Aortic Adventitial Fibroblasts Research Articles

Smad2/3 signaling involved in urotensin II-induced phenotypic differentiation, collagen synthesis and migration of rat aortic adventitial fibroblasts

Objective. To investigate whether Smad2/3 signaling is involved in urotensin II (UII) induced activation of aortic adventitial fibroblasts. Materials and Methods. Growth-arrested adventitial fibroblasts were stimulated with UII in the presence or absence of urotensin II receptor (UT) antagonist SB710411 or transfected with Smad2/3 small inhibitory RNA (siRNA). UII stimulated Smad2/3 phosphorylation, α-smooth muscle actin (α-SMA), and collagen I expression and migration of adventitial fibroblasts were evaluated by western blot analysis, real-time reverse transcription polymerase chain reaction, immunofluorescence, ELISA, and transwell migration assay, respectively. Results. In cultured adventitial fibroblasts, UII time- and dose-dependently stimulated Smad2/3 protein phosphorylation, with maximal effect at 10-8 mol/l (increased by 147.2%, P<0.001). UII stimulated Smad2/3 upregulation and nuclear translocation. SB710411 significantly inhibited these effects. In addition, UII potently induced α-SMA and procollagen 1 protein or mRNA expression (P<0.01), which were completely blocked by Smad2 (decreased by 75.1%, 54.2% in protein, and by 73.3% and 38.2% in mRNA, respectively, P<0.01) or Smad3 siRNA (decreased by 80.3% and 47.0% in protein, and by 72.3% and 47.7% in mRNA, respectively, P<0.01). Meanwhile, Smad2 or smad3 siRNA significantly inhibited the UII-induced collagen 1 secretion and cell migration. Conclusions. UII may stimulate adventitial-fibroblast phenotype conversion, migration, and collagen I synthesis via phosphorylated-Smad2/3 signal transduction pathways.

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.

PS-BPB01-5: LOX-1 DEFICIENCY INCREASES RUPTURED ABDOMINAL AORTIC ANEURYSM VIA THINNING OF ADVENTITIAL COLLAGEN

Objective: Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a key mediator of inflammation and plays an important role in the pathogenesis of atherosclerosis. Both inflammation and atherosclerosis have been proposed as contributors to abdominal aortic aneurysm (AAA) pathogenesis. However, the role of LOX-1 in AAA pathogenesis remains unknown. We aimed to investigate effects of Olr1, which encodes LOX-1, deletion on angiotensin II (Ang II)-induced AAA in apolipoprotein E knockout (ApoE KO) mice to determine whether LOX-1 deficiency mitigates AAA development. Design and method: Either Ang II (1.44 mg/kg/day) or saline (control) was administered continuously through a subcutaneously implanted osmotic pump for 4 weeks in the following 8-week-old male mice: Apoe-/-Olr1+/+ mice (Ang II, n = 40; saline, n = 10), Apoe-/-Olr1+/- mice (Ang II, n = 30; saline, n = 9), and Apoe-/-Olr1-/- mice (Ang II, n = 39; saline, n = 9). Results: Ang II infusion raised systolic blood pressure by tail-cuff methods in all three groups. Serial non-invasive assessment with ultrasound demonstrated that Ang II gradually increased the maximum area of the suprarenal abdominal aortas leading to AAAs. Although the incidence and expansion rate of AAA were comparable among genotypes, Olr1 deletion significantly increased the severity and rupture rate of AAAs (p = 0.01; incidence of type IV and ruptured AAA was 25% in Apoe-/-Olr1-/- mice vs 5% in Apoe-/-Olr1+/+ mice). Since atherosclerosis is considered a major contributor to the development of AAAs, the extent of the atheroma burden localized in aneurysmal lesions was evaluated by Oil Red O staining. Olr1 deletion did not decrease atherosclerosis formation in the aneurysmal wall. Interestingly, further histopathological analysis revealed that aneurysmal lesions in LOX-1-deficient mice had fewer fibroblasts and myofibroblasts, as well as thinner adventitial collagen, although the degree of elastin fragmentation or disruption was similar between LOX-1-deficient and control mice. These in vivo data suggested that adventitial fibroblasts play a pivotal role in the increased severity of AAA in LOX-1-deficient mice. To further determine the effects of Olr1 deletion on fibroblast differentiation, fibroblasts were isolated from the adventitia of abdominal aortas harvested from 8-week-old Apoe-/-Olr1+/+ and Apoe-/-Olr1-/- mice, and then cultured with Ang II stimulation. An in vitro study confirmed that Olr1 deletion completely abolished the Ang II-induced proliferative activity in the aortic adventitial fibroblasts (p = 0.02). Conclusions: Olr1 deletion may not mitigate aneurysm development but rather increase the vulnerability to rupture by suppressing adventitial fibroblast proliferation and collagen.

