce MCP-1, IL-6, and VEGF in Human Aortic Adventitial Fibroblasts and Smooth Muscle Cells

Accumulating evidence suggests that adventitial fibroblasts play a significant role in contributing to inflammation of the arterial wall and pathogenesis of atherosclerosis. The effects of gp130 cytokines on these cells (including oncostatin M-[OSM] and IL-6), some of which have been implicated in atherosclerosis, are currently unknown. Experiments were performed to determine whether gp130 cytokines regulate human aortic adventitial fibroblasts (HAoAFs) or smooth muscle cells (HAoSMCs) alone or in context of TLR-4 ligands (also implicated in atherosclerosis). HAoAFs and HAoSMCs were stimulated with LPS and/or one of OSM, IL-6, IL-11, IL-31, or LIF. ELISAs performed on cell supernatants showed that stimulation with OSM alone caused increased MCP-1, IL-6, and VEGF levels. When combined, LPS and OSM synergized to increase MCP-1, IL-6, VEGF protein, and mRNA expression as assessed by qRT-PCR, in both HAoAFs and HAoSMCs, while LPS-induced IL-8 levels were reduced. Such effects were not observed with other gp130 cytokines. Signalling pathways including STATs, MAPKinases, and NFκB were activated, and LPS induced steady state mRNA levels of the OSM receptor chains OSMRβ and gp130. The results suggest that OSM is able to synergize with TLR-4 ligands to induce proinflammatory responses by HAoAFs and HAoSMCs, supporting the notion that OSM regulation of these cells contributes to the pathogenesis of atherosclerosis.

Resilin‐Based Hybrid Hydrogels for Cardiovascular Tissue Engineering

The outstanding elastomeric properties of natural resilin, an insect protein, have motivated the engineering of resilin-like polypeptides (RLPs) as a potential material for cardiovascular tissue engineering. The RLPs, which incorporate biofunctional domains for cell-matrix interactions, are cross-linked into RLP-PEG hybrid hydrogels via a Michael-type addition of cysteine residues on the RLP with vinyl sulfones of an end-functionalized multi-arm star PEG. Oscillatory rheology indicated the useful mechanical properties of these materials. Assessments of cell viability via con-focal microscopy clearly show the successful encapsulation of human aortic adventitial fibroblasts in the three-dimensional matrices and the adoption of a spread morphology following 7 days of culture.

Transforming growth factor-β1 modulates nrf2 redox signalling and enhances migration of human aortic adventitial fibroblasts

Transforming growth factor-β1 modulates nrf2 redox signalling and enhances migration of human aortic adventitial fibroblasts

Abstract 82: TGF-β Regulates Matrix Production in Aortic Fibroblasts Through MicroRNA-29b

Abdominal aortic aneurysms (AAA) cause ∼15,000 annual deaths in the United States. The mechanisms of AAA expansion remain incompletely explored. MicroRNAs (miRs) regulate gene expression post-transcriptionally, and play crucial roles in vascular integrity. TGF-β can act as a profibrotic stimulus, is a known regulator of miR-29b expression in some cell types, and may play a protective role in AAA development. Numerous extracellular matrix genes are predicted targets of miR-29b. We have previously shown that miR-29b is decreased in both human AAA, and in aneurysmal aortic segments in murine AAA models. Further inhibition of miR-29b in vivo reduces abdominal aortic aneurysm progression in two murine AAA models: the porcine-pancreatic-elastase (PPE) model in C57Bl/6 mice, and the angiotensin II-infusion (AngII) model in ApoE -/- mice, and is associated with a pro-fibrotic response within the vessel wall. We performed vascular cell culture studies using human aortic adventitial fibroblasts (AAFs) and aortic smooth muscle cells (ASMCs) to evaluate the expression and regulation of miR-29b. Treatment with recombinant human TGFβ1 significantly decreased miR-29b levels in aortic adventitial fibroblasts, but not smooth muscle cells (vs. untreated cells). TGFβ1 treatment increased soluble collagen production by AAFs nearly 4-fold, and increased gene expression of COL1A1 and COL3A1 . In ASMCs, TGFβ1 primarily increased ELN expression, and COL1A1 and COL3A1 to a lesser extent. We altered miR-29b expression in vitro by transfecting AAFs and ASMCs with either an antagomir (anti-29b) to inhibit activity or a pre-miR (pre-29b) to enhance activity (vs. scrambled miR control). Successful transfection (&gt;50% of all cells) was confirmed by visual fluorescent microscopic analysis and FACS for labeled tag. Anti-29b significantly augmented the effects of TGFβ treatment upon soluble collagen protein levels and collagen gene expression in AAFs, while pre-29b inhibited this response nearly to untreated-control levels. A similar gene expression response was seen in ASMCs. These data suggest that TGFβ regulation of miR-29b in aortic fibroblasts leads to a profibrotic response, partly explaining its protective effects in AAA development.

Aortic Adventitial Fibroblasts Participate in Angiotensin-Induced Vascular Wall Inflammation and Remodeling

Background/Aims: The role of adventitial fibroblasts in the vascular inflammation observed in the adventitia of large vessels in numerous cardiovascular diseases remains unclear. Our objective was to explore the contribution of these cells to angiotensin II (Ang II)-induced aortic inflammation and adventitial expansion. Methods: Cytokine production by primary human aortic adventitial fibroblasts (AoAF) in tissue culture was detected using multiplex ELISA, and increases in cytokine mRNA following Ang II stimulation were quantitated by real-time PCR. The ability of AoAF-derived MCP-1 to attract monocytes was studied in vitro using Boyden assays, and the resulting effect of the monocyte-AoAF interaction on fibroblast proliferation was measured in vitro using proliferation and ³H-thymidine incorporation assays. Ang II-induced fibroblast proliferation was measured in vivo using aortic digestion of single cells followed by flow cytometric quantification of fibroblast numbers as well as fibroblast and PCNA immunostaining. The ability of monocytes to induce AoAF proliferation was demonstrated in vivo using CCR2^+/+ wild-type monocyte adoptive transfer into Ang II-stimulated CCR2-null mice which can produce MCP-1 but have cells lacking the MCP-1 receptor – CCR2. Results: AoAF constitutively secreted numerous proinflammatory cytokines, particularly IL-6 and MCP-1, whose gene expressions were further upregulated in response to Ang II stimulation. AoAF-derived MCP-1 was potent in recruiting THP-1 monocytes in vitro, and these monocytes stimulated AoAF proliferation based on a flow cytometric assessment of cell number and ³H-thymidine incorporation in tissue culture. In vivo, Ang II induced fibroblast proliferation, increased fibroblast and PCNA adventitial staining, and blunted inflammatory responses in the CCR2^–/– background. Injection of CCR2^+/+ monocytes into Ang II-treated CCR2^–/– mice restored adventitial thickening which resulted in increased fibrosis secondary to adventitial fibroblast proliferation. Conclusions: Our results suggest that Ang II-stimulates AoAF to recruit monocytes via fibroblast-derived MCP-1, and the recruited monocytes further activate fibroblast proliferation, adventitial thickening, and additional cytokine production. This fibroblast-monocyte amplification loop may critically mediate hallmarks of adventitial inflammation common to many cardiovascular diseases.