Metformin Attenuates Hyperglycaemia-Stimulated Pro-Fibrotic Gene Expression in Adventitial Fibroblasts via Inhibition of Discoidin Domain Receptor 2.

Molecular mechanisms underlying the diverse therapeutic effects of anti-diabetic metformin, beyond its anti-hyperglycaemic effects, remain largely unclear. Metformin is reported to reduce the long-term complications of diabetes, including cardiovascular fibrosis and remodelling. Our recent investigations show that Discoidin Domain Receptor 2 (DDR2), a Collagen receptor tyrosine kinase, has an obligate regulatory role in Collagen type I gene expression in cardiac and vascular adventitial fibroblasts, and that it may be a molecular link between arterial fibrosis and metabolic syndrome in rhesus monkeys. Using gene knockdown and overexpression approaches, the present study examined whether DDR2 is a target of metformin and whether, by targeting DDR2, it inhibits Fibronectin and Collagen type I expression in rat aortic adventitial fibroblasts exposed to hyperglycaemic conditions. Metformin was found to attenuate hyperglycaemia-induced increase in DDR2 mRNA and protein expression by inhibiting TGF-β1/SMAD2/3 signalling that mediates the stimulatory effect of hyperglycaemia on DDR2 expression. Metformin also inhibited DDR2-dependent expression of Fibronectin and Collagen type I, indicating that it regulates these matrix proteins via DDR2 inhibition. The findings identify DDR2, a mediator of cardiovascular remodelling, as a molecular target of metformin, thereby uncovering the molecular basis of its protective role in vascular fibrosis and possibly cardiac fibrosis associated with diabetic cardiomyopathy.

Elabela blunts doxorubicin-induced oxidative stress and ferroptosis in rat aortic adventitial fibroblasts by activating the KLF15/GPX4 signaling.

Doxorubicin (DOX) is a chemotherapeutic drug for a variety of malignancies, while its application is restricted by the cardiovascular toxic effects characterized by oxidative stress. Ferroptosis is a novel iron-dependent regulated cell death driven by lipid peroxidation. Our study aimed to investigate the role of Elabela (ELA) in DOX-induced oxidative stress and ferroptosis. In cultured rat aortic adventitial fibroblasts (AFs), stimulation with DOX dramatically induced cytotoxicity with reduced cell viability and migration ability, and enhanced lactate dehydrogenase (LDH) activity. Importantly, ELA and ferrostatin-1 (Fer-1) mitigated DOX-mediated augmentation of reactive oxygen species (ROS) in rat aortic AFs, accompanied by upregulated levels of Nrf2, SLC7A11, GPX4, and GSH. In addition, ELA reversed DOX-induced dysregulation of apoptosis- and inflammation-related factors including Bax, Bcl2, interleukin (IL)-1β, IL6, IL-10, and CXCL1. Intriguingly, knockdown of Krüppel-like factor 15 (KLF15) by siRNA abolished ELA-mediated alleviation of ROS production and inflammatory responses. More importanly, KLF15 siRNA impeded the beneficial roles of ELA in DOX-pretreated rat aortic AFs by suppressing the Nrf2/SLC7A11/GPX4 signaling. In conclusion, ELA prevents DOX-triggered promotion of cytotoxicity, and exerts anti-oxidative and anti-ferroptotic effects in rat aortic AFs via activation of the KLF15/GPX4 signaling, indicating a promising therapeutic value of ELA in antagonizing DOX-mediated cardiovascular abnormality and disorders.

C1q/gC1qR‐Mediated Activation of Extrinsic Coagulation

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. C1q has an affinity for gC1qR, a cell surface receptor molecule that is commonly found on cell types associated with cardiovascular disease development, including vascular smooth muscle cells and adventitial fibroblasts. When C1q interacts with gC1qR, many pathological pathways initiate including the inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. The intrinsic cascade plays a minor role in physiological and pathological thrombosis as compared with the extrinsic cascade; however, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation have not been studied. We hypothesized that C1q association with gC1qR would increase the amount of sub‐endothelial tissue factor expressed by both aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). We investigated these cells as they are the primary TF bearing cells. Further, we hypothesized that the newly expressed TF would bind Factor VII (FVII), which is a crucial step in the initiation of the extrinsic coagulation cascade. The objective of this study was to elucidate the role of C1q/gC1qR‐mediated TF expression and its ability to bind FVII. Using a solid‐phase ELISA, TF expression was observed on both HAAFs and HCASMCs. Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti‐gC1qR antibodies (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. A capture ELISA approach was used to evaluate the binding of FVII to TF following exposure after the same conditions. Further, a capture ELISA was used to assess released TF. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 15% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies prior to conditioning with C1q exhibited no change in TF expression when compared to unconditioned cells. Additionally, a 20‐30% increase in bound FVII was observed in both cell types when conditioned with C1q as compared to unconditioned cells. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying a convergence of inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF and the binding of FVII can support downstream extrinsic coagulation activity. Thanks to NIH for supporting this research.

MiRNA-122-5p inhibitor abolishes angiotensin II-mediated loss of autophagy and promotion of apoptosis in rat cardiofibroblasts by modulation of the apelin-AMPK-mTOR signaling.

MicroRNAs (miRNAs) have emerged as essential regulators that could have pivotal roles in cardiac homeostasis and pathological remodeling of various cardiovascular diseases. We previously demonstrated that miRNA-122-5p overexpression exacerbated the process of vascular hypertrophy, fibrosis, and dysfunction in hypertensive rats and rat aortic adventitial fibroblasts. However, the exact roles and underlyingmechanisms of miRNA-122-5p in myocardial fibroblasts remain largely unknown. In this work, neonatal rat cardiofibroblasts (CFs) were isolated and primarily cultured from the hearts of 2- to 3-d-old Sprague-Dawley rats. Stimulation of angiotensin II (Ang II) resulted in marked increases in cellular proliferation and migration and levels of collagen I, collagen III, CTGF, and TGF-β1 in cultured CFs. Furthermore, Ang II led to promoted expression of P62, Bax, and phosphorylated mTOR as well as downregulation of LC3II, beclin-1, and AMPK-phosphorylated levels, thereby contributing to imbalance of autophagyand apoptosis, and cellular injury in CFs, which were significantly ameliorated by treatment with miRNA-122-5p inhibitor. These changes were associated with decreased levels of collagen I, collagen III, CTGF, and TGF-β1. Furthermore, Ang II-induced loss of autophagy and promotion of apoptosis in CFs were prevented by the treatment with Pyr1-apelin-13 or AMPK agonist AICAR or mTOR inhibitor rapamycin, respectively. In contrast, administration of miRNA-122-5p mimics and autophagy inhibitor 3-methylademine reversed beneficial roles of Pyr1-apelin-13. Collectively, these data indicated that miRNA-122-5p is an essential regulator of autophagy and apoptosis in rat CFs via the apelin/AMPK/mTOR signaling pathway, which may be potentially used as a therapeutic target in myocardial fibrosis and related diseases.

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.

Elabela prevents angiotensin II-induced apoptosis and inflammation in rat aortic adventitial fibroblasts via the activation of FGF21-ACE2 signaling.

Apoptosis, inflammation, and fibrosis contribute to vascular remodeling and injury. Elabela (ELA) serves as a crucial regulator to maintain vascular function and has been implicated in the pathogenesis of hypertensive vascular remodeling. This study aims to explore regulatory roles and underlying mechanisms of ELA in rat aortic adventitial fibroblasts (AFs) in response to angiotensin II (ATII). In cultured AFs, exposure to ATII resulted in marked decreases in mRNA and protein levels of ELA, fibroblast growth factor 21 (FGF21), and angiotensin-converting enzyme 2 (ACE2) as well as increases in apoptosis, inflammation, oxidative stress, and cellular migration, which were partially blocked by the exogenous replenishment of ELA and recombinant FGF21, respectively. Moreover, treatment with ELA strikingly reversed ATII-mediated the loss of FGF21 and ACE2 levels in rat aortic AFs. FGF21 knockdown with small interfering RNA (siRNA) significantly counterbalanced protective effects of ELA on ATII-mediated the promotion of cell migration, apoptosis, inflammatory, and oxidative injury in rat aortic AFs. More importantly, pretreatment with recombinant FGF21 strikingly inhibited ATII-mediated the loss of ACE2 and the augmentation of cell apoptosis, oxidative stress, and inflammatory injury in rat aortic AFs, which were partially prevented by the knockdown of ACE2 with siRNA. In summary, ELA exerts its anti-apoptotic, anti-inflammatory, and anti-oxidant effects in rat aortic AFs via activation of the FGF21–ACE2 signaling. ELA may represent a potential candidate to predict vascular damage and targeting the FGF21–ACE2 signaling may be a promising therapeutic intervention for vascular adventitial remodeling and related disorders.

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.

Complement Component C1q Initiates Extrinsic Coagulation via gC1qR

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. When C1q interacts with gC1qR, many pathological pathways initiate and C1q/gC1qR plays a role in inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. While this link between inflammation and thrombosis is important, the intrinsic cascade plays a minor role in physiological and pathological thrombosis. The effects of C1 and the role of C1q/gC1qR on extrinsic coagulation, which is more physiologically relevant, has not been studied. Extrinsic coagulation initiates upon vessel damage, exposing tissue factor (TF) to the blood. Adventitial fibroblasts and vascular smooth muscle cells are the primary TF bearing cells. We hypothesized that C1q binding to gC1qR enhances the expression of sub-endothelial TF in these cells. The objective of this study was to elucidate the role of gC1qR-mediated TF expression. Using a solid-phase ELISA, TF expression was observed on aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti-gC1qR (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. Further, a capture ELISA was used to assess released TF under these same conditions. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 35% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies exhibited a marked decrease in TF expression (by approximately 10-20%) when compared to cells conditioned with C1q alone. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying common inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF can support extrinsic coagulation activity.

Acute Exposure of Atmospheric Ultrafine Particles Induced Inflammation Response and Dysregulated TGFβ/Smads Signaling Pathway in ApoE-/- Mice.

Ultrafine particles (UFPs) referred to particular matters with aerosol diameter less than 100nm. Because of the lightweight and small size, UFPs have become an occupational inhalation risk. The UFPs will be accumulated in the deep lung through inhalation, and then reach into all the organs via circulation system; some UFPs even stay in the brain. As previous study reported, UFPs exposure is usually associated with cardiovascular disease, such as atherosclerosis (AS). In our study, we tried to understand how acute UFP exposure caused the biological dysregulation in atherosclerosis. Acute exposure of UFPs were applied to mice for 6 consecutive days, mice were sacrificed after 3, 5, 7, and 10days post-exposure. Aorta and serum were collected for histological and biomarkers analysis. Mice aortic adventitial fibroblasts (MAFs) were isolated from mice and used to further study to understand the mechanism of UFPs induced atherosclerosis. Compared to the untreated control, the inflammation responses and nitrate stress were observed after acute exposure of UFPs, with increased IL-6, MCP-1, p47phox, and 3-NT levels in the mice serum. Besides, upregulation of microRNAs: miR-301b-3p and Let-7c-1-3p, and their downstream target: Smad2, Smad3, and TGFβ1 were also observed in mouse aorta after acute exposure of UFPs. Similar results were identified in vitro as well. Acute exposure of UFPs induced the systematic nitrate stress and inflammation responses, along with the changes of vascular permeability. Dysregulated miRNAs and TGFβ/Smads signaling pathway indicated the higher risk of atherosclerosis/vasculature remodeling when exposed to UFPs.

Regulation of neovasculogenesis in co-cultures of aortic adventitial fibroblasts and microvascular endothelial cells by cell-cell interactions and TGF-β/ALK5 signaling.

Adventitial fibroblasts (AFs) are critical mediators of vascular remodeling. However, the contributions of AFs towards development of vasculature and the specific mechanisms by which these cells regulate physiological expansion of the vasa vasorum, the specialized microvasculature that supplies nutrients to the vascular wall, are not well understood. To determine the regulatory role of AFs in microvascular endothelial cell (MVEC) neovasculogenesis and to investigate the regulatory pathways utilized for communication between the two cell types, AFs and MVECs were cultured together in poly(ethylene glycol)-based hydrogels. Following preliminary evaluation of a set of cell adhesion peptides (AG10, AG73, A2G78, YIGSR, RGD), 7.5wt% hydrogels containing 3 mM RGD were selected as these substrates did not initiate primitive tubule structures in 3D MVEC monocultures, thus providing a passive platform to study AF-MVEC interaction. The addition of AFs to hydrogels promoted MVEC viability; however, increasing AF density within hydrogels stimulated MVEC proliferation, increased microvessel density and size, and enhanced deposition of basement membrane proteins, collagen IV and laminin. Importantly, AF-MVEC communication through the transforming growth factor beta (TGF-β)/activin receptor-like kinase 5 (ALK5) signaling pathway was observed to mediate microvessel formation, as inhibition of ALK5 significantly decreased MVEC proliferation, microvessel formation, mural cell recruitment, and basement membrane production. These data indicate that AFs regulate MVEC neovasculogenesis and suggest that therapeutics targeting the TGF-β/ALK5 pathway may be useful for regulation of vasculogenic and anti-vasculogenic responses.

MicroRNA-122 aggravates angiotensin II-mediated apoptosis and autophagy imbalance in rat aortic adventitial fibroblasts via the modulation of SIRT6-elabela-ACE2 signaling

Abnormal aortic adventitial fibroblasts (AFs) play essential roles in the development of vascular remodeling and disorders. Previous studies revealed that microRNA-122 (miR-122) levels were elevated in the aortic adventitia of hypertensive rats with vascular injury. Here, we aim to evaluate the biological effects and underlying mechanisms of miR-122 in rat AFs. Exposure to angiotensin II (ATII) in rat AFs resulted in decreased levels of sirtuin 6 (SIRT6), elabela (ELA), and angiotensin-converting enzyme 2 (ACE2). Additionally, stimulation with ATII contributed to a decline in autophagic flux and obvious increases in cellular migration, oxidative stress, and apoptosis, which were exacerbated by the transfection of miR-122-5p mimic but were rescued by miR-122-5p inhibitor, exogenous replenishment of ELA, and recombinant adeno-associated virus expressing SIRT6 (rAAV-SIRT6), respectively. Moreover, stimulation with miR-122-5p mimic led to a marked reduction in the levels of SIRT6 and ELA in rat AFs, which were elevated by stimulation with rAAV-SIRT6. Furthermore, miR-122-5p inhibitor-mediated pro-autophagic, anti-oxidant and anti-apoptotic effects in rat AFs were partially suppressed by 3-methyladenine, SIRT6 small interfering RNA (siRNA) and ELA siRNA, which were linked with the downregulation in the protein levels of LC3-II, beclin-1, and ACE2 and the upregulation of p62 expression and bax/bcl-2 ratio. Our findings indicated that miR-122-5p inhibition prevented ATII-mediated loss of autophagy, and the promotion of apoptosis and oxidative stress via activating the SIRT6-ELA-ACE2 signaling. MiR-122-5p may be a novel predictive biomarker of adventitial injury, and targeting the SIRT6-ELA-ACE2 signaling may have the potential therapeutic importance of controlling vascular remodeling and disorders.

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.

MiR155-5p in adventitial fibroblasts-derived extracellular vesicles inhibits vascular smooth muscle cell proliferation via suppressing angiotensin-converting enzyme expression

ABSTRACT Proliferation of vascular smooth muscle cells (VSMCs) plays crucial roles in vascular remodelling and stiffening in hypertension. Vascular adventitial fibroblasts are a key regulator of vascular wall function and structure. This study is designed to investigate the roles of adventitial fibroblasts-derived extracellular vesicles (EVs) in VSMC proliferation and vascular remodelling in normotensive Wistar-Kyoto rat (WKY) and spontaneously hypertensive rat (SHR), an animal model of human essential hypertension. EVs were isolated from aortic adventitial fibroblasts of WKY (WKY-EVs) and SHR (SHR-EVs). Compared with WKY-EVs, miR155-5p content was reduced, while angiotensin-converting enzyme (ACE) content was increased in SHR-EVs. WKY-EVs inhibited VSMC proliferation of SHR, which was prevented by miR155-5p inhibitor. SHR-EVs promoted VSMC proliferation of both strains, which was enhanced by miR155-5p inhibitor, but abolished by captopril or losartan. Dual luciferase reporter assay showed that ACE was a target gene of miR155-5p. MiR155-5p mimic or overexpression inhibited VSMC proliferation and ACE upregulation of SHR. WKY-EVs reduced ACE mRNA and protein expressions while SHR-EVs only increased ACE protein level in VSMCs of both strains. However, the SHR-EVs-derived from the ACE knockdown-treated adventitial fibroblasts lost the roles in promoting VSMC proliferation and ACE upregulation. Systemic miR155-5p overexpression reduced vascular ACE, angiotensin II and proliferating cell nuclear antigen levels, and attenuated hypertension and vascular remodelling in SHR. Repetitive intravenous injection of SHR-EVs increased blood pressure and vascular ACE contents, and promoted vascular remodelling in both strains, while WKY-EVs reduced vascular ACE contents and attenuated hypertension and vascular remodelling in SHR. We concluded that WKY-EVs-mediated miR155-5p transfer attenuates VSMC proliferation and vascular remodelling in SHR via suppressing ACE expression, while SHR-EVs-mediated ACE transfer promotes VSMC proliferation and vascular remodelling.

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.